The modulation of collagen synthesis in cultured arterial smooth muscle cells by platelet-derived growth factor.

Changes on collagen synthetic activity of cultured arterial smooth muscle cells of rabbits induced with purified platelet-derived growth factor (PDGF) were examined. PDGF treatment (final concentration was 5 units/ml) decreased the total collagen synthesis per cell, while the rate of collagen synthesis against total protein synthesis was raised by PDGF. Type analysis of collagen revealed substantial reduction of type IV collagen and relative increase of type V collagen in the PDGF-treated cells. By immunofluorescence study using anti-type IV collagen antibody, the lacework fluorescence was decreased with PDGF supplement. These findings indicate that PDGF induces the decrease of type IV collagen synthesis with the simultaneous diminution of basement membrane formation probably in association with phenotypic modulation of smooth muscle cells.     

Comparison of the collagenous products synthesized in culture by pig aortic endothelial and smooth-muscle cells. Variability in endothelial-cell cultures.

In contrast with smooth-muscle cells from the same tissue, endothelial cells from pig aorta were found to exhibit in culture considerable variability in the pattern of collagen synthesis between one isolation of cells and the next. Synthesis varied from largely collagen type I to virtually all type III in the absence of type I but with small amounts still of collagens types IV and V, to, in one instance, synthesis basically of only type V. Synthesis usually by these cells of collagen predominantly of the interstitial type (I and III) rather than, as might be expected, that from basement membrane (type IV) was not attributable to the influence of subculture. All four collagen types were deposited in the cell layer to an increased extent in primary compared with secondary cultures of either smooth muscle or endothelial origin. Endothelial cells appeared sometimes to synthesize a large-Mr collagenous entity that might conceivably be related to 'short-chain' collagen. In addition, small-Mr hydroxyproline-containing peptides were detected that might reflect rapid collagen(s) turnover in endothelial cultures.     

Expression of contractile proteins α-actin and myosin of smooth muscle cells and of type IV collagen in human placenta at placental insufficiency in III trimester of pregnancy

Changes of expression of contractile proteins (smooth muscle cell α-actin and myosin) and of type IV collagen in villous stroma of human placenta were studied at the diagnosed placental insufficiency (PI) in III trimester of pregnancy. The study revealed pronounced disturbances of expression of contractile proteins and type IV collagen at PI. It is shown that in perivascular sheaths of vessels of stem and intermediate villi there is present a much greater amount of cells expressing smooth muscle actin and myosin. These cells are arranged by the denser concentric layers and more compactly than in norm and fill the intervascular space inside the villi. The width of perivascular sheaths of vessels is higher, while vascular lumens are lower than in norm. In terminal villi the capillary walls are thickened and the number of pericytes immunopositive against the smooth muscle cell α-actin and myosin as well as type IV collagen is increased. The change of synthesis of the cytoskeletal contractile proteins and type IV collagen is shown to lead to structural disturbances of villi of different types and of perivascular areas and vessels, which doubtlessly indicates their participation in pathogenesis of placental dysfunction and of disturbance of placental hemodynamics.     

Expression of extracellular matrix components is regulated by substratum

Reconstituted basement membranes and extracellular matrices have been demonstrated to affect, positively and dramatically, the production of milk proteins in cultured mammary epithelial cells. Here we show that both the expression and the deposition of extracellular matrix components themselves are regulated by substratum. The steady-state levels of the laminin, type IV collagen, and fibronectin mRNAs in mammary epithelial cells cultured on plastic dishes and on type I collagen gels have been examined, as has the ability of these cells to synthesize, secrete, and deposit laminin and other, extracellular matrix proteins. We demonstrate de novo synthesis of a basement membrane by cells cultured on type I collagen gels which have been floated into the medium. Expression of the mRNA and proteins of basement membranes, however, are quite low in these cultures. In contrast, the levels of laminin, type IV collagen, and fibronectin mRNAs are highest in cells cultured on plastic surfaces, where no basement membrane is deposited. It is suggested that the interaction between epithelial cells and both basement membrane and stromally derived matrices exerts a negative influence on the expression of mRNA for extracellular matrix components. In addition, we show that the capacity for lactational differentiation correlates with conditions that favor the deposition of a continuous basement membrane, and argue that the interaction between specialized epithelial cells and stroma enables them to create their own microenvironment for accurate signal transduction and phenotypic function.     

Differential expression of type IV collagen isoforms, α5(IV) and α6(IV) chains, in basement membranes surrounding smooth muscle cells

 Smooth muscle is composed of cigar-shaped, non-striated cells, each of which is encapsulated by a basement membrane and forms the contractile portion of tubular organs such as the gastrointestinal tract, pulmonary tract, genitourinary tract, and vasculature, in which slow and sustained contractions are needed. We examined basement membranes produced by smooth muscle cells and, using α(IV) chain-specific monoclonal antibodies, analyzed type IV collagens in these organs. Detailed distribution analysis of the α chains in normal and Alport cases by use of specific antibodies indicated that there are at least three molecular forms of type IV collagen, [α1(IV)]₂α2(IV), α3(IV)α4(IV)α5(IV), and α5(IV)/α6(IV). Smooth muscle cells in the urinary bladder and uterus were enclosed by basement membranes composed of α1, α2, α5, and α6 chains. The same α chains were present around smooth muscle cells in the muscular layer of the fundus of the stomach, whereas those in the antrum and further distal side of the gastrointestinal tract expressed mostly α1 and α2 chains. In addition, immunostaining analysis of the vasculature also showed that most of the smooth muscle cells were positive for α1 and α2 chains; however, α5 and α6 chains were also expressed by smooth muscle cells in the aorta and some arteries where blood pressure changes significantly. These results suggest that the smooth muscle cells enclosed by α5/α6-containing basement membranes might have some particular function related to mechanical stress or tensile strength during the characteristic contractile activity of tubular organs. Accepted: 23 March 1998     

Expression and adhesive properties of basement membrane proteins in cerebral capillary endothelial cell cultures

Previous studies have indicated the importance of basement membrane components both for cellular differentiation in general and for the barrier properties of cerebral microvascular endothelial cells in particular. Therefore, we have examined the expression of basement membrane proteins in primary capillary endothelial cell cultures from adult porcine brain. By indirect immunofluorescence, we could detect type IV collagen, fibronectin, and laminin both in vivo (basal lamina of cerebral capillaries) and in vitro (primary culture of cerebral capillary endothelial cells). In culture, these proteins were secreted at the subcellular matrix. Moreover, the interaction between basement membrane constituents and cerebral capillary endothelial cells was studied in adhesion assays. Type IV collagen, fibronectin, and laminin proved to be good adhesive substrata for these cells. Although the number of adherent cells did not differ significantly between the individual proteins, spreading on fibronectin was more pronounced than on type IV collagen or laminin. Our results suggest that type IV collagen, fibronectin, and laminin are not only major components of the cerebral microvascular basal lamina, but also assemble into a protein network, which resembles basement membrane, in cerebral capillary endothelial cell cultures.     

In vitro studies on adult cardiac myocytes: attachment and biosynthesis of collagen type IV and laminin

The interactions between adult rat cardiac myocytes and the basement membrane components collagen type IV and laminin were investigated in attachment experiments and biosynthesis studies and by immunofluorescence staining. Adult myocytes attached equally well to native collagen type IV and laminin but did not attach to collagen type IV solubilized with pepsin (P-CIV) or to collagen type I. However, when laminin was used to coat P-CIV, attachment was enhanced. Affinity-purified antibodies against laminin inhibited the attachment of myocytes to dishes coated with native collagen type IV, indicating that cell surface-bound laminin mediated attachment of the cells to this substrate. Immunofluorescence staining of freshly isolated myocytes, using antibodies against laminin or collagen type IV, revealed the presence of laminin but not of collagen type IV on the surface of freshly isolated cells, indicating that during the isolation procedure collagen IV was removed from the cell surface. Metabolic labeling followed by immunoprecipitation demonstrated synthesis of both laminin and collagen type IV in cardiac myocytes as they progressed into culture over a 14-day period. This synthesis was accompanied by the deposition of the collagen type IV and laminin into distinctly different patterns as revealed by immunofluorescence staining. As the cells progressed into culture, newly synthesized laminin formed a network radiating from the center of the reorganizing cell into the pseudopods. The laminin was redistributed and remodeled with time in culture to form a dense layer beneath the cell. Collagen type IV was also synthesized with time in culture, but the pattern was a much finer network as opposed to the denser pattern of laminin staining. These studies demonstrate that adult cardiac myocytes synthesize and remodel the basement membrane as they adapt to the culture environment.     

Cells that emerge from embryonic explants produce fibers of type IV collagen

Double immunofluorescence staining experiments designed to examine the synthesis and deposition of collagen types I and IV in cultured explants of embryonic mouse lung revealed the presence of connective tissue-like fibers that were immunoreactive with anti-type IV collagen antibodies. This observation is contrary to the widely accepted belief that type IV collagen is found only in sheet-like arrangements beneath epithelia or as a sheath-like layer enveloping bundles of nerve or muscle cells. The extracellular matrix produced by cells that migrate from embryonic mouse lung rudiments in vitro was examined by double indirect immunofluorescence microscopy. Affinity-purified monospecific polyclonal antibodies were used to examine cells after growth on glass or native collagen substrata. The data show that embryonic mesenchymal cells can produce organized fibers of type IV collagen that are not contained within a basement membrane, and that embryonic epithelial cells deposit fibers and strands of type IV collagen beneath their basal surface when grown on glass; however, when grown on a rat tail collagen substratum the epithelial cells produce a fine meshwork. To our knowledge this work represents the first report that type IV collagen can be organized by cells into a fibrous extracellular matrix that is not a basement membrane.     

Contraction of cultured human uterine smooth muscle cells after stimulation with endothelin-1

To our knowledge, the problem of how to maintain isolated smooth cells in a "contractile" phenotypic state without deviation after subculturing has yet to be resolved. The present study characterized the in vitro contractile response of human uterine smooth muscle cell to endothelin-1, which induces contractions in isolated uterine strips. Contractile effects were qualitatively investigated using silicone rubber substrata. Endothelin-1 was able to distort and reduce the wrinkles in the silicone surface. Contractions were also quantified by measuring the resulting change in the collagen lattice area. Endothelin-1 significantly increased the contractile response in a dose-dependent manner by selectively activating endothelin A receptors. When myometrial cells were cultured within collagen lattices, a microfilament-disrupting agent, cytochalasin B, abolished contractions, and no change was observed in smooth muscle alpha-actin immunostaining. Taken together, these observations show that the uterine smooth muscle cells are contractile and respond appropriately to a potent uterotonic agent. Based on these findings, a cultured uterine smooth muscle cell model, which could be used to elucidate the mechanisms controlling uterine activity, is proposed.     

Diverse effects of fibronectin and laminin on phenotypic properties of cultured arterial smooth muscle cells

Plasma fibronectin promotes modulation of rat arterial smooth muscle cells from a contractile to a synthetic phenotype during the first few days in primary culture. This process includes cell adhesion and spreading, loss of myofilaments, and formation of a widespread rough endoplasmic reticulum and a prominent Golgi complex. The structural reorganization is accompanied by activation of overall RNA and protein synthesis. Moreover, the cells gain the ability to replicate their DNA and divide in response to platelet-derived growth factor. Here, it is demonstrated that the power of fibronectin to bring about this change in the differentiated properties of the smooth muscle cells resides in a 105-kD cell-binding fragment, whereas a 70-kD collagen-binding fragment and a 31-kD heparin-binding fragment are inactive in this respect. Laminin, another adhesive glycoprotein and a component of the basement membrane that normally surrounds arterial smooth muscle, was contrarily found to maintain the cells in a contractile phenotype. However, with increasing time more and more cells went through the modulation into a synthetic phenotype. This "catch-up" was counteracted by a peptide that contained the cell-attachment sequence of fibronectin (Arg-Gly-Asp-Ser). Hence, it is possible that the delayed modulation on laminin was due to production of fibronectin by the cells themselves. In support of this notion, fibronectin isolated from smooth muscle cultures was found to be as effective as plasma fibronectin in stimulating the phenotypic modulation. Moreover, using a combination of chemical, immunochemical, and immunocytochemical methods, it was demonstrated that the cells secreted fibronectin as well as laminin at an increasing rate during the first 4 d in primary culture and, notably, cells cultured on laminin produced more fibronectin than cells cultured on fibronectin. Newly synthesized fibronectin was incorporated into a network of pericellular and intercellular fibrils, whereas laminin formed a more diffuse layer covering the cells in a basement membrane-like manner. Taken together, the findings suggest diverse roles for fibronectin and laminin in the control of the differentiated properties of arterial smooth muscle cells. They further indicate that the ability of arterial smooth muscle cells to produce fibronectin and laminin early in primary culture is not directly related to the phenotypic state as determined morphologically and by measurement of overall rates of RNA and protein synthesis. This may be due to the cells being able to sense the macromolecular composition of the pericellular matrix and to modify their secretory activity accordingly.(ABSTRACT TRUNCATED AT 400 WORDS)     

Trichinella spiralis and Trichinella pseudospiralis induce collagen synthesis by host fibroblasts in vitro and in vivo

Immunoperoxidase staining of muscle infected with Trichinella spiralis for murine collagen types I and IV provided both qualitative and quantitative evidence of extensive synthesis of both types of collagen by fibroblasts in infected muscle compared to that seen uninfected muscle. Moreover, fibroblasts in muscle infected with T. pseudospiralis, a nonencapsulating species, showed significantly less staining for both types of collagen compared to muscle from mice infected with T. spiralis. Analysis of collagen composition of isolated nurse cells using an ELISA specific for either type I or type IV murine collagen suggested that of these 2 types of collagen, only type IV basement membrane collagen is found in Trichinella capsular collagen. Excretory/secretory products of T. spiralis and T. pseudospiralis induced extensive synthesis of exclusively type IV collagen by 3T3 murine fibroblasts in vitro.     

Antisense oligonucleotides to stromelysin mRNA inhibit injury-induced proliferation of arterial smooth muscle cells.

Smooth muscle cell migration and proliferation are important events in the formation of intimal lesions associated with atherosclerosis and restenosis following balloon angioplasty. To make this possible, the smooth muscle cell has to change from a contractile to an activated repair cell with capacity to synthesize DNA and extracellular matrix components. There is now considerable evidence that the extracellular matrix has important functions in modulating the phenotypic properties of smooth muscle cells, but less is known about the role of the matrix metalloproteinases. The present study investigates the role of stromelysin in the modulation of rat aortic smooth muscle cell morphology and function following mechanical injury in vitro and in vivo. Antisense mRNA oligonucleotides were used to investigate the role of stromelysin expression in injury-induced phenotypic modulation and the subsequent migration and proliferation of vascular smooth muscle cells. Cultured rat aortic smooth muscle cells and balloon-injured rat carotid arteries were used as experimental models. Light- and electron microscopy were used to follow changes in smooth muscle cell phenotype and lesion formation and incorporation of 3H-thymidine to detect DNA synthesis. Injury-induced DNA synthesis and migration in vitro were inhibited by 72% and 36%, respectively, by adding stromelysin antisense oligonucleotides to the medium prior to injury. In primary cultures, 67% of the smooth muscle cells treated with stromelysin antisense were retained in a contractile phenotype as judged by analysis of cell fine structure, compared to 15% untreated cells and 40% in cells treated with mismatched oligonucleotides. Examination of the carotid arteries one week after balloon injury likewise demonstrated a larger fraction of contractile cells in the inner parts of the media in vessels treated with antisense oligonucleotides compared to those treated with mismatched oligonucleotides. The neointima was also distinctly thinner in antisense-treated than in mismatched-treated and control arteries at this time. These findings indicate that stromelysin mRNA antisense oligonucleotides inhibited phenotypic modulation of rat arterial smooth muscle cells and so caused a decrease in migration and proliferation and neointima formation in response to vessel wall injury.     

Effect of heparin on synthesis of short chain collagen by chondrocytes and smooth muscle cells

The treatment of embryonic chick chondrocyte cultures with heparin results in a decrease in collagen synthesis. One of the collagens synthesized by hypertrophic chondrocytes, specifically type X collagen, may play an important role in cartilage mineralization and endochondral ossification. Recently a new short chain collagenous component was found in cultures of rat vascular smooth muscle cells (Majack, R. A., and P. Bornstein, 1985, J. Cell Biol., 100: 613-619). The present study was initiated to investigate heparin's effect on type X collagen in embryonic chick chondrocytes and to further evaluate the nature of the short chain component synthesized by rat vascular smooth muscle cells. Different tissues may respond differently to the administration of heparin. In chondrocyte cultures heparin decreased both total collagen synthesis as well as the synthesis of type X collagen. There was an accumulation of collagen precursors, found principally in the cell layer compartment, which appeared to be the result of heparin's inhibition of the NH2-terminal protease. In cultures of rat vascular smooth muscle cells heparin was found to increase the synthesis of a short chain collagenous component as previously reported. However, comparison with a type X collagen standard showed this to be different from type X. In all cases, the effect of heparin on collagen chain precursors, chondrocyte type X synthesis, and synthesis of a vascular smooth muscle short chain collagen was shown to be reversible. Similar effects were obtained by adding chondroitin sulfate to chondrocytes, suggesting a role for extracellular matrix components in the modulation of collagen synthesis. These findings are consistent with the concept of a group of short chain collagens with type X collagen being unique to hypertrophic chondrocytes.     

Basement membrane collagen requirements for attachment and growth of mammary epithelium.

In vivo mammary epithelial cells rest upon a basement membrane composed in part of type IV collagen which is synthesized by these cells. In this study, basement membrane collagen is shown to be selectively recognized by normal mammary ducts and alveoli for attachment and growth when compared to the types of collagen derived from stroma (types I or III) or cartilage (type II). Cell attachment and growth on type I collagen is inhibited by the proline analogue, cis-hydroxyproline, which blocks normal collagen production. These effects of cis-hydroxyproline are not apparent when a basement membrane collagen substratum is provided. Unlike normal mammary epithelium, mammary fibroblasts show little preference for the collagen to which they will attach. A requirement of type IV collagen synthesis for normal mammary epithelial cell attachment and growth on stromal collagen in vitro may have significance in vivo where a basement membrane scaffold may be necessary for normal mammary morphogenesis and growth.     

Laminin, fibronectin and type IV collagen in BM-like material from cultured arterial smooth muscle cells

1. The intra- and extracellular distribution of fibronectin and laminin was studied by immunofluorescence in cultures of rabbit and human arterial smooth muscle cells. 2. Basement membrane (BM)-like material was isolated from the cell layer of arterial smooth muscle cells cultures and analysed by sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) and immunoblotting. The major 220-240 kD component of arterial BM-like material was identified as fibronectin. Also a 200 kD fibronectin band was observed. 3. The 200 kD subunit of laminin was contained in isolated BM-like material, but no slower migrating laminin chains were detected. 4. Collagens were prepared from pepsinized BM-like material. The band pattern as resolved by SDS-PAGE and silver staining suggested that type IV collagen is the major collagen of arterial BM-like material.     

Modulation of types I and III procollagen synthesis at various stages of arterial smooth muscle cell growth in vitro

Collagen synthesis was monitored in cultures of rabbit arterial smooth muscle cells (SMC). Both the rate of collagen synthesis per cell and collagen synthesis as a percent of total protein synthesis were measured at specific intervals from 1 to 14 days after inoculation of smooth muscle cells. The proportions of types I and III collagen present in the conditioned incubation medium and in the cell layer were also examined. After inoculation the cells displayed population expansion typical of SMC in which growth slowed but did not cease after the cells attained confluence. Collagen synthesis rates, expressed as [14C]hydroxyproline per cell, were eight-fold higher in preconfluent cells. In these cultures collagen accounted for more than 20% of the newly synthesized, 14C-labeled protein present as trichloroacetic acid (TCA)-insoluble material in 24 h culture media. In post-confluent cultures, this percentage was reduced to about 7% of the total protein synthesized. Synthesis rates of both collagen and non-collagen protein decreased with increasing time after inoculation. However, the rate of decline of collagen synthesis was three times greater than that seen for non-collagen protein. Early cultures synthesized relatively more type I than type III procollagen. The type I to type III ratio was highest at day 3 and declined after that time to day 14. While the synthesis of both types decreased with increasing age, type I declined at a greater rate resulting in a predominance of type III procollagen secretion by older cultures. We conclude that protein synthesis in general and collagen synthesis in particular are quantitatively and qualitatively dependent upon the growth stage of SMC in vitro.     

Cell density and proliferation modulate collagen synthesis and procollagen mRNA levels in arterial smooth muscle cells.

Collagen synthesis and procollagen mRNA levels were determined and compared in (1) sparse, rapidly proliferating smooth muscle cells (SMC); (2) postconfluent, density-arrested SMC; and (3) sparse, nonproliferating (mitogen-deprived) rabbit arterial SMC. Collagen synthesis per SMC was decreased by 70% in postconfluent versus proliferating cells. However, relative collagen synthesis, expressed as the percentage of total protein synthesis, increased from 3.7% in sparse cultures to approximately 7% in postconfluent cultures. Slot blot analyses demonstrated that the relative steady state alpha 1(I) and alpha 1(III) procollagen mRNA levels were also increased in postconfluent cultures when compared to sparse cultures. As with collagen synthesis per cell, the mRNA levels per cell for types I and III procollagen in postconfluent cells, determined by densitometry of blots, were likewise approximately half that found in sparse, proliferating cells. In a separate study to determine if cell-cell contact was necessary for eliciting these changes in collagen synthesis, we determined collagen synthesis in mitogen-deprived and proliferating SMC cultures at low density. Mitogen-deprived cultures synthesized only 10% the amount of collagen produced (per cell) by proliferating cultures in 10% fetal bovine serum. Relative collagen synthesis in proliferating and nonproliferating cultures was 5.0 and 8.3%, respectively. These results demonstrate elevated collagen synthesis, per cell, by proliferating cultures compared with nonproliferating cultures, regardless of whether cells were rendered quiescent by density arrest or by mitogen deprivation. Results also suggest a pretranslational mechanism for the regulation of collagen synthesis in rabbit aortic smooth muscle cells.     

Smooth muscle trans-membrane sarcoglycan complex in partial bladder outlet obstruction

The urinary bladder experiences both distension and contraction as a part of the normal filling and emptying cycle. To empty properly, tension generated intracellularly in a smooth muscle cell must be smoothly and efficiently transferred across its sarcolemma to the basement membrane, which mediates its binding to both the extracellular matrix and to other cells. As a consequence of urethral obstruction, the bladder cannot generate appropriate force to contract the organ, thereby leading to inefficient emptying and associated sequelae. In this study, an animal model of urethral obstruction was utilized to study the membrane-associated structures that transfer tension across the sarcolemma of bladder smooth muscle cells. Immunohistochemical localization of key components of the smooth muscle tension transfer apparatus (TTA) was performed utilizing specific antibodies against:(1) the α-chains of type IV collagen, a basement membrane component, and (2) β-sarcoglycan, an integral membrane protein that is a participant in the physical linkage between the cytoskeleton and the basement membrane. We demonstrate, in obstructed animals, that there is a pronounced disruption of the TTA with a physical displacement of these two components that can be demonstrated at the level of the light microscope using scanning confocal microscopy. Electron microscopy further demonstrates significant increases in the size of the junctional plaques between smooth muscle cells.