Loss of Protease-Activated Receptor 4 Prevents Inflammation Resolution and Predisposes the Heart to Cardiac Rupture After Myocardial Infarction.

BACKGROUND: Cardiac rupture is a major lethal complication of acute myocardial infarction (MI). Despite significant advances in reperfusion strategies, mortality from cardiac rupture remains high. Studies suggest that cardiac rupture can be accelerated by thrombolytic therapy, but the relevance of this risk factor remains controversial. METHODS: We analyzed protease-activated receptor 4 (Par4) expression in mouse hearts with MI and investigated the effects of Par4 deletion on cardiac remodeling and function after MI by echocardiography, quantitative immunohistochemistry, and flow cytometry. RESULTS: Par4 mRNA and protein levels were increased in mouse hearts after MI and in isolated cardiomyocytes in response to hypertrophic and inflammatory stimuli. Par4-deficient mice showed less myocyte apoptosis, reduced infarct size, and improved functional recovery after acute MI relative to wild-type (WT). Conversely, Par4-/- mice showed impaired cardiac function, greater rates of myocardial rupture, and increased mortality after chronic MI relative to WT. Pathological evaluation of hearts from Par4-/- mice demonstrated a greater infarct expansion, increased cardiac hemorrhage, and delayed neutrophil accumulation, which resulted in impaired post-MI healing compared with WT. Par4 deficiency also attenuated neutrophil apoptosis in vitro and after MI in vivo and impaired inflammation resolution in infarcted myocardium. Transfer of Par4-/- neutrophils, but not of Par4-/- platelets, in WT recipient mice delayed inflammation resolution, increased cardiac hemorrhage, and enhanced cardiac dysfunction. In parallel, adoptive transfer of WT neutrophils into Par4-/- mice restored inflammation resolution, reduced cardiac rupture incidence, and improved cardiac function after MI. CONCLUSIONS: These findings reveal essential roles of Par4 in neutrophil apoptosis and inflammation resolution during myocardial healing and point to Par4 inhibition as a potential therapy that should be limited to the acute phases of ischemic insult and avoided for long-term treatment after MI.

Different Expression of Hypoxic and Angiogenic Factors in Human Endometriotic Lesions.

Endometriosis is associated with local angiogenic and hypoxic mechanisms. Indeed, peritoneal fluid of women with endometriosis generates a specific microenvironment to support the growth and development of ectopic endometrial tissues. The association between proangiogenic markers and hypoxic processes in different endometriosis phenotypes was investigated in the present study, analyzing the expression of several genes, related to hypoxic signaling pathway and involved in angiogenic processes, in nonpregnant women with different forms of endometriosis. Samples of ovarian endometrioma (OMA; n = 16) or deep infiltrating endometriosis (DIE; n = 11) were collected, and in addition, control endometrium was collected from healthy women by hysteroscopy. The gene expression of the hypoxia-inducible factors (HIF) 1/2α, protease-activated receptors (PARs) », and vascular endothelial growth factor (VEGF) A was evaluated by quantitative reverse-transcription polymerase chain reaction. Ovarian endometrioma expresses high levels of HIF-1/2α, PAR-1/4, and VEGF-A, while DIE did not show significantly different gene expression compared to endometrium from unaffected women. A positive correlation between the expression of HIF-1/2α and VEGF-A mRNA was observed in OMA. The overall data point out that the heterogeneity of the disease reflects differences in expression levels of genes associated with hypoxia and angiogenesis, suggesting that such conditions may have an active role in the development of the disease.

Expression, function and cooperating partners of protease-activated receptor type 3 in vascular endothelial cells and B lymphocytes studied with specific monoclonal antibody.

Receptor-specific antibodies can both prevent ligand-receptor interaction and initiate receptor signaling. Previously we generated monoclonal antibody 8E8 (mAb 8E8) against protease-activated receptor type 3 (PAR3) which inhibited proliferation of B cell hybridoma. Here we used mAb 8E8 and PAR1-specific polyclonal antibody to reveal the functions and cooperating partners of PAR3 in endothelial cells and in B lymphocytes. MAb 8E8 or PAR1 agonist peptide stimulated IL-6 and IL-8 production and VCAM-1 expression in HPMEC-ST1.6R cells. PAR1 antibody stimulated only VCAM-1 expression, while ICAM-1 expression was stimulated with mAB 8E8 or PAR3 peptide. MAb 8E8 stimulated weak mitogenic response, while PAR1 antibody inhibited it in normal but not in malignant B lymphocytes. Sandwich ELISA assay demonstrated the interaction of PAR3 with PAR1 in malignant cell lines and with IgM in normal B lymphocytes. It is concluded that PAR3 cooperates with PAR1 to mediate the effect of thrombin on cytokine production and VCAM-1 expression in endothelial cells and on cell proliferation in malignant B cells. ICAM-1 expression in endothelial cells requires PAR3 without PAR1. The inhibitory effect of thrombin in normal B lymphocytes is mediated by PAR1 alone, while mitogenic and pro-survival signaling in B lymphocytes is provided through PAR3 in cooperation with BCR.

TMZ-induced PrPc/par-4 interaction promotes the survival of human glioma cells.

Malignant gliomas recur even after extensive surgery and chemo-radiotherapy. Although a relatively novel chemotherapeutic agent, temozolomide (TMZ), has demonstrated promising activity against gliomas, the effects last only a few months and drug resistance develops thereafter in many cases. It has been acknowledged that glioma cells respond to TMZ treatment by undergoing G2/M arrest, but not apoptosis. Here we demonstrate a phase-specific chemotherapy resistance due to cellular prion protein (PrPc) in human glioma cells upon TMZ treatment. TMZ-induced G2/M-arrested cultures show an upregulation of PrPc expression and are more resistant, whereas G1/S-phase cells that show decreased levels of PrPc are more sensitive to apoptosis. Furthermore, an investigation into the biological significance of PrPc association with par-4 provided the first evidence of a relationship between the endogenous levels of PrPc and the resistance of glioma cells to the apoptotic effects of TMZ. Upon TMZ treatment, PrPc exerts its antiapoptotic activity by inhibiting PKA-mediated par-4 phosphorylation that are important for par-4 activation, nuclear entry and initiation of apoptosis. In context with cell cycle-dependent responses to chemotherapy, the data from this study suggest the possibility of exploiting the PrPc-dependent pathway to improve the efficacy of TMZ-based regimen for patients with gliomas.

Phosphatidylserine exposure and other apoptotic-like events in Bernard-Soulier syndrome platelets.

In the Bernard-Soulier syndrome (BSS), the giant platelets are said to have increased phosphatidylserine (PS) surface exposure in the resting state and shortened survival in the circulation. When normal platelets are activated, they undergo many biochemical and morphological changes, some of which are apoptotic. Herein, we investigated apoptotic-like events in BSS platelets upon activation, specifically, PS exposure, microparticle (MP) formation, cell shrinkage, and loss of mitochondrial inner membrane potential (DeltaPsi(m)). Platelets from two unrelated BSS patients were examined in whole blood; agonists used were collagen, thrombin, PAR1- or PAR4-activating peptides (APs), or combinations of collagen with thrombin, and the PAR-APs. Flow cytometry was used to measure PS exposure (annexin A5 binding), platelet-derived MPs (forward scatter; events <0.75 microm size), and DeltaPsi(m) (TMRM fluorescence). PS exposure was increased on resting and activated BSS platelets, and this was independent of the platelet size. MP formation by BSS platelets was generally enhanced. Cell shrinkage occurred on activation to form smaller, PS-exposing platelets in BSS and controls. A proportion of PS-exposing BSS and control platelets exhibited DeltaPsi(m) loss, but unlike controls, there was also loss of DeltaPsi(m) in the BSS platelets not exposing PS. Thus, BSS platelets undergo apoptotic-like events upon activation, with PS exposure and MP formation being enhanced. These events may play a role in the shortened survival in BSS, as well as affecting thrombin generation.

Expression of proteinase-activated receptor 1-4 (PAR 1-4) in human cancer.

Proteinase activated receptors (PAR 1-4) are membrane receptors with a unique way of activation by proteinases like thrombin, trypsin and matrix metalloproteinases which lead to a specific cellular response. To evaluate the significance of expression and co-expression of PAR in cancer we performed a survey on published data. A Pubmed literature search on "PAR, thrombin, cancer" was performed and 46 publications were selected for systematic review based on the availability of information on tumor type, material type, detection method and specification of positive cases. PAR-1 was found in 77.3% of malignant samples (n = 678), PAR-2 in 79.5% (n = 592), PAR-3 in 12.6% (n = 87) and PAR-4 in 54.9% (n = 153). PAR-1 and -2 were present in adenocarcinomas, melanomas, osteosarcomas, glioblastomas, meningiomas, leukaemias and squamous cell carcinomas. Presence of PAR-3 was limited to kidney and liver cancer. The data on PAR-4 expression was inconclusive. Those studies analysing PAR-1 and PAR-2 reported coexpression of the two receptors. PAR-1 and -2 are widely expressed in human tumors suggesting an important role in tumorigenesis and providing potential targets for therapy. PAR-3 and PAR-4 are less frequently detectable, their expression and potential role in tumorigenesis require further investigation.

Altered adhesive properties of cord blood endothelial outgrowth cells expressing IL-1ra.

The aim of this study was to examine the potential of endothelial outgrowth cells (EOCs) expanded from CD34(+) cord blood-derived cells (CB-EOCs) for overexpression of therapeutic transgenes. As proof of principle, we overexpressed icIL-1ra in CB-EOCs. Proinflammatory activation of CB-EOCs in response to cytokine stimulation (IL-1beta and tumor necrosis factor (TNF)) and during coculture with monocytes showed that icIL-1ra-expressing CB-EOCs express significantly reduced levels of ICAM-1, MCP-1 and thrombin receptor expression. Moreover, overexpression of icIL-1ra attenuated the IL-1beta-mediated proinflammatory activation by diminishing the expression of ICAM-1, SELE, MCP-1 and IL-1beta. Interestingly, overexpression of icIL-1ra also inhibited TNF-induced upregulation of ICAM-1. Expression of ICAM-1, VCAM-1, tissue factor and IL-1beta was also decreased on direct contact with monocytes. These changes in gene expression were accompanied by functional reduction in leukocyte rolling, adhesion of monocytes to CB-EOCs, as well as by a reduction in transendothelial migration of monocytes. Our findings show that CB-EOCs stably expressing transgenic icIL-1ra are protected against activation by not only IL-1beta but also TNFalpha-mediated proinflammatory stimuli and inhibit decisive pathomechanisms of inflammatory processes such as rolling, adhesion and transmigration of monocytes. Therefore, icIL-ra transgenic CB-EOCs may prove to be beneficial in the treatment of IL-1beta- and TNFalpha-mediated inflammatory vasculopathies.

Delivery of PAR-4 plasmid in vivo via nanoliposomes sensitizes colon tumor cells subcutaneously implanted into nude mice to 5-FU.

The prostate apoptosis response protein 4 (Par-4), a tumor suppressor, has been shown to induce apoptosis in cancer cells. While reduced Par-4 expression has been linked to survival of some cancers, its involvement in colon cancer has not been well documented. To explore the feasibility of increasing Par-4 in colon cancer to induce apoptosis, the human colon cancer cell line, HT29, was transfected to overexpress Par-4. In these cells, overexpressed Par-4 led to increased apoptosis in the presence of 5-fluorouracil. Subsequently, PAR-4 cDNA was packaged in nanoliposomal particles. Treating cells with the Par-4 nanoliposomes also increased susceptibility to 5-FU. These nanoliposomes were used to deliver Par-4 plasmid to tumors growing in nude mice from wild type HT29 cells. Results showed that nanoliposomes effectively delivered plasmid DNA to tumors in vivo. Again, tumors in mice treated with the Par-4 nanoliposomes were more susceptible to 5-FU treatment. This suggests that upregulation of Par-4 expression is a potentially useful mechanism to enhance the current chemotherapeutic regimen for colon cancer. Packaging Par-4 cDNA in nanoliposomal particles is a promising delivery method to increase response to chemotherapy.

Up-regulation of protease-activated receptor-1 in diabetic glomerulosclerosis.

Patients with diabetes are under a hypercoagulable state leading to generation of thrombin. It is not known whether thrombin plays a role in the progression of diabetic nephropathy. We analyzed gene expression of two thrombin receptors, protease-activated receptor-1 (PAR-1) and PAR-4 in the kidney of diabetic db/db mice. Mice developed hyperglycemia from 7 to 10 weeks of age and showed renal abnormalities such as mesangial expansion and urinary albumin excretion at 10 weeks of age. PAR-1 mRNA was up-regulated in isolated glomeruli in db/db mice compared with age-matched db/m littermates, but PAR-4 mRNA was not. In situ hybridization studies showed that PAR-1 mRNA was detected mainly at the glomerulus, and that intensive signals were observed in mesangial cells and podocytes. The up-regulation of PAR-1 in glomeruli in diabetic mice may play a role in the progression of glomerulosclerosis and abnormal urinary albumin excretion in diabetic nephropathy.

Cathepsin G recruits osteoclast precursors via proteolytic activation of protease-activated receptor-1.

Metastatic breast cancer shows extreme tropism for the bone microenvironment, leading to the establishment of osteolytic metastases. Perpetuation of tumor-induced osteolysis requires a continuous supply of osteoclast precursors migrating into the bone microenvironment that can subsequently differentiate into mature osteoclasts and resorb bone. Thus, identification and subsequent targeting of chemoattractants of osteoclast precursors that are up-regulated at the tumor-bone interface represents a potential avenue to interrupt osteolysis. We report that cathepsin G, a serine protease, plays a vital role in the bone microenvironment by modulating tumor-stromal interaction in a manner that favors tumor establishment and regulates chemotaxis of monocytes, a subset of which has the potential to differentiate into osteoclasts. Our data show that cathepsin G-induced chemotaxis of monocytes is mediated by proteolytic activation of protease-activated receptor-1 (PAR-1). Attenuation of PAR-1 activation abrogates cathepsin G-mediated induction of monocyte chemotaxis. We also show that in vivo inhibition of cathepsin G reduces the number of CD11b(+) osteoclast precursors and mature osteoclasts at the tumor-bone interface. Together, these data suggest that therapeutic targeting of both PAR-1 signaling in osteoclast precursors as well as cathepsin G at the tumor-bone interface has the potential to reduce osteolysis by inhibiting the recruitment, differentiation, and activation of osteoclast precursors.

Thrombin-stimulated proliferation of cultured human synovial fibroblasts through proteolytic activation of proteinase-activated receptor-1.

We examined the mechanism of thrombin on proliferation of synovial fibroblasts obtained from rheumatoid arthritis (RA). Thrombin concentration-dependently induced proliferation of synovial fibroblasts. Proliferation in response to thrombin (10 U/ml) was completely blocked by hirudin. TP367 and TP508, peptides corresponding to 2 noncatalytic regions of thrombin, failed to induce cell proliferation. Thrombin did not induce the production of basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF) in synovial fibroblasts. Expression of proteinase-activated receptor (PAR)-1 and PAR-3 mRNAs was observed in synovial fibroblasts. Thrombin and PAR-1 agonist peptide (AP), but not PAR-3 AP, induced intracellular calcium mobilization. PAR-1 AP induced cell proliferation whereas PAR-3 AP and PAR-4 AP had no effect on proliferation. Pertussis toxin (PTX), a Gialpha protein inhibitor; wortmannin, a PI (phosphatidylinositol) 3-kinase inhibitor; and PD98059, a specific MEK [mitogen-activated protein (MAK) kinase kinase] inhibitor, inhibited the thrombin-induced cell proliferation. Furthermore, the proliferation of synovial fibroblasts was suppressed by U-73122, a PLC (phospholipase C) inhibitor; 2-APB, an antagonist of InsP3 (inositol 1,4,5-triphosphate) receptor; and GF-109203X, a PKC (protein kinase C) inhibitor. These results suggest that thrombin induces the proliferation of RA synovial fibroblasts through the activation of PAR-1, leading to the PTX-sensitive G proteins - PI3 kinase pathway and PTX-insensitive G proteins - PLC (InsP3 receptor) Ca(2+)-PKC branch.

Cytokine upregulation of proteinase-activated-receptors 2 and 4 expression mediated by p38 MAP kinase and inhibitory kappa B kinase beta in human endothelial cells.

BACKGROUND AND PURPOSE: Up-regulation of proteinase-activated receptor-2 (PAR2) is a factor in a number of disease states and we have therefore examined the signalling pathways involved in the expression of the receptor. EXPERIMENTAL APPROACH: We investigated the effects of tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), trypsin and the PAR2 activating peptide, 2-furoyl(2f)-LIGKV-OH on both mRNA and functional expression of PAR2 in human umbilical vein endothelial cells (HUVECs). The effect of specific chemical inhibitors and dominant negative adenovirus constructs of the mitogen-activated protein kinase (MAPK) cascade and the nuclear factor kappa B (NF-kappaB) signalling pathway was assessed. Methods included semi-quantitative and quantitative RT-PCR, [(3)H]inositol phosphate (IP) accumulation and Ca(2+)-dependent fluorescence. KEY RESULTS: The above agonists induced both mRNA and functional expression of PAR2; PAR4 mRNA, but not that for PAR1 or PAR-3, also increased following TNFalpha treatment. Inhibition of p38 MAP kinase reduced PAR2 and PAR4 expression, whilst inhibition of MEK1/ERK/JNK was without effect. A similar dependency upon p38 MAP kinase was observed for the expression of PAR4. TNFalpha -induced enhancement of PAR2 stimulated [(3)H]-inositol phosphate accumulation (IP) and Ca(2+) signalling was abolished following SB203580 pre-treatment. Infection with adenovirus encoding dominant-negative IKKbeta (Ad.IKKbeta(+/-)) and to a lesser extent dominant-negative IKKalpha (Ad.IKKalpha(+/-)), substantially reduced both control and IL-1beta- induced expression of both PAR2 and PAR4 mRNA and enhancement of PAR2-stimulated IP accumulation and Ca(2+) mobilisation. CONCLUSIONS AND IMPLICATIONS: These data reveal for the first time the signalling events involved in the upregulation of both PAR2 and PAR4 during pro-inflammatory challenge.

Par-4 is a novel mediator of renal tubule cell death in models of ischemia-reperfusion injury.

Prostate apoptosis response-4 (Par-4) is a leucine zipper protein linked to apoptotic cell death in prostate cancer and neuronal tissues. The leucine zipper domain of Par-4 (Leu.zip) mediates protein-protein interactions that are essential for sensitization of cells to apoptosis, and overexpression of Leu.zip blocks Par-4 activity in a dominant negative fashion. Ischemia-reperfusion-induced renal injury (IRI) is clinically important because it typically damages renal tubular epithelial cells and glomerular cells, and it is the most common cause of acute renal failure (ARF). We now report that Par-4 is expressed in renal tubule cells and that aberrant expression of Par-4 activity plays a crucial role in activating apoptotic pathways in well-characterized models of renal IRI. Increased levels of Par-4 were observed following chemical ischemia-reperfusion in HK-2 cells in vitro and in mouse renal tubular cells following bilateral clamping of renal pedicles in vivo. Inhibition of Par-4 expression by specific par-4 antisense oligonucleotides largely prevented HK-2 cell apoptosis induced by IRI. Overexpression of Par-4 in these cells exacerbated mitochondrial dysfunction and caspase activation and conferred increased sensitivity to IRI-induced apoptosis. Expression of Leu.zip, a dominant negative regulator of Par-4, largely prevented mitochondrial dysfunction and caspase activation and significantly inhibited IRI-induced apoptosis in HK-2 cells. In addition, transfection of Par-4 increased while transfection of Leu.zip decreased necrosis in HK-2 cells following prolonged IRI. These results identify Par-4 as a novel and early mediator of renal tubule cell injury following IRI and provide a potential target for developing new therapeutic strategies for renal IRI and ARF.

Protease-activated receptor-4: a novel mechanism of inflammatory pain modulation.

BACKGROUND AND PURPOSE: Protease-activated receptor-4 (PAR(4)), the most recently discovered member of the PARs family, is activated by thrombin, trypsin and cathepsin G, but can also be selectively activated by small synthetic peptides (PAR(4)-activating peptide, PAR(4)-AP). PAR(4) is considered a potent mediator of platelet activation and inflammation. As both PAR(1) and PAR(2) have been implicated in the modulation of nociceptive mechanisms, we investigated the expression of PAR(4) in sensory neurons and the effects of its selective activation on nociception. EXPERIMENTAL APPROACH AND KEY RESULTS: We demonstrated the expression of PAR(4) in sensory neurons isolated from rat dorsal root ganglia by reverse transcription-polymerase chain reaction and immunofluorescence. We found that PAR(4) colocalized with calcitonin gene-related peptide and substance P. We also showed that a selective PAR(4)-AP was able to inhibit calcium mobilization evoked by KCl and capsaicin in rat sensory neurons. Moreover, the intraplantar injection of a PAR(4)-AP significantly increased nociceptive threshold in response to thermal and mechanical noxious stimuli, while a PAR(4) inactive control peptide had no effect. The anti-nociceptive effects of the PAR(4)-AP were dose-dependent and occurred at doses below the threshold needed to cause inflammation. Finally, co-injection of the PAR(4)-AP with carrageenan significantly reduced the carrageenan-induced inflammatory hyperalgesia and allodynia, but had no effect on inflammatory parameters such as oedema and granulocyte infiltration. CONCLUSIONS AND IMPLICATIONS: Taken together, these results identified PAR(4) as a novel potential endogenous analgesic factor, which can modulate nociceptive responses in normal and inflammatory conditions.

[Effects of puerarin on proliferation of vascular smooth muscle cells induced by thrombin].

OBJECTIVE: To investigate the effects of puerarin on the proliferation of vascular smooth muscle cells (VSMC) induced by thrombin and the mechanism thereof. METHODS: VSMCs were isolated from the thoracic aorta of a SD rat and cultured, then co-cultured with thrombin of the concentration 0.1, 0.3, 1.0, 3.0, and 10 U/L for 24 h, thrombin of the concentration of 1 U/L for 0, 6, 12, 24, 36, and 48 h respectively, or thrombin of the concentration of 1 U/L combined with puerarin of the concentrations of 1.5 x 10(-5), 1.5 x 10(-4), or 1.5 x 10(-3) mol/L for 24 h. Flow cytometry was used to detect the cell number and cell cycle. Western blotting was used to indicate the protein expression of the oncogenes c-fos and bcl-2 RT-PCR was used to evaluate the thrombin receptor (TR) mRNA expression. RESULTS: he numbers of the groups of VSMCs stimulated by 0.1, 0.3, 1.0, 3.0, and 10 U/L thrombin for 24 hours were 4.82 x 10(4)/ml +/- 0.11 x 10(4)/ml, 6.37 x 10(4)/ml +/- 0.09 x 10(4)/ml, 8.78 x 10(4)/ml +/- 0.08 x 10(4)/ml, 7.37 x 10(4)/ml +/- 0.07 x 10(4)/ml, and 5.28 x 10(4)/ml +/- 0.12 x 10(4)/ml respectively, all significantly higher than that of the control group (4.08 +/- 0.054 x 10(4)/ml, all P < 0.05). The effect of thrombin was in a dose-dependent manner within a concentration range of 0.1 - 1.0 U/L. The suppression rates of VSMC proliferation in the combination groups with puerarin of the concentrations of 1.5 x 10(-5), 1.5 x 10(-4), and 1.5 x 10(-3) mol/L were 10.9% +/- 1.6%, 32.1% +/- 3.3%, and 42.6% +/- 5.2% respectively in comparison with the thrombin group (all P < 0.05). The c-fos protein expression of the VSMCs after thrombin stimulation for 24 h increased by 156.0% +/- 11.3% (P < 0.05), and the bcl-2 protein expression of the VSMCs pretreated with puerarin of the concentrations of 1.5 x 10(-5), 1.5 x 10(-4), and 1.5 x 10(-3) mol/L, and then stimulated by thrombin was significantly lower than that of the VSMCs only stimulated by thrombin with the suppression rates of 20.7% +/- 2.1%, 31.6% +/- 5.2%, and 44.5% +/- 7.5% respectively (all P < 0.05). The bcl-2 protein expression of the VSMCs after thrombin stimulation for 24 h increased by 96.7% +/- 8.3% (P < 0.05), and the bcl-2 protein expression of the VSMCs pretreated with puerarin of the concentrations of 1.5 x 10(-5), 1.5 x 10(-4), and 1.5 x 10(-3) mol/L, and then stimulated by thrombin was significantly lower than that of the VSMCs only stimulated by thrombin with the suppression rates of 7.1% +/- 0.8%, 18.8% +/- 1.2%, and 39.6% +/- 6.4% respectively (all P < 0.05). The stimulation of thrombin increased the TR mRNA expression by 183.9% +/- 9.4%. The puerarin of the concentrations of 1.5 x 10(-5) mol/L and 1.5 x 10(-4) mol/L decreased the increase of TR mRNA expression induced by thrombin, however, without significant differences (both P > 0.05), and puerarin of the concentration of 1.5 x 10(-3) mol/L significantly suppressed the increase of TR mRNA expression induced by thrombin by 17.6% +/- 1.7% (P < 0.05). CONCLUSION: Puerarin suppresses the proliferation and DNA synthesis of VSMC induced by thrombin. The inhibitory effect of puerarin is closely related with the suppression of the protein expression of c-fos and bcl-2n, and partly related with the suppression of the TR mRNA expression.

Up-regulation of protease-activated receptor-2 by bFGF in cultured human synovial fibroblasts.

Protease-activated receptors (PARs) have been implicated in the development of acute and chronic inflammatory responses. We have examined the expression of mRNA for PARs and their regulation by growth factors and cytokines in synovial fibroblasts derived from patients with rheumatoid arthritis (RA). Messenger RNA for PAR-1, -2 and -3 was detected in these cells, but not that for PAR-4. Expression of mRNA for PAR-2 was up-regulated by bFGF in a concentration-dependent manner, whereas expression of mRNA for PAR-1 and PAR-3 was not affected. Levels of mRNA encoding PAR-1, PAR-2 and PAR-3 did not increase in response to IL-1beta and TNF-alpha. Expression of mRNA for PAR-2 was maximal 12 h after addition of bFGF, and maximal levels of immunoreactive PAR-2 were reached after 24 h. Furthermore, PAR-2 agonist peptide (SLIGKV-NH(2)), but not the inactive reverse peptide (VKGILS-NH(2)), induced transitory cytosolic Ca(2+) mobilization in cells, and its response was increased by pretreatment with bFGF. An important role could be played by bFGF in the regulation of functional PAR-2 expression in cultured RA synovial fibroblasts.

Immunohistochemical expression of Bcl2 is an independent predictor of time-to-biochemical failure in patients with clinically localized prostate cancer following radical prostatectomy.

BACKGROUND: Whether the immunohistochemical expression (IHCE) of the bcl2, the p53 and of the prostate apoptosis response-4 (PAR4) proteins is associated with pre-operative PSA levels, post-operative parameters of prostate cancer (PC) pathology, surgical staging or biochemical failure (BF) of patients with clinically localized PC who underwent radical prostatectomy (RP) of curative intent, was investigated. PATIENTS AND METHODS: A retrospective analysis of clinical data evaluating surgical specimens of 131 patients with PC, consecutively treated with RP for clinically localized disease, was performed. The IHC method of streptavidin biotin peroxidase on paraffin tissue sections was used to detect bcl2 and p53 oncoproteins and PAR4 pro-apoptotic protein expression in surgical specimens. RESULTS: Statistically significant relationships were detected between: (i) p53 IHC expression and infiltration of periprostatic tissue (IPT; p = 0.011); (ii) tumor volume (TV; p = 0.027); and (iii) bcl2 IHCE and absence of prostatic intraepithelial neoplasia (PIN) (p = 0.004). Biochemical failure (BF) was documented in 37% of these patients. Kaplan-Meier survival curves showed that the IHCE of bcl2 and p53 was significantly related to BF. Taking the hazard ratio (HR) estimated from the Cox proportional hazard regression model to be 1.00 for patients with negative bcl2 IHCE, a value of 2.82 was found for patients with positive bcl2 IHCE (p = 0.015, 95% CI = 1.22-6.47). The HR for patients with positive p53 IHCE was 2.05 (p = 0.048, 95% CI = 1.00-4.19). Multivariate analysis showed that only seminal vesicle invasion (SVI), pelvic lymph node metastasis (PLNM) and bcl2 IHCE were independent predictors for BF (HR = 3.06, 3.31 and 3.15; p = 0.048, p = 0.031 and p = 0.031 for SVI, PLNM and bcl2 IHCE, respectively). CONCLUSION: Bcl2 immunohistochemical overexpression in specimens of RP suggests high risk for BF in clinically localized PC.

Activation of PAR4 induces a distinct actin fiber formation via p38 MAPK in human lung endothelial cells.

Protease-activated receptors (PARs) are multifunctional G protein-coupled receptors. Among the four existing PARs, PAR4 is preferentially expressed in the human lung tissue. However, the function of PAR4 has not been defined in the lung endothelial cells. Because PAR1-mediated cellular effects are deeply related to the morphological changes, we focused on the actin fiber and p38 mitogen-activated protein kinase (MAPK) signaling involved in actin polymerization to elucidate the role of PAR4. RT-PCR and Western blot analyses identified PAR4 expression in human pulmonary artery endothelial cells and in human microvascular endothelial cells from lung. We then examined the changes in actin fibers in endothelial cells treated with PAR4-activating peptide. PAR1-activating peptide was used for comparison. Activation of PAR4 and PAR1 by their corresponding peptides induced actin fiber formation; however, the actin filaments were broadly bundled in PAR4 as compared with the ringlike actin filaments in PAR1 activation. Correspondingly, the magnitude of p38 MAPK phosphorylation was different between cells treated with PAR4 and PAR1, with PAR4-activating peptide showing a significantly higher sensitivity to p38 MAPK inhibitor, SB203580. Taken together, these results demonstrate that activation of PAR4 results in the formation of actin fiber distinct from that by PAR1 activation, suggesting PAR4 may play specific roles in the lung endothelial cells.

G-protein-activated phospholipase C-beta, new partners for cell polarity proteins Par3 and Par6.

Cell polarity and asymmetric cell division are fundamental traits of all living cells and play an essential role in embryonic development, neuronal cell chirality formation, and maintenance of mammalian epithelial cell morphology. Heterotrimeric GTP-binding proteins (G proteins) are involved in directing cell polarity and asymmetric cell division in different organisms. However, the mechanism for G-protein-mediated cell polarity and asymmetric cell division is poorly understood. In this study, we have demonstrated that G-protein-activated phospholipase C-beta (PLC-beta) interacts with cell polarity proteins Par3 and Par6 (Par: partition-defective) to form protein complexes and to mediate downstream signal transduction. The interactions between PLC-beta and Par proteins are direct and require the extreme C-terminal-specific sequence motifs of PLC-beta and the PDZ (PSD95/Dlg/ZO-1) domains of Par proteins. Binding of Par proteins with PLC-beta stimulates PLC-beta enzymatic activity, leading to the hydrolysis of phosphatidylinositol-4,5-bisphosphate, and the production of diacylglycerol and inositol 1,4,5-triphosphate, important mediators in cell polarity and cell asymmetric division processes. Furthermore, we have shown that coexpression of PLC-beta with Par proteins induces transcriptional activation coupled to intracellular Ca2+ and the Wnt signaling pathway. Therefore, our data suggest that the interaction of PLC-beta with cell polarity Par proteins may serve as a nexus to transduce extracellular signals to transcriptional regulation through G-protein-mediated signaling pathway in cell polarity and cell asymmetric division.

Gene expression profiling of inflamed human endothelial cells and influence of activated protein C.

BACKGROUND: During systemic inflammation, activation of vascular endothelium by proinflammatory cytokines leads to hypotension, microvascular thrombosis, and organ damage. Recent data suggest a link between coagulation and inflammation through the activated protein C (APC) pathway. We studied gene expression profiles in human coronary artery endothelial cells (HCAECs) exposed to proinflammatory stimuli and the influence of APC on expression of candidate genes regulated by these stimuli. METHODS AND RESULTS: HCAECs were stimulated with interleukin-1beta, interferon-gamma, and tumor necrosis factor-alpha. In gene expression profiling, 400 of 8400 genes were regulated >2-fold. Verification of selected candidate genes was achieved by measuring expression of mRNA species by real-time polymerase chain reaction, cytokine secretion by ELISA, and metabolites of tetrahydrobiopterin (BH4) biosynthesis by high-performance liquid chromatography. BH4 synthesis, interleukin-6, interleukin-8, monocyte chemotactic protein-1 (MCP-1), and intercellular adhesion molecule-1 (ICAM-1) were downregulated by APC at the transcriptional and protein level. Endothelial nitric oxide synthase, endothelial adhesion molecule, and vascular cell adhesion molecule-1 were not affected by APC. Activities of transcription factors c-Fos, FosB, and c-Rel were inhibited by APC in inflamed HCAECs. CONCLUSIONS: Our study revealed a novel antiinflammatory mechanism of APC-dependent gene regulation in HCAECs since c-Fos-dependent induction of MCP-1 and ICAM-1 was suppressed. APC downregulates expression and activity of genes related to inflammation, most pronounced under intermediate or mild inflammatory conditions.