Thrombin stimulates fibroblast chemotaxis and replication.

The serine protease alpha-thrombin, a product of the circulating zymogen prothrombin, plays multiple roles in homeostasis and coagulation. During blood clotting, it is present within fibrin matrices and is likely to be presented to local cell populations. It is known to be a fibroblast mitogen, but its effects on fibroblast recruitment have not been assessed. Here we compared the effect of human alpha-thrombin on chemotaxis and proliferation of human and rat skin fibroblasts and assessed the mechanism of these actions. Fibroblast chemotaxis was assayed using a 48-well Boyden chamber and replication assessed by a spectrophotometric method, based upon the uptake and subsequent elution of methylene blue by fibroblasts. Two fibroblast cell lines were used; fetal rat skin (FR) and newborn human foreskin (HS68). Human alpha-thrombin stimulated FR fibroblast chemotaxis over a wide range of doses (10(-12) M to 10(-7) M). Maximal migration was seen at 10(-10) M; 39 +/- 2.5 cells/high power field (h.p.f.) compared with 19 +/- 3 cells/h.p.f. for media control. In the same assay platelet-derived growth factor, a well characterized fibroblast chemoattractant, caused a maximal stimulation of 44 +/- 5 cells/h.p.f. at a concentration of 3 x 10(-9) M. A similar stimulation was observed with HS68 fibroblasts, although for this cell line maximal chemotaxis (190 +/- 12.5% of control) was seen at 10(-8) M thrombin. Fibroblast replication was optimal at 1.25 x 10(-9) M thrombin (134 +/- 4 and 127 +/- 5% of control for FR and HS68, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in collagen metabolism in response to endothelin-1: evidence for fibroblast heterogeneity.

Endothelin-1 (Et-1) is a 21-amino acid peptide primarily synthesized by endothelial cells. It was originally classified as a potent vasoconstrictor but recent evidence suggests that it also possesses a wide variety of non-vascular actions. It stimulates fibroblast and smooth muscle cell proliferation and it has been shown to stimulate fibroblast collagen metabolism. However, studies on its ability to regulate collagen production remain incomplete, and its effect on post-translational processing of procollagen has not been studied. This report details the effect of Et-1 on the rates of procollagen synthesis and degradation in two fibroblast cell lines; human foetal lung (HFL-1) and whole foetal rat fibroblasts (Rat 2). Fibroblast cultures were incubated for 24 hr in the presence or absence of Et-1 before procollagen metabolism was determined by measuring hydroxyproline. Non-collagen metabolism was also determined in these cultures from the uptake of tritiated phenylalanine. Et-1 stimulated procollagen synthesis in HFL-1 fibroblasts and reduced synthesis in Rat 2 cells. The response was dose dependent with the greatest effect at 1.10(-6) M Et-1 for both cell types (155 +/- 6% of control (mean +/- SD, n = 6, P < 0.01) and 61 +/- 4% of control (n = 4, P < 0.01) for HFL-1 and Rat 2 fibroblasts, respectively). Non-collagen protein synthesis was increased to 148 +/- 5% of control (P < 0.05) at 1.10(-6) M Et-1. Non-collagen protein synthesis remained unaffected in the HFL-1 fibroblast cultures. Procollagen degradation, expressed as a proportion of total procollagen synthesis, was decreased in HFL-1 fibroblasts (control, 29 +/- 2%; Et-1, 1.10(-6) M; 21 +/- 2%; P < 0.01), and increased in Rat 2 fibroblasts (control 42 +/- 1%; Et-1, 1.10(-6) M; 49 +/- 1%; P < 0.01). Blocking of the EtA receptor for Et-1, using the receptor antagonist-BQ123, abolished the effect of Et-1 on procollagen metabolism in both cell types. These results suggest that different populations of fibroblasts exhibit heterogeneous responses to Et-1. It is concluded that Et-1 may play an important role in the extent and distribution of fibrosis seen in diseases associated with the overproduction of Et-1.

[Endothelin-1 induces chemotaxis of pulmonary artery smooth muscle cells].

Migration of pulmonary artery smooth muscle cells (PASMC) and their precorsors might be an important mechanism of pulmonary vascular structural remodelling associated with hypoxic pulmonary hypertension (HPH). Endothelin-1 (ET-1) plays an important role in the pathogenesis of HPH. This study observed that ET-1 induced chemotaxis of PASMC in a dose dependent manner. The maxium chemotactic response of PASMC to ET-1 occured at a concentration of 10(-9) mol/L. The chemotactic activity of ET-1 is weakened, when its concentration is higher than 10(-9) mol/L. BQ123, a specific antagonist of ETRA, significantly inhibited the chemotaxis of PASMC induced by ET-1, which indicates that the chemotactic activity of ET-1 may be mediated by ETRA. Hypoxia enhanced the chemotaxis of PASMC induced by ET-1. This study suggests that ET-1 and hypoxia might stimulate the migration of PASMC and/or its precorsor. The further mechanism is under study.

[Study on the chemotaxis and DNA synthesis of pulmonary artery smooth muscle cells].

OBJECTIVE: To explore the effect of hypoxia on the proliferation and migration of pulmonary vascular smooth muscle cells (PVSMC), and whether migration of PASMC is involved in the pathogenesis of pulmonary vascular remodeling associated with hypoxia. METHODS: In this study, the effect of PDGF, ANP and hypoxia on DNA synthesis and chemotaxis of cultured neonatal calf pulmonary artery smooth muscle cells (PASMC) is investigated by 3H-thymidine incorporation and measurement of cell migration using a 48-well Boyden chamber respectively. RESULTS: The results demonstrated that hypoxia could stimulate DNA synthesis and chemotaxis of PASMC induced by PDGF; ANP could inhibit DNA synthesis and chemotaxis of PASMC by a cGMP dependent pathway. CONCLUSIONS: It is suggested that PDGF, ANP and hypoxia play important roles in regulating the proliferation and migration of PASMC, which is important in the pathogenesis of hypoxic pulmonary vascular remodeling.

Differential effects of platelet-derived growth factor BB on p125 focal adhesion kinase and paxillin tyrosine phosphorylation and on cell migration in rabbit aortic vascular smooth muscle cells and Swiss 3T3 fibroblasts.

In rabbit aortic vascular smooth muscle cells (VSMC) platelet-derived growth factor BB (PDGF-BB) stimulated the tyrosine phosphorylation of phospholipase C-gamma, p120 GTPase-activating protein, and the p85 alpha subunit of phosphatidylinositol 3'-kinase only at high concentrations (5-25 ng/ml). In contrast, PDGF-BB induced a rapid and concentration-dependent increase in p125 focal adhesion kinase (p125FAK) tyrosine phosphorylation, which was half-maximal and maximum at 1 and 2.5 ng/ml, respectively. Saliently, stimulation of p125FAK tyrosine phosphorylation was sustained at up to 100 ng/ml PDGF-BB and for prolonged times of treatment. With similar concentration dependence, PDGF-BB stimulated the tyrosine phosphorylation of the 68-kDa focal adhesion-associated protein, paxillin. PDGF-BB also induced p125FAK and paxillin tyrosine phosphorylation in human aortic VSMC. PDGF-BB caused no detectable disruption of the actin cytoskeleton in VSMC. PDGF-BB stimulated rabbit VSMC migration with a very similar concentration dependence to that for p125FAK and paxillin tyrosine phosphorylation. PDGF-BB was equally effective in stimulating p125FAK and paxillin tyrosine phosphorylation under conditions similar to those used for cell migration. In Swiss 3T3 fibroblasts, PDGF-BB and -AA stimulated p125FAK tyrosine phosphorylation and cell migration only at low concentrations, and stimulation was abolished at 10-25 ng/ml. PDGF-AA failed to stimulate tyrosine phosphorylation, mitogenesis, and chemotaxis in rabbit VSMC, and immunoblot analysis showed that rabbit VSMC expressed PDGF beta-receptors but no alpha-receptors. These results implicate p125FAK in the chemotactic response to PDGF-BB and suggest that the ability of PDGF-BB to trigger the p125FAK pathway may be dependent both upon cell type and receptor isotype expression.

Endothelin-1 and endothelin-3 induce chemotaxis and replication of pulmonary artery fibroblasts.

The remodeling of pulmonary vessels that occurs in association with pulmonary hypertension involves, in part, thickening of the adventitia. The stimulus for this process is not understood. One explanation is that endothelial cells secrete a growth factor that expands the local population of fibroblasts by acting as a chemoattractant and mitogen. Endothelins are a family of potent newly discovered vasoactive peptides. One of these compounds, endothelin-1 (ET-1), is secreted by endothelial cells and is known to constrict pulmonary vessels. Another, endothelin-3 (ET-3), is not secreted by endothelial cells and is less potent as a pulmonary vasoconstrictor. We hypothesized that the endothelins may have the capacity both to constrict these vessels and to initiate fibroblast chemotaxis and replication. Here we investigated the effects of both ET-1 and ET-3 on the chemotaxis and replication of fibroblasts derived from pulmonary vessels. Cells were isolated from rat pulmonary arteries, cultured in medium and 10% newborn calf serum, and used between passages 2 and 5. Chemotaxis was assessed using a modified Boyden chamber with a polycarbonate filter (pore size, 8 microns) separating cells in the upper chambers from endothelin in the lower chambers. Replication was assessed both by direct cell counts and by a colorimetric assay based on uptake and subsequent release of methylene blue. Both ET-1 and ET-3 induced chemotaxis of pulmonary artery fibroblasts and did so in a dose-dependent fashion. The maxima for both peptides occurred at a concentration of about 10(-7) M, when chemotaxis was greatest for ET-1 (22 +/- 1.4 versus 14 +/- 1.8 cells/grid [mean +/- SEM], (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of fibroblast mitogens and chemoattractants produced by endothelial cells exposed to hypoxia.

During pulmonary hypertension there is remodeling of the pulmonary vasculature, with enhanced fibroblast proliferation and connective tissue production. The stimulus for this process is not understood, but one explanation is that endothelial cells secrete moieties that expand local cell populations by acting as chemoattractants and mitogens. Here, we investigated the effect of hypoxia (35 mm Hg) on the production of chemoattractants and mitogens by human umbilical vein endothelial cells. Endothelial cells were subjected to hypoxia for up to 24 h and the resultant conditioned media tested for chemotactic and mitogenic activity. Chemotaxis of pulmonary artery fibroblasts were measured using a 48-well Boyden chamber and replication assessed by a spectrophotometric method, based upon the uptake and subsequent elution of methylene blue by fibroblasts. Within 6 h of culture, media derived from both hypoxic and normoxic endothelial cells stimulated fibroblast chemotaxis and replication. This activity increased with time, and by 24 h there was a significantly greater response toward media obtained from cells exposed to hypoxia compared with normoxic controls (P < 0.01). The addition of antibodies to endothelin-1 (Et-1) or platelet-derived growth factor (PDGF) reduced the chemotactic activity in hypoxic conditioned media by almost 50% (45 +/- 6 to 24 +/- 5 cells/h.p.f. and 45 +/- 6 to 26 +/- 4.5 cells/h.p.f. for anti-Et-1 and anti-PDGF, respectively; P < 0.001). Fibroblast proliferation in response to hypoxic conditioned media was also reduced in the presence of antibodies to PDGF (55 +/- 11% to 14 +/- 12% above media control; P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of neuropeptides on human lung fibroblast proliferation and chemotaxis.

An increase in subepithelial mesenchymal cells and associated connective tissue is a feature of bronchial asthma. We determined whether neuropeptides could modulate fibroblast activity, particularly with respect to proliferation and chemotaxis. Human lung fibroblasts were cultured with neurokinin A (NKA), substance P (SP), vasoactive intestinal peptide (VIP), and calcitonin-gene-related peptide (CGRP). After 48 h, fibroblast proliferation was measured by a colorimetric assay based on the uptake and subsequent release of methylene blue. The chemotactic response to neuropeptides was determined with the use of a modified Boyden chamber. Both NKA and SP (10(-7)-10(-4) M) stimulated human lung fibroblast proliferation in HFL1 and IMR-90 fibroblasts. VIP and CGRP had no effect on fibroblast proliferation. NKA alone stimulated fibroblast chemotaxis maximally at 10(-10) M. Neutral endopeptidase (NEP) activity of 0.52 and 5.2 pmol/10(6) cells was assayed in IMR-90 and Hs68 fibroblasts, respectively. Phosphoramidon (5 x 10(-6)-10(-5) M), an NEP inhibitor, enhanced fibroblast proliferation in a dose-dependent manner. Thus neuropeptides have the potential to cause activation of mesenchymal cells, and neuropeptide release may contribute to the structural abnormalities observed in asthmatic airways.

Increased levels of endothelin-1 in bronchoalveolar lavage fluid from patients with systemic sclerosis contribute to fibroblast mitogenic activity in vitro.

Pulmonary fibrosis is a major cause of morbidity and mortality in patients with systemic sclerosis (SSc). The pathogenesis of this condition is poorly understood, but one of the earliest pathologic features is endothelial and epithelial cell injury with subsequent regeneration. Endothelial and epithelial cells can release several mediators, including endothelin-1 (ET-1). In this study, we investigated the levels of ET-1 in bronchoalveolar lavage fluid (BALF) from patients with SSc and assessed the contribution of ET-1 to the fibroblast mitogenic activity induced by these fluids. A total of 26 patients were evaluated and divided into those with evidence of pulmonary fibrosis, assessed by thin-section computed tomography (group I, n = 16), and those with a normal scan (group II, n = 10). BALF from both groups of patients stimulated fibroblast proliferation. Values expressed as median (range) percentage increase above media controls were 25.5% (5.0 to 47.8%) and 27.6% (10.9 to 51.6%) for groups I and II, respectively (P < 0.02 in both cases). Mitogenic activity was inhibited by about 40% in the presence of either a neutralizing antibody to ET-1 or two synthetic ET-1 receptor ligands. Levels of ET-1 in BALF, expressed as medians (range) were 2.90 ng/mg albumin (0.68 to 5.75) in patients with SSc and 1.23 ng/mg albumin (0.84 to 2.0) in control patients (P < 0.02). Furthermore, ET-1 levels in BALF from patients in group II (3.83 ng/mg albumin, range 1.76 to 5.75) were elevated compared with those in group I (2.62 ng/mg albumin, range 0.68 to 3.81; P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)