Coronary Microvascular Reactivity Research Articles

Abstract Tu028: NADPH Oxidase Inhibition Antagonizes Endothelial Pro-inflammatory and Pro-oxidant Signaling Resulting in Enhanced Coronary Vasorelaxation in Diabetic Models

Introduction: NADPH oxidase (NOX) overactivation and reactive oxygen species amplification may underlie worse cardiac outcomes in patients with diabetes. Our group has shown impaired endothelium-dependent coronary vasodilation in diabetes, with restored endothelial function following NOX inhibition. Persistently high levels of oxidative stress may be driving this impaired coronary microvascular reactivity by altering small-conductance potassium channel activity. We hypothesized that NADPH oxidase inhibition would be vasoprotective through reductions in pro-oxidant and pro-inflammatory signaling. Methods: Human coronary arterial endothelial cells (HCAECs) were obtained from diabetic (D) and nondiabetic (C) patients (N = 4 per group). HCAECs were cultured in normo- or hyperglycemic conditions, with one hyperglycemic group receiving NADPH oxidase inhibitor apocynin (DT). Molecular signaling was assessed using immunoblotting. Results: Diabetes resulted in significantly increased pro-oxidant markers, including NOX 4 (p = 0.005), phosphorylated endothelial nitric oxide synthase (p-eNOS) (p = 0.007), and eNOS (p = 0.003). Pro-inflammatory Nrf2 was also increased (p = 0.0003), with a trend toward increased NFkB (p = 0.09) in D compared to C. Antioxidant peroxiredoxin (PRDX) 1 was significantly reduced in D compared to C (p = 0.02). NOX inhibition rescued these effects, with reversed trends in NOX 4 (p = 0.008), p-eNOS (p = 0.03), and PRDX 1 (p = 0.02) following apocynin treatment. NOX inhibition resulted in de novo decrease in NOX 1 in DT when compared to D (p = 0.008) and C (p = 0.02), and decrease in phosphorylated NFkB (p-NFkB) when compared to D (p = 0.02). There was a trend toward decreased total NFkB (p = 0.07) and p-NFkB:NFkB ratio (p = 0.06) and increased antioxidant thioredoxin (TXN) 2 (p = 0.05) in diabetic cells following treatment. Antioxidant superoxide dismutase (SOD) 2 (p = 0.01) and TXN 1 (p = 0.02) were increased in D when compared to C, while apocynin treatment reversed these changes (SOD 2, p = 0.0006; TXN 1, p = 0.04). There were no significant differences in NOX 2, SOD 1, catalase, PRDX 2, or PRDX 3 between the groups. Conclusions: Apocynin overall suppressed pro-oxidant and pro-inflammatory signaling in human diabetic coronary endothelial cells, resulting in improved coronary endothelium-dependent vasorelaxation. Cardioprotective strategies that incorporate NADPH oxidase inhibition in the setting of diabetes warrant further investigation.

Abstract 17167: Uncontrolled Hypertension Increases Endothelin-1-Induced Contractile Response of Coronary Microvasculature Both Before and After Cardioplegia and Cardiopulmonary Bypass

Introduction: Hypertensive patients undergoing cardiopulmonary bypass (CPB) have worse clinical outcomes, and coronary microvascular dysfunction may play a role. Endothelin-1 (ET-1), a vasoactive biopeptide, is increased in cardiomyocytes during the hypertrophic response to hypertension and in patients following CPB. Differential effects of hypertension on coronary microvascular reactivity and molecular signaling pathways following CPB have yet to be elucidated. Aims: This study investigated the effects of well-controlled and uncontrolled hypertension on CPB-induced changes in coronary microvascular vasoconstriction to ET-1 and associated signaling using unique in vitro microvascular reactivity studies, RNA deep sequencing, and established molecular methods. Methods: Coronary microvessels (<200um) were harvested from atrial samples from patients with no hypertension (NH), well-controlled hypertension (CH), and uncontrolled hypertension (UH) before and after CPB. In vitro contractile response to ET-1 was assessed using video microscopy. Protein expression and receptor localization were measured using immunoblotting and immunohistochemistry, respectively. Enzyme-linked immunosorbent assay was used to quantify plasma concentration of ET-1. Results: Pre-CPB contractile response of coronary microvessels to ET-1 in UH was increased compared to NH or CH (p <0.05). Post-CPB contractile response was impaired overall (p <0.05). Between groups, contractile response in CH and NH was comparable while response in UH was higher (p <0.05). Plasma ET-1 increased post-CPB (p = 0.18). Total protein levels and localization of ET A R and ET B R were unchanged post-CPB. Conclusions: While CPB overall blunted vasomotor contractile response of coronary microvessels to ET-1, UH was associated with relatively increased vasoconstriction compared to CH and NH. Changes in plasma ET-1, ET A R and ET B R levels or localization did not contribute to these effects.

Abstract 13713: Coronary Endothelial Dysfunction and Microvascular Vasospasm in Patients With Postural Orthostatic Tachycardia Syndrome

Background: Postural Orthostatic Tachycardia Syndrome (POTS) is characterized by autonomic dysfunction-related symptoms such as orthostatic intolerance, palpitations, dizziness, and chest pain. The mechanisms driving chest pain in POTS are unclear. Hypothesis: We hypothesized that coronary endothelial dysfunction (ED) and abnormal microvascular reactivity may be a contributor to chest pain in POTS. Methods: Patients (n=6) were diagnosed with POTS or had significant tachycardia despite medical therapy out of 67 patients in our cohort. All patients reported persistent chest pain and were referred for coronary function testing (CFT) to test macro- and micro-vascular function. CFT was performed to test endothelium-dependent and non-endothelium dependent function using graded doses of intracoronary acetylcholine (ACH), and intravenous adenosine (ADO). Coronary Flow Reserve<2.0 and Index of Microvascular Resistance>25 diagnosed coronary microvascular dysfunction. Coronary artery diameter narrowing to ACH (100 mcg) was used to diagnose ED. Epicardial coronary spasm was defined as >90% constriction to ACH. Microvascular spasm was diagnosed by ACH provoked-chest pain and EKG changes in the absence of epicardial spasm. Results: Mean age was 44.1 ± 15.2 years, 67% were female, and all tolerated CFT without complications. POTS symptoms, baseline medications, and CFT results are shown ( Table ). All had an abnormal response to ACH, indicating ED and microvascular spasm. Non-endothelium function was normal in all patients. Anti-anginal medications were changed based on CFT information, with improvement in chest pain with calcium channel blocker use. Conclusion: In all 6 pts, CP was associated with ED and microvascular spasm. Mechanistic links between ED and POTS need further investigation. Given that ED is a prognostic marker associated with recurrent angina hospitalization and events, investigation of therapies that target ED in POTS may be warranted.

Increased Coronary Contraction to Thromboxane A2 in Cardiac Surgery Patients With Poorly Controlled Hypertension

IntroductionCardioplegia and cardiopulmonary bypass (CP/CPB) alters coronary arteriolar response to thromboxane A2 (TXA2) in patients undergoing cardiac surgery. Comorbidities, including hypertension (HTN), can further alter coronary vasomotor tone. This study investigates the effects of HTN on coronary arteriolar response to TXA2 pre and post-CP/CPB and cardiac surgery. Materials and methodsCoronary arterioles pre and post-CP/CPB were dissected from atrial tissue samples in patients with no HTN (NH, n = 9), well-controlled HTN (WC, n = 12), or uncontrolled HTN (UC, n = 12). In-vitro coronary microvascular reactivity was examined in the presence of TXA2 analog U46619 (10−9-10−4M). Protein expression of TXA2 receptor in the harvested right atrial tissue samples were measured by immunoblotting. ResultsTXA2 analog U46619 induced dose-dependent contractile responses of coronary arterioles in all groups. Pre-CPB contractile responses to U46619 were significantly increased in microvessels in the UC group compared to the NH group (P < 0.05). The pre-CP/CPB contractile responses of coronary arterioles were significantly diminished post-CP/CPB among the three groups (P < 0.05), but there remained an increased contractile response in the microvessels of the UC group compared to the WC and NH groups (P < 0.05). There were no significant differences in U46619-induced vasomotor tone between patients in the NH and WC groups (P > 0.05). There were no differences in expression of TXA2R among groups. ConclusionsPoorly controlled HTN is associated with increased contractile response of coronary arterioles to TXA2. This alteration may contribute to worsened recovery of coronary microvascular function in patients with poorly controlled HTN after CP/CPB and cardiac surgery.

Impaired coronary microvascular reactivity in youth with bipolar disorder.

Cardiovascular disease (CVD) is excessively prevalent and premature in bipolar disorder (BD), even after controlling for traditional cardiovascular risk factors. The increased risk of CVD in BD may be subserved by microvascular dysfunction. We examined coronary microvascular function in relation to youth BD. Participants were 86 youth, ages 13-20 years (n = 39 BD, n = 47 controls). Coronary microvascular reactivity (CMVR) was assessed using quantitative T2 magnetic resonance imaging during a validated breathing-paradigm. Quantitative T2 maps were acquired at baseline, following 60-s of hyperventilation, and every 10-s thereafter during a 40-s breath-hold. Left ventricular structure and function were evaluated based on 12-15 short- and long-axis cardiac-gated cine images. A linear mixed-effects model that controlled for age, sex, and body mass index assessed for between-group differences in CMVR (time-by-group interaction). The breathing-paradigm induced a significant time-related increase in T2 relaxation time for all participants (i.e. CMVR; β = 0.36, p < 0.001). CMVR was significantly lower in BD v. controls (β = -0.11, p = 0.002). Post-hoc analyses found lower T2 relaxation time in BD youth after 20-, 30-, and 40 s of breath-holding (d = 0.48, d = 0.72, d = 0.91, respectively; all pFDR < 0.01). Gross left ventricular structure and function (e.g. mass, ejection fraction) were within normal ranges and did not differ between groups. Youth with BD showed evidence of subclinically impaired coronary microvascular function, despite normal gross cardiac structure and function. These results converge with prior findings in adults with major depressive disorder and post-traumatic stress disorder. Future studies integrating larger samples, prospective follow-up, and blood-based biomarkers are warranted.

Canagliflozin improves coronary microvascular vasodilation and increases absolute blood flow to the myocardium independent of angiogenesis

ObjectivesSodium-glucose cotransporter-2 inhibitor, canagliflozin, improves myocardial perfusion to ischemic territory without accompanying changes in vascular density. We aimed to (1) characterize effects on angiogenic pathways, (2) use multiomics to identify gene expression and metabolite profiles relevant to regulation of myocardial blood flow, and (3) investigate drug effect on coronary microvascular reactivity. MethodsA nondiabetic swine model of chronic myocardial ischemia and nondiabetic rat model were used to study functional and molecular effects of canagliflozin on myocardium and in vitro microvascular reactivity. ResultsCanagliflozin resulted in increased coronary microvascular vasodilation and decreased vasoconstriction (P < .05) without changes in microvascular density (P > .3). Expression of the angiogenic modulator, endostatin, increased (P = .008), along with its precursor, collagen 18 (P < .001), and factors that increase its production, including cathepsin L (P = .004). Endostatin and collagen 18 levels trended toward an inverse correlation with blood flow to ischemic territory at rest. Proangiogenic fibroblast growth factor receptor was increased (P = .03) and matrix metalloproteinase-9 was decreased (P < .001) with canagliflozin treatment. Proangiogenic vascular endothelial growth factor A (P = .13), Tie-2 (P = .10), and Ras (P = .18) were not significantly altered. Gene expression related to the cardiac renin-angiotensin system was significantly decreased. ConclusionsIn chronic myocardial ischemia, canagliflozin increased absolute blood flow to the myocardium without robustly increasing vascular density or proangiogenic signaling. Canagliflozin resulted in altered coronary microvascular reactivity to favor vasodilation, likely through direct effect on vascular smooth muscle. Downregulation of cardiac renin-angiotensin system demonstrated local regulation of perfusion. Video Abstract [Display omitted]

Metabolic regulation of endothelial SK channels and human coronary microvascular function

BackgroundDiabetic (DM) inactivation of small conductance calcium-activated potassium (SK) channels contributes to coronary endothelial dysfunction. However, the mechanisms responsible for this down-regulation of endothelial SK channels are poorly understood. Thus, we hypothesized that the altered metabolic signaling in diabetes regulates endothelial SK channels and human coronary microvascular function. MethodsHuman atrial tissue, coronary arterioles and coronary artery endothelial cells (HCAECs) obtained from DM and non-diabetic (ND) patients (n = 12/group) undergoing cardiac surgery were used to analyze metabolic alterations, endothelial SK channel function, coronary microvascular reactivity and SK gene/protein expression/localization. ResultsThe relaxation response of DM coronary arterioles to the selective SK channel activator SKA-31 and calcium ionophore A23187 was significantly decreased compared to that of ND arterioles (p < 0.05). Diabetes increases the level of NADH and the NADH/NAD+ ratio in human myocardium and HCAECs (p < 0.05). Increase in intracellular NADH (100 μM) in the HCAECs caused a significant decrease in endothelial SK channel currents (p < 0.05), whereas, intracellular application of NAD+ (500 μM) increased the endothelial SK channel currents (p < 0.05). Mitochondrial reactive oxygen species (mROS) of HCAECs and NADPH oxidase (NOX) and PKC protein expression in the human myocardium and coronary microvasculature were increased respectively (p < 0.05). ConclusionsDiabetes is associated with metabolic changes in the human myocardium, coronary microvasculature and HCAECs. Endothelial SK channel function is regulated by the metabolite pyridine nucleotides, NADH and NAD+, suggesting that metabolic regulation of endothelial SK channels may contribute to coronary endothelial dysfunction in the DM patients with diabetes.

Up-regulation of heme oxygenase-1 after infarct initiation reduces mortality, infarct size and left ventricular remodeling: experimental evidence and proof of concept

BackgroundUp-regulation of HO-1 by genetic manipulation or pharmacological pre-treatment has been reported to provide benefits in several animal models of myocardial infarction (MI). However, its efficacy following MI initiation (as in clinical reality) remains to be tested. Therefore, this study investigated whether HO-1 over-expression, by cobalt protoporphyrin (CoPP) administered after LAD ligation, is still able to improve functional and structural changes in left ventricle (LV) in a rat model of 4-week MI.MethodsA total of 144 adult male Wistar rats were subjected to either left anterior coronary artery ligation or sham-operation. The effect of CoPP treatment (5 mg/kg i.p. at the end of the surgical session and, then, once a week for 4 weeks) was evaluated on the basis of survival, electro- and echocardiography, plasma levels of B-type natriuretic peptide (BNP), endothelin-1 and prostaglandin E2, coronary microvascular reactivity, MI size, LV wall thickness and vascularity. Besides, the expression of HO-1 and connexin-43 in different LV territories was assessed by western blot analysis and immunohistochemistry, respectively.ResultsCoPP induced an increased expression of HO-1 protein with >16 h delay. CoPP treatment significantly reduced mortality, MI size, BNP concentration, ECG alterations, LV dysfunction, microvascular constriction, capillary rarefaction and restored connexin-43 expression as compared to untreated MI. These functional and structural changes were paralleled by increased HO-1 expression in all LV territories. HO activity inhibition by tin-mesoporphyrin abolished the differences between CoPP-treated and untreated MI animals.ConclusionsThis is the first report demonstrating the putative role of pharmacological induction of HO-1 following coronary occlusion to benefit infarcted and remote territories, leading to better cardiac function in a 4-week MI outcome.

Impaired coronary microvascular reactivity in women with apical ballooning syndrome (Takotsubo/stress cardiomyopathy)

The pathophysiology of apical ballooning syndrome (ABS) remains to be elucidated. The aim of this study was to evaluate the coronary vascular reactivity of patients who were previously diagnosed with ABS. A total of 228 cases of ABS were prospectively identified, and of these, 10 patients (median age 61 years (IQR 48-75); all females) who underwent coronary vasomotion testing were included in the study. Coronary epicardial and microvascular responses to intracoronary acetylcholine (ACH; % change in diameter and % change in blood flow at doses of 10(-6)-10(-4) mol/l), nitroglycerin (200-300 mg), and adenosine (36-60 µg) were evaluated. The median change in diameter with ACH was -9.3% (IQR -36.4, 3.2) with six patients (60%) demonstrating epicardial coronary constriction. The median increase in peak coronary blood flow in response to ACH was 13.1% (IQR -18.6, 55.0). This was markedly lower than the blood flow response seen in a reference group of 211 women from our laboratory (mean age 60 years) with normal microvascular responses to ACH: 103% (IQR 75, 149). Seven (70%) patients had <50% increase in coronary blood flow indicating abnormal microvascular response to ACH. 70% had either abnormal epicardial or microvascular response to ACH. Median coronary flow reserve was abnormal at 2.2% (IQR 2.0, 3.4; normal >2.5), and 90% had at least one abnormal measure of microvascular vasomotion. The novel observation is that coronary microvascular dysfunction is highly prevalent in patients with ABS. Thus, chronically impaired coronary vascular reactivity, especially involving the microcirculation, may be a central feature of the pathophysiology of ABS.

Preserved endothelium-dependent dilatation of the coronary microvasculature at the early phase of diabetes mellitus despite the increased oxidative stress and depressed cardiac mechanical function ex vivo

BackgroundThere has been accumulating evidence associating diabetes mellitus and cardiovascular dysfunctions. However, most of the studies are focused on the late stages of diabetes and on the function of large arteries. This study aimed at characterizing the effects of the early phase of diabetes mellitus on the cardiac and vascular function with focus on the intact coronary microvasculature and the oxidative stress involved.Materials and methodsZucker diabetic fatty rats and their lean littermates fed with standard diet A04 (Safe) were studied at the 11th week of age. Biochemical parameters such as glucose, insulin and triglycerides levels as well as their oxidative stress status were measured. Their hearts were perfused ex vivo according to Langendorff and their cardiac activity and coronary microvascular reactivity were evaluated.ResultsZucker fatty rats already exhibited a diabetic state at this age as demonstrated by the elevated levels of plasma glucose, insulin, glycated hemoglobin and triglycerides. The ex vivo perfusion of their hearts revealed a decreased cardiac mechanical function and coronary flow. This was accompanied by an increase in the overall oxidative stress of the organs. However, estimation of the active form of endothelial nitric oxide synthase and coronary reactivity indicated a preserved function of the coronary microvessels at this phase of the disease. Diabetes affected also the cardiac membrane phospholipid fatty acid composition by increasing the arachidonic acid and n-3 polyunsaturated fatty acids levels.ConclusionsThe presence of diabetes, even at its beginning, significantly increased the overall oxidative stress of the organs resulting to decreased cardiac mechanical activity ex vivo. However, adaptations were adopted at this early phase of the disease regarding the preserved coronary microvascular reactivity and the associated cardiac phospholipid composition in order to provide a certain protection to the heart.

Decreased coronary microvascular reactivity after cardioplegic arrest in patients with uncontrolled diabetes mellitus

We investigated the effects of cardioplegic arrest and reperfusion on coronary arteriolar responses to endothelium-dependent and -independent vasodilators and associated signaling pathways in uncontrolled diabetic, well controlled diabetic, and case-matched nondiabetic patients undergoing coronary artery bypass graft surgery. Coronary arterioles from harvested right atrial tissues were dissected pre- and post-cardioplegic arrest and reperfusion from uncontrolled diabetic (n=10; hemoglobin A1c=9.3±0.3), well controlled diabetic (n=10; hemoglobin A1c=6.2±0.2), and nondiabetic patients (n=10; hemoglobin A1c=5.1±0.1) undergoing coronary artery bypass graft surgery. The baseline microvascular response to adenosine 5'-diphosphate, substance P, and sodium nitroprusside of arterioles from uncontrolled diabetic patients were decreased compared to the respective response from nondiabetic or well controlled diabetic patients (P<.05). The vasodilatory responses to adenosine 5'-diphosphate and substance P after cardioplegic arrest and reperfusion were significantly decreased in all 3 groups compared to pre-cardioplegic arrest and reperfusion responses (P<.05). However, these decreases were more pronounced in the uncontrolled diabetic group (P<.05). The expression of protein kinase C-α, protein kinase C-β, and protein oxidation in atrial tissues was significantly increased in the uncontrolled diabetic group compared to the nondiabetic or controlled diabetes groups. Uncontrolled diabetes is associated with endothelium-dependent and -independent vascular dysfunction of coronary arterioles. In addition, uncontrolled diabetes worsens the recovery of coronary arteriolar function after cardioplegic arrest and reperfusion. These alterations are associated with an increased expression/activation of protein kinase C-α and protein kinase C-β and enhanced oxidative stress.

NON-INVASIVE CORONARY FLOW RESERVE ASSESSMENT PREDICTS ADVERSE OUTCOME IN WOMEN WITH SUSPECTED OR KNOWN CORONARY ARTERY DISEASE WITHOUT OBSTRUCTIVE CORONARY ARTERY STENOSIS

Evaluation of coronary flow reserve (CFR) is the physiological approach to assess the severity of coronary stenosis and microvascular dysfunction. Impaired CFR occurs frequently in women with suspected or known coronary artery disease (CAD) and recently invasive coronary microvascular reactivity to

Changes in Coronary Microvascular Reactivity After Cardioplegic Arrest in Patients With Uncontrolled Versus Controlled Diabetes

Changes in Coronary Microvascular Reactivity After Cardioplegic Arrest in Patients With Uncontrolled Versus Controlled Diabetes

Effect of Thrombin Fragment (TP508) on Myocardial Ischemia Reperfusion Injury in a Model of Type 1 Diabetes Mellitus

We investigated the efficacy of novel thrombin fragment TP508 on ischemia-reperfusion injury using a porcine model of type 1 diabetes mellitus. Alloxan-induced diabetic male Yucatan swine underwent 60 minutes of mid-left anterior descending coronary artery occlusion, followed by 120 minutes of reperfusion. Fifty minutes into ischemia, animals received either placebo (DM; n=8) or TP508 as a bolus of 1 mg/kg followed by infusion at 2.5 mg/kg per hour (DMT; n=8). Hemodynamic parameters and myocardial function were monitored. Monastryl blue/triphenyl tetrazolium chloride staining was used to assess sizes of the areas at risk and infarction. Coronary microvascular reactivity was measured and expression of cell survival and proapoptotic proteins quantified. Preoperative serum glucose values were similar between groups (309±57 mg/dL in DM versus 318±67 mg/dL in DMT; P=0.92). Infarct size was smaller in the TP508-treated group (5.3±1.9% in DMT versus 19.4±5.6% in DM; P=0.03). There was no statistically significant difference in global or regional left ventricular function between groups. Endothelium-dependent microvessel relaxation was moderately improved in the DMT group (P=0.09), whereas endothelium-independent relaxation was similar between groups. The expression of cell survival proteins Akt, phospho-p38, and mammalian target of rapamycin was higher in the areas at risk of DMT animals compared with DM animals (P<0.05), and expressions of proapoptotic glycogen synthase kinase 3β and caspase 3 were lower in the DMT group (P<0.05). This study demonstrates that, in type 1 diabetic swine, TP508 reduces infarct size after ischemia-reperfusion. Thus, TP508 may offer a novel approach in cardioprotection from ischemia-reperfusion injury in diabetic patients.

Coronary Microvascular Reactivity to Adenosine Predicts Adverse Outcome in Women Evaluated for Suspected Ischemia: Results From the National Heart, Lung and Blood Institute WISE (Women's Ischemia Syndrome Evaluation) Study

We investigated whether coronary microvascular dysfunction predicts major adverse outcomes during follow-up among women with signs and symptoms of ischemia. Altered coronary reactivity occurs frequently in women evaluated for suspected ischemia, and the endothelium-dependent component is linked with adverse outcomes. Possible links between endothelium-independent microvascular coronary reactivity and adverse outcomes remain uncertain. As part of the National Heart, Lung and Blood Institute-sponsored WISE (Women's Ischemia Syndrome Evaluation), we investigated relationships between major adverse outcomes and baseline coronary flow reserve (CFR) after intracoronary adenosine in 189 women referred to evaluate suspected ischemia. At a mean of 5.4 years, we observed significant associations between CFR and major adverse outcomes (death, nonfatal myocardial infarction, nonfatal stroke, or hospital stay for heart failure). An exploratory receiver-operator characteristic analysis identified CFR <2.32 as the best discriminating threshold for adverse outcomes (event rate 26.7%; and >or=2.32 event rate 12.2%; p = 0.01). Lower CFR was associated with increased risk for major adverse outcomes (hazard ratio: 1.16, 95% confidence interval: 1.04 to 1.30; p = 0.009). This held true among the 152 women without obstructive coronary artery disease (CAD) (hazard ratio: 1.20, 95% confidence interval: 1.05 to 1.38; p = 0.008). The CFR significantly improved prediction of adverse outcomes over angiographic CAD severity and other risk conditions. Among women with suspected ischemia and atherosclerosis risk factors, coronary microvascular reactivity to adenosine significantly improves prediction of major adverse outcomes over angiographic CAD severity and CAD risk factors. These findings suggest that coronary microvessels represent novel targets for diagnostic and therapeutic strategies to predict and limit adverse outcomes in women. (Women's Ischemia Syndrome Evaluation [WISE]; NCT00000554).

Improved myocardial perfusion in chronic diabetic mice by the up‐regulation of pLKB1 and AMPK signaling

Previous studies related impaired myocardial microcirculation in diabetes to oxidative stress and endothelial dysfunction. Thus, this study was aimed to determine the effect of up-regulating pAMPK-pAKT signaling on coronary microvascular reactivity in the isolated heart of diabetic mice. We measured coronary resistance in wild-type and streptozotocin (STZ)-treated mice, during perfusion pressure changes. Glucose, insulin, and adiponectin levels in plasma and superoxide formation, NOx levels and heme oxygenase (HO) activity in myocardial tissue were determined. In addition, the expression of HO-1, 3-nitrotyrosine, pLKB1, pAMPK, pAKT, and peNOS proteins in control and diabetic hearts were measured. Coronary response to changes in perfusion pressure diverged from control in a time-dependent manner following STZ administration. The responses observed at 28 weeks of diabetes (the maximum time examined) were mimicked by L-NAME administration to control animals and were associated with a decrease in serum adiponectin and myocardial pLKB1, pAMPK, pAKT, and pGSK-3 expression. Cobalt protoporphyrin treatment to induce HO-1 expression reversed the microvascular reactivity seen in diabetes towards that of controls. Up-regulation of HO-1 was associated with an increase in adiponectin, pLKB1, pAKT, pAMPK, pGSK-3, and peNOS levels and a decrease in myocardial superoxide and 3-nitrotyrosine levels. In the present study we describe the time course of microvascular functional changes during the development of diabetes and the existence of a unique relationship between the levels of serum adiponectin, pLKB1, pAKT, and pAMPK activation in diabetic hearts. The restoration of microvascular function suggests a new therapeutic approach to even advanced cardiac microvascular derangement in diabetes.

Hydrogen sulfide therapy attenuates the inflammatory response in a porcine model of myocardial ischemia/reperfusion injury

Hydrogen sulfide is produced endogenously in response to myocardial ischemia and thought to be cardioprotective. The mechanism underlying this protection has yet to be fully elucidated, but it may be related to sulfide's ability to limit inflammation. This study investigates the cardioprotection provided by exogenous hydrogen sulfide and its potential anti-inflammatory mechanism of action. The mid left anterior descending coronary artery in 14 Yorkshire swine was acutely occluded for 60 minutes, followed by reperfusion for 120 minutes. Controls (n = 7) received placebo, and treatment animals (n = 7) received sulfide 10 minutes before and throughout reperfusion. Hemodynamic and functional measurements were obtained. Evans blue and triphenyl tetrazolium chloride staining identified the area at risk and infarction. Coronary microvascular reactivity was assessed. Tissue was assayed for myeloperoxidase activity and proinflammatory cytokines. Pre-ischemia/reperfusion hemodynamics were similar between groups, whereas post-ischemia/reperfusion mean arterial pressure was reduced by 28.7 +/- 5.0 mm Hg in controls versus 6.7 +/- 6.2 mm Hg in treatment animals (P = .03). Positive first derivative of left ventricular pressure over time was reduced by 1325 +/- 455 mm Hg/s in controls versys 416 +/- 207 mm Hg/s in treatment animals (P = .002). Segmental shortening in the area at risk was better in treatment animals. Infarct size (percent of area at risk) in controls was 41.0% +/- 7.8% versus 21.2% +/- 2.5% in the treated group (P = .036). Tissue levels of interleukin 6, interleukin 8, tumor necrosis factor-alpha, and myeloperoxidase activity decreased in the treatment group. Treated animals demonstrated improved microvascular reactivity. Therapeutic sulfide provides protection in response to ischemia/reperfusion injury, improving myocardial function, reducing infarct size, and improving coronary microvascular reactivity, potentially through its anti-inflammatory properties. Exogenous sulfide may have therapeutic utility in clinical settings in which ischemia/reperfusion injury is encountered.

Effect of thrombin fragment (TP508) on myocardial ischemia-reperfusion injury in hypercholesterolemic pigs

Myocardial ischemia-reperfusion (IR) injury occurs frequently in the setting of hypercholesterolemia. We investigated the potential efficacy of a novel thrombin fragment (TP508) on IR injury in a hypercholesterolemic porcine model. Twenty-one hypercholesterolemic male Yucatan pigs underwent 60 min of mid-left anterior descending coronary artery occlusion followed by 120 min of reperfusion. Pigs received either placebo (control, n = 7) or TP508 in two doses (TP508 low dose, n = 7, as bolus of 0.5 mg/kg 50 min into ischemia and an infusion of 1.25 mg.kg(-1).h(-1) during reperfusion period or TP508 high dose, n = 7, a double dose of TP508 low-dose group). Myocardial function was monitored throughout the experiment. The area at risk and myocardial necrosis were determined by Monastryl blue/triphenyl tetrazolium chloride staining. Apoptosis in the ischemic territory was assessed. Coronary microvascular reactivity to endothelium-dependent and -independent factors was measured. Myocardial necrosis was lower in both TP508-treated groups vs. control (P < 0.05). Regional left ventricular function was improved only in the TP508 high-dose group (P < 0.05). Endothelium-dependent coronary microvascular reactivity was greater in both TP508-treated groups (P < 0.05) vs. control. The expression of proteins favoring cell survival, 90-kDa heat shock protein and phospho-Bad (Ser112) was higher in the TP508 high-dose group (P < 0.05). The expression of the cell death signaling proteins, cleaved caspase-3 (P < 0.05), apoptosis-inducing factor (P < 0.05), and poly-ADP ribose polymerase (P = 0.07) was lower in the TP508 low-dose group vs. TP508 high-dose and control. The terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling positive cell count was lower in both TP508 groups compared with the control (P < 0.05). This study demonstrates that, in hypercholesterolemic pigs, TP508 decreases myocardial necrosis and apoptosis after IR. Thus TP508 may offer a novel approach in protecting the myocardium from IR injury.

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

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. 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. 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. 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 Increases Myocardial Indices of Oxidative Stress in a Porcine Model of Hypercholesterolemia and Chronic Ischemia

Atorvastatin has previously been shown to reduce the endogenous angiogenic response to chronic ischemia in a porcine model. One possible mechanism for this effect is reduced bioavailability of nitric oxide, a key mediator of angiogenesis, secondary to increased oxygen free radicals. We sought to determine if atorvastatin modulates oxidative stress in myocardial tissue. Dietary induction of hypercholesterolemia was performed over 20 weeks in Yucatan swine with treated animals receiving atorvastatin 3 mg/kg/day. Chronic myocardial ischemia was induced via surgical placement of an ameroid constrictor ring around the proximal circumflex artery at age 20 weeks, followed by tissue harvest at age 27 weeks. Myocardial levels of protein, lipid, and DNA biomarkers of oxidative stress, serum levels of 8-isoprostane, nitric oxide (NO) dependent, and independent coronary microvascular reactivity, as well as isotope-labeled microsphere myocardial perfusion analysis and histologic analysis for endothelial cell density was performed. Atorvastatin treatment was associated with elevated levels of myocardial protein oxidation and lipid peroxidation. Conversely, serum oxidant stress biomarkers were not elevated. Atorvastatin treatment improved nitric oxide dependent and independent microvascular reactivity, and was associated with decreased perfusion in the ischemic myocardial territory. Treatment with atorvastatin was associated with increased levels of myocardial tissue protein and lipid oxidative stress biomarkers and a reduced functional endogenous angiogenic response, but improved coronary microvascular reactivity. Increased oxidative stress in tissues may play a role in the reduced angiogenic response seen with atorvastatin treatment in other studies.