Smooth Muscle—Specific Expression of CYP4A1 Induces Endothelial Sprouting in Renal Arterial Microvessels

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Abstract
Cytochrome P450 (CYP) 4A1 has been characterized as the most efficient arachidonic acid ω-hydroxylase catalyzing the formation of 20-hydroxyeicosatetraenoic acid (20-HETE), a potent constrictor of the renal and cerebral microcirculation and a mitogen for smooth muscle cells. We constructed adenoviruses expressing the CYP4A1 cDNA or LacZ under the control of the smooth muscle cell–specific promoter SM22α (Ad-SM22-4A1 and Ad-SM22-nLacZ, respectively). β-Galactosidase expression was detected in Ad-SM22-nLacZ–transduced vascular smooth muscle A7r5 and PAC1 cells, but not in Ad-SM22-nLacZ-transduced 3T3 fibroblasts or vascular endothelial cells. Likewise, CYP4A1 mRNA and protein were detected in Ad-SM22-4A1–transduced A7r5 and PAC1 cells. Ad-SM22-4A1–transduced A7r5 cells metabolized lauric acid to 12-hydroxy-lauric acid at a rate 5 times greater than that of cells transduced with Ad-SM22-nLacZ (4.79±1.77 versus 0.97±0.57 nmol 12-hydroxy lauric acid/106 cells per h). Smooth muscle–specific LacZ expression was also detected in microdissected renal interlobar arteries transduced with Ad-SM22-nLacZ. Arteries transduced with Ad-SM22-4A1 produced higher levels of 20-HETE (4.04±0.29 and 13.43±2.84 ng/mg protein in Ad-SM22-nLacZ–transduced and Ad-SM22-4A1–transduced arteries, respectively) and demonstrated a marked angiogenic activity measured as the total length of sprouting neovessels (12.63±3.66 mm in Ad-SM22-4A1–transduced vessels versus 1.79±0.89 mm in Ad-SM22-nLacZ–transduced vessels). This angiogenic activity represented endothelial cell sprouting and was fully blocked by treatment with HET0016, a selective inhibitor of CYP4A-catalyzed reactions. The inhibitory effect of HET0016 was reversed by addition of a 20-HETE agonist. We conclude that Ad-SM22-4A1 drives a smooth muscle–specific functional expression of CYP4A1 and demonstrates increased angiogenesis, presumably via increased production of 20-HETE.