Noe Gonzales

Vascular smooth muscle cells exhibit increased growth in response to elevated glucose

Diabetes mellitus is associated with an increased risk of cardiovascular disease. In order to elucidate the association between hyperglycemia and vascular complications, the growth patterns of vascular smooth muscle cells were studied under high glucose conditions. We examined the effect of culturing porcine aortic smooth muscle cells (PVSMC) in high glucose (25 mM, HG) on total cell protein, cell volume, DNA synthesis and cell number. We observed that cells cultured in HG had higher total cell protein content which was associated with increased cell volume as compared to the cells cultured under normoglycemic conditions (5.5 mM glucose, NG). PVSMC cultured in HG also had 1.4 fold increased growth rate and a greater fetal calf serum-induced DNA synthesis rate compared to cells cultured in NG. These observations suggest for the first time that elevated glucose could lead to both hypertrophic and hyperplastic effects in PVSMC. We also examined protein kinase C (PKC) activities as well as the cellular levels of the 12-lipoxygenase product, 12-hydroxyeicosatetraenoic acid (12-HETE) in NG and HG as possible mechanisms for the enhanced growth effects in HG. The results show that PVSMC cultured in HG have increased PKC activity as well as increased levels of 12-HETE. Therefore hyperglycemia may be linked to accelerated vascular disease by increasing smooth muscle cell growth and proliferation.

Evidence that pioglitazone, metformin and pentoxifylline are inhibitors of glycation

Enhanced formation and accumulation of advanced glycation end products (AGEs) have been proposed to play a major role in the pathogenesis of diabetic complications, and atherosclerosis, leading to the development of a range of diabetic complications including nephropathy, retinopathy and neuropathy. Several potential drug candidates as AGE inhibitors have been reported recently. Aminoguanidine is the first drug extensively studied. However, there are no currently available medications known to block AGE formation. We have previously reported a number of novel and structurally diverse compounds as potent inhibitors of glycation and AGE formation. We have now studied several of the existing drugs, which are in therapeutic practice for lowering blood sugar or the treatment of peripheral vascular disease in diabetic patients, for possible inhibitory effects on glycation. We show that that three compounds; pioglitazone, metformin and pentoxifylline are also inhibitors of glycation.

Effects of high glucose on vascular endothelial growth factor expression in vascular smooth muscle cells

Vascular endothelial growth factor (VEGF), in addition oto 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.

PLATELET-DERIVED GROWTH FACTOR BB MEDIATED REGULATION OF 12-LIPOXYGENASE IN PORCINE AORTIC SMOOTH MUSCLE CELLS

Platelet‐derived growth factor BB (PDGF) is a potent mitogen and chemoattractant for vascular smooth muscle cells (VSMC). In the present study, we have examined the effects of PDGF on the 12‐lipoxygenase (12‐LO) pathway of arachidonate metabolism in porcine aortic VSMC (PVSMC). The rationale for this is previous studies showing that LO products have growth and chemotactic effects in VSMC and that another VSMC growth factor, angiotensin II, is a potent positive regulator of 12‐LO activity and expression. We observed that PDGF causes a significant increase in the formation of the 12‐LO product, 12‐hydroxyeicosatetraenoic acid (12‐HETE) in PVSMC. In addition, PDGF also markedly increased leukocyte‐type 12‐LO messenger RNA and protein expression. PDGF‐induced PVSMC migration was inhibited significantly by two LO blockers but not by a cyclooxygenase blocker. Furthermore, although the proliferative effects of PDGF on PVSMC were not altered by cell culture under hyperglycemic conditions (25 mM glucose, HG), the chemotactic effects of PDGF as well as those of 10% fetal calf serum were significantly greater in cells cultured in HG as compared to normal glucose conditions (5.5 mM), thus indicating a potential new mechanism for the accelerated cardiovascular disease usually observed in diabetes. These results indicate a novel mechanism for the biological effects of PDGF in leading to cardiovascular disease.

Regulation of 12-Lipoxygenase by Cytokines in Vascular Smooth Muscle Cells

Increasing evidence suggests that cytokines such as interleukin-1beta (IL-1), IL-4, and IL-8 may play an important role in the chronic inflammation and cellular growth observed in cardiovascular diseases. The lipoxygenase (LO) pathway of arachidonate metabolism has also been related to the pathology of hypertension and atherosclerosis. LO products have chemotactic, hypertrophic, and mitogenic effects in vascular cells, and the LO enzyme has been implicated in the oxidation of LDL. Furthermore, earlier studies have shown that vascular smooth muscle cell (VSMC) growth factors such as angiotensin II and platelet-derived growth factor can increase LO activity and expression in VSMCs. In the present study, we have examined whether vasoactive and inflammatory cytokines such as IL-1, IL-4, and IL-8 can modulate 12-LO activity and expression in porcine VSMCs and also whether they have growth-promoting effects in these cells. Treatment of porcine VSMCs with these cytokines led to significant increases in the levels of a cell-associated 12-LO product, 12-hydroxyeicosatetraenoic acid, as well as intracellular 12-LO enzyme activity. Furthermore, each of these cytokines led to a dose-dependent increase in 12-LO mRNA expression (333-base pair PCR product) as well as 12-LO protein expression (72 kD). In addition, all three interleukins could induce significant increases in VSMC DNA synthesis as well as proliferation. These results suggest that these cytokines have mitogenic effects in VSMCs and are also potent positive regulators of the 12-LO pathway. Thus, enhanced 12-LO activity and expression may be a key mechanism for cytokine-induced VSMC migration and proliferation.

Evidence That Angiotensin II and Lipoxygenase Products Activate c-Jun NH2-Terminal Kinase

The effect of angiotensin II (Ang II) to activate c-Jun amino-terminal kinase (JNK) was studied in a Chinese hamster ovary fibroblast cell line overexpressing the rat vascular type-1a Ang II receptor (CHO-AT1a). Ang II treatment induced a time-dependent activation of JNK. Ang II (10(-7) mol/L) activated JNK activity, with a peak at 30 minutes (9.39 +/- 2.52-fold, n = 7, P < .02 versus control), which was maintained until 3 hours (2.7 +/- 0.65-fold, n = 3, P < .02 versus control). Ang II-induced JNK activation at 30 minutes was inhibited by a specific lipoxygenase (LO) pathway inhibitor, cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (1 mumol/L) by 87.5% (n = 4, P < .01 versus Ang II-induced JNK activity). The direct addition of 12-HETE also induced a time-dependent JNK activation. 12-HETE (10(-7) mol/L) activated JNK activity, with a peak at 10 minutes (3.43 +/- 0.87-fold, n = 6, P < .02 versus control), which remained elevated until 1 hour. These results suggest that the LO pathway is a mediator of Ang II-induced JNK activation. 15-HETE can also activate JNK at 5 minutes, but this activity was reduced at 30 minutes and could not be seen at 1 hour, indicating that the time course was different from that seen with 12-HETE. N-Acetylcysteine (NAC), an antioxidant, was used to perturb intracellular reactive oxygen intermediate (ROI) levels to assess the role of endogenous ROIs in regulating JNK activity. Pretreatment of cells with 500 mumol/L NAC for 1 hour attenuated approximately 50% of Aug II-induced JNK activation, suggesting that ROIs, at least partially, mediate Ang II-induced JNK activation. Furthermore, 12-HETE-induced JNK activation was reduced by approximately 90% by NAC. Finally, pertussis toxin completely blocked 12-HETE-induced JNK activation, suggesting that Gi-protein signaling participates in 12-HETE-induced effects. These results suggest that LO activation plays a role in mediating Ang II-induced JNK activation in part by altering the redox tone and Gi-protein signaling of cells.

Evidence that 12-lipoxygenase product 12-hydroxyeicosatetraenoic acid activates p21-activated kinase.

The effect of 12-hydroxyeicosatetraenoic acid (12-HETE), an arachidonic acid metabolite of 12-lipoxygenase, to activate p21(Rac/Cdc42)-activated kinase (PAK1) was studied in a Chinese hamster ovary fibroblast cell line overexpressing the rat vascular type-1a angiotensin II receptor (CHO-AT(1a)). 12-HETE (0.1 microM) treatment induced a time-dependent activation of PAK1, with a peak effect at 10 min (335 +/- 16% of control; n=3, P<0.001). The stimulatory effect of 12-HETE on PAK1 activity was dose-dependent, with the maximal activation at 0.01 microM (350+/-15% of control; n=3, P<0.001). A PAK1 fragment encoding the Cdc42/Rac binding domain (amino acid residues 67-150 of hPAK1 termed PBD), was transfected into CHO-AT(1a) cells. PBD transfection markedly reduced 12-HETE-induced PAK1 activation. Furthermore, transfection of dominant negative Cdc42 and Rac1 inhibited 12-HETE-induced PAK1, strongly suggesting that Cdc42 and Rac1 are the upstream activators of 12-HETE-induced PAK1 activation. Low concentrations (1.5 microM) of LY294002, a highly specific inhibitor of phosphoinositide 3-kinase (PI-3K), abolished 12-HETE-induced PAK1 activation, suggesting that PI-3K activation is upstream of 12-HETE-induced PAK1 activation. Transfection of dominant negative PAK1 blocked 12-HETE-induced PAK1, cJun N-terminal kinase (JNK1) and extracellular-signal-regulated kinase (ERK) activity, while transfection of constitutively active PAK1 stimulated PAK1, JNK1 and ERK activity, suggesting that PAK1 is an upstream activator of 12-HETE-induced JNK1 and ERK activation in these cells. We conclude that 12-HETE can activate Cdc42, Rac1 and PI-3K, which then participate as upstream signalling molecules for PAK1 and JNK1 activation.