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. Key Words: pericytes-based cell therapy Ⅲ myocardial infarction Ⅲ angiogenesis Ⅲ VEGF-B Ⅲ microRNA-132 W ith myocardial infarction (MI) remaining a major cause of morbidity and mortality worldwide, cell therapy now aims to offer a novel option for cardiac repair. 1 Clinical trials showed that administration of bone marrowderived progenitor cells (PCs) improves left ventricular (LV) function in patients with coronary artery disease. 2-4 However, more specialized cells are warranted to fulfill specific regenerative needs of the ischemic myocardium. ConclusionPericytes provide the physical strength and nurturing signals that instruct neovessels to organize in a stable and efficient tubular network. 5 On the other hand, ischemic disease and associated risk factors may impair pericyte recruitment. 6 -8 Therefore, a supply-side approach with fresh pericytes from exogenous sources could be helpful therapeutically. However, difficulties in isolating and expanding bona-fide pericytes from accessible human tissues have so far precluded clinical applications.Two main mural cell populations, probably originating from a common emb...

Rationale The impact of diabetes mellitus on bone marrow (BM) structure is incompletely understood. Objective Investigate the effect of type-2 diabetes mellitus (T2DM) on BM microvascular and hematopoietic cell composition in patients without vascular complications. Methods and Results Bone samples were obtained from T2DM patients and nondiabetic controls (C) during hip replacement surgery and from T2DM patients undergoing amputation for critical limb ischemia. BM composition was assessed by histomorphometry, immunostaining, and flow cytometry. Expressional studies were performed on CD34pos immunosorted BM progenitor cells (PCs). Diabetes mellitus causes a reduction of hematopoietic tissue, fat deposition, and microvascular rarefaction, especially when associated with critical limb ischemia. Immunohistochemistry documented increased apoptosis and reduced abundance of CD34pos-PCs in diabetic groups. Likewise, flow cytometry showed scarcity of BM PCs in T2DM and T2DM+critical limb ischemia compared with C, but similar levels of mature hematopoietic cells. Activation of apoptosis in CD34pos-PCs was associated with upregulation and nuclear localization of the proapoptotic factor FOXO3a and induction of FOXO3a targets, p21 and p27kip1. Moreover, microRNA-155, which regulates cell survival through inhibition of FOXO3a, was downregulated in diabetic CD34pos-PCs and inversely correlated with FOXO3a levels. The effect of diabetes mellitus on anatomic and molecular end points was confirmed when considering background covariates. Furthermore, exposure of healthy CD34pos-PCs to high glucose reproduced the transcriptional changes induced by diabetes mellitus, with this effect being reversed by forced expression of microRNA-155. Conclusions We provide new anatomic and molecular evidence for the damaging effect of diabetes mellitus on human BM, comprising microvascular rarefaction and shortage of PCs attributable to activation of proapoptotic pathway.

BackgroundLiving 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 ResultsCD34pos 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.ConclusionsThese 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.

Background-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. Methods and Results-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 mobilization, delayed blood flow recovery, and reduced neovascularization after ischemia. We next asked whether SP is instrumental to PC mobilization and homing in patients with ischemia. Human PC express NK1, and SP-induced migration provides enrichment for proangiogenic PC. Patients with acute myocardial infarction show high circulating levels of SP and NK1-positive cells that coexpress PC antigens, such as CD34, KDR, and CXCR4. Moreover, NK1-expressing PC are abundant in infarcted hearts but not in hearts that developed an infarct after transplantation. Conclusions-Our

Rationale: Studies in transgenic mice showed the key role of (Pim-1) (proviral integration site for Moloney murine leukemia virus-1) in the control of cardiomyocyte function and viability.Objective: We investigated whether Pim-1 represents a novel mechanistic target for the cure of diabetic cardiomyopathy, a steadily increasing cause of nonischemic heart failure. Methods and Results:In streptozotocin-induced type 1 diabetic mice, Pim-1 protein levels declined during progression of cardiomyopathy, along with upregulation of Pim-1 inhibitors, protein phosphatase 2A, and microRNA-1. Moreover, diabetic hearts showed low levels of antiapoptotic B-cell lymphoma-2 (Bcl-2) protein and increased proapoptotic caspase-3 activity. Studies on adult rat cardiomyocytes and murine cardiac progenitor cells challenged with high glucose confirmed the in vivo expressional changes. In rescue studies, anti-microRNA-1 boosted Pim-1 and Bcl-2 expression and promoted cardiomyocyte and cardiac progenitor cell survival under high glucose conditions. Similarly, transfection with Pim-1 plasmid prevented high glucoseinduced cardiomyocyte and cardiac progenitor cell apoptosis. Finally, a single intravenous injection of human PIM-1 via cardiotropic serotype-9 adeno-associated virus (1؋10 10 or 5؋10 10 genome copies per animal) at 4 weeks after diabetes induction led to sustained cardiac overexpression of Pim-1 and improved diastolic function and prevented left ventricular dilation and failure. Histological examination showed reduced cardiomyocyte apoptosis and fibrosis in association with increased c-kit ؉ cells and cardiomyocyte proliferation, whereas molecular analysis confirmed activation of the prosurvival pathway and conservation of sarcoendoplasmic reticulum Ca 2؉-ATPase and ␣-myosin heavy chain in Pim-1-treated hearts. Key Words: diabetic cardiomyopathies Ⅲ diastolic dysfunction Ⅲ Pim-1 kinase Ⅲ gene therapy Ⅲ cardiac stem cells A common form of cardiomyopathy directly related to diabetes mellitus (DM), ie, diabetic cardiomyopathy, typically progresses from diastolic dysfunction to heart failure in the absence of coronary artery disease or hypertension. [1][2][3] Studies in animal models illustrate the complexity of the underpinning pathogenic mechanisms (reviewed in Bugger and Abel 4 ). Hence, a deeper understanding of targets for early therapeutic interventions is critically needed. Conclusions:Recent work from Muraski and colleagues 5 uncovered the pivotal role of (Pim-1) (proviral integration site for Moloney murine leukemia virus-1), a member of the serine/threonine protein kinase family, in the cardiac cell response to stressors. Promotion of cardiomyocyte survival by Pim-1 is mediated by activation of B-cell lymphoma-2 (Bcl-2), phosphorylation/ inhibition of Bcl-2-associated death promoter (Bad), and maintenance of mitochondrial integrity. 6,7 By inducing c-Myc, nucleostemin, and cyclin E expression and p21 phosphorylation, Pim-1 increases the proliferative activity of cardiac progenitor cells (CPCs). 8,9 Furthermore, Pim-1 acts...

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a broad range of clinical responses including prominent microvascular damage. The capacity of SARS-CoV-2 to infect vascular cells is still debated. Additionally, the SARS-CoV-2 Spike (S) protein may act as a ligand to induce non-infective cellular stress. We tested this hypothesis in pericytes (PCs), which are reportedly reduced in the heart of patients with severe coronavirus disease-2019 (COVID-19). Here we newly show that the in vitro exposure of primary human cardiac PCs to the SARS-CoV-2 wild type strain or the Alpha and Delta variants caused rare infection events. Exposure to the recombinant S protein alone elicited signalling and functional alterations, including: (1) increased migration, (2) reduced ability to support endothelial cell (EC) network formation on Matrigel, (3) secretion of pro-inflammatory molecules typically involved in the cytokine storm, and (4) production of pro-apoptotic factors causing EC death. Next, adopting a blocking strategy against the S protein receptors angiotensin-converting enzyme 2 (ACE2) and CD147, we discovered that the S protein stimulates the phosphorylation/activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) through the CD147 receptor, but not ACE2, in PCs. The neutralisation of CD147, either using a blocking antibody or mRNA silencing, reduced ERK1/2 activation, and rescued PC function in the presence of the S protein. Immunoreactive S protein was detected in the peripheral blood of infected patients. In conclusion, our findings suggest that the S protein may prompt PC dysfunction, potentially contributing to microvascular injury. This mechanism may have clinical and therapeutic implications.

We provide new evidence of the negative impact of diabetes and high glucose on mechanisms controlling CPC redox state and survival. Boosting the pentose phosphate pathway might represent a novel mechanistic target for protection of CPC integrity.