Alan R. Olzinski

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.

Differential uptake of ferumoxtran-10 and ferumoxytol, ultrasmall superparamagnetic iron oxide contrast agents in rabbit: Critical determinants of atherosclerotic plaque labeling†

To compare atherosclerotic plaque uptake of a first (ferumoxtran‐10) and second generation (ferumoxytol) ultrasmall superparamagnetic iron oxide (USPIO) contrast agent with different pharmacokinetic/pharmacodynamic properties.

p38 MAPK Inhibition Reduces Aortic Ultrasmall Superparamagnetic Iron Oxide Uptake in a Mouse Model of Atherosclerosis. MRI Assessment

Objective—Ultrasmall superparamagnetic iron oxide (USPIO) contrast agents have been used for noninvasive MRI assessment of atherosclerotic plaque inflammation. The purpose of this study was to noninvasively evaluate USPIO uptake in aorta of apoE−/− mice and to determine the effects of Angiotensin II (Ang II) infusion and chronic antiinflammatory treatment with a p38 MAPK inhibitor on this uptake. Methods and Results—ApoE−/− mice were administered saline or Ang II (1.44 mg/kg/d) for 21 days. In vivo MRI assessment of USPIO uptake in the aortic arch was observed in all animals. However, although the Ang II group had significantly higher absolute iron content (↑103%, P<0.001) in the aortic arch compared with the saline group, the p38 MAPK inhibitor (SB-239063, 150 mg/kg/d) treatment group did not (↑6%, NS). The in vivo MRI signal intensity was significantly correlated to the absolute iron content in the aortic arch. Histological evaluation of the aortic root lesion area showed colocalization of USPIO with macrophages and a reduction in USPIO but not macrophage content with SB-239063 treatment. Conclusion—The present study demonstrates that noninvasive assessment of USPIO uptake, as a marker for inflammation in murine atherosclerotic plaque, is feasible and that p38 MAPK inhibition attenuates the uptake of USPIO in aorta of Ang II–infused apoE−/− mice.

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 Serial Assessment of Aortic Aneurysm Formation in Apolipoprotein E–Deficient Mice via MRI

Background—Hyperlipidimic mice administered angiotensin II have been used for the study of abdominal aortic aneurysms (AAAs). The purpose of this study was to examine the use of MRI for studying AAA development and for examining the effects of pharmacological intervention on AAA development in the apolipoprotein E–deficient mouse. Methods and Results—Suprarenal aortic aneurysms were generated in apolipoprotein E–deficient mice administered angiotensin II (1000 ng/kg per min) for up to 28 days. In vivo MRI was performed serially (once weekly) to assess AAA development and rupture. Comparison of AAA size as measured by in vivo and ex vivo MRI resulted in excellent agreement (r=0.96, P<0.0001). In addition, MRI correlated with histology-derived AAA area assessment (in vivo versus histology: r=0.84, P<0.0001; ex vivo versus histology: r=0.89, P<0.0001). In a separate study, angiotensin II–administered apolipoprotein E–deficient mice were treated with doxycycline (broad-based matrix metalloproteinase inhibitor; 30 mg/kg per day for 28 days). MRI was able to noninvasively assess a reduced rate of AAA development (46% versus 71%, P<0.05), a decreased AAA area (2.56 versus 4.02 mm2, P<0.01), and decreased incidence of rupture (43% versus 100%) in treated versus control animals. Inhibition of aorta matrix metalloproteinase 2/9 activity was observed in the treated animals. Conclusions—These results demonstrate the use of MRI to noninvasively and temporally assess AAA development on pharmacological intervention in this preclinical cardiovascular disease model.

PPAR?? Activation Normalizes Cardiac Substrate Metabolism and Reduces Right Ventricular Hypertrophy in Congestive Heart Failure

Previously, it was shown that selective deletion of peroxisome proliferator activated receptor δ (PPARδ) in the heart resulted in a cardiac lipotoxicity, hypertrophy, and heart failure. The aim of the present study was to determine the effects of chronic and selective pharmacological activation of PPARδ in a model of congestive heart failure. PPARδ-specific agonist treatment (GW610742X at 30 and 100 mg/kg/day for 6-9 weeks) was initiated immediately postmyocardial infarction (MI) in Sprague-Dawley rats. Magnetic resonance imaging/spectroscopy was used to assess cardiac function and energetics. A 1-13C glucose clamp was performed to assess relative cardiac carbohydrate versus fat oxidation. Additionally, cardiac hemodynamics and reverse-transcription polymerase chain reaction gene expression analysis was performed. MI rats had significantly reduced left ventricle (LV) ejection fractions and whole heart phosphocreatine/adenosine triphosphate ratio compared with Sham animals (reduction of 43% and 14%, respectively). However, GW610742X treatment had no effect on either parameter. In contrast, the decrease in relative fat oxidation rate observed in both LV and right ventricle (RV) following MI (decrease of 58% and 54%, respectively) was normalized in a dose-dependent manner following treatment with GW610742X. These metabolic changes were associated with an increase in lipid transport/metabolism target gene expression (eg, CD36, CPT1, UCP3). Although there was no difference between groups in LV weight or infarct size measured upon necropsy, there was a dramatic reduction in RV hypertrophy and lung congestion (decrease of 22-48%, P < 0.01) with treatment which was associated with a >7-fold decrease (P < 0.05) in aterial natriuretic peptide gene expression in RV. Diuretic effects were not observed with GW610742X. In conclusion, chronic treatment with a selective PPARδ agonist normalizes cardiac substrate metabolism and reduces RV hypertrophy and pulmonary congestion consistent with improvement in congestive heart failure.

Pharmacological Inhibition of C-C Chemokine Receptor 2 Decreases Macrophage Infiltration in the Aortic Root of the Human C-C Chemokine Receptor 2/Apolipoprotein E −/− Mouse: Magnetic Resonance Imaging Assessment

Purpose—This study assessed the pharmacological effect of a novel selective C-C chemokine receptor (CCR) 2 antagonist (GSK1344386B) on monocyte/macrophage infiltration into atherosclerotic plaque using magnetic resonance imaging (MRI) in an atherosclerotic mouse model. Methods and Results—Apolipoprotein E−/− mice expressing human CCR2 were fed a Western diet (vehicle group) or a Western diet plus10 mg/kg per day of GSK1344386B (GSK1344386B group). After the baseline MRI, mice were implanted with osmotic pumps containing angiotensin II, 1000 ng/kg per minute, to accelerate lesion formation. After five weeks of angiotensin II administration, mice received ultrasmall superparamagnetic iron oxide, an MRI contrast agent for the assessment of monocyte/macrophage infiltration to the plaque, and underwent imaging. After imaging, mice were euthanized, and the heart and aorta were harvested for ex vivo MRI and histopathological examination. After 5 weeks of dietary dosing, there were no significant differences between groups in body or liver weight or plasma cholesterol concentrations. An in vivo MRI reflected a decrease in ultrasmall superparamagnetic iron oxide contrast agent uptake in the aortic arch of the GSK1344386B group (P<0.05). An ex vivo MRI of the aortic root also reflected decreased ultrasmall superparamagnetic iron oxide uptake in the GSK1344386B group and was verified by absolute iron analysis (P<0.05). Although there was no difference in aortic root lesion area between groups, there was a 30% reduction in macrophage area observed in the GSK1344386B group (P<0.05). Conclusion—An MRI was used to noninvasively assess the decreased macrophage content in the atherosclerotic plaque after selective CCR2 inhibition.

Assessment of macrophage infiltration in a Murine model of abdominal aortic aneurysm

To evaluate the use of an ultrasmall superparamagnetic iron oxide (USPIO) contrast agent as a marker for the detection of macrophage in a preclinical abdominal aortic aneurysm animal (AAA) model.

Differential effects of p38 mitogen-activated protein kinase and cyclooxygenase 2 inhibitors in a model of cardiovascular disease.

The evidence is compelling for a role of inflammation in cardiovascular diseases; however, the chronic use of anti-inflammatory drugs for these indications has been disappointing. The recent study compares the effects of two anti-inflammatory agents [cyclooxygenase 2 (COX2) and p38 inhibitors] in a model of cardiovascular disease. The vascular, renal, and cardiac effects of 4-(4-methylsulfonylphenyl)-3-phenyl-5H-furan-2-one (rofecoxib; a COX2 inhibitor) and 6-{5-[(cyclopropylamino)carbonyl]-3-fluoro-2-methylphenyl}-N-(2,2-dimethylpropyl)-3-pyridinecarboxamide [GSK-AHAB, a selective p38 mitogen-activated protein kinase (MAPK) inhibitor], were examined in the spontaneously hypertensive stroke-prone rat (SHR-SP). In SHR-SPs receiving a salt-fat diet (SFD), chronic treatment with GSK-AHAB significantly and dose-dependently improved survival, endothelial-dependent and -independent vascular relaxation, and indices of renal function, and it attenuated dyslipidemia, hypertension, cardiac remodeling, plasma renin activity (PRA), aldosterone, and interleukin-1β (IL-1β). In contrast, chronic treatment with a COX2-selective dose of rofecoxib exaggerated the harmful effects of the SFD, i.e., increasing vascular and renal dysfunction, dyslipidemia, hypertension, cardiac hypertrophy, PRA, aldosterone, and IL-1β. The protective effects of a p38 MAPK inhibitor are clearly distinct from the deleterious effects of a selective COX2 inhibitor in the SHR-SP and suggest that anti-inflammatory agents can have differential effects in cardiovascular disease. The results also suggest a method for evaluating long-term cardiovascular efficacy and safety.

Comparison of Soluble Guanylate Cyclase Stimulators and Activators in Models of Cardiovascular Disease Associated with Oxidative Stress

Soluble guanylate cyclase (sGC), the primary mediator of nitric oxide (NO) bioactivity, exists as reduced (NO-sensitive) and oxidized (NO-insensitive) forms. We tested the hypothesis that the cardiovascular protective effects of NO-insensitive sGC activation would be potentiated under conditions of oxidative stress compared to those of NO-sensitive sGC stimulation. The cardiovascular effects of the NO-insensitive sGC activator GSK2181236A [a low, non-depressor dose, and a high dose which lowered mean arterial pressure (MAP) by 5–10 mmHg] and those of equi-efficacious doses of the NO-sensitive sGC stimulator BAY 60-4552 were assessed in (1) Sprague Dawley rats during coronary artery ischemia/reperfusion (I/R) and (2) spontaneously hypertensive stroke prone rats (SHR-SP) on a high salt/fat diet (HSFD). In I/R, neither compound reduced infarct size 24 h after reperfusion. In SHR-SP, HSFD increased MAP, urine output, microalbuminuria, and mortality, caused left ventricular hypertrophy with preserved ejection fraction, and impaired endothelium-dependent vasorelaxation. The low dose of BAY 60-4552, but not that of GSK2181236A, decreased urine output, and improved survival. Conversely, the low dose of GSK2181236A, but not that of BAY 60-4552, attenuated the development of cardiac hypertrophy. The high doses of both compounds similarly attenuated cardiac hypertrophy and improved survival. In addition to these effects, the high dose of BAY 60-4552 reduced urine output and microalbuminuria and attenuated the increase in MAP to a greater extent than did GSK2181236A. Neither compound improved endothelium-dependent vasorelaxation. In SHR-SP isolated aorta, the vasodilatory responses to the NO-dependent compounds carbachol and sodium nitroprusside were attenuated by HSFD. In contrast, the vasodilatory responses to both GSK2181236A and BAY 60-4552 were unaltered by HSFD, indicating that reduced NO-bioavailability and not changes in the oxidative state of sGC is responsible for the vascular dysfunction. In summary, GSK2181236A and BAY 60-4552 provide partial benefit against hypertension-induced end-organ damage. The differential beneficial effects observed between these compounds could reflect tissue-specific changes in the oxidative state of sGC and might help direct the clinical development of these novel classes of therapeutic agents.