Elisa Avolio

Chemokine-directed migration is crucial for homing of regenerative cells to the infarcted heart and correlates with outcomes of cell therapy trials. Hence, transplantation of chemokine-responsive bone marrow (BM) cells may be ideal for treatment of myocardial ischemia. To verify the therapeutic activity of BM mononuclear cells (BM-MNCs) selected by in vitro migration towards the chemokine stromal cell-derived factor-1 (SDF-1) in a mouse model of myocardial infarction (MI). We used for this purpose BM-MNCs from patients with previous large MI recruited in the TransACT-1&2 cell therapy trials. Un-fractioned BM-MNCs, SDF-1 responsive, and SDF-1 non-responsive BM-MNCs isolated by patients recruited in the TransACT-1&2 cell therapy trials were tested in Matrigel assay to evaluate angiogenic potential. Secretome and antigenic profile were characterized by flow cytometry. Angiogenin expression was measured by RT-PCR. Cells groups were also intra-myocardially injected in an in vivo model of...

Introduction: Current prostheses for correction of congenital heart disease (CHD) are unable to match the growth of an infant’s heart and deteriorate due to matrix degradation. Biological scaffolds integrated with progenitor cells able to grow and renew the prosthetic matrix may provide durable correction of CHD. We investigate the plasticity of cardiac pericyte-like cells obtained from CHD infants and their compatibility with a clinically certified prosthetic graft (CorMatrix). Methods&Results: CD34+ CD31- Pericytes (CPs) were immuno-sorted from myocardial specimen leftovers (n=13) of neonates and infants undergoing repairs of CHD. CPs were expanded and characterized for surface antigens, plasticity toward cardiovascular lineages, clonogenicity and ability to colonize a CorMatrix patch. We successfully expanded CPs in culture for several passages to reach a high number of cells (>20 million at P5). Flow cytometry of expanded cells at P4 (n=7) indicates a mesenchymal phenotype (C...

Background-—Living grafts produced by combining autologous heart-resident stem/progenitor cells and tissue engineering could provide a new therapeutic option for definitive correction of congenital heart disease. The aim of the study was to investigate the antigenic profile, expansion/differentiation capacity, paracrine activity, and pro-angiogenic potential of cardiac pericytes and to assess their engrafting capacity in clinically certified prosthetic grafts.
Methods and Results-—CD34pos cells, negative for the endothelial markers CD31 and CD146, were identified by immunohis- tochemistry in cardiac leftovers from infants and children undergoing palliative repair of congenital cardiac defects. Following isolation by immunomagnetic bead-sorting and culture on plastic in EGM-2 medium supplemented with growth factors and serum, CD34pos/CD31neg cells gave rise to a clonogenic, highly proliferative (>20 million at P5), spindle-shape cell population. The following populations were shown to expresses pericyte/mesenchymal and stemness markers. After exposure to differentiation media, the expanded cardiac pericytes acquired markers of vascular smooth muscle cells, but failed to differentiate into endothelial cells or cardiomyocytes. However, in Matrigel, cardiac pericytes form networks and enhance the network capacity of endothelial cells. Moreover, they produce collagen-1 and release chemo-attractants that stimulate the migration of c-Kitpos cardiac stem cells. Cardiac pericytes were then seeded onto clinically approved xenograft scaffolds and cultured in a bioreactor. After 3 weeks, fluorescent microscopy showed that cardiac pericytes had penetrated into and colonized the graft.
Conclusions-—These findings open new avenues for cellular functionalization of prosthetic grafts to be applied in reconstructive surgery of congenital heart disease.

Rationale: Optimization of cell therapy for cardiac repair may require the association of different cell populations with complementary activities.
Objective: Compare the reparative potential of saphenous vein–derived pericytes (SVPs) with that of cardiac stem cells (CSCs) in a model of myocardial infarction, and investigate whether combined cell transplantation provides further improvements.
Methods and Results: SVPs and CSCs were isolated from vein leftovers of coronary artery bypass graft surgery and discarded atrial specimens of transplanted hearts, respectively. Single or dual cell therapy (300 000 cells of each type per heart) was tested in infarcted SCID (severe combined immunodeficiency)-Beige mice. SVPs and CSCs alone improved cardiac contractility as assessed by echocardiography at 14 days post myocardial infarction. The effect was maintained, although attenuated at 42 days. At histological level, SVPs and CSCs similarly inhibited infarct size and interstitial fibrosis, SVPs were superior in inducing angiogenesis and CSCs in promoting cardiomyocyte proliferation and recruitment of endogenous stem cells. The combination of cells additively reduced the infarct size and promoted vascular proliferation and arteriogenesis, but did not surpass single therapies with regard to contractility indexes. SVPs and CSCs secrete similar amounts of hepatocyte growth factor, vascular endothelial growth factor, fibroblast growth factor, stem cell factor, and stromal cell–derived factor-1, whereas SVPs release higher quantities of angiopoietins and microRNA-132. Coculture of the 2 cell populations results in competitive as well as enhancing paracrine activities. In particular, the release of stromal cell–derived factor-1 was synergistically augmented along with downregulation of stromal cell–derived factor-1–degrading enzyme dipeptidyl peptidase 4.
Conclusions: Combinatory therapy with SVPs and CSCs may complementarily help the repair of infarcted hearts.

This review article reports on the new field of stem cell therapy and tissue engineering and its potential on the management of congenital heart disease. To date, stem cell therapy has mainly focused on treatment of ischemic heart disease and heart failure, with initial indication of safety and mild-to-moderate efficacy. Preclinical studies and initial clinical trials suggest that the approach could be uniquely suited for the correction of congenital defects of the heart. The basic concept is to create living material made by cellularized grafts that, once implanted into the heart, grows and remodels in parallel with the recipient organ. This would make a substantial improvement in current clinical management, which often requires repeated surgical corrections for failure of implanted grafts. Different types of stem cells have been considered and the identification of specific cardiac stem cells within the heterogeneous population of mesenchymal and stromal cells offers opportunities for de novo cardiomyogenesis. In addition, endothelial cells and vascular progenitors, including cells with pericyte characteristics, may be necessary to generate efficiently perfused grafts. The implementation of current surgical grafts by stem cell engineering could address the unmet clinical needs of patients with congenital heart defects.

Cardiac stem cells (CSC) from explanted decompensated hearts (E-CSC) are, with respect to those obtained from healthy donors (D-CSC), senescent and functionally impaired. We aimed to identify alterations in signaling pathways that are associated with CSC senescence. Additionally, we investigated if pharmacological modulation of altered pathways can reduce CSC senescence in vitro and enhance their reparative ability in vivo. Measurement of secreted factors showed that E-CSC release larger amounts of proinflammatory cytokine IL1b compared with D-CSC. Using blocking antibodies, we verified that IL1b hampers the paracrine protective action of E-CSC on cardiomyocyte viability. IL1b acts intracranially inducing IKKb signaling, a mechanism that via nuclear factor-jB upregulates the expression of IL1b itself. Moreover, E-CSC show reduced levels of AMP protein kinase (AMPK) activating phosphorylation. This latter event, together with enhanced IKKb signaling, increases TORC1 activity, thereby impairing the autophagic flux and inhibiting the phosphorylation of Akt and cAMP response element-binding protein. The combined use of rapamycin and resveratrol enhanced AMPK, thereby restoring downstream signaling and reducing IL1b secretion. These molecular corrections reduced E-CSC senescence, re establishing their protective activity on cardiomyocytes. Moreover ex vivo treatment with rapamycin and resveratrol improved E-CSC capacity to induce cardiac repair upon injection in the mouse infarcted heart, leading to reduced cardiomyocyte senescence and apoptosis and increased abundance of endogenous c-Kit+ CSC in the peri-infarct area. Molecular rejuvenation of patient-derived CSC by short pharmacologic conditioning boosts their in vivo reparative abilities. This approach might prove useful for refinement of CSC-based therapies.

INTRODUCTION: Chemokine-directed migration is crucial for homing of regenerative cells to the infarcted heart and correlates with outcomes of cell therapy trials. Hence, transplantation of chemokine-responsive bone marrow cells may be ideal for treatment of myocardial ischemia. To verify the therapeutic activity of bone marrow mononuclear cells (BM-MNCs) selected by in vitro migration towards the chemokine stromal cell-derived factor-1 (SDF-1) in a mouse model of myocardial infarction (MI), we used BM-MNCs from patients with previous large MI recruited in the TransACT-1&2 cell therapy trials.
METHODS:
Unfractioned BM-MNCs, SDF-1-responsive, and SDF-1-nonresponsive BM-MNCs isolated by patients recruited in the TransACT-1&2 cell therapy trials were tested in Matrigel assay to evaluate angiogenic potential. Secretome and antigenic profile were characterized by flow cytometry. Angiogenin expression was measured by RT-PCR. Cells groups were also intramyocardially injected in an in vivo model of MI (8-week-old immune deficient CD1-FOXN1(nu/nu) mice). Echocardiography and hemodynamic measurements were performed before and at 14 days post-MI. Arterioles and capillaries density, infiltration of inflammatory cells, interstitial fibrosis, and cardiomyocyte proliferation and apoptosis were assessed by immunohistochemistry.
RESULTS:
In vitro migration enriched for monocytes, while CD34(+) and CD133(+) cells and T lymphocytes remained mainly confined in the non-migrated fraction. Unfractioned total BM-MNCs promoted angiogenesis on Matrigel more efficiently than migrated or non-migrated cells. In mice with induced MI, intramyocardial injection of unfractionated or migrated BM-MNCs was more effective in preserving cardiac contractility and pressure indexes than vehicle or non-migrated BM-MNCs. Moreover, unfractioned BM-MNCs enhanced neovascularization, whereas the migrated fraction was unique in reducing the infarct size and interstitial fibrosis. In vitro studies on isolated cardiomyocytes suggest participation of angiogenin, a secreted ribonuclease that inhibits protein translation under stress conditions, in promotion of cardiomyocyte survival by migrated BM-MNCs.
CONCLUSIONS:
Transplantation of bone marrow cells helps post-MI healing through distinct actions on vascular cells and cardiomyocytes. In addition, the SDF-1-responsive fraction is enriched with angiogenin-expressing monocytes, which may improve cardiac recovery through activation of cardiomyocyte response to stress. Identification of factors linking migratory and therapeutic outcomes could help refine regenerative approaches.

Background: Cardiac progenitor cells (CPCs) are foundation to spontaneous and therapeutic cardiac repair. We investigate if progenitor cells from accessible human tissues exert benefits similar to human CPCs and interact with endogenous CPCs to improve recovery in a murine myocardial infarction (MI) model. Methods&Results: CPCs were isolated from discarded atrial specimens of transplanted hearts and adventitial progenitor cells (APCs) were immunosorted from vein leftovers of patients undergoing coronary artery bypass surgery. Both CPCs and APCs express mesenchymal (CD44/CD90/CD105/vimentin) and pericyte markers (NG2/PDGFRb), but are negative for endothelial and hematopoietic antigens. They secrete similar paracrine factors, including HGF, SCF, VEGF, FGF and angiopoietin1. Moreover, APCs acquire cardiomyocyte antigens (alpha-sarcomeric actinin/NKX2.5/TNNI3/CACNAIC) using 2 different differentiation protocols. Transplantation of APCs or CPCs (300,000/heart) in immunodeficient mice wit...

Chemokine-directed migration is crucial for homing of regenerative cells to the infarcted heart and correlates with outcomes of cell therapy trials. Hence, transplantation of chemokine-responsive bone marrow (BM) cells may be ideal for treatment of myocardial ischemia. To verify the therapeutic activity of BM mononuclear cells (BM-MNCs) selected by in vitro migration towards the chemokine stromal cell-derived factor-1 (SDF-1) in a mouse model of myocardial infarction (MI). We used for this purpose BM-MNCs from patients with previous large MI recruited in the TransACT-1&2 cell therapy trials. Un-fractioned BM-MNCs, SDF-1 responsive, and SDF-1 non-responsive BM-MNCs isolated by patients recruited in the TransACT-1&2 cell therapy trials were tested in Matrigel assay to evaluate angiogenic potential. Secretome and antigenic profile were characterized by flow cytometry. Angiogenin expression was measured by RT-PCR. Cells groups were also intra-myocardially injected in an in vivo model of...

Pain triggers a homeostatic alarm reaction to injury. It remains unknown, however, whether nociceptive signaling activated by ischemia is relevant for progenitor cells (PC) release from bone marrow. To this end, we investigated the role of the neuropeptide substance P (SP) and cognate neurokinin 1 (NK1) nociceptor in PC activation and angiogenesis during ischemia in mice and in human subjects. The mouse bone marrow contains sensory fibers and PC that express SP. Moreover, SP-induced migration provides enrichment for PC that express NK1 and promote reparative angiogenesis after transplantation in a mouse model of limb ischemia. Acute myocardial infarction and limb ischemia increase SP levels in peripheral blood, decrease SP levels in bone marrow, and stimulate the mobilization of NK1-expressing PC, with these effects being abrogated by systemic administration of the opioid receptor agonist morphine. Moreover, bone marrow reconstitution with NK1-knockout cells results in depressed PC ...

Background: We demonstrated that both age and pathology exert detrimental effects on human cardiac stem cells (CSC) and are associated with reduced telomerase activity and telomere length, telomere erosion, telomere induced dysfunction foci and CSC dysfunction in vitro. Our aims were to investigate whether CSC senescence is associated with their reduced reparative ability in vivo, to identify the molecular determinants possibly responsible for CSC senescence, to screen drugs (i.e. rapamycin, resveratrol, and DETA/NO) able to interfere with CSC senescence, and to verify if CSC drug treatment in vitro is effective in restoring the reparative potential of senescent CSC in vivo. Methods: CSCs were isolated both from normal (D) and failing (F) human hearts. The reparative capacity of CSC was evaluated in a SCID/beige mouse model of acute myocardial infarction (AMI). Echocardiography and cardiac catheterization were performed 2 weeks post-AMI. Fibrosis, angiogenesis, myocyte growth, myocy...