Biomedical subjects

U R Pendurthi

Publications and source records attributed to U R Pendurthi.

At least 19 recordsLinked to original sources

PubMed2000

Factor VIIa and thrombin induce the expression of Cyr61 and connective tissue growth factor, extracellular matrix signaling proteins that could act as possible downstream mediators in factor VIIa x tissue factor-induced signal transduction.

Extracellular interactions of plasma clotting factor VIIa (FVIIa) with tissue factor (TF) on cell surfaces trigger the intracellular signaling events. At present, it is unclear how these signals influence phenotype. To elucidate this, we have used cDNA microarray technology to examine changes in transcriptional program in human fibroblasts in response to exposure to FVIIa. cDNA microarrays revealed that FVIIa binding to TF up-regulated the expression of Cyr61 and CTGF (connective tissue growth factor), the genes that encode extracellular matrix signaling proteins Cyr61 and CTGF, respectively. Northern blot analysis confirmed that FVIIa binding to TF markedly increased the expression of Cyr61 and CTGF in a time- and dose-dependent manner. FVIIa catalytic activity is required for the gene induction. In addition to FVIIa, thrombin also induced the expression of Cyr61 and CTGF. Hirudin abolished the thrombin-induced expression of these mRNAs but not the FVIIa-induced expression. FVIIa-induced expression of Cyr61 appears not to involve the currently known protease-activated receptors (PARs), whereas thrombin-induced expression involves the activation of PAR1 and possibly an additional PAR. Various intracellular signaling pathway inhibitors exhibited different inhibitory pattern on FVIIa and thrombin-induced up-regulation of Cyr61. Cyr61 and CTGF could act as downstream mediators of FVIIa x TF in affecting various biological processes.

Cell Line↗

PubMed2000

Suppression of transcription factor Egr-1 by curcumin.

The transcription factor early growth response-1 gene product (Egr-1) is a member of the family of immediate early response genes and regulates a number of pathophysiologically relevant genes in vasculature that are involved in growth, differentiation, immune response, wound healing, and blood clotting. In the present study, we investigated the effect of curcumin, a natural plant phenolic compound known to exhibit anticarcinogenic, antioxidant, and antiinflammatory properties, on Egr-1 expression in endothelial cells and fibroblasts. Gel mobility shift assays showed that pretreatment of endothelial cells and fibroblasts with curcumin suppressed phorbol 12-myristate 13-acetate and serum-induced Egr-1 binding activity to the consensus Egr-1 binding site and also to the Egr-1 binding site present in the promoter of tissue factor gene. Western blot analysis revealed that curcumin inhibited phorbol 12-myristate 13-acetate-induced de novo synthesis of Egr-1 protein in endothelial cells. Suppression of Egr-1 protein expression in curcumin-treated cells stemmed from the suppression of Egr-1 mRNA. Northern blot analysis showed that curcumin inhibited serum and phorbol 12-myristate 13-acetate induced expression of tissue factor and urokinase-type plasminogen activator receptor mRNA in fibroblasts. Cumulatively, the data show that curcumin suppresses the induction of transcription factor Egr-1 and thereby modulates the expression of Egr-1-regulated genes in endothelial cells and fibroblasts.

Binding Sites↗

PubMed1999

Catabolism of factor VIIa bound to tissue factor in fibroblasts in the presence and absence of tissue factor pathway inhibitor.

Vascular injury leads to the exposure of blood to fibroblasts and smooth muscle cells within the vessel wall. These cells constitutively express tissue factor (TF), the cellular receptor for plasma clotting factor VIIa (FVIIa). Formation of TF.FVIIa complexes on cell surfaces triggers the blood coagulation cascade. In the present study, we have investigated the fate of TF.FVIIa complexes formed on the cell surface of fibroblasts in the presence and absence of plasma inhibitor, tissue factor pathway inhibitor (TFPI). FVIIa bound to TF on the cell surface was internalized and degraded without depleting the cell surface TF antigen and activity. TFPI significantly enhanced the TF-specific internalization and degradation of FVIIa. TFPI-enhanced internalization and degradation of FVIIa requires the C-terminal domain of TFPI and factor Xa. TFPI. Xa-mediated internalization of FVIIa was associated with the depletion of TF from the cell surface. A majority of the internalized FVIIa was degraded, but a small portion of the internalized FVIIa recycles back to the cell surface as an intact protein. In addition to TF, other cell surface components, such as low density lipoprotein receptor-related protein (LRP) and heparan sulfates, are essential for TFPI.Xa-induced internalization of FVIIa. Acidification of cytosol, which selectively inhibits the endocytotic pathway via coated pits, inhibited TFPI.Xa-mediated internalization but not the basal internalization of FVIIa. Overall, our data support the concept that FVIIa bound to cell surface TF was endocytosed by two different pathways. FVIIa complexed with TF in the absence of the inhibitor was internalized via a LRP-independent and probably noncoated pit pathway, whereas FVIIa complexed with TF along with the inhibitor was internalized via LRP-dependent coated pit pathway.

Cell Line↗

PubMed1999

Regulation of tissue factor pathway inhibitor expression in smooth muscle cells.

Tissue factor pathway inhibitor (TFPI) is the primary physiological inhibitor that regulates tissue factor-induced blood coagulation. TFPI is thought to be synthesized, in vivo, primarily by microvascular endothelial cells. Little is known about how TFPI is regulated under pathophysiological conditions. In this study, we determined mechanisms by which TFPI expression is regulated by human pulmonary artery smooth muscle cells (PASMC), because these cells contribute to remodeling of the pulmonary vasculature in disease. PASMC in culture constitutively synthesize and secrete TFPI. Exposure of PASMC to phorbol myristate acetate, lipopolysaccharide, tumor necrosis factor alpha, thrombin, interleukin-1, and transforming growth factor-beta had no significant effect on expression of TFPI by PASMC. By contrast, treatment of PASMC with serum and basic fibroblast growth factor (bFGF)/heparin markedly upregulated the expression of TFPI activity and antigen. On Western blot analysis, a protein consistent with full-length TFPI (42 kD) was identified in the conditioned media of PASMC, and the levels of the protein were much higher in the conditioned media of serum and bFGF/heparin-treated cells. Northern blot analysis showed that PASMC constitutively express TFPI mRNA, and treatment of cells with serum and bFGF/heparin had a minimal effect on the steady-state levels of TFPI mRNA. Nuclear run-on analysis did not show a significant increase in the transcriptional rate of TFPI gene in PASMC treated with serum or bFGF/heparin. Cycloheximide, but not actinomycin-D, treatment inhibited the serum and bFGF/heparin-induced increase in TFPI activity in PASMC. In conclusion, our data demonstrate that PASMC constitutively synthesize and secrete TFPI and serum or bFGF upregulate its expression, suggesting that growth factors that can stimulate the vessel wall in vivo might locally regulate TFPI expression. Our study also suggests that control of TFPI expression by serum or bFGF occurs via translational rather than transcriptional regulation.

Animals↗

PubMed1999

Resveratrol, a polyphenolic compound found in wine, inhibits tissue factor expression in vascular cells : A possible mechanism for the cardiovascular benefits associated with moderate consumption of wine.

A number of studies suggest that moderate consumption of red wine may be more effective than other alcoholic beverages in decreasing the risk of coronary heart disease mortality. The phytochemical resveratrol found in wine, derived from grapes, has been thought to be responsible for cardiovascular benefits associated with wine consumption because it was shown to have antioxidant and antiplatelet activities. In the present investigation, we examined the effect of resveratrol on induction of tissue factor (TF) expression in vascular cells that were exposed to pathophysiological stimuli. The data presented herein show that resveratrol, in a dose-dependent manner, inhibited the expression of TF in endothelial cells stimulated with a variety of agonists, including interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNFalpha) and lipopolysaccharide (LPS). A similar inhibition of TF induction was also seen in LPS stimulated monocytes that were pretreated with resveratrol before their stimulation with LPS. In addition, resveratrol was shown to inhibit the LPS-induced expression of TNFalpha mRNA in endothelial cells and of TNFalpha and IL-1beta mRNA in monocytes. Nuclear run-on analysis in endothelial cells showed that resveratrol inhibited TF expression at the level of transcription. However, resveratrol did not significantly alter the binding of the transcription factors c-Fos/c-Jun and c-Rel/p65, the transcription factors required for the induction of TF promoter in both endothelial cells and monocytes. Similarly, resveratrol had no significant effect on the binding of NF-kappaB in endothelial cells stimulated with IL-1beta, TNFalpha, and LPS. Overall, our data show that resveratrol could effectively suppress the aberrant expression of TF and cytokines in vascular cells, but it requires further investigation to understand how resveratrol exerts its inhibitory effect.

Alcohol Drinking↗

PubMed1998

Tissue factor on cells.

Tissue factor (TF), a cell surface glycoprotein, serves as the cellular receptor for either activated or non-activated factor VII [FVII(a)] and it is the formation of TF-FVII(a) complexes on cell surfaces which triggers the coagulation cascade in vivo. TF procoagulant functional expression on cell surfaces can be regulated by at least three distinct major mechanisms: (1) transcriptional regulation of TF gene expression; (2) cell membrane alterations in cells expressing TF; and (3) neutralization of TF-activated factor VII (FVIIa) activity by plasma inhibitors. The TF gene, which is not normally expressed in vascular cell types, can be induced by several pathophysiological stimuli, particularly those elaborated upon in inflammation and cancer. However, some of the stimuli elaborated in these pathological processes, e.g. basic fibroblast growth factor, suppress the induced expression of TF in endothelium. Not all TF molecules expressed on cell surfaces are functional even though they have the ability to bind to FVII(a). The availability of anionic phospholipids on cell membranes in the vicinity of TF and the spatial localization of TF within the cell membrane influence the functional activity of TF. Once TF-FVII(a) complexes are assembled on cell surfaces, at least two plasma inhibitors, TF pathway inhibitor and antithrombin III play an important role in regulating the TF-FVII(a) functional activity by inhibiting the activation of factor VII bound to TF and by inhibiting the catalytic activity of TF-FVIIa complexes. The availability of heparan sulphate proteoglycans with anticoagulant activity on cell surfaces plays an important role in enhancing the activity of the inhibitors. This manuscript summarizes the mechanisms by which TF functional expression on cells is regulated with a particular emphasis on the recent findings of the authors and their collaborators.

Cell Membrane↗

PubMed1997

Binding of factor VIIa to tissue factor induces alterations in gene expression in human fibroblast cells: up-regulation of poly(A) polymerase.

Tissue factor (TF) is the cellular receptor for an activated form of clotting factor VII (VIIa) and the binding of factor VII(a) to TF initiates the coagulation cascade. Sequence and structural patterns extracted from a global alignment of TF confers homology with interferon receptors of the cytokine receptor super family. Several recent studies suggested that TF could function as a genuine signal transducing receptor. However, it is unknown which biological function(s) of cells are altered upon the ligand, VIIa, binding to TF. In the present study, we examined the effect of VIIa binding to cell surface TF on cellular gene expression in fibroblasts. Differential mRNA display PCR technique was used to identify transcriptional changes in fibroblasts upon VIIa binding to TF. The display showed that VIIa binding to TF either up or down-regulated several mRNA species. The differential expression of one such transcript, VIIa-induced up-regulation, was confirmed by Northern blot analysis. Isolation of a full-length cDNA corresponding to the differentially expressed transcript revealed that VIIa-up-regulated gene was poly(A) polymerase. Northern blot analysis of various carcinomas and normal human tissues revealed an over expression of PAP in cancer tissues. Enhanced expression of PAP upon VIIa binding to tumor cell TF may potentially play an important role in tumor metastasis.

Base Sequence↗

PubMed1997

Characterization of a full-length cDNA for rabbit factor X.

A 300 bp probe generated by the PCR was derived from rabbit genomic DNA using primers from a highly conserved region of the DNA for human factor X (HFX). The probe was used in northern blot analysis of liver RNA to demonstrate an mRNA species of 1.6 kb for the rabbit factor X and subsequently for isolation and characterization of the cDNA for rabbit factor X (RFX) from a lambda Zap II cDNA library generated from rabbit liver mRNA. The cDNA contains 22 bases upstream from the 5'-translation initiation codon, 1470 nucleotides of open reading frame, a stop codon and a 3' poly (A) tail. The cDNA codes for a 40-residue signal/propeptide region, followed by a 447-residue mature protein. The deduced amino acid sequence shows a high degree of homology with the sequence of HFX. Inhibitory peptides derived from interactive sites of HFX for activators, cofactor and substrate exerted degrees of inhibition of RFX activation which showed a dependence on extent of homology with the corresponding regions of RFX.

Amino Acid Sequence↗

PubMed1997

Inhibition of tissue factor gene activation in cultured endothelial cells by curcumin. Suppression of activation of transcription factors Egr-1, AP-1, and NF-kappa B.

Binding of plasma factor VII(a) to tissue factor (TF) initiates the coagulation cascade. In health, TF is not expressed in endothelial cells. However, endothelial cells express TF in response to lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF alpha), and other biological stimuli. TF expression by endothelial cells is implicated in thrombotic disorders in patients with a variety of clinical disorders. In the present study, we demonstrate that curcumin (diferulolylmethane), a known anticarcinogenic and anti-inflammatory agent, inhibited phorbol 12-myristate 13-acetate (PMA), LPS, TNF alpha, and thrombin-induced TF activity and TF gene transcription in human endothelial cells. The present data show that curcumin prevented the activation of c-Rel/p65, which is essential for TF gene activation in endothelial cells, by impairing the proteolytic degradation inhibitor protein, I kappa B alpha. The data also show that curcumin downregulated AP-1 binding activity. The present studies are the first to demonstrate that PMA, but not LPS, TNF alpha, and thrombin, induced Egr-1 binding to the second serum-responsive region (SRR-2) of TF promoter and that curcumin inhibited the PMA-induced Egr-1 binding to SRR-2. Overall, the data suggest that the anticarcinogenic and anti-inflammatory properties of curcumin may be related to its ability to inhibit cellular gene expression regulated by transcription factors NF-kappa B, AP-1, and Egr-1.

Cells, Cultured↗

PubMed1997

Acidic and basic fibroblast growth factors suppress transcriptional activation of tissue factor and other inflammatory genes in endothelial cells.

Tissue factor (TF) is a transmembrane receptor that serves as a cofactor for factor VIIa and initiates the extrinsic pathway of blood coagulation. Under normal physiological conditions, TF is expressed in extravascular and perivascular cells but not in vascular endothelial cells and monocytes. TF can be induced in these cells by inflammatory regulators and other stimulators, such as LPS, thrombin, oxidized lipoproteins, and certain growth factors. An earlier study showed that growing primary cultures of human umbilical vein endothelial cells (HUVECs) with endothelial cell growth supplement (ECGS) and heparin had impaired the ability of monolayers to express surface membrane TF activity after perturbation. The mechanism by which ECGS suppressed TF activity was not known. In the present study, we investigated the effect of recombinant acidic and basic fibroblast growth factors (aFGF and bFGF) on the induction of TF in a HUVEC cell line and a fibroblast cell line. Both aFGF and bFGF suppressed the phorbol myristate acetate-induced expression of TF in endothelial cells but not the serum-induced expression of TF in fibroblast cells. Diminished expression of the cell surface TF activity observed in endothelial cells grown with aFGF or bFGF was due to the accumulation of a lower number of TF mRNA transcripts. TF mRNA stability was not altered in HUVECs grown with aFGF or bFGF. Nuclear run-on experiments revealed that the transcription of TF and several other genes that play an important role in inflammation and angiogenesis was reduced in the endothelial cells that were cultured with aFGF or bFGF. The diminished expression of TF may be part of a generalized response of endothelial cells to FGF that facilitates migration of endothelial cells during angiogenesis.

Cells, Cultured↗

PubMed1995

Mechanism of antithrombin III inhibition of factor VIIa/tissue factor activity on cell surfaces. Comparison with tissue factor pathway inhibitor/factor Xa-induced inhibition of factor VIIa/tissue factor activity.

Recent studies have shown that antithrombin III (AT III)/heparin is capable of inhibiting the catalytic activity of factor VIIa bound either to relipidated tissue factor (TF) in suspension or to TF expressed on cell surfaces. We report studies of the mechanism of which by AT III inhibits factor VIIa bound to cell surface TF and compare this inhibitory mechanism with that of tissue factor pathway inhibitor (TFPI)-induced inhibition of factor VIIa/TF. AT III alone and AT III/heparin to a greater extent reduced factor VIIa bound to cell surface TF. Our data show that the decrease in the amount of factor VIIa associated with cell surface TF in the presence of AT III was the result of (1) accelerated dissociation of factor VIIa from cell surface TF after the binding of AT III to factor VIIa/TF complexes and (2) the inability of the resultant free factor VIIa-AT III complexes to bind effectively to a new cell surface TF site. Binding of TFPI/factor Xa to cell surface factor VIIa/TF complexes markedly decreased the dissociation of factor VIIa from the resultant quaternary complex of factor VIIa/TF/TFPI/factor Xa. Addition of high concentrations of factor VIIa could reverse the AT III-induced inhibition of cell surface factor VIIa/TF activity but not TFPI/factor Xa-induced inhibition of factor VIIa/TF activity.

Antithrombin III↗

PubMed1993

Cloning and characterization of rabbit liver UDP-glucuronosyltransferase cDNAs. Developmental and inducible expression of 4-hydroxybiphenyl UGT2B13.

A polyclonal antibody generated against rabbit liver p-nitrophenol UDP-glucuronosyltransferase (UGT) was used to screen a rabbit liver cDNA expression library constructed in lambda gt11. A 500-base pair cDNA clone, termed pPNP, generated a fusion protein that was antigenic with the antibody. Clone pPNP encoded the 3' region of a UGT. To identify larger recombinants, clone pPNP was used as a probe to screen a second cDNA library constructed in lambda ZAP. Two different cDNA clones were identified by DNA sequence analysis. Based upon their predicted amino acid sequence analysis, the clones encode transferases belonging to the UGT2 subfamily, and have been identified as UGT2B13 and UGT2B14. The predicted N-terminal sequence of UGT2B13 is identical to that determined for the purified rabbit liver estrone UGT. However, expression of the UGT2B13 cDNA in COS-1 cells displayed no activity in the presence of estrone but efficiently conjugated 4-hydroxybiphenyl. Results of Southern blot analysis using the 5' divergent region of the UGT2B13 cDNA that encodes exon 1 demonstrates that multiple genes share sequence homology to UGT2B13, an observation which indicates that the estrone UGT and UGT2B13 genes are encoded by separate alleles. When the 5' variable regions of the cDNAs where used in Northern blot analysis, the expression of UGT2B13 and UGT2B14 were shown to be expressed primarily in adult rabbits. However, when neonatal rabbits were treated with either dexamethasone or rifampicin, UGT2B13 mRNA levels were induced. The neonatal induction of UGT2B13 mRNA corresponded with similar increases in 4-hydroxybiphenyl UGT activity. The expression and induction of UGT2B13 paralleled that of the developmentally regulated rabbit liver progesterone 6 beta-hydroxylase P4503A6.

Amino Acid Sequence↗

PubMed1993

Accumulation of the nuclear dioxin (Ah) receptor and transcriptional activation of the mouse Cyp1a-1 and Cyp1a-2 genes.

The treatment of C57BL/6 mice with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads to the nuclear uptake of the arylhydrocarbon receptor (AhR) and transcriptional activation of Cyp1a-1 and Cyp1a-2 (S. T. Okino, et al., J. Biol. Chem. 267, 6991, 1992). In the present study, early nuclear uptake of the AhR and its role in transcriptional activation of the Cyp1 genes have been evaluated. After 30 min following a dose of TCDD to C57BL/6 mice, the AhR could be detected in liver nuclei. The effect of TCDD treatment within 30 min enhanced the transcriptional rate of the Cyp1a-2 gene to 70% of its maximal rate, with maximal levels of transcription occurring after 1 h. Early increases in 1a-2 mRNA were also observed by 30 min and increased to maximal levels by 12 h. In contrast, the levels of Cyp1a-1 transcription were 5 to 10% of maximal levels at 30 min, and gradually increased to maximal levels by 2 h. Concordant with the levels of transcription, 1a-1 mRNA was not detected until 1 h following TCDD treatment. While the AhR is responsible for transcriptional activation of the Cyp1a-1 gene, the concordant increase in the nuclear accumulation of the ligand-dependent AhR and Cyp1a-2 gene transcription suggests that the receptor plays an important role in the regulation of the Cyp1a-2 gene.

Animals↗

PubMed1993

2,3,7,8-Tetrachlorodibenzo-p-dioxin induces the nuclear translocation of two XRE binding proteins in mice.

The administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene (3MC) to mice results in their binding to the ligand binding portion of the cytosolic dioxin-(Ah)-receptor, followed by translocation of the Ah receptor complex to the nucleus where the DNA binding form of the receptor can be measured by gel retardation analysis. In this report, extended electrophoresis of the nuclear DNA binding proteins isolated from liver demonstrate that TCDD and 3MC induce two nuclear DNA binding proteins in Ah-responsive C57BL/6 mice, while only TCDD induces these proteins in the Ah-nonresponsive DBA/2 mice. The two TCDD inducible (TI) nuclear DNA binding proteins, identified as TI-1 and TI-2, bind specifically to the Cypla-1 gene dioxin-(Ah)-receptor enhancer sequences (XREs) concordant with the properties of the Ah receptor. TI-1 is the predominant inducible form that is present in liver and extrahepatic tissues and most likely represents what is thought to be the Ah receptor, while TI-2 represents a minor form that is found only in liver. The nuclear induction of the Ah receptor by TCDD can be inhibited by phorbol esters such as TPA (Okino et al., 1992), but analysis of nuclear TI-1 and TI-2 shows that TPA can selectively inhibit the appearance of TI-1. The results of differential expression with regard to tissue and also inhibition by TPA suggests that TI-1 and TI-2 are under different modes of regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Phorbol esters inhibit the dioxin receptor-mediated transcriptional activation of the mouse Cyp1a-1 and Cyp1a-2 genes by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Tetradecanoyl phorbol acetate (TPA) has been shown to inhibit 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced mouse P450IA1 benzo[a]pyrene hydroxylase activity (Raunio, H., and Pelkonen, O. (1983) Cancer Res. 43, 782-786). When we co-administered TPA and TCDD to C57BL/6 mice, the accumulation of TCDD-inducible liver P450IA1 and P450IA2 mRNA, as well as kidney P450IA1 mRNA, was greatly inhibited. When nuclear run-on assays were conducted, maximal levels of transcriptional activation were achieved for both liver Cyp1a-1 and Cyp1a-2 with 1 micrograms/kg (approximately equal to 3.0 nmol/kg) TCDD. TCDD elicited a dose-dependent increase in the rates of gene transcription, which paralleled the induction of P450IA1 and P450IA2 mRNA. Only Cyp1a-1 gene transcription was elevated in kidney. When these experiments were repeated following the co-administration of TPA with TCDD, the levels of TCDD-mediated transcriptional increases in liver Cyp1a-1 and Cyp1a-2 and P450IA1 and P450IA2 mRNAs were dramatically inhibited. The reduction in Cyp1a gene transcription by TPA could be accounted for by reduced DNA binding of the dioxin receptor to the xenobiotic-responsive element (XRE) sequences, as measured by gel-retardation analysis. Analysis of nuclear [3H]TCDD dioxin receptor by sucrose density gradients demonstrated that the inhibition of Cyp1a gene transcription and DNA binding by TPA resulted from a reduction in nuclear dioxin receptor concentration.

Animals↗

PubMed1992

Characterization of a rabbit factor IX cDNA.

A rabbit factor IX cDNA was isolated and characterized. The cDNA was 2,676 bp in length and contained the coding region for the leader peptide, the entire mature factor IX protein and a long 3' untranslated region. The deduced amino acid sequence shows a high degree of homology with the sequences of human factor IX and factor IX from other species. Northern blot analysis of liver RNA showed a single mRNA species of 2.8 kb for the rabbit factor IX.

Amino Acid Sequence↗

PubMed1991

Mechanism for diminished tissue factor expression by endothelial cells cultured with heparin binding growth factor-1 and heparin.

We have extended our earlier observation that growing primary cultures of human umbilical vein endothelial cells (HUVEC) with heparin binding growth factor 1 (HBGF-1) 20 micrograms/mL and heparin 12 U/mL inhibits expression of tissue factor (TF) activity on HUVC monolayers perturbed with thrombin. TF activity was measured as the ability of monolayers or cell lysates to support FVIIa-catalyzed activation peptide release from 3H-FX. TF antigen in HUVEC extracts was measured in an enzyme-linked immunosorbent assay (ELISA) that uses a double-antibody sandwich technique with rabbit and goat antibodies to human TF. TF-mRNA was measured by Northern blot hybridization with a 32P-TF cDNA probe. Cells growth with HBGF-1/heparin had both decreased surface and total TF activity as compared with HUVEC from the same endothelial cell pool grown without HBGF-1/heparin. Means +/- SD for TF antigen for four primary cultures were 4.4 +/- 0.9 ng/10(6) cells without HBGF-1/heparin and 0.6 +/- 0.3 ng/10(6) cells with HBGF-1/heparin. TF mRNA 4 hours after incubation with thrombin of HUVEC grown without HBGF-1/heparin was about sevenfold higher than TF mRNA of HUVEC grown with HBGF-1/heparin. These data establish that growing primary cultures of HUVEC with HBGF-1/heparin impairs their ability to synthesize TF apoprotein after perturbation. This may be part of a generalized response of endothelial cells to HBGF-1/heparin facilitating migration during angiogenesis.

Antigens↗

PubMed1990

Characterization of the CYP2C5 gene in 21L III/J rabbits. Allelic variation affects the expression of P450IIC5.

Rabbits exhibit two heritable phenotypes, 21L and 21H, with the latter displaying roughly 10-fold higher liver microsomal progesterone 21-hydroxylase activity. The higher activity of the 21H animals reflects the elevated expression of cytochrome P-450 1 (P450IIC5). Breeding studies indicate that this phenotypic difference is linked to allelic CYP2C5 genes (Johnson, E. F., Finlayson, M., Husjsak, C. M., Pendurthi, U. R., and Tukey, R. H. (1989) Arch. Biochem. Biophys. 273, 273-280). In this study, we report on the expression and structure of the CYP2C5 gene in the 21L inbred strain III/J. A cDNA encoding P450IIC5 was generated from III/J rabbit liver mRNA using polymerase chain reaction to amplify this sequence. DNA sequence analysis revealed that the cDNA encoded the same protein as the cDNA cloned previously from a 21H rabbit, demonstrating that structural differences in P450IIC5 do not underlie the phenotypic difference in the expression of 21-hydroxylase activity. DNA sequence analysis of the exons and surrounding intron regions in two III/J genomic clones that encode portions of the CYP2C5 gene showed that the exon sequences were identical to that of the P450IIC5 mRNA and that the codon positions of the exon/intron junctions are conserved with other class II genes. Southern blot analysis of DNA digested with BamHI and probed with a portion of DNA isolated from intron 5 revealed that the gene characterized in this study is an allele of the gene conferring the 21H phenotype. Primer extension analysis with a P450IIC5-specific oligonucleotide demonstrated that the CYP2C5 gene uses several transcriptional start sites in both 21H and 21L rabbits. Analysis of mRNA from livers of 21H and 21L rabbits with the P450IIC5-specific oligonucleotide showed a greater than 20-fold difference in the relative abundance of mRNA, which accounts quantitatively for the phenotypic difference in progesterone 21-hydroxylase activity.

Alleles↗