Bernadette Lyons-Giordano

Expression of plasminogen activator inhibitor type 2 in normal and psoriatic epidermis.

The plasminogen activator (PA) proteolytic cascade has been implicated in the regulation of cell activities, including proliferation and differentiation, both of which occur continuously in normal human epidermis and are aberrant in psoriatic epidermis. To elucidate further the mechanisms by which PA is regulated in epidermis, we evaluated the levels of PA inhibitors type 1 (PAI-1) and type 2 (PAI-2) in normal and psoriatic epidermis. PAI-2, but not PAI-1, was detectable by mRNA, antigen, and activity assays, indicating that PAI-2 is the predominant epidermal PA inhibitor. In situ hybridization revealed that PAI-2 mRNA occurred throughout normal epidermis, although the signal was most intense in the granular layers. Similarly, PAI-2 antigen was most prominent in the granular layers; its distribution in these differential layers was along the cell periphery. Diffuse, fainter staining for PAI-2 was also detected in the basal cells and in some spinous layers of normal epidermis. Extracts of normal epidermis contained PA inhibitory activity identified as PAI-2 by immunoprecipitation with specific antibody. In psoriatic epidermis, PAI-2 mRNA and antigen were most prominent in the more superficial layers beneath the cornified cells. As with normal epidermis, PAI-2 assumed a pericellular distribution in the psoriatic cells. These data demonstrate that PAI-2 is constitutively expressed in vivo by keratinocytes in human epidermis and indicate that this protein is the predominant inhibitor of PA activity in normal and psoriatic human epidermis.

EXPRESSION OF PLASMINOGEN ACTIVATORS IN BASAL CELL CARCINOMA

The plasminogen activators, tissue type and urokinase type (tPA and uPA, respectively), have been identified in various malignancies and have been implicated in both local growth and metastatic spread. To characterize plasminogen activator expression more fully in human basal cell carcinoma, the localization of uPA and tPA mRNAs was evaluated byin situ hybridization. Nodular basal cell carcinomas demonstrated uPA expression in most cases, whereas the non‐nodular subtypes were negative. Message for uPA was identified within tumour islands (11/12 cases), scattered fibroblast‐like stromal cells (6/12 cases), and the basal layer of the overlying epidermis (10/12 cases). In addition, signal for uPA was elevated and pronounced in areas where the epidermis merged into invasive basal cell carcinoma in the superficial papillary dermis in some cases. Message for uPA was often associated with ulceration or erosion of the overlying epithelium. Expression of tPA was noted in the epidermis (3/12 cases) and in tumour cells (4/12 cases), but tended to be focal and sparse. These results suggest that complex interactions involving uPA expression occur between the tumour, the stroma, and the overlying epidermis. Both the stroma and the epidermis may contribute to local spread of the tumour through production of uPA and consequent plasmin‐mediated activation of collagenases and metalloproteinases.

The effect of heparin on fibronectin and thrombospondin synthesis by human smooth muscle cells

Heparin causes increased synthesis of fibronectin and thrombospondin by human vascular smooth muscle cells as assessed by immunoprecipitation and ELISA techniques. More fibronectin and thrombospondin were immunoprecipitated from the medium of cells treated with 180 micrograms/ml heparin than from that of control cells. Heparin did not effect levels of fibronectin and thrombospondin immunoprecipitated from the cell-matrix fractions. By ELISA, heparin was found to cause a 1.7 fold increase in medium fibronectin levels/cell and a 10 fold increase in medium thrombospondin levels/cell. Concomitantly, smooth muscle cells treated with 180 g/ml heparin for 48 h exhibited 55% decrease in proliferation relative to controls.

The effects of interleukin-1 on the expression of thrombospondin and fibronectin by rabbit articular chondrocytes

Studies to evaluate the effects of recombinant interleukin-1 beta (IL-1) on the expression of matrix proteins by rabbit articular chondrocytes were conducted. Chondrocytes expressed high levels of message for thrombospondin (Tsp) and fibronectin (Fn). RNA slot-blot analysis demonstrated that treatment of the cultures with IL-1 (100 ng/ml) for 24 h caused a 70% suppression of their steady-state Tsp mRNA levels whereas those of Fn were not affected. Steady-state mRNA levels for the intracellular protein, actin, were not modulated by treatment with IL-1. The suppression of Tsp mRNA levels by IL-1 (100 ng/ml) was maximal by 4 h and was concentration dependent; half-maximal suppression was estimated to require 0.12 ng/ml IL-1. Cycloheximide treatment enhanced Tsp mRNA levels, but did not modulate IL-1 suppression of Tsp mRNA. Using pulse-labeling and immunoprecipitation techniques, we found that IL-1 suppression of Tsp mRNA levels was reflected in a coordinate inhibition of Tsp protein synthesis. Chondrocyte synthesis of Fn was not affected by IL-1. These data suggest that IL-1 specifically regulates chondrocyte expression of Tsp at least in part by decreasing the amount of Tsp mRNA available for translation.

The effect of heparin on fibronectin and thrombospondin synthesis and mRNA levels in cultured human endothelial cells.

Studies to eludicate the effect of heparin on the synthesis of extracellular matrix components by cultured human umbilical vein endothelial cells (EC) were conducted. Using pulse-labeling and ELISA techniques, we found that EC grown in the presence of heparin (90 micrograms/ml) and endothelial cell growth factor (ECGF) synthesized 50% less fibronectin (FN) than did ECGF-treated control cultures. No change in the synthesis of thrombospondin (TSP) was induced by heparin. The effect of heparin on EC FN synthesis was independent of whether the cells were cultivated on plastic or gelatin substrates. However, ECGF modulates the effect of heparin on EC synthesis of FN. RNA slot-blot analysis demonstrated that heparin treatment specifically decreased the steady-state mRNA levels for both FN and TSP in the cells. Steady-state levels of mRNA for two intracellular proteins, actin and tubulin, were unchanged. These data suggest that heparin decreases EC expression of FN at least in part by decreasing the amount of FN mRNA available for translation. The failure of heparin to inhibit TSP expression, although it reduces TSP mRNA levels, points to the possibility that the rate of EC synthesis of TSP is translationally or post-translationally regulated.

Heparin increases mRNA levels of thrombospondin but not fibronectin in human vascular smooth muscle cells

The effects of heparin (180 micrograms/ml) on steady state mRNA levels for fibronectin, thrombospondin, actin and collagen types I and III were investigated in human umbilical artery smooth muscle cells. Heparin caused a 120% increase in thrombospondin mRNA levels and a 60% and 180% increase in the mRNA levels of procollagen chains alpha 2(I) and alpha 1(III), respectively. No change in fibronectin or actin mRNA levels resulted from heparin treatment. We reported earlier (Biochem. Biophys. Res. Comm. 148:1264, 1987) that heparin increases smooth muscle cell synthesis of both fibronectin and thrombospondin. These data show that heparin coordinately regulates thrombospondin mRNA and protein levels. The heparin induced increase in fibronectin biosynthesis apparently reflects control at the translational or post-translational level.

Modulation of Fibronectin and Thrombospondin Synthesis and mRNA Levels by Heparin in Human Endothelial and Smooth Muscle Cell Cultures

Since the initial observations that mast cells containing heparin accumulate at tumor sites prior to neovascularization,1 numerous studies have underscored the potential importance of heparin in the regulation of new vessel formation. In vitro heparin potentiates human umbilical vein endothelial cell (EC)* chemotactic and growth responses to endothelial cell growth factor (ECGF), a member of the fibroblast growth factor (FGF) family of mitogens.2,3 The growth and migration stimulatory activity of heparin is neutralized by protamine, an arginine-rich basic protein which is known to bind avidly to heparin.2,4 In vivo local administration of protamine inhibits neovascularization induced by inflammatory agents or by immune reactions. Coadministration of heparin reverses the effect of protamine.5

Heparin Coordinately Suppresses Endothelial Cell Plasminogen Activator Inhibitor-1, Fibronectin and Thrombospondin mRNA Levels

The studies described herein were undertaken to define the effects of heparin, an angiogenic factor, on endothelial cell (EC) expression of extracellular matrix components. In vitro heparin is known to enhance EC chemotactic and proliferative responses to endothelial cell growth factor (ECGF), a member of the fibroblast growth factor family of mitogens (1, 2). While numerous studies have demonstrated the importance of matrix composition in the regulation of EC attachment (3), migration (4), proliferation (5), and multicellular organization (6), little is known about the effects of angiogenic factors on EC biosynthesis of matrix molecules. In culture, EC synthesize a matrix which contains proteoglycans (7), collagens (8) and other glycoproteins including fibronectin (FN) (9), laminin (10), thrombospondin (11), and plasminogen activator inhibitor-1 (PAI-1) (12). In addition, EC synthesize fibroblast growth factor which they sequester in their matrix (13). In this report, we present data which demonstrate that heparin, in the presence of ECGF, coordinately suppresses EC steady-state mRNA levels for FN, TSP and PAI-1. The suppression of these messages correlates with the potentiation of EC proliferative response to ECGF by heparin. These data suggest a potential role for heparin in the remodeling of extracellular matrix concomitant with neovascularization and further highlight the potential role of matrix components in the autocrine regulation of cell growth.