KPC1 alleviates hypoxia/reoxygenation-induced apoptosis in rat cardiomyocyte cells though BAX degradation.

Bax triggers cell apoptosis by permeabilizing the outer mitochondrial membrane, leading to membrane potential loss and cytochrome c release. However, it is unclear if proteasomal degradation of Bax is involved in the apoptotic process, especially in heart ischemia-reperfusion (I/R)-induced injury. In the present study, KPC1 expression was heightened in left ventricular cardiomyocytes of patients with coronary heart disease (CHD), in I/R-myocardium in vivo and in hypoxia and reoxygenation (H/R)-induced cardiomyocytes in vitro. Overexpression of KPC1 reduced infarction size and cell apoptosis in I/R rat hearts. Similarly, the forced expression of KPC1 restored mitochondrial membrane potential (MMP) and cytochrome c release driven by H/R in H9c2 cells, whereas reducing cell apoptosis, and knockdown of KPC1 by short-hairpin RNA (shRNA) deteriorated cell apoptosis induced by H/R. Mechanistically, forced expression of KPC1 promoted Bax protein degradation, which was abolished by proteasome inhibitor MG132, suggesting that KPC1 promoted proteasomal degradation of Bax. Furthermore, KPC1 prevented basal and apoptotic stress-induced Bax translocation to mitochondria. Bax can be a novel target for the antiapoptotic effects of KPC1 on I/R-induced cardiomyocyte apoptosis and render mechanistic penetration into at least a subset of the mitochondrial effects of KPC1.

VEGF-A and VEGF-B Coordinate the Arteriogenesis to Repair the Infarcted Heart with Vagus Nerve Stimulation.

BACKGROUND/AIMS: Vagus nerve stimulation (VNS) suppresses arrhythmic activity and minimizes cardiomyocyte injury. However, how VNS affects angiogenesis/arteriogenesis in infarcted hearts, is poorly understood. METHODS: Myocardial infarction (MI) was achieved by ligation of the left anterior descending coronary artery (LAD) in rats. 7 days after LAD, stainless-steel wires were looped around the left and right vagal nerve in the neck for vagus nerve stimulation (VNS). The vagal nerve was stimulated with regular pulses of 0.2ms duration at 20 Hz for 10 seconds every minute for 4 hours, and then ACh levels by ELISA in cardiac tissue and serum were evaluated for its release after VNS. Three and 14 days after VNS, Real-time PCR, immunostaining and western blot were respectively used to determine VEGF-A/B expressions and α-SMA- and CD31-postive vessels in VNS-hearts with pretreatment of α7-nAChR blocker mecamylamine (10 mg/kg, ip) or mACh-R blocker atropine (10 mg/kg, ip) for 1 hour. The coronary function and left ventricular performance were analyzed by Langendorff system and hemodynamic parameters in VNS-hearts with pretreatment of VEGF-A/B-knockdown or VEGFR blocker AMG706. Coronary arterial endothelial cells proliferation, migration and tube formation were evaluated for angiogenesis following the stimulation of VNS in coronary arterial smooth muscle cells (VSMCs). RESULTS: VNS has been shown to stimulate VEGF-A and VEGF-B expressions in coronary arterial smooth muscle cells (VSMCs) and endothelial cells (ECs) with an increase of α-SMA- and CD31-postive vessel number in infarcted hearts. The VNS-induced VEGF-A/B expressions and angiogenesis were abolished by m-AChR inhibitor atropine and α7-nAChR blocker mecamylamine in vivo. Interestingly, knockdown of VEGF-A by shRNA mainly reduced VNS-mediated formation of CD31+ microvessels. In contrast, knockdown of VEGF-B powerfully abrogated VNS-induced formation of α-SMA+ vessels. Consistently, VNS-induced VEGF-A showed a greater effect on EC tube formation as compared to VNS-induced VEGF-B. Moreover, VEGF-A promoted EC proliferation and VSMC migration while VEGF-B induced VSMC proliferation and EC migration in vitro. Mechanistically, vagal neurotransmitter acetylcholine stimulated VEGF-A/B expressions through m/nACh-R/PI3K/Akt/Sp1 pathway in EC. Functionally, VNS improved the coronary function and left ventricular performance. However, blockade of VEGF receptor by antagonist AMG706 or knockdown of VEGF-A or VEGF-B by shRNA significantly diminished the beneficial effects of VNS on ventricular performance. CONCLUSION: VNS promoted angiogenesis/arteriogenesis to repair the infracted heart through the synergistic effects of VEGF-A and VEGF-B.

S100B promotes injury-induced vascular remodeling through modulating smooth muscle phenotype.

S100B is a biomarker of nervous system injury, but it is unknown if it is also involved in vascular injury. In the present study, we investigated S100B function in vascular remodeling following injury. Balloon injury in rat carotid artery progressively induced neointima formation while increasing S100B expression in both neointimal vascular smooth muscle (VSMC) and serum along with an induction of proliferating cell nuclear antigen (PCNA). Knockdown of S100B by its shRNA delivered by adenoviral transduction attenuated the PCNA expression and neointimal hyperplasia in vivo and suppressed PDGF-BB-induced VSMC proliferation and migration in vitro. Conversely, overexpression of S100B promoted VSMC proliferation and migration. Mechanistically, S100B altered VSMC phenotype by decreasing the contractile protein expression, which appeared to be mediated by NF-κB activity. S100B induced NF-κB-p65 gene transcription, protein expression and nuclear translocation. Blockade of NF-κB activity by its inhibitor reversed S100B-mediated downregulation of VSMC contractile protein and increase in VSMC proliferation and migration. It appeared that S100B regulated NF-κB expression through, at least partially, the Receptor for Advanced Glycation End products (RAGE) because RAGE inhibitor attenuated S100B-mediated NF-κB promoter activity as well as VSMC proliferation. Most importantly, S100B secreted from VSMC impaired endothelial tube formation in vitro, and knockdown of S100B promoted re-endothelialization of injury-denuded arteries in vivo. These data indicated that S100B is a novel regulator for vascular remodeling following injury and may serve as a potential biomarker for vascular damage or drug target for treating proliferative vascular diseases.

Vascular endothelial growth factor-C protects heart from ischemia/reperfusion injury by inhibiting cardiomyocyte apoptosis.

VEGF-C is a newly identified proangiogenic protein playing an important role in vascular disease and angiogenesis. However, its role in myocardial ischemia/reperfusion (I/R) injury remains unknown. The objective of this study was to determine the role and mechanism of VEGF-C in myocardial ischemia-reperfusion injury. Rat left ventricle myocardium was injected with recombinant human VEGF-C protein (0.1 or 1.0 µg/kg b.w.) 1 h prior to myocardial ischemia-reperfusion (I/R) injury. 24 h later, the myocardial infarction size, the number of TUNEL-positive cardiomyocytes, the levels of creatine kinase (CK), CK-MB, cardiac troponin, malondialdehyde (MDA) content, and apoptosis protein Bax expression were decreased, while Bcl2 and pAkt expression were increased in VEGF-C-treated myocardium as compared to the saline-treated I/R hearts. VEGF-C also improved the function of I/R-injured hearts. In the H2O2-induced H9c2 cardiomyocytes, which mimicked the I/R injury in vivo, VEGF-C pre-treatment decreased the LDH release and MDA content, blocked H2O2-induced apoptosis by inhibiting the pro-apoptotic protein Bax expression and its translocation to the mitochondrial membrane, and consequently attenuated H2O2-induced decrease of mitochondrial membrane potential and increase of cytochrome c release from mitochondria. Mechanistically, VEGF-C activated Akt signaling pathway via VEGF receptor 2, leading to a blockade of Bax expression and mitochondrial membrane translocation and thus protected cardiomyocyte from H2O2-induced activation of intrinsic apoptotic pathway. VEGF-C exerts its cardiac protection following I/R injury via its anti-apoptotic effect.

LUBAC promotes angiogenesis and lung tumorigenesis by ubiquitinating and antagonizing autophagic degradation of HIF1α.

Hypoxia-inducible factor 1 (HIF1) is critically important for driving angiogenesis and tumorigenesis. Linear ubiquitin chain assembly complex (LUBAC), the only known ubiquitin ligase capable of catalyzing protein linear ubiquitination to date, is implicated in cell signaling and associated with cancers. However, the role and mechanism of LUBAC in regulating the expression and function of HIF1α, the labile subunit of HIF1, remain to be elucidated. Herein we showed that LUBAC increases HIF1α protein expression in cultured cells and tissues of human lung cancer and enhances HIF1α DNA-binding and transcriptional activities, which are dependent upon LUBAC enzymatic activity. Mechanistically, LUBAC increases HIF1α stability through antagonizing HIF1α decay by the chaperone-mediated autophagy (CMA)-lysosome pathway, thereby potentiating HIF1α activity. We further demonstrated that HIF1α selectively interacts with HOIP (the catalytic subunit of LUBAC) primarily in the cytoplasm. LUBAC catalyzes linear ubiquitination of HIF1α at lysine 362. Linear ubiquitination shields HIF1α from interacting with heat-shock cognate protein of 70 kDa and lysosome-associated membrane protein type 2 A, two components of CMA. Consequently, linear ubiquitination confers protection against CMA-mediated destruction of HIF1α, increasing HIF1α stability and activity. We found that prolyl hydroxylation is not a perquisite for LUBAC's effects on HIF1α. Functionally, LUBAC facilitates proliferation, clonogenic formation, invasion and migration of lung cancer cells. LUBAC also boosts angiogenesis and exacerbates lung cancer growth in mice, which are greatly compromised by inhibition of HIF1α. This work provides novel mechanistic insights into the role of LUBAC in regulating HIF1α homeostasis, tumor angiogenesis and tumorigenesis of lung cancer, making LUBAC an attractive therapeutic target for cancers.

Inactivation of EGLN3 hydroxylase facilitates Erk3 degradation via autophagy and impedes lung cancer growth.

EGLN3 is critically important for growth of various cancers including lung cancer. However, virtually nothing is known about the role and mechanism for EGLN3 hydroxylase activity in cancers. EGLN3 catalyzes the hydroxylation of extracellular signal-regulated kinase 3 (Erk3), a potent driver of cancers. The role and mechanism for EGLN3-induced stabilization of Erk3 remain to be defined. Here, we show that Erk3 interacts with heat shock cognate protein of 70 kDa (HSC70) and lysosome-associated membrane protein type 2 A (LAMP2A), two core components of chaperone-mediated autophagy (CMA). As a consequence, Erk3 is degraded by the CMA-lysosome pathway. EGLN3-catalyzed hydroxylation antagonizes CMA-dependent destruction of Erk3. Mechanistically, hydroxylation blunts the interaction of Erk3 with LAMP2A, thereby blocking lysosomal decay of Erk3. EGLN3 inactivation inhibits macrophage migration, efferocytosis, and M2 polarization. Studies using EGLN3 catalytically inactive knock-in mice indicate that inactivation of EGLN3 hydroxylase in host cells ameliorates LLC cancer growth through reprogramming the tumor microenvironment (TME). Adoptive transfer of macrophages with inactivated EGLN3 restrains tumor growth by mounting anti-tumor immunity and restricting angiogenesis. Administration of EGLN3 hydroxylase pharmacologic inhibitor to mice bearing LLC carcinoma impedes cancer growth by targeting the TME. LLC cells harboring inactivated EGLN3 exhibit reduced tumor burden via mitigating immunosuppressive milieu and inducing cancer senescence. This study provides novel insights into the role of CMA in regulating Erk3 stability and the mechanism behind EGLN3-enhanced stability of Erk3. This work demonstrates that inactivation of EGLN3 in malignant and stromal cells suppresses tumor by orchestrating reciprocal interplays between cancer cells and the TME. This work sheds new light on the role and mechanism for EGLN3 catalytic activity in regulating cancer growth. Manipulating EGLN3 activity holds promise for cancer treatment.

Novel Role for Tranilast in Regulating NLRP3 Ubiquitination, Vascular Inflammation, and Atherosclerosis.

Background Aberrant activation of the NLRP3 (nucleotide-binding oligomerization domain, leucine-rich repeat-containing receptor family pyrin domain-containing 3) inflammasome is thought to play a causative role in atherosclerosis. NLRP3 is kept in an inactive ubiquitinated state to avoid unwanted NLRP3 inflammasome activation. This study aimed to test the hypothesis that pharmacologic manipulating of NLRP3 ubiquitination blunts the assembly and activation of the NLRP3 inflammasome and protects against vascular inflammation and atherosclerosis. Since genetic studies yielded mixed results about the role for this inflammasome in atherosclerosis in low-density lipoprotein receptor- or apolipoprotein E-deficient mice, this study attempted to clarify the discrepancy with the pharmacologic approach using both models. Methods and Results We provided the first evidence demonstrating that tranilast facilitates NLRP3 ubiquitination. We showed that tranilast restricted NLRP3 oligomerization and inhibited NLRP3 inflammasome assembly. Tranilast markedly suppressed NLRP3 inflammasome activation in low-density lipoprotein receptor- and apolipoprotein E-deficient macrophages. Through reconstitution of the NLRP3 inflammasome in human embryonic kidney 293T cells, we found that tranilast directly limited NLRP3 inflammasome activation. By adopting different regimens for tranilast treatment of low-density lipoprotein receptor- and apolipoprotein E-deficient mice, we demonstrated that tranilast blunted the initiation and progression of atherosclerosis. Mice receiving tranilast displayed a significant reduction in atherosclerotic lesion size, concomitant with a pronounced decline in macrophage content and expression of inflammatory molecules in the plaques compared with the control group. Moreover, tranilast treatment of mice substantially hindered the expression and activation of the NLRP3 inflammasome in the atherosclerotic lesions. Conclusions Tranilast potently enhances NLRP3 ubiquitination, blunts the assembly and activation of the NLRP3 inflammasome, and ameliorates vascular inflammation and atherosclerosis in both low-density lipoprotein receptor- and apolipoprotein E-deficient mice.

Continuous exposure of isoprenaline inhibits myoblast differentiation and fusion through PKA/ERK1/2-FOXO1 signaling pathway.

AIM: The objective of this study is to determine if exuberant sympathetic nerve activity is involved in muscle satellite cell differentiation and myoblast fusion. METHODS AND RESULTS: By using immunoassaying and western blot analyses, we found that ß1 and ß2-adrenergic receptors (AdR) were expressed in C2C12 cells. The differentiated satellite cells exhibited an increased expression of ß2-AdR, as compared with the proliferating cells. Continuous exposure of isoprenaline (ISO), a ß-AdR agonist, delayed C2C12 cell differentiation, and myoblast fusion in time- and dose-dependent manner. ISO also increased short myotube numbers while decreasing long myotube numbers, consistent with the greater reduction in MyHC1, MyHC2a, and MyHC2x expression. Moreover, continuous exposure of ISO gradually decreased the ratio of PKA RI/RII, and PKA RI activator efficiently reversed the ISO effect on C2C12 cell differentiation and myoblast fusion while PKA inhibitor H-89 deteriorated the effects. Continuous single-dose ISO increased ß1-AdR expression in C2C12 cells. More importantly, the cells showed enhanced phospho-ERK1/2 levels, resulting in increasing phospho-ß2-AdR levels while decreasing ß2-AdR levels, and the specific effects could be abolished by ERK1/2 inhibitor. Furthermore, continuous exposure of ISO induced FOXO1 nuclear translocation and increased the levels of FOXO1 in nuclear extracts while reducing pAKT, p-p38MAPK, and pFOXO1 levels. Conversely, blockade of ERK1/2 signaling partially abrogated ISO effects on AKT, p38MAPK, and FOXO1signaling, which partially restored C2C12 cell differentiation and myoblast fusion, leading to an increase in the numbers of medium myotube along with the increased expression of MyHC1 and MyHC2a. CONCLUSION: Continuous exposure of ISO impedes satellite cell differentiation and myoblast fusion, at least in part, through PKA-ERK1/2-FOXO1 signaling pathways, which were associated with the reduced ß2-AdR and increased ß1-AdR levels.