A new in vitro model to study endothelial injury.
BACKGROUND
Endothelial dysfunction or "endothelialitis" is a prominent feature in several disease states ranging from atherosclerosis to transplant rejection. This dysfunction is also caused by drugs such as cyclosporin A (CyA) and leads to allograft vasculopathy and eventual graft loss. Despite the frequency and importance of this injury, there is no model to study the morphological effects of endothelial injury and dysfunction in vitro.


METHODS
We utilized a model in which mouse endothelial cells (SVEC 4-10) can be induced to form capillary tubes by culturing on a laminin-rich matrix (Matrigel). In this morphological model of endothelial cell function, we studied the effect of varying doses of CyA on two parameters of tube formation: initiation of tube formation and disruption of mature capillary tubes. As a positive control we used IFN-gamma, which inhibited capillary tube formation. We developed this assay in 96-well culture plates to test several samples simultaneously.


RESULTS
The assay could be adapted to a 96-well format by optimizing the cell density. Endothelial dysfunction was seen when the endothelial cells were incubated with cyclosporin A, which affected both morphological parameters of tube formation. At higher doses (2-20 microg/ml) CyA both inhibited capillary tube formation and disrupted mature capillary tubes. At lower doses CyA only inhibited the initiation of tube formation; it did not disrupt mature capillary tubes. IL-2 (400-1000 pg/ml) and IFN-gamma (10-400 pg/ml) inhibited initiation of tube formation but did not disrupt mature capillary tubes. None of these agents, including high doses of CyA, impaired endothelial cell viability.


CONCLUSIONS
CyA-induced endothelial dysfunction can be modeled in vitro by this novel morphological assay of capillary tube formation. This assay can discern mild and severe degrees of endothelial dysfunction. The different effects of low and high levels of CyA on capillary tube formation imply that similar dysfunction in vivo may be responsible for allograft vasculopathy caused by CyA. This novel model can also be utilized to study other forms of vasculitis.
Wilasrusmee
Chumpon
C
Department of Surgery, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, New York 13210, USA.
Da Silva
Monica
M
Singh
Bhupinder
B
Kittur
Smita
S
Siddiqui
Josephine
J
Bruch
David
D
Wilasrusmee
Skuntala
S
Kittur
Dilip S
DS
D000818:Animals; D002196:Capillaries / Q000187:drug effects / Q000473:pathology; D002452:Cell Count; D002461:Cell Line, Transformed; D003094:Collagen; D003470:Culture Media; D016572:Cyclosporine / Q000009:adverse effects / Q000494:pharmacology; D004195:Disease Models, Animal; D004338:Drug Combinations; D004730:Endothelium, Vascular / Q000187:drug effects / Q000473:pathology*; D007166:Immunosuppressive Agents / Q000009:adverse effects / Q000494:pharmacology; D007371:Interferon-gamma / Q000494:pharmacology; D007376:Interleukin-2 / Q000494:pharmacology; D007797:Laminin; D051379:Mice; D011509:Proteoglycans; D014657:Vasculitis / Q000139:chemically induced / Q000473:pathology*
D003470:Culture Media; D004338:Drug Combinations; D007166:Immunosuppressive Agents; D007376:Interleukin-2; D007797:Laminin; D011509:Proteoglycans; C059485:matrigel; D007371:Interferon-gamma; D016572:Cyclosporine; D003094:Collagen