Modulation of the angiogenic phenotype of normal and systemic sclerosis endothelial cells by gain–loss of function of pentraxin 3 and matrix metalloproteinase 12

Abstract

Objective. Studies have shown that in systemic sclerosis (SSc) endothelial cells, overproduction of matrix metalloproteinase 12 (MMP-12) and pentraxin 3 (PTX3) is associated with defective angiogenesis. This study was undertaken to examine whether overexpression of the relevant molecules could inhibit angiogenesis of normal microvascular endothelial cells (MVECs), and whether silencing of these molecules in SSc MVECs could restore the lost angiogenic properties of the cells in vitro and in vivo.Methods. Transient transfection of MVECs with human MMP12 and PTX3 was performed by electroporation. Silencing of MMP12 and PTX3 was obtained by treatment with small interfering RNA, and treatment effects were validated by Western blotting with specific antibodies and a fluorimetric assay. In vitro cell migration and capillary morphogenesis were studied on Matrigel substrates. In vivo angiogenesis was studied using a Matrigel sponge assay in mice.Results Many human diseases that involve alterations of the angiogenic process, including cancer and inflammatory conditions, are characterized by excess angiogenesis (1-4). In contrast, in systemic sclerosis (SSc), the pathologic abnormalities affecting microvessels and the breakdown of their patency leads to chronic tissue hypoxia and to capillary loss, which in turn leads to vital organ failure (5). These features suggest that there is an insufficient angiogenic response occurring in SSc (6), despite the tissue hypoxia that characterizes the disease.

Endothelial progenitor cell–dependent angiogenesis requires localization of the full-length form of uPAR in caveolae

“…Our aim to investigate a possible correlation between caveolar rafts, distribution of full-length and truncated forms of uPAR, and angiogenesis in ECFCs relies on its identification in caveolae 13 and on the recognized role of full-length uPAR in angiogenesis. 10,11,31,32,35 Here, we used UCB ECFCs because of their biologic properties 5 : ECFCs display a higher proliferative rate compared with mature ECs, form secondary and tertiary colonies on replating and de novo blood vessel formation in vivo, 36 and can be obtained by a noninvasive method and then expanded ex vivo to achieve a considerable number of cells.…”

“…Production of recombinant MMP12 vector and the expression of MMP12 gene and protein were previously described. 11 Expression of MMP12 gene was measured by RT-PCR, and the level of released protein was analyzed by Western blotting of culture medium.…”

“…Fractions were assayed by Western blotting for the presence of MMP-12 enzymatic activity was measured in ECFC conditioned medium using a fluorimetric kit (EnzoLyte 520 MMP-12 assay kit with 5-FAM/QXL 520 fluorescence resonance energy transfer; AnaSpec, DBA-Italia), as previously described. 11 *ECFC conditioned medium under basal conditions. †ECFC conditioned medium under basal conditions in the presence of anti-MMP12 antibody.…”

“…Indeed, in systemic sclerosis, a disease characterized by defective angiogenesis, the impairment of vessel formation is related also to overproduction of matrix metalloprotease-12 (MMP12), which truncates uPAR. 10,11 The role of uPAR in ECFC-dependent vessel formation is less known. A single study indicated that EPCs display higher uPA activity and uPAR levels than mature ECs.…”

“…The latter one is suggested by the observation that truncated uPAR is not found in lipid rafts and that uPAR needs to be in its full-length to recycle through this pathway. 38 On the basis of our previous studies 10,11 showing that the MMP12-dependent uPAR cleavage severely impaired angiogenesis, we evaluated a possible role of MMP12 in ECFC angiogenesis. We demonstrated that the increased angiogenic ability of VEGF-treated ECFCs is related both to uPAR up-regulation and to its lower cleavage because of the decreased levels of MMP12 and that MMP12 overproduced by transfected ECFCs cleaves uPAR and reduces ECFC angiogenic performance in vitro.…”