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 ϩ endothelial cells. Cultured endothelial cells from BM of diabetic mice showed higher levels of oxidative stress, increased activity of the senescence marker ␤-galactosidase, reduced migratory and network-formation capacities, and increased permeability and adhesiveness to BM mononuclear cells. Flow cytometry analysis of lineage Ϫ c-Kit ϩ Sca-1 ϩ cell distribution along an in vivo Hoechst-33342 dye perfusion gradient documented that diabetes depletes lineage Ϫ c-Kit ϩ Sca-1 ϩ 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 Ϫ c-Kit ϩ Sca-1 ϩ 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. (Arterioscler Thromb Vasc Biol. 2010;30:498-508.)

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

Background-The increasing incidence of diabetes mellitus will result in a new epidemic of heart failure unless novel treatments able to halt diabetic cardiomyopathy early in its course are introduced. This study aimed to determine whether the activity of the Akt/Pim-1 signaling pathway is altered at critical stages of diabetic cardiomyopathy and whether supplementation with vitamin B1 analog benfotiamine (BFT) helps to sustain the above prosurvival mechanism, thereby preserving cardiomyocyte viability and function. Methods and Results-Untreated streptozotocin-induced type 1 or leptin-receptor mutant type 2 diabetic mice showed diastolic dysfunction evolving to contractile impairment and cardiac dilatation and failure. BFT (70 mg/kg Ϫ1 /d Ϫ1 ) improved diastolic and systolic function and prevented left ventricular end-diastolic pressure increase and chamber dilatation in both diabetic models. Moreover, BFT improved cardiac perfusion and reduced cardiomyocyte apoptosis and interstitial fibrosis. In hearts of untreated diabetic mice, the expression and activity of Akt/Pim-1 signaling declined along with O-N-acetylglucosamine modification of Akt, inhibition of pentose phosphate pathway, activation of oxidative stress, and accumulation of glycation end products. Furthermore, diabetes reduced pSTAT3 independently of Akt. BFT inhibited these effects of diabetes mellitus, thereby conferring cardiomyocytes with improved resistance to high glucose-induced damage. The phosphoinositide-3-kinase inhibitor LY294002 and dominant-negative Akt inhibited antiapoptotic action of BFT-induced and Pim-1 upregulation in high glucose-challenged cardiomyocytes. Conclusions-These results show that BFT protects from diabetes mellitus-induced cardiac dysfunction through pleiotropic mechanisms, culminating in the activation of prosurvival signaling pathway. Thus, BFT merits attention for application in clinical practice. (Circ Heart Fail. 2010;3:294-305.)Key Words: diabetes mellitus Ⅲ cardiomyopathy Ⅲ diastolic dysfunction Ⅲ benfotiamine Ⅲ apoptosis D iabetes mellitus (DM) is a potent and prevalent risk factor for heart failure independent of coronary artery disease or hypertension. 1 Diabetic cardiomyopathy has an insidious onset and remains, therefore, undiagnosed and untreated in a large number of patients. Furthermore, recent studies have shown evidence of diastolic dysfunction in up to 75% of young, asymptomatic patients with type 1 or type 2 DM. 2 The association of diastolic dysfunction and microangiopathy synergistically increases the risk of heart failure, thus pointing out the urgent need of early mechanistic treatment. 3,4 Clinical Perspective on p 305A variety of molecular alterations have been associated with diabetic cardiomyopathy, including defects in calcium homeostasis 5 and substrate metabolism, 6 accumulation of advanced glycation end products (AGE), 7 activation of the hexosamine pathway, 8 and oxidative stress leading to cardiomyocyte apoptosis. 9 However, early stage mechanisms remain mostly unknown.The pivota...

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...

Objective-We evaluated whether phosphatidylinositol 3-kinase ␥ (PI3K␥) plays a role in reparative neovascularization and endothelial progenitor cell (EPC) function. Methods and Results-Unilateral limb ischemia was induced in mice lacking the PI3K␥ gene (PI3K␥ Ϫ/Ϫ ) or expressing a catalytically inactive mutant (PI3K␥ KD/KD ) and wild-type controls (WT). Capillarization and arteriogenesis were reduced in PI3K␥ Ϫ/Ϫ ischemic muscles resulting in delayed reperfusion compared with WT, whereas reparative neovascularization was preserved in PI3K␥ KD/KD . In PI3K␥ Ϫ/Ϫ muscles, endothelial cell proliferation was reduced, apoptosis was increased, and interstitial space was infiltrated with leukocytes but lacked cKit ϩ progenitor cells that in WT muscles typically surrounded arterioles. PI3K␥ is constitutively expressed by WT EPCs, with expression levels being upregulated by hypoxia. PI3K␥ Ϫ/Ϫ EPCs showed a defect in proliferation, survival, integration into endothelial networks, and migration toward SDF-1. The dysfunctional phenotype was associated with nuclear constraining of FOXO1, reduced Akt and eNOS phosphorylation, and decreased nitric oxide (NO) production. Pretreatment with an NO donor corrected the migratory defect of PI3K␥

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