Toll-like receptors (TLRs) are important in innate immune responses, which are crucial in collateral artery formation (arteriogenesis). TLR4⁻/⁻ mice undergoing hind limb ischemia show decreased perfusion recovery accompanied by an... more
Toll-like receptors (TLRs) are important in innate immune responses, which are crucial in collateral artery formation (arteriogenesis). TLR4⁻/⁻ mice undergoing hind limb ischemia show decreased perfusion recovery accompanied by an impaired infiltration of inflammatory cells. TLR antagonists are currently developed and tested with the objective to inhibit acute exacerbation of organ damaging immune responses. However, systemic inhibition of innate immune responses may negatively influence arteriogenesis. In this study, we evaluated if TLR4 inhibition by a potent TLR4 inhibitor (TAK-242) would negatively influence perfusion recovery in a mouse model for arteriogenesis. Whole blood from human and mouse origin was stimulated with the TLR4 ligand lipopolysaccharide following TAK-242 incubation. After stimulation, cellular TLR4 activation was measured using fluorescence-activated cell sorting and tumor necrosis factor alpha release was measured using enzyme-linked immunosorbent assay. Next, the effect of TAK-242 was tested in a mouse model for arteriogenesis on perfusion recovery. TLR4 responses measured by tumor necrosis factor alpha levels were inhibited by TAK-242 in human and mouse blood after long-term stimulation. TAK-242 attenuated TLR4 responses in vivo but did not inhibit perfusion recovery in mice. In conclusion, TAK-242 does not negatively influence perfusion recovery following hind limb ischemia despite its TLR4 inhibiting properties.
Research Interests: Pharmacology, Flow Cytometry, Biology, Medicine, Lipopolysaccharide, and 15 moreHumans, Ischemia, Mice, Cardiovascular Pharmacology, Animals, Male, Inhibition, Perfusion, Receptor, Reperfusion injury, Enzyme Linked Immunosorbent Assay, Arteriogenesis, Cardiovascular medicine and haematology, Pharmacology and pharmaceutical sciences, and In Vitro Techniques
Research Interests: Technology, Immunology, Biomarkers, Atherosclerosis, Medicine, and 15 moreSignal Transduction, Biological Sciences, Humans, Mice, Animals, Biomed, Chemokine, Myocardial Infarction, Cardiovascular Diseases, Aneurysm, Lymphocytes, Knockout Mice, Leukocytes, Leukocyte trafficking, and Gene Expression Regulation
Research Interests: Flow Cytometry, Biology, Cytokines, Medicine, Ischemia, and 15 moreMice, Animals, Bone marrow, Perfusion, Clinical Sciences, European, Analysis of Variance, Leukocytes, Bone Marrow Cells, Temporal Change, Arteriogenesis, Arterial Occlusive Diseases, Extravasation, Peripheral blood, and leukocyte Count
Drug delivery systems are useful tools for local drug delivery in the enhancement of collateral growth (angiogenesis and arteriogenesis). These systems have major clinical potential for patients suffering from coronary artery disease or... more
Drug delivery systems are useful tools for local drug delivery in the enhancement of collateral growth (angiogenesis and arteriogenesis). These systems have major clinical potential for patients suffering from coronary artery disease or peripheral artery disease for whom current therapies do not seem to work sufficiently. Administration of drugs for enhancing collateral growth has been used in the past. However, free drug administration may lead to harmful side effects, such as local edema. Therefore, encapsulation of drugs into carriers leads to improved local delivery with its therapeutic effects on collateral growth. Different nano- and microparticles have been developed in the past for drug loading and local drug delivery. These carriers can move through the vasculature without being obstructed. Therefore, they can reach any desired area for stimulation of collateral growth. One type of microparticle is the microbubble (MB), which has been frequently tested in animal models. Different constructs have been developed already for carrying different drug types, such as proteins and gene constructs for cell transfection. Lipid-coated and polymer-coated MBs have been developed in the past. In this thesis, different drug delivery systems will be evaluated for the enhancement of collateral growth. Current therapies tested in experimental studies and clinical trials will be evaluated. The main focus is on new therapeutic approaches for local and sustained drug delivery. Furthermore, suggestions are made for MB constructs to optimize drug delivery for the enhancement of collateral growth
Research Interests:
Cardiovascular disease (CVD) is the leading cause of death globally and it is predicted this will remain to increase throughout 2030 to an estimated 23,3 million patients per year. This trend is accompanied by a steep increase in... more
Cardiovascular disease (CVD) is the leading cause of death globally and it is predicted this will remain to increase throughout 2030 to an estimated 23,3 million patients per year. This trend is accompanied by a steep increase in healthcare costs, making it a great health and socio-economic burden. The underlying pathology of CVD is often atherosclerosis, characterized by the development of atherosclerotic plaques in middle- and larger-sized arteries. Peripheral artery disease (PAD) is a disease of the peripheral vasculature, most often in the lower extremities, resulting from significant development of atherosclerotic plaques. Patients suffer from arterial occlusion in different parts of the lower extremities. Around 10 to 20% of the patients suffer from intermittent claudication and up to 2% will suffer from critical limb ischemia with leg amputation as a consequence. Unfortunately, not all patients are eligible for current available treatment options, such as surgical interventions, to restore blood flow and prevent tissue ischemia and tissue loss. There is a great need for new therapeutic options for these patients. A possible option is to stimulate arteriogenesis (collateral artery growth) in patients to recover hampered blood flow in their lower extremities. In order to develop new treatments, there is a need to better understand the process for arteriogenesis. The inflammatory local environment is shown crucial for this process. The research described in this thesis focuses on different inflammatory components involved in arteriogenesis for the development of new therapeutic options. First, we focused on CXCL10, a possible inflammatory target, and its role in cardiovascular disease. Furthermore, we investigated the involvement of CXCL10 in arteriogenesis in a murine hindlimb ischemia model. Next, we focused on the CD200-CD200 receptor inhibitory axis in arteriogenesis using this model, since this axis is described before as critical in infectious diseases. Next, we studied the involvement of small particles, called exosomes, derived from human mesenchymal stem cells and its possible therapeutic effects on arteriogenesis in a murine hindlimb model. In addition, we studied the possible negative effects of a Toll-like receptor 4 inhibitor in arteriogenesis, using the murine hindlimb model. Lastly, we focused on the role of another inflammatory target, leukotriene b4, for its role in two different CVDs, being advanced human atherosclerotic disease and abdominal aortic aneurysm.
Research Interests: Atherosclerosis, Inflammation, Medicine, Ischemia, Disease, and 8 moreAmputation, Exosomes, Blood Flow, AAA, LTB, Critical Limb Ischemia , Arteriogenesis, and TLR
Drug delivery systems are useful tools for local drug delivery in the enhancement of collateral growth (angiogenesis and arteriogenesis). These systems have major clinical potential for patients suffering from coronary artery disease or... more
Drug delivery systems are useful tools for local drug delivery in the enhancement of collateral growth (angiogenesis and arteriogenesis). These systems have major clinical potential for patients suffering from coronary artery disease or peripheral artery disease for whom current therapies do not seem to work sufficiently. Administration of drugs for enhancing collateral growth has been used in the past. However, free drug administration may lead to harmful side effects, such as local edema. Therefore, encapsulation of drugs into carriers leads to improved local delivery with its therapeutic effects on collateral growth. Different nano- and microparticles have been developed in the past for drug loading and local drug delivery. These carriers can move through the vasculature without being obstructed. Therefore, they can reach any desired area for stimulation of collateral growth. One type of microparticle is the microbubble (MB), which has been frequently tested in animal models. Different constructs have been developed already for carrying different drug types, such as proteins and gene constructs for cell transfection. Lipid-coated and polymer-coated MBs have been developed in the past. In this thesis, different drug delivery systems will be evaluated for the enhancement of collateral growth. Current therapies tested in experimental studies and clinical trials will be evaluated. The main focus is on new therapeutic approaches for local and sustained drug delivery. Furthermore, suggestions are made for MB constructs to optimize drug delivery for the enhancement of collateral growth
Research Interests:
Cardiovascular disease (CVD) is the leading cause of death globally and it is predicted this will remain to increase throughout 2030 to an estimated 23,3 million patients per year. This trend is accompanied by a steep increase in... more
Cardiovascular disease (CVD) is the leading cause of death globally and it is predicted this will remain to increase throughout 2030 to an estimated 23,3 million patients per year. This trend is accompanied by a steep increase in healthcare costs, making it a great health and socio-economic burden. The underlying pathology of CVD is often atherosclerosis, characterized by the development of atherosclerotic plaques in middle- and larger-sized arteries. Peripheral artery disease (PAD) is a disease of the peripheral vasculature, most often in the lower extremities, resulting from significant development of atherosclerotic plaques. Patients suffer from arterial occlusion in different parts of the lower extremities. Around 10 to 20% of the patients suffer from intermittent claudication and up to 2% will suffer from critical limb ischemia with leg amputation as a consequence. Unfortunately, not all patients are eligible for current available treatment options, such as surgical interventio...
Research Interests: Atherosclerosis, Inflammation, Medicine, Ischemia, Disease, and 8 moreAmputation, Exosomes, Blood Flow, AAA, LTB, Critical Limb Ischemia , Arteriogenesis, and TLR
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<p>LTB4 plaque expression levels are expressed as mean ± standard deviation and as pg/mg protein. P-values were calculated using Student’s T test. Differences with p-value <0.05 was regarded as being... more
<p>LTB4 plaque expression levels are expressed as mean ± standard deviation and as pg/mg protein. P-values were calculated using Student’s T test. Differences with p-value <0.05 was regarded as being significant.</p><p>Abbreviations: CEA: carotid endarterectomy, FU: follow-up, LTB4: leukotriene B4, SD: standard deviation.</p
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<p>Box-plots of LTB4 abdominal aortic aneurysm expression levels in relation with semi-quantative histological AAA characteristics. All characteristics have been correlated with continuous LTB4 expression levels and analyzed by... more
<p>Box-plots of LTB4 abdominal aortic aneurysm expression levels in relation with semi-quantative histological AAA characteristics. All characteristics have been correlated with continuous LTB4 expression levels and analyzed by Spearman’s Bivariate correlation test. Differences with p-value <0.05 were regarded as being significant. All data is expressed as median with interquartile range [IQR] and in pg/mg. Abbreviations: SMC: smooth muscle cell content.</p
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<p>LTB4 levels in atherosclerotic plaques were measured using ELISA and corrected for total protein content. Uncorrected and corrected levels were related to the three plaque phenotypes: fibrous (n = 113), fibroatheromatous... more
<p>LTB4 levels in atherosclerotic plaques were measured using ELISA and corrected for total protein content. Uncorrected and corrected levels were related to the three plaque phenotypes: fibrous (n = 113), fibroatheromatous (n = 131) and atheromatous (n = 130). Uncorrected LTB4 levels were positively related with an atheromatous plaque phenotype (A, p = 0.005). After correction, this association was not present (B, p = 0.791). Data is presented as median with interquartile range [IQR] and in pg/ul for uncorrected data and mean ± standard deviation and in pg/mg for corrected data.</p
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<p>Histology was performed at baseline, day 3 and day 7 in wildtype (black bars) and <i>Cd200<sup>−/−</sup></i> mice (white bars). Total number of alpha-Smooth Muscle Actin (<b>α</b>-SMA) positive... more
<p>Histology was performed at baseline, day 3 and day 7 in wildtype (black bars) and <i>Cd200<sup>−/−</sup></i> mice (white bars). Total number of alpha-Smooth Muscle Actin (<b>α</b>-SMA) positive vessels per section in the adductor muscle of the operated hindlimb (n = 9–11 per time point and genotype) (A). Number of CD31 positive vessels corrected for muscle fiber number in the ischemic peroneus muscle of the operated hindlimb (n = 9–11 per time point and genotype) (B). T lymphocyte (C) and macrophage number (D) was determined in the perivascular space around growing collateral vessels in the adductor muscle of the operated hindlimb (n = 9–11 at baseline and day 3; n = 23–26 at day 7, both wildtype and <i>Cd200<sup>−/−</sup></i>). Photos are representative images of CD3 (E) and Mac-3 (F) staining. Scale bar indicates 100 µm. Data are presented as mean ± Standard Error of the Mean (SEM). *P = 0.01 for T lymphocytes; *P = 0.02, **P<0.001 for CD31 positive vessels (<i>Cd200<sup>−/−</sup></i> versus wildtype).</p
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<p>Histology was performed after PBS (black bars), IgG (white bars) or CD200R agonist (grey bars) treatment (n = 10 per treatment) at day 7. Total number of alpha-Smooth Muscle Actin (<b>α</b>-SMA) positive vessels per... more
<p>Histology was performed after PBS (black bars), IgG (white bars) or CD200R agonist (grey bars) treatment (n = 10 per treatment) at day 7. Total number of alpha-Smooth Muscle Actin (<b>α</b>-SMA) positive vessels per section in the adductor muscle of the operated hindlimb (A). Number of CD31 positive vessels corrected for muscle fiber number in the ischemic calf muscle of the operated hindlimb (B). T lymphocyte (C) and macrophage number (D) was determined in the perivascular space around growing collateral vessels in the adductor muscle of the operated hindlimb. Photos are representative images of CD3 (E) and Mac-3 (F) staining. Scale bar indicates 100 µm. Data are presented as mean ± Standard Error of the Mean (SEM). *P = 0.03 (CD200R agonist versus IgG).</p
Research Interests: Cardiology, Immunology, Immune response, Inflammation, Cell Biology, and 15 moreImmunoregulation, Numbers, Biological Sciences, Anatomy, Immunity, T cells, Peripheral Vascular Disease, Monocytes, Vascular Medicine, Blood cells, Agonist, Vascular Diseases, White Blood Cells, inflammatory, and Immune cells
<p>Collateral vessel geometry was performed at baseline, day 3 and day 7 in wildtype (black bars) and <i>Cd200<sup>−/−</sup></i> mice (white bars) (A). Vessel wall thickness is calculated in the average... more
<p>Collateral vessel geometry was performed at baseline, day 3 and day 7 in wildtype (black bars) and <i>Cd200<sup>−/−</sup></i> mice (white bars) (A). Vessel wall thickness is calculated in the average diameter of the vessel wall and expressed in micrometer (µm). Vessel wall area per vessel is expressed in square micrometer (µm<sup>2</sup>) and calculated as area of total vessel minus lumen area. Lumen area per vessel is expressed in square micrometer (µm<sup>2</sup>). Outer and inner perimeter is expressed in micrometer (µm). Photos are representative images of alpha-Smooth Muscle Actin (<b>α</b>-SMA) staining for wildtype and <i>Cd200<sup>−/−</sup></i> mice at baseline and day 7. Scale bar indicates 100 µm. Number of animals: n = 9–11 per time point for both wildtype mice (black bars) and <i>Cd200<sup>−/−</sup></i> mice (white bars). Perfusion recovery was performed at baseline, day 3 and day 7 and was calculated as percentage perfusion in operated (ischemic) of the control (nonischemic) paw (B). Number of animals: n = 35 at baseline, n = 25 at day 3, n = 23 at day 7 for wildtype mice (black squares) and n = 35 at baseline day 3, n = 26 at day 7 for Cd200−/− mice (white circles). Data are presented as mean ± Standard Error of the Mean (SEM); *P<0.05; **P = 0.001 (<i>Cd200<sup>−/−</sup></i> versus wildtype).</p
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<p>Validation of <i>ex vivo</i> LTB4 production after whole blood collection by EDTA (black bars, n = 4, duplo) or Sodium Citrate (white bars, n = 4, duplo) Vacutainers. Directly after collection, whole blood was... more
<p>Validation of <i>ex vivo</i> LTB4 production after whole blood collection by EDTA (black bars, n = 4, duplo) or Sodium Citrate (white bars, n = 4, duplo) Vacutainers. Directly after collection, whole blood was incubated with a specific 5-LOX inhibitor Zileuton (100 µM for 30 minutes) and plasma was stored. LTB4 was measured using ELISA and compared with levels of CEA patients (n = 35). Data is expressed as mean ± standard deviation and in pg/ml.</p
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<p>Collateral vessel geometry was performed after PBS (black bars) IgG (white bars) and CD200R agonist (grey bars) treatment (n = 10 per treatment) (A). Vessel wall thickness is calculated in the average diameter of the vessel wall... more
<p>Collateral vessel geometry was performed after PBS (black bars) IgG (white bars) and CD200R agonist (grey bars) treatment (n = 10 per treatment) (A). Vessel wall thickness is calculated in the average diameter of the vessel wall and expressed in micrometer (µm). Vessel wall area per vessel is expressed in square micrometer (µm<sup>2</sup>) and calculated as area of total vessel minus lumen area. Lumen area per vessel is expressed in square micrometer (µm<sup>2</sup>). Outer and inner perimeter is expressed in micrometer (µm). Photos are representative images of alpha-Smooth Muscle Actin (α-SMA) staining for PBS, IgG and CD200R agonist treatment at day 7. Scale bar indicates 100 µm. Number of animals: n = 10 for PBS (black bar), IgG (white bar) and CD200R agonist (grey bar). Perfusion recovery was performed at baseline, day 3 and day 7 and was calculated as percentage perfusion in operated (ischemic) of the control (nonischemic) paw (B). Number of animals: n = 10 for PBS (black square), IgG (black triangle) and CD200R agonist (white circle) at all time points. Data are presented as mean ± Standard Error of the Mean (SEM); * P<0.05 (CD200R agonist versus IgG).</p
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<p>LTB4 abdominal aortic aneurysm expression levels are expressed as median [IQR] and as pg/mg protein. P-values were calculated using Mann-Whitney U test. Differences with p-value <0.05 was regarded as being... more
<p>LTB4 abdominal aortic aneurysm expression levels are expressed as median [IQR] and as pg/mg protein. P-values were calculated using Mann-Whitney U test. Differences with p-value <0.05 was regarded as being significant.</p><p>Abbreviations: AAA: Abdominal Aortic Aneurysm, FU: follow-up, IQR: interquartile range, LTB4: leukotriene B4 SD: standard deviation.</p
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<p>LTB4 levels in atherosclerotic plaques and AAA tissue were measured using ELISA and corrected for total protein content. For validation purposes, also uncorrected data are depicted in these graphs. Uncorrected and corrected LTB4... more
<p>LTB4 levels in atherosclerotic plaques and AAA tissue were measured using ELISA and corrected for total protein content. For validation purposes, also uncorrected data are depicted in these graphs. Uncorrected and corrected LTB4 levels were related to control versus events (no secondary outcome versus secondary outcome during follow-up). Both corrected and uncorrected LTB4 levels were not significantly different between controls and events for both CEA (A and B) and AAA (C and D) patients. Data is presented as mean ± standard deviation or median with interquartile range [IQR] and in pg/ul for uncorrected data and in pg/mg for corrected data.</p
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Copyright © 2014 Pleunie van den Borne et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original... more
Copyright © 2014 Pleunie van den Borne et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. C-X-Cmotif ligand 10 (CXCL10), or interferon-inducible protein-10, is a small chemokine belonging to the CXC chemokine family. Its members are responsible for leukocyte trafficking and act on tissue cells, like endothelial and vascular smooth muscle cells. CXCL10 is secreted by leukocytes and tissue cells and functions as a chemoattractant, mainly for lymphocytes. After binding to its receptor CXCR3, CXCL10 evokes a range of inflammatory responses: key features in cardiovascular disease (CVD). The role of CXCL10 in CVD has been extensively described, for example for atherosclerosis, aneurysm formation, and myocardial infarction. However, there seems to be a discrepancy between experimental and clinical settings. This discrepan...
Research Interests: Medicine and Circulation
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Objective—To score the number of plaque neutrophils and relate the score to plaque morphology and inflammatory status. Methods and Results—Neutrophils are inflammatory cells with tissue destruction capabilities that have been found at the... more
Objective—To score the number of plaque neutrophils and relate the score to plaque morphology and inflammatory status. Methods and Results—Neutrophils are inflammatory cells with tissue destruction capabilities that have been found at the site of an atherosclerotic plaque rupture or erosion. Poor evidence exists for neutrophil infiltration in human carotid atherosclerotic plaques, and its association with plaque morphology has not yet been described. A set of 355 human carotid plaques was stained for the neutrophil marker CD66b. High neutrophil numbers were found in plaques with a large lipid core, high macrophage numbers, and low collagen amount and smooth muscle cell numbers. High neutrophil numbers were associated with high interleukin 8 (P0.001) and matrix metalloproteases 8 (P0.005) and 9 (P0.001) plaque levels. High microvessel density within plaques was correlated with high neutrophil numbers (P0.01). In addition, low numbers of neutrophils were associated with female sex and...
Research Interests: Immunohistochemistry, Macrophages, Lipids, Humans, Collagen, and 15 moreFemale, Male, Clinical Sciences, Aged, Middle Aged, Biological markers, Disease Progression, Logistic Models, Microvessels, Cell Adhesion Molecules, Matrix Metalloproteinase, Interleukin, Carotid Arteries, Cardiovascular medicine and haematology, and leukocyte Count
The role of the CD200 ligand-CD200 receptor (CD200-CD200R) inhibitory axis is highly important in controlling myeloid cell function. Since the activation of myeloid cells is crucial in arteriogenesis, we hypothesized that disruption of... more
The role of the CD200 ligand-CD200 receptor (CD200-CD200R) inhibitory axis is highly important in controlling myeloid cell function. Since the activation of myeloid cells is crucial in arteriogenesis, we hypothesized that disruption of the CD200-CD200R axis promotes arteriogenesis in a murine hindlimb ischemia model. Female Cd2002/2 and wildtype (C57Bl/6J) mice underwent unilateral femoral artery ligation. Perfusion recovery was monitored over 7 days using Laser-Doppler analysis and was increased in Cd2002/2 mice at day 3 and 7 after femoral artery ligation, compared to wildtype. Histology was performed on hindlimb muscles at baseline, day 3 and 7 to assess vessel geometry and number and inflammatory cell influx. Vessel geometry in non-ischemic muscles was larger, and vessel numbers in ischemic muscles were increased in Cd2002/2 mice compared to wildtype. Furthermore, T lymphocyte influx was increased in Cd2002/2 compared to wildtype. CD200R agonist treatment was performed in male C...