Weike Bao

Systemic Activation of the Transient Receptor Potential Vanilloid Subtype 4 Channel Causes Endothelial Failure and Circulatory Collapse: Part 2

The transient receptor potential (TRP) vanilloid subtype 4 (V4) is a nonselective cation channel that exhibits polymodal activation and is expressed in the endothelium, where it contributes to intracellular Ca2+ homeostasis and regulation of cell volume. The purpose of the present study was to evaluate the systemic cardiovascular effects of GSK1016790A, a novel TRPV4 activator, and to examine its mechanism of action. In three species (mouse, rat, and dog), the i.v. administration of GSK1016790A induced a dose-dependent reduction in blood pressure, followed by profound circulatory collapse. In contrast, GSK1016790A had no acute cardiovascular effects in the TRPV4−/− null mouse. Hemodynamic analyses in the dog and rat demonstrate a profound reduction in cardiac output. However, GSK1016790A had no effect on rate or contractility in the isolated, buffer-perfused rat heart, and it produced potent endothelial-dependent relaxation of rodent-isolated vascular ring segments that were abolished by nitric-oxide synthase (NOS) inhibition (N-nitro-l-arginine methyl ester; l-NAME), ruthenium red, and endothelial NOS (eNOS) gene deletion. However, the in vivo circulatory collapse was not altered by NOS inhibition (l-NAME) or eNOS gene deletion but was associated with (concentration and time appropriate) profound vascular leakage and tissue hemorrhage in the lung, intestine, and kidney. TRPV4 immunoreactivity was localized in the endothelium and epithelium in the affected organs. GSK1016790A potently induced rapid electrophysiological and morphological changes (retraction/condensation) in cultured endothelial cells. In summary, inappropriate activation of TRPV4 produces acute circulatory collapse associated with endothelial activation/injury and failure of the pulmonary microvascular permeability barrier. It will be important to determine the role of TRPV4 in disorders associated with edema and microvascular congestion.

Myocardial protection from ischemia/reperfusion injury by targeted deletion of matrix metalloproteinase-9

OBJECTIVE Matrix metalloproteinase-9 (MMP-9) activity is up regulated in the heart subjected to ischemic insult. Whether increased MMP-9 activity contributes to acute myocardial injury after ischemia-reperfusion remains unknown. To investigate the role of MMP-9 in myocardial infarction, we utilized a MMP-9 knockout mouse. METHODS AND RESULTS Standard homologous recombination in embryonic stem cells was used to generate a mouse lacking MMP-9. The left anterior descending coronary artery was occluded for 30 min followed by 24 h reperfusion, and the ischemic and infarct sizes were determined. Targeted deletion of MMP-9 protected the heart from no-flow ischemia-reperfusion-induced myocardial injury. The myocardial infarct size was reduced by 17.5% in MMP-9 heterozygotes (+/-) (P<0.01) and 35.4% in MMP-9 knockout (-/-) mice (P<0.01) versus the wild-type (+/+) mice, respectively. Analysis of MMP activity in myocardial extracts by zymography demonstrated that ischemia-reperfusion-induced expression of proMMP-9 and active MMP-9 was reduced by 77.8% (P<0.01) and 69.1% (P<0.001), respectively, in (+/-) mice compared to (+/+) mice, and was absent in (-/-) animals. The expression of TIMP-1, an endogenous inhibitor of MMP-9, was elevated 4.7-fold (P<0.05) and 21.4-fold (P<0.05) in the (+/-) and (-/-) mice, respectively, compared to (+/+) mice. Immunohistochemical analysis revealed that neutrophils were the primary cellular source of MMP-9, and less neutrophils were detected in the ischemic region of the heart following ischemia-reperfusion in (-/-) mice compared to (+/+) mice. Measurement of myeloperoxidase activity, a marker enzyme of neutrophils, demonstrated a 44% reduction in neutrophils infiltrated into the ischemic myocardium in the (-/-) mice compared to the (+/+) mice (P<0.05). CONCLUSION These results suggest that MMP-9 plays an important role in ischemia-reperfusion-induced myocardial infarction and MMP-9 could be a target for prevention or treatment of acute ischemic myocardial injury.

An Orally Active TRPV4 Channel Blocker Prevents and Resolves Pulmonary Edema Induced by Heart Failure

Transient receptor potential vanilloid 4 (TRPV4) channels are expressed in human heart failure lungs, which can be blocked to prevent and resolve heart failure–induced pulmonary edema. Ion Channel Blockade Prevents Pulmonary Edema Heart failure affects not only the heart and vessels but also the lungs. As blood pressure builds up in the lung’s vessels, fluid leaks into the lungs. Treatment options are limited for these patients, mostly because the mechanism underlying pulmonary edema is unclear. Here, Thorneloe and colleagues implicate the activation of the transient receptor potential vanilloid 4 (TRPV4) ion channel in the onset of edema during heart failure and show that a small-molecule drug can prevent such leakage. Activation of the ion channel TRPV4 results in pulmonary edema in animal lungs. The authors first confirmed that TRPV4 was expressed in normal human lungs and then demonstrated that it was increased in lung tissue from patients with a history of congestive heart failure. Using a small-molecule screen, Thorneloe et al. discovered GSK2193874. In human cells in vitro and mouse lungs ex vivo, the small molecule effectively blocked TRPV4 channels to maintain endothelial (vessel) layer integrity. A related study by Huh et al. (this issue) shows that the drug indeed prevents vascular leakage of human cell cultures in vitro. The GSK2193874 analog GSK2263095 displayed similar activity in canine lungs ex vivo. In vivo in rat models of heart failure, the authors found that the drug was effective in both preventing and reversing pulmonary edema. The molecule only protected against lung permeability at high (pathological) pulmonary venous pressure. Thorneloe and colleagues showed that GSK2193874 blocked TRPV4 activity across species, including in human cells, without adversely affecting heart rate or arterial pressure. This suggests that TRPV4 blockers might be used therapeutically to treat patients with heart failure–induced pulmonary edema. Pulmonary edema resulting from high pulmonary venous pressure (PVP) is a major cause of morbidity and mortality in heart failure (HF) patients, but current treatment options demonstrate substantial limitations. Recent evidence from rodent lungs suggests that PVP-induced edema is driven by activation of pulmonary capillary endothelial transient receptor potential vanilloid 4 (TRPV4) channels. To examine the therapeutic potential of this mechanism, we evaluated TRPV4 expression in human congestive HF lungs and developed small-molecule TRPV4 channel blockers for testing in animal models of HF. TRPV4 immunolabeling of human lung sections demonstrated expression of TRPV4 in the pulmonary vasculature that was enhanced in sections from HF patients compared to controls. GSK2193874 was identified as a selective, orally active TRPV4 blocker that inhibits Ca2+ influx through recombinant TRPV4 channels and native endothelial TRPV4 currents. In isolated rodent and canine lungs, TRPV4 blockade prevented the increased vascular permeability and resultant pulmonary edema associated with elevated PVP. Furthermore, in both acute and chronic HF models, GSK2193874 pretreatment inhibited the formation of pulmonary edema and enhanced arterial oxygenation. Finally, GSK2193874 treatment resolved pulmonary edema already established by myocardial infarction in mice. These findings identify a crucial role for TRPV4 in the formation of HF-induced pulmonary edema and suggest that TRPV4 blockade is a potential therapeutic strategy for HF patients.

Activation of Peroxisome Proliferator–Activated Receptor-α Protects the Heart From Ischemia/Reperfusion Injury

Background—Peroxisome proliferator–activated receptor-&agr; (PPAR-&agr;) is expressed in the heart and regulates genes involved in myocardial fatty acid oxidation (FAO). The role of PPAR-&agr; in acute ischemia/reperfusion myocardial injury remains unclear. Methods and Results—The coronary arteries of male mice were ligated for 30 minutes. After reperfusion for 24 hours, ischemic and infarct sizes were determined. A highly selective and potent PPAR-&agr; agonist, GW7647, was administered by mouth for 2 days, and the third dose was given 1 hour before ischemia. GW7647 at 1 and 3 mg · kg−1 · d−1 reduced infarct size by 28% and 35%, respectively (P <0.01), and myocardial contractile dysfunction was also improved. Cardioprotection by GW7647 was completely abolished in PPAR-&agr;–null mice. Ischemia/reperfusion downregulated mRNA expression of cardiac PPAR-&agr; and FAO enzyme genes, decreased myocardial FAO enzyme activity and in vivo cardiac fat oxidation, and increased serum levels of free fatty acids. All of these changes were reversed by GW7647. Moreover, GW7647 attenuated ischemia/reperfusion-induced release of multiple proinflammatory cytokines and inhibited neutrophil accumulation and myocardial expression of matrix metalloproteinases-9 and -2. Furthermore, GW7647 inhibited nuclear factor-&kgr;B activation in the heart, accompanied by enhanced levels of inhibitor-&kgr;B&agr;. Conclusions—Activation of PPAR-&agr; protected the heart from reperfusion injury. This cardioprotection might be mediated through metabolic and antiinflammatory mechanisms. This novel effect of the PPAR-&agr; agonist could provide an added benefit to patients treated with PPAR-&agr; activators for dyslipidemia.

Activation of peroxisome proliferator-activated receptor-alpha protects the heart from ischemia/reperfusion injury.

Background—Peroxisome proliferator–activated receptor-&agr; (PPAR-&agr;) is expressed in the heart and regulates genes involved in myocardial fatty acid oxidation (FAO). The role of PPAR-&agr; in acute ischemia/reperfusion myocardial injury remains unclear. Methods and Results—The coronary arteries of male mice were ligated for 30 minutes. After reperfusion for 24 hours, ischemic and infarct sizes were determined. A highly selective and potent PPAR-&agr; agonist, GW7647, was administered by mouth for 2 days, and the third dose was given 1 hour before ischemia. GW7647 at 1 and 3 mg · kg−1 · d−1 reduced infarct size by 28% and 35%, respectively (P <0.01), and myocardial contractile dysfunction was also improved. Cardioprotection by GW7647 was completely abolished in PPAR-&agr;–null mice. Ischemia/reperfusion downregulated mRNA expression of cardiac PPAR-&agr; and FAO enzyme genes, decreased myocardial FAO enzyme activity and in vivo cardiac fat oxidation, and increased serum levels of free fatty acids. All of these changes were reversed by GW7647. Moreover, GW7647 attenuated ischemia/reperfusion-induced release of multiple proinflammatory cytokines and inhibited neutrophil accumulation and myocardial expression of matrix metalloproteinases-9 and -2. Furthermore, GW7647 inhibited nuclear factor-&kgr;B activation in the heart, accompanied by enhanced levels of inhibitor-&kgr;B&agr;. Conclusions—Activation of PPAR-&agr; protected the heart from reperfusion injury. This cardioprotection might be mediated through metabolic and antiinflammatory mechanisms. This novel effect of the PPAR-&agr; agonist could provide an added benefit to patients treated with PPAR-&agr; activators for dyslipidemia.

Effects of p38 MAPK Inhibitor on angiotensin II-dependent hypertension, organ damage, and superoxide anion production.

Angiotensin II (Ang II) activates p38 mitogen-activated protein kinase (p38 MAPK) and increases reactive oxygen species (ROS), but the nature of the relationship in vivo is not fully understood. We assess the effect of SB239063AN, a highly selective, orally active, p38 MAPK inhibitor, on Ang II-dependent hypertension, target-organ damage and ROS production. Sprague-Dawley rats and MAPKAP kinase-2 knockout mice were infused with Ang II. Ang II infusion increased the levels of phosphorylated p38 MAPK in the heart and aorta. Production of superoxide anion and expression of NAD(P)H oxidase subunit gp91phox in the aorta were increased 4- and 5-fold, respectively. In addition, Ang II infusion led to endothelial dysfunction, progressive and sustained hypertension, and cardiac hypertrophy. Treatment with SB239063AN (800 ppm in the diet) significantly attenuated the levels of phosphorylated p38 MAPK in the heart and aorta, reduced superoxide anion generation by 57% (P < 0.01), markedly suppressed gp91phox mRNA expression, prevented endothelial dysfunction, and blunted both the hypertension and cardiac hypertrophy. Ang II-dependent hypertension was also significantly attenuated in MAPKAP kinase-2 knockout mice. The results suggest that Ang II induced hypertension, organ damage, and ROS production are possibly mediated by p38 MAPK and inhibition of p38 MAPK may offer a therapeutic approach for cardiovascular disease.

Albiglutide, a Long Lasting Glucagon-Like Peptide-1 Analog, Protects the Rat Heart against Ischemia/Reperfusion Injury: Evidence for Improving Cardiac Metabolic Efficiency

Background The cardioprotective effects of glucagon-like peptide-1 (GLP-1) and analogs have been previously reported. We tested the hypothesis that albiglutide, a novel long half-life analog of GLP-1, may protect the heart against I/R injury by increasing carbohydrate utilization and improving cardiac energetic efficiency. Methods/Principal Findings Sprague-Dawley rats were treated with albiglutide and subjected to 30 min myocardial ischemia followed by 24 h reperfusion. Left ventricle infarct size, hemodynamics, function and energetics were determined. In addition, cardiac glucose disposal, carbohydrate metabolism and metabolic gene expression were assessed. Albiglutide significantly reduced infarct size and concomitantly improved post-ischemic hemodynamics, cardiac function and energetic parameters. Albiglutide markedly increased both in vivo and ex vivo cardiac glucose uptake while reducing lactate efflux. Analysis of metabolic substrate utilization directly in the heart showed that albiglutide increased the relative carbohydrate versus fat oxidation which in part was due to an increase in both glucose and lactate oxidation. Metabolic gene expression analysis indicated upregulation of key glucose metabolism genes in the non-ischemic myocardium by albiglutide. Conclusion/Significance Albiglutide reduced myocardial infarct size and improved cardiac function and energetics following myocardial I/R injury. The observed benefits were associated with enhanced myocardial glucose uptake and a shift toward a more energetically favorable substrate metabolism by increasing both glucose and lactate oxidation. These findings suggest that albiglutide may have direct therapeutic potential for improving cardiac energetics and function.

Chronic Inhibition of Hypoxia-inducible Factor Prolyl 4-hydroxylase Improves Ventricular Performance, Remodeling, and Vascularity After Myocardial Infarction in the Rat

Background: Hypoxia inducible factors (HIFs) are transcription factors that are regulated by HIF-prolyl 4-hydroxylases (PHDs) in response to changes in oxygen tension. Once activated, HIFs play an important role in angiogenesis, erythropoiesis, proliferation, cell survival, inflammation, and energy metabolism. We hypothesized that GSK360A, a novel orally active HIF-PHD inhibitor, could facilitate local and systemic HIF-1α signaling and protect the failing heart after myocardial infarction (MI). Methods and Results: GSK360A is a potent (nanomolar) inhibitor of HIF-PHDs (PHD1>PHD2 = PHD3) capable of activating the HIF-1α pathway in a variety of cell types including neonatal rat ventricular myocytes and H9C2 cells. Male rats treated orally with GSK360A (30 mg·kg−1·d−1) had a sustained elevation in circulating levels of erythropoietin and hemoglobin and increased hemoxygenase-1 expression in the heart and skeletal muscle. In a rat model of established heart failure with systolic dysfunction induced by ligation of left anterior descending coronary artery, chronic treatment with GSK360A for 28 days prevented the progressive reduction in ejection fraction, ventricular dilation, and increased lung weight, which were observed in the vehicle-treated animals, for up to 3 months. In addition, the microvascular density in the periinfarct region was increased (>2-fold) in GSK360A-treated animals. Treatment was well tolerated (survival was 89% in the GSK360A group vs. 82% in the placebo group). Conclusions: Chronic post-myocardial infarction treatment with a selective HIF PHD inhibitor (GSK360A) exerts systemic and local effects by stabilizing HIF-1α signaling and improves long-term ventricular function, remodeling, and vascularity in a model of established ventricular dysfunction. These results suggest that HIF-PHD inhibitors may be suitable for the treatment of post-MI remodeling and heart failure.

In Vivo Activation of Peroxisome Proliferator-Activated Receptor-δ Protects the Heart from Ischemia/Reperfusion Injury in Zucker Fatty Rats

Peroxisome proliferator-activated receptor (PPAR)-δ is a transcription factor that belongs to the PPAR family. PPAR-δ is abundantly expressed in the heart, and its role in the heart is largely unknown. We tested whether pharmacological activation of PPAR-δ protects the heart from ischemia/reperfusion (I/R) injury in male Zucker fatty rats, a rodent model of obesity and dyslipidemia. A highly selective PPAR-δ agonist, [4-[[[2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5-thiazolyl]methyl] thio]-2-methylphenoxy]acetic acid (GW0742), was administered for 7 days at 10 mg/kg/day (p.o., once a day). Ischemic injury was produced by occlusion of the left anterior descending artery for 30 min followed by reperfusion for up to 24 h. Treatment with GW0742 reduced serum levels of cardiac troponin-I and infarct size by 63% (p < 0.01) and 32% (p < 0.01), respectively, and improved left ventricular function. Treatment with GW0742 up-regulated gene expression involved in cardiac fatty acid oxidation, increased fat use in the heart, and reduced serum levels of free fatty acids. The enhanced cardiac expression of interleukin (IL)-6, IL-8, intercellular adhesion molecule-1, and monocyte chemoattractant protein-1 induced by I/R were significantly attenuated by GW0742. Treatment with GW0742 also reduced apoptotic cardiomyocytes by 34% and cardiac caspase-3 activity by 61% (both p < 0.01 versus vehicle). GW0742 differentially regulated Bcl family members, favoring cell survival, and attenuated I/R-induced cardiac mitochondrial damage. In addition, GW0742 treatment augmented the cardiac Akt signaling pathway, as reflected by enhanced phospho-3-phosphoinositide-dependent kinase-1 and p-Akt. The results indicate that activation of PPAR-δ protected the heart from I/R injury in Zucker fatty rats, and multiple mechanisms including amelioration of lipotoxicity, anti-inflammation, and up-regulation of prosurvival signaling contribute together to the cardioprotection.

Cardioprotection by systemic dosing of thymosin beta four following ischemic myocardial injury

Thymosin beta 4 (Tβ4) was previously shown to reduce infarct size and improve contractile performance in chronic myocardial ischemic injury via two phases of action: an acute phase, just after injury, when Tβ4 preserves ischemic myocardium via antiapoptotic or anti-inflammatory mechanisms; and a chronic phase, when Tβ4 activates the growth of vascular or cardiac progenitor cells. In order to differentiate between the effects of Tβ4 during the acute and during the chronic phases, and also in order to obtain detailed hemodynamic and biomarker data on the effects of Tβ4 treatment suitable for use in clinical studies, we tested Tβ4 in a rat model of chronic myocardial ischemia using two dosing regimens: short term dosing (Tβ4 administered only during the first 3 days following injury), and long term dosing (Tβ4 administered during the first 3 days following injury and also every third day until the end of the study). Tβ4 administered throughout the study reduced infarct size and resulted in significant improvements in hemodynamic performance; however, chamber volumes and ejection fractions were not significantly improved. Tβ4 administered only during the first 3 days following injury tended to reduce infarct size, chamber volumes and improve hemodynamic performance. Plasma biomarkers of myocyte injury were significantly reduced by Tβ4 treatment during the acute injury period, and plasma ANP levels were significantly reduced in both dosing groups. Surprisingly, neither acute nor chronic Tβ4 treatment significantly increased blood vessel density in peri-infarct regions. These results suggest the following: repeated dosing may be required to achieve clinically measureable improvements in cardiac function post-myocardial infarction (MI); improvement in cardiac function may be observed in the absence of a high degree of angiogenesis; and that plasma biomarkers of cardiac function and myocardial injury are sensitive pharmacodynamic biomarkers of the effects of Tβ4.