Lipodermatosclerosis refers to skin induration of the lower extremities and is associated with patients preceding venous ulcerations. To better understand the pathogenesis of ulcer formation we investigated the expression of matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in lipodermatosclerosis. By preparing biopsies from healthy skin and liposclerotic lesions, MMP-1, MMP-2, MMP-9, TIMP-1, and TIMP-2 were analyzed by using reverse transcriptase-polymerase chain reaction, western blot, zymography, hydrolysis of [3H]labeled collagens, and immunohistochemistry. Our investigations provide evidence that mRNA and protein expression of MMP-1, MMP-2, and TIMP-1 were significantly increased in lipodermatosclerosis, whereas the total amount of MMP-9 and TIMP-2 mRNA and protein was not altered. Western blot of liposclerotic lesions revealed an inactive proMMP-1-TIMP-1 complex, whereas MMP-2 was prominent as an active 66 kDa band. Increased proteolytic activity of MMP-2 could be proven in lesional in comparison with healthy skin by zymography and [3H] collagen degradation. Increased diffuse staining was found for MMP-1 in the epidermis and dermis in comparison with controls. In lipodermatosclerosis, MMP-2 was predominantly localized in the basal and suprabasal layers of the epidermis, in perivascular regions, and in the reticular part of the dermis. Furthermore, MMP-2 was imbalanced by locally reduced expression of TIMP-2 in the basement membrane zone of lesional skin. Our findings indicate lipodermatosclerosis to be characterized by elevated matrix turnover.

Growth factors produced by a variety of cells act as signalling peptides through specific cell surface receptor pathways. Functions such as cell proliferation, migration and differentiation have been assigned to each of them. Here, we report alterations of platelet-derived growth factor receptor alpha (PDGFR-alpha) and beta (PDGFR-beta) and vascular endothelial growth factor (VEGF) expression patterns in the progressive clinical stages of chronic venous insufficiency (CVI). A total of 30 punch biopsies were taken from patients with CVI, and VEGF and PDGFR were detected by indirect immunofluorescence and immunoperoxidase techniques. PDGFR-alpha and PDGFR-beta expression was strongly increased in endothelial cells of capillaries, pericapillary cells and connective tissue cells in the stroma of the skin of venous eczema and venous leg ulcer patients, and to a smaller extend in the dermis of those with lipodermatosclerosis. VEGF staining showed a similar expression pattern in the progressive CVI stages. However, staining of vessels in particular might simply reflect binding of VEGF, secreted by keratinocytes or fibroblasts, to its receptors. Growth factor and receptor expression in specimens from telangiectases and reticular veins, and from pigmented areas, resembled that of normal skin. We conclude that PDGFR-alpha, PDGFR-beta and VEGF play an important role in mediating inflammation and epithelial hyperproliferation in venous eczema, inducing connective tissue sclerosis in lipodermatosclerosis, and causing the reduced reepithelialization tendency in venous ulcers. We speculate that endothelial proliferation with chronic venous hypertension might be mediated by these growth factors.

Cultured kidney glomerular mesangial cells (MCs) allow the role of extracellular matrix (ECM) and growth factors in glomerular inflammatory disease to be studied. To investigate the potential of MCs to interact with matrix components, the expression of integrin mRNA in cultured MCs was examined by polymerase chain reaction (PCR), by Northern blotting and by immunofluorescence. In addition, the effect of matrix substrates on mRNA expression was assessed by PCR. Northern blots with cDNA probes to integrin alpha-chains revealed that MCs expressed alpha 1, alpha 3 and alpha 5 integrin mRNA. alpha 1 and alpha 3 were the major messages. No alpha 2, alpha 4 or alpha 6 were detectable. RT-PCR revealed that alpha 2 and alpha 6 were also expressed at low levels. The control cells, HT1080, expressed alpha 2, alpha 3, alpha 4, alpha 5 and alpha 6 mRNA, and Rugli expressed alpha 1, alpha 3 and alpha 5, supporting previous studies. Immunocytochemistry confirmed that alpha 1 beta 1, alpha 2 beta 1, and alpha 5 beta 1 integrins were expressed and that they were concentrated into focal adhesions (alpha 1 beta 1 on type I collagen and laminin; alpha 2 beta 1 on type I collagen; alpha 3 beta 1 on type I collagen, laminin and fibronectin; alpha 5 beta 1 on fibronectin). alpha 6 beta 1 was not detected in focal contacts. Attachment, spreading, and formation of talin and integrin containing focal contacts still occurred when endogenous protein synthesis was blocked with 30 micrograms.ml-1 cycloheximide.(ABSTRACT TRUNCATED AT 250 WORDS)

The movement of integrins into focal adhesive structures accompanies cell attachment to extracellular matrix. The kinetics of incorporation of integrins into focal contacts was studied during attachment to matrix of mesangial cells of the kidney glomerulus. On collagen, fibronectin, laminin and vitronectin, the number and intensity of talin-focal contacts increased with time. Talin-containing focal contacts were present in mesangial cells within 2 h of plating and in control cells (HT1080 and Rugli) within 1 h. Integrin alpha-chains colocalized with talin, dependent on the matrix substrate. The attachment, spreading and organization of integrin into focal contacts was not affected when endogenous protein synthesis was suppressed with cycloheximide. In Rugli, alpha 1 beta 1 organized into focal contacts on collagen and laminin, while in HT1080 alpha 2 beta 1 organized on collagen type I, alpha 5 beta 1 on fibronectin, alpha 6 beta 1 on laminin, and alpha 3 beta 1 and alpha 4 beta 1 were diffusely distributed on all substrates. These distributions mirrored the usage and expression patterns previously established for integrins in these cells and was as predicted from the literature. In mesangial cells, however, alpha 3 beta 1 was also organized into prominent focal contact arrays on collagen, fibronectin, EHS and human placental laminins, but not on vitronectin, while alpha 6 beta 1 was not organized. Initial attachment and spreading of mesangial cells was absolutely dependent on divalent cations. Mg2+ and Mn2+ supported attachment on all substrates, while Ca2+ stimulated attachment on laminin (E8), fibronectin and vitronectin. The data suggest that the functional integrins on mesangial cells include alpha 1 beta 1 (on collagen and laminin) alpha 2 beta 1 (on collagen), alpha 5 beta 1 (on fibronectin) and alpha V beta 3 (on vitronectin). However, mesangial cells do not use alpha 6 beta 1 on laminin, and the data support a role for alpha 3 beta 1 as putative receptor for fibronectin, collagen and laminin.