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.

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 (8×10(5) 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.

Diabetes causes bone marrow endothelial barrier dysfunction by activation of the RhoA-Rho-associated kinase signaling pathway.

OBJECTIVE: Diabetes mellitus causes bone marrow (BM) microangiopathy. This study aimed to investigate the mechanisms responsible for BM endothelial dysfunction in diabetes mellitus. METHODS AND RESULTS: The analysis of differentially expressed transcripts in BM endothelial cells (BMECs) from type-1 diabetic and nondiabetic mice showed an effect of diabetes mellitus on signaling pathways controlling cell death, migration, and cytoskeletal rearrangement. Type-1 diabetic-BMECs displayed high reactive oxygen species levels, increased expression and activity of RhoA and its associated protein kinases Rho-associated kinase 1/Rho-associated kinase 2, and reduced Akt phosphorylation/activity. Likewise, diabetes mellitus impaired Akt-related BMEC functions, such as migration, network formation, and angiocrine factor-releasing activity, and increased vascular permeability. Moreover, high glucose disrupted BMEC contacts through Src tyrosine kinase phosphorylation of vascular endothelial cadherin. These alterations were prevented by constitutively active Akt (myristoylated Akt), Rho-associated kinase inhibitor Y-27632, and Src inhibitors. Insulin replacement restored BMEC abundance, as assessed by flow cytometry analysis of the endothelial marker MECA32, and endothelial barrier function in BM of type-1 diabetic mice. CONCLUSIONS: Redox-dependent activation of RhoA/Rho-associated kinase and Src/vascular endothelial cadherin signaling pathways, together with Akt inactivation, contribute to endothelial dysfunction in diabetic BM. Metabolic control is crucial for maintenance of endothelial cell homeostasis and endothelial barrier function in BM of diabetic mice.

Role for substance p-based nociceptive signaling in progenitor cell activation and angiogenesis during ischemia in mice and in human subjects.

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 data highlight the role of SP in reparative neovascularization. Nociceptive signaling may represent a novel target of regenerative medicine.

Intravenous gene therapy with PIM-1 via a cardiotropic viral vector halts the progression of diabetic cardiomyopathy through promotion of prosurvival signaling.

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 glucose-induced 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) plaque-forming units 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 α-myosin heavy chain in Pim-1-treated hearts. CONCLUSIONS: Pim-1 downregulation contributes in the pathogenesis of diabetic cardiomyopathy. Systemic delivery of human PIM-1 via cardiotropic adeno-associated virus serotype-9 represents a novel and effective approach to treat diabetic cardiomyopathy.

Inhibition of delta-like-4-mediated signaling impairs reparative angiogenesis after ischemia.

RATIONALE: Notch signaling regulates vascular development. However, the implication of the Notch ligand Delta-like 4 (Dll4) in postischemic angiogenesis remains unclear. OBJECTIVE: We investigated the role of Dll4/Notch signaling in reparative angiogenesis using a mouse model of ischemia. METHODS AND RESULTS: We found Dll4 weakly expressed in microvascular endothelial cells of normoperfused muscles. Conversely, Dll4 is upregulated following ischemia and localized at the forefront of sprouting capillaries. We analyzed the effect of inhibiting endogenous Dll4 by intramuscular injection of an adenovirus encoding the soluble form of Dll4 extracellular domain (Ad-sDll4). Dll4 inhibition caused the formation of a disorganized, low-perfused capillary network in ischemic muscles. This structural abnormality was associated to delayed blood flow recovery and muscle hypoxia and degeneration. Analysis of microvasculature at early stages of repair revealed that Dll4 inhibition enhances capillary sprouting in a chaotic fashion and causes excessive leukocyte infiltration of ischemic muscles. Furthermore, Dll4 inhibition potentiated the elevation of the leukocyte chemoattractant CXCL1 (chemokine [C-X-C motif] ligand 1) following ischemia, without altering peripheral blood levels of stromal cell-derived factor-1 and monocyte chemoattractant protein-1. In cultured human monocytes, Dll4 induces the transcription of Notch target gene Hes-1 and inhibits the basal and tumor necrosis factor-alpha-stimulated production of interleukin-8, the human functional homolog of murine CXCL1. The inhibitory effect of Dll4 on interleukin-8 was abolished by DAPT, a Notch inhibitor, or by coculturing activated human monocytes with Ad-sDll4-infected endothelial cells. CONCLUSIONS: Dll4/Notch interaction is essential for proper reparative angiogenesis. Moreover, Dll4/Notch signaling regulates sprouting angiogenesis and coordinates the interaction between inflammation and angiogenesis under ischemic conditions.

Diabetes mellitus induces bone marrow microangiopathy.

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 beta-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. CONCLUSIONS: 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.

Neurotrophin p75 receptor (p75NTR) promotes endothelial cell apoptosis and inhibits angiogenesis: implications for diabetes-induced impaired neovascularization in ischemic limb muscles.

Diabetes impairs endothelial function and reparative neovascularization. The p75 receptor of neurotrophins (p75(NTR)), which is scarcely present in healthy endothelial cells (ECs), becomes strongly expressed by capillary ECs after induction of peripheral ischemia in type-1 diabetic mice. Here, we show that gene transfer-induced p75(NTR) expression impairs the survival, proliferation, migration, and adhesion capacities of cultured ECs and endothelial progenitor cells (EPCs) and inhibits angiogenesis in vitro. Moreover, intramuscular p75(NTR) gene delivery impairs neovascularization and blood flow recovery in a mouse model of limb ischemia. These disturbed functions are associated with suppression of signaling mechanisms implicated in EC survival and angiogenesis. In fact, p75(NTR) depresses the VEGF-A/Akt/eNOS/NO pathway and additionally reduces the mRNA levels of ITGB1 [beta (1) integrin], BIRC5 (survivin), PTTG1 (securin) and VEZF1. Diabetic mice, which typically show impaired postischemic muscular neovascularization and blood perfusion recovery, have these defects corrected by intramuscular gene transfer of a dominant negative mutant form of p75(NTR). Collectively, our data newly demonstrate the antiangiogenic action of p75(NTR) and open new avenues for the therapeutic use of p75(NTR) inhibition to combat diabetes-induced microvascular liabilities.

Bone marrow-derived EP3-expressing stromal cells enhance tumor-associated angiogenesis and tumor growth.

Recent results suggest that bone marrow (BM)-derived hematopoietic cells are major components of tumor stroma and play crucial roles in tumor growth and angiogenesis. An E-type prostaglandin is known to regulate angiogenesis. We examined the role of BM-derived cells expressing an E-type prostaglandin receptor subtype (EP3) in tumor-induced angiogenesis and tumor growth. The replacement of wild-type (WT) BM with BM cells (BMCs) from green fluorescent protein (GFP) transgenic mice revealed that the stroma developed via the recruitment of BMCs. Selective knockdown of EP3 by recruitment of genetically modified BMCs lacking EP3 receptors was performed by transplantation of BMCs from EP3 knockout (EP3(-/-)) mice. Tumor growth and tumor-associated angiogenesis were suppressed in WT mice transplanted with BMCs from EP3(-/-) mice, but not in mice transplanted with BMCs from either EP1(-/-), EP2(-/-), or EP4(-/-) mice. Immunohistochemical analysis revealed that vascular endothelial growth factor (VEGF) expression was suppressed in the stroma of mice transplanted with BMCs from EP3(-/-) mice. EP3 signaling played a significant role in the recruitment of VEGFR-1- and VEGFR-2-positive cells from the BM to the stroma. These results indicate that the EP3 signaling expressed in bone marrow-derived cells has a crucial role in tumor-associated angiogenesis and tumor growth with upregulation of the expression of the host stromal VEGF together with the recruitment of VEGFR-1/VEGFR-2-positive. The present study suggests that the blockade of EP3 signaling and the recruitment of EP3-expressing stromal cells may become a novel strategy to treat solid tumors.

Phosphoinositide 3-kinase gamma gene knockout impairs postischemic neovascularization and endothelial progenitor cell functions.

OBJECTIVE: We evaluated whether phosphatidylinositol 3-kinase gamma (PI3Kgamma) plays a role in reparative neovascularization and endothelial progenitor cell (EPC) function. METHODS AND RESULTS: Unilateral limb ischemia was induced in mice lacking the PI3Kgamma gene (PI3Kgamma-/-) or expressing a catalytically inactive mutant (PI3Kgamma(KD/KD)) and wild-type controls (WT). Capillarization and arteriogenesis were reduced in PI3Kgamma-/- ischemic muscles resulting in delayed reperfusion compared with WT, whereas reparative neovascularization was preserved in PI3Kgamma(KD/KD). In PI3Kgamma-/- muscles, endothelial cell proliferation was reduced, apoptosis was increased, and interstitial space was infiltrated with leukocytes but lacked cKit+ progenitor cells that in WT muscles typically surrounded arterioles. PI3Kgamma is constitutively expressed by WT EPCs, with expression levels being upregulated by hypoxia. PI3Kgamma-/- 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 PI3Kgamma-/- EPCs. PI3Kgamma(KD/KD) EPCs showed reduced Akt phosphorylation, but constitutive activation of eNOS and preserved proliferation, survival, and migration. CONCLUSIONS: We newly demonstrated that PI3Kgamma modulates angiogenesis, arteriogenesis, and vasculogenesis by mechanisms independent from its kinase activity.

Development of immunocompetent lymphocytes in vivo from murine umbilical cord blood cells.

BACKGROUND: Reports concerning the immunological functions of lymphocytes derived from umbilical cord blood cell (UCBC) have been limited. METHODS: In murine syngeneic transplantation system using green fluorescent protein transgenic donors, UCBC-derived lymphocytes were studied for their immunological competence. RESULTS: Hematopoietic stem cells (HSC) among UCBC differentiated in the recipients into phenotypically mature T and B lymphocytes. The T lymphocytes were capable of specific recognition of major histocompatibility complex/peptide complex and of the subsequent activation. The antigen-induced CD4(+) T cells produced lymphokine upon in vitro antigen stimulation. CD8(+) T cells simulated in the mixed lymphocyte culture could lyze specific target cells. Furthermore, RAG2(-/-) mice reconstituted with UCBC mounted specific antibody responses to T-dependent antigen comparable to those by bone marrow chimeras and also rejected allogeneic skin grafts. CONCLUSION: Collectively the data indicated that T and B lymphocytes derived from UCBC-HSC are fully competent in immunological terms.

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: CD34(pos) 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, CD34(pos)/CD31(neg) 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-Kit(pos) 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.

Full reconstitution of hematopoietic system by murine umbilical cord blood.

BACKGROUND: Murine umbilical cord blood cells (UCBCs) were studied for their ability to reconstitute the hematopoietic system. METHODS: On average, 150 microL of cord blood per fetus containing 1.2 to 2 x 10(4) nucleated cells were collected from day 18.5 fetal umbilical cord, and 3 to 6 x 10(3) cells per fetus were obtained after separation by gradient centrifugation. RESULTS: Although lineage marker-, c-Kit+, and Sca-1+ cells were detectable among UCBCs, cells designated to be in the side population (SP) by Hoechst 33342 staining were hardly detectable within this population; the frequency of cells of this phenotype was less than 1 of 105. Instead, the lineage marker-, c-Kit+, and Sca-1- population contained a considerable number of SP cells. Nevertheless, UCBCs obtained from fetuses of green fluorescent protein-transgenic mice successfully reconstituted the blood cells of lethally irradiated recipients. Fluorescent cells could be readily detected in every blood cell lineage and among immature cell populations. Furthermore, fluorescent SP cells sorted from the recipient bone marrow cells could also reconstitute the blood cells in the secondary recipients, indicating that UCBCs also replenished bone marrow stem cells. CONCLUSION: Murine UCBC could fully reconstitute the hematopoietic system of lethally irradiated recipients including hematopoietic stem cells in bone marrow.

Increased antioxidant defense mechanism in human adventitia-derived progenitor cells is associated with therapeutic benefit in ischemia.

AIMS: Vascular wall-resident progenitor cells hold great promise for cardiovascular regenerative therapy. This study evaluates the impact of oxidative stress on the viability and functionality of adventitia-derived progenitor cells (APCs) from vein remnants of coronary artery bypass graft (CABG) surgery. We also investigated the antioxidant enzymes implicated in the resistance of APCs to oxidative stress-induced damage and the effect of interfering with one of them, the extracellular superoxide dismutase (EC-SOD/SOD3), on APC therapeutic action in a model of peripheral ischemia. RESULTS: After exposure to hydrogen peroxide, APCs undergo apoptosis to a smaller extent than endothelial cells (ECs). This was attributed to up-regulation of antioxidant enzymes, especially SODs and catalase. Pharmacological inhibition of SODs increases reactive oxygen species (ROS) levels in APCs and impairs their survival. Likewise, APC differentiation results in SOD down-regulation and ROS-induced apoptosis. Oxidative stress increases APC migratory activity, while being inhibitory for ECs. In addition, oxidative stress does not impair APC capacity to promote angiogenesis in vitro. In a mouse limb ischemia model, an injection of naïve APCs, but not SOD3-silenced APCs, helps perfusion recovery and neovascularization, thus underlining the importance of this soluble isoform in protection from ischemia. INNOVATION: This study newly demonstrates that APCs are endowed with enhanced detoxifier and antioxidant systems and that SOD3 plays an important role in their therapeutic activity in ischemia. CONCLUSIONS: APCs from vein remnants of CABG patients express antioxidant defense mechanisms, which enable them to resist stress. These properties highlight the potential of APCs in cardiovascular regenerative medicine.

Boosting the pentose phosphate pathway restores cardiac progenitor cell availability in diabetes.

AIMS: Diabetes impinges upon mechanisms of cardiovascular repair. However, the biochemical adaptation of cardiac stem cells to sustained hyperglycaemia remains largely unknown. Here, we investigate the molecular targets of high glucose-induced damage in cardiac progenitor cells (CPCs) from murine and human hearts and attempt safeguarding CPC viability and function through reactivation of the pentose phosphate pathway. METHODS AND RESULTS: Type-1 diabetes was induced by streptozotocin. CPC abundance was determined by flow cytometry. Proliferating CPCs were identified in situ by immunostaining for the proliferation marker Ki67. Diabetic hearts showed marked reduction in CPC abundance and proliferation when compared with controls. Moreover, Sca-1(pos) CPCs isolated from hearts of diabetic mice displayed reduced activity of key enzymes of the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PD), and transketolase, increased levels of superoxide and advanced glucose end-products (AGE), and inhibition of the Akt/Pim-1/Bcl-2 signalling pathway. Similarly, culture of murine CPCs or human CD105(pos) progenitor cells in high glucose inhibits the pentose phosphate and pro-survival signalling pathways, leading to the activation of apoptosis. In vivo and in vitro supplementation with benfotiamine reactivates the pentose phosphate pathway and rescues CPC availability and function. This benefit is abrogated by either G6PD silencing by small interfering RNA (siRNA) or Akt inhibition by dominant-negative Akt. CONCLUSION: 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.

Bone marrow microenvironment: a newly recognized target for diabetes-induced cellular damage.

Diabetes mellitus is considered a cardiovascular disease owing to its prevalent association with cardiovascular morbidity and mortality. Cardiovascular events are not only more frequent but also complicated with more severe outcomes in diabetic patients as compared with non-diabetic patients. One mechanism accounting for this difference consists of the impairment of the regenerative cellular machinery, which contributes to tissue healing. Recent evidence indicates the contribution of resident progenitor cells in post-ischemic tissue remodeling. In addition, a wide spectrum of cells from distant sources, including the bone marrow, is attracted and home to the healing tissue. Diabetes affects the process of mobilization and recruitment as well as intrinsic functional properties of bone marrow-derived progenitor cells. This review highlights current evidence for diabetes-induced damage of bone marrow hematopoietic progenitor cells in the endosteal and vascular niches.

Vitamin B1 analog benfotiamine prevents diabetes-induced diastolic dysfunction and heart failure through Akt/Pim-1-mediated survival pathway.

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(-1)/d(-1)) 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.

Recruitment of a prostaglandin E receptor subtype, EP3-expressing bone marrow cells is crucial in wound-induced angiogenesis.

E-type prostaglandins have been reported to be proangiogenic in vivo. Thus, we examined prostaglandin receptor signaling relevant to wound-induced angiogenesis. Full-thickness skin wounds were created on the backs of mice, and angiogenesis in wound granulation tissues was estimated. Wound closure and re-epithelization in EP3 receptor knockout mice (EP3-/-) were significantly delayed compared with their wild-type (WT) mice, whereas those in EP1-/-, EP2-/-, and EP4-/- were not delayed. Wound-induced angiogenesis estimated with CD31 immunohistochemistry in EP3-/- mice was significantly inhibited compared with that in WT mice. Immunoreactive vascular endothelial growth factor (VEGF) in wound granulation tissues in EP3-/- mice was markedly less than that in WT mice. Wound closure in WT mice was delayed significantly by VEGF neutralizing antibody compared with control IgG. Wound-induced angiogenesis and wound closure were significantly suppressed in EP3-/- bone marrow transplantation mice compared with those in WT bone marrow transplantation mice. These were accompanied with the reductions in accumulation of VEGF-expressing cells in wound granulation tissues and in mobilization of VEGF receptor 1-expressing leukocytes in peripheral circulation. These results indicate that the recruitment of EP3-expressing cells to wound granulation tissues is critical for surgical wound healing and angiogenesis via up-regulation of VEGF.

Cell-mediated fas-based lysis of dendritic cells which are apparently resistant to anti-Fas antibody.

In this report, the controversy concerning the sensitivity of dendritic cells (DCs) to Fas-dependent induction of apoptosis was examined using murine DCs. Although DCs could not be lysed when exposed to an anti-Fas antibody, Jo2, the observed resistance turned out to reflect their lack of the expression of Fc(gamma)R necessary for crosslinking the antibody, rather than their intrinsic resistance. Thus, at least a fraction of DCs was sensitive to Jo2 in the presence of Fc(gamma)R-expressing by-standers. Consistently, a significant fraction of DCs was sensitive to Fas-dependent lysis mediated by T cells including the antigen-specific killing by CD4+ T cells. Both immature (class II MHClow) and mature (class II MHChigh) DCs were sensitive to the Fas-based induction of apoptosis.