Rationale:: Pericytes are key regulators of vascular maturation, but their value for cardiac repair remains unknown. Objective:: We investigated the therapeutic activity and mechanistic targets of saphenous vein-derived pericyte progenitor cells (SVPs) in a mouse myocardial infarction (MI) model. Methods and Results:: SVPs have a low immunogenic profile and are resistant to hypoxia/starvation (H/S). Transplantation of SVPs into the peri-infarct zone of immunodeficient CD1/Foxn-1 ^nu/nu or immunocompetent CD1 mice attenuated left ventricular dilatation and improved ejection fraction compared to vehicle. Moreover, SVPs reduced myocardial scar, cardiomyocyte apoptosis and interstitial fibrosis, improved myocardial blood flow and neovascularization, and attenuated vascular permeability. SVPs secrete vascular endothelial growth factor A, angiopoietin-1, and chemokines and induce an endogenous angiocrine response by the host, through recruitment of vascular endothelial growth factor B expressing monocytes. The association of donor- and recipient-derived stimuli activates the proangiogenic and prosurvival Akt/eNOS/Bcl-2 signaling pathway. Moreover, microRNA-132 (miR-132) was constitutively expressed and secreted by SVPs and remarkably upregulated, together with its transcriptional activator cyclic AMP response element-binding protein, on stimulation by H/S or vascular endothelial growth factor B. We next investigated if SVP-secreted miR-132 acts as a paracrine activator of cardiac healing. In vitro studies showed that SVP conditioned medium stimulates endothelial tube formation and reduces myofibroblast differentiation, through inhibition of Ras-GTPase activating protein and methyl-CpG-binding protein 2, which are validated miR-132 targets. Furthermore, miR-132 inhibition by antimiR-132 decreased SVP capacity to improve contractility, reparative angiogenesis, and interstitial fibrosis in infarcted hearts. Conclusion:: SVP transplantation produces long-term improvement of cardiac function through a novel paracrine mechanism involving the secretion of miR-132 and inhibition of its target genes.

Background— Clinical trials in ischemic patients showed the safety and benefit of autologous bone marrow progenitor cell transplantation. Non–bone marrow progenitor cells with proangiogenic capacities have been described, yet they remain clinically unexploited owing to their scarcity, difficulty of access, and low ex vivo expansibility. We investigated the presence, antigenic profile, expansion capacity, and proangiogenic potential of progenitor cells from the saphenous vein of patients undergoing coronary artery bypass surgery. Methods and Results— CD34-positive cells, negative for the endothelial marker von Willebrand factor, were localized around adventitial vasa vasorum. After dissection of the vein from surrounding tissues and enzymatic digestion, CD34-positive/CD31-negative cells were isolated by selective culture, immunomagnetic beads, or fluorescence-assisted cell sorting. In the presence of serum, CD34-positive/CD31-negative cells gave rise to a highly proliferative population that expressed pericyte/mesenchymal antigens together with the stem cell marker Sox2 and showed clonogenic and multilineage differentiation capacities. We called this population “saphenous vein–derived progenitor cells” (SVPs). In culture, SVPs integrated into networks formed by endothelial cells and supported angiogenesis through paracrine mechanisms. Reciprocally, endothelial cell–released factors facilitated SVP migration. These interactive responses were inhibited by Tie-2 or platelet-derived growth factor-BB blockade. Intramuscular injection of SVPs in ischemic limbs of immunodeficient mice improved neovascularization and blood flow recovery. At 14 days after transplantation, proliferating SVPs were still detectable in the recipient muscles, where they established N-cadherin–mediated physical contact with the capillary endothelium. Conclusions— SVPs generated from human vein CD34-positive/CD31-negative progenitor cells might represent a new therapeutic tool for angiogenic therapy in ischemic patients.

We evaluated the healing potential of human fetal aorta-derived CD133 ⁺ progenitor cells and their conditioned medium (CD133 ⁺ CCM) in a new model of ischemic diabetic ulcer. Streptozotocin-induced diabetic mice underwent bilateral limb ischemia and wounding. One wound was covered with collagen containing 2×10 ⁴ CD133 ⁺ or CD133 ⁻ cells or vehicle. The contralateral wound, covered with only collagen, served as control. Fetal CD133 ⁺ cells expressed high levels of wingless ( Wnt ) genes, which were downregulated following differentiation into CD133 ⁻ cells along with upregulation of Wnt antagonists secreted frizzled-related protein (sFRP)-1, -3, and -4. CD133 ⁺ cells accelerated wound closure as compared with CD133 ⁻ or vehicle and promoted angiogenesis through stimulation of endothelial cell proliferation, migration, and survival by paracrine effects. CD133 ⁺ cells secreted high levels of vascular endothelial growth factor (VEGF)-A and interleukin (IL)-8. Consistently, CD133 ⁺ CCM accelerated wound closure and reparative angiogenesis, with this action abrogated by coadministering the Wnt antagonist sFRP-1 or neutralizing antibodies against VEGF-A or IL-8. In vitro, these effects were recapitulated following exposure of high-glucose-primed human umbilical vein endothelial cells to CD133 ⁺ CCM, resulting in stimulation of migration, angiogenesis-like network formation and induction of Wnt expression. The promigratory and proangiogenic effect of CD133 ⁺ CCM was blunted by sFRP-1, as well as antibodies against VEGF-A or IL-8. CD133 ⁺ cells stimulate wound healing by paracrine mechanisms that activate Wnt signaling pathway in recipients. These preclinical findings open new perspectives for the cure of diabetic ulcers.

Objective— The impact of diabetes on the bone marrow (BM) microenvironment was not adequately explored. We investigated whether diabetes induces microvascular remodeling with negative consequence for BM homeostasis. Methods and Results— We found profound structural alterations in BM from mice with type 1 diabetes with depletion of the hematopoietic component and fatty degeneration. Blood flow (fluorescent microspheres) and microvascular density (immunohistochemistry) were remarkably reduced. Flow cytometry verified the depletion of MECA-32 ⁺ endothelial cells. Cultured endothelial cells from BM of diabetic mice showed higher levels of oxidative stress, increased activity of the senescence marker β-galactosidase, reduced migratory and network-formation capacities, and increased permeability and adhesiveness to BM mononuclear cells. Flow cytometry analysis of lineage ⁻ c-Kit ⁺ Sca-1 ⁺ cell distribution along an in vivo Hoechst-33342 dye perfusion gradient documented that diabetes depletes lineage ⁻ c-Kit ⁺ Sca-1 ⁺ cells predominantly in the low-perfused part of the marrow. Cell depletion was associated to increased oxidative stress, DNA damage, and activation of apoptosis. Boosting the antioxidative pentose phosphate pathway by benfotiamine supplementation prevented microangiopathy, hypoperfusion, and lineage ⁻ c-Kit ⁺ Sca-1 ⁺ cell depletion. Conclusion— We provide novel evidence for the presence of microangiopathy impinging on the integrity of diabetic BM. These discoveries offer the framework for mechanistic solutions of BM dysfunction in diabetes.

Background— Myocardial ischemia (MI) leads to derangements in cellular electrical stability and the generation of lethal arrhythmias. Vagal nerve stimulation has been postulated to contribute to the antifibrillatory effect. Here, we suggest a novel mechanism for the antiarrhythmogenic properties of vagal stimulation during acute MI. Methods and Results— Under anesthesia, Wistar rats underwent 30 minutes of left coronary artery (LCA) ligation with vagal stimulation (MI-VS group, n=11) and with sham stimulation (MI-SS group, n=12). Eight of the 12 rats in the MI-SS group had ventricular tachyarrhythmia (VT) during 30-minute LCA ligation; on the other hand, VT occurred in only 1 of the 11 rats in the MI-VS group (67% versus 9%, respectively). Atropine administration abolished the antiarrhythmogenic effect of vagal stimulation. Immunoblotting revealed that the MI-SS group showed a marked reduction in the amount of phosphorylated connexin43 (Cx43), whereas the MI-VS group showed only a slight reduction compared with the sham operation and sham stimulation group (37±20% versus 79±18%). Immunohistochemistry confirmed that the MI-induced loss of Cx43 from intercellular junctions was prevented by vagal stimulation. In addition, studies with rat primary-cultured cardiomyocytes demonstrated that acetylcholine effectively prevented the hypoxia-induced loss of phosphorylated Cx43 and ameliorated the loss of cell-to-cell communication as determined by Lucifer Yellow dye transfer assay, which supports the in vivo results. Conclusions— Vagal nerve stimulation exerts antiarrhythmogenic effects accompanied by prevention of the loss of phosphorylated Cx43 during acute MI and thus plays a critical role in improving ischemia-induced electrical instability.