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Objective: Clinical evidence suggests that the vascular abnormalities of systemic sclerosis (SSc) precede the onset of fibrosis, but molecular cues accounting for a possible vascular connection of SSc fibrosis have been elusive, although they have been searched for intensively. Since we had previously shown that connective tissue growth factor (CCN2), endowed with fibroblast-oriented activities, was overexpressed by endothelial cells (ECs) from SSc patients, we undertook this study to investigate its role and mechanisms in fibroblast activation., Methods: Normal fibroblasts were challenged with conditioned medium of normal microvascular ECs (MVECs) and MVECs obtained from SSc patients with the diffuse form of the disease. Fibroblast invasion was studied using the Boyden chamber Matrigel assay. Fibroblast activation was evaluated by Western blotting and immunofluorescence of α-smooth muscle actin (α-SMA), vimentin, and type I collagen. Matrix metalloproteinase (MMP) production was evaluated by zymography and reverse transcription-polymerase chain reaction. Signal transduction and activation of the small GTPases RhoA and Rac1 were studied by Western blotting. Inhibition of SSc MVEC conditioned medium-dependent fibroblast activation was obtained by anti-CCN2 antibodies and the transforming growth factor β (TGFβ) antagonist peptide p17., Results: SSc MVEC CCN2 stimulated fibroblast activation and invasion. Such activities depended on CCN2-induced overexpression of TGFβ and its type I, II, and III receptors combined with overproduction of MMP-2 and MMP-9 gelatinases. All of these effects were reversed by the TGFβ antagonist peptide p17. Motility increase required Rac1 activation and RhoA inhibition and was inhibited by an MMP inhibitor. These features connoted a mesenchymal style of cell invasion. Since fibroblast activation also fostered overexpression of α-SMA, vimentin, and type I collagen, the CCN2-dependent increase in fibroblast activities recapitulated the characteristics of a mesenchymal-to-mesenchymal transition., Conclusion: SSc MVECs recruit and activate dermal fibroblasts by induction of a CCN2/TGFβ-dependent mesenchymal-to-mesenchymal transition., (Copyright © 2013 by the American College of Rheumatology.)

Objective: Urokinase plasminogen activator (uPA), uPA receptor (uPAR), and PA inhibitor 1 (PAI-1) have pivotal roles in the proliferation and invasion of several cell types, including synovial fibroblasts (SFs). The aim of this study was to investigate the possibility of controlling the invasion of rheumatoid arthritis (RA) SFs in vitro and in vivo by inhibiting uPA and uPAR., Methods: Normal SFs, SFs from patients with RA, and SFs from patients with psoriatic arthritis (PsA) were used. The levels of uPA, uPAR, and PAI-1 were measured by enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction analysis of messenger RNA. The activity of uPA was studied by zymography. Proliferation was measured by cell counting, and cell invasion was measured with a Boyden chamber assembled with Matrigel-coated porous filters. Human cartilage and RA SF implantation in the SCID mouse model of RA were used to study cartilage invasion in vivo., Results: RA SFs and PsA SFs overexpressed uPAR and as a result were more active than their normal counterparts in terms of both Matrigel invasion and proliferation. This effect was counteracted by a specific inhibitor of uPA enzymatic activity (WX-340) and by uPAR antisense treatment. The use of both WX-340 and uPAR antisense treatment in vitro showed cooperative effects in RA SFs that were more intense than the effects of either treatment alone. Significant inhibition of cartilage invasion was obtained in vivo with uPAR antisense treatment, while uPA inhibition was inefficient, either alone or in combination with antisense treatment., Conclusion: The decrease in uPAR expression in RA SFs reduced invasion of human cartilage in vitro and in the SCID mouse model., (Copyright © 2011 by the American College of Rheumatology.)

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: Transfection of MMP12 and PTX3 in normal MVECs resulted in loss of proliferation, invasion, and capillary morphogenesis in vitro, attributed to truncation of the urokinase-type plasminogen activator receptor by MMP12 and to the anti-fibroblast growth factor 2/anti-vascular endothelial growth factor activity of PTX3. These effects were particularly evident in mixed populations of transfected normal MVECs (50% transfected with MMP12 and 50% with PTX3). Silencing of the same molecules in SSc MVECs increased their invasion in Matrigel. Single-gene silencing did not increase the capillary morphogenesis of SSc MVECs, whereas double-gene-silenced cells showed a burst of capillary tube formation. Culture medium of silenced SSc MVECs stimulated angiogenesis in assays of Matrigel sponge invasion in mice., Conclusion: Overexpression of either MMP12 or PTX3 in normal MVECs blunts their angiogenic properties. Loss of function of MMP12 and PTX3 in SSc MVECs restores the ability of the cells to produce capillaries in vitro and induces vascularization in vivo on a Matrigel sponge.

Objective: In systemic sclerosis (SSc) microvascular endothelial cells (MVECs), angiogenesis is blocked by matrix metalloproteinase 12-dependent cleavage of domain 1 of the urokinase-type plasminogen activator receptor (uPAR). Since integrins are associated with the invasive activity of uPAR in angiogenesis, this study was undertaken to show whether full-size and truncated uPAR are differentially associated with integrins and with motor components of the cytoskeleton., Methods: SSc and normal MVECs were isolated from human skin biopsy specimens and studied by confocal laser scanning microscopy and immunoprecipitation to assess the mechanisms of association of truncated and full-size uPAR with integrins and the actin cytoskeleton. The integrin composition of the MVECs was studied by reverse transcription-polymerasechain reaction. Cell migration and capillary morphogenesis were studied on fibrinogen substrates. Involvement of Rac and Cdc42 was evaluated by Western blotting., Results: Only full-size uPAR showed a connection with the actin cytoskeleton in ECs. This connection was mediated by the uPAR-associated alphaMu- and alphaX-subunits of beta2 integrin, and was absent from SSc MVECs. The cleaved uPAR was not associated with beta2 integrins or with actin. beta3 integrins were associated with both the full-size and cleaved uPAR at focal contacts. The uncoupling of uPAR from beta2 integrins in SSc MVECs impaired the activation of Rac and Cdc42 (thus inhibiting their mediation of uPAR-dependent cytoskeletal rearrangements and cell motility) and blocked the integrin-engagement-delivered signals to the actin cytoskeleton. Invasion and capillary morphogenesis on fibrinogen-coated substrates indicated that ligation of uPAR by uPA empowers the beta2/beta3 integrin-dependent invasion of fibrinogen, and that this system is impaired in SSc MVECs., Conclusion: The reduced angiogenic properties of SSc MVECs can be explained by the effects of uPAR truncation and the subsequent loss of the beta2 integrin-mediated connection of uPAR with the actin cytoskeleton in these ECs.

Objective: Postnatal angiogenesis relies on a proper response of endothelial cells to angiogenic stimuli. In systemic sclerosis (SSc), endothelial cells are unresponsive to angiogenic factors. Since circumstantial and experimental evidence points to tissue kallikreins as powerful effectors of the angiogenic response, we undertook this study to investigate the kallikrein pattern of normal and SSc endothelial cells in order to identify differences that can account for defective angiogenesis., Methods: Expression of 14 tissue kallikreins was studied by a microarray approach, by reverse transcription-polymerase chain reaction, and by Western blotting in endothelial cells isolated from the skin of clinically healthy subjects and SSc patients. Cell proliferation was quantified by direct cell counting. Invasion and capillary morphogenesis were evaluated in a Boyden chamber and in culture flasks layered with Matrigel. Cyclic nucleotide production was measured by enzyme immunoassay. MAP kinase and ERK activation were measured by Western blotting., Results: Endothelial cells from SSc patients showed poor expression of kallikreins 9, 11, and 12 compared with endothelial cells from normal subjects. Antibodies against the relevant kallikreins on normal endothelial cells revealed that while kallikreins 9, 11, and 12 induced cell growth, only kallikrein 12 regulated invasion and capillary morphogenesis. Buffering of kallikrein 12 with antibodies resulted in the acquisition of an SSc-like pattern by normal cells in in vitro angiogenesis. Reduction of cAMP and cGMP production and of ERK phosphorylation upon administration of antikallikrein antibodies revealed that the activity of kallikreins 9, 11, and 12 was mediated by kinins., Conclusion: Reduction of tissue kallikreins 9, 11, and 12 may be relevant to reduced angiogenesis in SSc patients.

Objective: Defective angiogenesis, resulting in tissue ischemia, is particularly prominent in the diffuse form of systemic sclerosis (SSc). The present study was undertaken to identify possible differences between normal and SSc microvascular endothelial cells (MVECs) in the expression of the cell-associated urokinase-type plasminogen activator (uPA)/uPA receptor (uPAR) system, which is critical in the angiogenic process., Methods: MVECs were isolated from the dermis of healthy individuals and from the dermis of patients with diffuse SSc. The uPA/uPAR system was examined at the protein and messenger RNA levels. Angiogenesis was assayed on Matrigel-coated porous filters and plates to evaluate cell proliferation, invasion, and capillary morphogenesis. Cleavage of uPAR and the activity of matrix metalloproteinase 12 (MMP-12) were evaluated by Western blotting., Results: Compared with MVECs from healthy skin, MVECs from SSc patients showed higher expression of uPAR. However, in SSc MVECs, uPAR undergoes truncation between domain 1 and domain 2, as shown by flow cytometry, enzyme-linked immunosorbent assay, and Western blotting, a cleavage that is known to impair uPAR functions. These properties of SSc MVECs were associated with poor spontaneous and uPA-dependent invasion, proliferation, and capillary morphogenesis. The uPAR cleavage occurring in SSc MVECs was associated with overexpression of MMP-12. SSc MVEC-conditioned medium impaired uPA-dependent proliferation and invasion as well as capillary morphogenesis in normal MVECs in vitro. Both a general hydroxamate inhibitor of MMP activity and anti-MMP-12 antibodies restored this SSc MVEC-induced impaired functioning., Conclusion: Overproduction of MMP-12 by SSc MVECs accounts for the cleavage of uPAR and the impairment of angiogenesis in vitro and may contribute to reduced angiogenesis in SSc patients., (Copyright 2004 American College of Rheumatology)