Angiogenesis plays a critical role in physiology and pathophysiology of the human body; hence, it is important to explore the methods to study angiogenesis under in vitro and in vivo settings. Here, we describe three different methods to assess angiogenesis using Matrigel: an in vitro two- or three-dimensional (2D/3D) tube formation or angiogenesis assay using endothelial cells with growth factor supplemented Matrigel, an ex vivo sprouting angiogenesis assay embedding aortic rings in the Matrigel, and finally, Matrigel plug assays wherein Matrigels are implanted into the flanks of mice to assess the recruitment of endothelial cells to form new blood vessels in vivo.

BACKGROUND: Left ventricular hypertrophy is a bipolar response, starting as an adaptive response to the hemodynamic challenge, but over time develops maladaptive pathology partly due to microvascular rarefaction and impaired coronary angiogenesis. Despite the profound influence on cardiac function, the mechanotransduction mechanisms that regulate coronary angiogenesis, leading to heart failure, are not well known. METHODS: We subjected endothelial-specific knockout mice of mechanically activated ion channel, TRPV4 (transient receptor potential cation channel subfamily V member 4; TRPV4 ECKO ) to pressure overload via transverse aortic constriction and examined cardiac function, cardiomyocyte hypertrophy, cardiac fibrosis, and apoptosis. Further, we measured microvascular density and underlying TRPV4 mechanotransduction mechanisms using human microvascular endothelial cells, extracellular matrix gels of varying stiffness, unbiased RNA sequencing, small interfering RNA, Western blot, quantitative-PCR, and confocal immunofluorescence techniques. RESULTS: We demonstrate that endothelial-specific deletion of TRPV4 preserved cardiac function, cardiomyocyte structure, and reduced cardiac fibrosis compared with TRPV4 lox/lox mice, 28 days post–transverse aortic constriction. Interestingly, comprehensive RNA sequencing analysis revealed an upregulation of proangiogenic factors (VEGFα [vascular endothelial growth factor α], NOS3 [nitric oxide synthase 3], and FGF2 [fibroblast growth factor 2]) with concomitant increase in microvascular density in TRPV4 ECKO hearts after transverse aortic constriction compared with TRPV4 lox/lox . Further, an increased expression of VEGFR2 (vascular endothelial growth factor receptor 2) and activation of the YAP (yes-associated protein) pathway were observed in TRPV4 ECKO hearts. Mechanistically, we found that downregulation of TRPV4 in endothelial cells induced matrix stiffness–dependent activation of YAP and VEGFR2 via the Rho/Rho kinase/large tumor suppressor kinase pathway. CONCLUSIONS: Our results suggest that endothelial TRPV4 acts as a mechanical break for coronary angiogenesis, and uncoupling endothelial TRPV4 mechanotransduction attenuates pathological cardiac hypertrophy by enhancing coronary angiogenesis.

Transient receptor potential vanilloid 4 (TRPV4) channels are mechanosensitive ion channels that regulate systemic endothelial cell (EC) functions such as vasodilation, permeability, and angiogenesis. TRPV4 is expressed in retinal ganglion cells, Müller glia, pigment epithelium, microvascular ECs, and modulates cell volume regulation, calcium homeostasis, and survival. TRPV4‐mediated physiological or pathological retinal angiogenesis remains poorly understood. Here, we demonstrate that TRPV4 is expressed, functional, and mechanosensitive in retinal ECs. The genetic deletion of TRPV4 did not affect postnatal developmental angiogenesis but increased pathological neovascularization in response to oxygen‐induced retinopathy (OIR). Retinal vessels from TRPV4 knockout mice subjected to OIR exhibited neovascular tufts that projected into the vitreous humor and displayed reduced pericyte coverage compared with wild‐type mice. These results suggest that TRPV4 is a regulator of retinal angiogenesis, its deletion augments pathological retinal angiogenesis, and that TRPV4 could be a novel target for the development of therapies against neovascular ocular diseases.

Angiogenesis, the formation of new blood vessels from existing ones, is a normal physiological process. However, deregulation of angiogenesis can lead to pathological states such as cancer, that is characterized by hyper‐permeable and tortuous vessels. We have recently shown a significant decrease in functional expression of the mechanosensitive ion channel, transient potential receptor vanilloid 4 (TRPV4), in tumor endothelial cells (TEC). Further, pharmacological activation of TRPV4 induced normalization of tumor vasculature and improved cancer therapy. However, the molecular mechanisms by which TRPV4 is downregulated in TEC is not yet known. To determine this mechanism, we focused on extracellular vesicles (EVs) derived from tumor cells. We first collected conditioned media (TCM) from tumor cells with and without pre‐treatment of an exosome inhibitor, GW4869. We found that treatment of human normal endothelial cells (hNEC) with TCM transformed them into tumor‐endothelial like (hTEC) phenotype as revealed by expression of TEM8, VEGFR2 membrane translocation, and abnormal tube formation. However, TCM from exosome inhibitor‐treated cells, failed to induce endothelial transformation. Further, we found that EVs isolated from TCM induced hNEC transformation to hTEC. Mechanistically, we found that tumor derived EVs induced functional downregulation of TRPV4 in hNEC as assessed by calcium imaging. Taken together, our results suggest that tumor derived EVs transforms normal endothelial cells via downregulation of TRPV4 channels.Support or Funding InformationNational Institutes of Health R15CA202847 and R01HL119705

Cardiac fibrosis caused by adverse cardiac remodeling following myocardial infarction can eventually lead to heart failure. Although the role of soluble factors such as TGF-β is well studied in cardiac fibrosis following myocardial injury, the physiological role of mechanotransduction is not fully understood. Here, we investigated the molecular mechanism and functional role of TRPV4 mechanotransduction in cardiac fibrosis. TRPV4KO mice, 8 weeks following myocardial infarction (MI), exhibited preserved cardiac function compared to WT mice. Histological analysis demonstrated reduced cardiac fibrosis in TRPV4KO mice. We found that WT CF exhibited hypotonicity-induced calcium influx and extracellular matrix (ECM)-stiffness-dependent differentiation in response to TGF-β1. In contrast, TRPV4KO CF did not display hypotonicity-induced calcium influx and failed to differentiate on high-stiffness ECM gels even in the presence of saturating amounts of TGF-β1. Mechanistically, TRPV4 mediated cardiac fibrotic gene promoter activity and fibroblast differentiation through the activation of the Rho/Rho kinase pathway and the mechanosensitive transcription factor MRTF-A. Our findings suggest that genetic deletion of TRPV4 channels protects heart from adverse cardiac remodeling following MI by modulating Rho/MRTF-A pathway-mediated cardiac fibroblast differentiation and cardiac fibrosis.

Coronary microvascular dysfunction has been identified as one of the underlying causes for progression of heart failure following hypertrophy or myocardial infarction. However, the mechanisms underlying microvascular dysfunction during the progression of heart failure are unknown. Recently, we found that transient receptor potential vanilloid 4 (TRPV4) channel acts as mechanosensor in endothelial cells and negatively regulates angiogenesis. Therefore, to determine the role of TRPV4 in coronary microvascular function during pathological hypertrophy, we have induced pressure-overload in WT and TRPV4KO mice via transverse aortic constriction (TAC). We found that TAC-induced cardiomyocyte hypertrophy and reduced cardiac function in WT mice, after 28 days. In contrast, both myocyte structure and cardiac function were preserved in TRPV4KO-TAC compared to WT-TAC mice. Notably, WT-TAC hearts exhibited increased ECM deposition and reduced microvascular density compared to TRPV4KO-TAC hearts, suggesting that the absence of TRPV4 may protect myocardium from pressure-overload-induced stress. To evaluate the specific role of endothelial TRPV4 in coronary microvascular function, endothelial specific TRPV4KO (TRPV4 ECKO ) mice were generated by crossing TRPV4 lox/lox mice with Tie2-Cre mice. After confirming endothelial deletion of TRPV4 through RT-PCR and immunostaining, we have subjected TRPV4 lox/lox and TRPV4 ECKO mice to TAC. Immuno-histological analysis revealed that TRPV4 ECKO hearts exhibited increased microvascular density compared to TRPV4 lox/lox mice, 28 days post TAC. Further, we found preserved cardiac structure (myocyte cross sectional area) and cardiac function (% ejection fraction and fractional shortening) with reduced cardiac fibrosis in TRPV4 ECKO mice compared to TRPV4 lox/lox , post TAC. Thus, our results suggest that endothelial TRPV4 channels are key regulators of coronary microvasculature function and deletion of endothelial TRPV4 offers cardio-protection via increased coronary angiogenesis following pressure overload-induced by TAC.

The soluble and mechanical microenvironment surrounding endothelial cells influences and instructs them to form new blood vessels. The cells in the pathological tumor microenvironment release extracellular vesicles (EVs) for paracrine signaling. EVs have been shown to induce angiogenesis by communicating with endothelial cells, but the underlying molecular mechanisms are not well known. We have recently shown that the mechanosensitive ion channel transient receptor vanilloid 4 (TRPV4) expression and activity is significantly reduced in tumor endothelial cells (TEC), and that activation of TRPV4 normalized the tumor vasculature and improved cancer therapy. However, whether and how the tumor microenvironment downregulates TRPV4 and transforms the normal endothelial cell phenotype remains unknown. To explore this, we exposed normal human endothelial cells (hNEC) to human lung tumor cell conditioned media (TCM) and measured phenotypic changes and angiogenesis. We found that treatment with TCM transformed hNEC to a TEC-like phenotype (hTEC) as evidenced by increased expression of tumor endothelial cell marker 8 (TEM8) and exhibition of abnormal angiogenesis on 2D-Matrigels compared to normal hNEC. Mechanistically, expression and activity of TRPV4 was decreased in hTEC. Further, when pre-treated with exosome inhibitor GW4869, TCM failed to induce hNEC transformation to hTEC. Finally, addition of purified EVs from TCM induced transformation of hNEC to hTEC as evidenced by abnormal angiogenesis in vitro. Taken together, our results suggest that the pathological (tumor) microenvironment transforms normal endothelial cells into a tumor endothelial cell-like phenotype through EVs via the downregulation of TRPV4.

The transient receptor potential vanilloid 4 (TRPV4) channel is a mechanosensor in endothelial cells (EC) that regulates cyclic strain-induced reorientation and flow-mediated nitric oxide production. We have recently demonstrated that TRPV4 expression is reduced in tumor EC and tumors grown in TRPV4KO mice exhibited enhanced growth and immature leaky vessels. However, the mechanism by which TRPV4 regulates tumor vascular integrity and metastasis is not known. Here, we demonstrate that VE-cadherin expression at the cell-cell contacts is significantly reduced in TRPV4-deficient tumor EC and TRPV4KO EC. In vivo angiogenesis assays with Matrigel of varying stiffness (700-900 Pa) revealed a significant stiffness-dependent reduction in VE-cadherin-positive vessels in Matrigel plugs from TRPV4KO mice compared with WT mice, despite an increase in vessel growth. Further, syngeneic Lewis Lung Carcinomatumor experiments demonstrated a significant decrease in VE-cadherin positive vessels in TRPV4KO tumors compared with WT. Functionally, enhanced tumor cell metastasis to the lung was observed in TRPV4KO mice. Our findings demonstrate that TRPV4 channels regulate tumor vessel integrity by maintaining VE-cadherin expression at cell-cell contacts and identifies TRPV4 as a novel target for metastasis.

Anticancer drugs exert their effects on cancer cells by deregulating many pathways linked to cell cycle, apoptosis, etc. but cancer cells gradually become resistive against anticancer drugs, thereby necessitating the development of newer generation anticancer molecules. N-end rule pathway has been shown to be involved in the degradation of many cell cycle and apoptosis-related proteins. However, the involvements of this pathway in cancer are not well established. Recently, we developed a non-peptide-based N-end rule pathway inhibitor, RF-C11 for type 1 and 2 recognition domains of E3 ubiquitin ligases. The inhibitor significantly increased the half-life of potential N-degrons leading to significant physiological changes in vivo. We hypothesized RF-C11 may be used to decipher the N-end rule pathway's role in cancer towards the development of anticancer therapeutics. In this study, we showed that RF-C11, barring noncancer cells, significantly sensitizes cancer cells towards different anticancer agents tested. We further find that the profound cellular sensitization to anticancer drugs was affected by (a) downregulation of X-linked inhibitor of apoptosis protein, an antiapoptotic protein and (b) by stabilization of RAD21, and thereby inhibiting metaphase to anaphase promotion. The study shows that RF-C11 or its analogs may be used as a novel additive in combination therapy against cancer.

Abnormal angiogenesis and hyper‐permeable vessels characterize the tumor vasculature. Together, these attributes contribute to atypical growth rate and metastasis of tumor cells. We have recently shown that the mechanosensitive ion channel, transient receptor vanilloid 4 (TRPV4) expression and activity is significantly reduced in tumor endothelial cells (TEC), and that activation of TRPV4 normalized tumor vasculature and improved cancer therapy. However, whether and how the tumor microenvironment downregulates TRPV4 and transforms normal endothelial cell phenotype remains unknown. To explore this, we repeatedly exposed normal human endothelial cells (NhEC) to human lung adenocarcinoma (tumor) cell conditioned media and measured phenotypic changes, angiogenesis, and functional expression of TRPV4. We found that treatment with cancer cell conditioned media changed NhEC to a tumor endothelial‐like phenotype (ThEC) as evidenced by; increased expression of tumor endothelial cell marker 8 (TEM8) and exhibition of abnormal angiogenesis on 2D‐Matrigels compared to normal NhEC. Mechanistically, we observed that expression and activity of TRPV4 was reduced in ThEC. Finally, we found that treatment with a Rho kinase inhibitor, Y‐27632 normalized abnormal tube formation exhibited by ThEC. Taken together, our results suggest that the tumor microenvironment transforms normal endothelial cells into tumor endothelial cell‐like phenotype through the downregulation of TRPV4.Support or Funding InformationNIH (1RO1HL119705), NIH (1R15CA202847‐01), and start‐up funds from NEOMED (CKT).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Left ventricular hypertrophy (LVH) starts as an adaptive response of the heart to a hemodynamic challenge, e.g., hypertension, that normalizes wall stress. However, over time this adaptive response often progresses towards a maladaptive pathology with a progressive loss of ventricular function frequently leading to heart failure which is the cause of 1 of 8 deaths in the United States. Although impaired perfusion is one of the major reasons for heart failure, the mechanisms underlying this insidious progression from adaptation to maladaptation are unknown. Importantly, the role of mechanical factors and their molecular mechanisms associated with mechanotransduction in coronary angiogenesis and heart failure are understudied. We have previously shown that transient receptor potential vanilloid 4 (TRPV4) channel is an endothelial mechanosensor and negatively regulates tumor angiogenesis. Therefore, we investigated if TRPV4 mechanotransduction regulates coronary angiogenesis during myocardial stress. To determine this, we subjected WT and global TRPV4KO mice to pressure overload‐induced hypertrophy through transverse aortic construction (TAC). 2D echocardiography analysis revealed that cardiac structure and function is preserved in TRPV4KO‐TAC mice compared to WT‐TAC. Histologically, hearts from global TRPV4KO mice exhibited decreased myocyte cross‐sectional area and fibrosis compared to WT‐TAC. Notably, we found that TRPV4KO hearts exhibited increased capillary density than WT hearts, post‐TAC suggesting that absence of TRPV4 may contribute to these protective effects. To elucidate the specific role of endothelial TRPV4 in coronary angiogenesis, we have generated the endothelial‐specific TRPV4KO (TRPV4ECKO) mice by crossing TRPV4lox/lox mice with Tie2‐Cre mice. First, we have characterized and confirmed the deletion of TRPV4 by genotyping and functional assays in isolated endothelial cells. We then subjected TRPV4ECKO and TRPV4lox/lox mice to pressure overload and assessed the cardiac function up to 28 days using echocardiography. Confocal microscopy revealed larger and properly aligned vessels in TRPV4ECKO hearts compared to TRPV4lox/lox hearts. Further, we found preserved cardiac structure (myocyte cross‐sectional area) and cardiac function (% ejection fraction and fractional shortening) with reduced cardiac fibrosis in TRPV4ECKO mice compared to TRPV4lox/lox post‐TAC, like the global TRPV4KO TAC hearts. Thus, our results suggest that deletion of endothelial TRPV4 preserves cardiac function, myocyte structure and reduces fibrosis via increased coronary angiogenesis following pressure overload‐induced by TAC.Support or Funding InformationNational Institutes of Health (NIH) (R01HL119705 and R15CA202847), American Heart Association (AHA) Grant‐in‐Aid (14GRNT20380935), and start‐up funds from NEOMED (CKT).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ion channels have been implicated in tumor growth and metastasis and are considered as one of the promising therapeutic targets. Previously, we have shown that transient receptor potential vanilloid 4 (TRPV4) ion channel is a mechanosensor in endothelial cells (EC) which is downregulated in tumor endothelial cells (TEC). TEC exhibit high basal Rho activity, abnormal migration and angiogenesis in vitro. Mechanistically, we found that TRPV4 regulates EC migration and angiogenesis by integrating mechanical (Rho) and soluble (VEGF) signaling. Importantly, we found that tumor implantation in global TRPV4KO mice resulted in enhanced tumor growth, angiogenesis and lung metastasis compared to wild type mice suggesting that TRPV4 in endothelium negatively regulates tumor angiogenesis. However, the specific role of endothelial TRPV4 in tumor growth, angiogenesis and metastasis is not known. To determine this, we have generated endothelial specific TRPV4KO (TRPV4 ECKO ) mice by crossing TRPV4 lox/lox mice with Tie2‐Cre mice. By employing syngeneic Lewis lung carcinoma tumor model, we found that deletion of endothelial TRPV4 enhanced tumor growth in TRPV4 ECKO mice compared to TRPV4 lox/lox mice. Multiphoton microscopic analysis of tumor sections infused with Alexa‐Fluor isolectin‐B4 revealed tortuous tumor vasculature in TRPV4 ECKO mice compared to TRPV4 lox/lox mice. Further, we found that these vessels show reduced pericyte coverage with concomitant leakiness as evidenced by colocalization of CD31/α‐SMA and dextran leakage, respectively. Western blot analysis revealed increased tyrosine phosphorylation of VEGFR2 at Y1175 (p‐VEGFR2‐Y 1175 ) in tumors from TRPV4 ECKO mice compared to TRPV4 lox/lox . Finally, we found increased metastasis of tumor cells to the lung in TRPV4 ECKO mice. Our results thus confirm that endothelial TRPV4 is a negative regulator of tumor angiogenesis and metastasis and identifies TRPV4 is a novel VEGF‐independent target for tumor angiogenesis and metastasis or vascular normalization. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Ischemic heart disease (IHD) is the major underlying cause of myocardial infarction (MI), scarring, and hypertrophy leading to heart failure. Cardiac remodeling following myocardial infarction involves scar formation by synthesis and reorganization of ECM which is mediated by myofibroblasts. We have previously shown that the mechanosensitive ion channel TRPV4 (transient receptor potential vanilloid channel 4) regulates cardiac fibroblast differentiation into myofibroblasts via integration of soluble and mechanical signaling. However, the physiological or translational significance of TRPV4 in cardiac remodeling following MI is unknown. To determine this, we have subjected WT and TRPV4KO mice to MI (permanent LAD ligation). 2D‐echocardiography revealed that the cardiac function (ejection fraction and fractional shortening) is preserved post‐MI in TRPV4KO mice compared to WT mice. Further, we found reduced fibrosis at infarcted and remote zones in TRPV4KO‐MI hearts compared to WT‐MI and sham hearts. Furthermore, TRPV4KO hearts exhibited decreased cardiomyocyte apoptosis (TUNEL assay) and increased capillary density (CD31 staining) post‐MI compared to WT hearts. To explore the translational significance of these findings, in separate experiments, we have given an orally active TRPV4 antagonist GSK2193874, immediately after MI surgery and followed for 5 weeks. Cardiac function analysis revealed that both ejection fraction and fractional shortening were preserved in GSK2193874‐treated WT mice compared to either WT or vehicle treated mice. Our results thus suggest that targeting TRPV4 protects the heart from myocardial infarction‐induced damage by preserving cardiac structure and function via reduced myocyte apoptosis, diminished fibrosis and increased revascularization, and identifies TRPV4 as a novel therapeutic target for heart failure.Support or Funding InformationNIH (1RO1HL119705), NIH (1R15CA202847‐01).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

VEGF signaling via VEGF receptor-2 (VEGFR2) is a major regulator of endothelial cell (EC) functions, including angiogenesis. Although most studies of angiogenesis focus on soluble VEGF signaling, mechanical signaling also plays a critical role. Here, we examined the consequence of disruption of mechanical signaling on soluble signaling pathways. Specifically, we observed that small interfering RNA (siRNA) knockdown of a mechanosensitive ion channel, transient receptor potential vanilloid 4 (TRPV4), significantly reduced perinuclear (Golgi) VEGFR2 in human ECs with a concomitant increase in phosphorylation at Y1175 and membrane translocation. TRPV4 knockout (KO) ECs exhibited increased plasma membrane localization of phospho-VEGFR2 compared with normal ECs. The knockdown also increased phospho-VEGFR2 in whole cell lysates and membrane fractions compared with control siRNA-treated cells. siRNA knockdown of TRPV4 enhanced nuclear localization of mechanosensitive transcription factors, yes-associated protein/transcriptional coactivator with PDZ-binding motif via rho kinase, which were shown to increase VEGFR2 trafficking to the plasma membrane. Furthermore, TRPV4 deletion/knockdown enhanced VEGF-mediated migration in vitro and increased expression of VEGFR2 in vivo in the vasculature of TRPV4 KO tumors compared with wild-type tumors. Our results thus show that TRPV4 channels regulate VEGFR2 trafficking and activation to identify novel cross-talk between mechanical (TRPV4) and soluble (VEGF) signaling that controls EC migration and angiogenesis.-Kanugula, A. K., Adapala, R. K., Midha, P., Cappelli, H. C., Meszaros, J. G., Paruchuri, S., Chilian, W. M., Thodeti, C. K., Novel noncanonical regulation of soluble VEGF/VEGFR2 signaling by mechanosensitive ion channel TRPV4.

VEGF and VEGFR2 signaling is the major regulator of endothelial functions such as proliferation, migration, and angiogenesis. While many studies on the modulation of angiogenesis focus on targeting VEGF signaling, mechanical forces also play critical role. We have recently shown that the mechanosensitive ion channel transient receptor potential vanilloid 4 (TRPV4) negatively regulates angiogenesis via modulation of Rho/Rho kinase‐dependent endothelial mechanosensing. Nevertheless, it is not known if a cross‐talk exists between TRPV4 and VEGF signaling. To explore this nexus, we silenced TRPV4 in endothelial cells (EC) and measured VEGFR2 expression, localization, and phosphorylation. Immunofluorescence analysis revealed significant localization of total‐VEGFR2 around the perinuclear (Golgi) compartment in control EC. Small interfering RNA (siRNA) knockdown of TRPV4 significantly decreased perinuclear localization of VEGFR2. Interestingly, we found increased phosphorylation of VEGFR2 at Y1175 and its localization to the plasma membrane in TRPV4 knocked‐down cells. We also found increased plasma membrane localization of phospho‐VEGFR2 Y1175 in TRPV4 null EC. Western blot analysis further revealed a significant increase in phospho‐VEGFR2 at Y1175 in whole cell lysates and membrane fractions of TRPV4 knocked‐down cells compared to control siRNA‐treated cells. Finally, TRPV4 deletion/knockdown also increased VEGF‐mediated migration with localization of phospho‐VEGFR2 at the leading edge of migrating EC. Taken together, our results suggest that TRPV4 channels regulate VEGFR2 localization and activation to identify novel cross‐talk between mechanical (TRPV4) and soluble (VEGF) signaling in EC.Support or Funding InformationThis work was supported by National Institutes of Health (NIH) R01HL119705 and National Cancer Institute (NCI) R15CA202847This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ischemic heart disease (IHD) is one of the leading causes of death in the world. Developing novel strategies to limit the extent of ischemic injury is a promising therapeutic approaches for IHD, including myocardial infarction and heart failure. Oxidative stress, generated by ischemia/reperfusion (I/R), often contributes to cellular death due to the perturbations in the powerhouse of the cell, the "mitochondria". Ischemia-induced cell death is mediated through apoptosis, necrosis, or autophagy. Apoptosis, a tightly coordinated process of "programmed" cell death, involves cell shrinkage, fragmentation, followed by phagocytosis. Apoptosis is mediated via both extrinsic death receptor (DR) and intrinsic mitochondrial pathways leading to caspase activation and cytochrome C release by Bcl-2 family proteins, respectively. In contrast, necrosis, considered a random series of uncoordinated events, involves cell swelling, rupture of the plasma membrane, and breakdown of the cell. Recent evidence suggests that necrosis is carried out by concerted or programmed molecular events, termed necroptosis. Necrosis is also mediated by extrinsic caspase-independent pathways and intrinsic mitochondrial pathways leading to the opening of mitochondrial permeability transition pore (mPTP) [ [1] Konstantinidis K. Whelan R.S. Kitsis R.N. Mechanisms of cell death in heart disease. Arterioscler. Thromb. Vasc. Biol. 2012; 32: 1552-1562 Crossref PubMed Scopus (271) Google Scholar ]. However, the molecular mechanisms that mediate necroptosis are not clearly known. AMP-activated protein kinase protects against necroptosis via regulation of Keap1-PGAM5 complexInternational Journal of CardiologyVol. 259PreviewThe AMP-activated protein kinase (AMPK) plays critical roles in growth regulation and metabolism reprogramming. AMPK activation protects cells against apoptosis from injury in different cell and animal models. However, its function in necroptosis remains largely unclear. Full-Text PDF

Abnormal angiogenesis is a hallmark of pathological conditions characterized by leaky immature vessels. Most of the studies on pathological angiogenesis including vascular normalization strategies are focused on the inhibition of pro‐angiogenic growth factor signaling, which has been met with limited success. However, there are few studies on endogenous regulators and/or mechanical signaling in the regulation of pathological angiogenesis. We have recently found mechanosensitive ion channel transient receptor potential vanilloid 4 (TRPV4) to be functionally low in tumor‐derived endothelial cells which exhibit aberrant mechanosensitivity and abnormal angiogenesis that may be imparted due to high basal Rho activity. Further, we demonstrated that tumors grown in TRPV4KO animals show enhanced growth as well as leaky immature vasculature. In the present study, we investigated the molecular mechanisms by which TRPV4 modulates vascular integrity. First, immunostaining revealed that normal EC exhibited distinct VE‐cadherin expression at the cell‐cell contacts in vitro which is significantly reduced in TRPV4KO EC. Next, we measured vascular growth in varying stiffness Matrigel plugs (700–900 Pa) implanted in WT and TRPV4KO mice. Interestingly, we found increased vascular growth with increasing stiffness from TRPV4KO mice. In contrast, Matrigel plugs from WT mice showed no significant vascular growth with increasing stiffness. Surprisingly, we found a significant stiffness‐dependent reduction in VE‐cadherin positive vessels in Matrigel plugs from TRPV4KO mice compared to WT mice, despite an increase in vascular growth. Further, using an in vivo tumor model with WT and TRPV4KO mice, we found a significant increase in VE‐cadherin negative vessels in TRPV4KO mice compared to their WT counterparts. Functionally, we found tumor vessels are leaky which enhanced Lewis lung carcinoma (LLC) tumor metastasis to lung in TRPV4KO mice. Taken together, our findings suggest mechanosensitive ion channel TRPV4 regulates vessel integrity by maintaining VE‐cadherin expression at cell‐cell contacts and identifies TRPV4 as a novel target for vascular normalization therapies. Support or Funding Information This work is supported by American Heart Association (AHA) Grant‐in‐aid (14GRNT20380935), NIH‐R15CA202847, and start‐up funds from NEOMED (CKT).

Angiogenesis is critical for cardiovascular function, however, insufficient or excessive angiogenesis has been implicated in various diseases. Although both soluble (VEGF) and mechanical factors regulate angiogenesis, most of the studies on angiogenesis are focused on targeting soluble signaling. We have recently shown that mechanosensitive ion channel TRPV4 (transient receptor potential vanilloid 4) negatively regulates angiogenesis via modulation of Rho/Rho kinase‐dependent endothelial mechanosensing. We also found that pharmacological activation of TRPV4 normalized abnormal tumor vasculature and improved cancer therapy. However, it is not known if there is a cross‐talk exists between TRPV4 and VEGF signaling. To explore this cross‐talk, we knocked down TRPV4 in human endothelial cells and measured VEGFR2 expression, localization and phosphorylation. We found a significant increase in VEGFR2 phosphorylation at Y951 (kinase insert domain) and Y1175 (c‐terminal domain) in TRPV4 siRNA knocked down cells. Further, we found that knockdown of TRPV4 increased VEGFR2 expression with concomitant increase in angiogenic transcriptional factor GATA2. Importantly, immunofluorescence analysis revealed reduced accumulation of VEGFR2 in perinuclear Golgi compartment suggesting increased trafficking of VEGFR2. Taken together, our results suggest that TRPV4 mechanotransduction regulates VEGFR2 expression and trafficking and identifies a novel cross‐talk between mechanical (TRPV4) and soluble (VEGF) signaling in human endothelial cells.Support or Funding InformationNIH‐NHLBI (R01), NIH‐NCI (R15), AHA (Grant‐in‐Aid), NIH‐NHLBI (R15)