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


CONCLUSIONS
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.
Margheri
Francesca
F
University of Florence, Florence, Italy.
Serratì
Simona
S
Lapucci
Andrea
A
Chillà
Anastasia
A
Bazzichi
Laura
L
Bombardieri
Stefano
S
Kahaleh
Bashar
B
Calorini
Lido
L
Bianchini
Francesca
F
Fibbi
Gabriella
G
Del Rosso
Mario
M
D015153:Blotting, Western; D002097:C-Reactive Protein / Q000235:genetics / Q000378:metabolism*; D002465:Cell Movement / Q000502:physiology; D049109:Cell Proliferation; D042783:Endothelial Cells / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D053508:Matrix Metalloproteinase 12 / Q000235:genetics / Q000378:metabolism*; D009389:Neovascularization, Pathologic / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000502:physiology*; D012595:Scleroderma, Systemic / Q000378:metabolism*; D000683:Serum Amyloid P-Component / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D000099153:Pentraxins
D002097:C-Reactive Protein; D053508:Matrix Metalloproteinase 12; D000683:Serum Amyloid P-Component; D000099153:Pentraxins