Syamantak Majumder

YAP/TAZ Are Mechanoregulators of TGF-β-Smad Signaling and Renal Fibrogenesis

Like many organs, the kidney stiffens after injury, a process that is increasingly recognized as an important driver of fibrogenesis. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are related mechanosensory proteins that bind to Smad transcription factors, the canonical mediators of profibrotic TGF-β responses. Here, we investigated the role of YAP/TAZ in the matrix stiffness dependence of fibroblast responses to TGF-β In contrast to growth on a stiff surface, fibroblast growth on a soft matrix led to YAP/TAZ sequestration in the cytosol and impaired TGF-β-induced Smad2/3 nuclear accumulation and transcriptional activity. YAP knockdown or treatment with verteporfin, a drug that was recently identified as a potent YAP inhibitor, elicited similar changes. Furthermore, verteporfin reduced YAP/TAZ levels and decreased the total cellular levels of Smad2/3 after TGF-β stimulation. Verteporfin treatment of mice subjected to unilateral ureteral obstruction similarly reduced YAP/TAZ levels and nuclear Smad accumulation in the kidney, and attenuated renal fibrosis. Our data suggest that organ stiffening cooperates with TGF-β to induce fibrosis in a YAP/TAZ- and Smad2/3-dependent manner. Interference with this YAP/TAZ and TGF-β/Smad crosstalk likely underlies the antifibrotic activity of verteporfin. Finally, through repurposing of a clinically used drug, we illustrate the therapeutic potential of a novel mechanointerference strategy that blocks TGF-β signaling and renal fibrogenesis.

Secreted Frizzled-Related Protein 4: An Angiogenesis Inhibitor

Wnt signaling is involved in developmental processes, cell proliferation, and cell migration. Secreted frizzled-related protein 4 (sFRP4) has been demonstrated to be a Wnt antagonist; however, its effects on endothelial cell migration and angiogenesis have not yet been reported. Using various in vitro assays, we show that sFRP4 inhibits endothelial cell migration and the development of sprouts and pseudopodia as well as disrupts the stability of endothelial rings in addition to inhibiting proliferation. sFRP4 interfered with endothelial cell functions by antagonizing the canonical Wnt/beta-catenin signaling pathway and the Wnt/planar cell polarity pathway. Furthermore, sFRP4 blocked the effect of vascular endothelial growth factor on endothelial cells. sFRP4 also selectively induced apoptotic events in endothelial cells by increasing cellular levels of reactive oxygen species. In vivo assays demonstrated a reduction in vascularity after sFRP4 treatment. Most importantly, sFRP4 restricted tumor growth in mice by interfering with endothelial cell function. The data demonstrate sFRP4 to be a potent angiogenesis inhibitor that warrants further investigation as a therapeutic agent in the control of angiogenesis-associated pathology.

Thalidomide attenuates nitric oxide-driven angiogenesis by interacting with soluble guanylyl cyclase

Background and purpose:  Nitric oxide (NO) promotes angiogenesis by activating endothelial cells. Thalidomide arrests angiogenesis by interacting with the NO pathway, but its putative targets are not known. Here, we have attempted to identify these targets.

Shear stress promotes nitric oxide production in endothelial cells by sub-cellular delocalization of eNOS: A basis for shear stress mediated angiogenesis

This study aims to investigate the role of shear stress in cellular remodeling and angiogenesis with relation to nitric oxide (NO). We observed a 2-fold increase in endothelial cell (EC) migration in relation to actin re-arrangements under 15 dyne/cm(2) shear stress. Blocking NO production inhibited the migration and ring formation of ECs by 6-fold and 5-fold, respectively under shear stress. eNOS-siRNA knockdown technique also ascertained a 3-fold reduction in shear stress mediated ring formation. In ovo artery ligation model with a half and complete flow block for 30 min showed a reduction of angiogenesis by 50% and 70%, respectively. External stimulation with NO donor showed a 2-fold recovery in angiogenesis under both half and complete flow block conditions. NO intensity clustering studies by using Diaminofluorescein diacetate (DAF-2DA) probed endothelial monolayer depicted pattern-changes in NO distribution and cluster formation of ECs under shear stress. Immunofluorescence and live cell studies revealed an altered sub-cellular localization pattern of eNOS and phospho-eNOS under shear stress. In conclusion, shear-induced angiogenesis is mediated by nitric oxide dependent EC migration.

Simulated microgravity perturbs actin polymerization to promote nitric oxide-associated migration in human immortalized Eahy926 cells

Microgravity causes endothelium dysfunctions and vascular endothelium remodeling in astronauts returning from space flight. Cardiovascular deconditioning occurs as a consequence of an adaptive response to microgravity partially due to the effects exerted at cellular level. Directional migration of endothelial cell which are central in maintaining the structural integrity of vascular walls is regulated by chemotactic, haptotactic, and mechanotactic stimuli which are essential for vasculogenesis. We explored the migration property of transformed endothelial cells (EC) exposed to 2-h microgravity, simulated using a three-dimensional clinostat constructed based on blueprint published by the Fokker Space, Netherlands. Migration of EC was measured using the scrap wound healing in the presence or absence of actin polymerization inhibitor—cytochalasin D (CD) in Eahy926 cell lines. Simulated microgravity increased cellular migration by 25% while CD-blocked microgravity induced cellular migration. The key migratory structures of cells, filopodia and lamellipodia, formed by EC were more in simulated microgravity compared to gravity. Parallel experiments with phalloidin and diaminorhodamine-4M (DAR-4M) showed that simulated microgravity caused actin rearrangements that lead to 25% increase in nitric oxide production. Further nitric oxide measurements showed a higher nitric oxide production which was not abrogated by phosphoinositol 3 kinase inhibitor (Wortmanin). Bradykinin, an inducer of nitric oxide, prompted two folds higher nitric oxide production along with simulated microgravity in a synergistic manner. We suggest that limited exposure to simulated microgravity increases Eahy926 cell migration by modulating actin and releasing nitric oxide.

Simulated microgravity promoted differentiation of bipotential murine oval liver stem cells by modulating BMP4/Notch1 signaling.

Faster growth and differentiation of liver stem cells to hepatocyte is one of the key factors during liver regeneration. In recent years, simulated microgravity, a physical force has shown to differentially regulate the differentiation and proliferation of stem cells. In the present work, we studied the effect of simulated microgravity on differentiation and proliferation of liver stem cells. The cells were subjected to microgravity, which was simulated using indigenously fabricated 3D clinostat. Proliferation, apoptosis, immunofluorescence assays and Western blot analysis were carried out to study the effects of simulated microgravity on liver stem cells. Microgravity treatment for 2 h enhanced proliferation of stem cells by twofold without inducing apoptosis and compromising cell viability. Analysis of hepatocyte nuclear factor 4‐α (HNF4‐α) expression after 2 h of microgravity treatment revealed that microgravity alone can induce the differentiation of stem cells within 2–3 days. Probing bone morphogenic protein 4 (BMP4) and Notch1 in microgravity treated stem cells elaborated downregulation of Notch1 and upregulation of BMP4 after 2 days of incubation. Further, blocking BMP4 using dorsomorphin and chordin conditioned media from chordin plasmid transfected cells attenuated microgravity mediated differentiation of liver stem cells. In conclusion, microgravity interplays with BMP4/Notch1 signaling in stem cells thus inducing differentiation of stem cells to hepatocytes. Present findings can be implicated in clinical studies where microgravity activated stem cells can regenerate the liver efficiently after liver injury. J. Cell. Biochem. 112: 1898–1908, 2011.

Simulated microgravity promotes nitric oxide-supported angiogenesis via the iNOS–cGMP–PKG pathway in macrovascular endothelial cells

Angiogenesis is a physiological process involving the growth of blood vessel in response to specific stimuli. The present study shows that limited microgravity treatments induce angiogenesis by activating macrovascular endothelial cells. Inhibition of nitric oxide production using pharmacological inhibitors and inducible nitric oxide synthase (iNOS) small interfering ribo nucleic acid (siRNA) abrogated microgravity induced nitric oxide production in macrovascular cells. The study further delineates that iNOS acts as a molecular switch for the heterogeneous effects of microgravity on macrovascular, endocardial and microvascular endothelial cells. Further dissection of nitric oxide downstream signaling confirms that simulated microgravity induces angiogenesis via the cyclic guanosine monophosphate (cGMP)–PKG dependent pathway.

The Histone Methyltransferase Enzyme Enhancer of Zeste Homolog 2 Protects against Podocyte Oxidative Stress and Renal Injury in Diabetes

Epigenetic regulation of oxidative stress is emerging as a critical mediator of diabetic nephropathy. In diabetes, oxidative damage occurs when there is an imbalance between reactive oxygen species generation and enzymatic antioxidant repair. Here, we investigated the function of the histone methyltransferase enzyme enhancer of zeste homolog 2 (EZH2) in attenuating oxidative injury in podocytes, focusing on its regulation of the endogenous antioxidant inhibitor thioredoxin interacting protein (TxnIP). Pharmacologic or genetic depletion of EZH2 augmented TxnIP expression and oxidative stress in podocytes cultured under high-glucose conditions. Conversely, EZH2 upregulation through inhibition of its regulatory microRNA, microRNA-101, downregulated TxnIP and attenuated oxidative stress. In diabetic rats, depletion of EZH2 decreased histone 3 lysine 27 trimethylation (H3K27me3), increased glomerular TxnIP expression, induced podocyte injury, and augmented oxidative stress and proteinuria. Chromatin immunoprecipitation sequencing revealed H3K27me3 enrichment at the promoter of the transcription factor Pax6, which was upregulated on EZH2 depletion and bound to the TxnIP promoter, controlling expression of its gene product. In high glucose-exposed podocytes and the kidneys of diabetic rats, the lower EZH2 expression detected coincided with upregulation of Pax6 and TxnIP. Finally, in a gene expression array, TxnIP was among seven of 30,854 genes upregulated by high glucose, EZH2 depletion, and the combination thereof. Thus, EZH2 represses the transcription factor Pax6, which controls expression of the antioxidant inhibitor TxnIP, and in diabetes, downregulation of EZH2 promotes oxidative stress. These findings expand the extent to which epigenetic processes affect the diabetic kidney to include antioxidant repair.

Evaluation of the role of nitric oxide in acid sensing ion channel mediated cell death

Acid sensing ion channels (ASICs) are widely expressed in central and peripheral nervous system. They are involved in a variety of physiological and pathophysiological processes: synaptic transmission, learning and memory, pain perception, ischemia, etc. During ischemia, metabolic acidosis causes the drop of extracellular pH (pHe) which in turn activates ASICs. Activation of calcium permeable ASIC1a has been implicated in neuronal death. ASICs are modulated by several redox reagents, divalent cations and nitric oxide (NO). Although NO potentiates ASIC mediated currents, the physiological significance of such modulation has not been studied in detail. We have evaluated the role of endogenous NO in cell death at different pH, mediated by the activation of ASICs. At pH 6.1, death rates of ASIC1 expressing Neuro2A (N2A) cells are significantly higher in comparison to the cells that do not express ASICs. Amiloride, a blocker of ASICs protects the cell from acid-injury. Sodium nitroprusside, a potent NO donor not only increases the ASIC mediated currents but also increases cell death at low pH. L-Arg, the precursor of NO also potentiates ASICs in a pH dependent manner. L-Arg-induced NO production and potentiation of ASICs were observed at pHs 7.4, 7.2, 7.0 and 6.8. Lowering the pH below 6.8 did not result in significant production of NO or potentiation of ASICs upon L-Arg stimulation. Our results suggest that potentiation of ASICs by NO and subsequent cell death in vivo depends on the severity of acidosis. During mild and moderate acidosis, NO promotes cell death by potentiating ASICs, whereas this potentiation subsides in severe acidosis due to inhibition of NO synthase.

G-Protein–Coupled Receptor-2–Interacting Protein-1 Is Required for Endothelial Cell Directional Migration and Tumor Angiogenesis via Cortactin-Dependent Lamellipodia Formation

Objective—Recent evidence suggests G-protein–coupled receptor-2–interacting protein-1 (GIT1) overexpression in several human metastatic tumors, including breast, lung, and prostate. Tumor metastasis is associated with an increase in angiogenesis. We have showed previously that GIT1 is required for postnatal angiogenesis during lung development. However, the functional role of GIT1 in pathological angiogenesis during tumor growth is unknown. Approach and Results—In the present study, we show inhibition of angiogenesis in matrigel implants as well as reduced tumor angiogenesis and melanoma tumor growth in GIT1-knockout mice. We demonstrate that this is a result of impaired directional migration of GIT1-depleted endothelial cells toward a vascular endothelial growth factor gradient. Cortactin-mediated lamellipodia formation in the leading edge is critical for directional migration. We observed a significant reduction in cortactin localization and lamellipodia formation in the leading edge of GIT1-depleted endothelial cells. We specifically identified that the Spa homology domain (aa 250–420) of GIT1 is required for GIT1–cortactin complex localization to the leading edge. The mechanisms involved extracellular signal-regulated kinases 1 and 2–mediated Cortactin-S405 phosphorylation and activation of Rac1/Cdc42. Finally, using gain of function studies, we show that a constitutively active mutant of cortactin restored directional migration of GIT1-depleted cells. Conclusion—Our data demonstrated that a GIT1–cortactin association through GIT1-Spa homology domain is required for cortactin localization to the leading edge and is essential for endothelial cell directional migration and tumor angiogenesis.