Jonathan Rowlinson

Combined Intramyocardial Delivery of Human Pericytes and Cardiac Stem Cells Additively Improves the Healing of Mouse Infarcted Hearts Through Stimulation of Vascular and Muscular Repair

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.

Perivascular Delivery of Encapsulated Mesenchymal Stem Cells Improves Postischemic Angiogenesis Via Paracrine Activation of VEGF-A

Objective—To test the therapeutic activity of perivascular transplantation of encapsulated human mesenchymal stem cells (MSCs) in an immunocompetent mouse model of limb ischemia. Approach and Results—CD1 mice underwent unilateral limb ischemia, followed by randomized treatment with vehicle, alginate microbeads (MBs), MB-encapsulated MSCs (MB-MSCs), or MB-MSCs engineered with glucagon-like peptide-1. Treatments were applied directly in the perivascular space around the femoral artery. Laser Doppler and fluorescent microsphere assessment of blood flow showed a marked improvement of perfusion in the MB-MSCs and MB-MSCs engineered with glucagon-like peptide-1 groups, which was associated with increased foot salvage particularly in MB-MSCs engineered with glucagon-like peptide-1–treated mice. Histological analysis revealed increased capillary and arteriole density in limb muscles of the 2 MSC groups. Furthermore, MB-MSCs engineered with glucagon-like peptide-1 and, to a lesser extent, MB-MSC treatment increased functional arterial collaterals alongside the femoral artery occlusion. Analysis of expressional changes in ischemic muscles showed that MB-MSC transplantation activates a proangiogenic signaling pathway centered on vascular endothelial growth factor A. In contrast, intramuscular MB-MSCs caused inflammatory reaction, but no improvement of reparative vascularization. Importantly, nonencapsulated MSCs were ineffective either by intramuscular or perivascular route. Conclusions—Perivascular delivery of encapsulated MSCs helps postischemic reperfusion. This novel biological bypass method might be useful in patients not amenable to conventional revascularization approaches.

Hey2 acts upstream of Notch in hematopoietic stem cell specification in zebrafish embryos.

Hematopoietic stem cells (HSCs) are essential for homeostasis and injury-induced regeneration of the vertebrate blood system. Although HSC transplantations constitute the most common type of stem cell therapy applied in the clinic, we know relatively little about the molecular programming of HSCs during vertebrate embryogenesis. In vertebrate embryos, HSCs form in close association with the ventral wall of the dorsal aorta. We have shown previously that in zebrafish, HSC formation depends on the presence of a signaling cascade that involves Hedgehog, vascular endothelial growth factor, and Notch signaling. Here, we reveal that Hey2, a hairy/enhancer-of-split-related basic helix-loop-helix transcription factor often believed to act downstream of Notch, is also required for HSC formation. In dorsal aorta progenitors, Hey2 expression is induced downstream of cloche and the transcription factor Scl/Tal1, and is maintained by Hedgehog and vascular endothelial growth factor signaling. Whereas knockdown of Hey2 expression results in a loss of Notch receptor expression in dorsal aorta angioblasts, activation of Notch signaling in hey2 morphants rescues HSC formation in zebrafish embryos. These results establish an essential role for Hey2 upstream of Notch in HSC formation.

Epigenetic Profile of Human Adventitial Progenitor Cells Correlates With Therapeutic Outcomes in a Mouse Model of Limb Ischemia

Objective— We investigated the association between the functional, epigenetic, and expressional profile of human adventitial progenitor cells (APCs) and therapeutic activity in a model of limb ischemia. Approach and Results— Antigenic and functional features were analyzed throughout passaging in 15 saphenous vein (SV)–derived APC lines, of which 10 from SV leftovers of coronary artery bypass graft surgery and 5 from varicose SV removal. Moreover, 5 SV-APC lines were transplanted (8×105 cells, IM) in mice with limb ischemia. Blood flow and capillary and arteriole density were correlated with functional characteristics and DNA methylation/expressional markers of transplanted cells. We report successful expansion of tested lines, which reached the therapeutic target of 30 to 50 million cells in ≈10 weeks. Typical antigenic profile, viability, and migratory and proangiogenic activities were conserved through passaging, with low levels of replicative senescence. In vivo, SV-APC transplantation improved blood flow recovery and revascularization of ischemic limbs. Whole genome screening showed an association between DNA methylation at the promoter or gene body level and microvascular density and to a lesser extent with blood flow recovery. Expressional studies highlighted the implication of an angiogenic network centered on the vascular endothelial growth factor receptor as a predictor of microvascular outcomes. FLT-1 gene silencing in SV-APCs remarkably reduced their ability to form tubes in vitro and support tube formation by human umbilical vein endothelial cells, thus confirming the importance of this signaling in SV-APC angiogenic function. Conclusions— DNA methylation landscape illustrates different therapeutic activities of human APCs. Epigenetic screening may help identify determinants of therapeutic vasculogenesis in ischemic disease.

Expansion and Characterization of Neonatal Cardiac Pericytes Provides a Novel Cellular Option for Tissue Engineering in Congenital Heart Disease

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 immunohistochemistry 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.

Migration towards SDF-1 selects angiogenin-expressing bone marrow monocytes endowed with cardiac reparative activity in patients with previous myocardial infarction

IntroductionChemokine-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.MethodsUnfractioned 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-FOXN1nu/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.ResultsIn 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.ConclusionsTransplantation 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.