Tissue Factor Pathway Inhibitor Gene Research Articles

Human stool vimentin, oncostatin M receptor and tissue factor pathway inhibitor 2 gene methylation analysis for the detection of colorectal neoplasms

To explore the feasibility of detecting vimentin, oncostatin M receptor (OMSR) and tissue factor pathway inhibitor 2 (TFPI2) gene methylation status in stool samples as a noninvasive screening tool for colorectal cancer and precancerous lesions. Stool samples were collected from 60 patients with colorectal cancer, 17 patients with adenoma and 30 normal volunteers. And fecal DNA was extracted and vimentin, OMSR and TFPI2 gene methylation status was analyzed by methylation-specific polymerase chain reaction (MSP). The methylated vimentin, OMSR and TFPI2 was detected in 53.3% (32/60), 68.3% (41/60) and 75.0% (45/60) of colorectal cancer, and 5, 7 and 11 cases of colorectal adenoma respectively. The sensitivities of combined study, using three markers for the detection of colorectal cancer and colorectal adenomas, were 86.7% (26/30) and 76.5% (13/17) respectively. And the specificity was 86.7% (52/60). As a feasible epigenetic marker, promoter hypermethylation for vimentin, OMSR and TFPI2 in stool samples is a sensitive, specific and noninvasive alternative for colorectal cancer screening.

Melatonin stimulates release of tissue factor pathway inhibitor from the vascular endothelium

We previously found an association between the circadian variation of free tissue factor pathway inhibitor (TFPI) and melatonin in able-bodied males and in men with complete cervical spinal cord injuries. We therefore examined whether melatonin modifies production and/or secretion of TFPI in endothelial cells. We sampled supernatants from cultures of primary human umbilical vein endothelial cells (HUVECs) and of human coronary artery endothelial cells (HCAECs), that had been exposed to varying doses (0-300 pg/ml) of melatonin for 0.5-24 h. We then measured the protein concentrations of free TFPI, tissue factor and plasminogen activator inhibitor type 1 (PAI-1). We also measured endothelial TFPI, tissue factor and PAI-1 transcripts using quantitative real-time PCR. Melatonin dose dependently increased free TFPI levels about 25-30-fold in supernatants of both HUVEC and HCAEC, and independent of incubation duration. In contrast, TF and PAI-1 remained unaltered upon increasing doses of melatonin. Neither TFPI mRNAs nor tissue factor mRNAs nor PAI-1-mRNAs were changed in cell cultures added melatonin. The ratio of free TFPI in cell supernatants to free TFPI in cell lysates about doubled upon addition of melatonin, indicating that melatonin increased release from intracellular storages of free TFPI or from membrane-bound free TFPI. Our data indicate that melatonin stimulates vascular endothelial cells to secrete TFPI without altering transcription of the TFPI gene. If melatonin increases TFPI release in a similar fashion in vivo as in vitro, this could have potential clinical implications in both prophylaxis and treatment of thromboembolic events.

Impact of the tissue factor pathway inhibitor gene on apoptosis in human vascular smooth muscle cells

Tissue factor pathway inhibitor (TFPI) plays a vitally important role in the blood coagulation pathway. Recent studies indicated that TFPI induces apoptosis in vascular smooth-muscle cells (VSMCs) in animals. The present study investigated whether the TFPI gene could also induce apoptosis in human vascular smooth-muscle cells (hVSMCs). Such cells were isolated from human umbilical arteries and subsequently transfected with pIRES-TFPI plasmid (2 μg/mL). MTT assaying and cell counting were applied to measure cell viability and proliferation, RT-PCR was utilized to analyze TFPI gene expression in the cells. Apoptosis was analyzed by fluorescence activated cell sorting (FACS). Several key proteins involved in apoptosis were examined through Western blotting. It was shown that TFPI gene transfer led to its increased cellular expression, with a subsequent reduction in hVSMC proliferation. Further investigation demonstrated that TFPI gene expression resulted in lesser amounts of procaspase-3, procaspase-8 and procascase-9, and an increased release of mitochondrial cytochrome c (cyt-c) into cytoplasm, thereby implying the involvement of both extrinsic and intrinsic pathways in TFPI gene-induced apoptosis in hVSMCs.

Tissue factor pathway inhibitor suppresses the growth of human vascular smooth muscle cells through regulating cell cycle

Tissue factor pathway inhibitor (TFPI) was reported to suppress the proliferation and migration of vascular smooth muscle cells (VSMCs) which play an important role in several vascular proliferative disorders including restenosis. Our preliminary studies demonstrated that TFPI gene induced apoptosis in human vascular smooth muscle cells (hVSMCs). The current study was designed to address the role TFPI gene plays in the cell cycle of hVSMCs. hVSMCs isolated from human umbilical artery were transfected with pIRES-TFPI plasmid which expresses TFPI in eukaryotic cells. As measured by RT-PCR, the expression of TFPI was elevated in the TFPI treated cells, leading to the arrest of the cells at G1 phase as analyzed by flow cytometry. Further study by Western blotting demonstrated that TFPI gene transfer increased the amount of p21 and p53 and decreased the amount of cyclin D and phosphorylated cdk4 and cdk6 in the cells.

Isolation and culture of human umbilical artery smooth muscle cells and gene transfection of TFPI gene

Objective The aim of the present study is to investigate the effect of tissue factor pathway inhibitor （TFPI） gene on cell cycle of human vascular smooth muscle cells.Methods Human vascular smooth muscle cells were separated from human umbilical artery and identified by immunohistochemical staining.The cells were transfected with various amount of pIRES-TFPI plasmid （1,2,and 3 μg/ml,respectively）and the TFPI expression in the cells were analyzed by RT-PCR.MTT assay was employed to detect the effect of TFPI gene on the proliferation of human vascular smooth muscle cells.Results The proliferation of vascular smooth muscle cells was inhibited in pIRES-TFPI group 5 and 7 days after gene transfection when compared with that of pIRES 1-neo transfection group.Conclusion The overexpression of TFPI gene in human umbilical artery vascular smooth muscle cells may contribute to the suppression of the proliferation of cells by gene transfection. Key words: Human smooth muscle cell; Tissue factor pathway inhibitor; Restenosis

Tissue factor pathway inhibitor polymorphisms in women with and without a history of venous thrombosis and the effects of postmenopausal hormone therapy

Postmenopausal hormone therapy is associated with marked reduction in tissue factor pathway inhibitor (TFPI) levels, and low TFPI levels have been associated with increased risk of venous thrombosis. Polymorphisms in the TFPI gene may affect the expression of TFPI. We aimed to investigate the influences of such polymorphisms on plasma TFPI levels and to investigate the effect of hormone therapy. Four single nucleotide polymorphisms in the TFPI gene (the -287T/C and the -399C/T polymorphisms in the 5' upstream region, and the intron 7 -33T/C and the exon 9 874G/A polymorphisms) were studied with regard to frequency, phenotype, and their influence on hormone therapy in postmenopausal women with a history of venous thrombosis (n = 138), in healthy postmenopausal women (n = 202), and in normal controls (n = 212). The frequencies of the -287C and the -33C variants were nonsignificantly lower in cases than in controls, and the polymorphisms were associated with slightly higher levels of free TFPI antigen (-287C; P = 0.076) and higher TFPI activity (-33C; P < 0.001). The -399T variant showed equal distribution in cases and controls, but was associated with lower levels of TFPI activity (P = 0.036). Conventional-dose hormone therapy induced significant reductions in TFPI levels irrespective of genotypes. In healthy women treated with low-dose hormone therapy, the reduction in TFPI levels was less pronounced with the -287C variant (P = 0.054). Our study indicates that polymorphisms in the TFPI gene may be of importance for plasma TFPI levels and for the effects of hormone therapy.

Functional characterization of polymorphisms in the human TFPI gene

Tissue factor pathway inhibitor (TFPI) is the primary physiological inhibitor of tissue factor (TF) induced coagulation. Low plasma TFPI levels have been shown to be associated with increased risk of arterial and venous thrombosis. Several clinical studies have reported that single nucleotide polymorphisms (SNPs) in the regulatory regions of the gene, such as the −287T/C, the −399C/T, and the −33T/C SNPs, may affect plasma TFPI levels. However, molecular studies investigating the functionality of the polymorphisms are lacking. In this study, we found that the −287C and −399T alleles affected the activity of the promoter using a reporter gene system. This was also the case for the −33T/C polymorphism. An association regarding the transcriptional activity of the reporter gene was detected between the −287C allele and the −33T/C polymorphism. Analysis of the polymorphic sites with electrophoretic mobility shift assay (EMSA) showed that all three polymorphisms potentially alter DNA–protein interactions. Based on these findings, we speculate that the −287C and the −33C alleles can be associated with lowered risk of thrombosis.

The relationship between plasma and placental tissue factor, and tissue factor pathway inhibitors in severe pre-eclampsia patients

Objective To investigate the mechanism underlying the hypercoagulable state in severe pre-eclampsia. Methods Plasma tissue factor (TF) and tissue factor pathway inhibitor (TFPI) expression from pre-eclampsia patients and healthy pregnant controls were determined by ELISA. Placental TF and TFPI gene and protein expression were detected by quantitative RT-PCR, immunohistochemistry, and Western analysis. Results The plasma TF level in the pre-eclampsia group was significantly higher than the control group (p < 0.01), and surprisingly, the plasma TFPI-1 and TFPI-2 in the pre-eclampsia group were significantly lower (p < 0.01). Placental TF gene and protein expression levels in the pre-eclampsia group was significantly higher than the control group, while TFPI-1 and TFPI-2 levels were significantly lower (p < 0.05). Lastly, a significant correlation was found between plasma and placental TF protein levels in the pre-eclampsia group (p < 0.01). Conclusion Higher expression and/or release of TF from the placenta may contribute towards a pathological hypercoagulable state in pre-eclampsia patients.

Gender differences of polymorphisms in the TF and TFPI genes, as related to phenotypes in patients with coronary heart disease and type-2 diabetes

BackgroundTissue factor (TF) and its inhibitor tissue factor pathway inhibitor (TFPI) are the main regulators of the initiation of the coagulation process, important in atherothrombosis. In this study we have investigated the frequency of six known TF and TFPI single nucleotide polymorphisms (SNPs) in CHD patients as compared to healthy individuals. These genotypes and the phenotypes (TF, TFPI free and total antigen) were evaluated with special reference to gender and diabetes in the CHD population.MethodsPatients with angiographically verified CHD (n = 1001; 22% women, 20% diabetics), and 204 healthy controls (28% women), were included. The investigated SNPs were: TF -1812C/T and TF -603A/G in the 5'upstream region, TF 5466A/G in intron 2, TFPI -399C/T and TFPI -287T/C in the 5'upstream region and the TFPI -33T/C in intron 7.ResultsNo significant differences in frequencies between the CHD population and the controls of any polymorphisms were observed. In the CHD population, the TF 5466 A/G SNP were significantly more frequent in women as compared to men (p < 0.001). The TF-1812C/T and the TF-603A/G SNPs were significantly more frequent in women without type-2 diabetes compared to those with diabetes (p < 0.018, both), and the heterozygous genotypes were associated with significantly lower TF plasma levels compared to the homozygous genotypes (p < 0.02, both).The TFPI-399C/T and the TFPI-33T/C SNPs were associated with lower and higher TFPI total antigen levels, respectively (p < 0.001, both).ConclusionGenetic variations in the TF and TFPI genes seem to be associated with gender and type-2 diabetes, partly affecting their respective phenotypes.

Involvement of Heparanase in early pregnancy losses

Background Heparanase cloned from and abundant in the placenta is implicated in cell invasion, tumor angiogenesis and metastasis. Recently, we demonstrated that heparanase is involved in the regulation of the hemostatic system. Heparanase was found to up-regulate tissue factor (TF) expression (Nadir et al., JTH, 2006) and interact with tissue factor pathway inhibitor (TFPI) on the cell surface, leading to dissociation of TFPI from the cell membrane resulting in increased cell surface coagulation activity (Nadir et al., TH, 2008). Herein, we investigated the role of heparanase in the placenta, focusing on its effect on TF, TFPI, TFPI-2, and vascular endothelial growth factor (VEGF)-A. Methods Twenty formalin embedded placenta samples of abortions (weeks 6-10) were studied applying real time RT-PCR and immunostaining. Ten cases were miscarriages of women with thrombophilia and recurrent fetal losses, and ten control cases were pregnancy terminations. JAR (human choriocarcinoma trophoblasts) cells were transfected with full-length heparanase cDNA or incubated with active (50+8 kDa) recombinant heparanase and the effects on TF, TFPI, TFPI-2 and VEGF-A were examined using real time RT-PCR and immunoblotting. Results Sections obtained from miscarriages revealed increased (2-3-folds) levels of heparanase, VEGF-A and TFPI-2 compared to placentas from controls in maternal as well as in fetal placenta elements. JAR cells overexpressing heparanase or incubated with exogenous recombinant heparanase exhibited a 2-3-fold increase in TFPI and TFPI-2 in cell lysates both at the protein and mRNA levels, with no detectable effect on VEGF-A and TF levels. Accumulation of TFPI and TFPI-2 in the cell culture medium was increased 4-6-folds, exceeding the observed induction of TFPI and TFPI-2 gene transcription. Conclusions These results indicate a regulatory effect of heparanase on TFPI and TFPI-2 in trophoblasts, suggesting a potential involvement of heparanase in early miscarriages.

Prognostic significance of tissue factor pathway inhibitor-2 in pancreatic carcinoma and its effect on tumor invasion and metastasis

Tissue factor pathway inhibitor-2 (TFPI-2) is a matrix-associated kunitz-type serine proteinase inhibitor that plays an important role in plasmin and trypsin-mediated activation of zymogen matrix metalloproteinases involved in tumor angiogenesis, invasion and metastasis. Earlier studies have shown that the production of TFPI-2 is downregulated during the progression of various tumors. To detect whether TFPI-2 can be expressed in human pancreatic carcinoma samples, to evaluate its prognostic significance on pancreatic carcinoma and to investigate its effect on tumor invasion and metastasis, we collected 9 normal pancreatic tissue samples and 41 pancreatic carcinoma samples and stably transfected the human pancreatic carcinoma cell line Panc-1 with a vector capable of expressing TFPI-2 gene. RT-PCR and Western blot analysis revealed that the expression of TFPI-2 in pancreatic carcinoma samples was markedly lower than that in normal pancreas samples, and there was no TFPI-2 expression in Panc-1 cell. Its expression was related with biological characters of pancreatic carcinoma. The results of Boyden chamber assay and orthotopic pancreatic carcinoma model showed that TFPI-2 could inhibit invasion and metastasis ability of pancreatic carcinoma in vitro and in vivo. Kaplan-Meier survival curve and Cox proportional hazards model assay identified TFPI-2 as an independent prognostic factor for pancreatic carcinoma. Our data suggest that TFPI-2 plays a significant role in the invasion and metastasis of pancreatic carcinoma cell in vitro and in vivo and is determined to be an important prognostic factor for pancreatic carcinoma patients.

Expression of Tissue Factor Pathway Inhibitor 2 in Human Pancreatic Carcinoma and its Effect on Tumor Growth, Invasion, and Migration In Vitro and In Vivo

Tissue factor pathway inhibitor 2 (TFPI-2), also known as placental protein and matrix-associated serine protease inhibitor, plays an important role in angiogenesis, intravascular fibrinolysis, wound healing, tumor invasion, metastasis by plasmin, and trypsin mediated activation of zymogen matrix metalloproteinases. To detect whether TFPI-2 can be expressed in human pancreatic carcinoma samples and to investigate its role in the growth, invasion, and metastasis of pancreatic carcinoma cell in vitro and in vivo, we collected eight normal pancreatic tissue samples and 50 pancreatic carcinoma samples, and stably transfected the human pancreatic carcinoma cell line Panc-1 with a vector capable of expressing TFPI-2 gene. RT-PCR and Western blot analysis revealed that the levels of TFPI-2 expression were markedly lower in pancreatic carcinoma samples compared with normal pancreas samples. The level of TFPI-2 protein was significantly higher in cells transfected with TFPI-2 gene than that in the untransfected cells. The results of MTT assay showed that TFPI-2 inhibited Panc-1 cells growth in vitro. The invasive capacity of the cells transfected with TFPI-2 gene was also markedly less than that ofuntransfected cells in vitro as determined by the Matrigel invasion/migration assay. Moreover, TFPI-2 inhibited tumor growth, invasion, and metastasis in vivo in an orthotopic pancreatic carcinoma model. Our findings suggest that TFPI-2 plays a significant role in the growth, invasion, and metastasis of pancreatic carcinoma cell in vitro and in vivo, and has potential in anticancer therapy.

EGCG inhibits growth and induces apoptosis in renal cell carcinoma through TFPI-2 overexpression

EGCG (epigallocatechin gallate), the major catechin found in green tea, has been shown to inhibit proliferation and induce apoptosis in many tumors. EGCG can inhibit DNMT (DNA methyltransferase) activity, and cause CpG demethylation and reactivation of methylation-silenced genes. Tissue factor pathway inhibitor-2 (TFPI-2), a member of the Kunitz-type serine proteinase inhibitor family, is inversely related to an increasing degree of malignancy. Our previous study showed that the expression of TFPI-2 and invasiveness of renal cell carcinoma had a negative correlation. Overexpression of TFPI-2 may induce tumor cell apoptosis in renal cell carcinoma. Lower expression of TFPI-2 in renal cell carcinoma was partly due to hypermethylation of the gene promoter. Herein, using MTT and flow cytometry, we demonstrated that EGCG can inhibit growth and induces apoptosis in renal cell carcinoma cell line 786-0. In addition, Western blotting and real-time RT-PCR showed that EGCG can upregulate expression of TFPI-2. Before and after EGCG treatment, real-time methylation specific PCR could not detect methylation status of TFPI-2 gene promoter in cell line 786-0. In vivo invasiveness and metastasis test did not indicate any significant differences between control and treatment group. Our results suggest that EGCG inhibits growth and induces apoptosis in renal cell carcinoma through TFPI-2 overexpression. This is the first report showing that EGCG is likely to be an effective agent for renal cell carcinoma.

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-alpha). Cultured human umbilical vein endothelial cells were incubated in the presence or absence of testosterone or TNF-alpha. 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-kappaB) translocation was confirmed by electrophoretic mobility shift assays. We found that after NF-kappaB was activated by TNF-alpha, 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-alpha-treated group. NF-kappaB 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-kappaB activity.

Involvement of Heparanase in Early Pregnancy Losses

Heparanase activity is implicated in cell invasion, tumor metastasis and angiogenesis. Recently, we have reported that heparanase stimulates tissue factor (TF) expression in endothelial and cancer cells, resulting in elevation of coagulation activity (Nadir et al. JTH, 2006). We also found that heparanase releases tissue factor pathway inhibitor (TFPI) from endothelial and tumor cells, contributing to the local procoagulant balance (Nadir et al. TH, 2008). Heparanase was cloned from and is abundant in the placenta. We investigated the role of heparanase in the placenta, focusing on its effect on TF, TFPI, TFPI-2, and vascular endothelial growth factor-A (VEGF-A). Twenty formalin embedded samples of early pregnancy losses (weeks 5–10) were studied. Ten cases were miscarriages of women with thrombophilia and recurrent fetal losses, and ten control cases were pregnancy terminations. Applying real time RT-PCR and immunostaining for heparanase, TF, TFPI, TFPI-2, and VEGF-A it was found that in fetal losses related to thrombophilia the levels of heparanase, VEGF-A and TFPI-2 were significantly increased (2–3 fold) compared to placentas from controls. Next, JAR (human choriocarcinoma throphoblast) cells were transfected to overexpress the full length (65 kDa) heparanase or incubated with active (50+8 kDa) recombinant heparanase. Immunoblotting and real time RT-PCR showed that throphoblasts overexpressing or incubated with exogenous heparanase exhibited a 2–3 fold increase in TFPI and TFPI-2 in cell lysates both at the protein and mRNA levels, with no detectable effect on VEGF-A and TF. Accumulation of TFPI and TFPI-2 in the cell culture medium was increased 5–6 fold, exceeding the observed induction of TFPI and TFPI-2 gene transcription. Extracellular accumulation of TFPI and TFPI-2 was already evident 60 min following heparanase addition, prior to protein induction, indicating an active release of preformed proteins. These results indicate a regulatory effect of heparanase on TFPI and TFPI-2 in throphoblasts, suggesting a potential involvement in early miscarriages.

Expression of the V264M TFPI mutant in endothelial cell cultures may involve mRNA stability

Introduction Tissue factor (TF) pathway inhibitor (TFPI) is the endogenous inhibitor regulating TF-induced blood coagulation. Several polymorphisms have been identified in the TFPI gene and some of them have been correlated with variations in plasma TFPI levels. The aim of the present study was to characterize the TFPI V264M mutant in comparison with the wild type protein (TFPI WT). Materials and Methods We have overexpressed the TFPI V264M mutant and TFPI WT in human coronary artery endothelial cells and compared the expression and activity levels of the mutated protein relative to the TFPI WT. The protein levels were determined by ELISA, the inhibitory activity of the proteins was assessed with a chromogenic substrate assay. The mRNA level of the two TFPI variants was determined using real time RT-PCR. MFOLD was used to predict mRNA secondary structure. Results and Conclusions TFPI V264M displayed increased protein levels and activity compared to TFPI WT accompanied by an increase in mRNA levels of TFPI V264M due to prolonged stability of TFPI V264M mRNA. The specific activity of the TFPI V264M was similar to TFPI WT, indicating that the mutation does not affect the enzymatic function of the protein.

Tissue Factor Pathway Inhibitor-2 gene methylation is associated with low expression in carotid atherosclerotic plaques

The tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine-protease inhibitor which is expressed in atherosclerotic plaques. Epigenetic regulation of the TFPI-2 gene, through methylation of CpG islands, has been advocated in cancer. We hypothesized that TFPI-2 gene methylation could regulate TFPI-2 expression in atherosclerosis. We used Methylation Specific PCR (MSP) and pyrosequencing in order to identify 18 CpG of the TFPI-2 promoter, in 59 carotid atherosclerotic plaques and 26 control mammary arteries. MSP showed methylation of the TFPI-2 gene (MSP+) in 16 plaques (27%), while no methylation (MSP-) was found in control arteries. Pyrosequencing confirmed that MSP+ plaques presented higher methylation levels than MSP- ones and arteries (p=0.03 and 0.01). Moreover, the TFPI-2 mRNA levels were lower in methylated plaques than in unmethylated ones and than in arteries (p=0.04 and <0.0001). The methylated plaques contained less lipids and macrophage infiltration than unmethylated ones. Their TFPI-2 immunoreactivity was mainly detected in the macrophages located in the media on the adventitial side, rather than in the lipid-rich core. Methylation of the TFPI-2 gene takes place in atherosclerotic plaques and is associated with decreased TFPI-2 expression. The place of this process in atherosclerosis progression remains to be investigated.

HVJ-AVE liposome-mediated Tissue Factor Pathway Inhibitor (TFPI) gene transfer with recombinant TFPI (rTFPI) irrigation attenuates restenosis in atherosclerotic arteries

In this study, we investigate whether the combination of HVJ-AVE liposome-mediated TFPI gene transfer and recombinant TFPI (rTFPI) irrigation would reduce restenosis safely and more effectively. The results indicated that at 4 weeks after angioplasty, the MLD, EELA, IELA and LA of TFPI group and rTFPI group were markedly greater than those of the control groups, and those in the combination group were even greater. The mean IA, I/M, and the percentage of stenosis in TFPI gene group and rTFPI group were significantly reduced compared with control groups, and those in the combination group were even further reduced. Thrombosis in the TFPI gene group, rTFPI group and combination group was significantly reduced compared with the other control groups. The systemic coagulation status of treated animals was not significantly changed and no toxicity was observed in each group. So combination of TFPI gene transfer using HVJ-AVE liposomes and rTFPI irrigation could inhibit thrombosis and neointimal hyperplasia, and attenuate vascular remodeling and luminal stenosis more effectively than using each method alone. The combination method may be a more effective and safe strategy for the prevention of restenosis after angioplasty in humans.

Genome-wide linkage analysis for identifying quantitative trait loci involved in the regulation of lipoprotein a (Lpa) levels

Lipoprotein Lp(a) levels are highly heritable and are associated with cardiovascular risk. We performed a genome-wide linkage analysis to delineate the genomic regions that influence the concentration of Lp(a) in families from the Genetic Analysis of Idiopathic Thrombophilia (GAIT) Project. Lp(a) levels were measured in 387 individuals belonging to 21 extended Spanish families. A total of 485 DNA microsatellite markers were genotyped to provide a 7.1 cM genetic map. The variance component linkage method was used to evaluate linkage and to detect quantitative trait loci (QTLs). The main QTL that showed strong evidence of linkage with Lp(a) levels was located at the structural gene for apo(a) on chromosome 6 (LOD score=13.8). Interestingly, another QTL influencing Lp(a) concentration was located on chromosome 2 with an LOD score of 2.01. This region contains several candidate genes. One of them is the tissue factor pathway inhibitor (TFPI), which has antithrombotic action and also has the ability to bind lipoproteins. However, quantitative trait association analyses performed with 12 SNPs in TFPI gene revealed no association with Lp(a) levels. Our study confirms previous results on the genetic basis of Lp(a) levels. In addition, we report a new QTL on chromosome 2 involved in the quantitative variation of Lp(a). These data should serve as the basis for further detection of candidate genes and to elucidate the relationship between the concentration of Lp(a) and cardiovascular risk.

Impact of Adenovirus-mediated Local Expression of Human Tissue Factor Pathway Inhibitor on Vascular Smooth Muscular Cell Proliferation and Apoptosis in the Stent-implanted Femoral Artery of the Rabbit

This study examined the effect of gene transfer of local tissue factor pathway inhibitor (TFPI) on vascular smooth muscle cells (VSMCs) in stent-implanted arteries. Rabbit femoral arteries were balloon-injured, stent-implanted and infected with the replication-defective recombinant adenovirus-mediated TFPI gene (Ad-TFPI) or the beta-galactosidase gene (Ad-LacZ), or treated with saline solution. Expression of TFPI at the site of the stent was confirmed after 3 days using reverse transcription-polymerase chain reaction (RT-PCR). After 7 days, proliferating cells were visualized by immunostaining with antibodies to proliferating cell nuclear antigen (PCNA) and apoptotic cells were detected using the terminal deoxynucleotidyl mediated nick end labelling (TUNEL) technique. Cell proliferation was significantly decreased and apoptosis significantly increased in the media in the Ad-TFPI group compared with the other two groups. In conclusion, Ad-TFPI gene transfer can significantly suppress VSMC proliferation and induce its apoptosis in the media at the site of an implanted stent and may have potential for the treatment of instent re-stenosis.