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L Vijaya Mohan Rao

Publications and source records attributed to L Vijaya Mohan Rao.

14 recordsLinked to original sources
PubMed2026

Thrombin concentration shapes endothelial extracellular vesicle profiles with divergent inflammatory functions.

Thrombin, a central enzyme in the coagulation cascade, also regulates diverse cellular processes, including inflammation and vascular barrier function, primarily by activating protease-activated receptor 1. Previous studies demonstrated that thrombin elicits concentration-dependent, opposing effects; low concentrations confer anti-inflammatory and barrier-protective responses, whereas high concentrations promote inflammation and barrier disruption. The underlying mechanisms, however, remain incompletely understood. Here, we showed that thrombin stimulates extracellular vesicle (EV) release from endothelial cells across a broad concentration range and that EVs generated at low vs high thrombin concentrations carry distinct microRNA (miR) cargo. Low-thrombin EVs mediate cytoprotective responses via the transfer of miR-409-5p, which targets ubiquitin-specific protease 7 that promotes inflammation via the NF-kB signaling pathway in recipient cells, whereas high-thrombin EVs disrupt barrier integrity and promote inflammation through delivery of miR-155-5p, a regulator of suppressor of cytokine signaling 1 that acts as a crucial negative regulator of the cytokine signaling pathway. Functional manipulation of these EVs confirmed the causal roles. Incorporation of anti-miR-409-5p abrogated the protective effects of low-thrombin EVs, whereas anti-miR-155-5p suppressed the cytopathic effects of high-thrombin EVs. Moreover, control EVs engineered to carry a miR-409-5p mimic reproduced the anti-inflammatory and barrier-protective phenotype of low-thrombin EVs. Collectively, these findings identified EV-associated miRs as key mediators of the concentration-dependent dual actions of thrombin, which may open the therapeutic potential of EVs engineered to deliver selective miRs or anti-miRs for the treatment of inflammatory vascular diseases.

Thrombin↗
PubMed2006

Cellular localization and trafficking of tissue factor.

Tissue factor (TF) is the cellular receptor for clotting factor VIIa (FVIIa). The formation of TF-FVIIa complexes on cell surfaces triggers the activation of coagulation cascade and cell signaling. In the present study, we characterized the subcellular distribution of TF and its transport in fibroblasts by dual immunofluorescence confocal microscopy and biochemical methods. Our data show that a majority of TF resides in various intracellular compartments, predominantly in the Golgi. Tissue factor at the cell surface is localized in cholesterol-rich lipid rafts and extensively colocalized with caveolin-1. FVIIa binding to TF induces the internalization of TF. Of interest, we found that TF-FVIIa complex formation at the cell surface leads to TF mobilization from the Golgi with a resultant increase in TF expression at the cell surface. This process is dependent on FVIIa protease activity. Overall, the present data suggest a novel mechanism for TF expression at the cell surface by FVIIa. This mechanism could play an important role in hemostasis in response to vascular injury by increasing TF activity where and when it is needed.

Blood Coagulation↗
PubMed2005

Proteolysis of CCN1 by plasmin: functional implications.

Plasmin is shown to play a crucial role in many pathophysiologic processes primarily through its ability to degrade extracellular matrix (ECM) and/or mobilizing growth factors that are sequestered in the ECM. Cysteine-rich 61 (CCN1) is a matricellular protein of which expression is up-regulated in cancer and various vascular diseases. The present study was undertaken to investigate whether plasmin liberates CCN1 from the ECM and whether the released growth factor modulates endothelial cell migration. Treatment of breast carcinoma cells (MDA-MB-231) with plasmin released a truncated form of CCN1 (28 kDa) into the overlying medium. Experiments with recombinant CCN1 confirmed that plasmin effectively cleaves CCN1. Thrombin and other clotting/fibrinolytic proteases are ineffective in cleaving CCN1. Further studies revealed that the conditioned medium of plasmin-treated carcinoma cells supports endothelial cell migration and that antibodies specific to CCN1 blocked this enhancing effect. These data were the first to show that plasmin can liberate a pluripotent matrix signaling protein, CCN1, from the ECM. Because both CCN1 and the components of the plasmin generation system are present in tumor cells and a variety of other cells, the proteolysis of CCN1 by plasmin may play a role in many pathophysiologic processes, including tumor cell-mediated angiogenesis.

Breast Neoplasms↗
PubMed2004

Tissue factor-factor VIIa signaling.

How does tissue factor (TF), whose principle role is to support clotting factor VIIa (FVIIa) in triggering the coagulation cascade, affect various pathophysiological processes? One of the answers is that TF interaction with FVIIa not only initiates clotting but also induces cell signaling via activation of G-protein-coupled protease activated receptors (PARs). Recent studies using various cell model systems and limited in vivo systems are beginning to define how TF-VIIa-induced signaling regulates cellular behavior. Signaling pathways initiated by both TF-VIIa protease activation of PARs and phosphorylation of the TF-cytoplasmic domain appear to regulate cellular functions. In the present article, we review the emerging data on the mechanism of TF-mediated cell signaling and how it regulates various cellular responses, with particular focus on TF-VIIa protease-dependent signaling.

Animals↗
PubMed2004

Acute cholesterol depletion impairs functional expression of tissue factor in fibroblasts: modulation of tissue factor activity by membrane cholesterol.

Cholesterol, in addition to providing rigidity to the fluid membrane, plays a critical role in receptor function, endocytosis, recycling, and signal transduction. In the present study, we examined the effect of membrane cholesterol on functional expression of tissue factor (TF), a cellular receptor for clotting factor VIIa. Depletion of cholesterol in human fibroblasts (WI-38) with methyl-beta-cyclodextrin-reduced TF activity at the cell surface. Binding studies with radiolabeled VIIa and TF monoclonal antibody (mAB) revealed that reduced TF activity in cholesterol-depleted cells stems from the impairment of VIIa interaction with TF rather than the loss of TF receptors at the cell surface. Repletion of cholesterol-depleted cells with cholesterol restored TF function. Loss of caveolar structure on cholesterol removal is not responsible for reduced TF activity. Solubilization of cellular TF in different detergents indicated that a substantial portion of TF in fibroblasts is associated with noncaveolar lipid rafts. Cholesterol depletion studies showed that the TF association with these rafts is cholesterol dependent. Overall, the data presented herein suggest that membrane cholesterol functions as a positive regulator of TF function by maintaining TF receptors, probably in noncaveolar lipid rafts, in a high-affinity state for VIIa binding.

Cell Line↗
PubMed2004

Tissue factor-factor VIIa-specific up-regulation of IL-8 expression in MDA-MB-231 cells is mediated by PAR-2 and results in increased cell migration.

Tissue factor (TF), the cellular receptor for factor VIIa (FVIIa), besides initiating blood coagulation, is believed to play an important role in tissue repair, inflammation, angiogenesis, and tumor metastasis. Like TF, the chemokine interleukin-8 (IL-8) is shown to play a critical role in these processes. To elucidate the potential mechanisms by which TF contributes to tumor invasion and metastasis, we investigated the effect of FVIIa on IL-8 expression and cell migration in a breast carcinoma cell line, MDA-MB-231, a cell line that constitutively expresses abundant TF. Expression of IL-8 mRNA in MDA-MB-231 cells was markedly up-regulated by plasma concentrations of FVII or an equivalent concentration of FVIIa (10 nM). Neither thrombin nor other proteases involved in hemostasis were effective in stimulating IL-8 in these cells. Increased transcriptional activation of the IL-8 gene is responsible for increased expression of IL-8 in FVIIa-treated cells. PAR-2-specific antibodies fully attenuated TF-FVIIa-induced IL-8 expression. Additional in vitro experiments showed that TF-FVIIa promoted tumor cell migration and invasion, active site-inactivated FVIIa, and specific antibodies against TF, PAR-2, and IL-8 inhibited TF-FVIIa-induced cell migration. In summary, the studies described herein provide insight into how TF may contribute to tumor invasion.

Breast Neoplasms↗
PubMed2003

Antiapoptotic effect of coagulation factor VIIa.

Binding of factor VIIa (FVIIa) to its cellular receptor tissue factor (TF) was previously shown to induce various intracellular signaling events, which were thought to be responsible for TF-mediated biologic effects, including angiogenesis, tumor metastasis, and restenosis. To understand the mechanisms behind these processes, we have examined the effect of FVIIa on apoptosis. Serum deprivation-induced apoptosis of BHK(+TF) cells was characterized by apoptotic blebs, nuclei with chromatin-condensed bodies, DNA degradation, and activation of caspase 3. FVIIa markedly decreased the number of cells with apoptotic morphology and prevented the DNA degradation as measured by means of TdT-mediated dUTP nick end labeling (TUNEL). The antiapoptotic effect of FVIIa was confirmed by the observation that FVIIa attenuated caspase 3 activation. FVIIa-induced antiapoptotic effect was dependent on its proteolytic activity and TF but independent of factor Xa and thrombin. FVIIa-induced cell survival correlated with the activation of Akt and was inhibited markedly by the specific PI3-kinase inhibitor, LY294002. Blocking the activation of p44/42 mitogen-activated protein kinase (MAPK) by the specific mitogen-induced extracellular kinase (MEK) inhibitor, U0126, impaired modestly the ability of FVIIa to promote cell survival. In conclusion, FVIIa binding to TF provided protection against apoptosis induced by growth factor deprivation, primarily through activation of PI3-kinase/Akt pathway, and to a lesser extent, p44/42 MAPK pathway.

Animals↗
PubMed2003

Regulation of tissue factor-factor VIIa expression on cell surfaces: a role for tissue factor-factor VIIa endocytosis.

Tissue factor (TF) is the cellular receptor for plasma clotting factor VIIa (FVIIa) and the formation of TF-VIIa complexes on cell surfaces triggers the coagulation cascade. Further, TF-VIIa, either directly or indirectly, influences various biological processes, such as development, inflammation and tumor metastasis. Therefore, a proper regulation of TF-VIIa expression is critical for the maintenance of hemostatic balance and health in general. TF-VIIa functional expression on cell surfaces is regulated primarily by transcriptional regulation of TF gene or by specific plasma inhibitors, particularly tissue factor pathway inhibitor (TFPI). However, a number of other mechanisms that are yet to be well defined also regulate TF-VIIa functional expression. One such mechanism is the endocytosis of TF-VIIa. In this article, we provide a comprehensive review on the regulation of TF-VIIa functional expression by these other mechanisms, with a particular emphasis on TF-VIIa endocytosis, and our perspective of these studies.

Animals↗
PubMed2002

Plasmin induces Cyr61 gene expression in fibroblasts via protease-activated receptor-1 and p44/42 mitogen-activated protein kinase-dependent signaling pathway.

OBJECTIVE: The plasminogen system has been proposed to participate in vascular remodeling and angiogenesis. Although plasmin-mediated proteolysis could contribute these processes, proteolytic targets for plasmin and their downstream effector molecules are yet to be fully defined. The aim of the present study was to elucidate potential mechanisms by which plasmin affects various cellular processes. METHODS AND RESULTS: Plasmin upregulated the expression of Cyr61, a growth factor-like gene that has been implicated in cell proliferation, adhesion, and migration. Plasmin-induced gene expression is dependent on its proteolytic activity and requires its binding to cells. Studies that used wild-type fibroblasts and fibroblasts derived from PAR-1- and PAR-2-deficient mice showed that plasmin induced Cyr61 gene expression in wild-type fibroblasts and PAR-2-deficient cells but not in PAR-1-deficient cells. Consistent with this, plasmin induced the activation of p44/42 mitogen-activated protein kinase in wild-type, PAR-2 -/- cells but not in PAR-1 -/- cells. In contrast with thrombin, plasmin failed to induce Ca2+ signaling in fibroblasts. CONCLUSIONS: Plasmin induced an angiogenic and wound-healing promoter, Cyr61, in fibroblasts through activation of PAR-1. Plasmin-induced Cyr61 expression is mediated via the p44/42 mitogen-activated protein kinase pathway independent of Ca2+ signaling.

Animals↗
PubMed2002

Factor VIIa/tissue factor-induced signaling: a link between clotting and disease.

Tissue factor is a cellular receptor for plasma clotting factor VII. In health, tissue factor is constitutively expressed in many cells, such as fibroblasts and keratinocytes, but is absent in vascular cells that come in contact with blood. However, tissue factor is induced in vascular cells in certain pathophysiological conditions. Thus, vessel wall injury or a disease condition allows blood to come in contact with tissue factor, resulting in factor VII binding to tissue factor. Once native factor VII complexed with tissue factor is converted to the enzyme factor VIIa, the complex triggers the clotting cascade that ultimately leads to fibrin formation. In addition to its established role in coagulation, molecular links between factor VIIa/tissue factor and various biological processes, such as development, inflammation, and tumor metastasis, are also evident. Recent studies suggest that factor VIIa/tissue factor affects various cellular processes by inducing intracellular signaling. Emerging evidence suggest that factor VIIa/tissue factor participates in cell signaling by two distinct mechanisms: proteolysis-independent signaling via the cytoplasmic domain of tissue factor, and proteolysis-dependent signaling, which is independent of tissue factor's cytoplasmic tail. In proteolysis-independent signaling, filamin 1 is recruited to tissue factor upon its ligation, and this probably provides an essential intracellular link in transmitting signals from tissue factor. In proteolysis-dependent signaling, factor VIIa/tissue factor activates one or more protease-activated receptors, which couple to G proteins, to impact multiple signaling pathways. In this chapter, we review various nonhemostatic functions attributed to factor VIIa and tissue factor, describe signaling mechanisms initiated upon factor VIIa binding to tissue factor, and discuss how factor VIIa/tissue factor-induced signaling could contribute to various pathophysiological processes. The relationship between increased clotting and manifestation of various diseases is well recognized. Although aberrant activation of the coagulation pathway is primarily the consequence of a disease, activation of the coagulation pathway during the disease process, in turn, could contribute to pathogenesis of the disease. Further, recent transgenic studies in mouse suggest that the coagulation system also plays a role in embryogenesis and development. Then the question arises, how do proteins involved in the clotting regulate cellular functions? The coagulant proteases can regulate cell behavior in a number of ways. They can function like hormones, activating multiple signaling pathways, as well as release bioactive fragments and growth factors by proteolysis of cell-surface and extracellular matrix (ECM) components. This chapter reviews our current understanding of the role of factor VIIa/tissue factor (VIIa/TF), an enzyme/cofactor complex that triggers the clotting cascade, in affecting various cellular functions not related to the clotting, and discusses potential mechanisms by which it regulates cellular functions.

Animals↗
PubMed2002

Mechanism of resveratrol-mediated suppression of tissue factor gene expression.

Tissue factor (TF) is a cell surface receptor for factor VII(a), and the binding of factor VII(a) to TF initiates the coagulation cascade. Inappropriate in vivo expression of TF in vascular cells has been shown to be responsible for thrombotic disorders associated with a variety of pathological conditions, including gram-negative sepsis, cancer and atherosclerosis. A number of epidemiological studies suggest that moderate consumption of red wine provides protective effects against coronary heart disease mortality. Recently, we have shown that resveratrol, a polyphenolic compound found in wine, inhibited the induction of TF expression in endothelial cells and mononuclear cells (Pendurthi UR, Williams JT, Rao LVM. Arterioscler Thromb Vasc Biol 1999: 19: 419-426). In the present study, we examined the mechanism by which resveratrol inhibits the expression of TF in monocytes by using a monocytic cell line, THP-1, as a model cell. Northern blot analysis, gel mobility shift assays and transfection studies with various TF promoter constructs, as well as other transcription regulatory constructs, were used to elucidate the inhibitory mechanism of resveratrol. The data show that resveratrol inhibited lipopolysaccharide (LPS)-induced expression of TF in human monocytes and monocytic cell line, THP-1 in a dose dependent manner. Resveratrol did not significantly alter the binding of various transcription factors involved in TF gene expression to DNA. However, resveratrol suppressed the transcription of cloned human TF promoter. Further experiments revealed that resveratrol reduced kappaB- but not AP-1-driven transcriptional activity. Additional experiments showed that resveratrol suppressed the phosphorylation of p65 and its transactivation. In summary, our results indicate that resveratrol does not inhibit the activation or translocation of NF-kappaB/Rel proteins but inhibits NF-kappaB/Rel-dependent transcription by impairing the transactivation potential of p65.

Dose-Response Relationship, Drug↗
PubMed1999

Catabolism of factor VIIa bound to tissue factor in fibroblasts in the presence and absence of tissue factor pathway inhibitor.

Vascular injury leads to the exposure of blood to fibroblasts and smooth muscle cells within the vessel wall. These cells constitutively express tissue factor (TF), the cellular receptor for plasma clotting factor VIIa (FVIIa). Formation of TF.FVIIa complexes on cell surfaces triggers the blood coagulation cascade. In the present study, we have investigated the fate of TF.FVIIa complexes formed on the cell surface of fibroblasts in the presence and absence of plasma inhibitor, tissue factor pathway inhibitor (TFPI). FVIIa bound to TF on the cell surface was internalized and degraded without depleting the cell surface TF antigen and activity. TFPI significantly enhanced the TF-specific internalization and degradation of FVIIa. TFPI-enhanced internalization and degradation of FVIIa requires the C-terminal domain of TFPI and factor Xa. TFPI. Xa-mediated internalization of FVIIa was associated with the depletion of TF from the cell surface. A majority of the internalized FVIIa was degraded, but a small portion of the internalized FVIIa recycles back to the cell surface as an intact protein. In addition to TF, other cell surface components, such as low density lipoprotein receptor-related protein (LRP) and heparan sulfates, are essential for TFPI.Xa-induced internalization of FVIIa. Acidification of cytosol, which selectively inhibits the endocytotic pathway via coated pits, inhibited TFPI.Xa-mediated internalization but not the basal internalization of FVIIa. Overall, our data support the concept that FVIIa bound to cell surface TF was endocytosed by two different pathways. FVIIa complexed with TF in the absence of the inhibitor was internalized via a LRP-independent and probably noncoated pit pathway, whereas FVIIa complexed with TF along with the inhibitor was internalized via LRP-dependent coated pit pathway.

Cell Line↗