Angiogenesis In Ischemic Myocardium Research Articles

Abstract 14422: Mitochondrial ROS Scavenger JP4-039 Improves Mitochondrial Oxidative Phosphorylation and Induces Angiogenesis in Murine and Human Coronary Artery and Atrial Endothelial Cells

Introduction: Reducing mitochondrial ROS (mito-ROS) has been a therapeutic goal to improve cardiovascular disease. Here, we evaluated the effects of mito-ROS scavenger JP4-039 with a focus on mouse heart and human coronary artery endothelial cell (MHEC, HCAEC). Hypothesis: We hypothesized that JP4-039 would improve oxidative phosphorylation (OXPHOS) in EC and induce angiogenesis in post-AMI (acute myocardial infarction) hearts. Methods: Human atrial explants, NOX2-overexpressing (OE) MHEC, and HCAEC were treated with 1-5μM JP4-039 and examined for sprouting, tube formation, mitochondrial complex expression (Western blot), and OXPHOS measurements (Seahorse). FVB mice (n=8/group) underwent AMI surgery and were assigned to vehicle (sunflower seed oil) or JP4-039 group (1mg/kg, i.p., 3x/week for 4 weeks). Left ventricle (LV) function and capillary density in infarcted LV were assessed by echocardiogram and immunofluorescence, respectively, 28 days after AMI. Data were analyzed by One-way ANOVA and Tukey’s post-hoc, or student’s t test, as appropriate. A p-value <0.05 was considered statistically significant. Results: HCAEC treated with JP4-039 showed significantly increased expression of mitochondrial complex I (0.7±0.1 vs. 1.1±0.1) and V (1.0±0.1 vs. 1.2±0.1) with corresponding increase in basal (19.8±0.4 vs. 23.0±1.0) and maximal respiration (31.2±1.6 vs. 56.0±2.6), ATP production (16.6±0.3 vs. 18.5±0.4) and spare respiratory capacity (11.3±1.6 vs. 33.0±2.9) as compared with vehicle-treated cells. JP4-039 increased tube formation in NOX2-OE MHEC (12.6±2.2 vs. 21.3±2.4 tubes), HCAEC (33.6±3.1 vs. 49.0±2.6) as well as EC sprouting in mouse atrium (4496±1857 vs. 19552±4352 pixel 2 ). In post-AMI animals, JP4-039 improved LV systolic function (EF 24.4±3.6 vs 41.3±5.2%) and capillary density (451.7 (379.9; 629.7) vs. 698.3 (503.6; 965.8) capillaries/mm 2 ) in vivo . Conclusions: JP4-039 improved OXPHOS in ECs in vitro, and induced coronary angiogenesis in post-AMI hearts in vivo and in atrial tissues ex vivo . These findings were associated with improved cardiac function in post-AMI animals, suggesting that a mito-ROS scavenger may have significant roles in coronary angiogenesis in ischemic myocardium.

Abstract 14338: Mitochondria-Targeted ROS Scavenger JP4-039 Induces Coronary Angiogenesis in Ischemic Myocardium Resulting in the Improvement of Cardiac Function in Post-MI Animals

Introduction: A reduction of mitochondrial reactive oxygen species (mito-ROS) has been sought as a potential approach for recovering energy supply and improving cardiac function in cardiovascular disease. Here, we evaluated JP4-039, a mito-ROS scavenger, for post-myocardial infarction (MI) recovery. Hypothesis: We hypothesized that mitochondrial antioxidant JP4-039 will induce coronary angiogenesis and improve post-MI cardiac function in mice. Methods: FVB mice (n=8/group) underwent MI surgery and were assigned to either vehicle (sunflower seed oil) or JP4-039 group (1mg/kg, i.p., 3x/week for 4 weeks). A SHAM surgery group received vehicle only. Left ventricle (LV) ejection fraction (EF), fractional shortening (FS), and fractional area change (FAC) were assessed by echocardiography 28 days post-MI. Capillary (CD31 antibody) and arteriole density (α-SMA antibody) were quantified by immunofluorescence of ischemic heart sections 28 days post-MI. Western blot of myocardial homogenates was conducted to evaluate activation of Akt, ERK1/2, and AMPKα. Data were analyzed by One-way ANOVA and Tukey’s post-hoc, and a p value <0.05 was taken as significant. Results: Mice treated with JP4-039 presented with a 35% decrease in infarction size, and a 57% increase in EF compared to the vehicle group (43.6±5.1 vs. 27.8±5.3). FS and FAC of JP4-039-treated mice increased by 65% (21.9±2.9 vs. 13.3±2.6) and 90% (38.0±5.6 vs. 20.0±4.0), respectively. In ischemic myocardium, the JP4-039 group showed an increase in capillary density of 39% in comparison to vehicle (208.6±21.7 vs 149.9±18.0). Interestingly, arteriogenesis was decreased in ischemic myocardium by 31% in JP4-039-treated mice compared to vehicle group (9.0±1.1 vs. 13.1±1.5). Although activation of Akt or ERK1/2 signaling were unchanged, p-AMPKα was decreased by 67% in JP4-039 vs. vehicle (0.39±0.07 vs. 0.65±0.03) in myocardium. Conclusions: The findings suggest that mitochondrial antioxidant JP4-039 improves capillary angiogenesis in ischemic myocardium, resulting in improved cardiac function in post-MI mice. Mechanisms involved in differential levels of capillary and arteriole densities in ischemic myocardium are being examined in human coronary ECs and will be presented at the meeting.

Cardiac telocytes inhibit cardiac microvascular endothelial cell apoptosis through exosomal miRNA-21-5p-targeted cdip1 silencing to improve angiogenesis following myocardial infarction.

Promotion of cardiac angiogenesis in ischemic myocardium is a critical strategy for repairing and regenerating the myocardium after myocardial infarction (MI). Currently, effective methods to aid in the survival of endothelial cells, to avoid apoptosis in ischemic myocardium and to achieve long-term cardiac angiogenesis are still being pursued. Here, we investigated whether cardiac telocyte (CT)-endothelial cell communication suppresses apoptosis and promotes the survival of endothelial cells to facilitate cardiac angiogenesis during MI.Methods: CT exosomes were isolated from CT conditioned medium, and their miRNA profile was characterized by small RNA sequencing. A rat model of left anterior descending coronary artery ligation (LAD)-mediated MI was assessed with histology for infarct size and fibrosis, immunostaining for angiogenesis and cell apoptosis and echocardiography to evaluate the therapeutic effects. Cardiac microvascular endothelial cells (CMECs) and the LAD-MI model treated with CT exosomes or CT exosomal miRNA-21-5p in vitro and in vivo were assessed with cellular and molecular techniques to demonstrate the underlying mechanism.Results: CTs exert therapeutic effects on MI via the potent paracrine effects of CT exosomes to facilitate the inhibition of apoptosis and survival of CMECs and promote cardiac angiogenesis. A novel mechanism of CTs is revealed, in which CT-endothelial cell communication suppresses apoptosis and promotes the survival of endothelial cells in the pathophysiological myocardium. CT exosomal miRNA-21-5p targeted and silenced the cell death inducing p53 target 1 (Cdip1) gene and thus down-regulated the activated caspase-3, which then inhibited the apoptosis of recipient endothelial cells under ischemic and hypoxic conditions, facilitating angiogenesis and regeneration following MI.Conclusions: The present study is the first to show that CTs inhibit cardiac microvascular endothelial cell apoptosis through exosomal miRNA-21-5p-targeted Cdip1 silencing to improve angiogenesis in myocardial infarction. It is believed that these novel findings and the discovery of cellular and molecular mechanisms will provide new opportunities to tailor novel cardiac cell therapies and cell-free therapies for the functional and structural regeneration of the injured myocardium.

Abstract 17169: Endothelium-Specific Reduction in Mitochondrial ROS Induces Coronary Angiogenesis in Ischemic Myocardium via Activation of PI3k/Akt/ERG, ERK1/2, and Jag-1 Signaling

Introduction: Global reduction in reactive oxygen species (ROS) failed to improve outcomes in cardiovascular disease patients. Recent reports suggest that subcellular, rather than global ROS, play a crucial role in endothelial cell (EC) health. To that end, we generated a novel transgenic mouse model that overexpresses mitochondrial antioxidant MnSOD in EC-specific manner (MnSODVE-OE). Hypothesis: We hypothesized that decreased EC mitochondrial-ROS will improve post-myocardial infarction (MI) cardiac function by inducing coronary angiogenesis in ischemic myocardium. Methods: MnSODVE mice were assigned to Tet-ON (control) or Tet-OFF (MnSODVE-OE) group. To turn off the transgene, Tetracycline (Tet) (2mg/kg) was added to the drinking water (Tet-ON), while Tet-OFF mice did not receive Tet. Both groups underwent left anterior descending coronary artery (LAD) ligation surgery to mimic acute MI. Echocardiography was done 28 days after LAD ligation. Capillaries, arteriole density, and proliferating ECs were measured in heart sections using anti-CD31, anti-αSMA, and anti-PCNA immunofluorescence. Western blot, proteomic and phosphoproteomic analyses of mouse heart ECs isolated from MnSODVE (Tet-ON and Tet-OFF) animals were performed to study modulation of signaling cascades. Results: MnSODVE-OE mice demonstrated improved cardiac function (EF and FS increased by 16±7.87% and 21.73±10.31%, respectively, p <0.01), increase in capillary and arteriole densities by 4.13±4.10-fold, p<0.05 and 5.48±3.51-fold, p<0.001, respectively, and in EC proliferation by 1.46±0.80-fold, p <0.01, suggesting de novo coronary angiogenesis. Western blots showed activation of Akt (80.46±3.95% increase in p-Akt/ t-Akt ratio, p<0.05) and ERK1/2 (21.05±4.45% increase in p-ERK/ t-ERK 1/2 ratio, p<0.05). Proteome and phosphoproteome analyses showed upregulation of mitochondrial complex I biogenesis, RNA metabolism, and upregulation of Notch-related proteins ERG and Jag-1 (p<0.05). Student’s t-test was used for data analyses. Conclusions: Taken together, these results suggest that decreased mito-ROS in ECs improves post-MI cardiac function by inducing coronary angiogenesis through activation of PI3k/Akt/ERG, ERK1/2, and Jag-1 signaling.

Thioredoxin1 Inactivation Mediates the Impairment of Ischemia-Induced Angiogenesis and Further Injury in Diabetic Myocardium

Diabetes mellitus (DM)-induced impairment of collateral formation has been demonstrated in subjects with coronary artery disease, which contributes to unfavorable prognosis among diabetic individuals. In our previous studies, thioredoxin1 (Trx1) activity was shown to be decreased in diabetic cardiac tissues, but the reason of Trx1 inactivation and whether it mediates the impaired angiogenesis in ischemic myocardium is still to be identified. As thioredoxin-interacting protein (TXNIP), an endogenous inhibitor of Trx, is overexpressed in DM due to carbohydrate response element within its promoter, we hypothesized that inhibition of Trx1 by enhanced TXNIP expression in endothelial cells may play a role in hyperglycemia-induced impairment of angiogenesis. In the present study, we found that high glucose-mediated increase of TXNIP expression and TXNIP-Trx1 interaction induced the impairment in endothelial cell function and survival, since these detrimental effects are rescued by silencing TXNIP with small interfering RNA. In diabetic mice, TXNIP knockdown or recombinant human Trx1 treatment counteracted the impairment of angiogenesis, alleviated myocardial ischemic injury, and improved survival rate. All these data implicate that TXNIP upregulation and subsequently the increased formation of TXNIP-Trx1 complex is a novel pathologic pathway by which DM induces insufficient angiogenesis and thereby exacerbates myocardial ischemia injury.

Altered microRNA dynamics in acute coronary syndrome.

IntroductionIn the course of acute myocardial infarction (AMI) cardiomyocyte injury, activation and destruction of endothelial cells together with inflammation lead to miRNA expression alterations.AimTo assess levels of circulating cardiac-specific (miR-1) and endothelial-specific (miR-126) miRNAs in the acute phase of AMI and after a follow-up period.Material and methodsSeventeen AMI patients (mean age: 64.24 ±13.83 years, mean left ventricle ejection fraction (LVEF): 42.6 ±9.65%), treated with primary percutaneous coronary intervention within the first 12 h, had plasma miRNAs isolated (quantitative real-time PCR, Exiqon) on admission and after 19.2 ±5.9 weeks. Measurements were also performed in a control group of healthy volunteers matched for age and sex.ResultsConcentrations of both miRNAs were significantly higher in AMI patients as compared to healthy controls: miR-1: 5.93 (3.15–14.92) vs. 1.46 (0.06–2.96), p = 0.04; miR-126: 4.5 (3.11–7.64) vs. 0.54 (0.36–0.99), p = 0.00003, respectively. Levels of both miRNAs significantly decreased after the follow-up period: miR-1: 5.93 (3.15–14.92) vs. 1.34 (0.04–2.34), p = 0.002; miR-126: 4.5 (3.11–7.64) vs. 1.18 (0.49–1.68), p = 0.0005). Moreover, miR-1 correlated positively with maximal troponin I concentration (r = 0.59, p = 0.02) and negatively with LVEF (r = –0.76, p = 0.0004).ConclusionsIn our study, miR-1 emerged as a marker of cardiomyocyte injury and loss of myocardial contractility, whereas dynamics of miR-126 concentration may reflect endothelial activation and damage in the most extreme stage of atherosclerosis, followed by angiogenesis in ischemic myocardium. However, to fully elucidate the role of miR-1 and miR-126 as biomarkers of AMI and future therapeutic targets, further research is required.

Buyang Huanwu Decoction Exerts Cardioprotective Effects through Targeting Angiogenesis via Caveolin-1/VEGF Signaling Pathway in Mice with Acute Myocardial Infarction.

Background Acute myocardial infarction (AMI) remains a leading cause of morbidity and mortality worldwide. The idea of therapeutic angiogenesis in ischemic myocardium is a promising strategy for MI patients. Buyang Huanwu decoction (BHD), a famous Chinese herbal prescription, exerted antioxidant, antiapoptotic, and anti-inflammatory effects, which contribute to cardio-/cerebral protection. Here, we aim to investigate the effects of BHD on angiogenesis through the caveolin-1 (Cav-1)/vascular endothelial growth factor (VEGF) pathway in MI model of mice. Materials and Methods C57BL/6 mice were randomly divided into 3 groups by the table of random number: (1) sham-operated group (sham, n = 15), (2) AMI group (AMI+sham, n = 20), and (3) BHD-treated group (AMI+BHD, n = 20). 2,3,5-Triphenyltetrazolium chloride solution stain was used to determine myocardial infarct size. Myocardial histopathology was tested using Masson staining and hematoxylin-eosin staining. CD31 immunofluorescence staining was used to analyze the angiogenesis in the infarction border zone. Western blot analysis, immunofluorescence staining, and/or real-time quantitative reverse transcription polymerase chain reaction was applied to test the expression of Cav-1, VEGF, vascular endothelial growth factor receptor 2 (VEGFR2), and/or phosphorylated extracellular signal-regulated kinase (p-ERK). All statistical analyses were performed using the SPSS 20.0 software and GraphPad Prism 6.05. Values of P < 0.05 were considered as statistically significant. Results and Conclusion Compared with the AMI group, the BHD-treated group showed a significant improvement in the heart weight/body weight ratio, echocardiography images, cardiac function, infarct size, Mason staining of the collagen deposition area, and density of microvessel in the infarction border zone (P < 0.05). Compared with the AMI group, BHD promoted the expression of Cav-1, VEGF, VEGFR2, and p-ERK in the infarction border zone after AMI. BHD could exert cardioprotective effects on the mouse model with AMI through targeting angiogenesis via Cav-1/VEGF signaling pathway.

Silica-coated magnetic nanoparticles labeled endothelial progenitor cells alleviate ischemic myocardial injury and improve long-term cardiac function with magnetic field guidance in rats with myocardial infarction.

Low retention of endothelial progenitor cells (EPCs) in the infarct area has been suggested to be responsible for the poor clinical efficacy of EPC therapy for myocardial infarction (MI). This study aimed to evaluate whether magnetized EPCs guided through an external magnetic field could augment the aggregation of EPCs in an ischemia area, thereby enhancing therapeutic efficacy. EPCs from male rats were isolated and labeled with silica‐coated magnetic iron oxide nanoparticles to form magnetized EPCs. Then, the proliferation, migration, vascularization, and cytophenotypic markers of magnetized EPCs were analyzed. Afterward, the magnetized EPCs (1 × 106) were transplanted into a female rat model of MI via the tail vein at 7 days after MI with or without the guidance of an external magnet above the infarct area. Cardiac function, myocardial fibrosis, and the apoptosis of cardiomyocytes were observed at 4 weeks after treatment. In addition, EPC retention and the angiogenesis of ischemic myocardium were evaluated. Labeling with magnetic nanoparticles exhibited minimal influence to the biological functions of EPCs. The transplantation of magnetized EPCs guided by an external magnet significantly improved the cardiac function, decreased infarction size, and reduced myocardial apoptosis in MI rats. Moreover, enhanced aggregations of magnetized EPCs in the infarcted border zone were observed in rats with external magnet‐guided transplantation, accompanied by the significantly increased density of microvessels and upregulated the expression of proangiogenic factors, when compared with non‐external‐magnet‐guided rats. The magnetic field‐guided transplantation of magnetized EPCs was associated with the enhanced aggregation of EPCs in the infarcted border zone, thereby improving the therapeutic efficacy of MI.

COA-Cl (2-Cl-C.OXT-A) can promote coronary collateral development following acute myocardial infarction in mice

2-Cl-C.OXT-A (COA-Cl) is a novel nucleic acid analogue that promotes tube-forming activity of human umbilical vein endothelial cells (HUVEC) through vascular endothelial growth factor (VEGF). The development of coronary collateral circulation is critical to rescue the ischemic myocardium and to prevent subsequent irreversible ischemic injury. We evaluated whether COA-Cl can promote angiogenesis in ischemic tissue, reduce infarct size and preserve cardiac contractility in vivo. Mice received COA-Cl or placebo daily for three days after myocardial infarction (MI) by coronary ligation. The degree of angiogenesis in ischemic myocardium was assessed by staining endothelial cells and vascular smooth muscle cells, and measuring infarct size/area-at-risk. In mice treated with COA-Cl, enhanced angiogenesis and smaller infarct size were recognized, even given a similar area at risk. We observed increases in the protein expression levels of VEGF and in the protein phosphorylation level of eNOS. In addition, the heart weight to body weight ratio and myocardial fibrosis in COA-Cl mice were decreased on Day 7. Administration of COA-Cl after MI promotes angiogenesis, which is associated with reduced infarct size and attenuated cardiac remodeling. This may help to prevent heart failure due to cardiac dysfunction after MI.

Combination of Ligusticum Chuanxiong and Radix Paeonia Promotes Angiogenesis in Ischemic Myocardium through Notch Signalling and Mobilization of Stem Cells.

Objective To study the cardioprotective mechanism by which the combination of Chuanxiong (CX) and Chishao (CS) promotes angiogenesis. Methods Myocardial infarction (MI) mouse models were induced by ligation of the left anterior descending coronary artery. The effects on cardiac function were evaluated in the perindopril tert-butylamine group (PB group) (3 mg/kg/d), CX group (55 mg/kg/d), CS group (55 mg/kg/d), and CX and CS combination (CX-CS) group (27.5 mg/kg/d CX plus 27.5 mg/kg/d CS). RO4929097, an inhibitor of Notch γ secretase, was used (10 mg/kg/d) to explore the role of Notch signalling in the CX-CS-induced promotion of angiogenesis in the myocardial infarcted border zone (IBZ). The left ventricular ejection fraction (LVEF) and percentage of MI area were evaluated with animal ultrasound and Masson staining. The average optical densities (AODs) of CD31 and vWF in the myocardial IBZ were detected by immunofluorescence. Angiogenesis-related proteins including hypoxia-inducible factor 1-alpha (HIF-1α), fibroblast growth factor receptor 1 (FGFR-1), Notch1 and Notch intracellular domain (NICD), and stem cell mobilization-related proteins including stromal cell-derived factor 1 (SDF-1), C-X-C chemokine receptor type 4 (CXCR-4), and cardiotrophin1 were detected by western blot analysis. Results Compared with the model group, the CX-CS and PB groups both showed markedly improved LVEF and decreased percentage of MI area after 21 days of treatment. Although the CX group and CS group showed increased LVEF and decreased MI areas compared with the model group, the difference was not significant. The AOD of CD31 in the IBZ in both the model and the CX-CS-I group was markedly reduced compared with that in the sham group. CX-CS significantly increased the CD31 AOD in the IBZ and decreased the AODs of CD31 and vWF in the infarct zone compared with those in the model group. The expression of HIF-1α in both the model group and the CX-CS group was higher than that in the sham group. Compared with the model group, the expression of FGFR-1, SDF-1, cardiotrophin1, Notch1, and NICD was increased in the CX-CS group. Notch1 and NICD expression in the CX-CS-I group was reduced compared with that in the CX-CS group. Conclusions The combination of CX and CS protected cardiomyocytes in the IBZ better than CX or CS alone. The mechanism by which CX-CS protects ischemic myocardium may be related to the proangiogenesis effect of CX-CS exerted through Notch signalling and the mobilization of stem cells to the IBZ.

Transplantation of Cardiac Mesenchymal Stem Cell-Derived Exosomes Promotes Repair in Ischemic Myocardium.

Our previous study demonstrated the beneficial effects of exosomes secreted by cardiac mesenchymal stem cells (C-MSC-Exo) in protecting acute ischemic myocardium from reperfusion injury. Here, we investigated the effect of exosomes from C-MSC on angiogenesis in ischemic myocardium. We intramyocardially injected C-MSC-Exo or PBS into the infarct border zone after induction of acute mouse myocardial infarction (MI). We observed that hearts treated with C-MSC-Exo exhibit improved cardiac function compared to control hearts treated with PBS at one month after MI. Capillary density and Ki67-postive cells were significantly higher following treatment with C-MSC-Exo as compared with PBS. Moreover, C-MSC-Exo treatment increased cardiomyocyte proliferation in infarcted hearts. In conclusion, intramyocardial delivery of C-MSC-Exo after myocardial infarction enhances cardiac angiogenesis, promotes cardiomyocyte proliferation, and preserves heart function. C-MSC-Exo constitute a novel form of cell-free therapy for cardiac repair.

Myocardial infarction stabilization by cell-based expression of controlled Vascular Endothelial Growth Factor levels.

Vascular Endothelial Growth Factor (VEGF) can induce normal or aberrant angiogenesis depending on the amount secreted in the microenvironment around each cell. Towards a possible clinical translation, we developed a Fluorescence Activated Cell Sorting (FACS)‐based technique to rapidly purify transduced progenitors that homogeneously express a desired specific VEGF level from heterogeneous primary populations. Here, we sought to induce safe and functional angiogenesis in ischaemic myocardium by cell‐based expression of controlled VEGF levels. Human adipose stromal cells (ASC) were transduced with retroviral vectors and FACS purified to generate two populations producing similar total VEGF doses, but with different distributions: one with cells homogeneously producing a specific VEGF level (SPEC), and one with cells heterogeneously producing widespread VEGF levels (ALL), but with an average similar to that of the SPEC population. A total of 70 nude rats underwent myocardial infarction by coronary artery ligation and 2 weeks later VEGF‐expressing or control cells, or saline were injected at the infarction border. Four weeks later, ventricular ejection fraction was significantly worsened with all treatments except for SPEC cells. Further, only SPEC cells significantly increased the density of homogeneously normal and mature microvascular networks. This was accompanied by a positive remodelling effect, with significantly reduced fibrosis in the infarcted area. We conclude that controlled homogeneous VEGF delivery by FACS‐purified transduced ASC is a promising strategy to achieve safe and functional angiogenesis in myocardial ischaemia.

Danshensu accelerates angiogenesis after myocardial infarction in rats and promotes the functions of endothelial progenitor cells through SDF-1α/CXCR4 axis

The present study was performed to investigate the potential role of Danshensu in therapeutic angiogenesis in ischemic myocardium and endothelial progenitor cells (EPCs) function. The rat model of myocardial infarction (MI) injury was induced by left anterior descending coronary artery ligation for 14 days. Danshensu significantly alleviated myocardial ischemia injury by ameliorating left ventricular function and reducing infarct size. Furthermore, Danshensu potentiated post-ischemia neovascularization as evidenced by increased microvessel density in infarction boundary zone, as well as the expression of marker proteins vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Moreover, Danshensu notably promoted stromal cell-derived factor-1α (SDF-1α) level in plasma and C-X-C chemokine receptor type 4 (CXCR4) expression in peri-infarction myocardium, and AMD3100 (CXCR4 antagonist) could reverse the angiogenic and cardioprotective effects of Danshensu. For in vitro study, EPCs were isolated from bone marrow of rats. On the one hand, Danshensu provided significant cytoprotection against hypoxia insult by boosting EPCs viability and inhibiting apoptosis, and upregulated Akt phosphorylation. On the other hand, Danshensu enhanced proangiogenic functions of EPCs on cell migration and tube formation, and increased SDF-1α and CXCR4 expression. Likewise, the cytoprotection and proangiogenic functions of Danshensu on EPCs were partly negated by LY294002 (PI3K antagonist) and CXCR4 siRNA, respectively. Taken together, our results suggested that the cardioprotection of Danshensu in MI rats may be related to promoting myocardial neovascularization. The possible mechanisms may involve improving EPCs survival in hypoxia condition through Akt phosphorylation, and accelerating EPCs proangiogenic functions through SDF-1α/CXCR4 axis.

Low-energy Shock Wave Treatment Induces Angiogenesis in Ischemic Muscle by Stimulation of Toll-like Receptor 3 Signaling

Objectives: Low energy shock waves (SW) have been shown to induce angiogenesis in ischemic myocardium. The mechanism translating the physical stimulus to a biological signal is unknown. Toll-like receptor (TLR)-3 is activated by RNA binding. It plays a key role in inflammation and angiogenesis. We therefore hypothesized that SW cause cellular cavitation, thus liberating cytoplasmic mRNA that activates TLR-3 as does the specific agonist Poly I:C. Effects are suppressed in TLR-3 silenced cells and in TLR-3 knock out mice.

Cardioprotective effects on ischemic myocardium induced by SVVYGLR peptide via its angiogenic-promoting activity

In ischemic heart disease, cardiomyocyte death results from both necrosis and apoptosis, and the area of infarction gradually increases. The induction of therapeutic angiogenesis is a promising therapy for this condition. In the present study, we investigated the actions of SV peptide—a 7-amino-acid sequence with angiogenic properties derived from osteopontin in the extracellular matrix—in a rat model of myocardial ischemia. We examined the angiogenesis activity of SV ex vivo using ring assay and dorsal air sac assay and in ischemic myocardium. We also conducted histological evaluations of left-ventricle remodeling and evaluated left ventricular (LV) function by echocardiography. The SV peptide had strong angiogenic effects both ex vivo and in vivo. In the experimental rats, this peptide stimulated the formation of new blood vessels in ischemic myocardium. Histological evaluation of the left ventricle in the SV-treated group showed a significant decrease in the size of the infarction, rate of myocardial fibrosis, and cardiomyocyte hypertrophy. Moreover, administration of the SV peptide significantly improved LV function. These results indicate that SV peptide induced angiogenesis in ischemic myocardium and improved cardiac function. Thus, it could serve as a potentially useful new therapy for ischemic heart disease.

Vasculogenesis and Angiogenesis in VEGF Receptor-1 Deficient Mice.

Vascular endothelial growth factor receptor-1 (VEGFR-1)/Flt-1 is a transmembrane tyrosine kinase receptor for VEGF-A, VEGF-B, and placental growth factor (PlGF). VEGFR-1 is an enigmatic molecule whose precise role in postnatal angiogenesis remains controversial. Although many postnatal and adult studies have been performed by manipulating VEGFR-1 ligands, including competitive binding by truncated VEGFR-1 protein, neutralization by antibodies, or specific ligand overexpression or knockout, much less is known at the level of the receptor per se, especially in vivo. Perplexingly, while VEGFR-1 negatively regulates endothelial cell differentiation during development, it has been implied in promoting angiogenesis under certain conditions in adult tissues, especially in tumors and ischemic tissues. Additionally, it is unclear how VEGFR-1 is involved in vascular maturation and maintenance of vascular quiescence in adult tissues. To facilitate further investigation, we generated a conditional knockout mouse line for VEGFR-1 and characterized angiogenesis in postnatal and adult mice, including angiogenesis in ischemic myocardium. These methods are briefly outlined in this chapter. We also discuss these findings in the context of the interplay between VEGF family members and their receptors, and summarize various mouse models in the VEGF pathway.

Abstract 15272: Wnt11 Delivered in Mesenchymal Stem Cell-Derived Media Promotes Angiogenesis Through Non-canonical Wnt-PKC-JNK Pathway

Background: Wnt11 is involved in many developmental and cellular processes. We have previously reported that Wnt11 overexpressing (MSC Wnt11 ) significantly improved cardiac function and increased angiogenesis. Here, we aimed to elucidate how Wnt11 delivered by MSC increase blood vessel formation. Methods and Results: An acute myocardial infarction (MI) model in SD rats was developed by ligation of the left anterior descending (LAD) coronary artery. Conditioned medium (CdM) collected from MSC Wnt11 (CdM Wnt11 ) was injected immediately into the ischemic border area. Rats treated with CdM Wnt11 showed a significantly improved cardiac function. Furthermore, CdM Wnt11 increased blood vessel density and regional blood flow in ischemic myocardium. Fluorescent immunostaining showed that both vWF-positive single endothelial cell numbers and microvessel numbers were significantly increased in CdM Wnt11 treated myocardium. In vitro studies showed that CdM Wnt11 significantly increased number and length of capillary-like structure (CLS) formation and promoted human umbilical vein endothelial cells (HUVECs) migration. These results were confirmed by directly treating HUVECs with recombinant Wnt11 proteins (5μg/ml) or over-expressing Wnt11 in HUVECs (H Wnt11 ). However, these effects could be abolished by using a Wnt11 neutralizing antibody. Immunostaining and Western blotting results revealed upregulation of p-pan-PKC and p-JNK in H Wnt11 . Real-time PCR analysis indicated that the expression of novel PKCs including PKCδ, PKCε, PKCη, and PKCθ was significantly upregulated in H Wnt11 . Moreover, the effect of Wnt11 on CLT formation and HUVECs migration were abrogated when the JNK inhibitor SP600125 at 5 μM and the PKC inhibitor Calphostin-C at 0.1 μM were added to the culture system. Conclusion: Our data suggests, for the first time, that Wnt11 in MSC-derived media improved cardiac function and promoted angiogenesis in ischemic myocardium. The effect of Wnt11 in angiogenesis is mediated by novel PKCs, with JNK as its downstream mediator.

Abstract 346: Involvement of Non-canonical Wnt-PKC-JNK Pathway in Angiogenesis Enhanced by MSC Overexpressing Wnt11

Wnt11 is a key regulator of cardiac muscle cell proliferation and differentiation during heart development. Here, we hypothesized that preprogramming mesenchymal stem cells (MSC) with Wnt11 enhanced angiogenesis in ischemic myocardium. Methods and Results: An acute myocardial infarction model in SD rats was developed by ligation of the left anterior descending (LAD) coronary artery. Rat bone marrow derived MSC were transduced with Wnt11 gene (MSC-Wnt11) and transplanted into ischemic border area. The animals treated with MSC-Wnt11 showed a significantly improved cardiac function. Furthermore, fluorescent microsphere and histological studies revealed an increased blood flow and blood vessel density in MSC-Wnt11 transplanted animals. In vitro studies using conditioned medium (CdM) obtained from MSC-Wnt11 (CdM-Wnt11) showed an increased the length and number of capillary-like structure (CLS) formation and promoted migration of human umbilical vein endothelial cells (HUVECs), which was similar to that HUVECs were directly treated with recombinant Wnt11 proteins. However, these effects could be abolished by using a Wnt11 neutralizing antibody. Real-time PCR analysis indicated that the expression of novel PKCs including PKCδ, PKCε, PKCη, and PKCθ was significantly upregulated in HUVECs transduced with Wnt11 (H-Wnt11). Immunostaining and western blotting results showed that the protein levels of p-pan-PKC and p-JNK as well as VEGFA in H-Wnt11 were significantly higher than that in control HUVECs. However, no difference was detected in the expression of either p-p38 or p-ERK between H-Wnt11 and its control. Furthermore, the enhanced CLS formation and migration of HUVECs mediated by CdM-Wnt11 were partially abolished by using JNK inhibitor, SP600125 (5 μM) and PKC inhibitor, Calphostin-C (0.1 μM). Conclusion: Our results demonstrated, for the first time, that Wnt11 delivered by MSC improved cardiac function and promotes angiogenesis in ischemic myocardium, which may be associated with the activation of the non-canonical Wnt-PKC-JNK pathway.

Abstract 18112: Low-Energy Shock Wave Treatment Induces Angiogenesis in Ischemic Muscle by Stimulation of Toll-Like Receptor 3 Signaling

Introduction: Low energy shock waves (SW) have been shown to induce angiogenesis in ischemic myocardium. The mechanism translating the physical stimulus to a biological signal is unknown. Toll-like receptor (TLR)-3 is activated by RNA binding. It plays a key role in inflammation and angiogenesis. Hypothesis: SW cause cellular cavitation, thus liberating cytoplasmic mRNA that activates TLR-3 as does the specific agonist Poly I:C. Effects are suppressed in TLR-3 silenced cells and in TLR-3 knock out mice. Methods: The effect of SW was tested in human umbilical vein endothelial cells (HUVECs): untreated (control) vs. SW treated (SW group) vs. treated with 200 μg/ml Poly I:C (agonist). TLR-3 gene silencing was done with siRNA. Hind limb ischemia was performed in wild type and TLR-3 kock-out mice. Expression of mRNA and proteins of the TLR-3 signaling pathway as well as typical angiogenic genes and proteins were measured. Laser doppler perfusion imaging and necrosis score were assessed for clinical outcome evaluation (n=6). Results: Shock wave treatment of HUVECs shows increase of mRNA expression (% of control) as does Poly I:C after 2 hours: TLR-3 (SW group 123.8 ± 8.0 and agonist group 237.7 ± 14.1, p&lt;0.0001), Tie-2 (SW group 154.3 ± 20.0 and agonist 125.7 ± 12.3, p&lt;0.008). TLR-3 gene silencing in SW treated HUVECs causes loss of response for TLR-3 mRNA (107.0 ± 13.3) as compared to SW group (378.3 ± 14.2) or agonist (1261 ± 72.1), both p&lt;0.0001. SW treated TLR-3 knock-out mice showed no improvement of perfusion ratio 4 weeks after hind limb ischemia (0.52 ± 0.07 vs. 0.53 ± 0.02 controls, p&gt;0.05), whereas SW treated wild type animals improved significantly (0.78 ± 0.03 vs. 0.48 ± 0.08 controls, p=0.015). Pro-angiogenic genes and proteins were up-regulated significantly. All known TLR-3 signaling pathways were involved as shown by significant increase of key proteins Trif, TRAF6 and IRF3. Conclusion: Low energy shock waves induce angiogenesis in ischemic muscle by stimulation of Toll-like receptor 3 signaling in endothelial cells. Effects are suppressed in TLR-3 silenced cells and in TLR-3 knock-out mice.

Ketanserin improves cardiac performance after myocardial infarction in spontaneously hypertensive rats partially through restoration of baroreflex function

Baroreflex dysfunction is associated with a higher rate of sudden death after myocardial infarction (MI). Ketanserin enhances baroreflex function in rats. The present work was designed to examine whether ketanserin improves the post-MI cardiac function and to explore the possible mechanism involved. Spontaneously hypertensive rats (SHR) were treated with ketanserin (0.3 mg·kg(-1)·d(-1)). Two weeks later, blood pressure and baroreflex function were measured, followed by a ligation of the left coronary artery. The expressions of vesicular acetylcholine transporter (VAChT) and α7 nicotinic acetylcholine receptor (α7-nAChR) in ischemic myocardium, angiogenesis, cardiac function, and left ventricular (LV) remodeling were evaluated subsequently. Ketanserin significantly improved baroreflex sensitivity (0.62±0.21 vs 0.34±0.12 ms/mmHg, P<0.01) and vagal tonic activity (heart rate changes in response to atropine, 54.8±16.2 vs 37.6±13.4 bpm, P<0.01) without affecting the blood pressure or basic heart rate in SHR. Treatment of SHR with ketanserin prominently improved cardiac function and alleviated LV remodeling, as reflected by increases in the ejection fraction, fractional shortening, and LV systolic pressure as well as decreases in LV internal diameter and LV relative weight. The capillary density, vascular endothelial growth factor expression, and blood flow in the ischemic myocardium were significantly higher in the ketanserin-treated group. In addition, ketanserin markedly increased the expression of VAChT and α7-nAChR in ischemic myocardium. Ketanserin improved post-MI cardiac function and angiogenesis in ischemic myocardium. The findings provide a mechanistic basis for restoring baroreflex function using ketanserin in the treatment of MI.