The antiangiogenic tissue kallikrein pattern of endothelial cells in systemic sclerosis.
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.


CONCLUSIONS
Reduction of tissue kallikreins 9, 11, and 12 may be relevant to reduced angiogenesis in SSc patients.
Giusti
Betti
B
Department of Experimental Pathology and Oncology, University of Florence, Viale G M Morgagni 50, 50134 Florence, Italy.
Serratì
Simona
S
Margheri
Francesca
F
Papucci
Laura
L
Rossi
Luciana
L
Poggi
Filippo
F
Magi
Alberto
A
Del Rosso
Angela
A
Cinelli
Marina
M
Guiducci
Serena
S
Kahaleh
Bashar
B
Matucci-Cerinic
Marco
M
Abbate
Rosanna
R
Fibbi
Gabriella
G
Del Rosso
Mario
M
D019138:Antibodies, Blocking / Q000494:pharmacology; D015153:Blotting, Western; D002452:Cell Count; D049109:Cell Proliferation; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005260:Female; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D008297:Male; D008833:Microcirculation / Q000166:cytology; D009389:Neovascularization, Pathologic / Q000378:metabolism* / Q000473:pathology; D009712:Nucleotides, Cyclic / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D010766:Phosphorylation; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012595:Scleroderma, Systemic / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D012867:Skin / Q000098:blood supply; D020840:Tissue Kallikreins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D019138:Antibodies, Blocking; D009712:Nucleotides, Cyclic; D012333:RNA, Messenger; D048049:Extracellular Signal-Regulated MAP Kinases; D020840:Tissue Kallikreins