Endorepellin, the C-terminal module of perlecan, has angiostatic activity. Here we provide definitive genetic and biochemical evidence that the functional endorepellin receptor is the ␣2␤1 integrin. Notably, the specific endorepellin binding to the receptor was cation-independent and was mediated by the ␣2 I domain. We show that the anti-angiogenic effects of endorepellin cannot occur in the absence of ␣2␤1. Microvascular endothelial cells from ␣2␤1 ؊/؊ mice, but not those isolated from either wild-type or ␣1␤1 ؊/؊ mice, did not respond to endorepellin. Moreover, syngeneic Lewis lung carcinoma xenografts in ␣2␤1 ؊/؊ mice failed to respond to systemic delivery of endorepellin. In contrast, endorepellin inhibited tumor growth and angiogenesis in the wild-type mice expressing integrin ␣2␤1. We conclude that the angiostatic effects of endorepellin in vivo are mediated by a specific interaction of endorepellin with the ␣2␤1 integrin receptor.The incorporation of new blood vessels into growing neoplasms is a prerequisite for tumor viability and progression. Accordingly, much attention has been invested in the search for and characterization of anti-angiogenic agents to enable regulated and inhibited tumor angiogenesis as part of cancer therapies (1). The proteoglycan perlecan plays a key role in the angiogenic process, primarily by modulating the availability and activity of growth factors involved in angiogenesis such as fibroblast growth factor 2, VEGF, 3 and platelet-derived growth factor (2-6). The most C-terminal part of perlecan (domain V), named endorepellin, is a powerful angiogenic inhibitor (7). Endorepellin carries three laminin-like globular (LG) domains separated by epidermal growth factor-like repeats (8) and binds to numerous extracellular matrix proteins, growth factors, and receptors including collagen XVIII, fibulin-2, nidogen, fibroblast growth factor 7, fibroblast growth factor-binding protein, ECM1 (7, 9 -12), ␣-dystroglycan, and integrin ␣2␤1 (9, 13-16). The endorepellin anti-angiogenic effect is parallel to several proteolytically released fragments from vascular basement membrane such as endostatin, the NC-1 domain of collagen type XVIII, and tumstatin, the NC-1 domain the of type IV collagen ␣3 chain (8,17,18). These fragments principally act on endothelial cells as "negative" ligands for specific integrin receptors. Endorepellin is a potent inhibitor in several angiogenesis assays such as endothelial cell migration, collagen-induced capillary morphogenesis, blood vessel recruitment into Matrigel plugs, and chicken chorioallontoic membrane (7,19). It also effectively retards in vivo tumor growth by specifically targeting tumor angiogenesis (20). We hypothesize that endorepellin takes effect via the LG3 domain binding to the integrin ␣2␤1 causing actin disassembly and therefore affecting three key steps of angiogenesis: endothelial cell adhesion, migration, and morphogenesis.Here we have further investigated the endorepellin-␣2␤1 integrin interactions by using cell-free experiments with a solub...

Rationale G protein-coupled receptor kinase 2 (GRK2) is an important molecule upregulated after myocardial injury and during heart failure. Myocyte-specific GRK2 loss before and after myocardial ischemic injury improves cardiac function and remodeling. The cardiac fibroblast plays an important role in the repair and remodeling events following cardiac ischemia; the importance of GRK2 in these events has not been investigated. Objective The aim of this study is to elucidate the in vivo implications of deleting GRK2 in the cardiac fibroblast after ischemia/reperfusion (I/R) injury. Methods and Results We demonstrate, using Tamoxifen inducible, fibroblast-specific GRK2 knockout mice, that GRK2 loss confers a protective advantage over control mice after myocardial I/R injury. Fibroblast GRK2 knockout mice presented with decreased infarct size and preserved cardiac function 24 hours post-I/R as demonstrated by increased ejection fraction (58.1±1.8% vs. 48.7±1.2% in controls, p<0.0005). GRK2 fibroblast knockout mice also had decreased fibrosis and fibrotic gene expression. Importantly, these protective effects correlated with decreased infiltration of neutrophils to the ischemia site and decreased levels of TNFα expression and secretion in GRK2 fibroblast knockout mice. Conclusions These novel data showing the benefits of inhibiting GRK2 in the cardiac fibroblast adds to previously published data showing the advantage of GRK2 ablation and reinforces the therapeutic potential of GRK2 inhibition in the heart after myocardial ischemia.

Heart failure (HF) causes a tremendous burden on the worldwide healthcare system, affecting more than 23 million people. There are many cardiovascular disorders that contribute to the development of HF and multiple risk factors that accelerate its occurrence, but regardless of its underlying cause, HF is characterized by a marked decrease in myocardial contractility and loss of pump function. One biomarker molecule consistently shown to be upregulated in human HF and several animal models is G protein-coupled receptor (GPCR) kinase 2 (GRK2), a kinase originally discovered to be involved in GPCR desensitization, especially β-adrenergic receptors (βARs). Indeed, higher levels of GRK2 can impair βAR-mediated inotropic reserve and its inhibition or molecular reduction has shown to improve pump function in several animal models including a pre-clinical pig model of HF. Recently, non-classical roles for GRK2 in cardiovascular disease have been described, including negative regulation of insulin signaling, a role in myocyte cell survival and apoptotic signaling, and it has been shown to be localized in/on mitochondria. These new roles of GRK2 suggest that GRK2 may be a nodal link in the myocyte, influencing both cardiac contractile function and cell metabolism and survival and contributing to HF independent of its canonical role on GPCR desensitization. In this review, classical and non-classical roles for GRK2 will be discussed, focusing on recently discovered roles for GRK2 in cardiomyocyte metabolism and the effects that these roles may have on myocardial contractile function and HF development.

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Rationale G protein-coupled receptor (GPCR) kinases (GRKs) are dynamic regulators of cellular signaling. GRK5 is highly expressed within myocardium and is up-regulated in heart failure (HF). Although GRK5 is a critical regulator of cardiac GPCR signaling, recent data has uncovered non-canonical activity of GRK5 within nuclei that plays a key role in pathological hypertrophy. Targeted cardiac elevation of GRK5 in mice leads to exaggerated hypertrophy and early HF after transverse aortic constriction (TAC) due to GRK5 nuclear accumulation. Objective In this study we investigated the role of GRK5 in physiological, swimming induced hypertrophy (SIH). Methods and Results Cardiac-specific GRK5 transgenic mice (TgGRK5) and non-transgenic littermate control (NLC) mice were subjected to a 21-day high intensity swim protocol (or no swim sham controls). SIH and specific molecular and genetic indices of physiological hypertrophy were assessed including nuclear localization of GRK5 and compared to TAC. Unlike after TAC, swim-trained TgGRK5 and NLC mice exhibited similar increases in cardiac growth. Mechanistically, SIH did not lead to GRK5 nuclear accumulation, which was confirmed in vitro as insulin-like growth factor-1, a known mediator of physiological hypertrophy, was unable to induce GRK5 nuclear translocation in myocytes. We found specific patterns of altered gene expression between TAC and SIH with GRK5 overexpression. Further, SIH in post-TAC TgGRK5 mice was able to preserve cardiac function. Conclusions These data suggest that while nuclear-localized GRK5 is a pathological mediator after stress, this non-canonical nuclear activity of GRK5 is not induced during physiological hypertrophy.

As average life expectancy continues to rise in the developed world, age-associated pathologies are increasing in prevalence. The hallmarks of cardiac ageing include cardiomyocyte loss, fibrosis and hypertrophy, all of which contribute to an increased incidence of cardiac disease. At the molecular level, cellular ageing is characterized by increased ROS production, mitochondrial dysfunction and the accumulation of damaged proteins and organelles. Cardiomyocytes and other senescent cell types rely upon autophagy, a lysosome-mediated degradation pathway, to remove potentially toxic protein aggregates and damaged organelles from the cellular milieu. However, increasing lines of evidence point to an age-associated decrease in cardiomyocyte autophagy, with predictably negative consequences for cardiac function and health. Conversely, stimulation of autophagy has been shown to improve cellular health and cardiac function and to increase lifespan in numerous model organisms. Clearly, autophagy represents a critical pathway for cellular vitality, as well as a promising therapeutic target for the treatment of age-related cardiac pathologies. In this review, we will discuss the mechanism of autophagy and its regulation in the cell, the role of autophagy in the ageing heart, and how the autophagy pathway might be targeted to improve cardiac health.

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A predominant concern following oil spills is toxicity to aquatic organisms. However, few data are available on effects in deep-sea cold water fishes. The present study had 3 major objectives. The first was to investigate the relative sensitivity of the deep-sea species Anoplopoma fimbria (sablefish) to acute effects of 3 aromatic compounds (toluene, 2-methylnaphthalene, and phenanthrene), dispersant alone, and chemically enhanced water accommodated fractions (CEWAFs) of Alaskan North Slope crude oil. The second was to determine the critical target lipid body burden (CTLBB) for sablefish by fitting aromatic hydrocarbon toxicity data to the target lipid model (TLM), which then allowed expression of CEWAF exposures in terms of dissolved oil toxic units. The final aim was to apply a passive sampling method that targets bioavailable, dissolved hydrocarbons as an alternative analytical technique for improved CEWAF exposure assessment. The results indicate that sablefish exhibit sensitivity to Corexit 9500 (96-h median lethal concentration [LC50] = 72.2 mg/L) within the range reported for other fish species. However, the acute CTLBB of 39.4 ± 2.1 μmol/g lies at the lower end of the sensitivity range established for aquatic species. The utility of both toxic units and passive sampling measurements for describing observed toxicity of dispersed oil is discussed. The present study is novel in that a new test species is investigated to address the uncertainty regarding the sensitivity of deep-sea fishes, while also employing modeling and measurements to improve exposure characterization in oil toxicity tests. Environ Toxicol Chem 2018;37:2210-2221. © 2018 SETAC.