Shiva Keshava

Factor VIIa bound to endothelial cell protein C receptor activates protease activated receptor-1 and mediates cell signaling and barrier protection

Recent studies have shown that factor VIIa (FVIIa) binds to the endothelial cell protein C receptor (EPCR), a cellular receptor for protein C and activated protein C, but the physiologic significance of this interaction is unclear. In the present study, we show that FVIIa, upon binding to EPCR on endothelial cells, activates endogenous protease activated receptor-1 (PAR1) and induces PAR1-mediated p44/42 mitogen-activated protein kinase (MAPK) activation. Pretreatment of endothelial cells with FVIIa protected against thrombin-induced barrier disruption. This FVIIa-induced, barrier-protective effect was EPCR dependent and did not involve PAR2. Pretreatment of confluent endothelial monolayers with FVIIa before thrombin reduced the development of thrombin-induced transcellular actin stress fibers, cellular contractions, and paracellular gap formation. FVIIa-induced p44/42 MAPK activation and the barrier-protective effect are mediated via Rac1 activation. Consistent with in vitro findings, in vivo studies using mice showed that administration of FVIIa before lipopolysaccharide (LPS) treatment attenuated LPS-induced vascular leakage in the lung and kidney. Overall, our present data provide evidence that FVIIa bound to EPCR on endothelial cells activates PAR1-mediated cell signaling and provides a barrier-protective effect. These findings are novel and of great clinical significance, because FVIIa is used clinically for the prevention of bleeding in hemophilia and other bleeding disorders.

Endothelial Cell Protein C Receptor Opposes Mesothelioma Growth Driven by Tissue Factor

The procoagulant protein tissue factor (F3) is a powerful growth promoter in many tumors, but its mechanism of action is not well understood. More generally, it is unknown whether hemostatic factors expressed on tumor cells influence tissue factor-mediated effects on cancer progression. In this study, we investigated the influence of tissue factor, endothelial cell protein C receptor (EPCR, PROCR), and protease activated receptor-1 (PAR1, F2R) on the growth of malignant pleural mesothelioma (MPM), using human MPM cells that lack or express tissue factor, EPCR or PAR1, and an orthotopic nude mouse model of MPM. Intrapleural administration of MPM cells expressing tissue factor and PAR1 but lacking EPCR and PAR2 (F2RL1) generated large tumors in the pleural cavity. Suppression of tissue factor or PAR1 expression in these cells markedly reduced tumor growth. In contrast, tissue factor overexpression in nonaggressive MPM cells that expressed EPCR and PAR1 with minimal levels of tissue factor did not increase their limited tumorigenicity. More importantly, ectopic expression of EPCR in aggressive MPM cells attenuated their growth potential, whereas EPCR silencing in nonaggressive MPM cells engineered to overexpress tissue factor increased their tumorigenicity. Immunohistochemical analyses revealed that EPCR expression in tumor cells reduced tumor cell proliferation and enhanced apoptosis. Overall, our results enlighten the mechanism by which tissue factor promotes tumor growth through PAR1, and they show how EPCR can attenuate the growth of tissue factor-expressing tumor cells.

Influence of endothelial cell protein C receptor on breast cancer development.

Tumor cells of several cancers are known to constitutively express the coagulation initiating factor, tissue factor (TF). TF-mediated coagulation generates thrombin, platelet activation, and fibrin formation which altogether orchestrate cancer cell survival and proliferative pathways. Thrombin also induces activation of natural anticoagulant protein C. Activated protein C (APC) not only inhibits subsequent thrombin generation, but also induces cellular signaling through activation of protease activated receptor-1 (PAR1) [1,2]. Endothelial cell protein C receptor (EPCR) plays a key role in supporting APC-mediated cell signaling [1,2]. EPCR-APC-mediated cytoprotection and other cellular effects may accelerate cancer progression and metastasis. EPCR-dependent APC activation of PAR1 was shown to stimulate cell migration of breast cancer cells [3]. Recent studies have shown that EPCR-APC axis conferred a significant survival advantage to lung adenocarcinoma cells and favored their prometastatic activity [4]. Interestingly, our recent studies suggested that EPCR may also function as a negative regulator of cancer progression [5]. The present study was carried out to investigate the influence of EPCR on human breast cancer development. MDA-231t cells (tumor cells established from in vivo tumor developed by injection of MDA-MB-231 cells to a nude mouse) were stably transfected with a control vector (CV) or EPCR expression vector in pZeoSV plasmid vector. After 48 h of transfection, Zeocin (100 μg/ml) was added to the cells. After 3 weeks, stable transfectant colonies were isolated, expanded, and EPCR stable transfectants exhibiting similar TF activity as that of parental MDA-231t cells were selected for the present study. MDA-231t(+CV) and MDA-231t(+EPCR) cells expressed similar levels of TF antigen and activity (Fig. 1 panels A to C). MDA-231t(+CV) cells expressed very little EPCR, whereas EPCR expression levels in MDA-231t(+EPCR) cells was similar to that of HUVEC (Fig. 1A, ​,1B1B). Fig. 1 Influence of EPCR on tumor growth in a murine breast carcinoma model. TF and EPCR expression levels in MDA-231t cells stably transfected with a control or EPCR expression vector were analyzed by Western blot analysis (A) or immunofluorescence microscopy ... MDA-231t(+CV) or MDA-231t(+EPCR) cells were injected into the mammary fat pad (m.f.p) of nude mice, and the growth of tumor in m.f.p. was monitored for 2 months. As shown in Fig. 1D (in set), tumor growth rate is statistically significantly higher in mice injected with MDA-231t(+EPCR) cells compared to MDA-231t(+CV) cells until 40 days following tumor cell implantation. However, in the last two weeks, tumors derived from MDA231t(+EPCR) cells grew less rapidly than tumors originating from MDA-231t(+CV) cells. At the end of 60 days, the tumor volume of MDA-231t(+EPCR) cell-derived tumors was about 30% lower than that of MDA-231t(+CV) cell-derived tumors (Fig. 1D). Although this difference did not reach statistical significance, it was substantial and consistent. At the time of euthanasia (day 60), the mice bearing MDA-231t(+CV) cell-derived tumors appeared to be lethargic, and developed swollen lymph nodes (Fig. 1E and ​and1F),1F), whereas mice bearing MDA-231t(+EPCR) cell-derived tumors exhibited no outward sickness and did not develop any swollen lymph nodes (Fig. 1F). Histological examination of lymph node sections showed extensive infiltration of cells into this region in mice injected with MDA-231t(+CV) cells and not in mice injected with MDA-231(+EPCR) cells (Fig. 1G). The skin over the tumors of the mice injected with MDA-231t(+CV) cells turned blood red and looked different from that of the tumors generated by MDA-231(+EPCR) cells, starting around 30 to 35 days following tumor cell inoculation. At the time of sacrifice (60 days), all tumors developed in mice injected with MDA-231t(+CV) cells were highly inflamed and necrotic, most of which developed hematogenous ulcers at the top skin of tumors (Fig. 1H). Some necrotic tumors collapsed and had leaky liquid centers. None of the tumors in mice bearing MDA-231t(+EPCR) cells showed necrotic ulcerations. Interestingly, analysis of tumor tissue sections for EPCR and TF expression showed that a majority of tumor cells stained negative for EPCR irrespective of whether MDA-231t(+CV) or MDA-231t(+EPCR) cells were used for implantation (Fig. 1I). In both cases, tumor cells stained intensively positive for TF. Analysis of tumor tissue sections for macrophage infiltration and angiogenesis by staining them for F4/80 antigen and CD31, respectively, showed significant reduction in macrophage infiltration (Fig. 1J) and microvessel density (Fig. 1K) in tumors derived from MDA-231t(+EPCR) cells compared to tumors derived from MDA-231t(+CV) cells. It may be pertinent to note here that tissue sections analyzed for tumors derived from both MDA-231t(+CV) and MDA-231t(+EPCR) cells represent the actively growing regions of the tumor. During the preparation of this manuscript, Schaffner et al. [6] reported that EPCR-expressing cells, selected from expansion of EPCR+ cancer stem cell-like population from MDA-MB-231 mfp cells, had markedly increased tumor cell-initiating activity compared to EPCR− cells. Although the experimental approach and MDA-MB-231 cells used for implantation in the present study and the recently published study [6] differed, the results obtained from both the studies are similar to some extent. Schaffner et al. [6] observed that MDA-MB-231 mfp cells exhibit two distinct populations, EPCR+ cells with moderate levels of TF and EPCR negative cells with high levels of TF expression, and cell sorting was used to select EPCR+ and EPCR− cells for their experiments. Although varied levels of EPCR and TF expression were also found in our MDA-MB-231t cell population, we did not find two clearly distinctive populations of cells (see Fig. 1C). Most of the cells expressed very low levels of EPCR and high levels of TF. Therefore, we genetically engineered them to express EPCR to obtain EPCR+ cells. As reported by Schaffner et al. [6], EPCR expression in breast cancer cells increased the tumor cell growth potential, although not drastically, but in a statistically significant fashion. However, we found that in the later stages of tumor progression, the differences in tumor growth between tumors derived from EPCR+ or EPCRlow cells vanished. In fact, at the end of experimental period, tumor volume in mice injected with EPCR+ cells was 30% lower than in mice injected with EPCRlow cells. It is difficult to predict whether the earlier study [6] could have found similar results if their observation was not terminated when the tumor size reached less than 1 cm3. It is interesting to note that, as found in the earlier study [6], irrespective of the EPCR status in tumor cells that were inoculated, the majority of outgrown tumor cells were EPCR negative, which indicates a conversion from EPCR+ cells to EPCR− cells in the tumor microenvironment. Here, it is important to point out that our observation on loss of EPCR expression in tumor tissues derived from EPCR+ tumor cells is not due to a lack of sensitivity to detect EPCR in tumor tissues by immunohistochemistry method. Loss of EPCR in tumors derived from EPCR+ cells is also confirmed by immunoblot analysis of tumor tissue extracts (data not shown). At present, it is unknown at which stage of tumor growth the EPCR expression was lost and the underlying mechanism for it. A notable finding of the present study is that while we observed solid and liquefaction necrosis in tumors originated from EPCRlow cells, no necrosis was found in tumors originated from EPCR+ cells. Necrosis is a common feature of aggressive breast cancer and has been associated with a poor prognosis [7]. Tumor necrosis is the direct result of chronic ischemia caused by vascular collapse when the rate of tumor cell growth exceeds that of angiogenesis [8]. Necrotic liquefaction occurs when the cellular structures are broken down by proteolytic enzymes released from ruptured lysosomes of tumor cells and/or similar enzymes released by infiltrating inflammatory cells [9]. Although it appears to be counterintuitive at first, necrosis resulting from chronic ischemia is associated with increased angiogenesis [8]. Prolonged hypoxic conditions were known to increase tumor progression and angiogenesis, and to promote metastatic potential [10]. Therefore, EPCR expression in breast cancer cells, despite having initial growth advantage, may limit cancer progression at an advanced stage.

Rab GTPases Regulate Endothelial Cell Protein C Receptor-Mediated Endocytosis and Trafficking of Factor VIIa

Recent studies have established that factor VIIa (FVIIa) binds to the endothelial cell protein C receptor (EPCR). FVIIa binding to EPCR may promote the endocytosis of this receptor/ligand complex. Rab GTPases are known to play a crucial role in the endocytic and exocytic pathways of receptors or receptor/ligand complexes. The present study was undertaken to investigate the role of Rab GTPases in the intracellular trafficking of EPCR and FVIIa. CHO-EPCR cells and human umbilical vein endothelial cells (HUVEC) were transduced with recombinant adenoviral vectors to express wild-type, constitutively active, or dominant negative mutant of various Rab GTPases. Cells were exposed to FVIIa conjugated with AF488 fluorescent probe (AF488-FVIIa), and intracellular trafficking of FVIIa, EPCR, and Rab proteins was evaluated by immunofluorescence confocal microscopy. In cells expressing wild-type or constitutively active Rab4A, internalized AF488-FVIIa accumulated in early/sorting endosomes and its entry into the recycling endosomal compartment (REC) was inhibited. Expression of constitutively active Rab5A induced large endosomal structures beneath the plasma membrane where EPCR and FVIIa accumulated. Dominant negative Rab5A inhibited the endocytosis of EPCR-FVIIa. Expression of constitutively active Rab11 resulted in retention of accumulated AF488-FVIIa in the REC, whereas expression of a dominant negative form of Rab11 led to accumulation of internalized FVIIa in the cytoplasm and prevented entry of internalized FVIIa into the REC. Expression of dominant negative Rab11 also inhibited the transport of FVIIa across the endothelium. Overall our data show that Rab GTPases regulate the internalization and intracellular trafficking of EPCR-FVIIa.

Pharmacological concentrations of recombinant factor VIIa restore hemostasis independent of tissue factor in antibody-induced hemophilia mice.

Essentials The role of tissue factor (TF) in recombinant factor VIIa (rFVIIa) therapy in hemophilia is unclear. An acquired mouse hemophilia model with very low or normal levels of human TF was used in the study. rFVIIa is equally effective in correcting the bleeding in mice expressing low or normal levels of TF. Pharmacological doses of rFVIIa restore hemostasis in hemophilia independent of TF.

Factor VIIa interaction with EPCR modulates the hemostatic effect of rFVIIa in hemophilia therapy: mode of its action

Recent studies established that clotting factor VIIa (FVIIa) binds endothelial cell protein C receptor (EPCR). It has been speculated that FVIIa interaction with EPCR might augment the hemostatic effect of rFVIIa in therapeutic conditions. The present study is carried out to investigate the mechanism by which FVIIa interaction with EPCR contributes to the hemostatic effect of rFVIIa in hemophilia therapy. Active-site inhibited FVIIa, which is capable of binding to EPCR but has no ability to activate factor X, reduced the concentration of rFVIIa required to correct the bleeding following the saphenous vein injury in mouse hemophilia model systems. Higher doses of rFVIIa were required to restore hemostasis in EPCR overexpressing hemophilia mice compared to hemophilia mice expressing normal levels of EPCR. Administration of FVIII antibody induced only mild hemophilic bleeding in EPCR-deficient mice, which was corrected completely with a low dose of rFVIIa. Administration of therapeutic concentrations of rFVIIa increased plasma protein C levels in EPCR overexpressing mice, indicating the displacement of protein C from EPCR by rFVIIa. EPCR levels did not significantly alter the bioavailability of rFVIIa in plasma. Overall, our data indicate that EPCR levels influence the hemostatic effect of rFVIIa in treating hemophilia. Our present findings suggest that FVIIa displacement of anticoagulant protein C from EPCR that results in down-regulation of activated protein C generation and not the direct effect of EPCR-FVIIa on FX activation is the mechanism by which FVIIa interaction with EPCR contributes to the hemostatic effect of rFVIIa in hemophilia therapy.

Restoring cigarette smoke-induced impairment of efferocytosis in alveolar macrophages

Cigarette smoke has been associated with susceptibility to different pulmonary and airway diseases. Impaired alveolar macrophages (AMs) that are major phagocytes in the lung have been associated with patients with airway diseases and active smokers. In the current report, we show that exposure to second-hand cigarette smoke (SHS) significantly reduced efferocytosis in vivo. More importantly, delivery of recombinant granulocyte–macrophage colony-stimulating factor (GM-CSF) to the alveolar space restored and refurbished the efferocytosis capability of AMs. Exposure to SHS significantly reduced expression of CD16/32 on AMs, and treatment with GM-CSF not only restored but also significantly increased the expression of CD16/32 on AMs. GM-CSF treatment increased uptake and digestion/removal of apoptotic cells by AMs. The latter was attributed to increased expression of Rab5 and Rab7. Increased efferocytosis of AMs was also tested in a disease condition. AMs from GM-CSF-treated, influenza-infected, SHS-exposed mice showed significantly better efferocytosis activity, and mice had significantly less morbidity compared with phosphate-buffered saline-treated group. GM-CSF-treated mice had increased amphiregulin levels in the lungs, which in addition to efferocytosis of AMs may have attributed to their protection against influenza. These results will have great implications for developing therapeutic approaches by harnessing mucosal innate immunity to treat lung and airway diseases and protect against pneumonia.

Boosting efferocytosis in alveolar space using BCG vaccine to protect host against influenza pneumonia

Efferocytosis by alveolar phagocytes (APs) is pivotal in maintenance of lung homeostasis. Increased efferocytosis by APs results in protection against lethal acute lung injury due to pulmonary infections whereas defective efferocytosis by APs results in chronic lung inflammation. In this report, we show that pulmonary delivery of Bacillus Calmette-Guerin (BCG) significantly enhances efferocytosis by APs. Increased efferocytosis by APs maintains lung homeostasis and protects mice against lethal influenza pneumonia. Intranasally treated wild type C57Bl/6 (WT) mice with BCG showed significant increase in APs efferocytosis in vivo compared to their PBS-treated counterparts. All BCG-treated WT mice survived lethal influenza A virus (IAV) infection whereas all PBS-treated mice succumbed. BCG-induced resistance was abrogated by depleting AP prior to IAV infection. BCG treatment increased uptake, and digestion/removal of apoptotic cells by APs. BCG significantly increased the expression of TIM4 on APs and increased expression of Rab5 and Rab7. We demonstrated that increased efferocytosis by APs through pulmonary delivery of BCG initiated rapid clearance of apoptotic cells from the alveolar space, maintained lung homeostasis, reduced inflammation and protected host against lethal IAV pneumonia.

Factor VIIa induces anti-inflammatory signaling via EPCR and PAR1

Recent studies show that endothelial cell protein C receptor (EPCR) interacts with diverse ligands, in addition to its known ligands protein C and activated protein C (APC). We showed in earlier studies that procoagulant clotting factor VIIa (FVIIa) binds EPCR and downregulates EPCR-mediated anticoagulation and induces an endothelial barrier protective effect. Here, we investigated the effect of FVIIas interaction with EPCR on endothelial cell inflammation and lipopolysaccharide (LPS)-induced inflammatory responses in vivo. Treatment of endothelial cells with FVIIa suppressed tumor necrosis factor α (TNF-α)- and LPS-induced expression of cellular adhesion molecules and adherence of monocytes to endothelial cells. Inhibition of EPCR or protease-activated receptor 1 (PAR1) by either specific antibodies or small interfering RNA abolished the FVIIa-induced suppression of TNF-α- and LPS-induced expression of cellular adhesion molecules and interleukin-6. β-Arrestin-1 silencing blocked the FVIIa-induced anti-inflammatory effect in endothelial cells. In vivo studies showed that FVIIa treatment markedly suppressed LPS-induced inflammatory cytokines and infiltration of innate immune cells into the lung in wild-type and EPCR-overexpressing mice, but not in EPCR-deficient mice. Mechanistic studies revealed that FVIIa treatment inhibited TNF-α-induced ERK1/2, p38 MAPK, JNK, NF-κB, and C-Jun activation indicating that FVIIa-mediated signaling blocks an upstream signaling event in TNFα-induced signaling cascade. FVIIa treatment impaired the recruitment of TNF-receptor-associated factor 2 into the TNF receptor 1 signaling complex. Overall, our present data provide convincing evidence that FVIIa binding to EPCR elicits anti-inflammatory signaling via a PAR1- and β-arrestin-1 dependent pathway. The present study suggests new therapeutic potentials for FVIIa, which is currently in clinical use for treating bleeding disorders.

Intrapleural Adenoviral-mediated Endothelial Cell Protein C Receptor Gene Transfer Suppresses the Progression of Malignant Pleural Mesothelioma in a Mouse Model

Malignant pleural mesothelioma (MPM) is an aggressive thoracic cancer with a high mortality rate as it responds poorly to standard therapeutic interventions. Our recent studies showed that expression of endothelial cell protein C receptor (EPCR) in MPM cells suppresses tumorigenicity. The present study was aimed to investigate the mechanism by which EPCR suppresses MPM tumor growth and evaluate whether EPCR gene therapy could suppress the progression of MPM in a mouse model of MPM. Measurement of cytokines from the pleural lavage showed that mice implanted with MPM cells expressing EPCR had elevated levels of IFNγ and TNFα compared to mice implanted with MPM cells lacking EPCR. In vitro studies demonstrated that EPCR expression renders MPM cells highly susceptible to IFNγ + TNFα-induced apoptosis. Intrapleural injection of Ad.EPCR into mice with an established MPM originating from MPM cells lacking EPCR reduced the progression of tumor growth. Ad.EPCR treatment elicited recruitment of macrophages and NK cells into the tumor microenvironment and increased IFNγ and TNFα levels in the pleural space. Ad.EPCR treatment resulted in a marked increase in tumor cell apoptosis. In summary, our data show that EPCR expression in MPM cells promotes tumor cell apoptosis, and intrapleural EPCR gene therapy suppresses MPM progression.