Matrix metalloproteinase 12-dependent cleavage of urokinase receptor in systemic sclerosis microvascular endothelial cells results in impaired angiogenesis.
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.


CONCLUSIONS
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.
D'Alessio
Silvia
S
University of Florence, Florence, Italy.
Fibbi
Gabriella
G
Cinelli
Marina
M
Guiducci
Serena
S
Del Rosso
Angela
A
Margheri
Francesca
F
Serratì
Simona
S
Pucci
Marco
M
Kahaleh
Bashar
B
Fan
Pansheng
P
Annunziato
Francesco
F
Cosmi
Lorenzo
L
Liotta
Francesco
F
Matucci-Cerinic
Marco
M
Del Rosso
Mario
M
D015153:Blotting, Western; D042783:Endothelial Cells / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D066298:In Vitro Techniques; D053508:Matrix Metalloproteinase 12; D008666:Metalloendopeptidases / Q000378:metabolism / Q000502:physiology*; D008833:Microcirculation / Q000166:cytology; D018919:Neovascularization, Physiologic / Q000502:physiology*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D012595:Scleroderma, Systemic / Q000378:metabolism / Q000503:physiopathology*
C524898:PLAUR protein, human; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D008666:Metalloendopeptidases; C507122:MMP12 protein, human; D053508:Matrix Metalloproteinase 12