Assembly and secretion of recombinant chains of human inter-alpha-trypsin inhibitor in COS-7 cells

The inter-alpha-trypsin inhibitor (ITI) family is a group of structurally related plasma serine protease inhibitors. The ITI family members consist of combinations of mature heavy chains named HC1, HC2, HC3 linked to bikunin (a Kunitz-type protease inhibitor) by a covalent interchain protein-glycosaminoglycan-protein cross-link. The biosynthesis of the ITI family members takes place in the liver. In this report we examine the biosynthesis of these proteins using transient transfected COS-7 cells expressing one or more combinations of human ITI chains. The processing and secretion of alpha1-microglobulin and bikunin does not require the ITI heavy chains. A small proportion of the H3 chain seems to be processed into the HC3 form in the absence of the other ITI chains. In contrast, the processing of H2 into HC2 needs the presence of the L chain. The COS-7 cells are able to link the HC2 and HC3 heavy chains with bikunin by means of a chondroitin sulfate bridge, and thus to generate 260-kDa ITI-like proteins as well as pre-alpha-trypsin inhibitor (PalphaI). However, the maturation of the Hl chain into HC1 and the assembly of HC1 inside multichain proteins may take place according to a mechanism which differs from that of the H2 and H3 chains. These results indicate that the assembly of the constituent chains of the ITI-like proteins and PalphaI is not dependent on the liver machinery.     

Expression of tissue factor pathway inhibitor in vascular smooth muscle cells and its regulation by growth factors

Tissue factor pathway inhibitor (TFPI) in vivo is thought to be synthesized mainly by endothelial cells. To date, no significant regulator of TFPI synthesis has been described. Vascular smooth muscle cells (VSMC) express tissue factor in vitro and in vivo, which may contribute to vascular thrombosis. We hypothesized that VSMC might also express TFPI. To determine this, we examined growth-arrested coronary VSMC in culture and found that VSMC secreted an amount of TFPI similar to that seen in endothelial cells. Immunohistochemistry of normal human coronary arteries showed TFPI staining throughout the media and intima of the vessel with localization to VSMC and endothelial cells. To determine regulation of TFPI expression in VSMC, we examined the effects of serum stimulation on TFPI secretion and found that FBS induced a 5-fold increase in TFPI antigen and activity levels in conditioned medium at 48 hours (P<0.001) when compared with serum-free conditions. A similar stimulatory effect was seen with 10% pooled human serum. Moreover, epidermal growth factor and platelet-derived growth factor-B increased TFPI secretion by 4- to 5-fold and 2- to 3-fold, respectively (P<0.05), and these growth factors accounted for approximately 50% of the TFPI secretion effects of human serum. The serum effect was associated with a 3-fold increase in TFPI mRNA 24 hours after release from growth arrest and a 50% decrease in TFPI secretion after treatment with actinomycin D. Taken together, this study suggests that there is significant TFPI expression in VSMC in culture and in VSMC within the intima and media of the normal coronary artery wall. We present the first evidence for TFPI regulation by serum in VSMC and more specifically by its constituent growth factors, epidermal growth factor and platelet-derived growth factor-B.     

Expression cloning and receptor pharmacology of human calcitonin receptors from MCF-7 cells and their relationship to amylin receptors

Human breast cell carcinoma MCF-7 cells were found to bind 125I-labeled rat amylin (rAmylin) and the peptide amylin antagonist radioligand 125I-AC512 with high affinity. This high affinity binding possessed characteristics unique to the already defined high affinity binding site for amylin in the rat nucleus accumbens [Mol. Pharmacol. 44:493-497 (1993); J. Pharmacol. Exp. Ther. 270:779-787 (1994); Eur. J. Pharmacol. 262:133-141 (1994)]. To further define this receptor, we report results of expression cloning studies from an MCF-7 cell library. We isolated two variants of a seven-transmembrane receptor that were identical to two previously described human calcitonin receptors (hCTR1 and hCTR2). These receptors were characterized by expression in different surrogate host cell systems. Transient expression of hCTR1 in COS cells yielded membranes that bound 125I-AC512 and 125I-salmon calcitonin with high affinity, but no high affinity binding was observed with 125I-human calcitonin (hCAL) or 125I-rAmylin. Stable expression of hCTR1 in HEK 293 cells produced similar data. In contrast, expression of hCTR2 in COS cells yielded membranes that bound 125I-AC512, 125I-hCAL, and 125I-rAmylin with high affinity. The agonists 125I-hCAL and 125I-rAmylin bound 65% and 1.5%, respectively, of the sites bound by the antagonist radioligand 125I-AC512 in this expression system. This pattern of binding was repeated in HEK 293 cells stably transfected with hCTR2 (125I-hCAL = 24.8% Bmax, 125I-rAmylin = 8% Bmax). In both expression systems, the agonists hCAL and rAmylin were much more potent in displacing their radioligand counterparts than was the antagonist radioligand 125I-AC512. For example, the pKi value for displacement of 125I-AC512 by rAmylin was 7.2 in HEK 293 cells but rose to 9.1 when displacing 125I-rAmylin. Finally, hCTR2 was expressed in baculovirus-infected Ti ni cells. In this system, only specific binding to the antagonist 125I-AC512 and agonist 125I-hCAL was observed; no binding to 125I-rAmylin could be detected. These data are discussed in terms of two working hypotheses. The first is that amylin is a weak agonist for hCTR2 and that this receptor is unrelated to the amylin receptor found in this cell line. The second is that hCTR2 couples to different G proteins for calcitonin and amylin function in different cells. At present, these data cannot be used to disprove conclusively either hypothesis.     

Generation and characterization of a human monoclonal autoantibody that acts as a high affinity interleukin-1 alpha specific inhibitor

Interleukin-1 (IL-1) defines two polypeptides, IL-1 alpha and IL-1 beta, that possess a wide spectrum of biological effects. Two natural antagonists of IL-1 action have been characterized: the IL-1 receptor antagonist (IL-1Ra) and a soluble form of the type II IL-1 receptor. Neutralizing autoantibodies to IL-1 alpha have also been detected in sera of healthy individuals and patients with autoimmune or inflammatory diseases. To characterize such antibodies molecularly, we attempted to generate B cell clones producing anti-IL-1 alpha human monoclonal antibody (HuMAb) by combining Epstein-Barr virus-immortalization and CD40-activation of B lymphocytes from individuals with circulating anti-IL-1 alpha. We describe herein the generation and properties of a natural IgG4/kappa anti-IL-1 alpha monoclonal autoantibody, HuMAb X3, that bound specifically to human IL-1 alpha, but not to IL-1 beta and IL-1Ra, with a high affinity (Kd = 1.2 x 10(-10)M). HuMAb X3 inhibited IL-1 alpha binding to IL-1 receptors and neutralized biological activities of both recombinant and natural forms of IL-1 alpha. A recombinant form of HuMAb X3 was found to display identical specific IL-1 alpha antagonism. The presence of somatic mutations within X3 variable regions suggests an antigen-driven affinity maturation. This study extends the demonstration of the presence of high affinity neutralizing anti-IL-1 alpha autoantibodies that can function as a third type of IL-1 antagonist.     

Thrombin stimulates expression of tissue-type plasminogen activator and plasminogen activator inhibitor type 1 in cultured human vascular smooth muscle cells

The effect of thrombin on the fibrinolytic potential of human vascular smooth muscle cells (SMC) in culture was studied. SMC of different origin responded to thrombin treatment with a dose and time dependent increase in tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type-1 (PAI-1) levels in both cell lysates and conditioned media with maximum effects achieved at 10-20 IU/ml thrombin. PAI-1 antigen levels also increased in the extracellular matrix of thrombin treated SMC. PAI-2 levels in cell lysates of such SMC were not affected by thrombin. The effect was restricted to active thrombin, since DFP-thrombin and thrombin treated with hirudin showed no increasing effect on t-PA and PAI-1 levels in SMC. Enzymatically active thrombin also caused a four-fold increase in specific PAI-1 mRNA and a three-fold increase in t-PA mRNA. Furthermore we demonstrated the presence of high and low affinity binding sites for thrombin on the surface of SMC with a KD = 4.3 x 10(-10)M and 9.0 x 10(4) sites per cell and a KD = 0.6 x 10(-8) M and 5.8 x 10(5) sites per cell respectively. Thrombin could come in contact with SMC in case of vascular injury or following gap formation between endothelial cells. Our data support the idea that besides its known proliferative effect for SMC, thrombin could also modulate their fibrinolytic system.     

Kinetic analysis of the binding of human matrix metalloproteinase-2 and -9 to tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2

The dissociation constants (Kd) of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 for the active and latent forms of matrix metalloproteinase (MMP)-2 and MMP-9 were evaluated using surface plasmon resonance (SPR) and enzyme inhibition studies. SPR analysis shows biphasic kinetics with high (nM) and low (microM) affinity binding sites of TIMP-2 and TIMP-1 for MMP-2 (72- and 62-kDa species) and MMP-9 (92- and 82-kDa species), respectively. In contrast, binding data of TIMP-2 to an MMP-2 45-kDa active form lacking the C-terminal domain and to an MMP-2 C-terminal domain (CTD) fragment displays monophasic kinetics with Kd values of 315 and 60 nM, respectively. This suggests that the CTD contains the high affinity binding site, whereas the catalytic domain contains the low affinity site. Also, binding of TIMP-2 to pro-MMP-2 is stronger at both the high and low affinity sites than the corresponding binding of TIMP-2 to the MMP-2 62-kDa form demonstrating the importance of the N-terminal prodomain. In addition, the Kd value of TIMP-1 for the MMP-2 62-kDa species is 28. 6 nM at the high affinity site, yet neither the MMP-2 45-kDa species nor the CTD interacts with TIMP-1. Enzyme inhibition studies demonstrate that TIMPs are slow binding inhibitors with monophasic inhibition kinetics. This suggests that a single binding event results in enzyme inhibition. The kinetic parameters for the onset of inhibition are fast (kon approximately 10(5) M-1 s-1) with slow off rates (koff approximately 10(-3) s-1). The inhibition constants (Ki) are in the 10(-7)-10(-9) M range and correlate with the values determined by SPR.     

Differential expression of the keratinocyte growth factor (KGF) and KGF receptor genes in human vascular smooth muscle cells and arteries

The objective of this study was to analyze the efficacy and correlation between clinical and histologic parameters used to evaluate oral implants. After extraction of the premolars and a healing time of 4 months in 16 Dutch goats, four Br?nemark implants were placed in the maxillary left and right premolar regions. After a healing time of 6 months, followed by another 4 months with the permucosal abutments, the goats were sacrificed and the jaws were block-resected. Before histologic preparation, long-cone radiographs were made and Periotest scores of the implants were recorded. Bone level measured histomorphometrically were found to be 0.85 mm more apically, compared to that measured radiologically (P = .001). Furthermore, statistically significant correlations (P > 0.2) were not found between the Periotest values of the calcium-phosphate-coated and uncoated implants for (1) the first thread in contact with bone, or (2) with the total number of threads in contact with bone. It was concluded that the radiologic data overscored the real marginal bone level around screw-shaped oral implants, and that the Periotest device is neither able to discriminate between the first thread nor between the total number of threads in contact with bone.     

Immunoelectron microscopic localization of plasminogen activator inhibitor type 1 (PAI-1) in smooth muscle cells from morphologically normal and atherosclerotic human arteries

Vascular wall fibrinolytic system proteins are believed to play a pivotal role in atherogenesis. Tissue-type plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA) influence persistence of luminal thrombi and proteolysis of extracellular matrix, respectively. The major physiologic inhibitor of t-PA and u-PA is plasminogen activator inhibitor type 1 (PAI-1). All three of these fibrinolytic system proteins have been detected in vascular endothelial cells, smooth muscle cells, and macrophages by light microscopic immunohistochemistry. This study was undertaken to delineate, by immunoelectron microscopy, the loci of PAI-1 in smooth muscle cells from intact morphologically normal and atherosclerotic human arteries as well as in isolated and cultured smooth muscle cells from arteries. In intact vessels, PAI-1 immunoreactivity was associated with contractile filaments in cells in both normal and atherosclerotic tissues. Lipid-laden smooth muscle cells in atherosclerotic vessels were mainly of the synthetic phenotype and displayed lesser amounts of PAI-1 associated with rough endoplasmic reticulum and contractile filaments. Isolated smooth muscle cells exhibited either a contractile or synthetic phenotype. In the cells with a contractile phenotype, PAI-1 was associated with the contractile elements, whereas in the cells with a synthetic phenotype, the PAI-1 was associated predominantly with elements of the endoplasmic reticulum. Because PAI-1 is associated predominantly with contractile filaments in smooth muscle cells, the net amount of immunodetectable PAI-1 appears to be greater in contractile compared with synthetic phenotype cells.     

Effect of human recombinant erythropoietin (Epo) on cellular Ca2+, Na+, and K+ regulation of vascular smooth muscle cells grown in vitro--a new insight into the pathogenesis of Epo induced hypertension

To gain an insight into the effect of erythropoietin (Epo) upon cation transporters and cytosolic free Ca2+ concentration ([Ca2+]i) of vascular smooth muscle cells (VSMC), we studied whether 1) Epo, per se, alters Ca2+ Na+, K+ fluxes and [Ca2+]i of VSMC, and 2) Epo may modify the effect of endothelin (ET-1). Using serially passaged quiescent cultured VSMC, the following results were obtained. 1) Epo had no direct effect on steady state Na(+)-K+ transporters (Na(+)-K+ pump, Na(+)-K+ cotransport and Na(+)-H+ antiport). 2) ET-1 alone substantially stimulated Na(+)-K+ pump, Na(+)-H+ antiport and 45Ca uptake, although these effects were not potentiated in the presence of Epo. 3) Epo alone substantially stimulated 45Ca uptake, leading to an increase in [Ca2+]i, which effect was not seen in Ca2+ deficient medium, and was partially inhibited with diltiazem but not with TMB-8. 4) Even in the presence of Epo, ET-1 and angiotensin II (A II) had substantial stimulatory effect on [Ca2+]i of cultured VSMC. The present data indicate that Epo, per se, elicits an increase in [Ca2+]i of VSMC through the stimulation of inward Ca2+ flux without affecting Na(+)-K+ transporters. In contrast, Epo did not potentiate ET-1's stimulatory effect on the transporters. Although the effect of Epo was subtle compared to ET-1 and A II, it may alter an overall characteristic of vascular smooth muscle cell contractility, possibly leading to blood pressure elevation in patients on maintenance dialysis.     

Cytokine-mediated regulation of 92-kilodalton type IV collagenase, tissue inhibitor or metalloproteinase-1 (TIMP-1), and TIMP-3 messenger ribonucleic acid expression in human endometrial stromal cells

Interleukin-1 (IL-1) is expressed in human endometrium and has been shown to play an integral role in local cellular interactions during implantation. In addition, the matrix metalloproteinase (MMP) and its inhibitor, the tissue inhibitor of metalloproteinase (TIMP), are crucial during implantation, mediating in vitro trophoblast penetration, and are regulated by several cytokines expressed by trophoblast cells. We have investigated the roles of IL-1 beta and transforming growth factor-beta (TGF beta) in regulating TIMP-1, TIMP-3, and 92-kDa type IV collagenase messenger ribonucleic acid (mRNA) expression in human endometrial stromal cells using quantitative competitive PCR. Confluent stromal cell cultures treated with progesterone and estradiol for 9 days were stimulated with IL-1 beta, IL-1 beta plus anti-IL-1 beta antibody, TGF beta, and TGF beta plus anti-TGF beta antibody for an additional 24 h. Competitive complementary DNA fragments were constructed by deletion of a defined fragment from each of the target complementary DNA sequences and coamplified in quantitative competitive PCR as an internal standard. TIMP-1 and TIMP-3, but not 92-kDa type IV collagenase mRNA, were expressed in stromal cells. The 92-kDa type IV collagenase mRNA was only expressed after stimulation with IL-1 beta. IL-1 beta both augmented 92-kDa type IV collagenase mRNA expression and decreased TIMP-1 and TIMP-3 mRNA expression in a dose-dependent manner. Conversely, TGF beta augmented TIMP-1 and TIMP-3 mRNA expression, but did not affect 92-kDa type IV collagenase expression. IL-1 and TGF beta-mediated changes were both neutralized by specific antibodies. These results provide indirect evidence that IL-1 and TGF beta may play crucial roles at the embryo-maternal interface during trophoblast invasion by regulating stromal cell expression of TIMP-1, TIMP-3, and 92-kDa type IV collagenase, all of which are known to be important in trophoblast invasion.