Role of Inflammation in 20-HETE Regulation of Ischemia-Induced Angiogenesis

Objective: 20-Hydroxyeicosatetraenoic acid (20-HETE), an important bioactive lipid metabolite, has recently been identified to be a novel contributor of angiogenesis secondary to ischemia. Moreover, an inflammatory response is required for the initiation of ischemic angiogenesis, in response to ischemic tissue injury. The goal of this study is to investigate the role of inflammation in 20-HETE regulation of ischemia-induced angiogenesis. Methods: We first established a mouse hind limb ischemia model for immunocompetent Balb/C mice and immunodeficient NOD-SCID mice by femoral artery ligation. Groups of Balb/C and NOD-SCID mice were administered a 20-HETE synthesis inhibitor, DDMS, or saline as a solvent control. Laser Doppler perfusion imaging (LDPI) was used to visualize and quantify blood perfusion on days 0, 1, 3, 7, 14, and 21 post ligation, confirmed by microvessel density analysis. LC/MS/MS analysis was performed on day 3 post ligation on ischemic and non-ischemic control gracilis muscles to measure 20-HETE levels. Additionally, an antibody to lymphocyte antigen 6 complex (Ly6G/C) was administered to neutralize the infiltration of neutrophils, macrophages, and monocytes. 20-HETE levels were again measured on day 3 post ligation in these mice. Results: Quantification of the compensatory blood perfusion recovery post ischemia by LDPI showed that immunocompetent Balb/C control mice demonstrated a normal course of the compensatory angiogenic response while NOD-SCID immunodeficient mice showed a significantly decreased response. Additionally, DDMS was shown to inhibit the compensatory response in Balb/C mice, while no inhibitory effect was observed in immunodeficient NOD-SCID mice. This observation is confirmed by a marked decrease in microvessel density in SCID mice (1.5±0.2) post ischemia compared to immunocompetent Balb/C mice (2.65±0.32). As expected, ischemia markedly increased 20-HETE levels in the ischemic gracilis muscle of Balb/C mice by 6-fold (6±2 in non-ischemic vs 27±5 pg/mg in ischemic), while levels in NOD-SCID mice showed no change between the ischemic and non-ischemic control. Lastly, Balb/C mice that were treated with Ly6G/C neutralizing antibody exhibited significantly decreased 20-HETE levels in their ischemic gracilis muscle compared to the non-ischemic control. Conclusion: Inflammation may be an essential contributor in 20-HETE regulation of the ischemia-induced angiogenic response

植込型補助人工心臓（VAD）患者の社会復帰への人工心臓管理技術認定士の取り組み

ハートセンター, 臨床工学部ハートセンター, 内科学（心臓・血管）ハートセンター, 内科学（循環器・腎臓）ハートセンター, 心臓血管外科学ハートセンター, 内科学（心臓・血管）ハートセンター, 心臓血管外科学ハートセンター, 内科学（心臓・血管

Low-molecular weight heparin protamine complex augmented the potential of adipose-derived stromal cells to ameliorate limb ischemia

Center for Regenerative Medicine, Research Support Center, Dokkyo Medical University, Mibu, Tochigi, JapanCenter for Regenerative Medicine, Research Support Center, Dokkyo Medical University, Mibu, Tochigi, JapanCenter for Regenerative Medicine, Department of Cardiovascular Medicine, Dokkyo Medical University, Mibu, Tochigi, JapanCenter for Regenerative Medicine, Department of Cardiovascular Medicine, Dokkyo Medical University, Mibu, Tochigi, JapanCenter for Regenerative Medicine, Research Support Center, Dokkyo Medical University, Mibu, Tochigi, Japan:Department of Cardiology, Koshigaya Hospital, Dokkyo Medical University, Koshigaya, Saitama, JapanCenter for Regenerative Medicine, Research Support Center, Department of Cardiovascular Medicine, Dokkyo Medical University, Mibu, Tochigi, JapanCenter for Regenerative Medicine, Dokkyo Medical University, Mibu, Tochigi, Japa

MicroRNA-24 targets Notch and other vascular morphogens to regulates post-ischemic microvascular responses in limb muscles

MicroRNAs (miRs) regulate complex processes, including angiogenesis, by targeting multiple mRNAs. miR-24-3p-3p directly represses eNOS, GATA2, and PAK4 in endothelial cells (ECs), thus inhibiting angiogenesis during development and in the infarcted heart. miR-24-3p is widely expressed in cardiovascular cells, suggesting that it could additionally regulate angiogenesis by acting on vascular mural cells. Here, we have investigated: (1) new miR-24-3p targets; (2) the expression and the function of miR-24-3p in human vascular ECs; (3) the impact of miR-24-3p inhibition in the angiogenesis reparative response to limb ischemia in mice. Using bioinformatics target prediction platforms and 3′-UTR luciferase assays, we newly identified Notch1 and its Delta-like ligand 1 (Dll1) to be directly targeted by miR-24-3p. miR-24-3p was expressed in human ECs and pericytes cultured under normal conditions. Exposure to hypoxia increased miR-24-3p in ECs but not in pericytes. Transfection with a miR-24-3p precursor (pre-miR-24-3p) increased miR-24-3p expression in ECs, reducing the cell survival, proliferation, and angiogenic capacity. Opposite effects were caused by miR-24-3p inhibition. The anti-angiogenic action of miR-24-3p overexpression could be prevented by simultaneous adenovirus (Ad)-mediated delivery of constitutively active Notch intracellular domain (NICD) into cultured ECs. We next demonstrated that reduced Notch signalling contributes to the anti-angiogenic effect of miR-24-3p in vitro. In a mouse unilateral limb ischemia model, local miR-24-3p inhibition (by adenovirus-mediated miR-24-3p decoy delivery) restored endothelial Notch signalling and increased capillary density. However, the new vessels appeared disorganised and twisted, worsening post-ischemic blood perfusion recovery. To better understand the underpinning mechanisms, we widened the search for miR-24-3p target genes, identifying several contributors to vascular morphogenesis, such as several members of the Wingless (Wnt) signalling pathway, β-catenin signalling components, and VE-cadherin, which synergise to regulate angiogenesis, pericytes recruitment to neoformed capillaries, maturation, and stabilization of newly formed vessels. Among those, we next focussed on β-catenin to demonstrate that miR-24-3p inhibition reduces β-catenin expression in hypoxic ECs, which is accompanied by reduced adhesion of pericytes to ECs. In summary, miR-24-3p differentially targets several angiogenesis modulators and contributes to autonomous and non-autonomous EC crosstalk. In ischemic limbs, miR-24-3p inhibition increases the production of dysfunctional microvessels, impairing perfusion. Caution should be observed in therapeutic targeting of miR-24-3p

Angiogenesis and Endothelial Dysfunction: Insights of Autophagy Machinery in Regulating Endothelial cell Biology

Autophagy is an intracellular degradation system that delivers cytoplasmic components to the lysosome for degradation. Autophagy is essential for cellular homeostasis and provides a mechanism to adapt to metabolic and stress cues. Endothelial autophagy regulates the response of ECs to a variety of stress factors related to EC homeostasis and plasticity. However, the precise role of autophagy in angiogenesis requires more detailed research. Although autophagy-related 7 (ATG7) is essential for classical degradative autophagy and cell cycle regulation, whether and how ATG7 influences endothelial cell (EC) function and regulates post-ischemic angiogenesis remain unknown. Endothelial dysfunction is a potential contributor to the pathogenesis of diabetic cardiovascular complications. However, little is known about disruptions of endothelial autophagy contributing to diabetes-induced endothelial dysfunction. This dissertation aims to address how ATG7 influences endothelial cell (EC) function and regulates post-ischemic angiogenesis, and to determine the role of autophagy in the development of endothelial dysfunction. EC-specific deletion of Atg7 significantly impaired angiogenesis, delayed the recovery of blood flow reperfusion, and displayed reduction in hypoxia inducible factor 1 subunit alpha (HIF1A) expression. Mechanistically, lack of ATG7 in the cytoplasm disrupted the association between ATG7 and transcription factor ZNF148/ZBP-89 that is required for STAT1 (signal transducer and activator of transcription1) constitutive expression, increased the binding between ZNF148/ZBP-89 and importin-β1 (KPNB1), which promoted ZNF148/ZBP-89 nuclear translocation, and increased STAT1 expression. STAT1 bond to HIF1A promotor and suppressed HIF1A mRNA expression, thereby preventing ischemia-induced angiogenesis. These results demonstrate that ATG7 deficiency is a novel suppressor of ischemia-induced angiogenesis. In addition, streptozotocin (STZ)-induced type 1 diabetes inhibits autophagic flux and reduced protein levels of autophagy gene related protein, including ULK1, ATG7, ATG5, and Beclin1, which was accompanied by an impairment of acetylcholine-induced relaxation of isolated mouse aortas. Inhibition of endothelial autophagy by the deletion of endothelial ULK1 exacerbated diabetes-induced endothelial dysfunction, reactive oxygen species (ROS) overproduction and impeded endothelial nitric oxide synthase (eNOS) phosphorylation. Mechanistically, suppression of autophagy by diabetes aggravated ROS overproduction. Downregulation of ULK1 reduced eNOS phosphorylation. Thus, promoting autophagy activity may be a potential strategy to prevent endothelial dysfunction in diabetes

Inhibition of GPR39 restores defects in endothelial cell–mediated neovascularization under the duress of chronic hyperglycemia: evidence for regulatory roles of the sonic hedgehog signaling axis

Impaired endothelial cell (EC)–mediated angiogenesis contributes to critical limb ischemia in diabetic patients. The sonic hedgehog (SHH) pathway participates in angiogenesis but is repressed in hyperglycemia by obscure mechanisms. We investigated the orphan G protein–coupled receptor GPR39 on SHH pathway activation in ECs and ischemia-induced angiogenesis in animals with chronic hyperglycemia. Human aortic ECs from healthy and type 2 diabetic (T2D) donors were cultured in vitro. GPR39 mRNA expression was significantly elevated in T2D. The EC proliferation, migration, and tube formation were attenuated by adenovirus-mediated GPR39 overexpression (Ad-GPR39) or GPR39 agonist TC-G-1008 in vitro. The production of proangiogenic factors was reduced by Ad-GPR39. Conversely, human ECs transfected with GPR39 siRNA or the mouse aortic ECs isolated from GPR39 global knockout (GPR39KO) mice displayed enhanced migration and proliferation compared with their respective controls. GPR39 suppressed the basal and ligand-dependent activation of the SHH effector GLI1, leading to attenuated EC migration. Coimmunoprecipitation revealed that the GPR39 direct binding of the suppressor of fused (SUFU), the SHH pathway endogenous inhibitor, may achieve this. Furthermore, in ECs with GPR39 knockdown, the robust GLI1 activation and EC migration were abolished by SUFU overexpression. In a chronic diabetic model of diet-induced obesity (DIO) and low-dose streptozotocin (STZ)-induced hyperglycemia, the GPR39KO mice demonstrated a faster pace of revascularization from hind limb ischemia and lower incidence of tissue necrosis than GPR39 wild-type (GPR39WT) counterparts. These findings have provided a conceptual framework for developing therapeutic tools that ablate or inhibit GPR39 for ischemic tissue repair under metabolic stress

Impaired Collateral Recruitment and Outward Remodeling in Experimental Diabetes

OBJECTIVE—In this study, the effect of chronic hyperglycemia on acute ligation-induced collateral vasodilation, on monocyte chemotaxis, and on structural outward remodeling of collaterals was investigated

Smooth muscle cell sheet transplantation preserve cardiac function and minimize cardiac remodeling in a rat myocardial infarction model

Background: We examined whether a vascular smooth muscle cell (SMC) sheet is effective in the treatment of a rat myocardial infarction (MI) model. Methods: We examined the effect of SMC sheet on the cardiac function and cardiac remodeling in a rat MI model in comparison with their effect of dermal fibroblast (DFB) sheet in vivo. Furthermore, we estimated the apoptosis and secretion of angiogenic factor of SMC under hypoxic condition in comparison with DFB. Seven days after MI, monolayer cell sheets were transplanted on the infarcted area (SMC transplantation group, SMC-Tx; DFB transplantation group, DFB-Tx; no cell sheet transplantation group, Untreated; neither MI nor cell sheet transplantation group, Sham). We evaluated cardiac function by echocardiogram, degree of cardiac remodeling by histological examination, and secretion of angiogenic growth factor by enzyme immunoassay. Results: Twenty-eight days after transplantation, SMC-Tx showed the following characteristics compared with the other groups: 1) significantly greater fractional area shortening (SMC-Tx, 32.3 ± 2.1 %; DFB-Tx, 23.3 ± 2.1 %; untreated, 25.1 ± 2.6 %), 2) suppressed left ventricular dilation, smaller scar expansion, and preserved wall thickness of the area at risk and the posterior wall, 3) decreased fibrosis, preserved myocardium in the scar area, and greater number of arterioles in border-zone, 4) tight attachment of SMC sheets on the scarred myocardium, and less apoptotic cell death. In in vitro experiments, SMCs secreted higher amounts of basic fibroblast growth factor (SMC, 157.7 ± 6.4 pg/ml; DFB, 3.1 ± 1.0 pg/ml), and showed less apoptotic cell death under hypoxia. Conclusions: Our results illustrate that transplantation of SMC sheets inhibited the progression of cardiac remodeling and improve cardiac function. These beneficial effects may be due to superior SMC survival

Mesenchymal Stem Cells from Bone Marrow Enhance Neovascularization and Stromal Cell Proliferation in Rat Ischemic Limb in the Early Phase after plantation

Accumulating evidence from animal studies shows that the administration of mesenchymal stem cells (MSCs) from adult bone marrow ameliorates tissue damage after ischemic injury. In the present study we investigated the efficacy of MSC implantation into a hindlimb ischemia model over a short-term period to elucidate the effects conferred within the early phase after treatment. MSCs from rats expressing green fluorescence protein (GFP) were injected into rat ischemic limbs. Laser Doppler perfusion imaging revealed significantly higher blood perfusion recovery in the MSC group than in the control group on days 3 and 7 after the treatment. The capillary / muscle fiber ratio in ischemic muscle was also significantly higher in the MSC group than in the controls in a histological study. In spite of these benefits, we found no evident engraftment of the GFP-positive cells, and instead, the MSC treatment induced a proliferation of resident stromal cells in the perivascular area of the ischemic muscle, some of which produced vascular endothelial growth factor. The present study suggested that MSC therapy promotes neovascularization even in the early phase, both directly through endothelial proliferation and indirectly through activation of the resident stromal cells