Metformin, Macrophage Dysfunction and Atherosclerosis

REVIEW
published: 07 June 2021

doi: 10.3389/fimmu.2021.682853
Metformin, Macrophage Dysfunction

and Atherosclerosis
Xiaojun Feng 1†, Wenxu Chen 1†, Xiayun Ni 1, Peter J. Little 2,3, Suowen Xu 4*, Liqin Tang 1*
and Jianping Weng 4*
1Department of Pharmacy, the First Affiliated Hospital of University of USTC, Division of Life Sciences and Medicine,
University of Science and Technology of China (USTC), Hefei, China, 2 Sunshine Coast Health Institute, University of the
Sunshine Coast, Birtinya, QLD, Australia, 3 School of Pharmacy, Pharmacy Australia Centre of Excellence, The University of
Queensland, Woolloongabba, QLD, Australia, 4 Department of Endocrinology, The First Affiliated Hospital of USTC, Division
of Life Sciences and Medicine, University of Science and Technology of China( USTC), Hefei, China
Edited by: Metformin is one of the most widely prescribed hypoglycemic drugs and has the potential
Giamila Fantuzzi,
University of Illinois at Chicago,
to treat many diseases. More and more evidence shows that metformin can regulate the
United States function of macrophages in atherosclerosis, including reducing the differentiation of
Reviewed by: monocytes and inhibiting the inflammation, oxidative stress, polarization, foam cell
Liam McKeever,
formation and apoptosis of macrophages. The mechanisms by which metformin
American Society for Parenteral and
Enteral Nutrition, regulates the function of macrophages include AMPK, AMPK independent targets, NF-
United States kB, ABCG5/8, Sirt1, FOXO1/FABP4 and HMGB1. On the basis of summarizing these
Rand T. Akasheh,
American University of Madaba,
studies, we further discussed the future research directions of metformin: single-cell RNA
Jordan sequencing, neutrophil extracellular traps (NETs), epigenetic modification, and metformin-
*Correspondence: based combination drugs. In short, macrophages play an important role in a variety of
Jianping Weng
diseases, and improving macrophage dysfunction may be an important mechanism for
wengjp@ustc.edu.cn
Liqin Tang metformin to expand its pleiotropic pharmacological profile. In addition, the combination
tangliqin@ustc.edu.cn of metformin with other drugs that improve the function of macrophages (such as SGLT2
Suowen Xu
sxu1984@ustc.edu.cn
inhibitors, statins and IL-1b inhibitors/monoclonal antibodies) may further enhance the
†
These authors have contributed
pleiotropic therapeutic potential of metformin in conditions such as atherosclerosis,
equally to this work obesity, cancer, dementia and aging.
Keywords: metformin, atherosclerosis, macrophage, NETs, combination medication
Specialty section:
This article was submitted to
Inflammation,
a section of the journal
Frontiers in Immunology
INTRODUCTION
Received: 19 March 2021 Type 2 diabetes mellitus (T2DM) is a global epidemic. T2DM is highly prevalent in Asia, with
Accepted: 07 May 2021 increased prevalence in India and China (1). Although genetic factors determine the susceptibility of
Published: 07 June 2021
individuals to T2DM to a considerable extent, a sedentary lifestyle and obesogenic diet are
Citation: important predisposing factors for the current global epidemic (1, 2). Studies show lifestyle
Feng XJ, Chen WX, Ni XY, Little PJ, changes can prevent T2DM, including maintaining a healthy weight and diet, maintaining
Xu SW, Tang LQ and Weng JP
physical activity, not smoking and drinking moderately (1–3). Most T2DM patients have at least
(2021) Metformin, Macrophage
Dysfunction and Atherosclerosis.
one morbidity, termed a complication (including cardiovascular, renal, retinal and neurological
Front. Immunol. 12:682853. complications) (1–3). Accelerated atherosclerosis (AS) is the main cause of the high incidence of
doi: 10.3389/fimmu.2021.682853 cardiovascular (CV) complications and premature death in patients with diabetes (4, 5).
Frontiers in Immunology www.frontiersin.org 1 June 2021 olume 12 Article 682853

Feng et al. Metformin Improves Macrophage Dysfunction
Atherosclerosis, depending on the vascular bed affected, can in all aspects of AS, including ECs dysfunction (19, 37), vascular
result in coronary artery disease (CAD), stroke, and peripheral smooth muscle cells (SMCs) proliferation and migration (38, 39),
arterial disease (6). monocyte/macrophage differentiation (40), macrophage
The oxidation of low-density lipoprotein cholesterol (LDL-c) mediated inflammation (41), and foam cell formation (42). In
is considered to be one of the most important of the plethora of addition, metformin improves vascular remodeling caused by
factors associated with the occurrence and development of AS (7, pulmonary hypertension (43). Macrophages play an important
8). Lowering LDL plasma levels is a first line clinical treatment to role in T2DM and its complications (AS), and numerous studies
decrease the risk profile of patients for cardiovascular and have shown that metformin has a preventative role in the
cerebrovascular diseases (7, 9). In the initial stage of the development of AS and in the regulation of the function of
development of atherosclerotic lesions, plasma lipoproteins macrophages. We have analyzed and summarized the research
trapped by modified proteoglycans accumulate in the about metformin in the modulation of macrophage function in
subendothelial space, which can cause endothelial cell (EC) AS: including roles in inflammation, polarization, oxidative
and platelet activation (8–12). Circulating monocytes adhere to stress, foam cell formation and apoptosis, and considered the
the activated endothelium, penetrate the endothelium and relevant mechanisms of action. Finally, we looked forward to the
differentiate into pro-inflammatory macrophages, which form future research on metformin in macrophage pathobiology,
foam cells by increasing the uptake of oxLDL. This aggravated including non-AMPK-dependent mechanisms, epigenetic
inflammation expands as foam cells spread over the growing modification, single-cell sequencing, neutrophil extracellular
atheroma (9, 10). The process of AS is accelerated by numerous traps (NETs), and actions of metformin when combined with
factors, such as the generation of ROS, as well as the release of other relevant drugs.
inflammatory chemokines and cytokines (9, 13). Regardless of We searched the Pubmed and Google databases for the
whether or not there is diabetes present, monocytes and following information: first, we searched all public publications
macrophages exhibit a very important role in all stages of the about metformin and macrophages, with the keyword
development of AS. The diabetic environment further changes “metformin and macrophages.” Second, we searched for other
the phenotype of monocytes and macrophages, such as related publications, including the function of macrophages and
promoting the recruitment of monocytes to the lesion, further neutrophils in AS, the effect of metformin on AS, non-
activating inflammation, increasing the accumulation and coding RNA.
metabolism of lipids in macrophages, and increasing the death
of macrophages (14–16). Macrophage pathobiology has become
a new potential target for anti-atherosclerosis therapy (14, 17). CLINICAL STUDIES OF METFORMIN IN
Metformin was prepared and synthesized in 1922, and the THE TREATMENT OF AS
hypoglycemic effect of metformin was confirmed through
clinical trials in 1957 (18). In 1995, metformin was approved Due to its high morbidity and mortality, CAD represents a major
for the therapy of T2DM in the United States (18). The discovery global health and economic burden (7, 44). Therefore, early
of metformin opened a new page in the emerging struggle recognition, diagnosis and timely treatment of risk factors is an
between humans and diabetes (18, 19). With the publication of essential component of patient care (35). Diabetes is an
a number of large-scale clinical studies with positive outcomes, important independent risk factor for the occurrence of CAD
metformin has been widely accepted as the first-line therapy for (45). Metformin, as the most widely used prescription
T2DM (20). Metformin mainly activates AMPK, thereby hypoglycemic agent, has been demonstrated to reduce CV
reducing hepatic glucose output (by inhibiting hepatic events in patients with diabetes in a number of clinical studies
gluconeogenesis) and increasing glucose utilization in (35, 36).
peripheral tissues (by increasing insulin receptor sensitivity For example, Petrie, JR et al. (24) conducted a double-blind,
and improving insulin resistance) (20, 21). In addition, the placebo-controlled trial (REMOVAL) to investigate whether or
hypoglycemic mechanism of metformin also involves not metformin (combined with insulin therapy) reduces AS in
promoting the increase of glucagon-like peptide-1 (GLP-1) patients with T1DM who are at higher risk of CVD. In 23
release, regulating the intestinal flora and promoting the hospital diabetes clinics in five countries (United Kingdom,
excretion of blood glucose from the intestine (21). Metformin Netherlands, Canada, Australia, and Denmark), subjects with
is a cardioprotective and is a hypoglycemic drug with clear age ≥ 40 years old, T1DM for at least 5 years and at least 10
evidence of cardiovascular benefits (22, 23). Mounting specific CV risk factors were randomized to receive either
evidence have shown that long-term treatment of metformin is placebo or metformin (1 g twice a day). Among the 428
related to a significantly reduced risk of cardiovascular disease randomly assigned subjects, there were 219 subjects in the
(CVD) in pre-T2DM patients and T2DM patients and even in placebo group and 209 subjects in the metformin group.
some T1DM patients (Table 1). Metformin is being explored for Although the average common carotid intima-media thickness
new functions and pharmacological mechanisms. (cIMT), a surrogate measure of AS, progression of the metformin
The results of various preclinical studies and clinical trials treatment group was not decreased, the maximum cIMT of the
have shown that metformin plays a protective role in CVD metformin group was decreased. In the metformin treated group,
(Tables 1 and 2) (35, 36). Metformin exhibits a protective effect the average insulin requirement within 3 years did not decrease
Frontiers in Immunology | www.frontiersin.org 2 June 2021 | Volume 12 | Article 682853

TABLE 1 | Clinical studies of metformin in diabetic patients with cardiovascular risk.
Subjects Grouping Results Conclusion
T1DM who are Metformin (1 g twice a day) + insulin ↓the maximum cIMT Metformin may exhibit a greater effect in CVD risk
at higher risk of (n=219) ↓body weight and LDL cholesterol management in patients with T1DM who are at higher risk of
CVD. Placebo + insulin (n=219) ↑eGFR CVD (24).
Children with Metformin (1 g twice a day) + insulin ↑GTN Metformin can improve HbA1c, the function of vascular
T1DM. (n=45) ↓insulin dose smooth muscle and reduce insulin doses in children with
Placebo + insulin (n=45) ↓HbA1c T1DM (25).
T2DM Metformin users (n = 7457) ↓mortality rates (HR=0.76, 0.65-0.89; P<0.001) Metformin treatment, as a means of secondary prevention,
Non-users (n = 12 234) ↓mortality rates of patients with a history of can reduce the mortality of diabetic patients (26).
congestive heart failure (HR=0.69, 0.54-0.90;
P=0.006).
Patients with Metformin users (n = 67749), ↓MACE rates (adjusted HR=0.80, 95% CI, In diabetic patients with decreased renal function treated with
T2DM and renal Sulfonylureas (n = 28976) 0.75-0.86) monotherapy, metformin treatment reduced the risk of MACE
insufficiency. Weighted cohort metformin compared with sulfonylureas (27).
(n=24679), sulfonylureas (n=24799)
T2DM Metformin group (n=20) (500 mg ↓LDL(3) mass and the LDL(3)-to-LDL ratio Compared with gliclazide, the content of HDL and LDL
twice a day) ↑HDL(2)-to -HDL(3) ratio subgroups in the pioglitazone or metformin group changed
Gliclazide group (n=20) favorably.Such changes may be related to reducing the risk of
Pioglitazone group (n=20) AS (28).
Patients with 1645 pairs of matched samples (534 LSM: ↑large HDL, ↓small HDL, small and LSM or metformin treatment has a good effect on lipoprotein
IGT LSM, 558 metformin(850 mg twice dense LDL as well as large and buoyant VLDL subcomponents, which may delay the development of
daily) and 553 placebo). Metformin: modestly ↑small and large HDL, diabetes and AS (29).
↓small and dense LDL
Pre-DM Placebo (n=1082) ↓CAC severity and presence of men (CAC Metformin can prevent CAD in men with prediabetes and
Metformin (850 mg twice daily) presence, 75% vs. 84%, CAC severity, 39.5 early diabetes (30).
(n=1073) vs. 66.9 agatston units)
Lifestyle (n=1079)
HIV patients No LSM (n=11), ↓CAC These results show that metformin can prevent plaque
with MetS LSM (n=11), improved HOMA-IR progression in HIV-infected MetS patients (31).
No LSM + metformin (n=13), LSM had no obvious effect on CAC
LSM +metformin (n=15) progression
(850 mg twice a day)
First-degree Placebo (n = 15) metformin (n = 16) ↓body weight, BMI, FPG and systolic blood Metformin can improve the vascular endothelial function of
relatives of (850 mg twice a day) pressure first-degree relatives with MetS in patients with T2DM,
T2DM patients Improved blood lipids and endothelium- regardless of the known hypoglycemic effect (32).
with MetS dependent FBF.
Patients with 86 normal blood glucose (NG) ↓percentage of endothelial LAD dysfunction Metformin treatment can decrease the high risk of MACE in
stable angina subjects, 86 pre-DM subjects and ↓MACE (predicted by a multivariable logistic pre-DM patients via improving coronary endothelial
pectoris and 86 metformin-treated pre-diabetes regression model) dysfunction (33).
NOCS (pre-DM +metformin) subjects
diabetes. (850 mg twice a day)
Patients with Placebo (n=34) ↓LVMI Metformin treatment obviously decreased LVMI, LVM, local
CAD, IR and/or Metformin (n=34) (2 g once a day) ↓LVM, office systolic blood pressure, systolic blood pressure, and body weight. Large trials of
pre-DM. subcutaneous adipose tissue, body weight, cardiovascular results require definitive evidence to prove the
and thiobarbituric acid reactive substance cardioprotective effects of metformin (34).
concentrations (oxidative stress biomarkers)
In this table, we describe the role of metformin in clinical research on atherosclerotic diseases, including subjects, grouping, results and conclusions. ↑Represents increase or activation.
↓Represents to reducion or inhibition. The corresponding abbreviations are as follows: AS, atherosclerosis; BMI, body mass index; CAC, coronary artery calcification; cIMT, carotid intima-
media thickness; CVD, cardiovascular disease; DM, diabetes mellitus; eGFR, estimated glomerular filtration rate; FBF, forearm blood flow; FPG, fasting blood glucose; GTN, glyceryl
trinitrate-mediated dilatation; HIV, The human immunodeficiency virus; HDL, high-density lipoprotein; HOMA-IR, homeostatic model of assessment-insulin resistance; IGT, impaired
glucose tolerance; IR, insulin resistance; LAD, left anterior descending coronary artery; LDL, low-density lipoprotein; LSM, lifestyle modification; LVH, left ventricular hypertrophy; LVM, left
ventricular mass; LVMI, left ventricular mass indexed to height; MACE, major adverse cardiac events; MetS, metabolic syndrome; NG, normal blood glucose; NOCS, non-obstructive
coronary artery stenosis; pre-DM, pre-diabetes; T1DM, type 1 diabetes;T2DM, type 2 diabetes.
significantly, but body weight and LDL-c decreased, and trial involving 90 participants found that metformin (1 g twice a
estimated glomerular filtration rate (eGFR) increased. These day) can ameliorate SMCs function and HbA1c in children with
results indicate that metformin may exhibit a greater effect in T1DM, and reduce insulin doses (25).
cardiovascular risk management (24). Children with T1DM have To assess whether metformin therapy affects the mortality
vascular dysfunction before AS. Early intervention is needed to caused by atherosclerotic thrombosis in patients with diabetes, a
prevent CVD. A 12-month randomized controlled double-blind study was conducted which included 19,691 diabetic patients with

TABLE 2 | Clinical, animal and cellular studies of metformin on macrophages associated with atherosclerotic diseases.
Functions Models or subjects Mechanisms Results Conclusions
Inflammation DM patients +metformin(n=498) or + ↓neutrophils to ↓inflammation in diabetic and The anti-inflammatory effect of
Sulfonylurea(n=172), non-diabetic patients lymphocytes (NLR), non-diabetic patients metformin has nothing to do with
with congestive heart failure+metformin (n=20) ↓CCL11 diabetes status. The study may
or + Placebo (n=13) accelerate the study of the effect of
metformin in non-diabetic CVD (55).
PCOS (n = 83), Controls (n = 39), and PCOS+ ↓ASAA ↓intima-media Metformin treatment can reduce serum
metformin (n=21) ASAA in women with PCOS (57).
ARDS mice Metformin (50 mg/kg) ↓miR-138-5p ↓LPS-induced deaths Metformin can reduce the LPS-induced
Alveolar macrophages (NR8383) cells, ↑ SIRT1 ↓IL-6, IL-1b, IL-17 and TNF-a lung damage via decreasing miR-138-
metformin (40 mg/mL) ↓p-p38, p-ERK and p-NF- 5p expression, increasing the
kB expression of its target gene SIRT1 and
inhibiting MAPK pathway (58).
LPS treated macrophages, metformin ↑AMPK, ↓CXCL10 and CXCL11 Metformin inhibits LPS-stimulated
(10 mM) ↓NF-kB chemokines expression via AMPK and
NF-kB signaling pathways (53).
ECs, macrophages and SMCs, Metformin ↓PI3K-Akt ↓IL-6 and IL-8 Metformin can block NF-kB by
(20 mM) ↓NF-kB inhibiting the PI3K-Akt pathway,
thereby exerting a direct vascular anti-
inflammatory effect (54).
HFD fed rabbit, Metformin (200 mg/kg/day) ↓inflammatory cytokines ↓AS Metformin may inhibit the development
monocytes, macrophages metformin and adhesion molecules ↓the adhesion of monocytes, of AS via inhibiting macrophage
(200 mg/mL) inflammatory response of infiltration and inflammation (62).
macrophages
Inflammation LPS-stimulated endotoxemia mice, metformin ↑AMPK and ATF3 ↓TNF-a and IL-6 AMPK activation and induction of ATF3
(250 or 500 mg/kg, twice daily), ob/ob mice NF-kB enrichment on TNF- are potential mechanisms for metformin
(250 mg/kg, twice daily) a and IL-6 promoters to exhibit anti-inflammatory effect in
LPS treated macrophages, metformin (0.5, 1, caused by LPS were macrophages (113).
2, 4 mM) replaced by ATF3
LPS induced macrophages, metformin ↑AMPK ↓CXCL10 and CXCL11, ↓IL-1 Metformin reduces LPS-stimulated
(10 mM) ↓NF-kB and IL-6 chemokine expression via AMPK and
NF-kB signaling pathways (53).
AGEs induced mouse BMDM ↑AMPK ↓CD86) (M1 marker), Metformin partially reduces AGEs-
Metformin (2mM) ↓NF-kB ↑CD206 (M2 marker) and IL-10 stimulated the inflammatory response of
mouse macrophages via AMPK
activation and RAGE/NFkB pathway
inhibition (114).
HFD fed mice, metformin (150 mg/kg/d) ↑AMPK ↓liver steatosis Metformin mainly prevents obesity-
LPS-induced BMDM, metformin (500 µM) ↓JNK1 and NF-kB ↓fat deposition ↓pro- related NAFLD by directly reducing liver
inflammatory cytokines and cell fat deposition and inhibiting the
lipogenic enzymes inflammatory response of liver cells and
macrophages (115).
Acetaminophen treated mice, metformin Binds directly to the C- ↓inflammatory response Metformin inhibits inflammation via
(350 mg/kg) terminal acid tail of reducing the extracellular activity of
HMGB1 treated macrophages, metformin HMGB1. HMGB1 (116).
(0.1~10 mM)
Inflammation Neutrophils from patients with ARDS, ↑ AMPK Neutralization of HMGB1 in Neutralizing HMGB1 or restoring AMPK
metformin (500 µM/L) Neutralization of HMGB1 in BAL fluid or activation of AMPK activity with metformin represents a
BAL fluid in macrophages in BAL fluid promising therapeutic strategy to
improved cell swelling and reduce persistent lung inflammation of
NETs clearance. ARDS (117).
LPS-treated mice, metformin (50, 100 mg/kg), ↑ AMPK ↓serum levels of HMGB1, IL- Metformin improves the survival rate of
LPS-treated macrophages, metformin ↓HMGB1. 1b, TNF-a and the lethal endotoxemia mouse model
(1, 5, 10 mM) myeloperoxidase activity in the via inhibiting the release of HMGB1.
lung. AMPK activation is one of the
↑the survival rate mechanisms causing HMGB1 secretion
inhibition (118).
Methionine treated mice, ↑CSE expression ↓levels of Hcy, TNF-a and IL- Metformin treatment reduces the
Hcy treated macrophages, metformin (12.5, 1b, harmful effects of methionine (63).
25 and 50 mM)
(Continued)

TABLE 2 | Continued
Inflammasome ATP treated macrophages, metformin (2 mM) ↑AMPK ↑mortality of mice with bacterial These results indicate that AMPK signal
bacterial sepsis mice, metformin (250 mg/kg) ↑inflammasomes sepsis. transduction positively regulates ATP-
↑the activation of systemic cuased inflammasome activation and
inflammasomes (such as pyrophosphorylation in macrophages
increased IL-1b level in blood (72).
and liver).
oxLDL treated macrophages, metformin ↑AMPK and PP2A, ↓NF- ↓NLRP3 inflammasome Metformin inhibited expression and
(80mM) kB activation of NLRP3 in oxLDL-induced
macrophages via AMPK and PP2A (71).
Inflammasome Diabetic mice, Metformin (300 mg/kg/d) ↓disorder of thioredoxin-1/ ↓metabolic disorders and AS Metformin can inhibit the NLRP3
thioredoxin interacting inflammasome activation in ApoE-/-
protein. mice and inhibit diabetes-accelerated
↓ROS and NLRP3 AS, at least in part by activating AMPK
inflammasome and regulating thioredoxin-1/thioredoxin
interacting protein (70).
Control (n=57), T2DM patients (n=47), Among ↑AMPK ↓the maturation of IL-1b in NLRP3 inflammasome is activated in
47 diabetic patients, 11 patients received MDM of T2DM patients MDM of T2DM patients, and metformin
metformin(500–1,000 mg/day). administration helps to regulate the
activation of inflammasomes in T2DM
(69).
Oxidative Macrophages (shPTEN cells), metformin ↓Akt and ROS ↓iNOS/NO and COX-2/PGE2, In shPTEN cells, Metformin can reduce
stress (5 to 40 mM) ↑apoptosis the diffusion of inflammatory mediators
and cell growth by inhibiting Akt
activation and ROS production (78).
LPS treated Peripheral blood monocytes, ↑AMPK ↓ROS and inflammatory Metformin can improve diabetes by
metformin (0.02 and 2 mM) ↑ superoxide dismutase, cytokines (such as iNOS) lowering blood glucose, reducing the
glutathione peroxidase, oxidative stress, inflammatory
catalase cytokines, and inducing phenotypic
↓malondialdehyde changes of macrophages (79).
macrophages, metformin (2-5 mM) ↓Glutathione ↓cholesterol content and The inhibitory effect of metformin on
↑paraoxonase 2 biosynthesis rate cholesterol biosynthesis is at least
↑ cellular ROS Antioxidants decreased partially related to metformin-induced
metformin-induced ROS and ROS in macrophages (81).
cancelled the inhibitory effect of
cholesterol biosynthesis.
macrophages, C2C12 skeletal muscle cells ↑calcineurin and Cn- ↑ ROS in mitochondria The retrograde signal induced by Mito-
and HCT116 adenocarcinoma cells, dependent retrograde Metformin is through the activation of
Mito-Metformin signaling pathway the Ca2/Cn pathway (82).
Foam cell ApoE-/- mice fed with HFD, metformin ↑AMPK, ↑ABCA1 and ↑RCT AMPK activators promote the anti-
formation (260 mg/kg) ABCG1 in macrophages, ↓blood lipids peroxidation, atherosclerotic properties of HDL and
↑LCAT and SR-B1 in liver. ↓inflammatory cytokines attenuate AS (104).
↑ M2 polarization expression
↑paraoxonase 1 ↓atherosclerotic plaque.
↓myeloperoxidase
3-DG incubated macrophages, metformin ↑the glycated HDL-mediated cholesterol efflux Glycated HDL particles cannot
(100 mM) Exogenous HDL reduces the expression of ABCG1 mRNA effectively act as ABCG1-mediated
and protein, but glycosylation makes HDL lose this effect. cholesterol efflux receptors. Metformin
may be a drug candidate to improve
cholesterol efflux (23).
LPS and oxLDL induced macrophages ↓ ADRP ↓LDs in the foam cells Metformin can reduce the formation of
Metformin (100 or 200 mM/L) THP-1 derived foam cells caused by
LPS, decrease the ADRP expression
and intracellular lipid accumulation (90).
palmitic acid (PA) treated macrophages, ↓FOXO1 ↓lipids accumulation in Metformin reduces the lipids
Metformin (100, 250, 500mM) ↓FABP4 macrophages accumulation in macrophages via
↑ CPT-1. reducing FOXO1-mediated FABP4
transcription (91).
oxLDL treated macrophages, metformin ↑ABCG1 ↓cholesterol accumulation and The study highlights the therapeutic
(15 mM) ↑ outflow of cholesterol to the formation of foam cell potential of metformin to target
(Continued)

TABLE 2 | Continued
HDL macrophage cholesterol efflux, which

↑IL-10 secretion may reduce foam cell formation (89).
Foam cell ApoE-/- mice, ↓macrophages/foam cells Co-administration increases the Co-administration of metformin and
formation Co-treatment with T317 and metformin in the arterial wall ↑ABCA1/ stability of the lesion T317 can improve AS without activating
(100 mg/day/kg) ABCG1. block T317-caused fatty liver adipogenesis, which indicates that this
Metformin activates combination may be a new method to
AMPKa and reduces inhibit AS (92).
T317-stimulated hepatic
LXRa activation and
lipogenic gene expression.
mouse BMDM and primary human MDMs, ↑AMPK Induced heme oxygenase and Heme (10 mM) activates AMPK, and the
metformin (10 mM) ↑ATF1. LXR jointly induce the Mhem downstream ATF1-induced heme
phenotype. oxygenase and LXR jointly induce the
Mhem phenotype. (119).
LPS induced macrophages, metformin ↓NF-kB apoE expression↑ Metformin is a potential adjuvant in the
(1-3 mM) treatment strategy of AS (120).
HFD fed mice, metformin (250mg/kg) ↓multiple binding sites of ↑expression of ABCG5/8 and Metformin may provide some support
primary hepatocytes, phase 2 (transcription BSEP for cardiovascular benefits by increasing
metformin(0.5 mmol/L) repressor) occupancy ↑initial clearance of 3H- RCT, and AMPK activation inhibition
within ABCG5/8 site cholesteryl ester HDL from the may mediate anti-atherosclerotic effects
blood. by increasing ABCG5/8 expression
(121).
M1/M2 PBMCs were isolate from 30 normal-weight ↑CD68 marker in obesity and in T2DM. PBMCs in T2DM patients express a
polarization healthy adult volunteers, 30 obese volunteers, ↓CD11b, CD11c, CD163 and CD169 in T2DM patients different pattern of phenotypic markers
20 obese newly diagnosed diabetic patients, ↑TNFa, iNOS, IL-6, CD16, CD36, and CD206 in the T2DM (represent metabolically activated
as well as 30 metformin-treated obese Metformin restored TNFa, iNOS, IL-6, CD11c, CD36, CD169 macrophage (MMe)-like cells), which is
diabetic patients. and CD206 in T2DM patients to levels equivalent to those of not the pattern normally found in M1 or
lean volunteers. M2-like macrophages, and metformin
can reduce circulating MMe-like cells
(42).
Olanzapine reated rats, metformin ↓body weight and IR, Metformin may reduce the IR caused
(300 mg/kg/day) ↓macrophage polarization and pro-inflammatory factors. by olanzapine by inhibiting the
polarization and inflammation of
macrophages in white AT (43).
M1/M2 HFD fed mice, metformin (300 mg/kg/d) ↑ AMPK, ↓IL-6 and TNF-a Metformin can regulate the polarization
polarization In palmitate stimulated BMDM, metformin ↓CD11c and MCP1 (M1 markers) in AT of macrophages to anti-inflammatory
(2 mM) ↓proportion of M1 macrophages, ↑the proportion of M2 M2 and improve low-grade
macrophages inflammation in obesity by activating
AMPK (23).
Obese mice, metformin (300 mg/kg) ↓SHP-1 and ↑insulin ↓CD80, CD86, TLR2, TLR4, Metformin exerts its insulin sensitization
sensitivity. NF-kB, STAT1 and other effect by inhibiting the activity and
↑anti-inflammatory inflammatory markers expression of SHP-1 (102).
macrophages AT ↓inflammation of AT
Ldlr-/- hyperlipidemia mice, metformin ↑AMPK/ATF1 ↓atherosclerotic lesions. metformin can activate the AMPK-
(in drinking water, 1mg/mL) ↑M2 marker genes, ↑LXRb, Hmox1, ApoE, ABCA1, ATF1-M2-like pathway in macrophages.
Mouse BMDM, metformin (10 mM) ↓ iNOS PDGF and IGF1 These findings support the clinical trials
of metformin in non-diabetic patients
with high risk of AS (122).
MCAO mice, ↑ AMPK, ↑ functional recovery, Chronic metformin treatment after
metformin (50 mg/kg/day) microglia/macrophages ↑neurogenesis and stroke improves functional recovery
tend to M2 phenotype angiogenesis through AMPK-induced M2 polarization
(103).
macrophages with/without LPS, metformin ↑IL-4, IL-10, arginase 1 (Arg1) and lectin-1 (Mgl1) Metformin regulates the M2 phenotype
(1, 5, 10 mM) ↓Notch1, TNF-a, IL-1b and IL-6. of RAW264.7 macrophages with/
without LPS. The Notch1 signal may
play a vital role (23).
Monocyte Forty-four subjects with AMI and T2DM ↓Akt , ↓sCD40L level Metformin therapy in patients with AMI
differentiation (metformin, n=21; short-acting insulin, n=23) and T2DM can cause a faster decline in
sCD40L, which may help improve the
prognosis of this cohort (109).
(Continued)

TABLE 2 | Continued
Monocyte HFD-fed ApoE -/-

mice, metformin (260 mg/kg) ↓CCR2 expression ↓number of Ly6Chi monocytes AMPK activation decreases the
differentiation in circulation as well as development of AS induced
atherosclerotic plaques. macrophages accumulation in ApoE-/-
mice via reudcing the CCR2
expression, thereby preventing CCR2-
induced migration of Ly6Chigh
monocytes (123).
Ang-II induced ApoE-/- mice, Metformin (100 ↑AMPK, ↓the infiltration of monocytes,AMPK activators reduce the
mg/kg/day) ↓ STAT3 ↓atherosclerotic plaques and differentiation of monocytes into
aortic aneurysms macrophages by regulating AMPK-
STAT3 axis (40).
Apoptosis oxLDL treated metformin, macrophages (0.1, ↓scavenger receptors, ↓lipid uptake Metformin can prevent oxLDL-caused
0.3, 0.5, 1 mM) including CD36 and SRA ↓the apoptosis of macrophages macrophage apoptosis and inhibit lipid
↓expression of ER stress uptake of macrophage (111).
marker proteins (such as
EIF2A and CHOP)
↓oxLDL-induced Dym loss
and cyto-c release.
In this table, we describe that metformin plays a role in atherosclerosis by regulating monocyte/macrophage function, including cell function, objects, mechanisms, results and conclusions.
↑Represents increase or activation. ↓Represents to reducion or inhibition. The corresponding abbreviations are as follows: ABCA1, ATP-binding cassette transporter A1; ABCG1, ATP-
binding cassette transporter G1;ADRP, adipose differentiation-related protein; AMPK, AMP-activated protein kinase; AGEs, advanced glycation end products; AMI, acute myocardial
infarction; Ang-II, angiotensin II; ApoE, apolipoprotein E; ARDS, acute respiratory distress syndrome; ASAA, acute-phase serum amyloid A; AS, atherosclerosis; AT, adipose tissue; ATF3,
transcription factor 3; BAL, bronchoalveolar lavage; BM, bone marrow; BMDM, bone marrow-derived macrophages; BSEP, bile salt export pump; CXCL10, C-X-C motif ligand; CCR2, CC
chemokine receptor 2; CD, cluster of differentiation; CHOP, C/EBP homologous protein; CSE, cystathionine g-lyase; COX-2, cyclooxygenase 2;CPT-1, carnitine palmitoyl transferase I; 3-
DG, 3-deoxyglucosone; DM, diabetes mellitus; ECs, endothelial cells; EIF2A, eukaryotic translation initiation factor 2A; ERK, extracellular signal-regulated kinase; ER, endoplasmic
reticulum; FABP4, fatty acid binding protein 4; FOXO1, forkhead box transcription factor O1; HFD, homocysteine (Hcy,; high-fat diet; HMGB1, high-mobility group box 1; Hmox1, Heme
oxygenase 1; H2S, hydrogen sulfide; IGF1, insulin growth factor 1 IL, Interleukin; iNOS, inducible nitric oxide synthase; JNK1, c-Jun N-terminal kinase 1; LCAT, lecithin:cholesterol
acyltransferase; Ldlr, low-density lipoprotein receptor; LPS, lipopolysaccharide; LXR, Liver X receptor; MAPK, mitogen activated protein kinase; MCAO, middle cerebral artery occlusion;
MCP1, monocyte chemoattractant protein 1; MDMs, monocyte-derived macrophages; Dym, mitochondrial membrane potential; NAFLD, non-alcoholic fatty liver disease; NETs, neutrophil
extracellular traps; NF-kB, nuclear factor-kB nucleotide-binding oligomerisation domain receptor, pyrin domain containing (NLRP)3, the ratio of neutrophils to lymphocytes (NLR), NO,
nitric oxide; oxLDL, oxidized low-density lipoprotein; PA, palmitic acid; PBMC, peripheral blood mononuclear cell; PCOS, Polycystic ovary syndrome; PDGF, platelet-derived growth factor;
PGE2, prostaglandin E2; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; PMA, phorbol 12-myristate 13-acetate; PTEN, phosphatase and tensin homolog; RAGE, receptor for
advanced glycation end products; ROS, reactive oxygen species; SH2, Src homology 2; domain-containing protein tyrosine phosphatase 1 (SHP-1); SIRT1, Sirtuin-1; SMCs, smooth
muscle cells; SRA, scavenger receptor class A; SR-B1, scavenger receptor class B type 1; STAT, signal transducer and activator of transcription; TNF-a, tumor necrosis factor-a; TIP47,
tail-interacting protein; TLR, Toll-like receptor.
AS in the Reduction of AS Continuing Health (REACH) 1394 events in sulfonylurea users (29.2 per thousand person-years)
registration between 2003 and 2004. They received or did not and 1048 MACE events in metformin users (23.0 per thousand
receive metformin treatment, and the 2-year mortality rate in person-years). Compared with sulfonylureas, the MACE adjusted
these two groups was analyzed. The results showed that the HR of metformin is 0.80 (95% CI, 0.75-0.86). This study shows that
mortality rates of the metformin group and non-metformin compared with sulfonylureas, in patients with diabetes with renal
group were 6.3% and 9.8%, respectively, and the adjusted hazard insufficiency receiving monotherapy, metformin treatment may be
ratio (HR) was 0.76 (0.65-0.89; P<0.001). The correlation of lower associated with a lower risk of MACE (27).
mortality among the subgroups was consistent. Patients with a Lawrence, JM et al. compared the effects of oral hypoglycemic
history of congestive heart failure benefit more, with an HR of 0.69 drugs on lipoprotein sub-components in T2DM subjects. Sixty
(0.54-0.90; P=0.006). These results show that metformin may be overweight T2DM subjects who did not receive lipid-lowering
used as a secondary prevention method to reduce the mortality of treatment were randomly assigned to metformin, pioglitazone or
diabetic patients (26). Roumie, CL et al. studied the cardiovascular gliclazide after three months of diet run-in with adjustment of the
clinical outcome of metformin in patients with T2DM and renal drug dosage to optimize blood glucose control, and continuing
insufficiency. Including new users of metformin or sulfonylureas, treatment for 3 months. Compared with gliclazide, the content of
follow-up starts with a lowered renal function threshold lasting until high-density lipoprotein (HDL) and LDL subgroups in the
the occurrence of major adverse cardiac events (MACE) (including pioglitazone or metformin group changed favorably (including
myocardial infarction, heart failure, transient ischemic attack (TIA), the increase in HDL (2)-to -HDL (3) ratio, and the decrease in
ischemic or hemorrhagic stroke and CVD death), treatment LDL (3) mass and the LDL (3)-to-LDL ratio) (28). Such changes
changes, loss of follow-up, death, or the end of the study. The may be related to reducing the risk of AS (28). Similarly, a
results showed that the number of patients who used metformin or randomized placebo-controlled clinical trial evaluated the effects
sulfonylureas alone for a long time was 67749 and 28976, of lifestyle modification (LSM) (a low-fat diet that reduces body
respectively; there were 24679 metformin and 24799 sulfonylurea weight by 7%) or metformin (850 mg twice a day) on patients with
users in the weighted cohort. During the follow-up period (median, impaired glucose tolerance (IGT). LSM increased large HDL and
1.0 year for metformin and 1.2 years for sulfonylurea), there were decreased small HDL, small and dense LDL as well as large and

buoyant Very Low Density Lipoproteins (VLDL). Metformin placebo or metformin (2 g once a day) for 12 months. The
modestly raised small and large HDL as well as decreasing small findings were that metformin therapy decreased weight, left
and dense LDL. Metformin modestly raised small and large HDL ventricular mass (LVM), left ventricular mass indexed to
and decreased small and dense LDL. Thus, metformin treatment height (LVMI), and systolic BP (34).
has beneficial effects on lipoprotein subcomponent, but LSM may be
more effective. Both interventions may delay the development and
progression of AS (29).
Patients with prediabetes (pre-DM) are at a higher risk of CAD METFORMIN IN REGULATING
and may require prevention interventions to decrease the risk of MACROPHAGE DYSFUNCTION
CAD. The Diabetes Prevention Program Outcome Study (DPPOS)
and the Diabetes Prevention Program (DPP) studied 3234 subjects Inflammation
with pre-DM. After an average follow-up time of 14 years, 2029 Chronic inflammation or defective inflammation resolution
participants were assessed for AS through coronary artery calcium plays a key role in the occurrence and development of AS (46).
(CAC) measurements. Men in the metformin group had lower Macrophages phagocytose modified LDL (such as oxLDL)
CAC severity and presence compared with the placebo group but through multiple cell membrane scavenger receptors
no effect of metformin was observed in women. Metformin (850 mg such as lectin-like oxidized LDL receptor-1 (LOX-1),
twice daily) can prevent CAD in men with pre-DM and early scavenger receptor class A (SRA) and CD36 (16). In the
diabetes (30). Human immunodeficiency virus (HIV) patients are lysosome, cholesterol ester (CE) in LDL particles is degraded
prone to metabolic abnormalities (such as DM, obesity, to free cholesterol (FC) and free fatty acids by lysosomal acid
hyperlipidemia, and hypertension), which can induce CAC. In 50 lipase (LAL) (16, 47, 48). FC is re-esterified in the endoplasmic
subjects with metabolic syndrome (MetS) infected with HIV, the reticulum (ER) which contributes to CE accumulation and lipid
effect of LSM and/or metformin (850 mg twice a day) treatment on droplet formation (16, 49). CE is hydrolyzed to release FC, and is
the parameters of the MetS was studied. Subjects receiving further transported to the outside of the cell through cholesterol
metformin therapy for more than 1 year showed a significant efflux transporters, ABCA1, ABCG1 and scavenger receptor class
reduction in CAC progression, while LSM had no effect on CAC B type 1 (SR-B1); these processes maintain cholesterol
progression. These results show that metformin therapy can prevent metabolism homeostasis (16, 47). During the development and
plaque development in HIV-infected MetS patients (31). progression of AS, excessive accumulation of FC induces the
An early feature of AS in patients with T2DM is endothelial formation of cholesterol crystals in the lysosome, which activates
dysfunction. In this regard, thirty-one volunteers who were first- the nucleotide-binding oligomerization domain receptor, pyrin
degree relatives of patients with T2DM, and had normal glucose domain containing (NLRP)3 inflammasome, induces ER stress,
tolerance and MetS were recruited. The volunteers were and ultimately leads to the formation of foam cells (49). OxLDL
randomly assigned to a metformin (850 mg twice a day) group can also activate the NF-kB signaling pathway through the
(n = 16) or a control group (n = 15). The weight, BMI, fasting CD36–Toll-like receptor (TLR)4/TLR6 trimer (49, 50). Foam
blood glucose (FPG) and systolic blood pressure (BP) of the cells secrete macrophage chemotaxis retention factors, causing
metformin group decreased, and the blood lipid profiles also pro-inflammatory cytokines and chemokines to amplify the
improved. The measurement of the endothelium-dependent inflammatory response (49, 50) (Figure 1). Excessive
forearm blood flow (FBF) response also improved. These inflammation may induce plaque rupture and increase the risk
results indicate that metformin can improve the vascular of coronary thrombosis (51). Currently, evidence indicates that
endothelial responsiveness of first-degree relatives of T2DM cytokines (such as IL-1b, IL-17 and TNF) are targets for reducing
patients with MetS (32). Similarly, the 258 stable angina the progression of CVD at least in some cases (52).
patients matched by propensity scores included 86 normal Cytokines and chemokines exhibit a very important effect in
blood glucose (NG) subjects, 86 pre-DM subjects and 86 inflammation, and some of them are therapeutic targets for
metformin-treated pre-diabetes (Met+pre-DM) subjects. At the attenuating chronic inflammatory diseases (53). Metformin
24th month of follow-up, MACE of NG subjects and Met+pre- may be beneficial for macrovascular complications of diabetes,
DM subjects were lower than those of pre-DM subjects. as a complement to its hypoglycemic effect (54). In large-scale
In addition, the percentage of left anterior descending treatment of newly diagnosed diabetic patients, the difference in
coronary artery (LAD) endothelial dysfunction in NG subjects systemic inflammatory markers, neutrophil-lymphocyte ratios
and Met+pre-DM subjects was also lower than that in pre-DM after treatment with sulfonylurea or metformin monotherapy
subjects. These findings show that metformin therapy can was observed. Compared to sulfonylurea therapy, metformin
decrease the high risk of MACE in patients with pre-DM via decreased the mean log-transformed neutrophil to lymphocyte
improving coronary endothelial function (33). In addition, ratio by 0.09 U after 8 to 16 months. Following these findings in a
metformin has a beneficial effect on the regression of left non-diabetic heart failure trial (registration: NCT00473876),
ventricular hypertrophy (LVH) in patients with CAD, insulin metformin inhibited blood cytokines, including the C-C motif
resistance (IR) and/or prediabetes. The study conducted by chemokine 11 (CCL11). These findings reveal that metformin
Mohan, M et al. randomly assigned 68 subjects without has anti-inflammatory effects in both diabetic and non-diabetic
diabetes but with CAD, IR and/or prediabetes to receive patients, a finding which may accelerate the study of the role of

Frontiers in Immunology | www.frontiersin.org
Feng et al.
OxLDL
Ch
AP ol
e
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SR-A
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1/H ux rol
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DL
Secondary Necrotic
1
CA
necrosis core
1
AB
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-B1
L AL
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Defective Weakening of
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ACAT1 NCEH1 Apoptosis Thrombus

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activation
Lipid Lipid
ER stress Foam cell
droplets Macrophage
9
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36
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Macrophage CCL5, CXCL1) Cadherins) migration

L
FIGURE 1 | Macrophage lipid metabolism disorder and thrombosis. Macrophages phagocytose accumulated LDL and modified LDL (such as oxLDL) through multiple cell membrane scavenger receptors such as
LOX1, SR-A and CD36. In the lysosome, cholesterol ester (CE) in LDL particles is degraded to free cholesterol (FC) and free fatty acids by lysosomal acid lipase (LAL). In the endoplasmic reticulum (ER), FC is re-
Metformin Improves Macrophage Dysfunction

esterified by acetyl-coenzyme A: cholesterol acetyltransferase 1 (ACAT1), which contributes to CE accumulation and lipid droplet formation. CE is hydrolyzed by neutral cholesterol ester hydrolase 1 (NCEH1) to
release FC, and is further transported to the outside of the cell through cholesterol efflux transporters, ABCA1, ABCG1 and SR-B1, thereby maintaining cholesterol metabolism homeostasis. Under pathological
June 2021 | Volume 12 | Article 682853
conditions (such as AS), excessive accumulation of FC induces the formation of cholesterol crystals in the lysosome, which activates the NLRP3 inflammasome, induces ER stress, and ultimately leads to the
formation of foam cells. OxLDL can also activate the NF-kB signaling pathway through the CD36–TLR4/TLR6 trimer. Foam cells secrete macrophage chemotaxis retention factors (including netrin 1 and its receptor
UNC5B, cadherins and semaphorin 3E), pro-inflammatory cytokines (such as IL-1, IL-6 and TNF) and chemokines (such as CCL2, CCL5 and CXCL1) amplify the inflammatory response. Over time, foam cells
undergo apoptosis. When these apoptotic cells cannot be effectively cleared by macrophages in advanced disease (defective erythrocytosis), secondary necrosis will result. Further development will promote the
formation of necrotic cores. At the same time, the living macrophages in the late plaque secrete cytokines, proteases and procoagulant thrombosis factors, as well as VSMCs death and protease degradation of the
extracellular matrix. These effects weaken the stability of the fibrous cap (easy to rupture). The rupture leads to exposure of the thrombogenic substances in these lesions, which eventually causes platelet
aggregation and thrombosis, resulting in MACE events. ABCA1, ATP-binding cassette transporter A1; ABCG1, ATP-binding cassette transporter G1; CCL, C−C motif chemokine; CCR, C-C chemokine receptor;
CD, cluster of differentiation; ER, endoplasmic reticulum; HDL, high-density lipoprotein; IL, Interleukin; LOX1, lectin-like oxidized LDL receptor 1; APOA1, lipid-poor apolipoprotein A1; NF-kB, nuclear factor kappa B;
NLRP3, NOD-like receptor family pyrin domain containing 3; oxLDL, oxidized low-density lipoprotein; SRA, scavenger receptor class A; TLR, Toll-like receptor; SR-B1, scavenger receptor class B type 1.
metformin in CVD in non-diabetic patients (55). Similarly, in T2DM, AS, rheumatoid arthritis, gout and neurological diseases
the mouse macrophage cell line RAW264.7, in macrophages (64, 65). Various signals, including microbial molecules and
treated with lipopolysaccharide (LPS), metformin inhibited LPS- abnormal accumulation of cholesterol crystals, can trigger
stimulated chemokine expression (including CCL2, CXCL10 NLRP3 assembly and activation, thereby promoting the
and CXCL11) by activating AMPK and inhibiting the conversion and secretion of proIL-1b and proIL-18 into mature
phosphorylation of I-kBa and p65 (53). A similar study forms (66). AS is a disease based on inflammation/lipid
showed that metformin can attenuate LPS-stimulated acute abnormalities (46). OxLDL and cholesterol crystals can activate
lung injury (ALI) capillary damage by activating AMPKa1, NLRP3 inflammasome, and thereby the NLRP3 inflammasome
including reducing inflammatory cytokine release, neutrophil may be a key intermediate link in the induction of inflammation
and macrophage infiltration, and reducing myeloperoxidase by lipid metabolism disorders (67). Some studies in mouse
activity (56). Acute phase serum amyloid A (ASAA) is a pro- hyperlipidemia models have shown that the NLRP3
inflammatory adipokine which is up-regulated in obese and IR inflammasome can cause AS, but it may require a second
patients. Polycystic ovary syndrome (PCOS) is one of the most insult, such as oxidized mitochondrial DNA accumulation or
common metabolic disorders in premenopausal women, and is impaired cholesterol efflux, which may cause serious systemic
related to inflammation and AS. ASAA in serum and adipose inflammatory (68). In macrophages or neutrophils, the activation
tissue (AT) is up-regulated in women with PCOS. Metformin of inflammasomes leads to the lysis of Gasdermin-D, which
therapy can reduce blood ASAA in these women (57). In causes membrane pore formation, releases IL-18 and IL-1b, and
addition, in the LPS-induced mouse acute respiratory distress ultimately leads to the formation of extracellular traps in
syndrome (ARDS) model, metformin reduced LPS-induced lung neutrophils (NETosis) (68).
injury and inflammatory factor expression and mortality in mice. Compared with healthy controls, a significant up-regulation
In cultured alveolar macrophages (NR8383), metformin inhibits in NLRP3 mRNA and protein levels was found in monocyte-
the activation of mitogen-activated protein kinase (MAPK) derived macrophages (MDM) of newly diagnosed T2DM
(including p38 and ERK) signaling pathways and increases the subjects. Treatment with metformin for 2 months inhibited IL-
expression of SIRT1 by decreasing mir-138-5p expression, which 1b maturation in MDM in patients with T2DM by AMPK
may be the mechanism by which metformin inhibits ARDS (58). activation. These data indicate that anti-diabetic treatment
Inflammation is closely related to the progression of T2DM with metformin helps reduce inflammasome activation in
and AS (17, 59, 60). Metformin dose-dependently reduces IL-1b- T2DM (69).
stimulated production of IL-6 and IL-8 in human ECs, Metformin can inhibit the NLRP3 inflammasome activation
macrophages (Mphis) and SMCs. Mechanistic studies have in apolipoprotein E (apoE) -/- mice and inhibit diabetes-
indicated that metformin exerts vascular anti-inflammatory accelerated AS, at least in part by activating AMPK and
effects via blocking the PI3K-Akt pathway and inhibiting NF- regulating thioredoxin-1/thioredoxin interacting protein (70).
kB activation and nuclear translocation (54). An inflammatory Furthermore, metformin can inhibit the expression and
vascular model was prepared by implanting polyester- activation of NLRP3 in oxLDL-induced macrophages via
polyurethane sponge into mice and which were then treated AMPK and protein phosphatase 2A (PP2A) (71). Adenosine
with metformin for 6 days. Metformin attenuated the main triphosphate (ATP) treatment results in AMPK activation, and
components of mouse fibrovascular tissue by regulating major host cells release ATP during bacterial infection as a stimulant of
components of inflammatory angiogenesis (macrophage inflammasome activation. In LPS-stimulated mouse
recruitment, Hb content, transforming growth factor (TGF-b1) macrophages, ATP-stimulated inflammasome activation and
and collagen deposition) (61). In the rabbit AS model, metformin pyrophosphorylation were inhibited by small interfering RNA-
treatment decreased mRNA expression of adhesion molecules mediated AMPKa knockdown or compound C treatment.
and inflammatory cytokines in the aorta. This result suggests that Moreover, the mortality of bacterial sepsis mice was increased
metformin may impede the development of AS by inhibiting by metformin administration, which may be because metformin
macrophage infiltration and inflammatory responses (62). In promoted the activation of systemic inflammasomes in mice, as
C57BL/6 mice, methionine significantly up-regulated the levels shown by increased serum and liver IL-1b levels (72).
of homocysteine (Hcy), TNF-a, H2S and IL-1b, and down- The above studies indicate that metformin has the potential to
regulated the level of cystathionine g-lyase (CSE). In THP-1 inhibit NLRP3 inflammasome activity in chronic diseases (such
and raw264.7 cells, Hcy up-regulated the expression of DNA as T2DM), while it may promote the activity of inflammasomes
methyltransferase and increased the CSE promoter methylation. in acute diseases (such as bacterial infections).
Whether in mice or in macrophages, metformin treatment can
reduce the deleterious effects of methionine, which provides new Oxidative Stress
insights into the actions of metformin to inhibit AS (63). Oxidative stress imbalance in tissues is closely related to the
progression of many diseases, including AS, stroke, chronic
NLRP3 Inflammasome wounds and cancer (73, 74). Oxidative stress and inflammation
NLRP3 inflammasome (an innate immune signal complex) is are two interrelated processes, forming a strong feedforward cycle,
mainly expressed as an inflammasome component in which promotes the development of atherosclerotic plaque (75, 76).
macrophages and is closely related to many diseases, including During AS, mitochondrial oxidative metabolism, NADPH-oxidase,

peroxidase, NO-synthase, cyclooxygenase and lipoxygenase Mitochondrial function declines with age. The destruction of
produce macrophage ROS (75, 76). The antioxidant response of mitochondrial function is the result of a variety of intracellular
macrophages is the key to reducing ROS levels and protecting and extracellular stresses (84). Many studies have shown that
nucleic acids, proteins and mitochondria from oxidative damage metformin improves mitochondrial function and inhibits
(75, 76). In plaque macrophages, the mitochondrial transport of oxidative stress (83, 85). Other studies reported that metformin
antioxidant glutathione (GSH) and the transcription of antioxidant inhibits mitochondrial function and enhances oxidative stress.
genes are inhibited, which amplifies inflammation in the arterial This however, may be attributed to metformin overdosing. For
wall (75, 76). It has been well established that NADPH-oxidase example, in a study where metformin reduced cholesterol
derived ROS from macrophages enhances the oxidation of LDL in biosynthesis while enhancing oxidative stress (81), the drug
the arterial wall, thereby promoting the formation of macrophage dosage was 2-5 mM, which is significantly higher than
foam cells (75, 76). Antioxidant treatment is considered a promising the typical.
therapy for the prevention of AS, but currently available
antioxidants show quite limited clinical utility (8). This may be Foam Cell Formation
due to the various ways of inducing and inhibiting oxidation in the Foam cells rich in cholesteryl esters are a sign of atherosclerotic
body and the single role of antioxidants, which did not achieve the plaque (86). Plasma-derived lipoproteins are modified in the
expected effect (8, 77). Broad-spectrum ROS scavenging inner membrane and are absorbed by macrophages to form
nanoparticles have a significant anti-atherosclerotic effect by lipid-filled foam cells, causing the formation of atherosclerotic
decreasing local and systemic oxidative stress and inflammation lesions. Foam cells lack endocytosis and have insufficient
(77). This suggests that pleiotropic antioxidants are more promising inflammatory ablation ability, which maintains the progression
and will need to be further investigated. of the disease. This results in the accumulation of secondary
Macrophages with Phosphatase and tensin homolog (PTEN) necrotic macrophages and foam cells, and the formation of late
deficiency produce a sustained inflammatory microenvironment lesions with necrotic lipid cores, causing plaques to be vulnerable
in which inducible nitric oxide synthase (iNOS)/nitric oxide to rupture (87).
(NO) as well as cyclooxygenase-2 (COX-2)/Prostaglandin E2 In macrophages, metformin inhibits the accumulation of
(PGE2) are produced in large amounts. Metformin can reduce cholesterol induced by 3-deoxyglucosone (3-DG) (88), oxLDL
inflammatory mediators in PTEN-deficient cells by inhibiting (89), acetate (81), LPS (90) and palmitic acid (91).
ROS production and Akt activation (78). Metformin reduces HDL-mediated cholesterol efflux is a rate-limiting step in
macrophage oxidative stress and inflammatory cytokine reverse cholesterol transport (RCT). In macrophages, glycated
production by AMPK activation, but even after treatment with HDL particles (glycosylated by 3-DG) cannot be effective as
compound C, the residual activity of metformin is still significant receptors for ATP-binding cassette transporter G1 (ABCG1)-
(79). In addition, knockout of electron transport or calcium release mediated cholesterol efflux. Metformin can restore cholesterol
activated channel (CRAC) in mouse alveolar macrophages can efflux mediated by glycated HDL (88). Another similar study
prevent particulate matter-induced inflammation and arterial indicated that metformin reduced oxLDL-stimulated cholesterol
thrombosis. Treatment of mouse or human alveolar macrophages accumulation and the formation of foam cells via promoting
with metformin can prevent the generation of complex III cholesterol efflux to HDL, which may be related to up-regulation
mitochondrial ROS induced by particulates, thereby inhibiting of ABCG1. In addition, metformin increases IL-10 secretion
CRAC activation and IL-6 release (80). These studies have shown inhibited by oxLDL, which is an important anti-foam cell
that metformin can inhibit oxidative stress in macrophages. forming factor in AS (89).
However, one study suggests that metformin may enhance In the J774A.1 macrophage cell line, metformin reduced
oxidative stress. In the J774A.1 macrophage cell line, metformin cholesterol biosynthesis rate from acetate (81). In addition,
reduced cholesterol biosynthesis from acetate, but at the same time metformin inhibits LPS-induced THP-1-derived foam cell
significantly increased cellular oxidative stress, such as increased formation and reduces adipogenic differentiation-associated
ROS production and decreased GSH level. Moreover, metformin protein (ADRP) expression (90). Metformin also reduces
inhibited cholesterol biosynthesis of macrophages which is at least palmitate-induced lipid accumulation in macrophages via
partially related to metformin-induced oxidative stress (81). reducing the transcription of fatty acid binding protein 4
Chowdhury, AR et al. conjugated metformin with cationic (FABP4) mediated by forkhead box transcription factor O1
triphenyl phosphate (TPP) to form Mito-Metformin to selectively (FOXO1) (91).
target mitochondria. In HCT116 adenocarcinoma cells, C2C12 Combined with other drugs, metformin may have enhanced
skeletal muscle cells and Raw264.7 macrophages, Mito-Metformin efficacy and reduced adverse reactions. Liver X receptor (LXR)
induced the generation of ROS in mitochondria by acting on agonist T317 can improve AS, but at the same time induces fatty
complex I (82). These studies indicate that metformin may have liver. In a high fat diet (HFD) fed apoE-/- mice, co-administration
the effect of activating oxidative stress in macrophages, but this may of metformin and T317 inhibits the development of AS,
not affect the protection of metformin on the function including down-regulation of monocyte adhesion and
of macrophages. macrophages cell proliferation, and up-regulation of ABCA1/
Mitochondrial damage plays an important role in the ABCG1 expression. Metformin blocks T317-induced fatty liver
pathogenesis of obesity, diabetes, and CVD (83, 84). via reducing T317-stimulated hepatic LXRa nuclear

translocation, adipogenic gene expression and activating 6, CD16, CD36, and CD206 suggests that the M1-like phenotype of
AMPKa. This suggests that treatment with metformin and macrophages is observed in the T2DM. With metformin treatment,
T317 may be a new strategy to decrease foam cell formation TNFa, iNOS, IL-6, CD11c, CD36, CD169 and CD206 levels in
and AS (92). T2DM patients were restored to levels of lean volunteers. The results
Although there is a positive effect on the reverse transport of of this study suggest that PBMCs in T2DM express a different
cholesterol in macrophages, metformin may have no significant pattern of phenotypic markers (representing metabolically activated
effect on the reverse transport of intestinal cholesterol. The effect macrophage (MMe)-like cells), which is not the pattern normally
of dipeptidyl peptidase-4 inhibitor (DPP-4i) sitagliptin and found in M1 or M2-like macrophages, and further that metformin
metformin on RCT was studied using obese insulin-resistant can reduce circulating MMe-like cells (100).
CETP-apoB100 transgenic mice. Sitagliptin (rather than In HFD-fed C57/6J male mice, metformin administration for
metformin) increased fecal cholesterol excretion by 132%, 7 weeks not only decreased blood levels of TNF-a and IL-6, but
indicating that sitagliptin promotes RCT by reducing intestinal also decreased M1 macrophage marker (MCP1 and CD11c) in
cholesterol absorption (93). AT. This study demonstrates that metformin regulates
Although there is no significant effect on the reverse transport macrophages phenotype to M2 by activating AMPK, which
of intestinal cholesterol, the above studies indicate that decreases low-grade inflammation in obesity (101). The Src
metformin may decrease lipid accumulation in macrophages homology 2 (SH2) domain-containing protein tyrosine
and exert a positive effect in AS in patients with the MetS. phosphatase 1 (SHP-1) is a negative mediator of inflammation.
In an obese mouse model, metformin effectively polarizes AT
M1/M2 Macrophage Polarization macrophages to an anti-inflammatory state via indirectly
In addition to the first line of defense, macrophages also exert a inhibiting SHP-1 expression, thereby downregulating NF-kB,
very important effect in maintaining the homeostasis of various STAT1, CD80, CD86, TLR2, TLR4 and inhibiting inflammation
tissues and organs (94, 95). In response to extrinsic factors from a of AT (102). Acute AMPK activation has exacerbated ischemic
given tissue, macrophages activate different functional programs brain injury, but the clinical application of metformin reduced
to generate polarized macrophage populations, which are the incidence of stroke. Chronic metformin administration after
responsible for inducing inflammation against microorganisms, stroke improves functional recovery after cerebral artery
removing cell debris, and tissue repair (96). The role of occlusion (MCAO) in mice by AMPK-dependent microglia/
macrophages in AS is thought to be inseparable from the macrophage M2 polarization. Regulation of microglia/
polarization and phenotypic expression of macrophages. In macrophage polarization may represent a promising treatment
addition, the role of macrophages in AS depends not only on for stroke (103). In apoE-/- mice, pharmacological AMPK
the function of different macrophage phenotypes, but also on the activation (such as metformin) inhibits the formation of
relative proportion of different phenotypes in atherosclerotic atherosclerotic plaque by inducing macrophage M2
plaques. Studies on AS therapy have shown that the decrease polarization, reducing plasma lipids peroxidation and
in plaque size and the increase in stability are partly attributable inflammatory cytokines expression (104).
to the regulation of macrophage polarization (97). M2 In a cellular model, metformin induces RAW264.7
macrophages are associated with regression of AS. The M2 macrophages with/without LPS stimulation to the M2
macrophages can produce the IL-10 and TGF-b, thereby phenotype, and Notch1 signaling may be involved in the
eliminating dying cells and debris through endocytosis, and regulation process of metformin on the polarization of
promoting tissue remodeling and repair through collagen macrophages (105). Metformin affects the phenotype of
formation (98). Platelets represent an important cell type that macrophages and ameliorates the activity of glutathione
mediates inflammation and immune processes in AS, mainly by peroxidase, superoxide dismutase and catalase (79). However,
secreting chemokines when platelets are activated. For example, studies have also shown that metformin specifically attenuates
CXCL4 binds to CCL5 to induce monocyte adhesion, thereby the production of pro-inflammatory cytokines without reducing
promoting the transdermal effect of monocytes into the M1/M2 differentiation (55).
subendothelial space. CXCL4 also induces monocyte
differentiation and forms a specific macrophage phenotype
(M4) (99). In summary, changing macrophage proportions Monocyte Differentiation Into Macrophage
and adjusting macrophage polarization in the plaque Monocytes are derived from the hematopoietic precursors of
represents a new treatment frontier for the therapy of AS. bone marrow or spleen and produce the classic lymphocyte
In a study involving 30 normal-weight healthy adult volunteers, antigen 6C (LY6C) high in mice (corresponding to human
30 obese volunteers, 20 obese newly diagnosed patients with CD14high CD16low monocytes), and nonclassical-LY6Clow in
diabetes, as well as 30 metformin-treated obese patients with mice (corresponding to human CD14low CD16high monocytes)
diabetes peripheral blood mononuclear cells (PBMCs) were (49, 106). LY6Chigh monocytes highly express CC chemokine
isolated and polarization markers were measured. The results receptor 2 (CCR2), and is considered to be the precursor of M1
showed CD68 marker was increased in obesity and in T2DM. macrophages. LY6C high monocytes can be recruited by
The levels of CD11b, CD11c, CD163 and CD169 in T2DM patients chemokines to atherosclerotic plaques and play a pro-
were reduced, and CD11c in obese volunteers was significantly inflammatory effect (49). Hypercholesterolemia promotes the
inhibited. In addition, the increased expression of TNFa, iNOS, IL- production of LY6Chigh monocytes by inducing the proliferation

of bone marrow precursors (107). LY6C low monocytes promotes the inflammatory environment in the blood vessel wall
(considered to be the precursor of M2 macrophages) highly to exacerbate AS (108) (Figure 2).
express chemokine receptor CX3CR1 (49). These monocyte Forty-four T2DM patients with acute myocardial infarction
subpopulations use different chemokine-chemokine receptor were divided into two groups according to the hypoglycemic
pairs to penetrate into the inner membrane, and then drugs taken, including 21 cases of metformin and 23 cases of
differentiate into macrophages in the inner membrane (106). short-acting insulin. Metformin treatment resulted in a
The differentiation of LY6Chigh monocytes to M1 macrophages faster reduction in sCD40L compared to insulin treatment,
CD14 + CD14 low

Monocytes in human CD16 - CD16 +
correspond to
Bone marrow CCR2
CD36 CX3CR1
LY6Chigh LY6Clow
Monocytes in mice
CD11c
CCL2/CCR2
Hyperlipidemia
Blood CCR2
CD36 CX3CR1
LY6Chigh LY6Clow
CD11c
VCAM1 CX3CL1/CX3CR1
Vascular endothelium P-selectin ICAM1

Tissue Monocyte-derived macrophages
3
Differentiation
AT
ST
Inflammatory Anti-inflammatory
macrophages Polarization macrophages
(M1) (M2)
Activation by LPS, oxLDL and INFγ Activation by IL-4 and IL-13

IL-1β, IL-12, TNF, iNOS/NO IL-10 and IL-1R antagonist
NF-κB, AP-1 and HIF1α Arg1, collagen
MHC class II molecules, CD80 KLF4, PPARγ, STAT6
and CD86 CD163, CD206, FIZZ1
Enriched in progressing plaques High endocytic activity
Enriched in regressing plaques
FIGURE 2 | Monocyte differentiation and macrophage polarization. Monocytes are derived from the hematopoietic precursors of bone marrow or spleen and
produce the classic lymphocyte antigen 6C (LY6C) high in mice (corresponding to human CD14high CD16low monocytes), and nonclassical-LY6Clow in mice
(corresponding to human CD14low CD16high monocytes). LY6Chigh monocytes highly express CCR2, which can be recruited by chemokines (such as CCL2) to
inflammatory sites (including atherosclerotic plaques) to play a pro-inflammatory effect (it is considered to be the precursor of M1 macrophages).
Hypercholesterolemia promotes the production of LY6Chigh monocytes by inducing the proliferation of bone marrow precursors. LY6Clow monocytes (considered to
be the precursor of M2 macrophages) highly express CX3CR1. These monocyte subpopulations use different chemokine-chemokine receptor pairs to penetrate into
the inner membrane, and then differentiate into macrophages in the inner membrane (LY6Chigh is more likely to differentiate into M1 macrophages, while LY6Clow is
more likely to differentiate M2 macrophages). M1 macrophages amplify the inflammatory effect by secreting pro-inflammatory cytokines. M2 macrophages help tissue
repair by secreting anti-inflammatory cytokines and collagen. AP-1, activator protein 1; Arg1, arginase 1; CCL, C−C motif chemokine; CCR, C-C chemokine
receptor; CD, cluster of differentiation; FIZZ1, found in inflammatory zone 1; HIF1a, hypoxia-inducible factor 1a; ICAM1, intercellular adhesion molecule-1; INFg,
interferon-g; iNOS, inducible nitric oxide synthase; IL, Interleukin; IL-1R, Interleukin 1 receptor; KLF4, Krüppel-like factor 4; LPS, lipopolysaccharide; NO, nitric oxide;
NF-kB, nuclear factor kappa B; PPAR, peroxisome proliferator activated receptor; STAT, Signal transducer and activator of transcription; VCAM1, vascular cell
adhesion protein 1.

which helped to improve the prognosis in this cohort. can produce off-target effects by inhibiting other protein kinases
Mechanistic studies suggest that the inhibitory effect of (124–126). As an AMPK agonist, AICAR may not be a
metformin on sCD40L levels may be due to reduction of Akt sufficiently specific enough tool to address the index questions
phosphorylation, which is responsible for activating immune (127). The M1/M2 polarization effect of AICAR on macrophages
+=+ −=−
response genes that cause platelet activation and monocyte has no difference in AMPKa2 and AMPKa2 , which
differentiation into macrophages cell capable of producing indicates that the M2 polarization effect induced by AICAR is
AS (109). at least partially independent of AMPK (128). Therefore, it is
In the AS model, metformin inhibits monocyte differentiation. necessary to use more specific AMPK activators and inhibitors to
In a diet-induced rabbit AS model, administration of metformin study the role of metformin. Importantly, the maximum dose of
inhibited rabbit monocytes from differentiating into macrophages oral metformin in clinical practice is 2.5–3 g per day, which is
and inflammatory responses and decreased aortic mRNA approximately 35–42 mg/kg (129, 130). In humans, the plasma
expression of adhesion molecules and inflammatory cytokines concentration range of metformin after passing through the liver
(62). In ApoE-/- mice, the AMPK-STAT3 axis exerts a key effect in is usually 10–40 mM (130). When we collated the studies of
modulating the differentiation of monocytes-macrophages. metformin and macrophage function in atherosclerotic diseases,
Metformin reduces STAT3 phosphorylation by increasing we found that the dose of metformin of some studies was much
AMPK activity and inhibits monocyte differentiation into higher than the highest clinical dose (Table 2) (72). Such studies
macrophages (40). have a role in exploring the mechanism of action of metformin,
but such studies may lack practical clinical significance.
Apoptosis
During the formation and development of atherosclerotic
lesions, lipid-filled macrophages form foam cells, accumulate
and eventually undergo apoptotic death. Further aggregation of MOLECULAR TARGETS OF METFORMIN
apoptotic foam cells may lead to secondary necrosis as well as the
formation of a necrotic lipid core, which makes the plaque AMPK
unstable and prone to rupture (Figure 1). Thus, non-lipid- Adenosine monophosphate activated protein kinase (AMPK)
filled macrophages, as the main phagocytic cells in plays a very critical role in the many beneficial effects of
atherosclerotic lesions, need to effectively remove apoptotic metformin, and in the protection from AS afforded by
foam cells (110). metformin. Studies have shown that long-term treatment with
Apoptosis of OxLDL-stimulated macrophages contributes to metformin to activate AMPK can reduce atherosclerotic
the development of AS. Metformin reduces oxLDL-stimulated calcification and inhibit Runt-related transcription factor
macrophage lipid uptake and prevents macrophage apoptosis. (Runx2) expression in ApoE-/- mice noting that Runx2 is an
Possible mechanisms involve inhibition of ER stress (reduction important promoting factor of vascular calcification in mice. On
of eukaryotic translation initiation factor 2A, C/EBP homologous the other hand, metformin has little effect on atherosclerotic
protein and glucose regulatory protein expression), reversal of calcification in ApoE-/-/AMPKa1-/- mice (39).
mitochondrial membrane potential loss and cytochrome c (cyto- The differentiation of monocytes into macrophages is a key
c) release, as well as regulation of scavenger receptor expression event that exacerbates AS via enhancing the inflammatory
(111). In addition, inflammatory aging is related to the environment in the blood vessel wall (40). Hyperlipidemia
progression of diabetes complications. Senescence-related usually reduces AMPK activity and increases CC chemokine
secretory phenotype (SASP) is the main factor resulting in receptor 2 (CCR2) expression. CCR2 controls the migration of
inflammatory senescence, and macrophages are important Ly6Chigh monocytes from the bone marrow (BM) to blood,
SASP-carrying cells. In macrophages, high glucose can induce which contributes in the accumulation of macrophages in the
cell senescence and secretion of SASP factors through the progression of AS. ApoE-/- mice were fed with HFD and then
phosphorylation of domain (CARD)-containing 4 (NLRC4), received AMPK activator (metformin, A769662 or AICAR)
and further stimulate the NF-kB/Caspase-1 cascade through treatment for 10 weeks. The AMPK activators decreased the
the interferon regulatory factor 8 (IRF8) pathway. Knockout of Ly6C high monocyte migration by reducing CCR2 protein
NLRC4 or IRF8 inhibited cell senescence and SASP caused by expression. At the same time, AMPK activators decreased AS-
hyperglycemia in macrophages. Under high glucose conditions, induced macrophage accumulation in ApoE-/- mice via reducing
metformin treatment can inhibit NLRC4 phosphorylation and CCR2 expression (113). Similarly, in ApoE-/- mice, metformin
significantly reduce cell senescence and SASP (112). and AICAR can partially reduce monocyte infiltration, thereby
Significant progress has been made in the study of metformin reducing Ang-II stimulated atherosclerotic plaque formation and
and macrophage function, but there are still some limitations to aortic aneurysms (40). During phorbol 12-myristate 13-acetate
these investigations. First of all, most of the studies on metformin (PMA) mediated differentiation of monocytes into macrophages,
and AS looking at improving the function of macrophages are AMPK activity decreases and pro-inflammatory cytokine levels
pre-clinical studies (Table 2). Compound C (an AMPK increase. AMPK activators metformin and AICAR reduce
inhibitor) and 5-amino-1-b-D-furanofuranosyl-imidazole-4- monocyte differentiation stimulated by PMA and the
carboxamide (AICAR, as AMPK activator) are often used in accompanying pro-inflammatory cytokine expression. By
mechanistic pharmacological research; however, compound C reducing the phosphorylation of STAT3, metformin and

AICAR inhibit the differentiation of monocytes to macrophages requires hematopoietic AMPK. The clinically relevant
by increasing AMPK activation (including in the absence of concentration (10mM) of metformin up-regulated LXRb,
PMA). These findings indicate that the AMPK-STAT3 axis Hmox1, ApoE, ABCA1, PDGF and IGF1, as well as increased
exerts a key role in modulating the differentiation of several M2 markers and decreased iNOS in mouse BMDM by
monocytes into macrophages, and AMPK activators reduce activating AMPK and ATF1. A similar effect was observed in
STAT3 phosphorylation by increasing AMPK activity and human blood-derived macrophages. These results indicate that
inhibiting monocyte differentiation (40). metformin can activate AMPK-ATF1-M2-like pathways in
In murine macrophages, metformin inhibits LPS-stimulated macrophages to inhibit AS in hyperlipidemia mice, and
IL-6 and TNF-a production, and simultaneously induces support the clinical study of metformin in people without DM
activation of transcription factor 3 (ATF3). ATF3 gene but with high risk of AS (115, 116). In HFD-fed C57/6J mice,
silencing reversed the inhibitory effect of metformin on LPS- metformin treatment for 7 weeks decreased the blood levels of
stimulated pro-inflammatory cytokine production, and at the TNF-a and IL-6 as well as the expression of CD11c and MCP-1
same time annulled the inhibitory effect of metformin on MAPK (M1 macrophage markers) in AT. In palmitate-stimulated
phosphorylation. After metformin treatment, the NF-kB RAW264.7 cells, metformin decreased the secretion of IL-6
enrichment on IL-6 and TNF-a promoters stimulated by LPS and TNF-a, while down-regulating M1 macrophages and up-
was replaced by ATF3. AMPK gene silencing attenuates all the regulating M2 macrophages. These findings show that
beneficial effects of metformin (including ATF3 activation, pro- metformin can regulate the polarization of macrophages to the
inflammatory cytokine suppression and MAPK inhibition). anti-inflammatory M2 phenotype and improve low-grade
These findings show that anti-inflammatory effect of inflammation in obesity by activating AMPK (101).
metformin in macrophages is at least partially through the Acute AMPK activation enhances ischemic brain injury, but
activation of the AMPK/ATF3 pathway (114). the clinical application metformin decreases the incidence of
In an animal model study, ApoE-/- mice were assigned to a stroke (103). The duration of AMPK activation plays a crucial
control group, streptozotocin treated group or metformin treated role for the effect of metformin on the prognosis of stroke (117).
group. Metformin treatment can reduce the metabolic disorder Mice received metformin treatment for 30 days after 24 hours of
and AS caused by streptozotocin via reducing NLRP3 MCAO. Chronic metformin treatment after stroke significantly
inflammasome activation as well as the disorder of enhances brain AMPK activation, increases angiogenesis and
thioredoxin-1/thioredoxin interacting protein. Metformin also neurogenesis, and makes microglia/macrophages tend to the M2
inhibits ROS accumulation and NLRP3 inflammasome state in the ischemic brain (103). Advanced glycation end
activation stimulated by high glucose in macrophages which is p r o d u c t s ( A G E s ) a r e t h e m a i n g l u c o s e -d e p e n d e n t
blocked by compound C. These results indicate that metformin inflammatory mediators of DM. In mouse macrophages,
can inhibit NLRP3 inflammasome activation in apoE-/- mice and metformin pretreatment can inhibit the expression of cluster
inhibit diabetes-accelerated AS, at least in part by activating marker 86 (CD86) (M1 marker) induced by AGEs by activating
AMPK and regulating thioredoxin-1/thioredoxin interaction AMPK, and promote the surface expression of CD206 (M2
protein (70). marker) and IL- 10 mRNA expression (118).
In oxLDL-stimulated macrophages, metformin treatment can In summary, AMPK activity is critical for the protective
attenuate the protein expression and activation of the NLRP3 effects of metformin, including inhibiting monocyte cell
inflammasome. AMPK gene knockdown partially restores the migration and differentiation, inhibiting oxidative stress,
activation of NLRP3 inflammasome, and PP2A inhibition inflammation and inflammasome activation, and macrophage
restores the metformin-mediated down-regulation of NLRP3 polarization. The diseases and disorders involved include AS,
and pre-IL-1b expression. Moreover, metformin-induced NF- stroke, diabetes, obesity and hyperlipidemia. These protective
kB inhibition also requires PP2A catalytic activity. These results effects and mechanisms are occurring at least partially through
show that metformin reduces NLRP3 protein expression and the activation of AMPK.
activation via AMPK and PP2A in oxLDL-induced macrophages
(71). In RAW264.7 macrophages, LPS treatment significantly LXR/ABCG1
induced the expression of CXCL10 and CXCL11. Metformin can The initial and rate-limiting step of RCT is the HDL-mediated
inhibit the phosphorylation of I-kBa and p65 by activating cholesterol efflux (88). ATP-binding cassette (ABC) cholesterol
AMPK and prevent the stimulation of these chemokines, as transporter-mediated cholesterol efflux from macrophages can
well as IL-1 and IL-6 induced by LPS (53). reduce the progression of AS in patients (89). HDL glycosylation
The heme produced by hemorrhage in the plaque may drive (3-DG incubation) obviously decreased HDL-mediated
the protective M2-like phenotype through AMPK and ATF1. cholesterol efflux from MDM. Glycated HDL particles cannot
Metformin has a similar effect in macrophages to inhibit AS. In effectively act as ABCG1-mediated cholesterol efflux receptors,
low-density lipoprotein receptor (Ldlr)-/- hyperlipidemia mice, which can partially explain the acceleration of AS in DM
oral metformin inhibited the development of atherosclerotic patients, and metformin treatment restores HDL-mediated
lesions by activating AMPK and ATF1 in macrophages. Bone cholesterol efflux (88). Similarly, RAW264.7 cells were
marrow transplantation experiments on AMPK knockout mice stimulated by oxLDL (50mg/ml) for 24 h, and then treated with
showed that the anti-atherosclerotic protection of metformin metformin (15mM) for 24 h. Metformin promotes the outflow of

cholesterol to HDL by increasing ABCG1 expression, which cholesterol. In human or mice primary hepatocytes, metformin
decreases foam cell formation induced by oxLDL. At the same treatment increased ABCG5/8 expression. Recent studies have
time, metformin increases the IL-10 secretion that is impaired by shown that metformin may increase the RCT of macrophages. In
oxLDL, which is an important anti-inflammatory cytokine in AS HFD-fed mice, metformin treatment reduced the occupation of the
(89). In HFD-fed apoE-/- mice and macrophages, metformin transcription repressor in Abcg5/8 and significantly increased
treatment also showed significant RCT. Mechanism studies have Abcg5/8 expression. These findings support that metformin may
shown that AMPK activates HDL in mice, with higher protect the cardiovascular system via up-regulating the reverse
paraoxonase 1 activity, lower myeloperoxidase activity and transport of cholesterol (131).
lower HDL inflammation index. Metformin also up-regulated
the expression of ABCG1 and ABCA1 in macrophages, as well as HMGB1
lecithin:cholesterol acyltransferase (LCAT) and SR-B1 in the High mobility group box-1 (HMGB1) protein was identified in
liver (104). 1973 and can be released from various cells. It is a typical
damage-related molecular model protein (122, 123). Several
NF-kB modifications of HMGB1 facilitate its transport to the
Numerous studies have shown that metformin can block the NF- cytoplasm and release from the cell either actively or passively.
kB signaling pathway in macrophages. For example, in BMDM, When released outside the cell, HMGB1 is essential for
metformin treatment attenuated the LPS-stimulated inflammation. Its biological function is through interaction
phosphorylation of p65 and JNK1 and the up-regulation of with receptors, such as receptor for advanced glycation end
p ro -i n flammatory c ytokine lev els (119). The a nt i- products (RAGE) (122, 123, 132). HMGB1 is released by
atherosclerotic effect of macrophage-derived apoE is well necrotic cells and leads to inflammatory responses through its
known. Metformin (1-3 mM) reversed the reduction of apoE cytokine-like activity; thus, HMGB1 is a potential target for the
expression in macrophages induced by LPS via reducing the development of anti-inflammatory therapies (133). By using a
nuclear translocation of NF-kB (120). In human SMCs, biotinylated metformin analog for affinity purification, it was
macrophages and ECs, metformin can dose-dependently found that metformin directly binds to the C-terminal acid tail of
inhibit the release of the IL-6 and IL-8 stimulated by IL-1b in HMGB1. Moreover, in the paracetamol-stimulated acute liver
these three cell types. Mechanism studies have indicated that damage model, HMGB1 released from damaged cells
metformin can block NF-kB nuclear translocation by inhibiting exacerbated liver injury, and metformin treatment inhibited
the PI3K-Akt pathway (54). In oxLDL-stimulated macrophages, the liver damage (133). In addition, the exaggerated release of
NF-kB inhibition induced by metformin requires PP2A catalytic NETs, reduced NET clearance as well as impaired clearance of
activity (71). Metformin may not require AMPK to inhibit NF- apoptotic cells may induce persistent inflammation of ARDS.
kB activation. For example, in AMPK null fibroblasts and Neutrophils from ARDS patients showed a decrease in apoptosis
macrophages, metformin inhibits the NF-kB signaling pathway and an increase in NETs formation. Incubation of neutrophils
induced by LPS (121). The anti-inflammatory role of metformin with bronchoalveolar lavage (BAL) fluid of ARDS can promote
may partially contribute to the effect of metformin in reducing NETs formation. In ARDS patients, macrophage phagocytosis of
MACE independently of its hypoglycemic effect (54). NETs and apoptotic neutrophils is decreased. Neutralization of
HMGB1 antibody in BAL fluid or activation of AMPK in
Sirt1 macrophages in BAL fluid improved cell swelling and NETs
The ARDS model was established by injecting LPS into mice clearance. These results suggest that using metformin to restore
treated with metformin in advance and mice in the control AMPK activity or specifically neutralize HMGB1 in BAL fluid
group. ARDS model mice developed neutrophil accumulation, may reduce persistent lung inflammation during ARDS (134).
vascular exudation and pulmonary edema, and the expression of Similarly, metformin can significantly reduce the LPS-stimulated
IL-1b, TNF-a, IL-6 and IL-17 were up-regulated. Metformin macrophage inflammatory response in vivo (mice) and in vitro
treatment partially reversed indicators of pulmonary arterial (RAW 264.7 cells) by inhibiting HMGB1 secretion, and improve
damage and reduced LPS-induced deaths. The expression of the survival rate of endotoxemic mice (135).
SIRT1 in metformin-treated alveolar macrophages (NR8383)
was higher than that of LPS alone, while the levels of p-p38, p- FOXO1/FABP4
ERK and p-NF-kB were significantly down-regulated. Further Lipid accumulation in macrophages induces the development of
research showed that metformin decreased miR-138-5p AS. In addition to reducing lipid accumulation in adipocytes,
expression, while miR-138-5p could target SIRT1 and inhibit metformin also reduces palmitic acid-stimulated lipid
its expression. These results indicate that metformin increases accumulation in macrophages. Mechanistic studies show that
SIRT1 expression and inhibits the MAPK pathway by reducing metformin reduces FABP4 expression and promotes ocarnitine
the expression of mir-138-5p, thereby reducing the lung injury palmitoyltransferase I (CPT-1) expression, which is related to
and the expression of inflammatory factors caused by LPS (58). palmitic acid-stimulated lipid accumulation in macrophages.
Further studies show that FOXO1 siRNA inhibits FOXO1
ABCG5/8 expression and significantly reduces basal and palmitic acid-
ATP-binding cassette transporter G5 and G8 (ABCG5/8) mediates induced FABP4 expression, while metformin reduces FABP4
the final step of RCT, which promotes the hepatobiliary transport of expression via reducing nuclear translocation of FOXO1. These

findings indicate that metformin decreases lipid accumulation insulin sensitivity, which is mediated by AMPK in mouse
via inhibiting FOXO1-induced FABP4 transcription in experiments. These results indicate that the acute and chronic
macrophages (91). pharmacological mechanism of action of metformin is both
AMPK-dependent and independent. Therefore, we speculate
The Mechanism of Metformin Actions that the AMPK-independent effect of metformin in
Independent of AMPK macrophages (such as the metformin inhibition of LPS-induced
AMPK consists of a catalytic subunit (a) and regulatory subunits NF-kB signaling pathway) may be an acute effect.
(b and g) (136, 137). The a subunit of AMPK has two subtypes, Metformin reduces macrophage oxidative stress and
AMPKa1 and AMPKa2, which differentially exist in different inflammatory cytokine production by AMPK activation, but
tissues (138). Among them, the AMPKa1 mainly exists in SMCs the residual effect of metformin remains significant even after
(139), macrophages (140) and AT (141), while AMPKa 2 compound C treatment (79). Although compound C has AMPK
expression is much higher in cardiomyocytes (142). AMPK off-target effects, the study also shows that metformin may not
exhibits a very important regulatory function in both completely rely on AMPK to inhibit macrophage inflammation.
metabolism and CVD (143). In addition, in AMPK null macrophages, metformin inhibited
−=−
AMPK a2 knockout (AMPKa2 ) mice have high ER stress the NF-kB signaling pathway induced by LPS. Therefore,
and atherosclerotic phenotypes of aortic ECs (144). The genetic metformin’s inhibition of macrophage inflammation may
defect of AMPKa1 (but not AMPKa2) enhances vascular partly directly depend on NF-kB. The molecular targets of
calcification. Long-term administration of metformin to activate metformin in ameliorating macrophage dysfunction was
AMPK can reduce vascular calcification in ApoE-/- mice, but not summarized in Figure 3.
in ApoE-/-/AMPKa1-/- mice. These studies reveal that both
AMPKa1 and AMPKa2 exhibit a very important effect in AS,
and metformin inhibits arteriosclerosis calcification mainly by
CURRENT PERSPECTIVES
activating AMPKa1 (39). As we mentioned earlier, metformin AND LIMITATIONS
plays a regulatory role in macrophage oxidative stress,
inflammation, macrophage polarization, foam cell formation, Research on Non-Coding RNA (Including
and apoptosis. However, the specific mechanisms are still not miRNA and lncRNA) Mechanisms in
clear. BM transplantation experiments on AMPK knockout mice the Pharmacological Actions of
showed that the anti-atherosclerotic protection of metformin Metformin in AS
requires hematopoietic AMPK, which suggests that AMPK Recent evidence has shown that AS is a disease with epigenetic
occupies a crucial position in the modulation of macrophage components. Various documented epigenetic modifications
function by metformin (115). However, in AMPK null (including histone methylation/acetylation and non-coding
fibroblasts and macrophages, metformin blocked LPS-stimulated RNA) exhibit an important regulatory effect in all aspects of
NF-kB signaling pathway (including preventing the nuclear the pathogenesis of AS, including EC damage and dysfunction,
translocation of NF-kB, and inhibiting IkB and IKKa/b SMC proliferation, migration and phenotypic modulation,
phosphorylation) (121). The differences in the mechanism of monocyte differentiation, macrophage-mediated inflammation
metformin in vivo and in vitro need to be further studied. It and polarization and foam cell formation, as well as platelet
may be impractical to perform macrophage-specific AMPK aggregation. Several small-molecule epigenetic drugs (including
knockout in vivo, because MDM are an important link in the histone deacetylase inhibitors, trichostatin A (TsA) and
development of AS. suberoylanilide hydroxamic acid (SAHA) as well as DNA
In the signaling of the regulation of liver glycogen homeostasis, methyltransferases inhibitors 5-Azacytidine and 5-aza-2’-
metformin inhibits complex I and prevents the production of deoxycytidine) approved by FDA have shown promise in
mitochondrial ATP, thereby increasing the ratio of cytoplasmic preclinical studies for the treatment of AS (149).
ADP: ATP and AMP: ATP, which leads to AMPK activation Monocyte/macrophages can develop long-term pro-
(145). However, the up-regulation of AMP: ATP also inhibits inflammatory and pro-atherosclerotic phenotypes after treatment
fructose 1, 6-bisphosphatase (FBPase), leading to an acute with inflammatory stimuli (including oxLDL). This inherent
reduction of gluconeogenesis (146), and at the same time immune memory is produced due to changes in metabolic
inhibited adenylate cyclase and reduced the production of pathways and epigenetic reprogramming of histone modifications.
cAMP (147). Acute treatment with AICAR or metformin can The persistence of blood hyperresponsive monocytes in the body
also inhibit glucose production in hepatocytes of wild type mice or may be due to training in myeloid progenitor cells in BM. In
mice lacking both AMPKa1 and AMPKa2 in the liver, and patients with AS and those with dyslipidemia, there are well-trained
metformin can improve glucose tolerance in the two mouse immunophenotypic monocytes (150). These findings indicate that
strains (148). This indicates that the acute inhibitory role of monocyte-transformed macrophages have amazing inflammatory
metformin on the production of hepatic glucose is independent plasticity, allowing chronic inflammation and atherosclerotic plaque
of AMPK, and hence that AMP inhibition of fructose-1,6- to persist (151).
bisphosphatase is a possible explanation. However, the main Non-coding RNA (ncRNA) include microRNA (miRNA), long
long-term hypoglycemic role of metformin is to increase hepatic non-coding RNA (lncRNA) as well as circular RNA (circRNA)

Foam cell Inflammation↓

formation↓
Neutrophil to lymphocyte ratio↓ CCL2↓

Cholesterol accumulation↓
CXCL10↓, CXCL11↓, IL-1β↓, IL-6↓, IL-8↓
cholesterol efflux↑
NLRP3↓, ASAA↓, iNOS↓, COX2↓, IL-10↑
APMK↑, NFκB↓, MAPK (p38 and ERK) ↓

ABCA1↑, ABCG1↑, ABCG5/8↑
mir-138-5p↓, SIRT1↑ , PI3K-Akt↓, ATF3↑
ADRP↓, FOXO1↓, FABP4↓, HDL↑
thioredoxin-1↑, PP2A↑ , HMGB1↓, CSE↑
Metformin
effects
AMPK↑, STAT3↓ APMK↑, SHP-1↓, ATF1↑
Akt/sCD40L↓, CCR2↓
SRA↓,CD36 ↓
M1 markers(CD86, CD11c and
Monocyte Lipid uptake↓
MCP1)↓, M2 markers(CD206)↑
differentiation↓ ER stress↓
Cyto-c release↓ M2
Apoptosis↓ Mitochondrial membrane polarization↑
potential loss↓
FIGURE 3 | The effect of metformin on monocyte/macrophage functions in AS. Metformin inhibits monocyte/macrophage dysfunction via modulating the expression
and activity of genes or proteins closely related to monocyte differentiation, macrophage inflammation, M1/M2 polarization, foam cell formation and macrophage
apoptosis. ↑indicates increase or activation, and ↓indicates decrease or suppression. ABCA1, ATP-binding cassette transporter A1; ABCG1, ATP-binding cassette
transporter G1; ABCG5/8, ATP-binding cassette transporter 5/8; ADRP, adipogenic differentiation-associated protein; Akt, protein kinase B; AMPK, 5’-adenosine
monophosphate-activated protein kinase; ASAA, acute-phase serum amyloid A; ATF, Activation of transcription factor; CCL2, C-C motif chemokine ligand 2; CCR2,
CC chemokine receptor 2; CD, cluster of differentiation; CXCL, Chemokine C-X-C ligand; CSE, Cystathionine g-lyase; cyto-c, cytochrome c; ER, endoplasmic
reticulum; ERK, Extracellular signal-regulated kinase; FABP4, Fatty acid binding protein 4; FOXO1, Forkhead box transcription factor O1; HDL, high-density
lipoprotein; HMGB1, high mobility group box-1; iNOS, inducible nitric oxide synthase; IL, Interleukin; MAPK, mitogen-activated protein kinase; MCP1, Monocyte
chemoattractant protein 1; NF-kB, nuclear factor kappa B; NLRP3, NOD-like receptor family pyrin domain containing 3; PI3K, phosphatidylinositol 3-kinase; PP2A,
protein phosphatase 2A; sCD40L soluble CD40 ligand; SH2, Src homology 2; domain-containing protein tyrosine phosphatase 1(SHP-1), SIRT1, Sirtuin-1; SRA,
scavenger receptor class A; STAT3, Signal transducer and activator of transcription 3.
(152). ncRNA can regulate the occurrence and development of AS inducing Dicer (a key miRNA biogenesis enzyme) and then up-
by interacting with proteins, DNA and RNA (153). Inhibiting the regulating miR-125b-5p and miR-34a-5p (155).
role of macrophages in inflammation is crucial for inducing the lncRNA is a non-protein coding RNA group whose length are
regression of AS, and miRNA has emerged as a key regulator of more than 200 nucleotides. lncRNA regulates the complex gene
macrophage phenotype. MiRNA which is a small non-coding RNA regulatory network in macrophages to participate in the process of AS
that modulates gene expression, is very stable in the circulation and (156–158). Many lncRNAs are involved in macrophage cholesterol
therefore has the potential as a therapeutic agent and/or biomarker deposition, inflammation and foam cell formation, such as LASER,
in the case of AS. miRNA (such as miR-155) is dysregulated during LeXis and CHROME. MANTIS, lncRNA-CCL2 and MALAT1 are
the development of AS and is an important mediator of macrophage associated with vascular inflammation. In addition, some lncRNAs
inflammation and polarization. Inhibiting macrophage-specific miR- are closely related to the response to statin therapy, such as NEXN-
155 can reduce atherosclerotic inflammation (154). Conjugated AS1 or LASER. Some lncRNAs can also serve as biomarkers of CVD
linoleic acid (CLA), a peroxisome proliferator-activated receptor (159). LncRNA is a potential new therapeutic target for CVD, but the
(PPAR)-g agonist, reduces inflammation of monocytes and research on lncRNA in AS is still in its infancy (159).
macrophages through regulation of miRNA, and exerts anti- NcRNA, including miRNA and lncRNA, exhibit an
atherosclerosis effect through this mechanism (154). In LPS- important effect in modulating the phenotype and function of
stimulated macrophages, metformin exhibits an anti-inflammatory macrophages through epigenetic modification of macrophages.
role (decrease the expression of IL-6 and TNFa) on macrophages by At present, whether metformin affects the function of

macrophages by acting on miRNA or lncRNA is still lacking, innate immune defense (171). NETs are reticular fiber structures
which may be an important direction for future research. consisted of DNA-histone complexes and proteins produced by
activated neutrophils (172). NETs were discovered ten years ago
Single Cell Sequencing Plays a Very as part of the first host defense system against invading
Important Role in Revealing the microorganisms (173). Accumulating evidence indicates that
Pathogenesis of AS NETs are also present in malignant tumors, AS and
Traditional genetic information research methods are carried out at autoimmune diseases, including systemic lupus erythematosus
the multicellular level. Therefore, the final signal value is actually the (SLE), rheumatoid arthritis (RA), gout and psoriasis. Recent
average of multiple cells, and the information of heterogeneity is evidence indicates that NETs are contributors to venous and
lost. Single-cell sequencing can detect heterogeneous information arterial thrombosis (174). The imbalance between NETosis (the
that cannot be obtained by sequencing mixed samples (160–162). process of NETs formation) and NETs degradation may be
Currently, barcode-based single-cell identification/combinatorial related to autoimmune diseases (175). NETs are found in
indexing is often adopted, and the information of hundreds of atherosclerotic lesions in human and animal models, and
single cells can be measured by building a database at a time (163, NETs are involved in multiple components of atherogenesis.
164). In recent years, single-cell sequencing has exerted an For example, NETs induce endothelial cell dysfunction and
important effect in revealing the pathogenesis of diseases and apoptosis, and promote the production of autoantibodies
targets for drug therapy. against double-stranded DNA. NETs may also play a role in
Atherosclerotic plaque contains autoantibodies, and there is a promoting thrombosis because they form a fibrin-like matrix
link between AS and autoimmunity (165–167). Through the high- where platelets adhere, become activated and aggregate. NETs
throughput single-cell analysis of AS-related antibody library, induce oxidative stress leading to oxidization of HDL particles,
including antibody gene sequencing for more than 1,700 B thereby reducing their beneficial cholesterol efflux ability (176).
lymphocytes in control mice and atherosclerotic Ldlr-/- mice, 56 In addition, NETs contain myeloperoxidase, which has the
antibodies were identified. In-depth proteomic analysis determined potential to modulate LDL oxidation (177, 178). In humans,
that ALDH4A1 is the target antigen for one of the above inflammasome activation and NETosis may result in
autoantibodies, A12. The circulating ALDH4A1 is increased in atherosclerotic plaque erosion and thrombosis, especially in
humans and mice with AS. Moreover, in Ldlr-/- mice, infusion of patients with T2DM, clonal hematopoiesis or chronic kidney
A12 antibody can delay atherosclerotic plaque formation and disease (179). In patients with diabetes, the in vitro studies show
reduce blood LDL and free cholesterol, which indicates that The that induction of NETosis and the circulating concentration of
ALDH4A1 antibody can prevent the development of AS and may NETs-related proteins appear to increase. NETosis seems to be
have therapeutic potential in CVD (168). part of the abnormal response to diabetes damage, which, in
Using single-cell RNA sequencing, Wirka, RC et al. turn, can promote or aggravate end-organ complications (180).
characterized the transcriptome phenotype of SMCs regulated NETs have been shown to exert an important effect in the
in atherosclerotic lesions of human and mice arteries, and found pathogenesis of SLE via inducing activation of plasmacytoid
that these SMCs transformed into unique fibroblast-like cells, dendritic cells (PDCs) and type I interferon (IFN) pathways
called “fibromyocytes”, rather than the classic macrophage (181). The incidence of MACE and subclinical AS is increased in
phenotype. The specific knockout of TCF21 (a basic helix- SLE patients (182). NETs can destroy and kill ECs, and increase
loop-helix transcription factor) inhibits the phenotypic inflammation in atherosclerotic plaques, and this may accelerate
regulation of SMC in mice, resulting in fewer fibromyocytes in AS in SLE patients. Anti-IFN-a therapy and other new drugs
the lesions. In addition, TCF21 expression is strongly correlated (including peptidylarginine deiminase inhibitor 4, N-
with SMC phenotype regulation in diseased human coronary acetylcysteine and DNase I) can target NETs and have
arteries, and higher expression of TCF21 is correlated with potential in the therapy of autoimmune diseases (183).
reduced CAD risk. These results show the protective effect of Metformin treatment promotes the differentiation of T cells
TCF21 and SMC phenotypic regulation in CAD (169). into memory and regulatory T cells and reduces the ability of
The above two studies show the important mechanism by neutrophils to engage in NETosis. Because metformin has an
which B lymphocytes and SMCs play a role in the progress of AS inhibitory effect on the pro-inflammatory phenotype of immune
through single cell sequencing. In future research, this cells, it has shown beneficial results in animal models of
technology should be used to analyze the plaques of patients autoimmune diseases as well as in some relevant clinical trials
with AS combined with CVD and treated with metformin and (184). For example, additional treatment of mild and moderate
we look forward to discovering specific molecular targets and SLE with metformin reduced clinical flares, body weight and
sensitive cell communities for macrophages. prednisone exposure. Further studies have shown that
metformin reduces PMA-induced neutrophil NETs formation
Neutrophil Extracellular Traps (NETs) May and CpG-induced PDC-IFNa production, indicating that
Play an Important Role in the Anti- metformin may be an adjuvant therapy for SLE (185). The
Atherosclerotic Effects of Metformin formation of NETs in ARDS patients is significantly enhanced.
The most abundant blood white blood cell of healthy humans is Incubation of neutrophils to BAL fluid of ARDS can promote
neutrophils (170). Neutrophils exert a very important effect in NETs formation. In ARDS patients, macrophages phagocytosis

of apoptotic neutrophils and NETs is reduced. The activation of increasing blood b-hydroxybutyrate (BHB). In vitro
AMPK in macrophages in BAL fluid improves cell swelling and experiments with macrophages supported the effect of low
NETs clearance. These results indicate that the administration of insulin and increased BHB levels on the inactivation of NLRP3
metformin to restore AMPK activity may reduce persistent lung inflammasome. SGLT2i attenuates the activation of NLRP3
inflammation during ARDS by improving macrophage function inflammasome, which might underlie its cardiovascular
and NETs clearance (134). protective effect (211). In addition, in a study in which C57BL/
In summary, NETs not only promote the occurrence and 6J mice were fed with HFD or HFD plus empagliflozin for 16
development of SLE and RA, but also play a very important weeks empagliflozin inhibited HFD-caused liver steatosis, insulin
role in diabetes and AS. This suggests that the study of the resistance and weight gain. In addition, empagliflozin decreases
effect of metformin on NETs is an important area of research the accumulation of M1 macrophages, simultaneously induces
to broaden the understanding of the full clinical potential the anti-inflammatory M2 macrophage phenotype in liver and
of metformin. white AT, and reduces obesity-related chronic inflammation
(such as blood TNFa level). Therefore, empagliflozin enhances
Combination Therapy Is an Important fat utilization, inhibits insulin resistance as well as reducing
Research Area for Metformin Treatment obesity-induced inflammation via polarizing M2 macrophages
Metformin, as an oral biguanide hypoglycemic drug, is mainly in liver and white AT, thereby inhibiting weight gain (212).
used in the clinical therapy of T2DM and it has efficacy and Critically, in patients with diabetes and the associated increased
safety as mono- and combination therapy with other anti- cardiovascular risk, metformin combined with SGLT2i
hyperglycemic medications (186, 187). In addition to its (empagliflozin) as well as specific GLP-1 receptor agonists
protective role in diabetes-associated CVD and potentially (semaglutide and liraglutide) reduced all-cause mortality and
CVD occurring in patients without diabetes, multiple other cardiovascular death (187).
therapeutic effects of metformin have been recognized, In addition, compared with metformin monotherapy,
including reducing the risk of dementia (improving cognitive atorvastatin and metformin co-treatment reduced the level of
impairment) (188), weight loss (189), anti-aging (190, 191), TNF-a after oral glucose in patients with T2DM, and partially
suppressing air pollution damage (inhibiting inflammation and prevented the increase in blood glucose caused by glucose load
thrombosis) (107), improving PCOS (192, 193), and reversing (213). The combination of colesevelam hydrochloride and
lung fibers (194), inhibiting hair loss (195), reducing the risk of metformin, a bile acid chelator, further improved the blood
death from kidney failure and kidney disease (196, 197), and glucose profile and lipid indicators [fasting blood glucose,
preventing and treating some cancers (198–201). Macrophages fructosamine, total cholesterol, LDL-c, high-sensitivity C-
are distributed in the circulation and tissues and aggregate under reactive protein (hsCRP)] in T2DM patients (214). Anti-
a variety of pathological conditions and play an important role in inflammatory drugs (such as canakinumab) have benefits in
many pathological processes by regulating inflammation, such as the treatment of AS. For example, in 10,061 patients with
cardiovascular disease (202), obesity (203), T2DM (203), T2DM previous myocardial infarction and elevated CRP levels, IL-1b-
(203), cancer (204), aging (205) and dementia (206). Therefore, targeted canakinumab (150 mg, every 3 months) treatment
we speculate that improving the function of macrophages is the (median follow-up time of 3.7 years) significantly reduced
cellular basis for the pleiotropic potential of metformin. To recurrence cardiovascular events (HR=0.85) (including non-
expand on this correlation, below we focus on the potential of fatal stroke, non-fatal myocardial infarction, or cardiovascular
metformin in combination with drugs that improve macrophage death), and this action was independent of changes in blood lipid
function in CVD, including hypoglycemic agent-sodium glucose profiles (215). Thus, the combination of metformin and
cotransporter 2 inhibitors (SGLT2i), lipid-lowering drug canakinumab may have a better role in the prevention and
(statins) and an anti-inflammatory drug (IL-1b inhibitor). treatment of CVD.
SGLT2i, including empagliflozin, dapagliflozin and
canagliflozin, have been shown recently to reduce the
hospitalization rate of patients with heart failure and decrease
the mortality rate from cardiovascular diseases (207–210). SAFETY AND SIDE EFFECTS
Inhibiting the inflammatory actions of macrophages is one of
the important mechanisms through which SGLT2is exert CVD Metformin has been used to treat diabetes for more than 60 years
protection. For example, subjects with T2DM at high (20).With its safety profile, effectiveness and significant low cost
cardiovascular risk were given sulfonylurea or empagliflozin advantage, metformin is the first-line treatment for most T2DM
(SGLT2i) treatment for 30 days, and the activation of NLRP3 patients (20). The common gastrointestinal side effects of
inflammasome in macrophages was analyzed. Although metformin are usually mild and transient. Lactic acidosis is the
the hypoglycemic ability of SGLT2i is close to that of only serious adverse reaction, but the incidence is very low (9
sulfonylurea, it has greater decrease of IL-1b production, times per 100,000 people per year) (216, 217). New drugs,
accompanied with decreasing blood insulin levels and including SGLT-2 inhibitors and GLP-1 receptor agonists, also

show significant cardiovascular benefits, but the long-term safety CONCLUSION

of these new drugs needs to be confirmed (218). Compared with
other hypoglycemic drugs, the clinical experience and safety data Metformin has evolved as the first-line hypoglycemic drug for the
of metformin are still significantly better (219, 220). For the treatment of diabetes and extensive laboratory and clinical research
common side effects of metformin, it is possible to use sustained- has revealed that it has the potential to treat many other diseases
release preparations, controlled (late) release products and to (such as CVD, cancer and aging). Macrophages, which are
control the dose of metformin and optimally mange patients and distributed in the circulation and tissues and aggregate under a
minimize adverse effects (20). Future prospects of metformin in variety of pathological conditions, can play an important role in a
atherosclerosis was summarized in Figure 4. variety of diseases by regulating inflammation. Considerable
Enhance the multi-effect therapeutic

potential of metformin
Hypoglycemic drugs Non-hyperglycemic drugs

(SGLT2i) (such as statins, canakinumab)
Combination therapy
Neutrophils Macrophages
Metformin
NETs Dysfunction
Single cell RNA

sequencing
Mechanism
AMPK Discover new Epigenetic

dependent and potential modification
non-dependent targets (non-coding RNA)
Looking for new treatments Immune memory
FIGURE 4 | Future directions of studying the pharmacological actions and molecular mechanisms of metformin in atherosclerosis. Numerous studies have
demonstrated the protective effects of metformin on macrophage function, however, the effect of metformin on atherosclerosis in experimental animal models and
humans are warranted. Recent studies have shown that neutrophils (the most abundant white blood cells in healthy people) can participate in the development of AS
through the formation of NETs. Moreover, single-cell RNA sequencing plays an increasingly important role in revealing various physiological and pathological
mechanisms, and can be further utilized to uncover the effects of metformin on monocytes/macrophages and neutrophils. This will provide important clues for the
study of immune memory and the exploration of new treatment methods. In addition, the combination of metformin and other drugs (such as statins and SGLT2i)
synergistically improves the function of macrophages, further enhancing the pleiotropic effects of metformin. NETs, neutrophil extracellular traps; SGLT2i, sodium-
glucose cotransporter 2 inhibitors.

evidence indicates that metformin can improve the dysfunction of providing input to aspects of diabetes, atherosclerosis and CVD.
macrophages which is a cause of AS. We speculate that improving All authors contributed to the article and approved the
the function of macrophages may be the basis for the expanding submitted version.
therapeutic potential of metformin. Combined with other drugs
that improve the function of macrophages (such as SGLT2i, statins
and IL-b inhibitor), this may help to further strengthen the
pleiotropic actions and thus the therapeutic potential of FUNDING
metformin. In addition, there is evidence that metformin can
inhibit the formation of NETs, which may be related to the effect This study was supported by grants from National Natural
of metformin on improving macrophage function. In terms of Science Foundation of China [Grant Nos. 81941022 to JW,
research depth, single-cell sequencing helps to further clarify the 81530025 to JW, 81773955 to LT, 82070464 to SX, 82003740 to
mechanism of metformin and help to discover new targets for XF]. This work was also supported by Strategic Priority
improving the function of macrophages and controlling or reducing Research Program of Chinese Academy of Sciences [Grant
the role of these cells in multiple disease processes and states. No. XDB38010100 to JW], Program for Innovative Research
Team of the First Affiliated Hospital of USTC (to JW), and the
N a t i o n a l K e y R & D P r o g r a m of C h i n a [ G r a n t N o .
AUTHOR CONTRIBUTIONS 2017YFC1309603 to JW]. This work was also supported by
Local Innovative and Research Teams Project of Guangdong
SX, LT, and JW conceived the manuscript. XF, and WC wrote Pearl River Talents Program [2017BT01S131], and the
the manuscript and designed the figures. XF, WC, XN, SX, LT Fundamental Research Funds for the Central Universities
and JW edited the manuscript. PL edited the manuscript while (WK9110000079 to XF).
13. Stojanovi SD, Fiedler J, Bauersachs J, Thum T, Sedding DG. Senescence-Induced

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published: 07 June 2021

Metformin, Macrophage Dysfunction

Frontiers in Immunology www.frontiersin.org 1 June 2021 olume 12 Article 682853

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TABLE 1 | Clinical studies of metformin in diabetic patients with cardiovascular risk.

Subjects Grouping Results Conclusion

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Functions Models or subjects Mechanisms Results Conclusions

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Functions Models or subjects Mechanisms Results Conclusions

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Functions Models or subjects Mechanisms Results Conclusions

HDL macrophage cholesterol efﬂux, which

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Functions Models or subjects Mechanisms Results Conclusions

Monocyte HFD-fed ApoE -/-

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ACAT1 NCEH1 Apoptosis Thrombus

TL Chemokines (CCL2, Reverse

Macrophage CCL5, CXCL1) Cadherins) migration

Metformin Improves Macrophage Dysfunction

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CD14 + CD14 low

Vascular endothelium P-selectin ICAM1

Activation by LPS, oxLDL and INFγ Activation by IL-4 and IL-13

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Foam cell Inflammation↓

Neutrophil to lymphocyte ratio↓ CCL2↓

APMK↑, NFκB↓, MAPK (p38 and ERK) ↓

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show signiﬁcant cardiovascular beneﬁts, but the long-term safety CONCLUSION

Enhance the multi-effect therapeutic

Hypoglycemic drugs Non-hyperglycemic drugs

Single cell RNA

AMPK Discover new Epigenetic

Looking for new treatments Immune memory

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13. Stojanovi SD, Fiedler J, Bauersachs J, Thum T, Sedding DG. Senescence-Induced

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