Reduced Growth Factor Responses in Vascular Smooth Muscle Cells Derived from 12/15-Lipoxygenase–Deficient Mice

Abstract

Abstract-Biochemical and genetic evidence support the involvement of leukocyte-type 12/15-lipoxygenase enzyme and its products in the atherogenic process. We recently showed that products of the 12/15-lipoxygenase pathway play an important role in mediating hypertrophy, matrix protein production, and inflammatory gene expression in vascular smooth muscle cells (VSMC) through activation of mitogen activated protein kinases and key transcription factors. The current study is aimed at establishing the in vivo role of 12/15-lipoxygenase in VSMC by comparing growth factor-induced responses in VSMC derived from 12/15-lipoxygenase knockout mice versus genetic control wild-type mice. In the lipoxygenase knockout cells, 12/15-lipoxygenase protein was not expressed, and levels of its product, 12(S)-hydroxyeicosatetraenoic acid, were reduced (51% of wild type). Knockout cells exhibited significantly lower rates of growth factor-induced migration, fibronectin production, and incorporation of 3 H-thymidine and 3 H-leucine (54%, 55%, 61%, and 57% of wild type, respectively). Growth factor-induced superoxide production and p38 mitogen-activated protein kinase activation were also reduced in knockout cells. Serum-stimulated AP-1 transcription factor activation was markedly reduced (50% of wild type), whereas cAMP response element binding protein activation was abrogated in knockout cells. Furthermore, growth factor-induced mRNA expression of immediate early genes and fibronectin were also greatly reduced. These results suggest that the modulation of specific signaling pathways and growth-responsive genes may be responsible for the altered growth factor responses in the lipoxygenase knockout cells. Leukocyte-type 12-LO has a wide tissue distribution, including vascular smooth muscle cells (VSMC). 6,7 In mice, both leukocyte 12/15-LO and platelet 12-LO are expressed. 8 Studies indicate that LO inhibition reduced blood pressure in renovascular hypertensive rats. 9 Furthermore, 12(S)-HETE levels were elevated in spontaneously hypertensive rats compared with age-matched Wistar-Kyoto rats, and 12(S)-HETE directly regulated calcium signals in VSMC. 10,11 Several lines of evidence implicate 12/15-LO in the development of atherosclerosis. 12/15-LO can mediate the oxidation of LDL, 12 and the enzyme and its products have been detected in atherosclerotic lesions. 13,14 Convincing evidence comes from recent data showing that disruption of 12/15-LO in apoE Ϫ/Ϫ or LDL-R Ϫ/Ϫ mice significantly reduced atherosclerosis in these mice models. 15,16 Additionally, growth factors (GFs) such as angiotensin II (Ang II) and platelet-derived growth factor (PDGF) and cytokines such as IL-1, IL-4, and IL-8 could induce 12/15-LO activity and expression in VSMC. 7,[17][18][19] Furthermore, the 12-LO product 12(S)-HETE could induce VSMC migration and extracellular matrix production. 20,21 Ang II-induced hypertrophy as well as PDGF-induced chemotactic effects were significantly blocked by pharmacological LO inhibitors and a molecular inhibitor, 12-LO ribozyme...

Enhanced Proatherogenic Responses in Macrophages and Vascular Smooth Muscle Cells Derived From Diabetic db/db Mice

“…Aortic VSMCs from 9-to 10-week-old male db/ϩ and db/db mice (MVSMCs) were isolated by enzymatic digestion as described earlier (34) and cultured in Dulbecco's modified Eagle's (DME)-F12 culture medium supplemented with 10% FCS. MVSMCs from db/ϩ and db/db mice were used between passages 5 and 8.…”

“…Oxidant stress. Intracellular superoxide production (index of oxidant stress) was evaluated with the use of a superoxide probe dihydroethidium (Molecular Probes, Eugene, OR) as described earlier (34). Cell migration.…”

A novel role for activating transcription factor-2 in 15(S)-hydroxyeicosatetraenoic acid-induced angiogenesis

“…With regard to their tissue distribution, while 15-LOX1 shows a narrow cell-specific expression, including human reticulocytes and airway epithelial cells, 15-LOX2 appears to be expressed in epithelial cell types in cornea, lung, prostate, and skin (5). Although the presence of 15-LOX2 in the vessel wall has yet to be reported, vascular smooth muscle cells (VSMC) and endothelial cells (EC) express 15-LOX1 (also known as 12/15-LOX in murines) and when exposed to AA, these cells produce 15(S)-HETE and 12(S)-HETE (6)(7)(8)(9). A number of studies have demonstrated that 15-LOX1 and its murine ortholog 12/15-LOX play a role in atherosclerosis (10)(11)(12).…”

“…Previously we have shown that 15(S)-HETE stimulates JNK1 in HRMVEC (7). To understand the mechanisms of JNK1 activation by 15(S)-HETE, here we have examined the role of Src and Rac1.…”

Increased 12/15-Lipoxygenase Enhances Cell Growth, Fibronectin Deposition, and Neointimal Formation in Response to Carotid Injury

“…5,7,8 Compared with VSMCs from C57BL/6 (BL-6) mice, VSMCs from 12/15LO-overexpressing transgenic (12/15LO-tg) mice grow faster, and VSMCs from 12/15LO Ϫ/Ϫ mice grow slower and display decreased S-phase entry in culture. 9,10 12/15LO and its products are increased in the vascular wall of animal models of atherosclerosis and injury-induced restenosis. 11 Compared with uninjured carotids, 12/15LO expression is significantly increased in rat carotids on day 12 after injury.…”