Smooth Muscle-Specific Expression of CYP4A1 Induces Endothelial Sprouting in Renal Arterial Microvessels
Miao Jiang1, Alexandre Mezentsev1, Rowena Kemp1, Kihwan Byun2, John R. Falck2, Joseph M. Miano3, Alberto Nasjletti1, Nader G. Abraham1, Michal Laniado-Schwartzman1
1Department of Pharmacology, New York Medical College, Valhalla, NY; 2Departments of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX; 3Center for Cardiovascular Research, University of Rochester, Rochester, NY
Purpose
Cytochrome P450 (CYP) 4A1 has been characterized as the most efficient arachidonic acid -hydroxlase catalyzing the formation of 20-hydroxyeicosatetraenoic acid (20-HETE). We constructed adenoviruses expressing CYP4A1 or LacZ under the control of the smooth muscle cell-specific promoter-SM22 (Ad-SM22-4A1 and Ad-SM22-nLacZ) to determine the effect of smooth muscle-specific expression of CYP4A1 in renal vasculature.
Methods
Rat renal arterial microvessels were transduced with Ad-SM22-4A1 or Ad-SM22-nLacZ. 24 hours later the vessels were embedded in matrigel and cultured. Expression of -galactosidase was detected by staining with the X-gal. The angiogenic response of the renal arteries was determined by measuring the length of the neovessel sprouts and counting the branching points. Media was collected and 20-HETE levels were quantitated by GC/MS. Endothelial origin of neovessel sprouts was determined by using the endothelial cells specific lectin Ulex europeus.
Results
Blue staining of the smooth muscle cells in the media of microvessels indicated that the SM22 promoter specifically drove gene expression to smooth muscle cells. The levels of 20-HETE in the culture medium from Ad-SM22-4A1-transduced vessels were 3-fold higher than that in Ad-SM22-nLacZ-transduced vessels. Addition of HET0016, a CYP 4A1 inhibitor, reduced the level of 20-HETE by 60%. Vessels transduced with Ad-SM22-4A1 showed a 7-fold increase in microvessel sprouting and branching compared to vessels transduced with Ad-SM22-nLacZ. The majority (75±20%, n=10) of cells forming capillary tube-like network were endothelial cells. Addition of HET0016 completely blocked the angiogenic activity in Ad-SM22-4A1-transduced vessels, which was reversed by the addition of a 20-HETE analog.
Conclusions
A smooth muscle-specific expression of CYP4A1 mediated by Ad-SM22-4A1 induces endothelial sprouting in renal arterial microvessels, presumably via increased production of 20-HETE. This induced angiogenic acitivity may be an important component for maintaining endothelial function in aging, diabetes and hypertension, in which renal interstitium capillary density and angiogenic activity is reduced.