L. V. M. Rao

Transcriptional program induced by factor VIIa-tissue factor, PAR1 and PAR2 in MDA-MB-231 cells.

Summary.u2002 Background:u2002Factor VIIa (FVIIa) binding to tissue factor (TF) induces cell signaling via the protease activity of FVIIa and protease‐activated receptor 2 (PAR2). Objective:u2002We examined how the gene‐expression profile induced by FVIIa corresponds to the profiles induced by protease‐activated receptor 1 (PAR1) or PAR2 agonists using MDA‐MB‐231 breast carcinoma cells that constitutively express TF, PAR1 and PAR2. Results and conclusions:u2002Out of 8500 genes, FVIIa stimulation induced differential regulation of 39 genes most of which were not previously recognized as FVIIa regulated. All genes regulated by FVIIa were similarly regulated by a PAR2 agonist peptide confirming FVIIa signaling via PAR2. An appreciable fraction of the PAR2‐regulated genes was also regulated by a PAR1 agonist peptide suggesting extensive redundancy between FVIIa/PAR2 signaling and thrombin/PAR1 signaling. The FVIIa regulated genes encode cytokines, chemokines and growth factors, and the gene repertoire induced by FVIIa in MDA‐MB‐231 cells is consistent with a role for TF–FVIIa signaling in regulation of a wound healing type of response. Interestingly, a number of genes regulated exclusively by FVIIa/PAR2‐mediated cell signaling in MDA‐MB‐231 cells were regulated by thrombin and a PAR1 agonist, but not by FVIIa, in the TF‐expressing glioblastoma U373 cell line.

Activity and regulation of factor VIIa analogs with increased potency at the endothelial cell surface

Summary.u2002 Background:u2002Variants of recombinant factor VIIa (rFVIIa) with increased intrinsic activity have been developed to improve efficacy in the treatment of bleeding disorders in the future. The increased potency of FVIIa variants was demonstrated in limited in vitro and in vivo studies. However, further characterization of FVIIa variants is needed to evaluate their potential clinical use. Methods:u2002In the present study, we investigated the interactions of two FVIIa variants, FVIIaQ and FVIIaDVQ, with plasma inhibitors, tissue factor pathway inhibitor (TFPI) and antithrombin (AT), and vascular endothelium. TF‐FVIIa activity or its inhibition was measured directly in an amidolytic activity assay or for its ability to activate factor X. Results:u2002Both TFPI and AT/heparin inhibited the FVIIa variants more rapidly than the wild‐type (WT) FVIIa in the absence of tissue factor (TF). In the presence of TF, TFPI, TFPI–Xa, and AT/heparin inhibited FVIIa and FVIIa variants at similar rates. Although the WT FVIIa failed to generate significant amounts of FXa on unperturbed endothelial cells, FVIIa variants, particularly FVIIaDVQ, generated a substantial amount of FXa on unperturbed endothelium. Annexin V fully attenuated the FVIIa‐mediated activation of FX on unperturbed endothelial cells. On stimulated human umbilical vein endothelial cells, FVIIa and FVIIa variants activated FX at similar rates, and annexin V blocked the activation only partly. AT/heparin and TFPI–Xa inhibited the activity of FVIIa and FVIIa variants bound to endothelial cell TF in a similar fashion. Interestingly, despite significant differences observed in FXa generation on unperturbed endothelium exposed to FVIIa and FVIIa analogs, no differences were found in thrombin generation when cells were exposed to FVIIa or FVIIa analogs under plasma mimicking conditions. Conclusion:u2002Overall, the present data suggest that although FVIIa variants generate substantial amounts of FXa, they do not generate excessive thrombin on the surface of endothelium.

A novel mechanism of plasmin-induced mitogenesis in fibroblasts.

Summary.u2002 The plasminogen activator/plasmin system is believed to play an important role in diverse pathophysiological processes, including wound healing, vascular remodeling and pulmonary fibrosis. Our recent studies show that plasmin upregulates the expression of Cyr61, a growth factor‐like gene that has been implicated in cell proliferation and migration. In the present study, we investigated whether plasmin promotes fibroblast proliferation and, if so, determine the role of Cyr61 in the plasmin‐induced response. Human lung fibroblasts were exposed to varying concentrations of plasmin and DNA synthesis was monitored by measuring the incorporation of 3H‐thymidine into DNA. Plasmin increased DNA synthesis of fibroblasts in a dose‐dependent manner. Protease‐activated receptor‐1 (PAR‐1)‐specific antibodies, but not PAR‐2‐specific antibodies, reduced the plasmin‐induced DNA synthesis. Consistent with this, plasmin had no substantial effect on the DNA synthesis in PAR‐1‐deficient mouse fibroblasts. Plasmin activated both p38 and p44/42 MAPKs and specific inhibitors of these pathways inhibited the plasmin‐induced DNA synthesis. Plasmin‐induced increase in the DNA synthesis was completely abrogated by anti‐Cyr61 antibodies. Interestingly, thrombin, which is a potent inducer of Cyr61, had only a minimal effect on fibroblast proliferation. Additional experiments suggested that plasmin cleaved cell/extracellular matrix‐associated Cyr61 and the conditioned media from plasmin‐treated cells could support the cell proliferation. Overall, these data suggest that plasmin promotes fibroblast proliferation by a novel pathway, involving two independent steps. In the first step, plasmin induces Cyr61 expression via activation of PAR‐1, and in the second step, plasmin releases Cyr61 deposited in the extracellular matrix, thus making it accessible to act on cells.

Endothelial cell protein C receptor‐mediated redistribution and tissue‐level accumulation of factor VIIa

Summary.u2002 Background:u2002 Recent studies show that activated factor VII (FVIIa) binds to the endothelial cell protein C receptor (EPCR) on the vascular endothelium; however, the importance of this interaction in hemostasis or pathophysiology is unknown.

Effect of wine phenolics on cytokine-induced C-reactive protein expression

Summary.u2002 Background:u2002Elevation of C‐reactive protein (CRP) levels in blood was recognized as one of the cardiac disease risk factors. Consumption of wine is shown to reduce the risk from heart disease and improve longevity. Objectives:u2002In the present study, we evaluated the effect of various wine polyphenolic compounds and several active synthetic derivatives of resveratrol on the inflammatory cytokines (IL‐1ßu2003+u2003IL‐6)‐induced CRP expression in Hep3B cells. Results:u2002Among the wine phenolics tested, quercetin and resveratrol, in a dose‐dependent manner, suppressed cytokine‐induced CRP expression. Two of the synthetic derivatives of resveratrol, R3 and 7b, elicited a fiftyfold higher suppressive effect compared with resveratrol. The inhibitory effects of resveratrol and its derivatives on CRP expression were at the level of mRNA production. Investigation of signaling pathways showed that the cytokines induced the phosphorylation of p38 and p44/42 MAP kinases. Inhibitors of p38 and p44/42 mitogen‐activated protein kinase (MAPK) activation inhibited CRP expression, implicating the involvement of both pathways in cytokine‐induced CRP expression. These data revealed a previously unrecognized role of the p44/42 MAPK signaling pathway in CRP expression. Wine polyphenolics or the synthetic compounds of resveratrol did not affect cytokine‐activated phosphorylation of these MAPKs. Conclusions:u2002Wine phenolics inhibit CRP expression; however, to do so, they do not utilize the MAPK pathways.

Activity and regulation of glycoPEGylated factor VIIa analogs.

Summary.u2002 Background:u2002Recombinant coagulation factor VIIa (rFVIIa) has proven to be a safe and effective drug for treatment of bleeding episodes in hemophilic patients with inhibitors. However, rFVIIa is cleared from the circulation relatively quickly. Protein modification with poly(ethylene glycol) (PEG) can prolong the circulatory lifetime of proteins but it could also impair protein function by molecular shielding of the protein surface. Objectives:u2002 To characterize the interaction of glycoPEGylated rFVIIa – rFVIIa‐10K PEG and rFVIIa‐40K PEG – with tissue factor (TF), factor X (FX) and plasma inhibitors, tissue factor pathway inhibitor (TFPI) and antithrombin (AT). Methods:u2002 The amidolytic and FX activation assays were employed to investigate the interaction of glycoPEGylated rFVIIa with its macromolecular substrate and inhibitors. Results:u2002 Both the glycoPEGylated rFVIIa analogs exhibited similar amidolytic activity as that of rFVIIa in the absence or the presence of relipidated TF. The analogs were as effective as rFVIIa in activating FX in the absence of TF. In the presence of TF, the glycoPEGylated rFVIIa variants, relative to rFVIIa, were slightly less effective at lower concentrations, but no significant differences were found among them in activating FX at saturating concentrations. Both AT/heparin and TFPI effectively inhibited the glycoPEGylated rFVIIa bound to relipidated TF or TF on stimulated endothelial cells. In contrast to their normal interaction with TF, the glycoPEGylated rFVIIa variants appeared to interact poorly with phospholipids. Conclusions:u2002 The glycoPEGylated rFVIIa variants retained their catalytic activity and interacted efficiently with TF, FX and the plasma inhibitors. Further work with appropriate in vitro and in vivo model systems is needed to determine the feasibility of using glycoPEGylated rFVIIa to improve therapeutic options for bleeding disorders.

Analysis of tissue factor expression in various cell model systems: cryptic vs. active.

Tissue factor (TF) encryption plays an important role in regulating TF coagulant activity. Potential differences in experimental cell model systems and strategies hampered our understanding of the TF encryption mechanisms.

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]. n nThe 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, u200b,1B1B). n n n nFig. 1 n nInfluence 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 ... n n n nMDA-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 u200band1F),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. n nInterestingly, 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. n nDuring 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. n nA 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.

Glycosylation of tissue factor is not essential for its transport or functions.

See also Morrissey JH. Low‐carb tissue factor? This issue, pp 1508–10.DOI: 10.1111/j.1538‐7836.2011.04332.x.

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.