Shu-Hua Xu

Endostatin and angiostatin are increased in diabetic patients with coronary artery disease and associated with impaired coronary collateral formation

Coronary artery disease (CAD) is the leading cause of mortality in diabetic patients. Because of the diffuse nature of their disease, diabetic patients may be at risk for incomplete revascularization, highlighting a potential role for proangiogenic therapy in this group. This study investigates molecular mechanisms of angiogenesis in diabetic patients. Myocardial tissue was harvested from patients undergoing coronary artery bypass grafting [nondiabetic (ND) 11, type 2 diabetic (DM) 10]. Expression of angiostatin, endostatin, their precursors (plasminogen and collagen XVIII, respectively), enzymes leading to their production [matrix metalloprotease (MMP)-2 and -9, cathepsin L], and an inhibitor of MMPs (tissue inhibitor of metalloproteinase) was assessed with Western blotting. MMP activity was assessed. Coronary collateralization was graded by Rentrop scoring of angiograms. Plasminogen and collagen XVIII expression were similar between groups. Angiostatin expression trended to increase 1.24-fold (P = 0.07), and endostatin expression increased 2.02-fold in DM patients relative to ND (P = 0.02). MMP-9 expression was no different between groups, whereas MMP-2 expression decreased 1.8-fold in diabetics (P = 0.003). MMP-2 and -9 activity decreased 1.33-fold (P = 0.03) and 1.57-fold (P = 0.04), respectively, in diabetic patients. Cathepsin L expression was 1.38-fold higher in diabetic patients (P = 0.02). Coronary collateralization scores were ND 2.1 +/- 0.37 vs. DM 1.0 +/- 0.4 (P = 0.05). Myocardial endostatin expression correlated strongly with the percentage of hemoglobin A(1c) (r = 0.742, P = 0.0001). Myocardial expression of angiostatin and endostatin demonstrated significant negative linear correlations with coronary collateralization (angiostatin r = -0.531, P = 0.035, endostatin r = -0.794, P = 0.0002). Diabetic patients with CAD exhibit increased levels of the antiangiogenic proteins angiostatin and endostatin and differential regulation of the enzymes governing their production relative to ND patients. Myocardial levels of these proteins show significant correlation to coronary collateralization. These findings offer potential new therapeutic targets for enhancing proangiogenic therapy and insight into the angiogenic impairments seen in diabetes.

Role of Stromal-Derived Factor-1α in the Induction of Circulating CD34+CXCR4+ Progenitor Cells After Cardiac Surgery

Background— Cardioplegia and cardiopulmonary bypass (CP/CPB) leads to an increase in circulating progenitor cells. The role of stromal-derived factor-1&agr; (SDF-1&agr;), a key regulator of progenitor cell mobilization, and other cytokines in this process is not clear. Methods and Results— Peripheral blood (n=24), atrial and skeletal tissue (n=6) samples were taken from patients undergoing CP/CPB before (pre-CP/CPB), 4 hours (post-CP/CPB), and 4 days (POD4) after CP/CPB. The number of circulating CD34+CXCR4+ cells increased post-CP/CPB (442±53 versus 286±27; P=0.04 versus pre-CP/CPB), but not at POD4 (382±50; P=0.28 versus pre-CP/CPB). Plasma levels of SDF-1&agr; increased post-CP/CPB as compared with pre-CP/CPB (3325±325 versus 2911±165 pg/mL; P=0.046) but returned to baseline at POD4 (2838±224 pg/mL; P=0.90). Plasma levels of vascular endothelial growth factor were similar post-CP/CPB (P=0.90 versus pre-CP/CPB) but increased at POD4 (220±40 pg/mL versus 134±26 pg/mL; P=0.04 versus pre-CP/CPB). Serum levels of granulocyte-colony stimulating factor (G-CSF) increased early after CP/CPB as compared with pre-CP/CPB (265.0±41.7 versus 11.1±1.1 pg/mL; P<0.001) and returned to baseline at POD4 (P=0.84 versus pre-CP/CPB). The circulating CD34+CXCR4+ cells were positively correlated with plasma levels of SDF-1&agr; early after CP/CPB (r=0.56, P<0.01), but not at other times. Protein expression of SDF-1&agr; was elevated in the atrial myocardium after CP/CPB (9.4-fold; P=0.03). Conclusions— Exposure to CP/CPB leads to an increase in circulating CD34+CXCR4+ progenitor cells, which is associated with increased myocardial SDF-1&agr; expression. The numbers of CD34+CXCR4+ progenitor cells positively correlate with the plasma levels of SDF-1&agr; post-CP/CPB, suggesting an important role of SDF-1&agr; in progenitor cell mobilization.

Effects of neuropeptide Y on collateral development in a swine model of chronic myocardial ischemia

We investigated the role of neuropeptide Y (NPY), abundant in the myocardial sympathetic nervous system and endothelial cells, in angiogenesis during chronic myocardial ischemia. Adult male Yorkshire swine underwent ameroid constrictor placement on the proximal left circumflex coronary artery. After 3 weeks, an osmotic pump was placed to deliver either placebo (control, n=8) or NPY(3-36) (NPY, n=8) to the collateral dependent region. Five weeks after pump placement, after cardiac catheterization and hemodynamic assessment, the heart was harvested for analysis. NPY treated animals demonstrated increased mean arterial pressures and improved left ventricular function (+dP/dt). Cardiac catheterization demonstrated a significant increase in the blush score in the NPY group (p<0.001). Blood flow to the ischemic myocardium was not different between groups at rest or during ventricular pacing. Immunohistochemical double staining for CD-31 and smooth muscle actin demonstrated an increase in capillary and arteriole formation in NPY treated animals (p=0.02 and p<0.001). Immunoblotting showed a significant upregulation of DPPIV (p=0.009) and NPY receptors 1 (p=0.008), 2 (p=0.02) and 5 (p=0.03) in the NPY treated group. Additionally, there was significant upregulation of VEGF (p=0.04), eNOS (p=0.014), phospho-eNOS (ser1177) (p=0.02), and PDGF (p<0.001) in NPY treated group. The anti-angiogenic factors endostatin and angiostatin were significantly decreased in NPY treated animals (endostatin, p=0.03; angiostatin, p=0.04). Exogenous NPY(3-36) resulted in improved myocardial function and increased angiogenesis and arteriogenesis by stimulating growth factor, pro-angiogenic receptor upregulation, and decreasing anti-angiogenic expression, but did not increase blood flow to the ischemic myocardium. NPY may act as a good adjunct to primary agents of therapeutic angiogenesis.

Comparison of vascular endothelial growth factor and fibroblast growth factor-2 in a swine model of endothelial dysfunction

OBJECTIVE Growth factor based angiogenesis, with or without cell therapy, is a promising therapeutic modality for patients with coronary artery disease. We compared the relative efficacies of surgically delivered vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) in a swine model of hypercholesterolemia-induced endothelial dysfunction which captures many of the pathophysiologic abnormalities of human coronary disease. METHODS Yucatan mini-swine (20-30 kg), fed a high cholesterol diet (total 20 weeks), underwent circumflex ameroid placement to create chronic myocardial ischemia, followed three weeks later by perivascular administration of VEGF (2 microg; n=6), FGF-2 (100 microg; n=6), or placebo (n=7) in the ischemic territory. Normocholesterolemic animals (n=7) served as controls. Four weeks later, endothelial function, collateral-dependent perfusion, as well as myocardial protein and mRNA levels of angiogenic mediators were assessed. RESULTS Endothelial dysfunction was observed in all hypercholesterolemic animals as impaired microvessel relaxation in response to adenosine diphosphate and VEGF. VEGF administration improved baseline-adjusted collateral-dependent perfusion at rest (-0.03+/-0.05 vs -0.12+/-0.04, VEGF vs placebo, p=0.09), but FGF-2 delivery caused a significantly greater improvement in perfusion compared to either group (+0.15+/-0.03, p<0.05 vs HC-placebo and HC-VEGF) at rest. Molecular analysis revealed increased eNOS expression (135%+/-8%, p=0.03 vs placebo) in all growth factor treated animals and increased expression of FGF-2 receptor, FGFR1 (65+/-26%, p=0.04 vs placebo), in FGF-2 treated animals. No significant changes were demonstrated in other angiogenic mediators including Akt, Syndecan-4. CONCLUSIONS In the setting of hypercholesterolemic endothelial dysfunction, FGF-2 is more effective than VEGF at enhancing collateral-dependent perfusion and thus, may be a better candidate than VEGF for angiogenic therapy in patients with end-stage CAD.

Effects of l-Arginine on Fibroblast Growth Factor 2–Induced Angiogenesis in a Model of Endothelial Dysfunction

Background—Nitric oxide availability, which is decreased in advanced coronary artery disease associated with endothelial dysfunction, is an important mediator of fibroblast growth factor-2 (FGF-2)–induced angiogenesis. This could explain the disappointing results of FGF-2 therapy in clinical trials despite promising preclinical studies. We examined the influence of l-arginine supplementation to FGF-2 therapy on myocardial microvascular reactivity and perfusion in a porcine model of endothelial dysfunction. Methods and Results—Eighteen pigs were fed either a normal (NORM, n=6) or high cholesterol diet, with (HICHOL-ARG, n=6) or without (HICHOL, n=6) l-arginine. All pigs underwent ameroid placement on the circumflex artery and 3 weeks later received surgical FGF-2 treatment. Four weeks after treatment, endothelial-dependent coronary microvascular responses and lateral myocardial perfusion were assessed. Endothelial cell density was determined by immunohistochemistry. FGF-2, fibroblast growth receptor-1, endothelial-derived nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and syndecan-4 levels were determined by immunoblotting. Pigs from the HICHOL group showed endothelial dysfunction in the circumflex territory, which was normalized by l-arginine supplementation. FGF-2 treatment was ineffective in the HICHOL group (circumflex/left anterior descending blood flow ratios: 1.01 (rest) and 1.01 (pace), after and before treatment). Addition of l-arginine improved myocardial perfusion in response to FGF-2 at rest (ratio 1.13, P=0.02 versus HICHOL) but not during pacing (ratio 0.94, P=NS), and was associated with increased protein levels of iNOS and eNOS. Conclusion—l-arginine supplementation can partially restore the normal response to endothelium-dependent vasorelaxants and myocardial perfusion in response to FGF-2 treatment in a swine model of hypercholesterolemia-induced endothelial dysfunction. These findings suggest a role for l-arginine in combination with FGF-2 therapy for end-stage coronary artery disease.

Aprotinin Preserves Cellular Junctions and Reduces Myocardial Edema After Regional Ischemia and Cardioplegic Arrest

Background—Cardiac surgery with cardiopulmonary bypass (CPB) and cardioplegic arrest has been associated with myocardial edema attributable to vascular permeability, which is regulated in part by thrombin-induced alterations in cellular junctions. Aprotinin has been demonstrated to prevent activation of the thrombin protease-activated receptor, and we hypothesized that aprotinin preserves myocardial cellular junctions and prevents myocardial edema in a porcine model of regional ischemia and cardioplegic arrest. Methods and Results—Fourteen pigs were subjected to 30 minutes of regional ischemia, followed by 60 minutes of CPB, with 45 minutes of crystalloid cardioplegia, then 90 minutes of post-CPB reperfusion. The treatment group (n=7) was administered aprotinin (40 000 kallikrein inhibitor units [KIU]/kg loading dose, 40 000 KIU/kg pump prime, and 10 000 KIU/kg per hour continuous infusion). Control animals (n=7) received normal saline. Myocardial vascular endothelial (VE)-cadherin, &bgr;-catenin and &ggr;-catenin, and associated mitogen-activated protein kinase (MAPK) pathways were assessed by immunoblot and immunoprecipitation. Histologic analysis of the cellular junctions was done by immunofluorescence. Myocardial tissue water content was measured. VE-cadherin, &bgr;-catenin, and &ggr;-catenin levels were significantly greater in the aprotinin group (all P<0.05). Immunfluorescence confirmed that aprotinin prevented loss of coronary endothelial adherens junction continuity. Aprotinin reduced tyrosine phosphorylation in myocardial tissue sections. Phospho-p38 activity was ≈30% lower in the aprotinin group (P=0.007). The aprotinin group demonstrated decreased myocardial tissue water content (81.2±0.5% versus 83.5±0.3%; P=0.01) and reduced intravenous fluid requirements (2.9±0.2 L versus 4.0±0.4 L; P=0.03). Conclusions—Aprotinin preserves adherens junctions after regional ischemia and cardioplegic arrest through a mechanism potentially involving the p38 MAPK pathway, resulting in preservation of the VE barrier and reduced myocardial tissue edema.

Increased Antiangiogenic Protein Expression in the Skeletal Muscle of Diabetic Swine and Patients

HYPOTHESIS Antiangiogenic protein expression is increased in skeletal muscle in the setting of diabetes. DESIGN, SETTING, AND PARTICIPANTS In animal studies, diabetes was induced in 8 Yucatan miniswine via single alloxan injection at age 8 months, followed by skeletal muscle harvest 15 weeks later. Eight nondiabetic Yucatan miniswine served as controls. In patient studies, skeletal muscle was harvested from 11 nondiabetic patients and 10 patients with type 2 diabetes mellitus undergoing initial elective coronary artery bypass graft surgery. Skeletal muscle samples were analyzed via Western blotting and zymography for protein expression and enzyme activity. The study was performed in an academic medical center. MAIN OUTCOME MEASURES Skeletal muscle expression of plasminogen, collagen XVIII, angiostatin, endostatin, matrix metalloproteinases 2 and 9, and tissue inhibitor of metalloproteinase 2. RESULTS Skeletal muscle expression of plasminogen and collagen XVIII (precursors of angiostatin and endostatin, respectively) remained similar between nondiabetic and diabetic swine and patients. Expression of angiostatin and endostatin was increased 1.70-fold and 1.84-fold, respectively, in diabetic swine relative to control swine. Endostatin expression was increased 1.69-fold in diabetic patients relative to nondiabetic patients. Matrix metalloproteinase 2 expression and activity were significantly increased in the skeletal muscle of diabetic swine and patients. CONCLUSIONS Antiangiogenic protein levels are increased in the skeletal muscle in the setting of diabetes. Angiostatin, endostatin, and matrix metalloproteinases may offer novel therapeutic targets to improve collateral formation in patients with diabetes.

Thrombin Fragment (TP508) Decreases Myocardial Infarction and Apoptosis After Ischemia Reperfusion Injury

BACKGROUND Myocardial ischemia-reperfusion injury may lead to cardiac dysfunction or death. This study investigates the potential efficacy of a novel thrombin fragment (TP508) on myocardial ischemia-reperfusion injury. METHODS Fourteen male Yucatan pigs underwent 60 minutes of mid-left anterior descending coronary artery occlusion followed by 120 minutes of reperfusion. Pigs received either saline vehicle (control, n = 7) or thrombin fragment TP508 (n = 7) as a bolus (0.5 mg/kg) 50 minutes into the ischemic period, followed by continuous intravenous infusion (1.25 mg x kg(-1) x h(-1)) during reperfusion. Myocardial function was monitored throughout the experiments. Monastryl blue/triphenyl tetrazolium chloride staining was utilized to measure the area at risk and infarcted tissue. Apoptosis was assessed by Western blotting and dUTP nick-end labeling (TUNEL) staining. Coronary microvascular reactivity to endothelium-dependent factors (adenosine diphosphate, substance P, A23187) and endothelium-independent factor (sodium nitroprusside) was examined. RESULTS Global and regional left ventricular function was not significantly different between groups. Endothelium-dependent coronary microvascular relaxation was greater in the TP508 group and associated with higher endothelial nitric oxide synthase phosphorylation. Both infarct size and TUNEL staining was significantly decreased in the TP508 group compared with the control group (p < 0.05). Expression of the cell survival proteins B-cell lymphoma 2 (2.2-fold, p < 0.05) and heat shock protein-73 (1.6-fold, p < 0.05) was higher in the TP508 group. Expression of the cell-death-signaling proteins poly adenosine diphosphate-ribose polymerase (1.6-fold, p < 0.05), cleaved poly adenosine diphosphate-ribose polymerase (6.4-fold, p < 0.05), and B-cell lymphoma 2/adenovirus E1B 19 kDa-interacting protein 3 (3.8-fold, p < 0.05) was significantly higher in the TP508 group in the ischemic territory. CONCLUSIONS This study demonstrates that TP508 decreases infarct size, improves endothelial microvascular function, and induces cell-survival signaling in the setting of ischemia-reperfusion injury. Thus, TP508 may be a useful agent to attenuate myocardial reperfusion injury.

Atorvastatin impairs the myocardial angiogenic response to chronic ischemia in normocholesterolemic swine

OBJECTIVE Statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, used routinely in patients with coronary disease, can improve endothelial function but can have biphasic and dose-dependent effects on angiogenesis. In vitro evidence suggests that the proangiogenic effects of statins are linked to activation of Akt, a mediator of endothelial cell survival and an activator of endothelial nitric oxide synthase. We investigated the functional and molecular effects of atorvastatin supplementation on microvascular function and the endogenous angiogenic response to chronic myocardial ischemia in normocholesterolemic swine. METHODS Yucatan miniswine were fed a normal diet with (ATOR, n = 7) or without (control, n = 8) atorvastatin (1.5 mg/kg/d) for 20 weeks. Chronic ischemia was induced by ameroid constrictor placement around the circumflex artery. Myocardial perfusion was assessed at 3 and 7 weeks using isotope-labeled microspheres. In vitro microvessel relaxation responses and myocardial protein expression were evaluated. RESULTS Endothelium-dependent relaxation to adenosine diphosphate and endothelium-independent relaxation to sodium nitroprusside were intact in both groups. The ATOR group demonstrated impaired microvessel relaxation to vascular endothelial growth factor (53% +/- 3% vs 70% +/- 7%, ATOR vs NORM at 10(-10) mol/L, P = .05) and fibroblast growth factor-2 (35% +/- 3% vs 57% +/- 5%, ATOR vs NORM at 10(-10) mol/L, P = .04). Baseline-adjusted myocardial perfusion in the ischemic circumflex territory was significantly reduced in the ATOR group (-0.29 +/- 0.10 mL/min/g vs NORM, P = .009). Phosphorylation of Akt was significantly increased in the ATOR group (+235% +/- 72%, P = .009 vs NORM), as was the myocardial expression of endostatin, an antiangiogenic protein (+51% +/- 9%, P < .001 vs NORM). Expression of vascular endothelial growth factor, Tie-2, fibroblast growth factor receptor-1, and endothelial nitric oxide synthase was similar in both groups. CONCLUSIONS Atorvastatin supplementation is associated with impaired growth factor-mediated microvessel relaxation and a significant reduction in collateral-dependent perfusion. Chronic Akt activation, increased myocardial expression of endostatin, and impaired growth factor signaling may account for the diminished endogenous angiogenic response observed with atorvastatin treatment.

Effects of cyclooxygenase inhibition on cardiovascular function in a hypercholesterolemic swine model of chronic ischemia

The cardiovascular effects of cyclooxygenase (COX) inhibition remain controversial, especially in the setting of cardiovascular comorbidities. We examined the effects of nonselective and selective COX inhibition on cardiovascular function in a hypercholesterolemic swine model of chronic ischemia. Twenty-four intact male Yorkshire swine underwent left circumflex ameroid constrictor placement and were subsequently given either no drug (HCC; n = 8), a nonselective COX inhibitor (440 mg/day naproxen; HCNS; n = 8), or a selective COX-2 inhibitor (200 mg/day celecoxib; HCCX; n = 8). After 7 wk, myocardial functional was measured and myocardium from the nonischemic ventricle and ischemic area-at-risk (AAR) were analyzed. Regional function as measured by segmental shortening was improved in the AAR of HCCX compared with HCC. There was no significant difference in perfusion to the nonischemic ventricle between groups, but myocardial perfusion in the AAR was significantly improved in the HCCX group compared with controls at rest and during pacing. Endothelium-dependent microvessel relaxation was diminished by ischemia in HCC animals, but both naproxen and celecoxib improved vessel relaxation in the AAR compared with controls, and also decreased the vasoconstrictive response to serotonin. Thromboxane levels in the AAR were decreased in both HCNS and HCCX compared with HCC, whereas prostacyclin levels were decreased only in HCNS, corresponding to a decrease in prostacyclin synthase expression. Chronic ischemia increased apoptosis in Troponin T negative cells and intramyocardial fibrosis, both of which were reduced by celecoxib administration in the AAR. Capillary density was decreased in both the HCNS and HCCX groups. Protein oxidative stress was decreased in both HCNS and HCCX, whereas lipid oxidative stress was decreased only in the HCCX group. Thus nonselective and especially selective COX inhibition may have beneficial myocardial effects in the setting of hypercholesterolemia and chronic ischemia. Whether these effects modulate cardiovascular risk in patients taking these drugs remains to be seen, but evidence to date suggests that they do not.