Publications by authors named "Sailaja Paruchuri"

42 Publications

Spatial survey of non-collagenous proteins in mineralizing and non-mineralizing vertebrate tissues .

Bone Rep 2021 Jun 10;14:100754. Epub 2021 Feb 10.

School of Polymer Science and Polymer Engineering, The University of Akron, Akron, OH 44325, USA.

Bone biomineralization is a complex process in which type I collagen and associated non-collagenous proteins (NCPs), including glycoproteins and proteoglycans, interact closely with inorganic calcium and phosphate ions to control the precipitation of nanosized, non-stoichiometric hydroxyapatite (HAP, idealized stoichiometry Ca(PO)(OH)) within the organic matrix of a tissue. The ability of certain vertebrate tissues to mineralize is critically related to several aspects of their function. The goal of this study was to identify specific NCPs in mineralizing and non-mineralizing tissues of two animal models, rat and turkey, and to determine whether some NCPs are unique to each type of tissue. The tissues investigated were rat femur (mineralizing) and tail tendon (non-mineralizing) and turkey leg tendon (having both mineralizing and non-mineralizing regions in the same individual specimen). An experimental approach was designed for this investigation by combining sequential protein extraction with comprehensive protein mapping using proteomics and Western blotting. The extraction method enabled separation of various NCPs based on their association with either the extracellular organic collagenous matrix phases or the inorganic mineral phases of the tissues. The proteomics work generated a complete picture of NCPs in different tissues and animal species. Subsequently, Western blotting provided validation for some of the proteomics findings. The survey then yielded generalized results relevant to various protein families, rather than only individual NCPs. This study focused primarily on the NCPs belonging to the small leucine-rich proteoglycan (SLRP) family and the small integrin-binding ligand N-linked glycoproteins (SIBLINGs). SLRPs were found to be associated only with the collagenous matrix, a result suggesting that they are mainly involved in structural matrix organization and not in mineralization. SIBLINGs as well as matrix Gla (γ-carboxyglutamate) protein were strictly localized within the inorganic mineral phase of mineralizing tissues, a finding suggesting that their roles are limited to mineralization. The results from this study indicated that osteocalcin was closely involved in mineralization but did not preclude possible additional roles as a hormone. This report provides for the first time a spatial survey and comparison of NCPs from mineralizing and non-mineralizing tissues and defines the proteome of turkey leg tendons as a model for vertebrate mineralization.
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http://dx.doi.org/10.1016/j.bonr.2021.100754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900015PMC
June 2021

Endothelial TRPV4 channels prevent tumor growth and metastasis via modulation of tumor angiogenesis and vascular integrity.

Angiogenesis 2021 Mar 3. Epub 2021 Mar 3.

Department of Integrative Medical Sciences, Northeast Ohio Medical University, Rootstown, OH, 44272, USA.

Transient receptor potential vanilloid 4 (TRPV4) is a ubiquitously expressed polymodally activated ion channel. TRPV4 has been implicated in tumor progression; however, the cell-specific role of TRPV4 in tumor growth, angiogenesis, and metastasis is unknown. Here, we generated endothelial-specific TRPV4 knockout (TRPV4) mice by crossing TRPV4 mice with Tie2-Cre mice. Tumor growth and metastasis were significantly increased in a syngeneic Lewis lung carcinoma tumor model of TRPV4 mice compared to TRPV4 mice. Multiphoton microscopy, dextran leakage, and immunohistochemical analysis revealed increased tumor angiogenesis and metastasis that were correlated with aberrant leaky vessels (increased width and reduced pericyte and VE-cadherin coverage). Mechanistically, increases in VEGFR2, p-ERK, and MMP-9 expression and DQ gelatinase activity were observed in the TRPV4 mouse tumors. Our results demonstrated that endothelial TRPV4 is a critical modulator of vascular integrity and tumor angiogenesis and that deletion of TRPV4 promotes tumor angiogenesis, growth, and metastasis.
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http://dx.doi.org/10.1007/s10456-021-09775-9DOI Listing
March 2021

Transient receptor potential vanilloid 4 channel deletion regulates pathological but not developmental retinal angiogenesis.

J Cell Physiol 2021 May 20;236(5):3770-3779. Epub 2020 Oct 20.

Department of Integrative Medical Sciences, Northeast Ohio Medical University, Rootstown, Ohio, USA.

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.
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http://dx.doi.org/10.1002/jcp.30116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7920906PMC
May 2021

The role of TRPV4 channels in ocular function and pathologies.

Exp Eye Res 2020 12 29;201:108257. Epub 2020 Sep 29.

Department of Integrative Medical Sciences, Northeast Ohio Medical University, Rootstown, OH, 44272, USA. Electronic address:

Transient potential receptor vanilloid 4 (TRPV4) is an ion channel responsible for sensing osmotic and mechanical signals, which in turn regulates calcium signaling across cell membranes. TRPV4 is widely expressed throughout the body, and plays an important role in normal physiological function, as well as different pathologies, however, its role in the eye is not well known. In the eye, TRPV4 is expressed in various tissues, such as the retina, corneal epithelium, ciliary body, and the lens. In this review, we provide an overview on TRPV4 structure, activation, mutations, and summarize the current knowledge of TRPV4 function and signaling mechanisms in various locations throughout the eye, as well as its role in ocular diseases, such as glaucoma and diabetic retinopathy. Based on the available data, we highlight the therapeutic potential of TRPV4 as well as the shortcomings of current research. Finally, we provide future perspectives on the implications of targeting TRPV4 to treat various ocular pathologies.
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http://dx.doi.org/10.1016/j.exer.2020.108257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7736234PMC
December 2020

Transient Receptor Potential Vanilloid channel regulates fibroblast differentiation and airway remodeling by modulating redox signals through NADPH Oxidase 4.

Sci Rep 2020 06 17;10(1):9827. Epub 2020 Jun 17.

Department of Chemistry, University of Akron, Akron, OH, US.

Asthma is characterized by pathological airway remodeling resulting from persistent myofibroblast activation. Although transforming growth factor beta 1 (TGFβ1), mechanical signals, and reactive oxygen species (ROS) are implicated in fibroblast differentiation, their integration is still elusive. We identified that Transient Receptor Potential Vanilloid 4 (TRPV4), a mechanosensitive ion channel mediates lung fibroblast (LF) differentiation and D. farinae-induced airway remodeling via a novel TRPV4-NADPH Oxidase 4 (NOX4) interaction. NOX4-mediated ROS production is essential for TGFβ1-induced LF differentiation via myocardin-related transcription factor-A (MRTF-A) and plasminogen activator inhibitor 1 (PAI-1). Importantly, TRPV4 inhibition prevented TGFβ1-induced NOX4 expression and ROS production. Both TRPV4 and NOX4 are activated by phosphatidylinositol 3-kinase (PI3K) downstream of TGFβ1, and signals from both TRPV4 and Rac are necessary for NOX4 upregulation. Notably, NOX4 expression is higher in fibroblasts derived from asthmatic patients (disease human LF; DHLF) in comparison to non-asthmatics (normal human LF; NHLF). Further, NOX4 expression is up-regulated in the lungs of D.farinae-treated wild type mice (WT) relative to saline-treated WT, which was attenuated in TRPV4 knockout (KO) mice. Our findings suggest that TRPV4 integrates TGFβ1 and ROS signaling through NOX4 and, TRPV4-NOX4 interaction is amenable to target lung remodeling during asthma.
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http://dx.doi.org/10.1038/s41598-020-66617-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7299963PMC
June 2020

TRPV4 deletion protects heart from myocardial infarction-induced adverse remodeling via modulation of cardiac fibroblast differentiation.

Basic Res Cardiol 2020 01 10;115(2):14. Epub 2020 Jan 10.

Department of Integrative Medical Sciences, Northeast Ohio Medical University, Rootstown, OH, 44272, USA.

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.
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http://dx.doi.org/10.1007/s00395-020-0775-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7322630PMC
January 2020

Extracellular Vesicles From Pathological Microenvironment Induce Endothelial Cell Transformation and Abnormal Angiogenesis via Modulation of TRPV4 Channels.

Front Cell Dev Biol 2019 17;7:344. Epub 2019 Dec 17.

Department of Integrative Medical Sciences, College of Medicine, Northeast Ohio Medical University, Rootstown, OH, United States.

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 . 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.
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http://dx.doi.org/10.3389/fcell.2019.00344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6928002PMC
December 2019

A NIR-emitting cyanine with large Stokes shifts for live cell imaging: large impact of the phenol group on emission.

Chem Commun (Camb) 2019 Oct;55(88):13223-13226

Department of Chemistry, The University of Akron, Akron, OH 44325, USA.

There are a limited number of near-infrared (NIR) emitting (λem = 700-900 nm) molecular probes for imaging applications. A NIR-emitting probe that exhibits emission at ∼800 nm with a large Stokes shift was synthesized and found to exhibit excellent selectivity towards mitochondria for live-cell imaging. The photophysical properties were attributed to an excited "cyanine structure" via intramolecular charge transfer (ICT) involving a phenol group.
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http://dx.doi.org/10.1039/c9cc06831gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6918678PMC
October 2019

Cysteinyl leukotriene 2 receptor promotes endothelial permeability, tumor angiogenesis, and metastasis.

Proc Natl Acad Sci U S A 2019 01 17;116(1):199-204. Epub 2018 Dec 17.

Department of Chemistry, University of Akron, Akron, OH 44325;

Cysteinyl leukotrienes (cys-LTs) are proinflammatory mediators that enhance vascular permeability through distinct receptors (CysLTRs). We found that CysLTR regulates angiogenesis in isolated mouse endothelial cells (ECs) and in Matrigel implants in WT mice and enhances EC contraction and permeability via the Rho-dependent myosin light chain 2 and vascular endothelial (VE)-cadherin axis. Since solid tumors utilize aberrant angiogenesis for their growth and metastasis and their vessels exhibit vascular hyperpermeability, we hypothesized that CysLTR, via its actions on the endothelium, might regulate tumor growth. Both tumor growth and metastases of adoptively transferred syngeneic Lewis lung carcinoma (LLC) cells are significantly reduced in CysLTR-null mice () compared with WT and CysLTR-null mice (). In WT recipients of LLC cells, CysLTR expression is significantly increased in the tumor vasculature, compared with CysLTR. Further, the tumor vasculature in recipients exhibited significantly improved integrity, as revealed by increased pericyte coverage and decreased leakage of i.v.-administered Texas Red-conjugated dextran. Administration of a selective CysLTR antagonist significantly reduced LLC tumor volume, vessel density, dextran leakage, and metastases in WT mice, highlighting CysLTR as a VEGF-independent regulator of the vasculature promoting risk of metastasis. Thus, both genetic and pharmacological findings establish CysLTR as a gateway for angiogenesis and EC dysregulation in vitro and ex vivo and in an in vivo model with a mouse tumor. Our data suggest CysLTR as a possible target for intervention.
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http://dx.doi.org/10.1073/pnas.1817325115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6320507PMC
January 2019

Mechanosensitive TRPV4 channels stabilize VE-cadherin junctions to regulate tumor vascular integrity and metastasis.

Cancer Lett 2019 02 27;442:15-20. Epub 2018 Oct 27.

Department of Integrative Medical Sciences, Northeast Ohio Medical University, Rootstown, OH, 44272, USA; School of Biomedical Sciences, Kent State University, Kent, OH, 44240, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.canlet.2018.07.042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6924277PMC
February 2019

Novel noncanonical regulation of soluble VEGF/VEGFR2 signaling by mechanosensitive ion channel TRPV4.

FASEB J 2019 01 29;33(1):195-203. Epub 2018 Jun 29.

Department of Integrative Medical Sciences, Northeast Ohio Medical University, Rootstown, Ohio, USA.

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.
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http://dx.doi.org/10.1096/fj.201800509RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6355071PMC
January 2019

Form follows function: polymorphisms in mAKAP alter cardiac cAMP/PKA signaling.

Am J Physiol Heart Circ Physiol 2018 09 4;315(3):H626-H628. Epub 2018 May 4.

Department of Integrative Medical Sciences, Northeast Ohio Medical University, Rootstown, Ohio.

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http://dx.doi.org/10.1152/ajpheart.00248.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6172640PMC
September 2018

An NIR-emitting lysosome-targeting probe with large Stokes shift via coupling cyanine and excited-state intramolecular proton transfer.

Chem Commun (Camb) 2017 Mar;53(26):3697-3700

Department of Chemistry, University of Akron, Akron, Ohio 44325, USA.

An NIR-emitting probe (λ ∼ 700 nm) with a large Stokes shift (Δλ ≈ 234 nm) is synthesized by using excited-state intramolecular proton transfer (ESIPT). The phenolic proton, which controls ESIPT, acts as a switch to give strong fluorescence at pH ≈ 5. The probe can selectively show lysosome organelles, therefore leading to a lysosome probe without exhibiting "an alkalinizing effect".
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http://dx.doi.org/10.1039/c7cc00700kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5492890PMC
March 2017

Synthesis, characterization, in vitro SAR and in vivo evaluation of N,N'bisnaphthylmethyl 2-alkyl substituted imidazolium salts against NSCLC.

Bioorg Med Chem Lett 2017 02 16;27(4):764-775. Epub 2017 Jan 16.

Department of Chemistry, University of Akron, Akron, OH 44325, United States. Electronic address:

Alkyl- and N,N'-bisnaphthyl-substituted imidazolium salts were tested in vitro for their anti-cancer activity against four non-small cell lung cancer cell lines (NCI-H460, NCI-H1975, HCC827, A549). All compounds had potent anticancer activity with 2 having IC values in the nanomolar range for three of the four cell lines, a 17-fold increase in activity against NCI-H1975 cells when compared to cisplatin. Compounds 1-4 also showed high anti-cancer activity against nine NSCLC cell lines in the NCI-60 human tumor cell line screen. In vitro studies performed using the Annexin V and JC-1 assays suggested that NCI-H460 cells treated with 2 undergo an apoptotic cell death pathway and that mitochondria could be the cellular target of 2 with the mechanism of action possibly related to a disruption of the mitochondrial membrane potential. The water solubilities of 1-4 was over 4.4mg/mL using 2-hydroxypropyl-β-cyclodextrin as a chemical excipient, thereby providing sufficient solubility for systemic administration.
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http://dx.doi.org/10.1016/j.bmcl.2017.01.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5575737PMC
February 2017

Mechanosensitive transient receptor potential vanilloid 4 regulates -induced airway remodeling 2 distinct pathways modulating matrix synthesis and degradation.

FASEB J 2017 04 10;31(4):1556-1570. Epub 2017 Jan 10.

Department of Chemistry, University of Akron, Akron, Ohio, USA; and

Contributions of mechanical signals to airway remodeling during asthma are poorly understood. Transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive ion channel, has been implicated in cardiac and pulmonary fibrosis; however, its role in asthma remains elusive. Employing a -induced asthma model, we report here that TRPV4-knockout mice were protected from -induced airway remodeling. Furthermore, lung fibroblasts that were isolated from TRPV4-knockout mice showed diminished differentiation potential compared with wild-type mice. Fibroblasts from asthmatic lung exhibited increased TRPV4 activity and enhanced differentiation potential compared with normal human lung fibroblasts. Of interest, TGF-β1 treatment enhanced TRPV4 activation in a PI3K-dependent manner in normal human lung fibroblasts Mechanistically, TRPV4 modulated matrix remodeling in the lung 2 distinct but dependent pathways: one enhances matrix deposition by fibrotic gene activation, whereas the other slows down matrix degradation by increased plasminogen activator inhibitor 1. Of importance, both pathways are regulated by Rho/myocardin-related transcription factor-A and contribute to fibroblast differentiation and matrix remodeling in the lung. Thus, our results support a unique role for TRPV4 in -induced airway remodeling and warrant further studies in humans for it to be used as a novel therapeutic target in the treatment of asthma.-Gombedza, F., Kondeti, V., Al-Azzam, N., Koppes, S., Duah, E., Patil, P., Hexter, M., Phillips, D., Thodeti, C. K., Paruchuri, S. Mechanosensitive transient receptor potential vanilloid 4 regulates -induced airway remodeling 2 distinct pathways modulating matrix synthesis and degradation.
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http://dx.doi.org/10.1096/fj.201601045RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5349793PMC
April 2017

Molecular Analysis of Stromal Cells-Induced Neural Differentiation of Mouse Embryonic Stem Cells.

PLoS One 2016 10;11(11):e0166316. Epub 2016 Nov 10.

Department of Biomedical Engineering, The University of Akron, Akron, Ohio 44325, United States of America.

Deriving specific neural cells from embryonic stem cells (ESCs) is a promising approach for cell replacement therapies of neurodegenerative diseases. When co-cultured with certain stromal cells, mouse ESCs (mESCs) differentiate efficiently to neural cells. In this study, a comprehensive gene and protein expression analysis of differentiating mESCs is performed over a two-week culture period to track temporal progression of cells from a pluripotent state to specific terminally-differentiated neural cells such as neurons, astrocytes, and oligodendrocytes. Expression levels of 26 genes consisting of marker genes for pluripotency, neural progenitors, and specific neuronal, astroglial, and oligodendrocytic cells are tracked using real time q-PCR. The time-course gene expression analysis of differentiating mESCs is combined with the hierarchal clustering and functional principal component analysis (FPCA) to elucidate the evolution of specific neural cells from mESCs at a molecular level. These statistical analyses identify three major gene clusters representing distinct phases of transition of stem cells from a pluripotent state to a terminally-differentiated neuronal or glial state. Temporal protein expression studies using immunohistochemistry demonstrate the generation of neural stem/progenitor cells and specific neural lineages and show a close agreement with the gene expression profiles of selected markers. Importantly, parallel gene and protein expression analysis elucidates long-term stability of certain proteins compared to those with a quick turnover. Describing the molecular regulation of neural cells commitment of mESCs due to stromal signaling will help identify major promoters of differentiation into specific cell types for use in cell replacement therapy applications.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0166316PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5104328PMC
June 2017

TRPV4 channels regulate tumor angiogenesis via modulation of Rho/Rho kinase pathway.

Oncotarget 2016 May;7(18):25849-61

Department of Integrative Medical Sciences, Northeast Ohio Medical University, OH 44272, Rootstown, USA.

Targeting angiogenesis is considered a promising therapy for cancer. Besides curtailing soluble factor mediated tumor angiogenesis, understanding the unexplored regulation of angiogenesis by mechanical cues may lead to the identification of novel therapeutic targets. We have recently shown that expression and activity of mechanosensitive ion channel transient receptor potential vanilloid 4 (TRPV4) is suppressed in tumor endothelial cells and restoring TRPV4 expression or activation induces vascular normalization and improves cancer therapy. However, the molecular mechanism(s) by which TRPV4 modulates angiogenesis are still in their infancy. To explore how TRPV4 regulates angiogenesis, we have employed TRPV4 null endothelial cells (TRPV4KO EC) and TRPV4KO mice. We found that absence of TRPV4 (TRPV4KO EC) resulted in a significant increase in proliferation, migration, and abnormal tube formation in vitro when compared to WT EC. Concomitantly, sprouting angiogenesis ex vivo and vascular growth in vivo was enhanced in TRPV4KO mice. Mechanistically, we observed that loss of TRPV4 leads to a significant increase in basal Rho activity in TRPV4KO EC that corresponded to their aberrant mechanosensitivity on varying stiffness ECM gels. Importantly, pharmacological inhibition of the Rho/Rho kinase pathway by Y-27632 normalized abnormal mechanosensitivity and angiogenesis exhibited by TRPV4KO EC in vitro. Finally, Y-27632 treatment increased pericyte coverage and in conjunction with Cisplatin, significantly reduced tumor growth in TRPV4KO mice. Taken together, these data suggest that TRPV4 regulates angiogenesis endogenously via modulation of EC mechanosensitivity through the Rho/Rho kinase pathway and can serve as a potential therapeutic target for cancer therapy.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5041949PMC
http://dx.doi.org/10.18632/oncotarget.8405DOI Listing
May 2016

Facile rhenium-peptide conjugate synthesis using a one-pot derived Re(CO)3 reagent.

Dalton Trans 2016 Mar 10;45(11):4729-35. Epub 2016 Feb 10.

Department of Chemistry, University of Akron, OH 44325-3601, USA.

We have synthesized two Re(CO)3-modified lysine complexes (1 and 2), where the metal is attached to the amino acid at the Nε position, via a one-pot Schiff base formation reaction. These compounds can be used in the solid phase synthesis of peptides, and to date we have produced four conjugate systems incorporating neurotensin, bombesin, leutenizing hormone releasing hormone, and a nuclear localization sequence. We observed uptake into human umbilical vascular endothelial cells as well as differential uptake depending on peptide sequence identity, as characterized by fluorescence and rhenium elemental analysis.
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http://dx.doi.org/10.1039/c5dt04694gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4991029PMC
March 2016

Anti-tumor activity of lipophilic imidazolium salts on select NSCLC cell lines.

Med Chem Res 2015 Jul 13;24(7):2838-2861. Epub 2015 Feb 13.

Department of Chemistry, The University of Akron, Akron, OH 44325-3601, USA.

The anti-tumor activity of imidazolium salts is highly dependent upon the substituents on the nitrogen atoms of the imidazolium cation. We have synthesized and characterized a series of naphthalene-substituted imidazolium salts and tested them against a variety of non-smallcell lung cancer cell lines. Several of these complexes displayed anticancer activity comparable to cisplatin. These compounds induced apoptosis in the NCI-H460 cell line as determined by Annexin V staining, caspase-3, and PARP cleavage. These results strongly suggest that this class of compounds can serve as potent chemotherapeutic agents.
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http://dx.doi.org/10.1007/s00044-015-1330-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4593509PMC
July 2015

TRPV4 channel activation selectively inhibits tumor endothelial cell proliferation.

Sci Rep 2015 Sep 21;5:14257. Epub 2015 Sep 21.

Department of Integrative Medical Sciences, Northeast Ohio Medical University, Rootstown, OH 44272.

Endothelial cell proliferation is a critical event during angiogenesis, regulated by both soluble factors and mechanical forces. Although the proliferation of tumor cells is studied extensively, little is known about the proliferation of tumor endothelial cells (TEC) and its contribution to tumor angiogenesis. We have recently shown that reduced expression of the mechanosensitive ion channel TRPV4 in TEC causes aberrant mechanosensitivity that result in abnormal angiogenesis. Here, we show that TEC display increased proliferation compared to normal endothelial cells (NEC). Further, we found that TEC exhibit high basal ERK1/2 phosphorylation and increased expression of proliferative genes important in the G1/S phase of the cell cycle. Importantly, pharmacological activation of TRPV4, with a small molecular activator GSK1016790A (GSK), significantly inhibited TEC proliferation, but had no effect on the proliferation of NEC or the tumor cells (epithelial) themselves. This reduction in TEC proliferation by TRPV4 activation was correlated with a decrease in high basal ERK1/2 phosphorylation. Finally, using a syngeneic tumor model revealed that TRPV4 activation, with GSK, significantly reduced endothelial cell proliferation in vivo. Our findings suggest that TRPV4 channels regulate tumor angiogenesis by selectively inhibiting tumor endothelial cell proliferation.
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http://dx.doi.org/10.1038/srep14257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4585691PMC
September 2015

Leukotriene D4 and prostaglandin E2 signals synergize and potentiate vascular inflammation in a mast cell-dependent manner through cysteinyl leukotriene receptor 1 and E-prostanoid receptor 3.

J Allergy Clin Immunol 2016 Jan 5;137(1):289-298. Epub 2015 Aug 5.

Department of Chemistry, University of Akron, Akron, Ohio. Electronic address:

Background: Although arachidonic acid metabolites, cysteinyl leukotrienes (cys-LTs; leukotriene [LT] C4, LTD4, and LTE4), and prostaglandin (PG) E2 are generated at the site of inflammation, it is not known whether crosstalk exists between these 2 classes of inflammatory mediators.

Objective: We sought to determine the role of LTD4-PGE2 crosstalk in inducing vascular inflammation in vivo, identify effector cells, and ascertain specific receptors and pathways involved in vitro.

Methods: Vascular (ear) inflammation was assessed by injecting agonists into mouse ears, followed by measuring ear thickness and histology, calcium influx with Fura-2, phosphorylation and expression of signaling molecules by means of immunoblotting, PGD2 and macrophage inflammatory protein 1β generation by using ELISA, and expression of transcripts by using RT-PCR. Candidate receptors and signaling molecules were identified by using antagonists and inhibitors and confirmed by using small interfering RNA.

Results: LTD4 plus PGE2 potentiated vascular permeability and edema, gearing the system toward proinflammation in wild-type mice but not in Kit(W-sh) mice. Furthermore, LTD4 plus PGE2, through cysteinyl leukotriene receptor 1 (CysLT1R) and E-prostanoid receptor (EP) 3, enhanced extracellular signal-regulated kinase (Erk) and c-fos phosphorylation, inflammatory gene expression, macrophage inflammatory protein 1β secretion, COX-2 upregulation, and PGD2 generation in mast cells. Additionally, we uncovered that this synergism is mediated through Gi, protein kinase G, and Erk signaling. LTD4 plus PGE2-potentiated effects are partially sensitive to CysLT1R or EP3 antagonists but completely abolished by simultaneous treatment both in vitro and in vivo.

Conclusions: Our results unravel a unique LTD4-PGE2 interaction affecting mast cells through CysLT1R and EP3 involving Gi, protein kinase G, and Erk and contributing to vascular inflammation in vivo. Furthermore, current results also suggest an advantage of targeting both CysLT1R and EP3 in attenuating inflammation.
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http://dx.doi.org/10.1016/j.jaci.2015.06.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5839097PMC
January 2016

Phytochemicals potently inhibit migration of metastatic breast cancer cells.

Integr Biol (Camb) 2015 Jul;7(7):792-800

Department of Biomedical Engineering, The University of Akron, Akron, Ohio 44325, USA.

Cell migration is a major process that drives metastatic progression of cancers, the major cause of cancer death. Existing chemotherapeutic drugs have limited efficacy to prevent and/or treat metastasis, emphasizing the need for new treatments. We focus on triple negative breast cancer (TNBC), the subtype of breast cancer with worst prognosis and no standard chemotherapy protocols. Here we demonstrate that a group of natural compounds, known as phytochemicals, effectively block migration of metastatic TNBC cells. Using a novel cell micropatterning technology, we generate consistent migration niches in standard 96-well plates where each well contains a cell-excluded gap within a uniform monolayer of cells. Over time, cells migrate into and occupy the gap. Treating TNBC cells with non-toxic concentrations of phytochemicals significantly blocks motility of cells. Using a molecular analysis approach, we show that anti-migratory property of phytochemicals is partly due to their inhibitory effects on phosphorylation of ERK1/2. This study provides a framework for future studies to understand molecular targets of phytochemicals and evaluate their effectiveness in inhibiting metastasis in animal models of cancer.
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http://dx.doi.org/10.1039/c5ib00121hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5474751PMC
July 2015

A step toward simplified detection of serum albumin on SDS-PAGE using an environment-sensitive flavone sensor.

Chem Commun (Camb) 2015 Jul;51(55):11060-3

Department of Chemistry, University of Akron, Akron, Ohio 44325, USA.

In this study, we report a series of novel flavone-based sensors that exhibit a superior fluorescence response when interacting with serum albumin in real serum samples and in acrylamide gels. The detection limit of probe 4 for serum albumin solution is 0.09 μg mL(-1), and the detectable volume for monkey serum reaches as low as 0.03 μL.
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http://dx.doi.org/10.1039/c5cc03516cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4512168PMC
July 2015

A selective NIR-emitting zinc sensor by using Schiff base binding to turn-on excited-state intramolecular proton transfer.

J Mater Chem B 2014 ;2(14):2008-2012

Department of Chemistry, The University of Akron, Akron, Ohio 44325, USA ; Maurice Morton Institute of Polymer Science, The University of Akron, Akron, Ohio 44325, USA.

A rational design has led to a highly selective and cell-permeable zinc sensor, which exhibits a large fluorescence turn-on at ~545 nm the desirable NIR emission (~720 nm) with a large Stokes' shift, providing a practical sensor platform with two emission channels for reliable zinc detection.
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http://dx.doi.org/10.1039/C3TB21339KDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4233661PMC
January 2014

TRPV4 mediates myofibroblast differentiation and pulmonary fibrosis in mice.

J Clin Invest 2014 Dec 3;124(12):5225-38. Epub 2014 Nov 3.

Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disorder with no effective medical treatments available. The generation of myofibroblasts, which are critical for fibrogenesis, requires both a mechanical signal and activated TGF-β; however, it is not clear how fibroblasts sense and transmit the mechanical signal(s) that promote differentiation into myofibroblasts. As transient receptor potential vanilloid 4 (TRPV4) channels are activated in response to changes in plasma membrane stretch/matrix stiffness, we investigated whether TRPV4 contributes to generation of myofibroblasts and/or experimental lung fibrosis. We determined that TRPV4 activity is upregulated in lung fibroblasts derived from patients with IPF. Moreover, TRPV4-deficient mice were protected from fibrosis. Furthermore, genetic ablation or pharmacological inhibition of TRPV4 function abrogated myofibroblast differentiation, which was restored by TRPV4 reintroduction. TRPV4 channel activity was elevated when cells were plated on matrices of increasing stiffness or on fibrotic lung tissue, and matrix stiffness-dependent myofibroblast differentiation was reduced in response to TRVP4 inhibition. TRPV4 activity modulated TGF-β1-dependent actions in a SMAD-independent manner, enhanced actomyosin remodeling, and increased nuclear translocation of the α-SMA transcription coactivator (MRTF-A). Together, these data indicate that TRPV4 activity mediates pulmonary fibrogenesis and suggest that manipulation of TRPV4 channel activity has potential as a therapeutic approach for fibrotic diseases.
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http://dx.doi.org/10.1172/JCI75331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4348970PMC
December 2014

Modulation of mast cell proliferative and inflammatory responses by leukotriene d4 and stem cell factor signaling interactions.

J Cell Physiol 2015 Mar;230(3):595-602

Department of Chemistry, University of Akron, Akron, Ohio.

Mast cells (MCs) are important effector cells in asthma and pulmonary inflammation, and their proliferation and maturation is maintained by stem cell factor (SCF) via its receptor, c-Kit. Cysteinyl leukotrienes (cys-LTs) are potent inflammatory mediators that signal through CysLT1 R and CysLT2 R located on the MC surface, and they enhance MC inflammatory responses. However, it is not known if SCF and cys-LTs cross-talk and influence MC hyperplasia and activation in inflammation. Here, we report the concerted effort of the growth factor SCF and the inflammatory mediator LTD4 in MC activation. Stimulation of MCs by LTD4 in the presence of SCF enhances c-Kit-mediated proliferative responses. Similarly, SCF synergistically enhances LTD4 -induced calcium, c-fos expression and phosphorylation, as well as MIP1β generation in MCs. These findings suggest that integration of SCF and LTD4 signals may contribute to MC hyperplasia and hyper-reactivity during airway hyper-response and inflammation.
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http://dx.doi.org/10.1002/jcp.24777DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4244248PMC
March 2015

Facile solid phase peptide synthesis with a Re-lysine conjugate generated via a one-pot procedure.

Dalton Trans 2014 Aug 30;43(30):11452-5. Epub 2014 May 30.

Department of Chemistry, University of Akron, Akron, OH 44325-3601, USA.

We have synthesized a Re(CO)3-modified lysine via a one-pot Schiff base formation reaction that can be used in the solid phase peptide synthesis. To demonstrate its potential use, we have attached it to a neurotensin fragment and observed uptake into human umbilical vascular endothelial cells.
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http://dx.doi.org/10.1039/c4dt01129eDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4991030PMC
August 2014

Urokinase-type plasminogen activator receptor (uPAR) ligation induces a raft-localized integrin signaling switch that mediates the hypermotile phenotype of fibrotic fibroblasts.

J Biol Chem 2014 May 18;289(18):12791-804. Epub 2014 Mar 18.

From the Departments of Pathobiology and.

The urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol-linked membrane protein with no cytosolic domain that localizes to lipid raft microdomains. Our laboratory and others have documented that lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) exhibit a hypermotile phenotype. This study was undertaken to elucidate the molecular mechanism whereby uPAR ligation with its cognate ligand, urokinase, induces a motile phenotype in human lung fibroblasts. We found that uPAR ligation with the urokinase receptor binding domain (amino-terminal fragment) leads to enhanced migration of fibroblasts on fibronectin in a protease-independent, lipid raft-dependent manner. Ligation of uPAR with the amino-terminal fragment recruited α5β1 integrin and the acylated form of the Src family kinase, Fyn, to lipid rafts. The biological consequences of this translocation were an increase in fibroblast motility and a switch of the integrin-initiated signal pathway for migration away from the lipid raft-independent focal adhesion kinase pathway and toward a lipid raft-dependent caveolin-Fyn-Shc pathway. Furthermore, an integrin homologous peptide as well as an antibody that competes with β1 for uPAR binding have the ability to block this effect. In addition, its relative insensitivity to cholesterol depletion suggests that the interactions of α5β1 integrin and uPAR drive the translocation of α5β1 integrin-acylated Fyn signaling complexes into lipid rafts upon uPAR ligation through protein-protein interactions. This signal switch is a novel pathway leading to the hypermotile phenotype of IPF patient-derived fibroblasts, seen with uPAR ligation. This uPAR dependent, fibrotic matrix-selective, and profibrotic fibroblast phenotype may be amenable to targeted therapeutics designed to ameliorate IPF.
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http://dx.doi.org/10.1074/jbc.M113.498576DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4007467PMC
May 2014

Cysteinyl leukotrienes regulate endothelial cell inflammatory and proliferative signals through CysLT₂ and CysLT₁ receptors.

Sci Rep 2013 Nov 20;3:3274. Epub 2013 Nov 20.

Department of Chemistry, University of Akron, OH 44325.

Cysteinyl leukotrienes (cys-LTs), LTC₄, LTD₄, LTE₄ are potent inflammatory lipid mediators that act through two distinct G-protein-coupled receptors, CysLT₁R and CysLT₂R. Although cys-LTs are shown to induce vascular leakage and atherosclerosis, the molecular mechanism by which cys-LTs modulate endothelial function is not known. Here, we show that cys-LTs (LTC₄ and LTD₄) induce robust calcium influx in human umbilical vein endothelial cells (HUVECs) through CysLT₂R, but not CysLT₁R. Further, cys-LT treatment induced endothelial cell (EC) contraction leading to monolayer disruption via CysLT₂R/Rho kinase dependent pathway. Furthermore, stimulation with cys-LTs potentiated TNFα-induced VCAM-1 expression and leukocyte recruitment to ECs through CysLT₂R. In contrast, we found that both LTC₄ and LTD₄ stimulated EC proliferation through CysLT₁R. Taken together, these results suggest that cys-LTs induce endothelial inflammation and proliferation via CysLT₂R/Rho kinase and CysLT₁R/Erk dependent pathways, respectively, which play critical role in the etiology of cardiovascular diseases such as atherosclerosis and myocardial infarction.
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http://dx.doi.org/10.1038/srep03274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3834363PMC
November 2013

Differential regulation of cysteinyl leukotriene receptor signaling by protein kinase C in human mast cells.

PLoS One 2013 15;8(8):e71536. Epub 2013 Aug 15.

Department of Chemistry, University of Akron, Akron, Ohio, United States of America.

Cysteinyl leukotrienes (cys-LTs) are a group of lipid mediators that are potent bronchoconstrictors, powerful inducers of vascular leakage and potentiators of airway hyperresponsiveness. Cys-LTs play an essential role in asthma and are synthesized as well as activated in mast cells (MCs). Cys-LTs relay their effects mainly through two known GPCRs, CysLT1R and CysLT2R. Although protein kinase C (PKC) isoforms are implicated in the regulation of CysLT1R function, neither the role of PKCs in cys-LT-dependent MC inflammatory signaling nor the involvement of specific isoforms in MC function are known. Here, we show that PKC inhibition augmented LTD4 and LTE4-induced calcium influx through CysLT1R in MCs. In contrast, inhibition of PKCs suppressed c-fos expression as well MIP1β generation by cys-LTs. Interestingly, cys-LTs activated both PKCα and PKCε isoforms in MC. However, knockdown of PKCα augmented cys-LT mediated calcium flux, while knockdown of PKCε attenuated cys-LT induced c-fos expression and MIP1β generation. Taken together, these results demonstrate for the first time that cys-LT signaling downstream of CysLT1R in MCs is differentially regulated by two distinct PKCs which modulate inflammatory signals that have significant pathobiologic implications in allergic reactions and asthma pathology.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0071536PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3744564PMC
April 2014