SYSTEMIC SCLEROSIS (SSC) FIBROBLASTS REDUCE ANGIOGENESIS IN HEALTHY MICROVASCULAR ENDOTHELIAL CELLS (H-MVEC) BY CLEAVAGE OF UROKINASE RECEPTOR (CD87, U-PAR)

Background: Endothelial damage and defective neoangiogenesis are hallmarks of SSc. We previously showed that in SSc-MVEC the impaired angiogenesis is associated to the cleavage of u-PAR operated by the matrix metallo-protease12 (MMP12) (1). Objectives: To evaluate the effects of SSc fibroblasts on healthy MVEC (H-MVEC) angiogenic potential, particularly on 1) proliferation; 2) chemoinvasion; 3) angiogenesis in vitro; 4) integrity of u-PAR. Methods: The effects of SSc fibroblasts were evaluated incubating H-MVEC with the conditioned medium from 3 healthy (H-MC) and 3 SSc (S-MC) fibroblast lines. In H-MVEC angiogenesis was assayed by Chemoinvasion was measured by the Boyden chamber invasion assay, using Matrigel as invasion matrix. Proliferation was evaluated as increase of cell number. In vitro angiogenesis was performed by the capillary morphogenesis assay. These parameters were studied in basal conditions and under induction by urokinase (u-PA) 250 ng/ml or vascular endothelial growth factor (VEGF) 20 ng/ml. The domains of u-PAR were studied by western blotting. Results: In H-MVEC, H-MC had slight stimulatory effects on proliferation, chemoinvasion and capillary morphogenesis while S-MC caused a significant inhibition of all these phenomenon even when they were induced by u-PA or VEGF, blocking these factors pro-angiogenic effects and showing a pattern similar to basal unstimulated cells . U-PAR analysis revealed that S-MC, but not H-MC, induced the cleavage of u-PAR in H-MVEC, which accounts for a block of u-PAR related interaction required for angiogenesis. Conclusion: These data show that SSc fibroblasts conditionate the H-MVEC microenvironment, secreting a protease that induce u-PAR cleavage, impairing their angiogenic properties and counteracting the response to angiogenic factors like u-PA and VEGF. U-PAR truncation brings H-MVEC in the same condition of S-MVEC. Then, SSc fibroblasts alterations might be considered as an early feature of SSc progression that can impair H-MVEC angiogenesis, contributing to endothelial damage and to the so called desert-like pattern of SSc.