Basic fibroblast growth factor regulates type I collagen and collagenase gene expression in human smooth muscle cells.

Basic fibroblast growth factor (bFGF) is a multifunctional peptide well known for angiogenic, neurotropic, and mesoderm-inducing effects. In the present study, we have investigated the effects of bFGF on collagen and collagenase gene expression in human iliac arterial smooth muscle cells. We report that bFGF inhibits type I collagen gene expression and collagen biosynthesis, with concomitant stimulation of collagenase gene expression. The smooth muscle cells incubated with human recombinant bFGF decreased the mRNA steady state levels of pro-alpha 1(I) type I collagen by as much as 72%. [3H]Hydroxyproline synthesis was also suppressed by 59% compared with untreated control cultures. Indirect immunofluorescence confirmed corresponding changes at the protein level. In contrast to the down-regulation of type I collagen gene expression, collagenase gene expression was found to be up-regulated severalfold by bFGF. The data suggest that bFGF is capable of regulating collagen and collagenase gene expression divergently in human smooth muscle cells and that the effects appear to be mediated at a pretranslational level.     

Synthesis of type I homotrimer collagen molecules by cultured human lung adenocarcinoma cells.

Studies have been performed to evaluate both the relative amounts and molecular forms of the collagens synthesized by a new cell line (HU1) established from a human lung adenocarcinoma. The collagens secreted into the culture medium and extracted from the cell layers of cultured HU1 cells were isolated after limited pepsin digestion and differential salt fractionation. More than 70% of the collagen synthesized by HU1 cells was secreted into the culture medium rather than remaining in the cell layer. Polyacrylamide gel electrophoresis under denaturing conditions of the collagens indicated the presence of components with properties corresponding to those of the chains present in the types I homotrimer, III, IV, and V collagens. Carboxymethyl-trisacryl chromatographic analysis revealed that approximately 90% of the total collagen synthesized by HU1 cells corresponded to the type I homotrimer and that the cells did not synthesize the alpha 2(I) collagen chain. Of the remaining collagen, types III, IV, and V molecules represented 6, 1, and 4%, respectively, of the total produced. These data establish the relative proportions of the collagens synthesized by cultured HU1 cells and represent one of the initial documentations of a cell line established from a carcinoma of pulmonary origin that synthesizes type I homotrimer molecules. Furthermore, these findings suggest that HU1 cells may be a useful model for investigating the molecular basis of alterations in collagen biosynthesis associated with neoplasia.     

In vitro formation of hybrid fibrils of type V collagen and type I collagen. Limited growth of type I collagen into thick fibrils by type V collagen

Type V collagen and type I collagen were obtained from human placentas by pepsin treatment, followed by salt fractionation. The precipitates formed at 37 degrees C from a mixed solution of type V collagen and type I collagen, reacted with antibodies to either type V collagen or type I collagen. The precipitates seen by electron microscopy were fine flexible fibrils, with a D-periodic banding pattern. The average diameter of hybrid fibrils was smaller than 50 nm, when the proportion of type V collagen exceeded that of type I collagen. Type V collagen directly interacts with type I collagen in forming hybrid fibrils, resulting in limitation of the growth of type I collagen fibrils into thicker fibrils. We propose that the fibrils with a predominant type V collagen content may occur in the pericellular environment of various tissues, as a basic structure in connecting basal laminae with interstitial collagen fibrils.     

In vitro growth of human urinary tract smooth muscle cells on laminin and collagen type I-coated membranes under static and dynamic conditions

This study investigates in vitro growth of human urinary tract smooth muscle cells under static conditions and mechanical stimulation. The cells were cultured on collagen type I- and laminin-coated silicon membranes. Using a Flexcell device for mechanical stimulation, a cyclic strain of 0-20% was applied in a strain-stress-time model (stretch, 104 min relaxation, 15 s), imitating physiological bladder filling and voiding. Cell proliferation and alpha-actin, calponin, and caldesmon phenotype marker expression were analyzed. Nonstretched cells showed significant better growth on laminin during the first 8 days, thereafter becoming comparable to cells grown on collagen type I. Cyclic strain significantly reduced cell growth on both surfaces; however, better growth was observed on laminin. Neither the type of surface nor mechanical stimulation influenced the expression pattern of phenotype markers; alpha-actin was predominantly expressed. Coating with the extracellular matrix protein laminin improved in vitro growth of human urinary tract smooth muscle cells.     

Transformation of rabbit vascular smooth muscle cells by human cytomegalovirus morphological transforming region I.

The association of human cytomegalovirus with atherosclerosis and the monoclonal hypothesis of atherogenesis suggested that transformation of vascular smooth muscle cells may be an outcome of the virus-host cell interaction. To test this hypothesis, rabbit aorta smooth muscle cells were transfected with the morphological transforming region I (mtrI) of human cytomegalovirus (HCMV) linked to the neomycin resistance gene. Foci of neomycin-resistant and morphologically transformed cells were isolated and expanded into fourteen RCMV strains. Eight of these strains acquired immortalization, but only one strain (RCMV-21) retained recombined viral sequences integrated in the cellular DNA. RCMV strains were heterogeneous in their morphology, expression of smooth muscle alpha-actin, growth, and mitogenic response to serum and fibroblast growth factor (FGF)-2 and -4. All RCMV strains assayed except RCMV-3 showed DNA synthesis in low serum medium and, with the exception of RCMV-1 cells, all showed a significant mitogenic response to FGF-2 and FGF-4, Maintenance of the transformed phenotype appeared independent of the retention of the transforming viral sequences, which was suggestive of a "hit-and-run" mechanism. These results suggested that morphological transformation by HCMV DNA sequences could enhance the mitogenic response of vascular smooth muscle cells to fibroblast growth factors.     

Altered collagen homeostasis in human aortic smooth muscle cells (HAoSMCs) induced by aldosterone

The importance of aldosterone for cardiovascular diseases is well established. Most of the adverse effects seem to originate from its ability to produce vascular injury, including fibrosis. It is currently under debate whether aldosterone per se is able to induce fibrosis or whether it acts as a cofactor under pathological conditions. We tested whether aldosterone per se and in the presence of reactive oxygen stress (H₂O₂) enhances collagen abundance in human aortic smooth muscle cell (HAoSMC) media in primary culture and, if so, by which means. Collagen abundance, as well as epidermal growth factor receptor (EGFR) expression and ERK1/2 phosphorylation, was investigated by ELISA and Western blot. Collagenase activity and H₂O₂ formation were determined by fluorometry and luminometry. Aldosterone alone did not affect collagen abundance but potentiated the stimulatory effect of low concentrations of H₂O₂ (1-10 μmol/l). This effect disappeared when shedding of membrane-bound EGFR ligands was prevented by GM6001. EGFR expression and cellular EGF responsiveness were enhanced by aldosterone. Inhibition of the EGFR kinase (tyrphostin AG1478) prevented the increase of collagen. The increase in collagen abundance was prevented by blockade of the mineralocorticoid receptor (MR) and could be reproduced by MR transfection into Chinese hamster ovary cells. We conclude that aldosterone sensitizes HAoSMC for H₂O₂-induced increase of collagen abundance at least in part by enhanced EGFR expression.     

Effects of collagen matrix on proliferation and differentiation of vascular smooth muscle cells in vitro

In an attempt to better define the relationship between collagen matrices and vascular smooth muscle cells in vitro, proliferation of smooth muscle cells was observed in the early stages of culture. Cells spread on collagen gels had a longer doubling time and less incorporation of [3H]thymidine into DNA on the first day of culture than did cells grown on a plastic substrate. Cells on collagen gels were more elongated than were those on the plastic substrate and showed a "hills and valleys" arrangement from the first day in culture on the collagen type III gel. All cells were identified as smooth muscle having definite microfilaments, dense bodies, and pinocytotic vesicles. They had a distinct actin filament running from end to end when labeled with nitrobenzoxadiazole-phallacidin. Cells on the collagen gels had a larger number of actin filaments traveling parallel to the direction of the major axis of their cytoplasm than did those on the glass substrate. Therefore, cultured smooth muscle cells in the more physiological environment for cells in vitro, i.e., on collagen gels, show a suppression of cellular proliferation and an enhancement of differentiation in the early stages of culture. The effects of collagens on the differentiation of cells vary with the collagen phenotype.     

Monoclonality of smooth muscle cells in human atherosclerosis.

Atherosclerotic plaques contain a large monoclonal population of cells. Monoclonality could arise by somatic mutation, selection of a pre-existing lineage, or expansion of a pre-existing (developmental) clone. To determine the monoclonal cell type in plaque and learn when monoclonality arises, we studied X chromosome inactivation patterns using methylation of the X-linked human androgen receptor gene. Assays based on polymerase chain reaction were performed on samples of known cellular composition, microdissected from histological sections of human arteries. In atherosclerotic vessels, the majority of medial samples (7/11 coronary and 2/3 aortic) showed balanced (paternal and maternal) patterns of X inactivation, indicating polyclonality. In contrast, most samples of plaque smooth muscle cells showed a single pattern of X inactivation (3/4 aortic plaques and 9/11 coronary plaques; P < 0.01 versus media), indicating that plaque smooth muscle cells are monoclonal. Samples of plaque containing inflammatory or endothelial cells showed balanced X inactivation, also demonstrating polyclonality. Multiple plaques from a given patient showed no bias toward one allele, indicating there was no X-linked selection of cells during plaque growth. To determine whether plaques might arise from pre-existing clones (large X inactivation patches), we then studied 10 normal coronaries with diffuse intimal thickening. Six of the ten coronaries showed skewed X inactivation patterns in normal media and intima, suggesting the patch size in normal arteries is surprisingly large. Thus, smooth muscle cells constitute the monoclonal population in atherosclerotic plaques. The finding that normal arteries may have large X inactivation patches raises the possibility that plaque monoclonality may arise by expanding a pre-existing clone of cells rather than generating a new clone by mutation or selection.     

Extra-and intracellular collagen resorption by smooth muscle cells in postpartum uterine involution

Localization of cathepsin B in rat uterus during postpartum involution is studied using electron histochemical technique. High extracellular activity of cathepsin B and accumulation of products on the cytolemma of smooth muscle cells and adjacent collagen fibrils are revealed. These findings suggest that smooth muscle cells participate in the extracellular collagen degradation via secretion of cathepsin B. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 1, pp. 106–108, January, 1999     

平滑肌细胞对共培养体系滋养细胞侵袭力与MMP-2及MMP-9表达的影响

目的模拟体内环境,建立可保持平滑肌细胞与绒毛外细胞滋养层细胞(EVCT)生物学特性的共培养细胞模型,应用于研究平滑肌细胞与滋养细胞理化特性与滋养细胞的侵袭行为。方法利用组织块培养法培养脐动脉平滑肌细胞,组织块培养、胰酶消化和Percoll梯度沉降,收集纯化人早孕绒毛组织的滋养细胞,免疫组化检测细胞的纯度。将滋养细胞与平滑肌细胞分别放入Transwell的上下小室,观察该模型下滋养细胞形态变化、细胞活力、侵袭力改变与分泌功能等特性。结果免疫组化显示EVCT的细胞角蛋白7阳性表达的细胞数占95%以上,SMC a-actin阳性表达的细胞数也超过95%,证实共培养系统中EVCT和SMC纯度均在95%以上,且生物学特性得以维持。上室中的EVCT保持了其侵袭能力,且平滑肌细胞能促进滋养细胞增殖活性、侵蚀能力及MMP2、MMP9的表达。结论成功地建立了平滑肌细胞与滋养细胞原代共培养系统模型,便于研究滋养细胞侵袭和子宫螺旋动脉重铸障碍的分子机制。     

A neo-esophagus reconstructed by cultured human esophageal epithelial cells, smooth muscle cells, fibroblasts, and collagen

The scientists involved in this study attempted to develop an artificial esophagus constructed of autologous cells grown by cell culture methods on an extracellular matrix. An artificial esophagus consisting of human esophageal epithelial cells, dermal fibroblasts, and smooth muscle cells isolated from the aortic media, was attempted. The purpose of this study was to examine whether smooth muscle cells could be used in the transforming matrix. Human fibroblasts were embedded in Type I collagen superimposed on the collagen layer of smooth muscle cells. Next, human esophageal epithelial cells were cultured on the collagen layer of the fibroblasts. The resulting collagen sheets were cultured in vitro for 1 week, then transplanted on the latissimus dorsi muscles of athymic rats. The sheets were examined histologically at 1 and 2 weeks using hematoxylin eosin and immunologic stain methods (antiactin antibody). At the end of 2 weeks after transplantation, on microscopic observation of the collagen sheets, it appeared that the epithelial layer, the submucosal tissue layer, and the proper muscle layer had been reconstructed. Additionally, the authors successfully isolated smooth muscle cells from the media of the left gastric artery as a surgical specimen by explant cell culture. The ability to transform collagen sheets consisting of esophageal epithelial cells, fibroblasts, and smooth muscle cells from a surgical specimen into a luminal structure may enable clinical application of the artificial esophagus.     

Interaction of collagen and smooth muscle cells in aortic biomechanics

To elucidate the contribution of smooth muscle to the mechanical properties of artery and to study its interaction with other tissue components, an enzymolysis technique has been used. Fresh canine artery was cut into circumferential strips, and a stressrelaxation test was performed in a Krebs-Ringer solution while continuously bubbling an O₂−CO₂ gas mixture. An aortic smooth muscle stimulant, norepinephrine, was perfused during the stress-relaxation test, before and after removing collagen with collagenase. The collagen-free artery did not show any contraction due to the smooth muscle, in contrast to the intact artery. This indicates that there may be important interactions between the collagen matrix and smooth muscle. This new technique can be used to study the contribution of smooth muscle to the mechanical properties of aorta.     

Chondrogenic differentiation of human mesenchymal stem cells in collagen type I hydrogels

The chondrogenic differentiation of bone marrow-derived human mesenchymal stem cells (MSCs) in a collagen type I hydrogel, which is in clinical use for matrix-based autologous chondrocyte transplantation (ACT), was investigated. Collagen hydrogels with 2.5 x 10(5) MSCs/mL were fabricated and cultured for 3 weeks in a serum-free, defined, chondrogenic differentiation medium containing 10 ng/mL TGF-beta1 or 100 ng/mL BMP-2. Histochemistry revealed morphologically distinct, chondrocyte-like cells, surrounded by a sulfated proteoglycan-rich extracellular matrix in the TGF-beta1 and BMP-2 treated group, with more elongated cells seen in the BMP-2 treated group. Immunohistochemistry detected collagen type II (Col II) in the TGF-beta1 and BMP-2 treated group. Collagen type X (Col X) staining was positive in the TGF-beta1 but only very weak in the BMP-2 treated group. RT-PCR analyses revealed a specific chondrogenic differentiation with the expression of the cartilage specific marker genes Col II, Col X, and aggrecan (AGN) in the TGF-beta1 and the BMP-2 treated group, with earlier expression of these marker genes in the TGF-beta1 treated group. Interestingly, MSC-gels cultured in DMEM with 10% FBS (control) indicated few isolated chondrocyte-like cells but no expression of Col II or Col X could be detected. The results show, that MSCs cultured in a collagen type I hydrogel are able to undergo a distinct chondrogenic differentiation pathway, similar to that described for MSCs cultured in high-density pellet cultures. These findings are valuable in terms of ex vivo predifferentiation or in situ differentiation of MSCs in collagen hydrogels for articular cartilage repair.     

Modified collagen fleece, a scaffold for transplantation of human bladder smooth muscle cells

Several congenital and acquired diseases of the human genito-urinary tract may need, due to lack or destruction of functional tissues, mechanically stable biomaterials as cell carriers for the engineering of these tissues. When using collagen scaffolds, both their capacity to induce tissue regeneration and their biocompatibility are advantageous characteristics to render them apt for tissue engineering. The attachment of extracellular matrix or serum proteins to their surfaces does further improve these characteristics, mimicking a close to natural cell environment. In this study, equine collagen scaffolds (TissueFleece) were modified by coating fetal bovine serum proteins, before human bladder smooth muscle cells were seeded. Cell growth was evaluated by WST-1 proliferation assay and improved when using modified collagen scaffolds. However, cell penetration assessed by histology showed similar results on modified and native scaffolds. These cell-scaffold constructs were further implanted in the dorsal subcutaneous space of athymic mice. In vivo studies showed the presence of the fluorescent-labeled transplanted smooth muscle cells until day 3 and thereafter angiogenesis was induced and infiltration of mouse fibroblasts and polymorphonuclear cells were observed. The latter had completely disappeared after 3 weeks.     

Cyclic AMP inhibits increased collagen production by cyclically stretched smooth muscle cells

Rabbit arterial smooth muscle cells, grown on elastin membranes which were cyclically elongated and relaxed, responded by increasing their rates of synthesis of protein and, in particular, of collagen, compared to stationary controls. Raising intracellular cyclic AMP (cAMP) levels by adding theophylline or dibutyryl cAMP to the culture medium prevented the synthetic response to cyclic stretching, but did not alter the rates of protein or collagen synthesis by stationary controls. Both synthesis and degradation of collagen by cyclically stretched cells increased in parallel such that the proportion of synthesized collagen that was degraded was similar to that found in the stationary cultures. Collagen degradation was not affected by theophylline administration to stationary cell cultures but the drug increased degradation of collagen by cyclically stretched cells. We conclude that the net production of protein, and in particular of a structural protein, collagen, by arterial smooth muscle cells subjected to the mechanical force of stretching was inhibited when intracellular levels of cAMP were raised. The results suggest that cAMP may play a role in the modulation of structural protein content of artery walls in response to changes in tensile stress.     

Effects of angiotensin II and vasopressin on human smooth muscle cells in vitro

Arterial smooth muscle cell hyperplasia is a histopathological marker of both hypertension and the initial stages of atherogenesis. In order to determine if vasoactive agents could promote arterial smooth muscle cell proliferation, two potent pressor hormones, angiotensin II and vasopressin, were tested in cell culture. Either one of these agents was added daily to cultures of human aortic smooth muscle cells maintained in homologous serum (HS) or fetal bovine serum (FBS). After 7 days, angiotensin II significantly enhanced cell proliferation in both types of sera. While vasopressin was found to be inhibitory in FBS, it also stimulated cell growth in HS. Furthermore, both peptides in HS induced an increase in average cell size. Comparative studies were conducted with human uterine smooth muscle cells and 3T3 mouse fibroblasts in order to determine the specificity of the growth effects for human vascular smooth muscle. The above in vitro studies suggest that angiotensin II and vasopressin, by modulating both number and size of arterial smooth muscle cells, may play a direct and until now unexpected role in the development of chronic vascular disease in man.     

High-density collagen gel tubes as a matrix for primary human bladder smooth muscle cells

Tissue-engineered grafts for the urinary tract are being investigated for the potential treatment of several urologic diseases. These grafts, predominantly tubular-shaped, usually require in vitro culture prior to implantation to allow cell engraftment on initially cell-free scaffolds. We have developed a method to produce tubular-shaped collagen scaffolds based on plastic compression. Our approach produces a ready cell-seeded graft that does not need further in vitro culture prior to implantation. The tubular collagen scaffolds were in particular investigated for their structural, mechanical and biological properties. The resulting construct showed an especially high collagen density, and was characterized by favorable mechanical properties assessed by axial extension and radial dilation. Young modulus in particular was greater than non-compressed collagen tubes. Seeding densities affected proliferation rate of primary human bladder smooth muscle cells. An optimal seeding density of 10(6) cells per construct resulted in a 25-fold increase in Alamar blue-based fluorescence after 2?wk in culture. These high-density collagen gel tubes, ready seeded with smooth muscle cells could be further seeded with urothelial cells, drastically shortening the production time of graft for urinary tract regeneration.     

培养的人主动脉平滑肌细胞异常表达HLA—DR,DP抗原

用γ-IFN、淋巴细胞条件培养液(LCM)对培养的人主动脉平滑肌细胞(SMC)进行诱导,观察其是否异常表达DR、DP抗原,以探讨在动脉粥样硬化(AS)发病中的意义。结果表明,γ-IFN组DR、DP阳性率分别为80%和73%;LCM组DR、DP阳性率为74%和70%。两组均明显高于正常对照组DR、DP的阳性率(11%,7%)。提示激活的T淋巴细胞通过分泌γ-IFN(或合并其它因子)诱导SMC异常表达DR、DP抗原,可能在AS的发生发展中起一定作用。     

Synthesis of type III collagen by cultured kidney epithelial cells

Studies have been performed to evaluate both the relative amounts and molecular forms of the collagens synthesized by an established line of cultured rat kidney epithelial (clone NRK52E) cells. The collagens secreted into the culture medium and extracted from the cell layers of cultured NRK52E cells were isolated after limited pepsin digestion and differential salt fractionation. Greater than 95% of the collagenous proteins synthesized by NRK52E cells were found to be associated with the cells and not secreted. Polyacrylamide gel electrophoresis under denaturing conditions of the NRK52E cell collagens indicated the presence of components exhibiting properties corresponding to those of the chains present in types I, III, IV and V collagen. Analysis of each fraction by carboxymethyl-trisacryl chromatography revealed that approximately two-thirds of the total collagen synthesized by NRK52E cells was type III. Of the remaining collagen types I, IV and V molecules represented 20%, 4% and 10% respectively, of the total produced. Essentially all of the type I collagen produced by NRK52E cells was recovered as the type I-trimer, whereas the type V molecules synthesized by NRK52E cells had the molecular compositions of [alpha 1(V)]2 alpha 2(V) and alpha 1(V)alpha 2(V)alpha 3(V). These data establish the relative proportions and molecular forms of the collagens synthesized by cultured NRK52E cells. Furthermore, these findings suggest that NRK52E cells may be a useful in vitro model for investigating the regulation of changes in collagen biosynthesis occurring under situations of renal epithelial cell injury.     

Inhibition of type I procollagen synthesis by damaged collagen in photoaged skin and by collagenase-degraded collagen in vitro

Type I and type III procollagen are reduced in photodamaged human skin. This reduction could result from increased degradation by metalloproteinases and/or from reduced procollagen synthesis. In the present study, we investigated type I procollagen production in photodamaged and sun-protected human skin. Skin samples from severely sun-damaged forearm skin and matched sun-protected hip skin from the same individuals were assessed for type I procollagen gene expression by in situ hybridization and for type I procollagen protein by immunostaining. Both mRNA and protein were reduced ( approximately 65 and 57%, respectively) in photodamaged forearm skin compared to sun-protected hip skin. We next investigated whether reduced type I procollagen production was because of inherently reduced capacity of skin fibroblasts in severely photodamaged forearm skin to synthesize procollagen, or whether contextual influences within photodamaged skin act to down-regulate type I procollagen synthesis. For these studies, fibroblasts from photodamaged skin and matched sun-protected skin were established in culture. Equivalent numbers of fibroblasts were isolated from the two skin sites. Fibroblasts from the two sites had similar growth capacities and produced virtually identical amounts of type I procollagen protein. These findings indicate that the lack of type I procollagen synthesis in sun-damaged skin is not because of irreversible damage to fibroblast collagen-synthetic capacity. It follows, therefore, that factors within the severely photodamaged skin may act in some manner to inhibit procollagen production by cells that are inherently capable of doing so. Interactions between fibroblasts and the collagenous extracellular matrix regulate type I procollagen synthesis. In sun-protected skin, collagen fibrils exist as a highly organized matrix. Fibroblasts are found within the matrix, in close apposition with collagen fibers. In photodamaged skin, collagen fibrils are shortened, thinned, and disorganized. The level of partially degraded collagen is approximately 3.6-fold greater in photodamaged skin than in sun-protected skin, and some fibroblasts are surrounded by debris. To model this situation, skin fibroblasts were cultured in vitro on intact collagen or on collagen that had been partially degraded by exposure to collagenolytic enzymes. Collagen that had been partially degraded by exposure to collagenolytic enzymes from either bacteria or human skin underwent contraction in the presence of dermal fibroblasts, whereas intact collagen did not. Fibroblasts cultured on collagen that had been exposed to either source of collagenolytic enzyme demonstrated reduced proliferative capacity (22 and 17% reduction on collagen degraded by bacterial collagenase or human skin collagenase, respectively) and synthesized less type I procollagen (36 and 88% reduction, respectively, on a per cell basis). Taken together, these findings indicate that 1) fibroblasts from photoaged and sun-protected skin are similar in their capacities for growth and type I procollagen production; and 2) the accumulation of partially degraded collagen observed in photodamaged skin may inhibit, by an as yet unidentified mechanism, type I procollagen synthesis.