Vascular endothelial growth factor/connective tissue growth factor and proteomic analysis of aqueous humor after intravitreal conbercept for proliferative diabetes retinopathy

AIM:To investigate the role of connective tissue growth factor(CTGF)and vascular endothelial growth factor(VEGF)in the protein profile of the aqueous humor in patients with proliferative diabetic retinopathy(PDR)following intravitreal injection of conbercept.METHODS:This study included 72 PDR patients and 8 cataract patients as controls.PDR patients were divided into 3 groups according to the intervals of 3,5,and 7d between intravitreal conbercept(IVC,0.5 mg/0.05 mL)injection and pars plana vitrectomy(PPV)performed.Aqueous humor samples were collected before and after IVC and PPV for VEGF and CTGF levels detected with enzyme-linked immunosorbent assay(ELISA).The differential proteomics of 10 patients who underwent PPV surgery 5d after IVC and 8 normal controls was studied,Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis were performed on the data,and the protein interaction network of 23 differential proteins was studied.RESULTS:Post-IVC,VEGF levels decreased and CTGF levels increased significantly in aqueous humor,with the CTGF/VEGF ratio rising significantly at all intervals.Liquid chromatography tandem mass spectrometry(LC-MS/MS)identified differentially expressed proteins between preand post-IVC samples.GO and KEGG analyses revealed involvement in immune response,stress response,complement and coagulation cascades,ferroptosis,and PPAR signaling pathways.PPI analysis highlighted key proteins like APOA1,C3,and transferrin(TF).ELISA assay confirmed the differential expression of proteins such as HBA1,SERPINA1,COL1A1,and ACTB,with significant changes in the IVC groups.CONCLUSION:The study demonstrates that IVC effectively reduces VEGF levels while increasing CTGF levels,thereby modifying the CTGF/VEGF ratio,and IVC significantly alters the protein profile in the aqueous humor of patients with PDR.Proteomic analysis reveals that these changes are associated with critical biological pathways and protein interactions involved in immune response,stress response,and cellular metabolism.

Role of Tissue Factor Pathway Inhibitor-2 in Ovarian Tumor Migration and Invasion

Objective: To elucidate the relation between human tissue factor pathwayinhibitor-2 (TFPI-2) expression and ovarian tumor migration and invasion. Methods: Human TFPI-2expression vector pBos-Cite-neo/TFPI-2 was transfected into ovarian tumor cells line A2780- Afterthe transfected cells were selected by G418, transfected and nontransfected cells were screened forTFPI-2 mRNA and protein by reverse transcription-polymerase chain reaction and Western blotanalysis, respectively. The number of transfected or nontransfected cells passing through membraneof Boyden chamber was counted as the basis assessing tumor cells migratory and invasive behaviors.Results: Expression of mRNA and protein of TFPI-2 was detectable in transfected cells. In invasionassay, the number of TFPI-2-expressing cells to traverse a Matrigel-coated membrane was obviouslydecreased compared with that of nonexpressing cells (59.3±6.5 vs 109.7±5.5, P 【 0.01); While inmigration assay, no significant difference through a noncoated membrane was observed amongtransfected and nontransfected cells (114.7±8.6 vs 127.3±7.1, P 】 0.05). Conclusion: Expression ofTFPI-2 may strongly inhibit the invasive ability of ovarian tumor cells in vitro, but has no effecton the migratory ability which provides an experimental basis for genotherapy of human ovariantumor.

Expression of tissue factor in pancreatic adenocarcinoma is associated with activation of coagulation

AIM： To study expression of tissue factor （TF） in pancreatic cancer and its role in the development of thromboembolism.METHODS： TF expression was studied in eight human pancreatic carcinoma cell lines by Northern blot and indirect immunofluorescence. Expression of alternatively spliced TF （asTF） was assessed by RT-PCR. In addition, TF expression was determined by immunofluorescence in pancreatic tissues of 19 patients with pancreatic adenocarcinoma （PCa）, 9 patients with chronic pancreatitis （CP） and 20 normal controls. Plasma samples （30 PCa-patients, 13 CP-patients and 20 controls） were investigated for soluble TF levels and coagulation activation markers [thrombin-antithrombin Ⅲ complex （TAT）, prothrombin fragment 1 ＋ 2 （F1 ＋ 2）]. RESULTS： All pancreatic carcinoma cell lines expressed TF （8/8） and most of them expressed asTF （6/8）. TF expression at the protein level did not correlate with the differentiation of the carcinoma cell line. All but two pancreatic cancer tissue samples stained positive for TF （17/19）. In all samples of CP weak staining was restricted to pancreatic duct cells, whereas only a few subendothelial cells were positive in 9/20 of normal controls. TF and TAT levels in PCa patients were significantly elevated compared to controls whereas elevated F1 ＋ 2 levels did not reach statistical significance compared to controls. In CP patients TAT and F1 ＋ 2 levels proved to be significantly elevated compared to controls, although TAT elevation was less pronounced than in PCa patients. CONCLUSION： We conclude that in addition to the upregulated expression of TF on the cell membrane, soluble TF might contribute to activation of the coagulation system in pancreatic cancer.

Expression of connective tissue growth factor in tumor tissues is an independent predictor of poor prognosis in patients with gastric cancer

AIM: To examine the expression of connective tissue growth factor (CTGF), also known as CCN2, in gastric carcinoma (GC), and the correlation between the expression of CTGF, clinicopathologic features and clinical outcomes of patients with GC. METHODS: One hundred and twenty-two GC patients were included in the present study. All patients were followed up for at least 5 years. Proteins of CTGF were detected using the Powervision two-step immunostaining method. RESULTS: Of the specimens from 122 GC patients analyzed for CTGF expression, 58 (58/122, 47.5%) had a high CTGF expression in cytoplasm of gastric carcinoma cells and 64 (64/122, 52.5%) had a low CTGF expression. Patients with a high CTGF expression showed a higher incidence of lymph node metastasis than those with a low CTGF expression (P = 0.032). Patients with a high CTGF expression had significantly lower 5-year survival rate than those with a low CTGF expression (27.6% vs 46.9%, P = 0.0178), especially those staging Ⅰ+Ⅱ+Ⅲ (35.7% vs 65.2%, P = 0.0027). CONCLUSION: GC patients with an elevated CTGF expression have more lymph node metastases and a shorter survival time. CTGF seems to be an independent prognostic factor for the successful differentiation of high-risk GC patients staging Ⅰ+Ⅱ+Ⅲ. Over-expression of CTGF in human GC cells results in an increased aggressive ability.

A fusion protein containing murine vascular endothelial growth factor and tissue factor induces thrombogenesis and suppression of tumor growth in a colon carcinoma model

Induction of tumor vasculature occlusion by targeting a thrombogen to newly formed blood vessels in tumor tissues represents an intriguing approach to the eradication of primary solid tumors. In the current study, we construct and express a fusion protein containing vascular endothelial growth factor (VEGF) and tissue factor (TF) to explore whether this fusion protein has the capability of inhibiting tumor growth in a colon carcinoma model. The murine cDNA of VEGF A and TF were amplified by reverse transcriptase polymerase chain reaction (RT-PCR), and then cloned into prokaryotic expression plasmid pQE30 with a linker. The expression product recombinant VEGF-TF (rVEGF-TF) was purified and proved to have comparable enzyme activity to a commercial TF and the capability of specific binding to tumor vessels. Significant decrease of tumor growth was found in the mice administered with rVEGF-TF on Day 6 after initiated rVEGF-TF treatment (P<0.05), and the tumor masses in 2 of 10 mice were almost disappeared on Day 14 after the first treatment. In addition, valid thrombogenesis and tumor necrosis were observed in the tumor tissues injected with rVEGF-TF. Our results demonstrate that occlusion of tumor vasculature with rVEGF-TF is potentially an effective approach for cancer therapy.

Connective tissue growth factor is overexpressed in human hepatocellular carcinoma and promotes cell invasion and growth

AIM:To determine the expression characteristics of connective tissue growth factor(CTGF/CCN2) in human hepatocellular carcinoma(HCC) in histology and to elucidate the roles of CCN2 on hepatoma cell cycle progression and metastasis in vitro.METHODS:Liver samples from 36 patients(who underwent hepatic resection for the first HCC between 2006 and 2011) and 6 normal individuals were examined for transforming growth factor β1(TGF-β1) or CCN2 mRNA by in situ hybridization.Computer image analysis was performed to measure integrated optimal density of CCN2 mRNA-positive cells in carcinoma foci and the surrounding stroma.Fibroblast-specific protein-1(FSP-1) and E-cadherin were examined to evaluate the process of epithelial to mesenchymal transition,α-smooth muscle actin and FSP-1 were detected to identify hepatic stellate cells,and CD34 was measured to evaluate the extent of vascularization in liver tissues by immunohistochemical staining.CCN2 was assessed for its stimulation of HepG2 cell migration and invasion using commercial kits while flow cytometry was used to determine CCN2 effects on HepG2 cell-cycle.RESULTS:In situ hybridization analysis showed that TGF-β1 mRNA was mainly detected in connective tissues and vasculature around carcinoma foci.In comparison to normal controls,CCN2 mRNA was enhanced 1.9-fold in carcinoma foci(12.36 ± 6.08 vs 6.42 ± 2.35) or 9.4-fold in the surrounding stroma(60.27 ± 28.71 vs 6.42 ± 2.35),with concomitant expression of CCN2 and TGF-β1 mRNA in those areas.Epithelial-mesenchymal transition phenotype related with CCN2 was detected in 12/36(33.3%) of HCC liver samples at the edges between carcinoma foci and vasculature.Incubation of HepG2 cells with CCN2(100 ng/mL) resulted in more of the cells transitioning into S phase(23.85 ± 2.35 vs 10.94 ± 0.23),and induced a significant migratory(4.0-fold) and invasive(5.7-fold) effect.TGF-β1-induced cell invasion was abrogated by a neutralizing CCN2 antibody showing that CCN2 is a downstream mediator of TGF-β1-induced hepatoma cell invasion.CONCLUSION:These data support a role for CCN2 in the growth and metastasis of HCC and highlight CCN2 as a potential novel therapeutic target.

Connective tissue growth factor hammerhead ribozyme attenuates human hepatic stellate cell function

AIM： To determine the effect of hammerhead ribozyme targeting connective tissue growth factor （CCN2） on human hepatic stellate cell （HSC） function.METHODS： CCN2 hammerhead ribozyme cDNA plus two self-cleaving sequences were inserted into pTriEx2 to produce pTriCCN2-Rz. Each vector was individually transfected into cultured LX-2 human HSCs, which were then stimulated by addition of transforming growth factor （TGF）-β1 to the culture medium. Semiquantitative RT-PCR was used to determine mRNA levels for CCN2 or collagen I, while protein levels of each molecule in cell /ysates and conditioned medium were measured by ELISA. Cell-cycle progression of the transfected cells was assessed by flow cytometry.RESULTS： In pTriEx2-transfected LX-2 cells, TGF-β1 treatment caused an increase in the mRNA level for CCN2 or collagen I, and an increase in produced and secreted CCN2 or extracellular collagen I protein levels, pTriCCN2-Rz-transfected LX-2 cells showed decreased basal CCN2 or collagen mRNA levels, as well as produced and secreted CCN2 or collagen I protein. Furthermore, the TGF-β1-induced increase in mRNA or protein for CCN2 or collagen I was inhibited partially in pTriCCN2-Rz-transfected LX-2 cells. Inhibition of CCN2 using hammerhead ribozyme cDNA resulted in fewer of the cells transitioning into S phase.CONCLUSION： Endogenous CCN2 is a mediator of basal or TGF-β1-induced collagen I production in human HSCs and regulates entry of the cells into S phase.

Homocysteine-induced Enhanced Expression of Tissue Factor in Human Vascular Smooth Muscle Cells

The homocysteine （Hcy）-induced tissue factor （TF） expression in human vascular smooth muscle cells （VSMCs） and the effect of Hcy on the activity of nuclear factor-kappaB （NF-кB） and the expression of inducible nitric oxide synthase （iNOS） were investigated. Human umbilical artery VSMCs were cultured by tissue explanting method, identified by α-actin immunohistochemistry, and incubated with different concentrations of Hcy/PTDC （NF-кB inhibitor）. Semi-quantitative RT-PCR was performed to detect the expression of TF mRNA in VSMCs. Flow cytometry was used to assay the expression of TF protein on the surface of VSMCs and the expression of iNOS in VSMCs. Western blot was carried out to detect the expression of NF-кB protein in nuclei. The results showed that Hcy could induce VSMCs expressing TF mRNA significantly after the VSMCs were incubated with Hcy at concentrations of 10, 100, 500 μmol/L respectively. There was low expression level of TF protein on the surface of the resting VSMCs and Hcy could also induce VSMCs expressing TF pro- tein on the cell surface in different concentrations. Additionally, Hcy could rapidly induce the activation of NF-кB and this effect could be significantly inhibited by PDTC. Hcy alone could not induce the expression of iNOS in VSMCs. It was concluded that Hcy could significantly induce the expression of TF in VSMCs and enhance the activation of NF-ΚB, subsequently mediate TF gene expression and protein synthesis. NF-кB-mediated expression of TF in VSMCs might be the important mechanism of atherosclerosis and thrombosis induced by Hcy.

Positive correlation between plasma PCSK9 and tissue factors levels in patients with angiographically diagnosed coronary artery disease and diabetes mellitus

Background Pro-protein convertase subtilisin/kexin type 9 （PCSK9） is a secreted protein that influences plasma levels of low-density lipoprotein cholesterol （LDL-C）. Both oxidized LDL and tissue factor （TF） contributed to the development of prothrombofic state. The pre- sent study aims to explore the relationship between plasma level of PCSK9 and that of TF in patient with coronary artery disease （CAD）. Methods From July 2013 to March 2014, we enrolled 197 consecutive patients who underwent coronary angiography because of suspected CAD at Beijing Anzhen Hospital in this study. All patients had no history of using lipid-lowering medication. Of these 197 patients （1B 1 male and 66 female, mean age 56.9 ± 11.8 years）, 81 had angiographically diagnosed CAD. Clinical data were collected. Plasma PCSK9 and TF were measured using enzyme-linked immunosorbent assay （ELISA）. Levels of plasma PCSK9 and TF were compared and their correlation analyzed among different patient groups. Results Both plasma levels of PCSK9 （279.8 ± 60.4μg/L vs. 216.5 ± 45.3μg/L, P 〈 0.01） and TF （156.4 ± 26.6 μg/mL vs. 112.1 ± 38.3 μg/L, P 〈 0.01） were significantly higher in patients with CAD, as compared with those with- out CAD. Correlation analysis showed plasma level of PCSK9 was significantly correlated with that of TF in both patients with and without CAD. However, multivariate regression analysis after adjustment for age, gender, smoking, alcohol, hypertension and hyperlipidemia showed that only in CAD patients with type 2 diabetes mellitus, there was significant positive correlation between plasma levels of PCSK9 and TF （β = 0.353, P 〈 0.01）. Coneluslons The plasma level of PCSK9 is independently and positively associated with that of TF in CAD patients with diabetes mellitus, but not in those without diabetes mellitus. Further study is needed to investigate the underlying mechanism.

Clinical significance of expression of tissue factor and tissue factor pathway inhibitor in ulcerative colitis

AIM: To investigate the clinical significance of expression of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) in ulcerative colitis (UC).

cytoplasmic domain of tissue factor promotes liver fibrosis in mice

To evaluate the role of tissue factor (TF) and protease activated receptor (PAR)-2 in liver fibrosis. METHODSUsing CCl4 administration for eight weeks, we induced hepatic fibrosis in wild-type C57BL/6 mice and in mice with deletion of the cytoplasmic signalling domain of TF (TF§CT/§CT), deletion of PAR-2 (PAR-2-/-) and combined deletion of TF signalling domain and PAR-2 (TF§CT/§CT/PAR-2-/-). Hepatic fibrosis area was assessed by quantitative imaging of picrosirius red staining. Hepatic collagen content was assessed by hydroxyproline levels. Hepatic stellate cells (αSMA positive) and hepatic macrophages (CD68 positive) were identified by immunohistochemistry. Hepatic gene expression was determined by PCR and liver TGFβ1 content by ELISA. RESULTSCCl4 treated mice with deletion of the PAR-2 gene (PAR-2-/-) and the cytoplasmic domain of TF (TF§CT/§CT) developed significantly less hepatic fibrosis, characterised by reduced liver fibrosis area and hydroxyproline content, compared to control wildtype mice treated with CCl4. The observed reduction in histological fibrosis was accompanied by a significant decrease in the hepatic content of TGFβ, the prototypic fibrogenic cytokine, as well as fewer activated hepatic stellate cells and hepatic macrophages. Deletion of the TF cytoplasmic signalling domain reduced hepatic fibrosis to levels similar to those observed in mice lacking PAR-2 signalling but combined deletion provided no added protection against fibrosis indicating a lack of mutual modulating effects that have been observed in other contexts such as angiogenic responses. CONCLUSIONTissue factor cytoplasmic domain is involved in TF-PAR-2 signalling initiating hepatic fibrosis and is a potential therapeutic target, as its deletion would not impact coagulation.

Research progress on the role of connective tissue growth factor in fibrosis of diabetic retinopathy

Diabetic retinopathy（DR） is one of the most important types of diabetic microangiopathy, which is a specific change of fundus lesions and is one of the most serious complications of diabetes. When DR develops to proliferative DR, the main factors of decreasing vision, and even blindness, include retinal detachment and vitreous hemorrhage caused by contraction of blood vessels by fiber membrane. Recent studies reported that the formation of fiber vascular membrane is closely related to retinal fibrosis. The connective tissue growth factor（CTGF） is a cytokine that is closely related to DR fibrosis. However, its mechanism is poorly understood. This paper summarizes the recent studies about CTGF on DR fibrosis for a comprehensive understanding of the role and mechanism of CTGF in PDR.

Binding of EGF1 Domain Peptide in Coagulation Factor Ⅶ with Tissue Factor and Its Implications for the Triggering of Coagulation

The binding function of EGF1 domain peptide with tissue factor(TF)and its ability of triggering coagulation were explored.The TF expression model in vitro was established by lipopolysaccha-ride induction.The affinity of EGFP-EGF1 and TF expressing cells was analyzed by fluorescence microscopy and flow cytometry(FCM).The affinity of EGFP-EGF1 and rat soluble TF was quantitated by surface plasmon resonance(SPR).The ability of EGFP-EGF1 in triggering coagulation was tested by prothrombin time assay.The FCM res...

Connective tissue growth factor reacts as an IL-6/STAT3-regulated hepatic negative acute phase protein

AIM:To investigate the mechanisms involved in a possible modulator role of interleukin(IL) -6 signalling on CYR61-CTGF-NOV(CCN) 2/connective tissue growth factor(CTGF) expression in hepatocytes(PC) and to look for a relation between serum concentrations of these two parameters in patients with acute inflammation. METHODS:Expression of CCN2/CTGF,p-STAT3,p-Smad 3/1 and p-Smad2 was examined in primary freshly isolated rat or cryo-preserved human PC exposed to various stimuli by Western blotting,electrophoretic mobility shift assay(EMSA) ,reporter-gene-assays and reversetranscriptase polymerase chain reaction. RESULTS:IL-6 strongly down-regulated CCN2/CTGF protein and mRNA expression in PC,enhanceable by extracellular presence of the soluble IL-6 receptor gp80,and supported by an inverse relation between IL-6 and CCN2/CTGF concentrations in patients'sera.The inhi-bition of TGFβ1 driven CCN2/CTGF expression by IL-6 did not involve a modulation of Smad2(and Smad1/3) signalling.However,the STAT3 SH2 domain binding peptide,a selective inhibitor of STAT3 DNA binding activity,counteracted the inhibitory effect of IL-6 on CCN2/CTGF expression much more pronounced than pyrrolidine-dithiocarbamate,an inhibitor primarily of STAT3 phosphorylation.An EMSA confirmed STAT3 binding to the proposed proximal STAT binding site in the CCN2/CTGF promoter. CONCLUSION:CCN2/CTGF is identified as a hepatocellular negative acute phase protein which is downregulated by IL-6 via the STAT3 pathway through interaction on the DNA binding level.

Association of Plasma Connective Tissue Growth Factor Levels with Hyperthyroid Heart Disease

Hyperthyroid heart disease(HHD)is one of the most severe complications of overt hyperthyroidism and increases the risk of mortality in affected patients.Early identification of patients at a higher risk of developing HHD can improve clinical outcomes through active surveillance and management.Connective tissue growth factor(CTGF),a secreted extracellular protein,plays a significant role in cardiac remodeling and dysfunction.We aimed to investigate the association between plasma CTGF level and the risk of HHD in this study.A total of 142 overt hyperthyroid patients without HHD and 99 patients with HHD were included.The plasma CTGF levels were measured using ELISA kits.Routine clinical medical data and echocardiography parameters were recorded for analysis.The plasma CTGF level was significantly higher in patients with HHD than in those without HHD(P=0.002).The plasma CTGF level was positively correlated with free triiodothyronin,tryrotropin receptor antibody,troponin I and lactate dehydrogenase levels and the left atrium diameters,right atrium diameters,and right ventricular end-diastolic diameters(all P<0.05).Logistic regression analysis showed that quartiles 3 and 4 of plasma CTGF levels were significantly associated with the increased risk of HHD(crude OR:2.529;95%CI:1.188-5.387).However,after adjustment for the potentially confounding variables,quartile 4 alone was significantly associated with the higher risk of HHD relative to quartile I.Hyperthyroid patients with HHD display higher plasma CTGF levels.Furthermore,CTGF is an independent risk factor for HHD.Therefore,the plasma CTGF level may be a potential biomarker for the risk of HHD.

Role of Connective Tissue Growth Factor in Extracellular Matrix Degradation in Renal Tubular Epithelial Cells

In order to investigate the effects of connective tissue growth factor （CTGF） antisense oligodeoxynucleotide （ODN） on plasminogen activator inhibitor-1 （PAI-1） expression in renal tubular cells induced by transforming growth factor β1 （TGF-β1） and to explore the role of CTGF in the degradation of renal extracellular matrix （ECM）, a human proximal tubular epithelial cell line （HKC） was cultured in vitro. Cationic lipid-mediated CTGF antisense ODN was transfected into HKC. After HKC were stimulated with TGF-β1 （5 μg/L）, the mRNA level of PAI-1 was detected by RT-PCR. Intracellular PAI-1 protein synthesis was assessed by flow cytometry. The secreted PAI-1 in the media was determined by Western blot. The results showed that TGF-β1 could induce tubular CTGF and PAI-1 mRNA expression. The PAI-1 mRNA expression induced by TGF-β1 was significantly inhibited by CTGF antisense ODN. CTGF antisense ODN also inhibited intracellular PAI-1 protein synthesis and lowered the levels of PAI-1 protein secreted into the media. It was concluded that CTGF might play a crucial role in the degradation of excessive ECM during tubulointerstitial fibrosis, and blocking the biological effect of CTGF may he a novel way in preventing renal fibrosis.

Down-regulation of Tissue Factor by siRNA Increased Doxorubi-cin-induced Apoptosis in Human Neuroblastoma

The effects of tissue factor （TF） on doxorubicin-induced apoptosis in human neuroblastoma were investigated. The expression of TF was examined by Western blotting. TFsiRNA-pSUPER plasmid was constructed by inserting specific 19-nt silencing sequence targeting TF gene into pSUPER vector. Transfection of TFsiRNA-pSUPER was performed using lipofectamine^2000. The cytotoxicity of doxorubicin was determined by WST assay. The activation of Caspase-3 and PARP induced by doxorubicin was tested by Western blotting. The apoptotic cells were stained by Hochest33342 and counted under fluorescence inverted microscope. It was found that human neuroblastoma cell line SK-N-MC expressed high level of TE Knockdown of the TF expression was achieved by transfection of TFsiRNA-pSUPER on SK-N-MC cells in a dose-dependent manner. Inhibition of TF significantly decreased the viability of transfected SK-N-MC cells treated with different concentrations of doxorubicin. Cleavage of Caspase-3 and PARP was enhanced in transfected SK-N-MC cells with down-regulation of TF. TFsiRNA treatment significantly increased the number of apoptotic cells in transfected SK-N-MC cells as compared with those control cells （P〈0.05） when these cells were exposed to 1 μg/mL doxorubicin for 8 h. These results suggested that knockdown of the TF expression by specific siRNA vector could increase the cytotoxicity of doxorubicin and enhance doxorubicin-induced apoptosis in human neuroblastoma cells. Over-expression of TF might contribute to chemotherapy resistance in human neuroblastoma and its progression, at lest in part, by regulating doxorubicin-induced apoptosis.

Testosterone alleviates tumor necrosis factor-alpha-mediated tissue factor pathway inhibitor downregulation via suppression of nuclear factor-kappa B in endothelial cells

We have observed earlier that testosterone at physiological concentrations can stimulate tissue factor pathway inhibitor(TFPI)gene expression through the androgen receptor in endothelial cells.This study further investigated the impact of testosterone on TFPI levels in response to inflammatory cytokine tumor necrosis factor-alpha(TNF-α).Cultured human umbilical vein endothelial cells were incubated in the presence or absence of testosterone or TNF-α.TFPI protein and mRNA levels were assessed by enzyme-linked immunosorbent assay and quantitative real-time reverse transcription polymerase chain reaction.To study the cellular mechanism of testosterone’s action,nuclear factor-kappa B(NF-κB)translocation was confirmed by electrophoretic mobility shift assays.We found that after NF-κB was activated by TNF-α,TFPI protein levels declined significantly by 37.3%compared with controls(P<0.001),and the mRNA levels of TFPI also decreased greatly(P<0.001).A concentration of 30 nmol L-1 testosterone increased the secretion of TFPI compared with the TNF-α-treated group.NF-κB DNA-binding activity was significantly suppressed by testosterone(P<0.05).This suggests that physiological testosterone concentrations may exert their antithrombotic effects on TFPI expression during inflammation by downregulating NF-κB activity.

Expression of Connective Tissue Growth Factor in Renal Tubulointerstitial Fibrosis in Rats and Its Pathogenic Role

Summary： In order to explore the role of connective tissue growth factor （CTGF） in the pathogenesis of renal tubulointerstitial fibrosis, 48 Wistar rats were randomly divided into sham-operated and unilateral ureteral obstruction （UUO） group. On the postoperative day 1, 3, 7 and 14, the rats were killed and the kidneys were removed. The renal tubulointerstitial injury index was evaluated according to the MASSON staining. The mRNA levels of CTGF, transforming growth factor β1 （TGF-β1）. collagen [ （col I ）, and plasminogen activator inhibitor-1 （PAI 1） were detected using rexerse transcriptional-polymerase chain reaction （RT PCR）. Immunohistochemistry was performed to evaluale the protein expression of the above factors, and the relations among them were analyzed. Quantitative expression of CTGF protein in the kidneys was also assessed using Western blot. The results showed that TGF-β1 mRNA level was increased at first day after UUO, followed by a marked elevation of CTGF mRNA level, which began to increase 3 days after UUO （P〈0.01）. With the progression of the disease, the mRNA expression of CTGF, col I and PAI-1 was increased progressively. Immunohistochemistry revealed that the CTGF protein expression was significantly increased in fibrotic areas and tubular epithelial cells 3 days after UUO. On the post-UUO day 7, the protein level of CTGF was positively related to the renal tubulointerstitial injury index （r =0.62, P〈0.01）, the expression of TGF-β1 （r=0.85, P〈0.01）, colI （r=0.78, P〈0.01）, and PAI-1（r=0.76, P〈0.01）. Upon Western blot analysis, CTGF protein expression began to increase 3 days after UUO, and appeared progressively throughout the time course （P〈0.01, as compared with sham-operated group）. It is concluded that CTGF can be induced by TGF-β and mediate various profibrotic actions of this cytokine, such as increasing extracellular matrix （ECM） synthesis and decreasing ECM degradation. The increased expression of CTGF may play a crucial role in the development and progression of tubulointerstitial fibrosis.

Clinical significance of connective tissue growth factor in hepatitis B virus-induced hepatic fibrosis

AIM:To determine the utility of connective tissue growth factor(CCN2/CTGF) for assessing hepatic fibrosis in hepatitis B virus(HBV)-induced chronic liver diseases(CLD-B).METHODS:Enzyme-linked immunosorbent assay was used to measure CCN2 in sera from 107 patients with chronic hepatitis B(CHB) and 39 patients with HBVinduced active liver cirrhosis and 30 healthy individuals.Liver samples from 31 patients with CHB,8 patients with HBV-induced liver cirrhosis and 8 HBV carriers with normal liver histology were examined for transforming growth factor β-1(TGF-β1) or CCN2 mRNA levels by in situ hybridization,and computer image analysis was performed to measure integrated optimal density(IOD) of CCN2 mRNA-positive cells in liver tissues.Histological inflammation grading and fibrosis staging were evaluated by H and E staining and Van Gieson's method.RESULTS:Serum CCN2 concentrations were,respectively,4.0-or 4.9-fold higher in patients with CHB or active liver cirrhosis as compared to healthy individuals(P < 0.01).There was good consistency between the levels of CCN2 in sera and CCN2 mRNA expression in liver tissues(r = 0.87,P < 0.01).The levels of CCN2 in sera were increased with the enhancement of histological fibrosis staging in patients with CLD-B(r = 0.85,P < 0.01).Serum CCN2 was a reliable marker for the assessment of liver fibrosis,with areas under the receiver operating characteristic(ROC) curves(AUC) of 0.94 or 0.85 for,respectively,distinguishing normal liver controls from patients with F1 stage liver fibrosis or discriminating between mild and significant fibrosis.CONCLUSION:Detection of serum CCN2 in patients with CLD-B may have clinical significance for assessment of severity of hepatic fibrosis.