Umbilical Vein Smooth Muscle Cells Research Articles

Phosphatidylinositol 3-Kinase Activity Is Required for the Activation Process of Focal Adhesion Kinase by Platelet-Derived Growth Factor

Platelet-derived growth factor (PDGF) is one of the agents which stimulate increase in phosphotyrosine content of focal adhesion kinase (FAK) in cultured cells. In the present study we report that wortmannin, a highly specific and potent inhibitor of the catalytic subunit of mammalian phosphatidylinositol (PI) 3-kinase, completely abolishes PDGF-BB-mediated increase in tyrosine phosphorylation of FAK in human umbilical vein smooth muscle cells. Furthermore, analysis of the wild-type and mutant human PDGF β-receptors stably expressed in porcine aortic endothelial cells also demonstrates that the Y740/751F mutant receptor, which cannot interact with PI 3-kinase due to the mutational alteration of its binding sites for PI 3-kinase, fails to increase FAK phosphorylation after PDGF-BB stimulation. These data suggest the requirement for PI 3-kinase activity in the activation process of FAK downstream of the PDGF receptor.

Human Umbilical Vein Smooth Muscle Cells as a Model to Study Thrombin Generation and Function: Effect of Thrombin Inhibitors

The aim of the present work was to study how human umbilical vein smooth muscle cells (HUVSMC) can initiate the coagulation process and to investigate the responses of these cells to thrombin. Exposure of HUVSMC to recalcified human plasma led to a time-dependent production of thrombin, measured both as amidolytic activity and as release of fibrinopeptide A. Thrombin activity was dose-dependently reduced by an anti-human tissue factor antibody (76 +/- 3% at 10 micrograms/ml) and by inhibitors like heparin, rec-hirudin, hirulog 1, Napap and hirunorm, a novel hirudin-like thrombin inhibitor (IC50 = 2 +/- 0.4, 8 +/- 1, 130 +/- 22, 199 +/- 29 and 68 +/- 8 nM, respectively). The release of fibrinopeptide A was similarly prevented (IC50 = 14 +/- 1, 132 +/- 25 and 50 +/- 8 nM for rec-hirudin, Napap and hirunorm, respectively). Exogenously added thrombin increased thymidine incorporation into HUVSMC to 240 +/- 30% of basal (EC50 = 0.49 +/- 0.09 nM) and thrombin inhibitors blocked this effect (IC50 = 10 +/- 3, 37 +/- 17, 343 +/- 165 and 1402 +/- 758 nM for rec-hirudin, hirunorm, Napap and hirulog-1, respectively). Also recalcified human plasma was mitogenic for HUVSMC and its effect was mainly due to endogenously generated thrombin, as shown by the use of thrombin inhibitors. In conclusion, HUVSMC are capable of initiating the extrinsic coagulation cascade, leading to the formation of thrombin which promotes clotting and stimulates DNA synthesis. Thrombin inhibitors prevent both coagulative and cellular effects of thrombin.

Oxidized LDL induce hsp70 expression in human smooth muscle cells

Heat shock protein 70 (hsp70) has been detected in atherosclerotic lesions, in which endothelial cells and smooth muscle cells are involved. In a previous report we showed that Ox-LDL, a causal factor in atherosclerosis, could induce hsp70 expression in cultured human endothelial cells [Zhu et al. B.B.R.C 1994, 200: 389]. Here, with immunofluorescence and immunoblotting techniques, we show that Ox-LDL are capable of inducing hsp70 expression also in human smooth muscle cells, and that this induction is dependent on cell density and on the concentration of Ox-LDL. The induced expression of hsp70 was higher in human umbilical vein smooth muscle cells than in a human smooth muscle cell line. Conversely, Ox-LDL was cytotoxic to both types of cells, more so to the human smooth muscle cell line. These observations indicate that Ox-LDL may be a stress responsible for hsp70 expression in atherosclerotic plaques and the presence of hsp70 in plaques may be a useful marker for continuous oxidative damage in the arterial wall.

Regulation of Endothelin Receptor Expression in Vascular Smooth-Muscle Cells

Endothelin-1 (ET-1) has been implicated in atherosclerosis, hypertension, and restenosis, all of which involve abnormal vascular smooth muscle cell function and/or proliferation. We have previously established that human umbilical vein smooth-muscle cells (HUVSMCs) can secrete ET, (1) whereas A10 cells do not. Therefore, we investigated the effect of exogenously added ET-1 on receptor density (Bmax) of A10 cells. Compared to controls, A10s exposed to ET-1 for 48 h showed a significant decrease (79%) in receptor density, with no change in affinity. In contrast, incubation of A10s with the ETA-selective antagonist FR139317 (at a concentration blocking 99% of ET receptors) for 48 h caused a significant increase (245%) in Bmax and a significant decrease in affinity. These changes persisted after coincubation with both ET-1 and FR139317, indicating that the antagonist was able to block the effects of exogenous ET-1. In concordance, incubation of HUVSMCs with FR139317 for 24 h caused a significant increase in receptor density (> 1,000%), although there was no change in levels of immunoreactive (IR) ET or big ET-1. However, after a shorter incubation (1 h), there was no change in Bmax but IR ET was significantly elevated by 878%, whereas IR big ET-1 was depressed by 86% compared to controls. Incubation of HUVSMCs with FR139317 did not affect receptor affinity. Our observations suggest that whereas the application of exogenous ET to A10s causes downregulation of ET receptor expression, the ETA antagonist FR139317 causes upregulation of ET receptor expression in VSMCs regardless of their ability to secrete ET. In VSMCs capable of secreting ET, acute antagonism of the ETA receptor causes a significant increase in IR-ET and a decrease of IR-big ET-1 levels.

The time course of intracellular calcium movements in single human umbilical vein smooth muscle cells.

Intracellular Ca2+ ([Ca2+]i) was measured in single isolated human umbilical vein smooth muscle cells. Stimulation with histamine, in the absence of external Ca2+, mobilised Ca2+ from intracellular stores. When repeated brief applications of agonist were used, the time to onset, amplitude and rate of rise of the Ca2+ transients were found to change. Two components could often be discerned in the rising phase of the transients, an initial slow "pacemaker" and a second faster and larger component. Following the first histamine-activated transient the basal level of [Ca2+]i was invariably lower than that prior to stimulation. This lower value was maintained whilst the cell remained in Ca(2+)-free solution, but could be returned to a higher level if the cell was exposed to external Ca2+. When the mobilisation of the intracellular store was reduced to undetectable levels, re-exposure to Ca(2+)-containing medium reactivated responses. In the absence of external Ca2+, continuous application of histamine activated a series of transient increases in intracellular Ca2+, which decreased progressively in amplitude and rate of rise. The interval between transients also increased. These findings are discussed in terms of the activation of inositol trisphosphate-sensitive intracellular Ca2+ stores and their sensitivity to cytoplasmic Ca2+ and intrasarcoplasmic reticulum Ca2+.

Regulation of cytosolic free Ca2+ concentration in vascular smooth muscle cells by A- and C-kinases.

The cytosolic free Ca2+ concentration [( Ca2+]i) was measured in cultured human umbilical vein smooth muscle cells using fura-2 as a Ca2+ indicator and microscopic digital image analysis system. Activation of cells with histamine and vasopressin resulted in a prompt though transient rise in [Ca2+]i 10- to 12-fold higher than the resting [Ca2+]i. The [Ca2+]i then declined rapidly during the first 30-40 seconds after hormonal stimulation and then gradually decreased to near resting levels in 2-3 minutes in the continued presence of hormones. The magnitude of the increase in peak [Ca2+]i was similar in buffered salt solution containing 1.8 mM Ca2+, zero Ca2+, and zero Ca2+ buffered salt solution containing 10 mM La3+, suggesting that receptor-mediated increase in [Ca2+]i is primarily due to the release of Ca2+ from the intracellular stores. Addition of La3+ produced oscillations in [Ca2+]i in approximately half the cells in response to both hormones. Addition of 10 microM forskolin did not significantly affect the resting [Ca2+]i, the hormone-stimulated peak [Ca2+]i, or the time course of hormone-stimulated [Ca2+]i transients. These data suggest that mechanisms involved in A-kinase-mediated smooth muscle relaxation may be subsequent to the changes in [Ca2+]i. Activation of C-kinase by 1 microM 12 deoxyphorbol 13-isobutyrate-20 acetate (DPBA) did not affect the resting [Ca2+]i, though it attenuated the histamine and vasopressin-mediated peak elevation in [Ca2+]i. Since DPBA inhibited the peak [Ca2+]i response to both the hormones to the same extent, it would appear that C-kinase activation may uncouple the receptor-mediated activation of phospholipase C.

Specific binding of endothelin on human vascular smooth muscle cells in culture.

Endothelin is a newly discovered, potent vasoconstrictor peptide secreted by endothelial cells. The binding of endothelin was studied on cultured human vascular smooth muscle cells obtained from umbilical veins. A single specific binding site for 125I-endothelin was identified, with an apparent Kd of 126 pM and a maximal binding capacity of approximately 10,000 sites per smooth muscle cell. At room temperature the binding was saturable, reached equilibrium at 2 h (using 20 pM endothelin), and was slowly and only partially reversed by unlabeled endothelin. The calcium antagonists nifedipine, nicardipine, and diltiazem did not compete for the same binding site. Conditioned medium from cultured human umbilical vein endothelial cells inhibited the binding of 125I-endothelin dose dependently. This effect was antagonized by anti-endothelin antiserum. We conclude that human umbilical vein smooth muscle cells possess specific binding sites for endothelin, and that human endothelial cells secrete an endothelinlike material.

Human Vascular Smooth Muscle Cells Have at Least Two Distinct PDGF Receptors and Can Secrete PDGF-AA

Platelet-derived growth factor (PDGF), a potent mitogen and chemoattractant for smooth muscle cells and fibroblasts in culture, is believed to play an important role in the formation of proliferative lesions of arterio-sclerosis. PDGF appears as three different dimeric isoforms: AA, AB, and BB. These were recently found to bind to two different receptors, the A/B receptor (which binds all three isoforms) and the B receptor (which binds only PDGF-BB). To find out whether these receptors exhibit functional differences, we have monitored the binding and mitogenic activities of PDGF-AA and -BB in human umbilical vein smooth muscle cells (HSMCs), human dermal fibroblasts (HFs), and Swiss mouse 3T3 cells. With each cell type, there was a good correlation between the maximal levels of DNA synthesis achieved by these isoforms and the numbers of the appropriate receptor present on the cell surface: HMSCs, which have at least 32,000 B receptors but only 8,000 A/B receptors, responded well to PDGF-BB but responded poorly to PDGF-AA; whereas Swiss 3T3 cells, which have about equal numbers of B and A/B receptors (70,000 and 90,000, respectively), responded equally well to both isoforms. PDGF-AB was a more efficacious mitogen of HSMCs and HFs than was PDGF-AA and inhibited [125I]-PDGF-BB binding to HSMCs more effectively than PDGF-AA. This indicates that there may exist a third PDGF receptor type to which PDGF-BB and -AB but not PDGF-AA can bind.(ABSTRACT TRUNCATED AT 250 WORDS)

Type V collagen selectively inhibits human endothelial cell proliferation

Type V collagen from human placenta remarkably inhibited human umbilical vein endothelial cell (HUVEC) proliferation in a dose-dependent manner when coated on the culture dishes. Other types of collagen (I, III, IV) and fibronectin enhanced HUVEC proliferation under the same conditions. The inhibitory activity of type V collagen was seen not only when it was coated on the dishes, but also when it was directly added into cell culture. The attachment effect of type V collagen did not differ from that of type I collagen. The inhibitory activity is a phenomenon selective for endothelial cells, since type V collagen did not affect the proliferation of human umbilical vein smooth muscle cells, aortic smooth muscle cells, or nasal mucosa fibroblasts.

Glycosphingolipids of human umbilical vein endothelial cells and smooth muscle cells

Glycosphingolipids (GSLs) represent an important class of immunogens and receptors. Although cell surface antigens and receptors of endothelial cells (ECs) have been the subject of extensive biochemical investigation, no information is available about their GSLs. We report here the characterization by chromatographic and immunological techniques of GSLs of cultured human umbilical vein ECs and, for comparison, umbilical vein smooth muscle cells (SMCs). The most abundant neutral GSLs of both cell types were lactosylceramide, Gb 3, and Gb 4, and both cells contained complex lacto and globo series compounds. Immunostaining revealed that ECs, but not SMCs, contained long chain GSLs bearing a type 2 blood group H determinant. ECs also contained more long chain GSLs bearing an unsubstituted terminal lactosamine structure than SMCs. Labeling with galactose oxidase/NaB 3H 4 demonstrated that neutral glycolipids that contained three or more sugars were accessible on the cell surface. The major gangliosides of both cell types were G M3 and IV 3NeuAcnLc 4. Immunostaining following neuraminidase treatment revealed that most of the long chain gangliosides in both types of cells contained a lacto core structure, and that ganglio series compounds were more abundant in SMCs than ECs. Gangliosides that contain a polyfucosyllactosamine core and a globo core were also present in both cell types. These results demonstrate that endothelial and smooth muscle cells contain a large diversity of GSL structures, and provide the basis for investigation of the role of these GSLs as cell surface antigens and receptors for blood components.

Human umbilical cord vein smooth muscle cells lack receptors for C3b and the Fc portion of immunoglobulin G

Under apparently normal conditions, the smooth muscle cells obtained from veins of human umbilical cord do not possess receptors for C3b or the Fc portion of IgG. These receptors were not expressed even after exposure to neutrophil lysate or a superoxide-generating system. The lack of these receptors argues against the possibility that smooth muscle cells participate in the pathogenesis of immune complex diseases through binding of C3b or Fc fragments.

Analysis of lipids and endothelial and smooth muscle cells of umbilical cord in familial homozygous hypercholesterolemia

Umbilical cord lipids from a familial hypercholesterolemia homozygote were extracted and quantitated. Free cholesterol was increased 26% and esterified cholesterol was increased 379% compared to normal, whereas phospholipid content was not altered. Analysis of homozygote umbilical artery revealed no excess cholesterol and the vessels were histologically normal. Thus, in the the homozygote cord, accumulation of cholesterol occurs first in Wharton's jelly, the hypocellular glycosaminoglycan-enriched cord matrix. Skin fibroblasts, umbilical vein endothelial cells, and umbilical vein smooth muscle cells were cultured from the homozygote. Low density lipoprotein receptor activity was reduced to 9%–38% of normal in all cell types from the homozygote, demonstrating that vessel cells express the LDL receptor defect seen in skin fibroblasts.

Stimulation of prostaglandin E synthesis in cultured human umbilical vein smooth muscle cells.

Cultured human umbilical vein smooth muscle cells secreted 1.26 +/- 0.20 mug of immunoreactive prostaglandin E (iPGE) per mg of cell protein per 24 hr (mean +/- SEM). In indomethacin, an inhibitor of prostaglandin synthesis, abolished greater than 95% of this basal iPGE production (ID50 = 1.8 nM).