Malory Weber

Integrin β3 Crosstalk with VEGFR Accommodating Tyrosine Phosphorylation as a Regulatory Switch

Integrins mediate cell adhesion, migration, and survival by connecting intracellular machinery with the surrounding extracellular matrix. Previous studies demonstrated the importance of the interaction between β3 integrin and VEGF type 2 receptor (VEGFR2) in VEGF-induced angiogenesis. Here we present in vitro evidence of the direct association between the cytoplasmic tails (CTs) of β3 and VEGFR2. Specifically, the membrane-proximal motif around 801YLSI in VEGFR2 mediates its binding to non-phosphorylated β3CT, accommodating an α-helical turn in integrin bound conformation. We also show that Y747 phosphorylation of β3 enhances the above interaction. To demonstrate the importance of β3 phosphorylation in endothelial cell functions, we synthesized β3CT-mimicking Y747 phosphorylated and unphosphorylated membrane permeable peptides. We show that a peptide containing phospho-Y747 but not F747 significantly inhibits VEGF-induced signaling and angiogenesis. Moreover, phospho-Y747 peptide exhibits inhibitory effect only in WT but not in β3 integrin knock-out or β3 integrin knock-in cells expressing β3 with two tyrosines substituted for phenylalanines, demonstrating its specificity. Importantly, these peptides have no effect on fibroblast growth factor receptor signaling. Collectively these data provide novel mechanistic insights into phosphorylation dependent cross-talk between integrin and VEGFR2.

Interference with akt signaling protects against myocardial infarction and death by limiting the consequences of oxidative stress

Reducing Akt activity might alleviate cardiovascular complications and mortality associated with atherosclerosis. Protecting Against Oxidized Lipids Excessive blood concentrations of oxidized cholesterol lead to the development of fatty plaques in blood vessels, a process called atherosclerosis. Blockage of blood vessels by fragments of ruptured plaques can lead to heart attacks, a major cause of mortality in developed nations. Mice that cannot efficiently clear lipoprotein-bound cholesterol from the bloodstream are a model for spontaneous atherosclerosis-induced myocardial infarctions. Kerr et al. found that the kinase Akt1 was activated in these mice, which was associated with accumulation of oxidized lipids. Deletion of the gene encoding Akt1 in these mice improved survival, reduced the severity of various cardiovascular complications, and decreased the incidence of spontaneous myocardial infarctions. Thus, using clinically available Akt inhibitors to normalize Akt activity could help to decrease some of the pathological effects that result from excessive lipid oxidation and atherosclerosis. The intricacy of multiple feedback loops in the pathways downstream of Akt allows this kinase to control multiple cellular processes in the cardiovascular system and precludes inferring consequences of its activation in specific pathological conditions. Akt1, the major Akt isoform in the heart and vasculature, has a protective role in the endothelium during atherosclerosis. However, Akt1 activation may also have detrimental consequences in the cardiovascular system. Mice lacking both the high-density lipoprotein receptor SR-BI (scavenger receptor class B type I) and ApoE (apolipoprotein E), which promotes clearance of remnant lipoproteins, are a model of severe dyslipidemia and spontaneous myocardial infarction. We found that Akt1 was activated in these mice, and this activation correlated with cardiac dysfunction, hypertrophy, and fibrosis; increased infarct area; cholesterol accumulation in macrophages and atherosclerosis; and reduced life span. Akt1 activation was associated with inflammation, oxidative stress, accumulation of oxidized lipids, and increased abundance of CD36, a major sensor of oxidative stress, and these events created a positive feedback loop that exacerbated the consequences of oxidative stress. Genetic deletion of Akt1 in this mouse model resulted in decreased mortality, alleviation of multiple complications of heart disease, and reduced occurrence of spontaneous myocardial infarction. Thus, interference with Akt1 signaling in vivo could be protective and improve survival under dyslipidemic conditions by reducing oxidative stress and responses to oxidized lipids.

Elevated Soluble Fms-Like Tyrosine Kinase-1 and Placental-Like Growth Factor Levels Are Associated With Development and Mortality Risk in Heart Failure

Background—Vascular endothelial dysfunction may play an important role in the progression of heart failure (HF). We hypothesize that elevated levels of vascular markers, placental-like growth factor, and soluble Fms-like tyrosine kinase-1 (sFlt-1) are associated with adverse outcomes in patients with HF. We also assessed possible triggers of sFlt-1 elevation in animal HF models. Methods and Results—We measured plasma placental-like growth factor and sFlt-1 in 791 HF patients undergoing elective coronary angiogram. Median (interquartile range) placental-like growth factor and sFlt-1 levels were 24 (20–29) and 382 (277–953) pg/mL, respectively. After 5 years of follow-up, and after using receiver operator characteristic curves to determine optimal cutoffs, high levels of sFlt-1 (≥280 pg/mL; adjusted hazard ratio, 1.47; 95% confidence interval, 1.03–2.09; P=0.035) but not placental-like growth factor (≥25 pg/mL; adjusted hazard ratio, 1.26; 95% confidence interval, 0.94–1.71, P=0.12) were associated with adverse cardiovascular outcomes. In addition, significant elevation of sFlt-1 levels was observed in left anterior descending artery ligation and transverse aortic constriction HF mouse models after 4 and 8 weeks of follow-up, suggesting vascular stress and ischemia as triggers for sFlt-1 elevation in HF. Conclusions—Circulating sFlt-1 is generated as a result of myocardial injury and subsequent HF development. Elevated levels of sFlt-1 are associated with adverse outcomes in stable patients with HF.

High-density lipoprotein-associated paraoxonase-1 activity for prediction of adverse outcomes in outpatients with chronic heart failure.

Decreased arylesterase (ArylE) activity of paraoxonase‐1, a HDL‐associated protein with anti‐inflammatory and antioxidant properties, has been associated with increased risk of cardiac events in patients with ischaemic heart failure (HF). We aim to investigate the prognostic significance of changes in serum ArylE activity over time.

Telocinobufagin, a Novel Cardiotonic Steroid, Promotes Renal Fibrosis via Na+/K+-ATPase Profibrotic Signaling Pathways

Cardiotonic steroids (CTS) are Na+/K+-ATPase (NKA) ligands that are elevated in volume-expanded states and associated with cardiac and renal dysfunction in both clinical and experimental settings. We test the hypothesis that the CTS telocinobufagin (TCB) promotes renal dysfunction in a process involving signaling through the NKA α-1 in the following studies. First, we infuse TCB (4 weeks at 0.1 µg/g/day) or a vehicle into mice expressing wild-type (WT) NKA α-1, as well as mice with a genetic reduction (~40%) of NKA α-1 (NKA α-1+/−). Continuous TCB infusion results in increased proteinuria and cystatin C in WT mice which are significantly attenuated in NKA α-1+/− mice (all p < 0.05), despite similar increases in blood pressure. In a series of in vitro experiments, 24-h treatment of HK2 renal proximal tubular cells with TCB results in significant dose-dependent increases in both Collagens 1 and 3 mRNA (2-fold increases at 10 nM, 5-fold increases at 100 nM, p < 0.05). Similar effects are seen in primary human renal mesangial cells. TCB treatment (100 nM) of SYF fibroblasts reconstituted with cSrc results in a 1.5-fold increase in Collagens 1 and 3 mRNA (p < 0.05), as well as increases in both Transforming Growth factor beta (TGFb, 1.5 fold, p < 0.05) and Connective Tissue Growth Factor (CTGF, 2 fold, p < 0.05), while these effects are absent in SYF cells without Src kinase. In a patient study of subjects with chronic kidney disease, TCB is elevated compared to healthy volunteers. These studies suggest that the pro-fibrotic effects of TCB in the kidney are mediated though the NKA-Src kinase signaling pathway and may have relevance to volume-overloaded conditions, such as chronic kidney disease where TCB is elevated.

Paraoxonase 2 prevents the development of heart failure

Background: Mitochondrial oxidation is a major source of reactive oxygen species (ROS) and mitochondrial dysfunction plays a central role in development of heart failure (HF). Paraoxonase 2 deficient (PON2‐def) mitochondria are impaired in function. In this study, we tested whether PON2‐def aggravates HF progression. Methods and results: Using qPCR, immunoblotting and lactonase activity assay, we demonstrate that PON2 activity was significantly decreased in failing hearts despite increased PON2 expression. To determine the cardiac‐specific function of PON2, we performed heart transplantations in which PON2‐def and wild type (WT) donor hearts were implanted into WT recipient mice. Beating scores of the donor hearts, assessed at 4 weeks post‐transplantation, were significantly decreased in PON2‐def hearts when compared to WT donor hearts. By using a transverse aortic constriction (TAC) model, we found PON2 deficiency significantly exacerbated left ventricular remodeling and cardiac fibrosis post‐TAC. We further demonstrated PON2 deficiency significantly enhanced ROS generation in heart tissues post‐TAC. ROS generation was measured through dihydroethidium (DHE) using high‐pressure liquid chromatography (HPLC) with a fluorescent detector. By using neonatal cardiomyocytes treated with CoCl2 to mimic hypoxia, we found PON2 deficiency dramatically increased ROS generation in the cardiomyocytes upon CoCl2 treatment. In response to a short CoCl2 exposure, cell viability and succinate dehydrogenase (SDH) activity assessed by MTT assay were significantly diminished in PON2‐def cardiomyocytes compared to those in WT cardiomyocytes. PON2‐def cardiomyocytes also had lower baseline SDH activity. By using adult mouse cardiomyocytes and mitochondrial ToxGlo assay, we found impaired cellular ATP generation in PON2‐def cells compared to that in WT cells, suggesting that PON2 is necessary for proper mitochondrial function. Conclusion: Our study suggests a cardioprotective role for PON2 in both experimental and human heart failure, which may be associated with the ability of PON2 to improve mitochondrial function and diminish ROS generation. HIGHLIGHTSHuman heart primarily expresses PON2 while lactonase activity was decreased.Mice with genetic deficiency of PON2 had dilated cardiac remodeling upon insults.PON2 deficiency lowered baseline SDH activity and increased ROS production in hypoxia.Strategies to upregulate PON2 function might lead to novel cardioprotective.

INTERFERENCE WITH AKT SIGNALLING IN DYSLIPIDEMIA DIMINISHES MYOCARDIAL INFARCTION AND PROMOTES SURVIVAL BY INHIBITING OXIDATIVE STRESS.

Objectives Cardiovascular disease resulting in myocardial infarction (MI) is the leading cause of mortality in developed countries; however, the mechanisms controlling this complex disorder remain elusive. Since Akt activation was shown to be increased in human and mouse atherosclerotic lesions and failing hearts, we hypothesised that Akt signalling might contribute to several aspects of the pathogenesis of cardiovascular disease. Methods C57BL/6, ApoE−/− (C57BL/6 background), and SR-BI+/−ApoE−/− (mixed C57BL/6xS129 background) mice were purchased from Jackson Laboratories (Bar Harbor, ME). SR-BI+/−ApoE−/− mice were backcrossed to C57BL/6 background for 10 generations. Akt1−/− mice were generated as previously described) and backcrossed to the C57BL/6 background for 10 generations. SR-BI−/−ApoE−/− (DKO) mice were generated by intercrossing SR-BI+/−ApoE−/− mice. SR-BI−/−ApoE−/−Akt1−/− (TKO) mice were generated by first crossing Akt1−/− mice with SR-BI+/−ApoE−/− mice. Mice were sacrificed at 42 days of age for most experiments. Immunoblotting Hearts were homogenised in sample buffer. Proteins were detected with anti-Akt1, anti-phospho-Akt, anti-phospho-GSK-3α/β or anti-GSK-3β antibodies. Cardiac function was evaluated with a Sequoia C256 (Acuson) and M mode analysis was performed to measure ejection fraction (EF) and fraction of shortening (FS). Mice were sacrificed at 6 weeks of age when cardiac dysfunction was just becoming apparent to assess the role of Akt1 deletion in cardiac pathogenesis. Hearts were cut at the mid-level transversely, as reported previously, and embedded in paraffin. Serial sections (8 µm) were cut and stained with H&E or Massons trichrome to quantify fibrosis. Five images were taken of four sections per Massons trichrome stained heart on an Olympus BX51 microscope. Cardiac fibrosis was quantified by measuring the total stained area the total area of the heart using Image Pro. Atherosclerotic lesions were quantified by en face aortic coverage measured by computer-assisted planimetry. Aortae were cut open longitudinally, stained with Oil red O, and digitally scanned. VCAM-1 expression was assessed in endothelial cells, as demonstrated by CD31 staining, in the aortic arch and descending thoracic aorta by en face staining, followed by laser-scanning confocal microscopy (Leica TCS-SP) as previously described. Hearts were embedded in OCT freezing medium and sectioned at 7 µm. Sections were then fixed in 4% PFA and analysed for TUNEL staining using the In Situ Cell Death Detection Kit. Dihydroethidium (DHE, Invitrogen) staining for superoxide was carried out as previously described. Results Akt1 deletion under dyslipidemia alleviates cardiac dysfunction (EF37.67%±6.60 in TKO vs 21.94%±3.78 in DKO, FS 39.25%±3.23 in TKO vs 16.22%±3.09 in DKO), diminishes MI size (3.78%±0.864 in TKO vs 22.02%±5.926 in DKO), and, most importantly, prolongs lifespan (44.23±7.43 days in DKO vs 51.43±6.29 days in TKO). TKO mice exhibit reduced atherosclerosis. While dyslipidemia was equal, ROS generation and consequent oxidised lipid accumulation was dramatically reduced in TKO. Simultaneously, Akt1 deletion diminished CD36 expression, the main oxidised lipid receptor responsible for foam cell formation. Conclusions Interference with Akt activation improves survival during dyslipidemia by reducing oxidative stress and oxidised lipid responses thus providing a protective effect. Normalisation or prevention of Akt overactivation during atherogenesis might be beneficial for the treatment of atherosclerosis and heart failure.