Cardiac Vascular Endothelial Growth Factor Research Articles

Investigation of impacts of decellularized heart extracellular matrix and VEGF on cardiomyogenic differentiation of mesenchymal stem cell through Notch/Hedgehog signaling pathways

ObjectiveDecellularization is the process to obtain natural scaffolds with tissue integrity and extracellular matrix components, and recellularization is used to produce tissue-like constructs with specific cell types. In this study, rat bone marrow-derived mesenchymal stem cells (rBM-MSCs) were cultured on decellularized heart extracellular matrix. These cells were then induced to differentiate into cardiomyogenic cells under the stimulatory effect of vascular endothelial growth factor (VEGF) and other chemicals. This study aimed to investigate the effect of the cardiac extracellular matrix and VEGF on cardiomyogenic differentiation in the context of the Notch and Hedgehog signaling pathways. MethodsHeart samples extracted from rats were decellularized by serial application of detergent to remove cells from the tissue, and then recellularized with rBM-MSCs. The recellularized tissue matrices were then analyzed for cardiomyogenesis. Cardiomyogenic differentiation was performed on decellularized heart extracellular matrix (ECM; three-dimensional scaffolds) and culture plates (two-dimensional cell culture system) for 28 days to understand the effects of the heart extracellular matrix. In addition, differentiation was induced with and without the stimulatory effect of VEGF to understand the effect of VEGF on cardiomyogenic differentiation of rBM-MSCs. ResultsImmunofluorescence staining showed that decellularization of the heart was performed effectively and successfully. After decellularization process, the heart extracellular matrix was completely free of cells. It was observed that rBM-MSCs transplanted onto the heart extracellular matrix remained viable and proliferated for 21 days after recellularization. The rBM-MSCs promoted cardiomyogenic differentiation in the conventional differentiation medium but were inversely affected by both VEGF and heart extracellular matrix proteins. Lower expression of connexin43 and cardiac troponin I genes was observed in cells induced by either matrix proteins or VEGF, compared to cells differentiated by chemical agents alone. ConclusionIn this study, we investigated the effect of decellularized heart extracellular matrix and VEGF on cardiomyogenic differentiation of rBM-MSCs. On the decellularized cardiac extracellular matrix, rBM-MSCs maintained their viability by adhering to the matrix and proliferating further. The adhesion of the cells to the matrix also produced a physical stimulus that led to the formation of histological structures resembling myocardial layers. Chemical stimulation of the decellularized heart extracellular matrix and cardiomyogenic differentiation supplements resulted in increased expression of cardiomyogenic biomarkers through modulation of the Notch and Hedgehog signaling pathways.

Myocardial angiogenesis induced by concurrent vitamin D supplementation and aerobic-resistance training is mediated by inhibiting miRNA-15a, and miRNA-146a and upregulating VEGF/PI3K/eNOS signaling pathway.

This study aimed to investigate the effects of co-treatment of aerobic-resistance training (ART), vitamin D3 (VD3) on cardiovascular function considering the involvement of microRNA-15a and microRNA-146a, vascular endothelial growth factor (VEGF), phosphatidylinositol-3 kinase (PI3K), and endothelial nitric oxide synthase (eNOS) after myocardial infarction (MI) in rats. To induce MI, male Wistar rats subcutaneously received isoproterenol for 2 days, then MI was confirmed by echocardiography. MI rats were divided into six groups (n = 8/group). MI + VD3, MI + sesame oil (Veh), MI + ART, MI + VD3 + ART, and MI + Veh + ART, and received the related treatments for 8 weeks. Exercise tests, echocardiography, real-time quantitative polymerase chain reaction (qRT-PCR), western blotting, and histological staining were performed after the end of treatments. The highest ejection fraction (EF%), fractional shortening (FS%), exercise capacity (EC), and maximal load test (MLT) amounts were observed in the groups treated with VD3, ART, and VD3 + ART (P < 0.05). These were accompanied by a significantly increased angiogenesis post-MI. Furthermore, the levels of circulating microRNA-15a and microRNA-146a were significantly decreased in these groups compared to MI rats that were together with a significant upregulation of cardiac VEGF, PI3K, and eNOS expression. Overall, the best results were observed in the group treated with VD3 + ART. Concurrent VD3 supplementation and ART attenuated microRNA-15a and microRNA-146a and induced angiogenesis via VEGF/PI3K/eNOS axis. This data demonstrate that concurrent VD3 supplementation and ART is a more efficient strategy than monotherapy to improve cardiac function post-MI.

Cardiac epigenetic changes in VEGF signaling genes associate with myocardial microvascular rarefaction in experimental chronic kidney disease.

Chronic kidney disease (CKD) is common in patients with heart failure and often results in left ventricular diastolic dysfunction (LVDD). However, the mechanisms responsible for cardiac damage in CKD-LVDD remain to be elucidated. Epigenetic alterations may impose long-lasting effects on cellular transcription and function, but their exact role in CKD-LVDD is unknown. We investigate whether changes in cardiac site-specific DNA methylation profiles might be implicated in cardiac abnormalities in CKD-LVDD. CKD-LVDD and normal control pigs (n = 6 each) were studied for 14 wk. Renal and cardiac hemodynamics were quantified by multidetector CT and echocardiography. In randomly selected pigs (n = 3/group), cardiac site-specific 5-methylcytosine (5mC) immunoprecipitation (MeDIP)- and mRNA-sequencing (seq) were performed, followed by integrated (MeDiP-seq/mRNA-seq analysis), and confirmatory ex vivo studies. MeDIP-seq analysis revealed 261 genes with higher (fold change > 1.4; P < 0.05) and 162 genes with lower (fold change < 0.7; P < 0.05) 5mC levels in CKD-LVDD versus normal pigs, which were primarily implicated in vascular endothelial growth factor (VEGF)-related signaling and angiogenesis. Integrated MeDiP-seq/mRNA-seq analysis identified a select group of VEGF-related genes in which 5mC levels were higher, but mRNA expression was lower in CKD-LVDD versus normal pigs. Cardiac VEGF signaling gene and VEGF protein expression were blunted in CKD-LVDD compared with controls and were associated with decreased subendocardial microvascular density. Cardiac epigenetic changes in VEGF-related genes are associated with impaired angiogenesis and cardiac microvascular rarefaction in swine CKD-LVDD. These observations may assist in developing novel therapies to ameliorate cardiac damage in CKD-LVDD.NEW & NOTEWORTHY Chronic kidney disease (CKD) often leads to left ventricular diastolic dysfunction (LVDD) and heart failure. Using a novel translational swine model of CKD-LVDD, we characterize the cardiac epigenetic landscape, identifying site-specific 5-methylcytosine changes in vascular endothelial growth factor (VEGF)-related genes associated with impaired angiogenesis and cardiac microvascular rarefaction. These observations shed light on the mechanisms of cardiac microvascular damage in CKD-LVDD and may assist in developing novel therapies for these patients.

MO421: Chronic Kidney Disease is Associated With Cardiac Downregulation of Vascular Endothelial Growth Factor a (VEGFA) and Development of Diastolic Dysfunction: Epigenetics and Micrornas

Abstract BACKGROUND AND AIMS Patients with chronic kidney disease (CKD) have a higher risk to develop heart failure with preserved ejection fraction (HFpEF). We showed in a swine model of CKD with diastolic dysfunction and cardiac remodelling that these cardiac features are associated with blunted cardiac mRNA and protein expression of vascular endothelial growth factor (VEGF), a pro-angiogenic cytokine that plays important roles in cardiac vascular development, morphogenesis and contractility. Hence, we aim to identify potential underlying mechanisms of cardiac VEGF downregulation in CKD. METHOD CKD and normal pigs (n = 3 each) were studied for 14 weeks. Cardiac morphology and function (echocardiography) were quantified in vivo and microvascular density (3D micro-CT) ex vivo. Then, cardiac micro-RNA (miRNA) and 5-hydroxymethylcytosine (5-hmC) profiles of VEGFA gene were examined by hydroxymethylated DNA immunoprecipitation and miRNA sequencing (hMeDIP-seq and miRNA-seq, respectively) and validated by qPCR and Western blotting. RESULTS Blunted renal function in CKD pigs associated with left ventricular (LV) hypertrophy, and abnormal LV strain and diastolic dysfunction with pEF (Figure 1A) and reduced subendocardial microvascular density (Figure 1B) compared to normal. miRNA-seq showed that mir-183 was the only miRNA capable of targeting VEGFA and upregulated in CKD hearts (Figure 1C), accompanied by downregulation of VEGFA gene and VEGF protein (Figure 1D), whereas hMeDIP-seq revealed reduced VEGFA 5hmC levels in the hearts of CKD versus normal pigs (Figure 1E). CONCLUSION Our study suggests that miRNA-modulation and cardiac epigenetic changes in VEGF might contribute to cardiac damage and the development of HF in CKD. These observations may assist in developing novel therapies, like miRNA antagonists or epigenetic modulators, to preserve VEGF signalling and protect the heart in patients with CKD.

Sitagliptin protects diabetic rats with acute myocardial infarction through induction of angiogenesis: role of IGF-1 and VEGF.

Angiogenesis is regulated in a tissue-specific manner in all patients, especially those with diabetes. In this study, we describe a novel molecular pathway of angiogenesis regulation in diabetic rats with myocardial infarction (MI) and examine the cardioprotective effects of different doses of sitagliptin. Male rats were divided into 5 groups: normal vehicle group, diabetic group, diabetic + MI, diabetic + MI + 5 mg/kg sitagliptin, and diabetic + MI + 10 mg/kg sitagliptin. Isoproterenol in diabetic rats resulted in significant (p < 0.05) disturbance to the electrocardiogram, cardiac histopathological manifestations, and an increase in inflammatory markers compared with the vehicle and diabetic groups. Treatment with sitagliptin improved the electrocardiogram and histopathological sections, upregulated vascular endothelial growth factor (VEGF) and transmembrane phosphoglycoprotein protein (CD34) in cardiac tissues, and increased serum insulin-like growth factor 1 (IGF-1) and decreased cardiac tissue homogenate for interleukin 6 (IL-6) and cyclooxygenase 2 (COX-2). A relationship was found between serum IGF-1 and cardiac VEGF and CD34 accompanied by an improvement in cardiac function of diabetic rats with MI. Therefore, the observed effects of sitagliptin occurred at least partly through an improvement in angiogenesis and the mitigation of inflammation. Consequently, these data suggest that sitagliptin may contribute, in a dose-dependent manner, to protection against acute MI in diabetic individuals.

Hydrogen sulfide enhances the effectiveness of mesenchymal stem cell therapy in rats with heart failure: In vitro preconditioning versus in vivo co-delivery

Stem cell therapy represents a promising therapeutic avenue for cardiac disorders, including heart failure. Although stem cell transplantation showed encouraging preliminary results, the outcomes of clinical studies are still unsatisfactory. This study aimed to compare the outcomes of two therapeutic approaches, in vivo co-delivery of sodium hydrogen sulfide (NaHS) concomitant with bone marrow-derived mesenchymal stem cell (BMSC) transplantation and in vitro preconditioning of BMSCs with NaHS, both of which are intended to promote the success of stem cell therapy in rats with isoprenaline-induced heart failure. Heart failure developed 4 weeks after the subcutaneous injection of isoprenaline (170 mg/kg) for 4 consecutive days. The in vivo approach involved the co-delivery of intraperitoneally administered NaHS concomitant with BMSC transplantation for a period of 14 days. The in vitro approach involved preconditioning BMSCs with NaHS for 30 min before transplantation. Compared to treatment with BMSCs alone, in vitro preconditioning of BMSCs with NaHS improved left ventricular function as measured by echocardiography and electrocardiography and enhanced stem cell homing, proliferation and differentiation as manifested by higher cardiac expression of GATA-4 and myocyte enhancer factor 2. Moreover, the measurement of cardiac transforming growth factor beta 1 levels and histopathological investigation revealed mitigated fibrosis and myocardial injury scores. Compared with BMSC therapy alone, the in vivo approach enhanced stem cell homing and differentiation, alleviated fibrosis and augmented vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) expression. In conclusion, NaHS can potentiate the efficiency of BMSC therapy for heart failure by in vitro preconditioning or in vivo co-delivery. The in vitro approach is superior with regard to improving cardiac function in addition to enhancing stem cell proliferation, while the in vivo approach is superior with regard to increasing cardiac VEGF and eNOS expression.

Modulation of cardiac vascular endothelial growth factor and PGC-1α with regular postexercise cold-water immersion of rats.

Myocardial mitochondrial biogenesis and vascular angiogenesis biomarker responses to postexercise cold-water immersion (CWI) have not been reported. Therefore, to determine those cardiac adaptations, adult male Sprague-Dawley rats were divided into three groups: postexercise CWI (CWI; n = 13), exercise only (Ex; n = 12), and untreated control (CON; n = 10). CWI and Ex were trained for 10 wk, 5 sessions/wk, 30-60 min/session. CWI rats were immersed after each session in cold water (15 min at ~12°C). CON remained sedentary. Left ventricle tissue was obtained 48 h after the last exercise session and analyzed for peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), vascular endothelial growth factor (VEGF), and heat shock protein 70 kDa (Hsp70) protein content and mRNA expression levels. In addition, superoxide dismutase activity and mRNA and malondialdehyde levels were evaluated. Ex and CWI induced higher PGC-1α protein content compared with CON (1.8 ± 0.6-fold, P < 0.001), which was significantly higher in CWI than Ex rats (P = 0.01). VEGF protein (4.3 ± 3.7-fold) and mRNA (10.1 ± 1.1-fold) were markedly increased only in CWI (P < 0.001) relative to CON. CWI and Ex augmented cardiac Hsp70 protein to a similar level relative to CON (P < 0.05); however, Hsp70 mRNA increased only in Ex (P = 0.002). No further differences were observed between groups. These results suggest that postexercise CWI may further enhance cardiac oxidative capacity by increasing the angiogenic and mitochondrial biogenic factors. In addition, CWI does not seem to worsen exercise-induced cardioprotection and oxidative stress. NEW & NOTEWORTHY A regular postexercise cold-water immersion for 10 wk of endurance training augmented the myocardial mitochondrial biogenesis and vascular angiogenesis coactivators peroxisome proliferator-activated receptor γ coactivator-1α and vascular endothelial growth factor, respectively. In addition, postexercise cold-water immersion did not attenuate the exercise-induced increase in the cardioprotective biomarker heat shock protein 70 kDa or increase exercise-induced oxidative stress.

DPP4 Inhibition Ameliorates Cardiac Function by Blocking the Cleavage of HMGB1 in Diabetic Mice After Myocardial Infarction.

High mobility group box 1 (HMGB1), a ubiquitous DNA-binding protein, promotes angiogenesis and tissue repair, resulting in restored cardiac function after myocardial infarction (MI). Although dipeptidyl peptidase 4 (DPP4) degrades certain peptides, it remains unclear as to whether HMGB1 is a substrate of DPP4 and whether DPP4 inhibition prevents the cleavage of HMGB1.In transgenic mice with cardiac-specific overexpression of HMGB1 (TG) and wild-type mice (WT), a diabetic state was induced by streptozotocin, and MI was created by ligation of the left anterior descending coronary artery. To inhibit DPP4 activity, a DPP4 inhibitor anagliptin was used. The plasma levels of HMGB1, infarct size, echocardiographic data, angiogenesis, and vascular endothelial growth factor (VEGF) expression in the peri-infarct area were compared among non-diabetic MI WT/TG, diabetic MI WT/TG, and anagliptin-treated diabetic MI WT/TG mice.DPP4 activity was increased in the diabetic state and blocked by anagliptin administration. The HMGB1 plasma levels were reduced in the diabetic TG compared with the non-diabetic TG mice, but DPP4 inhibition with anagliptin increased HMGB1 plasma levels in the diabetic TG mice. The infarct area was significantly larger in the diabetic TG than in the non-diabetic TG mice, and it was reduced by DPP4 inhibition. Cardiac function, angiogenesis, and VEGF expression were impaired in the diabetic TG mice, but they were ameliorated by the DPP4 inhibition to levels similar to those found in the non-diabetic TG mice.The DPP4 inhibitor ameliorated cardiac function by inhibiting the inactivation of HMGB1 in diabetic mice after MI.

Endothelial deletion of protein tyrosine phosphatase-1B protects against pressure overload-induced heart failure in mice.

Cardiac angiogenesis is an important determinant of heart failure. We examined the hypothesis that protein tyrosine phosphatase (PTP)-1B, a negative regulator of vascular endothelial growth factor (VEGF) receptor-2 activation, is causally involved in the cardiac microvasculature rarefaction during hypertrophy and that deletion of PTP1B in endothelial cells prevents the development of heart failure. Cardiac hypertrophy was induced by transverse aortic constriction (TAC) in mice with endothelial-specific deletion of PTP1B (End.PTP1B-KO) and controls (End.PTP1B-WT). Survival up to 20 weeks after TAC was significantly improved in mice lacking endothelial PTP1B. Serial echocardiography revealed a better systolic pump function, less pronounced cardiac hypertrophy, and left ventricular dilation compared with End.PTP1B-WT controls. Histologically, banded hearts from End.PTP1B-KO mice exhibited increased numbers of PCNA-positive, proliferating endothelial cells resulting in preserved cardiac capillary density and improved perfusion as well as reduced hypoxia, apoptotic cell death, and fibrosis. Increased relative VEGFR2 and ERK1/2 phosphorylation and greater eNOS expression were present in the hearts of End.PTP1B-KO mice. The absence of PTP1B in endothelial cells also promoted neovascularization following peripheral ischaemia, and bone marrow transplantation excluded a major contribution of Tie2-positive haematopoietic cells to the improved angiogenesis in End.PTP1B-KO mice. Increased expression of caveolin-1 as well as reduced NADPH oxidase-4 expression, ROS generation and TGFβ signalling were observed and may have mediated the cardioprotective effects of endothelial PTP1B deletion. Endothelial PTP1B deletion improves cardiac VEGF signalling and angiogenesis and protects against chronic afterload-induced heart failure. PTP1B may represent a useful target to preserve cardiac function during hypertrophy.

Bone marrow mononuclear cells enhance anti-inflammatory effects of pravastatin against isoproterenol-induced myocardial infarction in rats

The current study investigated the combinatorial effect of pravastatin (PRAV) and bone marrow mononuclear cells (BM-MNC) on acute myocardial infarction (AMI) induced experimentally in rats. After induction of MI, rats were given oral PRAV (20 mg/kg/day) for 28 days or a bolus intravenous injection (via lateral vein) of a total of 14 × 106 autologous BM-MNC or a combination of both. Serum brain natriuretic peptide (BNP) and histologic changes in cardiac tissues were assessed. Cardiac contents of lipid peroxides, superoxide dismutase (SOD) and inflammatory biomarkers including tumor necrosis factor (TNF)-α and interleukin (IL)-1β as well as vascular endothelial growth factor (VEGF) and nitric oxide (NO) were also measured. Combined PRAV and BM-MNC treatment significantly suppressed serum BNP. Cardiac cell apoptosis and inflammatory cell infiltration in heart tissue decreased significantly in both the PRAV and the PRAV + BM-MNC groups. Cardiac lipid peroxides along with TNFα and IL-1β levels were significantly reduced in both the PRAV and PRAV + BM-MNC hosts with an increase in SOD levels. However, the combined treatment increased cardiac NO levels and did not modify cardiac VEGF levels. The current results indicated that administration of BM-MNC improved the therapeutic efficacy of PRAV treatment by improving the morphology of infarcted hearts as well as decreasing inflammation in a host, but did not do so by inducing therapeutic angiogenesis.

Autoantibodies in dilated cardiomyopathy induce vascular endothelial growth factor expression in cardiomyocytes

BackgroundAutoantibodies have been identified as major predisposing factors for dilated cardiomyopathy (DCM). Patients with DCM show elevated serum levels of vascular endothelial growth factor (VEGF) whose source is unknown. Besides its well-investigated effects on angiogenesis, evidence is present that VEGF signaling is additionally involved in fibroblast proliferation and cardiomyocyte hypertrophy, hence in cardiac remodeling. Whether autoimmune effects in DCM impact cardiac VEGF signaling needs to be elucidated. MethodsFive DCM patients were treated by the immunoadsorption (IA) therapy on five consecutive days. The eluents from the IA columns were collected and prepared for cell culture. Cardiomyocytes from neonatal rats (NRCM) were incubated with increasing DCM-immunoglobulin-G (IgG) concentrations for 48 h. Polyclonal IgG (Venimmun N), which was used to restore IgG plasma levels in DCM patients after the IA therapy was additionally used for control cell culture purposes. ResultsElevated serum levels of VEGF decreased significantly after IA (Serum VEGF (ng/ml); DCM pre-IA: 45 ± 9.1 vs. DCM post–IA: 29 ± 6.7; P < 0.05). In cell culture, pretreatment of NRCM by DCM-IgG induced VEGF expression in a time and dose dependent manner. Biologically active VEGF that was secreted by NRCM significantly increased BNP mRNA levels in control cardiomyocytes and induced cell-proliferation of cultured cardiac fibroblast (Fibroblast proliferation; NRCM medium/HC-IgG: 1 ± 0.0 vs. NRCM medium/DCM-IgG 100 ng/ml: 5.6 ± 0.9; P < 0.05). ConclusionThe present study extends the knowledge about the possible link between autoimmune signaling in DCM and VEGF induction. Whether this observation plays a considerable role in cardiac remodeling during DCM development needs to be further elucidated.

Pretreatment of mesenchymal stem cells with angiotensin II enhances paracrine effects, angiogenesis, gap junction formation and therapeutic efficacy for myocardial infarction

BackgroundPretreatment of mesenchymal stem cells (MSCs) with growth factors is reported to be an effective route for improving cell-based therapy of myocardial infarction (MI). Angiotensin II (Ang II) triggers vascular endothelial growth factor (VEGF) synthesis in MSCs. This study aimed to investigate the effects and mechanisms of Ang II pretreatment in enhancing the therapeutic efficacy of MSCs in MI. MethodsMSCs and endothelial cells (ECs) were isolated from Sprague-Dawley rats. After pretreated with or without 100nM of Ang II for 24h, the MSCs were directly injected into the border zones of the ischemic heart. Cardiac function, fibrosis, infarct size, VEGF expression, angiogenesis, and cell differentiation in the infarcted myocardium were determined after 30days. The cell apoptosis of MSCs post hypoxia was assessed using flow cytometry. The angiogenic activity of MSCs was analyzed using tube formation assay. The gap junction protein connexin-43 (Cx43) expression was detected. ResultsCompared with the MSC group, pretreatment of MSCs with Ang II resulted in better cardiac function, less cardiac fibrosis, smaller infarct size, and higher expression of VEGF and Von Willebrand Factor in ischemic myocardium, but no promotion of cardiomyocyte-like differentiation of MSCs. Ang II pretreatment enhanced the survival of MSCs and the H9c2 cells surrounding MSCs, and augmented the tube formation of ECs and MSCs. Ang II pretreatment up-regulated the Cx43 expression. ConclusionsThe pretreatment of MSCs with Ang II improved the outcome of MSC-based therapy for MI via the mechanisms of enhancing the paracrine production of VEGF, angiogenesis, and gap junction formation.

High Mobility Group Box 1 Promotes Angiogenesis from Bone Marrow-derived Endothelial Progenitor Cells after Myocardial Infarction

High mobility group box 1 (HMGB1) is a DNA-binding protein secreted into the extracellular space from necrotic cells that acts as a cytokine. We examined the role of HMGB1 in angiogenesis from bone marrow-derived cells in the heart using transgenic mice exhibiting the cardiac-specific overexpression of HMGB1 (HMGB1-TG). HMGB1-TG mice and wild-type littermate (WT) mice were lethally irradiated and injected with bone marrow cells from green fluorescent protein mice through the tail vein. After bone marrow transplantation, the left anterior descending artery was ligated to induce myocardial infarction (MI). Flow cytometry revealed that the levels of circulating endothelial progenitor cells (EPCs) mobilized from the bone marrow increased after MI in the HMGB-TG mice versus the WT mice. In addition, the size of MI was smaller in the HMGB1-TG mice than in the WT mice, and immunofluorescence staining demonstrated that the number of engrafted vascular endothelial cells derived from bone marrow in the border zones of the MI areas was increased in the HMGB1-TG mice compared to that observed in the WT mice. Moreover, the levels of cardiac vascular endothelial growth factor after MI were higher in the HMGB1-TG mice than in the WT mice. The present study demonstrated that HMGB1 promotes angiogenesis and reduces the MI size by enhancing the mobilization and differentiation of bone marrow cells to EPCs as well as their migration to the border zones of the MI areas and engraftment as vascular endothelial cells in new capillaries or arterioles in the infarcted heart.

Landiolol Hydrochloride Normalizes Diminished Levels of Cardiac Vascular Endothelial Growth Factor (VEGF) Signaling System Components in Lipopolysaccharide-Induced Sepsis Independent of Inflammatory Markers

Aim: Myocardial dysfunction is one of the complications associated with sepsis during its (sepsis) pathogenesis. To date, very few studies have investigated whether angiogenic factors in the heart, such as vascular endothelial growth factor (VEGF) and components of its signaling system are involved in myocardial dysfunction during the early phases of sepsis. Therefore, the present study aims to examine: 1) the expression pattern of VEGF and its signaling molecules in the heart during the early hours of LPS-induced sepsis and 2) whether landiolol hydrochloride, an ultrashort-acting beta- blocker, can ameliorate alterations in the expression of cardiac VEGF signaling system components in the rats under these (sepsis) conditions. Method: Eight (8)-week-old male Wistar rats were administered for three hours with either LPS only once, or continuously with LPS plus landiolol. Result: At 3 h after LPS (only) administration, circulatory levels of tumor necrosis factor (TNF)-α, IL-6, iNOS, lactate concentration and percentage of fractional shortening of the heart were significantly increased. However, levels of cardiac VEGF and its downstream signaling components were significantly down regulated. Treatment of LPS-administered rats with landiolol for 3 h normalized LPS-induced blood lactate levels, cardiac functional compensatory events, as well as VEGF and its signaling molecules, but did not alter levels of plasma TNF-α, IL-6 and iNOS. Conclusion: Taken together, these data led us to conclude that landiolol may be cardio-protective in septic rats by normalizing coronary microcirculation through blockage of sepsis-induced decrease in expression of VEGF signaling system but independent of inflammatory cytokines.

Abstract 9483: High Mobility Group Box 1 Promotes Angiogenesis From Bone Marrow-Derived Endothelial Progenitor Cells After Myocardial Infarction

Background: High mobility group box 1 (HMGB1) is a DNA-binding protein secreted into extracellular space from necrotic cells and acts as a cytokine. We have reported that HMGB1 attenuates cardiac damage and restores cardiac function by enhancing angiogenesis after myocardial infarction (MI). We examined the role of HMGB1 in angiogenesis from bone marrow (BM) -derived cells in the heart, using transgenic mice with cardiac-specific overexpression of HMGB1 (HMGB1-TG). Methods and Results: HMGB1-TG mice and wild-type littermate (WT) mice were lethally irradiated and injected with BM cells from green fluorescent protein (GFP) mice through the tail vein. Two weeks after BM transplantation, the left anterior descending artery was ligated to create MI. In flow cytometry analysis, GFP-positive cells were identified as donor BM cells-derived endothelial progenitor cells (EPC) if they were positive for both CD34 and CD144 in granulocyte differentiation antigen-1-negative fraction. Circulating EPC mobilized from BM was increased at 1 week after MI in HMGB1-TG mice compared with WT mice (41.9% vs. 24.5%, P &lt; 0.01). Histological examination showed the size of MI was smaller in HMGB1-TG mice than in WT mice (42.9% vs. 59.1%, P &lt; 0.01) at 4 weeks after MI. In myocardial immunofluorescence staining, GFP and CD31 double-positive cells were BM-derived cells engrafted within myocardial tissue as vascular endothelial cells of new capillaries or arterioles. The ratio of these double positive cells to all cardiac cells was significantly higher in the HMGB1-TG mice than in the WT mice (8.3% vs. 2.9%, P &lt; 0.01). Enzyme-linked immunosorbent assay revealed that the levels of cardiac vascular endothelial growth factor at 1 week after MI were higher in HMGB1-TG mice than in WT mice (642.1 vs. 390.7 pg/dl, P &lt; 0.05). Conclusions: The present study demonstrated the direct in vivo evidence that HMGB1 promoted angiogenesis and reduced MI size by enhancing mobilization and differentiation of BM cells to EPC, migration to the border zone of MI, and engraftment as vascular endothelial cells of new capillaries or arterioles in the infarcted heart.

Dual blockade of endothelin action exacerbates up-regulated VEGF angiogenic signaling in the heart of lipopolysaccharide-induced endotoxemic rat model

AimsSepsis is a cluster of heterogeneous syndromes associated with progressive endotoxemic developments, ultimately leading to damage of multiple organs, including the heart. However, the pathogenesis of sepsis-induced myocardial dysfunction is still not fully understood. The present study is the first to examine alterations in expression of key angiogenic signaling system mediated by vascular endothelial growth factor (VEGF) in septic heart and the effects of endothelin dual blocker (ETDB) on it. Main methodsNormal Wistar rats were either administered with: a) vehicle only (control group), b) lipopolysaccharide only (LPS: 15mg/kg) and then sacrificed at different time points (1h, 3h, 6h and 10h), and c) the last group was co-administered with LPS and ETDB (SB-209670, 1mg/kg body weight) for 6h and then sacrificed. Key findingsAdministration of LPS resulted in increases in levels of: a) serum tumor necrosis factor (TNF)-α, b) serum VEGF and c) serum endothelin (ET)-1 levels accompanied by up-regulation of cardiac VEGF and its downstream angiogenic signaling molecules. While cardiac TNF-α level was unchanged among experimental groups, cardiac ET-1 level was significantly higher in LPS-administered group. SignificanceWe conclude that elevation in VEGF angiogenic signaling may be triggered by diminished oxygenation in the myocardium following LPS administration as a consequence of sepsis-induced microvascular dysfunction. Because of this cardiac dysfunction, oxygen supply may be inadequate at microregional level to support the normal heart metabolism and function. ETDB at 6h further increased the elevated levels of VEGF angiogenic signaling in endotoxemic heart.

Vascular Endothelial Growth Factor Blockade Prevents the Beneficial Effects of β-Blocker Therapy on Cardiac Function, Angiogenesis, and Remodeling in Heart Failure

Impaired angiogenesis in the post-myocardial infarction heart contributes to the progression to heart failure. The inhibition of vascular endothelial growth factor (VEGF) signaling has been shown to be crucial for the transition from compensatory hypertrophy to cardiac failure. Importantly, β-adrenergic receptor blocker therapy has been also shown to improve myocardial perfusion by enhancing neoangiogenesis in the failing heart. Eight weeks from surgically induced myocardial infarction, heart failure rats were randomized to receive bisoprolol (B) or vehicle. At the end of a 10-week treatment period, echocardiography revealed reduced cardiac diameters and improved cardiac function in B-treated compared with vehicle-treated rats. Moreover, B treatment was associated with increased cardiac angiogenesis and in vivo coronary perfusion and reduced cardiac fibrosis. Importantly, 2 weeks after B treatment was started, increased cardiac VEGF expression and Akt and endothelial NO synthase activation were observed by comparing B-treated with drug-untreated failing hearts. To test whether the proangiogenic effects of B act via activation of VEGF pathway, rats were intravenously injected with adenoviral vector encoding a decoy VEGF receptor (Ad-Flk) or a control adenovirus (Ad-C), at the start of the treatment with B. After 10 weeks, histological analysis revealed reduced capillary and coronary perfusion in B-treated plus Ad-Flk rats compared with B-treated plus Ad-C rats. Moreover, VEGF inhibition counteracted the positive effects of B on cardiac function and remodeling. β-Blockade promotes cardiac angiogenesis in heart failure via activation of VEGF signaling pathway. β-Blocker-induced enhancement of cardiac angiogenesis is essential for the favorable effects of this therapy on cardiac function and remodeling.

Muscle-Specific Vascular Endothelial Growth Factor Deletion Induces Muscle Capillary Rarefaction Creating Muscle Insulin Resistance

Muscle insulin resistance is associated with a reduction in vascular endothelial growth factor (VEGF) action and muscle capillary density. We tested the hypothesis that muscle capillary rarefaction critically contributes to the etiology of muscle insulin resistance in chow-fed mice with skeletal and cardiac muscle VEGF deletion (mVEGF−/−) and wild-type littermates (mVEGF+/+) on a C57BL/6 background. The mVEGF−/− mice had an ∼60% and ∼50% decrease in capillaries in skeletal and cardiac muscle, respectively. The mVEGF−/− mice had augmented fasting glucose turnover. Insulin-stimulated whole-body glucose disappearance was blunted in mVEGF−/− mice. The reduced peripheral glucose utilization during insulin stimulation was due to diminished in vivo cardiac and skeletal muscle insulin action and signaling. The decreased insulin-stimulated muscle glucose uptake was independent of defects in insulin action at the myocyte, suggesting that the impairment in insulin-stimulated muscle glucose uptake was due to poor muscle perfusion. The deletion of VEGF in cardiac muscle did not affect cardiac output. These studies emphasize the importance for novel therapeutic approaches that target the vasculature in the treatment of insulin-resistant muscle.

Bosentan ameliorates the expression of fibrotic related growth factors and collagen-1 in diabetic mice

To investigate the potential beneficial effect of bosentan in ameliorating fibrotic agents in diabetic mice. Male 6-week old C57BL/6 mice were divided into 3 groups (N=20): Control group, diabetes mellitus (DM) group and DM-B group (diabetes with bosentan group). Streptozotocin (STZ) was injected as 200 mg/Kg for single dose, i.p. (intraperitoneal injection). Fasting blood glucose (FBG) was measured at 0-, 1-, 2-week after STZ injection to confirm that diabetes was induced in the mice. Bosentan (100mg/Kg) and placebo was given i.g. (intragastric administration) once a day immediately after STZ injection for 18 weeks. The mRNA expression of tissue growth factor beta (TGF-b), connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF) and collagen-1 were evaluated by RT-PCR and real-time PCR. Differences in the data between the groups were compared by Student t-test for independent samples. After 18 weeks of diabetic situation, FBG of DM-B mice was significantly higher than that of control mice and was similar with that of DM mice (DM mice vs. control mice, p<0.001; DM-B vs. control mice, p<0.001; DM mice vs. DM-B mice, p>0.05). The cardiac VEGF mRNA (a potent angiogenic factor) level in DM-B mice was significantly higher than DM mice (p<0.01). The heart of DM-B mice also showed lower expression of fibrotic genes (TGF-b, CTGF and collagen-1) than DM mice (p<0.01). These findings indicate the potential usefulness of an ET receptor antagonist bosentan in the amelioration of fibrotic agents, which may promote tissue fibrosis. This may provide a promising therapeutical strategy for diabetic cardiac fibrosis.

CITED2 mutation links congenital heart defects to dysregulation of the cardiac gene VEGF and PITX2C expression

CITED2, a cardiac transcription factor, plays an important role in cardiac development. CITED2 mutations lead to a constellation of cardiac defects, which include tetralogy of Fallot and outflow tract malformations. However, the mechanisms underlying these mutations are poorly understood. We investigated the function and mechanism of two missense mutations, G184S and S192G, responsible for tetralogy of Fallot and aortic stenosis, respectively. We found that CITED2 variants decreased its ability to mediate the expression of vascular endothelial growth factor (VEGF) and the expression of the paired-like homeodomain transcription factor 2-gamma (PITX2C), both of which are closely related to cardiac development. Luciferase reporter and mammalian two-hybrid assays showed that G184S and S192G in CITED2 restored the expression of VEGF, which was due to a reduction in its competitiveness with hypoxia inducible factor 1-alpha (HIF1-α) for binding to CBP/p300. In addition, we found that the G184S and S192G mutant decreased cooperation between CITED2 and transcription factor AP2-gamma (TFAP2C) in the transactivation of the PITX2C gene. These results provide important evidence that the mutation of CITED2 may play a role in the development of congenital heart disease (CHD) in humans.