Alexandre Mezentsev

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

Abstract— Cytochrome P450 (CYP) 4A1 has been characterized as the most efficient arachidonic acid &ohgr;-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 SM22&agr; (Ad-SM22-4A1 and Ad-SM22-nLacZ, respectively). &bgr;-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.

Promoter activity and regulation of the corneal CYP4B1 gene by hypoxia

Hypoxic injury to the ocular surface provokes an inflammatory response that is mediated, in part, by corneal epithelial‐derived 12‐hydroxyeicosanoids. Recent studies indicate that a cytochrome P450 (CYP) monooxygenase, identified as CYP4B1, is involved in the production of these eicosanoids which exhibit potent inflammatory and angiogenic properties. We have isolated and cloned a corneal epithelial CYP4B1 full‐length cDNA and demonstrated that the CYP4B1 mRNA is induced by hypoxia in vitro and in vivo. To further understand the molecular regulation that underlies the synthesis of these potent inflammatory eicosanoids in response to hypoxic injury, we isolated and cloned the CYP4B1 promoter region. GenomeWalker libraries constructed from rabbit corneal epithelial genomic DNA were used as templates for primary and nested PCR amplifications with gene‐ and adaptor‐specific primers. A 3.41‐kb DNA fragment of the 5′‐flanking region of the CYP4B1 promoter was isolated, cloned, sequenced, and analyzed by computer software for the presence of known cis‐acting elements. Analysis of the promoter sequence revealed the presence of consensus DNA binding sequences for factors known to activate gene transcription in response to hypoxia including HIF‐1, NFκB, and AP‐1. Transient transfection of luciferase reporter (pGL3‐Basic) vectors containing different lengths of the CYP4B1 promoter fragment demonstrated hypoxia‐induced transcription in rabbit corneal epithelial (RCE) cells. Electrophoretic mobility shift assay (EMSA) revealed a marked induction of nuclear binding activity for the labeled HIF‐1 probe from the CYP4B1 promoter in nuclear extracts of cells exposed to hypoxia. This binding activity was due to sequence‐specific binding to the HIF‐1 oligonucleotide probe as shown by competition with excess unlabeled probe for the HIF‐1 but not with unlabeled NFκB probe. The nuclear binding activity of AP‐1 and NFκB probes from the CYP4B1 promoter was also enhanced in response to hypoxia suggesting that these transcription factors contribute to the hypoxic induction of CYP4B1 expression. The results of this study provide the first molecular mechanistic explanation for the induction of CYP4B1 and, thereby, the production of inflammatory eicosanoids in response to hypoxic injury. Further studies are needed to fully evaluate the molecular regulation of this gene during inflammation.

COX-2 lack of function in hypoxia-induced ocular surface inflammation.

Injury to the ocular surface increases corneal epithelial production of cyclooxygenase (COX)-derived eicosanoids but this increase correlates poorly to the inflammatory sequelae. Moreover, corticosteroids are effective in treatment of this inflammation but NSAIDs are not. The discovery of COX-2 that is differentially affected by common NSAIDs reopened the question of the role of prostaglandins in ocular surface inflammation. We examined the presence and inducibility of COX-2 in the corneal epithelium following hypoxia-induced injury in vivo and in vitro. COX-2, but not COX-1, protein levels markedly increased following hypoxia. Use of the selective COX-2 inhibitor, NS-398, indicated that COX activity in hypoxic corneas or cells is essentially that of COX-2; in control cells, both COX-1 and COX-2 contributed equally to the production of PGE2. COX-2 protein overexpression induced by hypoxia was not associated with a parallel increase in PGE2 accumulation but the enzyme regained full catalytic activity when cells were re-exposed to normoxia in the presence of heme and arachidonic acid. Decreases in the levels of oxygen and heme, essential substrates/cofactors for COX catalytic activity, contributed to a diminished prostanoid production during hypoxia. These results suggest that in hypoxic injury, molecules other than COX-derived prostanoids play a major pro-inflammatory role. Furthermore, this study provides an explanation for the ineffectiveness of classical NSAIDs in the treatment of hypoxia-related ocular surface inflammation.