ET－1，NO和缺氧对肺动脉平滑肌细胞钙内流及胶原合成的影响

肺血管平滑肌细胞是肺血管收缩反应的主要执行者,也是肺血管结构重建的重要参与者。本研究观察了内皮素１（ＥＴ１）,一氧化氮（ＮＯ）和缺氧对培养的新生小牛肺动肺平滑肌细胞（ＰＡＳＭＣ）钙内流以及胶原合成的影响。结果表明ＥＴ１和缺氧可促进ＰＡＳＭＣ的钙内流,ＮＯ供应剂硝普钠（ＳＭＰ）可抑制ＥＴＩ诱导的钙内流,其作用呈剂量依赖性,ＳＮＰ还可以剂量依赖地抑制ＰＡＳＭＣ的胶原合成,而缺氧可促进ＰＡＳＭＣ的胶原合成。     

Mucin synthesis and secretion by cultured tracheal cells: effects of collagen gel substratum thickness

Summary We previously demonstrated that human tracheobronchial epithelial (TBE) cells synthesize mucin and form mucous granules in culture when they are maintained on a collagen gel (CG) substratum, but not on a plastic tissue culture surface or a thin collagen-coated surface (Wu et al., Am. J. Respir. Cell Mol. Biol., 3:467–478; 1990). This observation led us to examine the effects of CG thickness on cell growth and differentiation in primary human/monkey TBE cell cultures. Using the same CG preparation, culture dishes with different thicknesses of CG substratum were prepared. In general, equivalent degrees of cell attachment and proliferation were observed in all cultures maintained on a collagen gel, independent of the thicknesses of CG substratum. However, a greater degree of mucin synthesis and secretion by the cells was observed as the thickness of the CG substratum was increased. Cultures maintained on a thick collagen gel (1 mm) exhibited greater apical membrane complexity, more pseudostratification, and more mucous granules than did cultures maintained on a thin CG substratum. The optimal culture surface for airway mucous cell differentiation contains more than 1-mm thickness of collagen gel substratum.     

Activation of type I collagen genes in cultured scleroderma fibroblasts

Fibroblasts cultured from affected skin areas of five patients with cutaneous scleroderma were found to produce increased amounts of collagen when compared with nonaffected control cells. Total RNA was isolated from the cultures and analyzed for its level of pro alpha 1 (I)collagen mRNA by hybridization of RNA blots with a cloned cDNA probe. The levels of pro alpha 1 (I)collagen mRNAs relative to total RNA were two- to sixfold higher in the samples from affected cells, accounting for the increased synthesis of type I collagen. Cytoplasmic dot hybridizations were performed to measure the cellular content of pro alpha 1 (I)collagen mRNA: up to ninefold increases in the level of this mRNA per cell were found. Upon subculturing, scleroderma fibroblasts were found to reduce gradually the increased synthesis of collagen to the level of nonaffected controls by the tenth passage. The levels of type I collagen mRNAs were also reduced, but more slowly. The results suggest that in scleroderma fibroblasts the genes for type I collagen are activated at procollagen mRNA level or that they are more stable and that the activating factors are lost during prolonged cell culture because cells from affected areas lose their activated state.     

Effects of 1alpha,25-dihydroxycholecalciferol and cortisol on the growth and differentiation of primary cultures of mouse mammary epithelial cells in collagen gel

We examined the effects of 1alpha,25-dihydroxycholecalciferol (1,25-DHCC) and the glucocorticoid, cortisol, on primary mouse mammary epithelial cells in collagen gel cell culture systems. Physiological low concentrations (10(-11)-10(-9) M) of 1,25-DHCC stimulated growth of the cells in a collagen gel matrix culture in serum-free DMEM+Ham's F12 (1:1) medium containing BSA, EGF and cholera toxin, and the cell number reached 1.8-fold the control after 6 d in culture. In contrast, supraphysiological concentrations (10(-8)-10(-7) M) of 1,25-DHCC suppressed cell growth. Cortisol produced similar, but smaller, dose-dependent effects. The addition of serum to the culture medium masked the stimulatory effect of 1,25-DHCC and both the stimulatory and inhibitory effects of cortisol. 1,25-DHCC also affected casein synthesis by cells cultured in a serum-free floating collagen gel culture containing prolactin, insulin and cortisol, enhancing synthesis at low concentrations (10(-11)-10(-9) M) and inhibiting it above 10(-8) M. In the absence of cortisol, no detectable change in casein synthesis was induced by 1,25-DHCC. These results suggest a physiological role for 1,25-DHCC in stimulating both growth and differentiation of mouse mammary epithelial cells, though 1,25-DHCC does not substitute for glucocorticoids in the differentiation of the cells.     

Osteogenin (bone morphogenetic protein-3) stimulates cartilage formation by chick limb bud cells in vitro.

Osteogenin is a protein isolated from demineralized bovine bone matrix. When implanted in rats, osteogenin induces the differentiation of cartilage and formation of endochondral bone. When added to stage 24 and 25 chick limb bud mesoderm cells in culture, it stimulated synthesis of sulfated proteoglycans by over 10-fold without stimulating cell division. The increase was detected after only 2 days in culture. Morphologically, in the presence of osteogenin, all cells in the culture appeared to form cartilage, rather than the nodules of cartilage surrounded by noncartilage areas in control cultures. The distribution of type II collagen correlated with the morphological differentiation of cartilage. When nonchondrocyte and chondrocyte cell populations were separated, osteogenin stimulated sulfated proteoglycan synthesis in all populations of cells. However, the greatest stimulation (24-fold) was seen in the originally nonchondrocyte population, which apparently still had some potential to form cartilage. In this study, chick limb bud mesoderm cells in vitro responded to osteogenin, a protein derived from adult bovine bone matrix. The cells that were responsive included those that initially did not form cartilage. Osteogenin belongs to a superfamily of proteins, many of which are important in development. It is possible that osteogenin has a role in embryonic cartilage development.     

Suppression of Growth-Associated Phosphorylation of Proteins of Fibroblasts by Collagen Fibrils

Collagen fibrils suppressed serum- or epidermal growth factor (EGF)-inducible DNA synthesis of human fibroblasts. The phosphorylation of cellular proteins upon these mitogenic stimulation was analyzed by two-dimentional polyacrylamide gel electrophoresis in order to reveal a possible interference of collagen fibrils with the cellular mitogenic signal transduction pathway coupled with the protein phosphorylation-dephosphorylation reaction. Spots of phosphorylated proteins numbered 192 on plain plastic which were reduced to 143 on collagen fibrils. More than half of them were matched between the two substrates, most of which were much more weakly phosphorylated on collagen fibrils. EGF stimulated the phosphorylation of these proteins of cells on plastic. Among them a protein with an approximate molecular weight of 27K and an isoelectric point of 5.3 was early and highly responsive to EGF, phosphorylation of which seemed to be catalyzed mainly by protein kinase C and tyrosine kinase. Collagen fibrils significantly suppressed this phosphorylation. The present study demonstrates that collagen fibrils modulate the growth-associated protein phosphorylation of cells, which seems to lead to the suppression of DNA synthesis.     

Fibroblast heterogeneity in glucocorticoid regulation of collagen metabolism: Genetic or epigenetic?

Summary Cultured fibroblasts derived from normal human dermis show a consistent 62% inhibition of collagen synthesis by hydrocortisone, whereas cultures derived from keloids average only 30% inhibition and show a much larger strain to strain variation ranging from 75% inhibition to 49% stimulation. Examination of fibroblast clones indicates that this high variation among keloid strains is not due to differences in the proportion of normal and keloid cells in the mass culture populations. Small but significant differences in the effect of hydrocortisone on collagen deposition are also seen among these clonal populations, but are not related to the type of tissue from which cultures were derived. Two to three-fold differences among clones derived from a single individual were observed, possibly suggesting functional heterogeneity of dermal fibroblasts with regard to collagen metabolism under control conditions and in response to hydrocortisone. However, this variation among clones may simply reflect differences in clonal growth, inasmuch as both collagen synthesis and deposition, and the effect of hydrocortisone on these processes, are strongly affected by population density. This work was supported in part by PHS grants, CA-17229 from the National Cancer Institute and AG-02046 from the National Institute on Aging, DHHS; and by Grant RIM 78-17313 from the National Science Foundation.     

Growth factor interactions between mouse mammary cell lines cocultured in collagen gels

Summary Three related mouse mammary cell lines were cultured in collagen gels and assayed for growth factor responsiveness and interaction via soluble factors. The CL-S1 cell line is nontumorigenic and grows poorly in collagen gel culture. The +SA and −SA cell lines exhibit different degrees of malignant behavior in vivo and have different growth properties in vitro. In collagen gel culture, +SA growth was stimulated by serum but not by epidermal growth factor (EGF), whereas both serum and EGF were required for optimal growth of −SA cells of early passage number as well as CL-S1 cells. −SA cells of later passage repeatedly exhibited a change so as to no longer require serum while retaining EGF responsiveness. [¹²⁵I]EGF binding analyses indicated that CL-S1 cells bound EGF with less affinity than did −SA cells whereas +SA cells bound almost to ligand. When cell lines were maintained in separate collagen gels but shared the same culture medium, growth of +SA or −SA cells was slightly enhanced in the presence of CL-S1 cells and −SA cell growth was enhanced by the presence of +SA cells. Using the normal rat kidney fibroblast line NRK (clone 49F) as an indicator, serum-containing conditioned media from each cell line and from each pair of cell lines cultured in collagen gels were tested for transforming growth factor (TGF) activity. Both the −SA and CL-S1 lines tested positive for TGF-α production and possibly released a TGF-β activity. These results suggest mechanisms by which cell populations in and around tumors can modify one another’s growth characteristics. The work was supported by a grant from the American Institute for Cancer Research, by American Cancer Society Institutional grant IN-119, by funds from the Poncin Trust (Seattle-First National Bank), and by grants CA-39611 and CA46885 from the National Institutes of Health, Bethesda, MD.     

Insulin-like growth factor-I (IGF-I) stimulates protein synthesis and collagen gene expression in monolayer and lattice cultures of fibroblasts.

Fibroblasts cultivated in three-dimensional tissue-like matrices are characterized by a slowed metabolism and a decrease of protein synthesis, unless they are submitted to physical tensions. We checked the effects of insulin like growth factor-I (IGF-I), known as a potent stimulator of mitogenesis and protein synthesis for many cell types, in various models of cultures: confluent monolayers, collagen lattices, non-retracting or retracting fibrin lattices. IGF-I (1-100 ng.ml-1) had no effect on cell divisions in lattice cultures. It was able to stimulate collagen lattice retraction when the medium was supplemented with low concentrations of serum. IGF-I at 10 or 100 ng.ml-1 stimulated collagen and non-collagen syntheses in all culture systems, but stimulation of collagen synthesis only began at the highest concentration (100 ng.ml-1) in retracted lattices. Northern blot and dot-blot analyses of mRNAs extracted from monolayer cultures of fibroblasts showed that IGF-I stimulated pro alpha 1(I) collagen synthesis at the pretranslational level. Cycloheximide (7.5 micrograms.ml-1) completely inhibited pro alpha 1(I) collagen gene expression induced by IGF-I. These results show that IGF-I is a potent stimulus for protein synthesis and collagen gene expression in monolayers and tridimensional cultures of fibroblasts, but that it exerts no mitogenic activity in tridimensional lattices. Synergistic associations of IGF-I with other growth factors will have to be found in order to reverse the quiescent status of fibroblasts in lattices.     

On the regulation of DNA synthesis in a line of adrenocortical tumor cells: effect of serum, adrenocorticotropin and pituitary factors.

Y-1 adrenal cells responded to serum step down by a several fold decrease in DNA synthesis. Serum starved cells resumed DNA synthesis upon serum step up. ACTH and cAMP inhibited DNA synthesis both at low and high serum concentrations, a fact previously known. Pituitary, brain and liver crude extracts stimulated DNA synthesis in serum starved cells. Purified pituitary factors preparations contained two activities: one specific for Y-1 cells and another active with both fibroblasts and Y-1 cells. The kinetics of restimulation of DNA synthesis by serum and pituitary factors was studied. DNA synthesis restimulation occurred after a lag of 11 hours. This lag did not vary irrespective of the type of stimulator or its concentration. Cells entered S phase continuously at a rate which increased with increasing concentrations of the stimulator. Cells became refractory to the inhibitory action of ACTH five hours before entering S phase. The implications of these data to the understanding of cell growth control are considered.     

The control of DNA synthesis in primary cultures of hepatocytes from adult and young rats: interactions of extracellular matrix components, epidermal growth factor, and the cell cycle

Hepatocytes from adult and 4-week-old rats cultured on one of several extracellular matrix components were stimulated to replicate by epidermal growth factor (EGF). DNA synthesis was increased at 44-48 hr in adult hepatocytes and at 24, 48, and 72 hr in hepatocytes from young rats when EGF was added 2 hr after explantation. When EGF was added at 24 hr, maximal DNA synthesis of adult hepatocytes was observed at 48 hr, whereas that of 4-week-old hepatocytes was seen at 48 and 72 hr. Ten ng EGF per ml was the optimal concentration for maximal DNA synthesis in both adult and young cells. DNA synthesis decreased with increasing cell density, but this effect was less in hepatocytes from young than in those from adults. When hepatocytes were cultured on substrata consisting of individual extracellular matrix components, neither the time that adult cells needed to respond to EGF nor the time from stimulation by EGF to the peak of maximal DNA synthesis was altered in either adult or young cells. The optimal EGF concentration for maximal DNA synthesis and the cell density control of replication were also not altered by the substrata used. Substrata made from each of the extracellular matrix components studied enhanced DNA synthesis of adult and young hepatocytes stimulated by EGF in the following decreasing order: fibronectin, type IV collagen, type I collagen, and laminin. In both adult and young hepatocytes the enhancement of DNA synthesis was greatest when cultured on fibronectin. Thus the initiation and magnitude of DNA synthesis in primary cultures of rat hepatocytes were altered both by the age of the donor and the substratum on which the cells were explanted.     

Cellular and metabolic specificity in the interaction of adhesion proteins with collagen and with cells

Fibronectin mediates the adhesion of fibroblasts to collagen substrates, binding first to the collagen and then to the cells. We report here that the interaction of the cells with the fibronectin-collagen complex is blocked by specific gangliosides, GD_{1 a} and GT₁, and that the sugar moieties of these gangliosides contain the inhibitory activity. The gangliosides act by binding to fibronectin, suggesting that they may be the cell surface receptor for fibronectin. Evidence is presented that other adhesion proteins or mechanisms of attachment exist for chondrocytes, epidermal cells, and transformed tumorigenic cells, since adhesion of these cells is not stimulated by fibronectin. Chondrocytes adhere via a serum factor that is more temperature-sensitive and less basic than fibronectin. Unlike that of fibroblasts chondrocyte adhesion is stimulated by low levels of gangliosides. Epidermal cells adhere preferentially to type IV (basement membrane) collagen but at a much slower rate than fibroblasts or chondrocytes. This suggests that these epidermal cells synthesize their own specific adhesion factor. Metastatic cells cultured from the T241 fibrosarcoma adhere rapidly to type IV collagen in the absence of fibronectin and do not synthesize significant amounts of collagen or fibronectin. Their growth, in contrast to that of normal fibroblasts, is unaffected by a specific inhibitor of collagen synthesis. These data indicate the importance of specific collagens and adhesion proteins in the adhesion of certain cells and suggest that a reduction in the synthesis of collagen and of fibronectin is related to some of the abnormalities observed in transformed cells.     

A comparison of the effects of different substrata on chondrocyte morphology and the synthesis of collagen types IX and X

Summary Embryonic chick sternal chondrocytes were cultured either within three dimensional gels of type I collagen, type II collagen or agar, or as monolayers on plastic dishes coated with air-dried films of these matrix macromolecules. It was observed that cell shape and cell growth varied markedly between the different culture conditions. Flattened monolayers of cells on plastic or films of type I or type II collagen, proliferated more rapidly and reached a higher final cell density per culture than the more rounded cells found in the cultures on agar films or within three-dimensional gels. Biosynthetic studies demonstrated that in addition to the synthesis of type II collagen, all the cultures were producing collagen types IX and X. Chondrocytes cultured on plastic or films of the different matrix macromolecules all showed a similar expression of types IX and X collagen, independent of whether they displayed a flattened or round cell morphology. In contrast, marked variations in the proportions of the minor collagens, particularly type X collagen, were observed when the cells were cultured within three-dimensional gels. The data suggest that direct interaction of the cell surface with matrix constituents displaying a particular spatial array could be an important aspect in the control of type IX and X collagen expression by chondrocytes. The financial support of the Arthritis & Rheumatism Council and the Medical Research Council is gratefully acknowledged.     

Differential effects of transforming growth factor-beta on the synthesis of extracellular matrix proteins by normal fetal rat calvarial bone cell populations

To determine the effects of transforming growth factor-beta (TGF-beta) on the different cell types that exist in bone, cell populations (I-IV), progressively enriched in osteoblastic cells relative to fibroblastic cells, were prepared from fetal rat calvaria using timed collagenase digestions. TGF-beta did not induce anchorage-independent growth of these cells, nor was anchorage-dependent growth stimulated in most populations studied, despite a two- to threefold increase in the synthesis of cellular proteins. In all populations the synthesis of secreted proteins increased 2-3.5-fold. In particular, collagen, fibronectin, and plasminogen activator inhibitor synthesis was stimulated. However, different degrees of stimulation of individual proteins were observed both within and between cell populations. A marked preferential stimulation of plasminogen activator inhibitor was observed in each population, together with a slight preferential stimulation of collagen; the effect on collagen expression being directed primarily at type I collagen. In contrast, the synthesis of SPARC (secreted protein acidic rich in cysteine/osteonectin was stimulated approximately two-fold by TGF-beta, but only in fibroblastic populations. Collectively, these results demonstrate that TGF-beta stimulates matrix production by bone cells and, through differential effects on individual matrix components, may also influence the nature of the matrix formed by different bone cell populations. In the presence of TGF-beta, osteoblastic cells lost their polygonal morphology and alkaline phosphatase activity was decreased, reflecting a suppression of osteoblastic features. The differential effects of TGF-beta on bone cell populations are likely to be important in bone remodeling and fracture repair.     

Leptin stimulates human osteoblastic cell proliferation, de novo collagen synthesis, and mineralization: Impact on differentiation markers, apoptosis, and osteoclastic signaling

Anabolic hormones, mechanical loading, and the obese protein leptin play separate roles in maintaining bone mass. We have previously shown that leptin, as well as its receptor, are expressed by normal human osteoblasts. Consequently, we have investigated how leptin affects proliferation, differentiation, and apoptosis of human osteoblasts. Iliac crest osteoblasts, incubated with either leptin (100 ng/ml), calcitriol (1,25(OH)(2)D(3); 10(-9) M) or 1-84 human parathyroid hormone (PTH; 10(-8) M), were cultured for 35 consecutive days and assayed for expression of various differentiation-related marker genes (as estimated by RT-PCR), de novo collagen synthesis, proliferation, in vitro mineralization, and osteoclast signaling. The effects of leptin on protection against retinoic acid (RA; 10(-7) M) induced apoptosis, as well as transition into preosteocytes, were also tested. Leptin exposure enhanced cell proliferation and collagen synthesis over both control condition and PTH exposure. Leptin inhibited in vitro calcified nodule production after 1-2 weeks in culture, however, subsequent to 4-5 weeks, leptin significantly stimulated mineralization. The mineralization profile throughout the entire incubation period was almost undistinguishable from the one induced by PTH. In comparison, 1,25(OH)(2)D(3) generally reduced proliferation and collagen production rates, whereas mineralization was markedly enhanced. Leptin exposure (at 2 and 5 weeks) significantly enhanced the expression of TGFbeta, IGF-I, collagen-Ialpha, ALP, and osteocalcin mRNA. Leptin also protected against RA-induced apoptosis, as estimated by soluble DNA fractions and DNA laddering patterns subsequent to 10 days of culture. The expression profiles of Bax-alpha and Bcl-2 mRNAs indicated that leptin per se significantly protected against apoptosis throughout the entire incubation period. Furthermore, the osteoblast marker OSF-2 was diminished, whereas the CD44 osteocyte marker gene expression was stimulated, indicating a transition into preosteocytes. In terms of osteoclastic signaling, leptin significantly augmented the mRNA levels of both interleukin-6 (IL-6) and osteoprotegerin (OPG). In summary, continuous leptin exposure of iliac crest osteoblasts, promotes collagen synthesis, cell differentiation and in vitro mineralization, as well as cell survival and transition into preosteocytes. Leptin may also facilitate osteoblastic signaling to the osteoclast.     

Effects of density and cellular aging on collagen synthesis and growth kinetics in keloid and normal skin fibroblasts

Summary Cell proliferation and collagen synthesis were analyzed in high, medium, and low density keloid and normal skin fibroblasts and also at various times during the course of in vitro aging to expand previous findings of increased collagen synthesis in keloid compared to normal skin fibroblasts. It was found that both keloid and normal fibroblasts (<20 population doublings) responded similarly to high, medium, and low initial plating densities; however, sparsely plated keloid fibroblasts exhibited a loss of replicative capacity earlier in their in vitro lifespan than did sparsely plated normal skin fibroblasts. When analyzed at population doubling levels 2 to 38, collagen synthesis was elevated in keloid compared to normal skin fibroblasts but decreased at the same rate in both cell types throughout this in vitro interval. Supported by NIH Grant GM-20298.     

Basic fibroblast growth factor regulates extracellular matrix and contractile protein expression independent of proliferation in vascular smooth muscle cells

Summary Basic fibroblast growth factor (bFGF) can influence proliferation and differentiation in vascular smooth muscle cells. Basic FGF promotes some features of the synthetic phenotype (proliferation) but is known to inhibit others (collagen synthesis). Whether bFGF availability influences smooth muscle cell phenotype independent of proliferation is not known. The purpose of this study was to determine if the effects of bFGF on extracellular matrix and contractile protein expression are dependent on changes in proliferation. Basic FGF availability was manipulated by adding bFGF to cultured cells or by inhibiting bFGF expression using antisense RNA, and adjusting culture conditions such that proliferation was held constant. Compared to cells cultured in serum alone, smooth muscle α-actin and myosin heavy chain expression was markedly reduced by added bFGF, but was not influenced by antisense inhibition of bFGF expression. Under the same conditions, collagen synthesis was inhibited by added bFGF, and was stimulated by reduced bFGF expression. These consequences of altering bFGF availability were not associated with changes in FGF receptor expression. These findings demonstrate that alterations in bFGF availability can regulate smooth muscle cell phenotype independent of proliferation, which may be related to the regulation of smooth muscle cell phenotype in vivo.