Raisa Serpi

p38 Kinase rescues failing myocardium after myocardial infarction: evidence for angiogenic and anti-apoptotic mechanisms

As a leading cause of heart failure, postinfarction left ventricular remodeling represents an important target for therapeutic interventions. Mitogen‐activated protein kinases regulate critical cellular processes including stress response and survival, but their role in left ventricular remodeling is unknown. In the present study, rats were subjected to myocardial infarction by ligating the left anterior descending coronary artery. Western blot and kinase assay analysis revealed an inactivation of p38 kinase after myocardial infarction. Local adenovirus‐mediated cotransfection of wild‐type (WT) p38 kinase and constitutively active MKK3b reduced infarct size (26±3% vs. 47±4%, P<0.05 vs. LacZ‐treated control) associated with improved ejection fraction (66.9±5.5% vs. 44.4±4.0%, P<0.001), fractional shortening (30.2±2.1% vs. 19.7±2.2%, P<0.001), and decreased left ventricular diastolic diameter (8.5±0.4 mm vs. 9.5±0.2 mm, P<0.01). p38 kinase gene transfer increased capillary density (2423±107/mm2 vs. 1934±86/mm2, P<0.001) and resulted in microvessel enlargement in the ischemic border zone. Apoptosis (35±7 vs. 69±13 cells, P<0.01) and fibrosis (16±3% vs. 34±8%, P<0.05) were reduced, while the number of c‐kit positive cardiac stem‐like cells remained unchanged. These results indicate that reduced p38 signaling predisposes to adverse postinfarction remodeling. The rescue of failing myocardium with p38 kinase may be a potential new therapy for heart failure after myocardial infarction. —Tenhunen, O., Soini, Y., Ilves, M., Rysä, J., Tuukkanen, J., Serpi, R., Pennanen, H., Ruskoaho, H., Leskinen, H. p38 Kinase rescues failing myocardium after myocardial infarction: evidence for angiogenic and anti‐apoptotic mechanisms. FASEB J. 20, E1276‐E1286 (2006)

GATA-4 Is an Angiogenic Survival Factor of the Infarcted Heart

Background—Recent data suggest that GATA-4 is an antiapoptotic factor required for adaptive responses and a key regulator of hypertrophy and hypertrophy-associated genes in the heart. As a leading cause of chronic heart failure, reversal of postinfarction left ventricular remodeling represents an important target for therapeutic interventions. Here, we studied the role of GATA-4 as a mediator of postinfarction remodeling in rats. Methods and Results—Myocardial infarction, caused by ligating the left anterior descending coronary artery, significantly decreased the DNA binding activity of GATA-4 at day 1, whereas at 2 weeks the GATA-4 DNA binding was significantly upregulated. To determine the functional role of GATA-4, peri-infarct intramyocardial delivery of adenoviral vector expressing GATA-4 was done before left anterior descending coronary artery ligation. Hearts treated with GATA-4 gene transfer exhibited significantly increased ejection fraction and fractional shortening. Accordingly, infarct size was significantly reduced. To determine the cardioprotective mechanisms of GATA-4, myocardial angiogenesis, rate of apoptosis, c-kit+ cardiac stemlike cells, and genes regulated by GATA-4 were studied. The number of capillaries and stemlike cells was significantly increased, and decreased apoptosis was observed. Conclusion—These results indicate that the reversal of reduced GATA-4 activity prevents adverse postinfarction remodeling through myocardial angiogenesis, antiapoptosis, and stem cell recruitment. GATA-4–based gene transfer may represent a novel, efficient therapeutic approach for heart failure.

Vascular endothelial growth factor-B gene transfer prevents angiotensin II-induced diastolic dysfunction via proliferation and capillary dilatation in rats

AIMS heart growth and function are angiogenesis-dependent, but little is known concerning the effects of key regulators of angiogenesis on diastolic heart failure. Here, we tested the hypothesis that local vascular endothelial growth factor-B (VEGF-B) gene therapy prevents left ventricular diastolic dysfunction. METHODS AND RESULTS rats were subjected to pressure overload by infusing angiotensin II (33.3 microg/kg/h) for 2 weeks using osmotic minipumps. Intramyocardial delivery of adenoviral vector expressing VEGF-B(167A) improved the angiotensin II-induced diastolic dysfunction compared with LacZ control virus. Local VEGF-B gene transfer increased the mean capillary area in the left ventricle in control and angiotensin II-infused animals, whereas the density of capillaries was not affected. Interestingly, significant increases were noted in Ki67(+) proliferating cells, expression of interleukin1β, and c-kit(+) cells in response to VEGF-B gene transfer. The increase in cardiac c-kit(+) cells was not associated with an induction of stromal cell-derived factor 1α, suggesting no mobilization of cells from bone marrow. Also, the phosphatidylinositol 3-kinase/Akt pathway was activated. CONCLUSION VEGF-B gene transfer resulted in prevention of the angiotensin II-induced diastolic dysfunction associated with induction of the Akt pathway, increased proliferation and number of c-kit(+) cells, as well as an increase in the capillary area in the left ventricle. VEGF-B may offer novel therapeutic possibilities for the prevention of the transition from compensated to decompensated cardiac hypertrophy and thereby for the treatment of heart failure.

HIF Prolyl 4-Hydroxylase-2 Inhibition Improves Glucose and Lipid Metabolism and Protects Against Obesity and Metabolic Dysfunction

Obesity is a major public health problem, predisposing subjects to metabolic syndrome, type 2 diabetes, and cardiovascular diseases. Specific prolyl 4-hydroxylases (P4Hs) regulate the stability of the hypoxia-inducible factor (HIF), a potent governor of metabolism, with isoenzyme 2 being the main regulator. We investigated whether HIF-P4H-2 inhibition could be used to treat obesity and its consequences. Hif-p4h-2–deficient mice, whether fed normal chow or a high-fat diet, had less adipose tissue, smaller adipocytes, and less adipose tissue inflammation than their littermates. They also had improved glucose tolerance and insulin sensitivity. Furthermore, the mRNA levels of the HIF-1 targets glucose transporters, glycolytic enzymes, and pyruvate dehydrogenase kinase-1 were increased in their tissues, whereas acetyl-CoA concentration was decreased. The hepatic mRNA level of the HIF-2 target insulin receptor substrate-2 was higher, whereas that of two key enzymes of fatty acid synthesis was lower. Serum cholesterol levels and de novo lipid synthesis were decreased, and the mice were protected against hepatic steatosis. Oral administration of an HIF-P4H inhibitor, FG-4497, to wild-type mice with metabolic dysfunction phenocopied these beneficial effects. HIF-P4H-2 inhibition may be a novel therapy that not only protects against the development of obesity and its consequences but also reverses these conditions.

In vivo biocompatibility of porous silicon biomaterials for drug delivery to the heart

Myocardial infarction (MI), commonly known as a heart attack, is the irreversible necrosis of heart muscle secondary to prolonged ischemia, which is an increasing problem in terms of morbidity, mortality and healthcare costs worldwide. Along with the idea to develop nanocarriers that efficiently deliver therapeutic agents to target the heart, in this study, we aimed to test the in vivo biocompatibility of different sizes of thermally hydrocarbonized porous silicon (THCPSi) microparticles and thermally oxidized porous silicon (TOPSi) micro and nanoparticles in the heart tissue. Despite the absence or low cytotoxicity, both particle types showed good in vivo biocompatibility, with no influence on hematological parameters and no considerable changes in cardiac function before and after MI. The local injection of THCPSi microparticles into the myocardium led to significant higher activation of inflammatory cytokine and fibrosis promoting genes compared to TOPSi micro and nanoparticles; however, both particles showed no significant effect on myocardial fibrosis at one week post-injection. Our results suggest that THCPSi and TOPSi micro and nanoparticles could be applied for cardiac delivery of therapeutic agents in the future, and the PSi biomaterials might serve as a promising platform for the specific treatment of heart diseases.

Intramyocardial BNP Gene Delivery Improves Cardiac Function Through Distinct Context-Dependent Mechanisms

Background—B-type natriuretic peptide (BNP) is an endogenous peptide produced under physiological and pathological conditions mainly by ventricular myocytes. It has natriuretic, diuretic, blood pressure–lowering, and antifibrotic actions that could mediate cardiorenal protection in cardiovascular diseases. In the present study, we used BNP gene transfer to examine functional and structural effects of BNP on left ventricular (LV) remodeling. Methods and Results—Human BNP was overexpressed by using adenovirus-mediated gene delivery in normal rat hearts and in hearts during the remodeling process after infarction and in an experimental model of angiotensin II–mediated hypertension. In healthy hearts, BNP gene delivery into the anterior wall of the LV decreased myocardial fibrosis (P<0.01, n=7 to 8) and increased capillary density (P<0.05, n=7 to 8) associated with a 7.3-fold increase in LV BNP peptide levels. Overexpression of BNP improved LV fractional shortening by 22% (P<0.05, n=6 to 7) and ejection fraction by 19% (P<0.05, n=6 to 7) after infarction. The favorable effect of BNP gene delivery on cardiac function after infarction was associated with normalization of cardiac sarcoplasmic reticulum Ca2+-ATPase expression and phospholamban Thr17-phosphorylation. BNP gene delivery also improved fractional shortening and ejection fraction in angiotensin II–mediated hypertension as well as decreased myocardial fibrosis and LV collagen III mRNA levels but had no effect on angiogenesis or Ca2+-ATPase expression and phospholamban phosphorylation. Conclusions—Local intramyocardial BNP gene delivery improves cardiac function and attenuates adverse postinfarction and angiotensin II–induced remodeling. These results also indicate that myocardial BNP has pleiotropic, context-dependent, favorable actions on cardiac function and suggest that BNP acts locally as a key mechanical load–activated regulator of angiogenesis and fibrosis.

Activation of Hypoxia Response in Endothelial Cells Contributes to Ischemic Cardioprotection

ABSTRACT Small-molecule inhibition of hypoxia-inducible factor prolyl 4-hydroxylases (HIF-P4Hs) is being explored for the treatment of anemia. Previous studies have suggested that HIF-P4H-2 inhibition may also protect the heart from an ischemic insult. Hif-p4h-2gt/gt mice, which have 76 to 93% knockdown of Hif-p4h-2 mRNA in endothelial cells, fibroblasts, and cardiomyocytes and normoxic stabilization of Hif-α, were subjected to ligation of the left anterior descending coronary artery (LAD). Hif-p4h-2 deficiency resulted in increased survival, better-preserved left ventricle (LV) systolic function, and a smaller infarct size. Surprisingly, a significantly larger area of the LV remained perfused during LAD ligation in Hif-p4h-2gt/gt hearts than in wild-type hearts. However, no difference was observed in collateral vessels, while the size of capillaries, but not their number, was significantly greater in Hif-p4h-2gt/gt hearts than in wild-type hearts. Hif-p4h-2gt/gt mice showed increased cardiac expression of endothelial Hif target genes for Tie-2, apelin, APJ, and endothelial nitric oxide (NO) synthase (eNOS) and increased serum NO concentrations. Remarkably, blockage of Tie-2 signaling was sufficient to normalize cardiac apelin and APJ expression and resulted in reversal of the enlarged-capillary phenotype and ischemic cardioprotection in Hif-p4h-2gt/gt hearts. Activation of the hypoxia response by HIF-P4H-2 inhibition in endothelial cells appears to be a major determinant of ischemic cardioprotection and justifies the exploration of systemic small-molecule HIF-P4H-2 inhibitors for ischemic heart disease.

(Pro)renin receptor triggers distinct angiotensin II-independent extracellular matrix remodeling and deterioration of cardiac function.

Background Activation of the renin-angiotensin-system (RAS) plays a key pathophysiological role in heart failure in patients with hypertension and myocardial infarction. However, the function of (pro)renin receptor ((P)RR) is not yet solved. We determined here the direct functional and structural effects of (P)RR in the heart. Methodology/Principal Findings (P)RR was overexpressed by using adenovirus-mediated gene delivery in normal adult rat hearts up to 2 weeks. (P)RR gene delivery into the anterior wall of the left ventricle decreased ejection fraction (P<0.01), fractional shortening (P<0.01), and intraventricular septum diastolic and systolic thickness, associated with approximately 2–fold increase in left ventricular (P)RR protein levels at 2 weeks. To test whether the worsening of cardiac function and structure by (P)RR gene overexpression was mediated by angiotensin II (Ang II), we infused an AT1 receptor blocker losartan via osmotic minipumps. Remarkably, cardiac function deteriorated in losartan-treated (P)RR overexpressing animals as well. Intramyocardial (P)RR gene delivery also resulted in Ang II-independent activation of extracellular-signal-regulated kinase1/2 phosphorylation and myocardial fibrosis, and the expression of transforming growth factor-β1 and connective tissue growth factor genes. In contrast, activation of heat shock protein 27 phosphorylation and apoptotic cell death by (P)RR gene delivery was Ang II-dependent. Finally, (P)RR overexpression significantly increased direct protein–protein interaction between (P)RR and promyelocytic zinc-finger protein. Conclusions/Significance These results indicate for the first time that (P)RR triggers distinct Ang II-independent myocardial fibrosis and deterioration of cardiac function in normal adult heart and identify (P)RR as a novel therapeutic target to optimize RAS blockade in failing hearts.

Inhibition of Let-7 microRNA attenuates myocardial remodeling and improves cardiac function postinfarction in mice

The members of lethal‐7 (Let‐7) microRNA (miRNA) family are involved in regulation of cell differentiation and reprogramming of somatic cells into induced pluripotent stem cells. However, their function in the heart is not known. In this study, we examined the effect of inhibiting the function of Let‐7c miRNA on the progression of postinfarction left ventricular (LV) remodeling in mice. Myocardial infarction was induced with permanent ligation of left anterior descending coronary artery with a 4‐week follow‐up period. Let‐7c miRNA was inhibited with a specific antagomir administered intravenously. The inhibition of Let‐7c miRNA downregulated the levels of mature Let‐7c miRNA and its other closely related members of Let‐7 family in the heart and resulted in increased expression of pluripotency‐associated genes Oct4 and Sox2 in cardiac fibroblasts in vitro and in adult mouse heart in vivo. Importantly, Let‐7c inhibitor prevented the deterioration of cardiac function postinfarction, as demonstrated by preserved LV ejection fraction and elevated cardiac output. Improvement in cardiac function by Let‐7c inhibitor postinfarction was associated with decreased apoptosis, reduced fibrosis, and reduction in the number of discoidin domain receptor 2–positive fibroblasts, while the number of c‐kit+ cardiac stem cells and Ki‐67+ proliferating cells remained unaltered. In conclusion, inhibition of Let‐7 miRNA may be beneficial for the prevention of postinfarction LV remodeling and progression of heart failure.

Parthenolide inhibits STAT3 signaling and attenuates angiotensin II-induced left ventricular hypertrophy via modulation of fibroblast activity

Parthenolide has shown promise in treatment of various cancers via inhibition of the transcription factor signal transducer and activator of transcription 3 (STAT3). Activation of STAT3 has been observed in left ventricular hypertrophy (LVH); however, its exact role is not known. The aim of the study was to examine the effects of parthenolide on pressure overload-induced LVH in rats. Pressure overload was induced by angiotensin II (Ang II) infusion (33 μg/kg/h) for 1 week in the presence or absence of parthenolide (0.5mg/kg/day, i.p.). Ang II infusion resulted in LVH associated with increased phosphorylation of STAT3 at Tyr705 and Ser727. Parthenolide treatment had no effect on ejection fraction, but abolished the activation of STAT3 and reduced the Ang II-induced LVH (LV posterior wall thickness in end-diastole: 2.28 ± 0.12 mm vs. 1.80 ± 0.06 mm, P<0.001). Importantly, parthenolide treatment had no effect on heart rate or blood pressure. Parthenolide treatment almost completely abolished the Ang II-induced increase in the number of cells positive for prolyl-4-hydroxylase, a marker for collagen-synthesizing cells, as well as Ang II-induced interstitial fibrosis in the left ventricles. This was associated with significant attenuation of Ang II-induced increase in mRNA levels of type 1 collagen and fibronectin. Moreover, parthenolide attenuated the Ang II-induced expression of interleukin-6, a potent pro-hypertrophic fibroblast-derived factor. We conclude that pharmacological inhibition of STAT3 signaling by parthenolide has favorable effects on pressure overload-induced LVH through attenuation of fibroblast activation. Therefore parthenolide may prove as a useful therapy for certain cardiovascular disease.