The lipoxygenase (LO) pathway of arachidonate metabolism has been suggested to play a key role in atherosclerosis and in mediating several actions of angiotensin II (AI). However, the relationship between LO activation and factors linked to accelerated diabetic vascular disease such as hyperglycemia and All is not known. We have investigated the effect of high glucose (HG; 25 mM) and All on LO activity as well as LO protein and mRNA expression in porcine aortic vascular smooth muscle cells (PVSMCs). We observed that cells cultured in HG had significantly higher levels of the cell-associated LO products 12-and 15-hydroxyeicosatetraenoic acids (HETEs LO inhibitors (5). Furthermore, increased levels of HETEs and HODEs were observed in the aortas of atherosclerotic rabbits (6). HETEs have also been suggested to play a key role in the pathogenesis of diabetic vascular disease, since vessels from infants of diabetic mothers had significantly elevated levels of 15-HETE and decreased formation of vasodilatory prostacyclin (7) and endothelial cells cultured under hyperglycemic conditions produced increased amounts of HETEs (8). Studies also suggest that HETEs have mitogenic properties (9) and can cause SMC migration at concentrations as low as 1 pM (10).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.It has been demonstrated that the vasopressor effects of angiotensin II (AII) are enhanced in the diabetic state (11). Increasing evidence also indicates that AII is an important local factor in leading to VSMC hypertrophy and neointimal SMC proliferation (12, 13). We have previously shown (14, 15) that the 12-LO pathway of arachidonic acid plays a key role in AII-induced aldosterone synthesis and adrenal cell proliferation. Furthermore, inhibition of the LO pathway can reduce vasopressor responses to All (16). In the present study, we have investigated the effects of HG and All on LO pathway activation in cultured PVSMCs.A leukocyte type of 12-LO has been purified and cloned from porcine leukocytes (17, 18). However, no studies have addressed whether this form of 12-LO is also present in VSMCs. Furthermore, the factor(s) regulating the expression of 12-LO is not known. In the present study, we determined whether the 12-LO enzyme is expressed in PVSMCs. We also investigated whether the 12-LO protein and mRNA levels were regulated by glucose and AII to determine whether the increases in 12-LO activity induced by HG or AII were at the translational and/or the transcriptional level. MATERIALS AND METHODSCulture of PVSMCs. Primary cultures of PVSMCs were obtained as described (2). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing NG (5.5 mM) and 10%o fetal calf serum (FCS). For studies under hyperglycemic conditions, the cells were cultured for at least two passages in DMEM HG (25 mM) before use. Controls for osmolality were cells gr...

The 12-lipoxygenase pathway is a key mediator of angiotensin II (Ang II)-induced effects in the adrenal cortex. We also recently demonstrated that Ang II increases 12-and 15-lipoxygenase product levels in vascular smooth muscle cells. However, the relation between lipoxygenase activation and Ang II-induced vascular smooth muscle cell hypertrophy is not known. We studied the effects of Ang II and 12-lipoxygenase products on both total cell protein content and the levels of the matrix protein fibronectin in quiescent porcine aortic smooth muscle cells. Ang II-induced increases in cellular protein content were attenuated by the specific 12-lipoxygenase inhibitor baicalein; in contrast, the cycloxygenase inhibitor ibuprofen had no effect. Direct addition of the 12-lipoxygenase product 12-S-hydroxyeicosatetraenoic acid increased total cell protein content. We have recently shown that porcine vascular smooth muscle cell growth is potentiated in high glucose (25 mmol/L) culture conditions. We observed that both Ang II and 12-5-hydroxyeicosatetraenoic acid induced a greater increase in protein content in cells cultured for two passages in high glucose. Furthermore, Ang II and 12-5-hydroxyeicosatetraenoic acid also markedly increased fibronectin levels in cells cultured in high glucose. These results suggest that 12-lipoxygenase activation plays a key role in Ang II-induced vascular smooth muscle cell hypertrophy.

Vascular endothelial growth factor (VEGF), in addition to its growth-promoting effects on endothelial cells, can also increase vascular permeability and monocyte migration. It has therefore been implicated in the pathogenic neovascularization associated with diabetic retinopathy and atherosclerosis. However, the factors regulating VEGF expression in the vascular wall are not fully understood. In this study, we examined the regulation of VEGF expression in vascular smooth muscle cells (VSMC) by hyperglycemia as well as by angiotensin II (ANG II). We also examined whether the 12-lipoxygenase (12-LO) product 12-hydroxyeicosatetraenoic acid (12-HETE) can alter VEGF expression, since 12-LO products of arachidonic acid have angiogenic properties, and ANG II as well as high glucose (HG, 25 mM) can increase 12-LO activity and expression in VSMC. Studies were carried out in human (HSMC) or porcine VSMC (PSMC), which were cultured for at least two passages under normal glucose (NG, 5.5 mM) or HG conditions. HG culture alone increased the expression of VEGF mRNA and protein in both HSMC and PSMC. Furthermore, ANG II treatment significantly induced VEGF mRNA and protein expression only in VSMC cultured in HG and not NG. In addition, 12-HETE significantly increased VEGF mRNA and protein expression in HSMC cultured in NG as well as in HG. Cells cultured in HG also secreted significantly greater amounts of VEGF into the culture medium. These results suggest that elevated VEGF production under HG conditions may play a role in the accelerated vascular disease observed in diabetes.

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