Increased serum concentrations of type IV collagen and laminin associated with granulosa cell tumour of the ovary.

A 65 year old woman presented with an ovarian mass. Ultrasonography and computed tomography findings, and an increased serum oestrogen concentration were suggestive of an oestrogen producing ovarian tumour. The tumour was removed surgically and weighed 460 g. The pathological diagnosis was adult-type granulosa cell tumour. Strong immunohistochemical staining specific for type IV collagen and laminin was observed, and these components were localised to the pericellular region of the granulosa cells. The serum concentrations of these basement membrane components (measured by radioimmunoassay) were very high before surgery, but decreased rapidly thereafter. Serial measurement of type IV collagen and laminin, in conjunction with other tumour markers and oestrogen concentrations, might be helpful in evaluating prognosis of ovarian granulosa cell tumours, in detecting metastatic or recurrent lesions and in monitoring response to treatment.     

Loss of type IV collagen alpha 5 and alpha 6 chains in human invasive prostate carcinomas.

Type IV collagen, a major component of basement membranes, is organized in a network responsible for the mechanical resistance of the basement membranes. It also plays a key role in epithelial cell adhesion to basement membranes. This study was designed to investigate the distribution of type IV collagen alpha-chains in normal, preneoplastic, and malignant prostate basement membranes. For this purpose, immunohistochemistry using specific antibodies raised against the different alpha-chains of type IV collagen was performed in eight normal samples, six prostatic intraepithelial neoplasia, and 20 malignant lesions of the prostate. Our results demonstrate the presence of the "novel" alpha 5 (IV) and alpha 6 (IV) chains along with the "classical" alpha 1 (IV)/alpha 2 (IV) chains in the basement membrane of the normal prostate gland. The alpha 3 (IV) chain was never detected in any prostate specimen. Prostatic intraepithelial neoplasia showed a similar immunostaining pattern to that found in normal glands. In cancer gland basement membranes, we demonstrate for the first time a specific loss of the alpha 5 (IV) and alpha 6 (IV) chains, whereas the classical alpha 1 (IV) and alpha 2 (IV) chains were consistently exhibited. Additionally, type VII collagen colocalized with alpha 5 (IV) collagen chain, and these two proteins, which were always observed in normal and prostatic intraepithelial neoplasia gland basement membranes, were lost in invasive carcinoma basement membranes. This observation raises questions about the possible association or cooperation between alpha 5 (IV)/alpha 6 (IV) chains and anchoring fibrils in prostate glands basement membrane.     

Differential distribution of basement membrane type IV collagen alpha1(IV), alpha2(IV), alpha5(IV) and alpha6(IV) chains in colorectal epithelial tumors

In this study, we examined the relationship between the histopathological grade and immunohistochemical localization of six genetically distinct type IV collagen alpha chains, the major component of basement membrane (BM), in normal and neoplastic colorectal tissues. In the normal colorectal mucosa, alpha1/alpha2(IV) and alpha5/alpha6(IV) chains were stained in all epithelial BM. However, alpha3/alpha4(IV) chains were restrictively immunostained in the BM of the apical surface epithelium. Similar immunostaining profiles for alpha1/alpha2(IV) and alpha5/alpha6(IV) chains were observed in tubular adenomas with mild/moderate atypia. However, in intramucosal carcinomas, both alpha1/alpha2(IV) chains were linearly stained in the BM of cancer cell nests, while the assembly of alpha5/alpha6(IV) chains into the BM was inhibited in a discontinuous or negatively stained pattern. The normal colorectal mucosa forms a second network of BM composed of alpha5/alpha6(IV), partly alpha3/alpha4(IV) chains, in addition to the classic network of alpha1/alpha2(IV) chains. The differential immunohistochemical localization of the type IV collagen alpha5/alpha6 chains could be one diagnostic marker for the invasiveness of colorectal cancer.     

Simultaneous expression of 70 kilodalton type IV collagenase and type IV collagen alpha 1 (IV) chain genes by cells of early human placenta and gestational endometrium.

BACKGROUND: In this study we used in situ hybridization to investigate the expression of the genes 70 kilodalton (kd) collagenase and the alpha 1(IV) collagen chain of type IV collagen in cells of early human placenta and gestational endometrium. EXPERIMENTAL DESIGN: The aim was to study the spatial distribution of these gene expressions within a developing tissue which possesses physiologic invasive potential. The results obtained for the 70 kd type IV collagenase mRNA expression were also compared with the immunohistochemical distribution of the corresponding antigen. RESULTS: Expression of mRNAs for these proteins was found in cells of trophoblastic columns, stromal cells of villi and in cells of decidua and endometrial stroma. The only differences between the expressions was the lower level of signals for 70 kd type IV collagenase in fibroblastic stromal cells and endothelial cells of villi and in the pericytic cells of spiral arteries. Otherwise the results for both types of mRNA were comparable. We also studied the immunohistochemical distribution of the 70 kd type IV collagenase using specific monoclonal antibodies against the enzyme. Immunohistochemistry supported well the findings obtained by in situ hybridization. CONCLUSIONS: The results indicate that the genes for the 70 kd type IV collagenase and for the alpha 1(IV) collagen chain are simultaneously active in cells of placenta and gestational endometrium and the same cells which produce type IV collagen also can produce the cleaving enzyme, the 70 kd type IV collagenase. The results also show that the cytotrophoblastic cells, which during early pregnancy invade the extracellular matrix and spiral arteries of uterine wall contain significant amount of mRNA for the 70 kd type IV collagenase. This finding supports the concept that the 70 kd type IV collagenase would be important for invasion, and in the case of this study, also for the physiologic invasion of placental cytotrophoblasts.     

Widespread histologic distribution of the alpha 2 beta 1 integrin cell-surface collagen receptor.

The alpha 2 beta 1 integrin (platelet membrane glycoprotein Ia-IIa, VLA-2, ECMR-II) functions as a cell surface receptor for collagen. The authors have determined the histologic distribution of the alpha 2 beta 1 receptor in normal tissues by immunohistochemical technique. The studies revealed that the alpha 2 beta 1 receptor was expressed on fibroblasts, endothelial cells, and epithelial cells from multiple sites including skin, tonsil, breast, sweat gland, gastrointestinal tract, lung, bladder, cervix, and prostate. Follicular dendritic cells of the lymph node, tonsil, and spleen and dendritic cells of the thymus also expressed the alpha 2 beta 1 receptor. The receptor also was present on Schwann cells of ganglia and on neuroglia. Greatly enhanced expression of the receptor in regions of proliferating epithelium suggests that enhanced expression of alpha 2 beta 1 is associated with orderly, regulated cell proliferation. The circumferential staining pattern of the alpha 2 beta 1 integrin within many epithelia is virtually identical to that observed for other adhesive receptors, such as the cadherins, which have been implicated in cell-cell adhesion.     

Vascular smooth muscle cells orchestrate the assembly of type I collagen via alpha2beta1 integrin,RhoA, and fibronectin polymerization

Assembly of collagen into fibrils is widely studied as a spontaneous and entropy-driven process. To determine whether vascular smooth muscle cells (SMCs) impact the formation of collagen fibrils, we microscopically tracked the conversion of soluble to insoluble collagen in human SMC cultures, using fluorescent type I collagen at concentrations less than that which supported self-assembly. Collagen microaggregates were found to form on the cell surface, initially as punctate collections and then as an increasingly intricate network of fibrils. These fibrils displayed 67-nm periodicity and were found in membrane-delimited cellular invaginations. Fibril assembly was inhibited by an anti-alpha2beta1 integrin antibody and accelerated by an alpha2beta1 integrin antibody that stimulates a high-affinity binding state. Newly assembled collagen fibrils were also found to co-localize with newly assembled fibronectin fibrils. Moreover, inhibition of fibronectin assembly with an anti-alpha5beta1 integrin antibody completely inhibited collagen assembly. Collagen fibril formation was also linked to the cytoskeleton. Fibrils formed on the stretched tails of SMCs, ran parallel to actin microfilament bundles, and formed poorly on SMCs transduced with retrovirus containing cDNA for dominant-negative RhoA and robustly on SMCs expressing constitutively active RhoA. Lysophosphatidic acid, which activates RhoA and stimulates fibronectin assembly, stimulated collagen fibril formation, establishing for the first time that collagen polymerization can be regulated by soluble agonists of cell function. Thus, collagen fibril formation is under close cellular control and is dynamically integrated with fibronectin assembly, opening new possibilities for modifying collagen deposition.     

Human type 1 and type 2 conventional dendritic cells express indoleamine 2,3-dioxygenase 1 with functional effects on T cell priming

Dendritic cells (DCs) are key regulators of the immune system that shape T cell responses. Regulation of T cell induction by DCs may occur via the intracellular enzyme indoleamine 2,3-dioxygenase 1 (IDO), which catalyzes conversion of the essential amino acid tryptophan into kynurenine. Here, we examined the role of IDO in human peripheral blood plasmacytoid DCs (pDCs), and type 1 and type 2 conventional DCs (cDC1s and cDC2s). Our data demonstrate that under homeostatic conditions, IDO is selectively expressed by cDC1s. IFN-γ or TLR ligation further increases IDO expression in cDC1s and induces modest expression of the enzyme in cDC2s, but not pDCs. IDO expressed by conventional DCs is functionally active as measured by kynurenine production. Furthermore, IDO activity in TLR-stimulated cDC1s and cDC2s inhibits T cell proliferation in settings were DC-T cell cell-cell contact does not play a role. Selective inhibition of IDO1 with epacadostat, an inhibitor currently tested in clinical trials, rescued T cell proliferation without affecting DC maturation status or their ability to cross-present soluble antigen. Our findings provide new insights into the functional specialization of human blood DC subsets and suggest a possible synergistic enhancement of therapeutic efficacy by combining DC-based cancer vaccines with IDO inhibition.     

Immunolocalization and distribution patterns of type IV collagen alpha chains in oral mucosal melanoma

The basement membrane (BM) is mainly composed of type IV collagen, which is composed of triple combinations of six distinct alpha (α) chains in a tissue-specific manner. The six collagen chain-specific antibodies (α1–α6) were used to examine the BMs of the oral epithelium (OE) and tumor clusters in oral mucosal melanoma (OMM). Eight OMM cases were examined. Results showed that the α1 and α2 chains were constantly detected at the BM of the normal OE as well as at the OE with atypical melanocytic proliferation and in invasive melanoma with nodular nests. The α1 and α2 chains were intermittently detected in in situ OMM, early invasive OMM and advanced invasive OMM with sheet-like nests. Gradual loss of α5 and α6 from the OE with atypical melanocyte through in situ OMM and early invasive OMM was observed. These findings suggest that changes in the immunolocalization and distribution patterns of type IV collagen α chains are associated with the progression of OMM. The distribution pattern of type IV collagen α chain varies depending on the architecture of the nest.     

Human melanocytes and melanoma cells constitutively express the Bcl-2 proto-oncogene in situ and in cell culture.

The Bcl-2 proto-oncogene regulates cell survival by antagonizing events that lead to apoptotic cell death and has been reported to be expressed in situ in lymphoid tissues, glandular epithelium, neurons, and basal epidermal cells. When we performed immunostaining on cryostat sections of normal skin, anti-Bcl-2 reactivity was confined to scattered dendritic cells in the basal epidermal layer. Double-staining experiments showed that the Bcl-2+ cells were positive for vimentin but negative for cytokeratins, CD1a, and CD45 antigens, excluding keratinocytes and Langerhans cells as possible candidates for constitutive Bcl-2 expression. Bcl-2+ epidermal cells also reacted with the monoclonal anti-melanocyte antibody NKI/beteb, and were absent from lesional skin in vitiligo, confirming that they represented epidermal melanocytes. Western blot analysis of cultured melanocytes and melanoma cell lines revealed a 26-kd protein specifically reacting with the anti-Bcl-2 monoclonal antibody. Immunostaining of pigmented lesions revealed strong expression of Bcl-2 by five of five nevocellular nevi and seven of seven melanomas. Our observations demonstrate that, within normal human epidermis, melanocytes are the only cells that express Bcl-2 constitutively and that Bcl-2 is expressed in benign and malignant pigmented tumors of the skin in situ.     

Differential tissular expression and localization of type IV collagen alpha1(IV), alpha2(IV), alpha5(IV), and alpha6(IV) chains and their mRNA in normal breast and in benign and malignant breast tumors

Type IV collagen, the major component of basement membrane (BM), is composed of six genetically distinct alpha chains. We investigated the cellular regulation and origin of these alpha(IV) chains in normal and neoplastic breast tissues by immunohistochemistry by using alpha(IV) chain-specific antibodies and by in situ hybridization. In normal breast, alpha1(IV) and alpha2(IV) chains were stained in all BM, whereas alpha5(IV) and alpha6(IV) chains were restrictively localized in a linear pattern in the BM of the mammary gland. Similar immunostaining profiles were observed in benign breast tumors and in the intraductal components of invasive ductal carcinoma. However, in invasive ductal carcinoma, alpha1(IV) and alpha2(1V) chains were discontinuously or negatively stained in the cancer cell nests, and the assembly of alpha5(IV) and alpha6(IV) chains into the BM was completely inhibited. Coexpression of alpha5(IV) and alpha6(IV) chains was related to the localization of alpha-smooth muscle actin (alpha-SMA)-positive myoepithelial cells. By in situ hybridization, in fibroadenoma and invasive ductal carcinoma, the signals for alpha1(IV) and alpha2(IV) mRNA were abundant in stromal cells. However, the signals for alpha5(IV) and alpha6(IV) mRNA were not seen in any of these cells. In contrast, in intraductal papilloma, coexpression of alpha1 (IV)/alpha2(IV) mRNA and alpha5(IV)/alpha6(IV) mRNA was identified in epithelial cells. The results indicate that the mammary gland forms a second network of BM composed of alpha5(IV)/alpha6(IV) chains, in addition to the classic network of alpha1(IV)/alpha2(IV) chains. The expression of type IV collagen alpha chains seems to be differentially regulated by the epithelial-myoepithelial interaction and to be associated with the invasive potential of breast cancer.     

Nervous system involvement in type IV glycogenosis.

A 30-month-old girl exhibited the 19th known case of type IV glycogenosis. Extensive involvement of the nervous system was found at autopsy. This represents only the second patient in whom the fine structure of the CNS and skeletal muscle has been described. We have also identified the abnormal polysaccharide in peripheral nerve, a finding that, to our knowledge, has not been reported previously. Our review of the literature indicates that approximately 50% of these patients exhibit signs or symptoms referable to the neuromuscular system. Most clinical and pathologic studies have focused on the severe liver involvement; insufficient attention has been directed toward the nervous system. This emphasizes the need for more detailed neurologic and neuropathologic examinations of children with type IV glycogenosis.     

Biosynthesis of type VI collagen by glioblastoma cells and possible function in cell invasion of three-dimensional matrices

The biosynthesis of type VI collagen was studied in human glioblastoma cell line, U-87 MG. The effects of ascorbic acid on type VI collagen synthesis and secretion were investigated. After ascorbic acid treatment, type VI collagen in cell layers increased from 4.48% in control to 6.63% in the ascorbic acid treated cultures, an increase of 48%. The effect of ascorbic acid on type VI collagen synthesized by glioblastoma cells was lower than that reported for osteosarcoma cells (Engvall et al., 1986). The reason for these differences is still under investigation. The function of type VI collagen in glioblastoma cells is still unknown. We utilized the collagen gel system to elucidate the possible roles of type VI collagen in glioblastoma cells in vitro. Glioblastoma cells in collagen gels showed a stellate shape with long, branched processes in all directions. The strong positive reactivity of type VI collagen detected on cell bodies and cell processes by anti-type VI collagen antibody indicated that this specific collagen was associated with cell surfaces and processes, without releasing or diffusing into the gels. Type VI collagen was directly involved in the cell process extension. When living cells were treated with anti-type VI collagen antibody, a variation of cell morphology was observed. Instead of a stellate shape with processes, cells formed clusters without or with very short processes. These data suggest that type VI collagen, synthesized and secreted by glioblastoma cells, may play a role in tumor cell adhesion and spreading, and enhance cell process extension, penetration, and invasion into collagen gels.     

Mature osteoblasts in human non-union fractures express collagen type III.

AIMS: High levels of collagen type III are biochemically detectable in biopsies of non-uniting fractures, and in the serum of patients suffering from this condition. The aim of this study was to determine whether the expression of collagen type III was limited to fibrous tissue in non-unions, or whether some was present in bone. METHODS: Biopsies from normally healing human fractures and non-unions were examined using in situ hybridisation and immunohistochemistry. RESULTS: The mesenchymal cell population, which includes fibroblast and osteoblast precursors, expressed mRNA for collagen type III. However, mature osteoblasts on the surface of woven bone varied profoundly between normally healing fractures (in which they were negative or occasionally weakly positive) and non-unions (in which they were strongly positive). Areas of woven bone that had osteoblasts positive for collagen type III mRNA also immunostained positively for the protein. CONCLUSIONS: This study shows that non-union fracture callus osteoblasts on the surfaces of woven bone exhibit an unusual phenotype: they express collagen type III, a molecule characteristic of an earlier stage of osteoblast differentiation, which is not expressed by osteoblasts on woven bone surfaces of bone that develops normally. This finding may be useful in developing an early clinical test for impending non-union.     

Crescentic glomerulonephritis and subepidermal blisters with autoantibodies to alpha5 and alpha6 chains of type IV collagen

We describe a novel autoimmune disease characterized by severe subepidermal bullous eruption and crescentic glomerulonephritis with autoantibodies directed against the noncollagenous domain of the alpha5 and alpha6 chains of type IV collagen. Biopsy of perilesional skin revealed a subepidermal blister with marked polymorphonuclear infiltrate with linear deposits of IgA and C3. Light microscopy of a kidney biopsy specimen revealed a crescentic glomerulonephritis, and immunofluorescence microscopy showed linear basement membrane staining for IgA (3+), C3 (1+), and IgG (1+). No electron-dense deposits were observed by transmission electron microscopy. The patient's autoantibodies reacted with normal human skin and kidney: IgA (3+) and IgG (1+) antibodies stained the basement membrane zones of skin, renal glomerulus, and some tubules. The identity of the target antigen was determined by immunochemical analyses of candidate antigens using the patient's autoantibodies. The patient's IgA and IgG autoantibodies reacted with a 185- to 190-kDa antigen from a human dermal extract that was distinguished from the other dermal or epidermal antigens, including the 145- to 290-kDa (type VII collagen) epidermolysis bullosa acquisita antigen, the 165- to 200-kDa alpha3 laminin mucous membrane cicatricial pemphigoid antigen, and the 230-kDa and the 180-kDa bullous pemphigoid antigens. Patient's IgA and IgG autoantibodies further reacted with the alpha5(IV) and weakly with the alpha6(IV) chains of type IV collagen by Western blot and ELISA. This report expands the repertoire of bullous skin disorders and provides an explanation for the association of anti-type IV collagen autoantibodies and glomerulonephritis with subepidermal blisters.     

Laminin production by murine melanoma cells: possible involvement in cell motility

Three lines of B16 melanoma cells (B16-F1, B16-F10 and B16-BL6) were examined for motility in the micropore filter assay and for synthesis in culture of the basal lamina glycoprotein laminin. All three lines synthesized laminin as judged by the incorporation of [³⁵S]methionine into immunoreactive laminin and secreted (or shed) laminin into the culture medium as indicated by biosynthetic labeling studies and enzyme-linked immunosorbent assays. Immunoreactive laminin was also seen on the surface of the cells as indicated by immunofluorescence staining and by complement-mediated killing. Analysis of [³⁵S]methionine-labeled laminin immunoprecipitates by sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) both with and without reduction of intersubunit disulfide bonds revealed that all three cell lines produced a similar array of laminin forms, and that the M _r=950kD laminin molecule (but not the uncombined subunits) was secreted into the culture medium. Laminin biosynthesis appeared to be limited by the availability of the M _r=400kD A subunit as shown by the intracellular accumulation of excess B subunit in the form of uncombined B subunit (M _r =200kD) and as a disulfide-linked B dimer (M _r=400 kD). The motility of all three cell lines was stimulated four- to five-fold by the addition of either exogenous laminin from the EHS sarcoma or culture medium from the B16 cells containing the secreted laminin. The stimulated motility was inhibited by antilaminin serum. These observations suggest that the laminin synthesized by the B16 melanoma cells themselves may facilitate their motility.     

T lymphocytes in giant cell arteritic lesions are polyclonal cells expressing alpha beta type antigen receptors and VLA-1 integrin receptors.

Giant cell arteritis (GCA) is a common disease in the elderly. It is characterized by focal inflammatory lesions dominated by T lymphocytes and macrophages. The etiology of GCA is, however, still unknown. The aim of the present study was to determine whether lesional T cells represent clonal proliferations, and to characterize adhesion receptors that could be important for recruitment of T cells and antigen receptors involved in their activation. Temporal artery biopsies were obtained from 13 patients presenting with clinical signs of GCA. Immunohistochemistry was used to characterize cell surface receptors on CD3+ T cells in situ in the lesions of eight patients with biopsy-verified GCA. The overwhelming majority of T cells in GCA lesions expressed the TCR alpha beta receptors. In sections from three of eight patients, a small proportion of cells expressing TCR gamma delta was also seen. Almost all T cells expressed the integrin receptors, LFA-1 and VLA-1, as determined by double-staining. To characterize the clonal composition of the lesional T cell population, cells were isolated by collagenase digestion of two lesions and T cells cloned by limiting dilution in the presence of mitogenic antibodies, IL-2 and autologous feeder cells. Rearrangements of the T cell receptor (TCR) genes of the clones were analysed by Southern hybridization using probes for TCR gamma and beta genes. T cell clones established from GCA lesions exhibited heterogeneous rearrangement patterns, indicating a polyclonal origin of the cells. We conclude that GCA lesions contain T lymphocytes that are of polyclonal origin and express integrin-type adhesion receptors. This supports the hypothesis that GCA involves an inflammatory response during which polyclonal T cells adhere to arterial tissue components and accumulate in the developing lesions.     

Origin of basement membrane type IV collagen in xenografted human epithelial tumor cell lines.

The origin of basement membrane (BM), deposited in epithelial neoplasms, was studied in xenografts of human tumor cell lines in nude mice and rats. Cell lines were chosen that in vitro do (WISH, KB) or do not (5583-E; HT-29) produce BM components, more specifically, type IV collagen. Basement membrane deposition was studied by immunohistochemistry, using species cross-reactive polyclonal anti-human type IV collagen antiserum, mouse- and rat-specific polyclonal anti-mouse type IV collagen antiserum, human-specific monoclonal anti-type-IV collagen antibody, and by in situ hybridization, using a 32S-labeled species cross-reactive cDNA probe, specific for type IV collagen mRNA. In the xenografts, species-cross-reactive anti-type-IV collagen antiserum demonstrated the presence of irregular and discontinuous BM. In 5583-E and HT-29 xenografts, only murine type IV collagen epitopes were detected. In contrast, in WISH and in KB xenografts, the BM stained human as well as murine type IV collagen epitopes. By in situ hybridization, type IV collagen mRNA was detectable in stromal cells only in 5583-E and HT-29 xenografts, but in both epithelial and stromal cells in WISH and KB xenografts. These results indicate that in this model system epithelial tumor cells and stromal (mesenchymal) cells are involved in the production and deposition of a BM.     

Genes of laminin B1 chain, alpha 1 (IV) chain of type IV collagen, and 72-kd type IV collagenase are mainly expressed by the stromal cells of lung carcinomas.

In this study, we analyzed the expression of messenger (m)RNAs for laminin B1 chain, alpha 1 (IV) chain of type IV collagen, and 72-kd type IV collagenase in 15 primary lung carcinomas and in two metastatic adenocarcinomas to the lung. The results show that the mRNA synthesis for these proteins mainly occurs in the stromal fibroblasts and endothelial cells. In a proportion of tumors, mRNAs for laminin B1 chain and 72-kd type IV collagenase could also be observed in carcinoma cells, but the amount of mRNAs was considerably lower in them than in the stromal cells. There were no convincing signals for the presence of the alpha 1 (IV) chain of type IV collagen mRNA in any of the carcinoma cells. A simultaneous expression or lack of expression of signals for laminin B1 chain and 72-kd type IV collagenase mRNAs could be observed in carcinoma cells of 12 cases, suggesting that the activation of these two genes may be somehow connected. There was no association between the mRNA expression and the differentiation degree or the size of the tumors. The occurrence of the mRNA synthesis for the 72-kd type IV collagenase in stromal fibroblasts and endothelial cells indicates that the stromal cells of tumors have a more pronounced impact on the spread of the neoplastic disease than previously thought. The results further show that in their ability to synthesize these proteins the stromal cells of tumors resemble those of developing embryonic tissues. This resemblance is probably connected with the constant remodeling of extracellular matrix in response to the proliferative activity of carcinoma cells.     

Dynamic expression of alpha 1 beta 1 and alpha 2 beta 1 integrin receptors by human vascular smooth muscle cells. Alpha 2 beta 1 integrin is required for chemotaxis across type I collagen-coated membranes.

Vascular smooth muscle cells (SMCs) in the media of normal arteries express alpha 1 beta 1 integrin with no detectable alpha 2 beta 1 as determined by immunocytochemistry. In contrast, immunoprecipitation of integrins expressed by human SMCs cultured from medial explants shows strong expression of alpha 2 beta 1 and no expression of alpha 1 beta 1. The apparent reciprocal expression of these two collagen and laminin receptors was confirmed by flow cytometric analysis of fluorescent labeled cells. Freshly isolated SMCs had detectable alpha 1, alpha 3, alpha 5, and alpha v subunits with low levels of detectable beta 3 and no detectable alpha 2. Cultured SMCs expressed alpha 2, alpha 3, alpha 5 and alpha v subunits with little or no alpha 1 or beta 3. Neither alpha 4 nor alpha 6 were detectable in freshly isolated or cultured cells. Expression of alpha 2 beta 1 receptors by cultured SMCs appears to be required for the migration of these cells on type I collagen. Migration of cultured SMCs across a type I collagen-coated membrane toward two different chemotactic stimuli, platelet-derived growth factor-BB (1 nmol/L) and insulin-like growth factor-(1 nmol/L), was Mg2+ dependent and inhibited by preincubation of cells with an affinity-purified polyclonal anti-alpha 2 beta 1 antibody or by monoclonal antibodies directed against the individual alpha 2 or beta 1 subunits. Attachment to type 1 collagen membranes was not affected by antibodies under conditions where migration was significantly impeded. The combined data show that SMC expression of alpha 1 beta 1 and alpha 2 beta 1 integrin receptors for collagen and laminin is dynamic and reciprocal and may be important with respect to SMC migration on type I collagen. These findings are potentially important in understanding the pathophysiology of vascular diseases, for example, atherosclerosis and restenosis following balloon angioplasty, where SMC migration is a contributing factor.