SYNOPSIS Vascular injury and chronic arterial diseases result in exposure of vascular smooth muscle cells (VSMCs) to increased concentrations of growth factors. The mechanisms by which growth factors trigger VSMC phenotype transitions remain unclear. Because cellular reprogramming initiated by growth factors requires not only the induction of genes involved in cell proliferation but also the removal of contractile proteins, we hypothesized that autophagy is an essential modulator of VSMC phenotype. Treatment of VSMCs with platelet-derived growth factor (PDGF)-BB resulted in decreased expression of the contractile phenotype markers calponin and α-smooth muscle actin and upregulation of the synthetic phenotype markers osteopontin and vimentin. Autophagy, as assessed by LC3-II abundance, LC3 puncta formation and electron microscopy, was activated by PDGF exposure. Inhibition of autophagy with 3-methyladenine, spautin-1, or bafilomycin stabilized the contractile phenotype. In particular, spautin-1 led to a remarkable stabilization α-smooth muscle cell actin and calponin in PDGF-treated cells and prevented actin filament disorganization, diminished production of extracellular matrix and abrogated VSMC hyperproliferation and migration. Interestingly, treatment of cells with PDGF prevented protein damage and cell death due to exposure to the lipid peroxidation product, 4-hydroxynonenal. These results demonstrate a distinct form of autophagy induced by PDGF that is essential for attaining the synthetic phenotype and for survival under conditions of high oxidative stress found to occur in vascular lesions.

Protein transduction with cell penetrating peptides over the past several years has been shown to be an effective way of delivering proteins in vitro and now several reports have also shown valuable in vivo applications in correcting disease states. An impressive bioinspired phenomenon of crossing biological barriers came from HIV transactivator Tat protein. Specifically, the protein transduction domain of HIV-Tat has been shown to be a potent pleiotropic peptide in protein delivery. Various approaches such as molecular modeling, arginine guanidinium head group structural strategy, multimerization of PTD sequence and phage display system have been applied for taming of the PTD. This has resulted in identification of PTD variants which are efficient in cell membrane penetration and cytoplasmic delivery. Inspite of these state of the art technologies, the dilemma of low protein transduction efficiency and target specific delivery of PTD fusion proteins remains unsolved. Moreover, some misconceptions about PTD of Tat in the literature require considerations. We have assembled critical information on secretory, plasma membrane penetration and transcellular properties of Tat and PTD using molecular analysis and available experimental evidences.

WE, Tseng MT, Tyagi SC. Mitochondrial matrix metalloproteinase activation decreases myocyte contractility in hyperhomocysteinemia. myocyte N-methyl-D-aspartate receptor-1 (NMDA-R1) activation induces mitochondrial dysfunction. Matrix metalloproteinase protease (MMP) induction is a negative regulator of mitochondrial function. Elevated levels of homocysteine [hyperhomocysteinemia (HHCY)] activate latent MMPs and causes myocardial contractile abnormalities. HHCY is associated with mitochondrial dysfunction. We tested the hypothesis that HHCY activates myocyte mitochondrial MMP (mtMMP), induces mitochondrial permeability transition (MPT), and causes contractile dysfunction by agonizing NMDA-R1. The C57BL/6J mice were administered homocystinemia (1.8 g/l) in drinking water to induce HHCY. NMDA-R1 expression was detected by Western blot and confocal microscopy. Localization of MMP-9 in the mitochondria was determined using confocal microscopy. Ultrastructural analysis of the isolated myocyte was determined by electron microscopy. Mitochondrial permeability was measured by a decrease in light absorbance at 540 nm using the spectrophotometer. The effect of MK-801 (NMDA-R1 inhibitor), GM-6001 (MMP inhibitor), and cyclosporine A (MPT inhibitor) on myocyte contractility and calcium transients was evaluated using the IonOptix video edge track detection system and fura 2-AM. Our results demonstrate that HHCY activated the mtMMP-9 and caused MPT by agonizing NMDA-R1. A significant decrease in percent cell shortening, maximal rate of contraction (ϪdL/dt), and maximal rate of relaxation (ϩdL/dt) was observed in HHCY. The decay of calcium transient amplitude was faster in the wild type compared with HHCY. Furthermore, the HHCY-induced decrease in percent cell shortening, ϪdL/dt, and ϩdL/dt was attenuated in the mice treated with MK-801, GM-6001, and cyclosporin A. We conclude that HHCY activates mtMMP-9 and induces MPT, leading to myocyte mechanical dysfunction by agonizing NMDA-R1. myocyte; calcium; mitochondrial permeability; N-methyl-D-aspartate receptor-1; arrhythmogenesis THE PATHOPHYSIOLOGY of chronic heart failure (CHF) involves abnormalities in systolic and/or diastolic function and increases the propensity for reentry arrhythmias (30, 6). Continued elevation of cardiac sympathetic drive contributes to myocardial toxicity, leading to the decline in cardiac contractility (29). Recent observations suggest an increase in glutamatergic activity on sympathetic regulation, due to the upregulation of hypothalamic N-methyl-D-aspartate receptor-1 subunits (NMDA-R1) during CHF (16). Ischemia-and reperfusion-induced arrhythmias are sensitive to NMDA-R1 blockade (8).Hyperhomocysteinemia (HHCY) is a graded risk factor for CHF (12, 7) and for sudden cardiac death (SCD) resulting from coronary fibrous plaques (4, 1, 5). Homocysteinemia (HCY) induces interstitial cardiac fibrosis leading to systolic/diastolic dysfunction (13). The antagonist to the NMDA-R protects against HCY-induced oxidative damage in neurons (10) and protects against...

. Homocysteine-mediated activation and mitochondrial translocation of calpain regulates MMP-9 in MVEC. Am J Physiol Heart Circ Physiol 291: H2825-H2835, 2006. First published July 28, 2006 doi:10.1152/ajpheart.00377.2006 is associated with atherosclerosis, stroke, and dementia. Hcy causes extracellular matrix remodeling by the activation of matrix metalloproteinase-9 (MMP-9), in part, by inducing redox signaling and modulating the intracellular calcium dynamics. Calpains are the calcium-dependent cysteine proteases that are implicated in mitochondrial damage via oxidative burst. Mitochondrial abnormalities have been identified in HHcy. The mechanism of Hcy-induced extracellular matrix remodeling by MMP-9 activation via mitochondrial pathway is largely unknown. We report a novel role of calpains in mitochondrial-mediated MMP-9 activation by Hcy in cultured rat heart microvascular endothelial cells. Our observations suggested that calpain regulates Hcy-induced MMP-9 expression and activity. We showed that Hcy activates calpain-1, but not calpain-2, in a calcium-dependent manner. Interestingly, the enhanced calpain activity was not mirrored by the decreased levels of its endogenous inhibitor calpastatin. We presented evidence that Hcy induces the translocation of active calpain from cytosol to mitochondria, leading to MMP-9 activation, in part, by causing intramitochondrial oxidative burst. Furthermore, studies with pharmacological inhibitors of calpain (calpeptin and calpain-1 inhibitor), ERK (PD-98059) and the mitochondrial uncoupler FCCP suggested that calpain and ERK-1/2 are the major events within the Hcy/MMP-9 signal axis and that intramitochondrial oxidative stress regulates MMP-9 via ERK-1/2 signal cascade. Taken together, these findings determine the novel role of mitochondrial translocation of calpain-1 in MMP-9 activation during HHcy, in part, by increasing mitochondrial oxidative tress. cysteine proteases; thioredoxin; Nicotinamide adenine dinucleotide phosphate-oxidase-4; mitochondrial redox signaling; cardiovascular remodeling; calcium; extracellular regulated kinase 1/2; mitogenactivating protein kinase; calpastatin; antiproteolytic therapy; microvascular endothelial cells; matrix metalloproteinase-9 A GROWING BODY OF LITERATURE indicates that elevated levels of homocysteine [hyperhomocysteinemia (HHcy)] are an independent risk factor for coronary, cerebrovascular, and peripheral atherosclerotic diseases (5, 11, 12). Matrix metalloproteinases (MMPs) are the members of Zn-containing endopeptidases that share structural domains but differ in the substrate specificity, cellular sources, and induciblity that are responsible for matrix turnover. It is well known that Hcy-induced vascular dysfunction is caused by extracellular matrix remodeling. Hcy induces extracellular matrix remodeling by MMP-9 activation, in part, by inducing the redox signaling and modulating the intracellular calcium homeostasis (13,14,19,20).Calpains are the family of calcium-dependent cysteine proteases that have previously been i...

Extensive research has shown that increased production of reactive oxygen species (ROS) results in tissue injury under a variety of pathological conditions and chronic degenerative diseases. While ROS are highly reactive and can incite significant injury, polyunsaturated lipids in membranes and lipoproteins are their main targets. ROS-triggered lipid peroxidation reactions generate a range of reactive carbonyl species (RCS), and these RCS spread and amplify ROS-related injury. Several RCS generated in oxidizing lipids, such as 4-hydroxy trans-2-nonenal (HNE), 4-oxo-2-(E)-nonenal (ONE), acrolein, malondialdehyde (MDA) and phospholipid aldehydes have been shown to be produced under conditions of oxidative stress and contribute to tissue injury and dysfunction by depleting glutathione and other reductants leading to the modification of proteins, lipids, and DNA. To prevent tissue injury, these RCS are metabolized by several oxidoreductases, including members of the aldo-keto reductase (AKR) superfamily, aldehyde dehydrogenases (ALDHs), and alcohol dehydrogenases (ADHs). Metabolism via these enzymes results in RCS inactivation and detoxification, although under some conditions, it can also lead to the generation of signaling molecules that trigger adaptive responses. Metabolic transformation and detoxification of RCS by oxidoreductases prevent indiscriminate ROS toxicity, while at the same time, preserving ROS signaling. A better understanding of RCS metabolism by oxidoreductases could lead to the development of novel therapeutic interventions to decrease oxidative injury in several disease states and to enhance resistance to ROS-induced toxicity.