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 signal transducer and activator of transcription 3 phosphorylation 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 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

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

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

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

OBJECTIVE: We evaluated whether phosphatidylinositol 3-kinase γ (PI3Kγ) plays a role in reparative neovascularization and endothelial progenitor cell (EPC) function. METHODS AND RESULTS: Unilateral limb ischemia was induced in mice lacking the PI3Kγ gene (PI3Kγ(−/−)) or expressing a catalytically inactive mutant (PI3Kγ(KD/KD)) and wild-type controls (WT). Capillarization and arteriogenesis were reduced in PI3Kγ(−/−) ischemic muscles resulting in delayed reperfusion compared with WT, whereas reparative neovascularization was preserved in PI3Kγ(KD/KD). In PI3Kγ(−/−) 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. PI3Kγ is constitutively expressed by WT EPCs, with expression levels being upregulated by hypoxia. PI3Kγ(−/−) 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 PI3Kγ(−/−) EPCs. PI3Kγ(KD/KD) EPCs showed reduced Akt phosphorylation, but constitutive activation of eNOS and preserved proliferation, survival, and migration. CONCLUSIONS: We newly demonstrated that PI3Kγ modulates angiogenesis, arteriogenesis, and vasculogenesis by mechanisms independent from its kinase activity

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. 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.

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<sup>NTR</sup>), 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<sup>NTR</sup> 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<sup>NTR</sup> 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<sup>NTR</sup> depresses the VEGF-A/Akt/eNOS/NO pathway and additionally reduces the mRNA levels ofITGB1 [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<sup>NTR</sup>. Collectively, our data newly demonstrate the antiangiogenic action of p75<sup>NTR</sup> and open new avenues for the therapeutic use of p75<sup>NTR</sup> inhibition to combat diabetes-induced microvascular liabilities

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. </jats:sec

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.&lt;p&gt;&lt;/p&gt; 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.&lt;p&gt;&lt;/p&gt; Conclusion—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.&lt;p&gt;&lt;/p&gt

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.<p></p> Objective: We investigated the role of Dll4/Notch signaling in reparative angiogenesis using a mouse model of ischemia.<p></p> 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-α–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.<p></p> 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.<p></p