José Martínez-González

Regulation of lysyl oxidase in vascular cells: lysyl oxidase as a new player in cardiovascular diseases.

Lysyl oxidase (LOX) plays a crucial role in the maintenance of extracellular matrix stability and could participate in vascular remodelling associated with cardiovascular diseases. Evidence from in vitro and in vivo studies shows that LOX downregulation is associated with the endothelial dysfunction characteristic of earlier stages of the atherosclerotic process. Conversely, upregulation of this enzyme in vascular cells could induce neointimal thickening in atherosclerosis and restenosis. In fact, LOX is chemotactic for vascular smooth muscle cells and monocytes, is modulated by proliferative stimulus in these cells, and could control other cellular processes such as gene expression and cell transformation. Furthermore, it is conceivable that LOX downregulation could underlie plaque instability and contribute to the destructive remodelling that takes place during aneurysm development. Overall, LOX could play a key role in vascular homeostasis and, hence, it emerges as a new player in cardiovascular diseases. This review addresses the experimental evidence related to the role of LOX in vascular disorders and the potential benefits of controlling its expression and function.

Neuron-Derived Orphan Receptor-1 (NOR-1) Modulates Vascular Smooth Muscle Cell Proliferation

Abstract— Vascular smooth muscle cells (VSMCs) migration and proliferation play a key role in the pathophysiology of cardiovascular disease. However, the transcription factors that regulate VSMC activation are not completely characterized. By a mRNA-differential display approach, we have identified neuron-derived orphan receptor-1 (NOR-1), a transcription factor within the NGFI-B subfamily of nuclear receptors, as a immediate-early gene in VSMCs. Two NOR-1 isoforms (&agr; and &bgr;) were identified and cloned from serum-induced porcine VSMC that shared high homology with the human isoforms. Northern blot analysis revealed a strong and transient (1 to 6 hours) upregulation of NOR-1 in both porcine and human coronary SMCs by growth factors (serum, platelet-derived growth factor-BB, and epidermal growth factor) and &agr;-thrombin but not by cytokines. NOR-1 upregulation is processed through G protein–coupled receptors and tyrosine kinase receptors, and involves Ca2+ mobilization, protein kinase C activation, and the mitogen-activated protein kinase pathway. This induction was closely dependent of the cAMP response elements present in NOR-1 promoter as transfection assays indicate. Human coronary atherosclerotic lesions overexpress NOR-1, and balloon angioplasty transiently induces NOR-1 in porcine coronary arteries with a pattern similar to that observed in VSMCs in culture. Antisense oligonucleotides against NOR-1 inhibited human coronary SMC proliferation (reduced de novo DNA synthesis, cell cycle progression, and VSMC wound repair) as efficiently as antisense against the protooncogene c-fos. These results show that NOR-1 modulates VSMC proliferation, and suggest that this transcription factor may play a role in both spontaneous and accelerated atherosclerosis.

3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibition Prevents Endothelial NO Synthase Downregulation by Atherogenic Levels of Native LDLs: Balance Between Transcriptional and Posttranscriptional Regulation

Abstract—Atherogenic levels of native low density lipoproteins (nLDLs) decrease the bioavailability of endothelium-derived NO and downregulate endothelial NO synthase (eNOS) expression in cultured human endothelial cells. Here, we show that simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, within the therapeutic range (0.01 to 1 &mgr;mol/L) prevented the downregulation of eNOS mRNA and protein promoted by nLDL (180 mg cholesterol/dL, 48 hours) in human umbilical vein endothelial cells. This effect of simvastatin was completely reversed by mevalonate, the product of the reaction, and to a lesser extent by farnesol and geranyl geraniol. Simvastatin significantly stabilized eNOS mRNA in cells treated with nLDL during 48 hours (eNOS mRNA half-life ≈11 hours in controls versus >24 hours in nLDL per 0.1 &mgr;mol/L simvastatin–treated cells). The downregulation of eNOS by nLDL was abrogated by cycloheximide, an inhibitor of protein synthesis, and by N-acetyl-leucyl-leucyl-norleucinal, a protease inhibitor that reduces the catabolism of sterol regulatory element binding proteins. Sterol deprivation increased the downregulation produced by nLDL on eNOS and sterol regulatory element binding protein-2 expression levels. However, no differential modulation of the retardation bands corresponding to the putative sterol-responsive element present in the eNOS promoter was detected by electrophoretic mobility shift assay. Our results suggest that nLDL promote eNOS downregulation operating at a transcriptional level, whereas simvastatin prevents such an effect through a posttranscriptional mechanism.

Endogenous Expression of C-Reactive Protein Is Increased in Active (Ulcerated Noncomplicated) Human Carotid Artery Plaques

Background and Purpose— There is growing evidence suggesting that C-reactive protein (CRP) is an effecter molecule able to induce and promote atherothrombosis. The presence of CRP in atherosclerotic plaques may reflect local production or infiltration from circulating CRP increased in general inflammatory responses. Our aim was to analyze the presence of CRP in human advanced carotid artery plaques with differential anatomo-pathological characteristics and to assess local expression of CRP and other proinflammatory genes in these lesions. Methods— Human carotid artery specimens from 38 patients undergoing scheduled endarterectomy were classified into 3 groups: ulcerated (noncomplicated) (UNC, n=19), fibrous (F, n=12) and ulcerated (complicated/hemorrhagic) plaques (UC, n=7). The presence of CRP was evaluated by immunohistochemistry, and plasma samples were screened for circulating high-sensitivity C-reactive protein. TaqMan Low-density Arrays were used for study of genes related to inflammation (CRP, interleukin-6, macrophage colony-stimulating factor-1, monocyte chemotactic protein-1, cyclooxygenase-2). Results— CRP mRNA levels were predominantly detected in UNC-high risk plaques but not in UC (P=0.001). UNC also exhibit the highest expression levels of other genes involved in the inflammatory responses: cyclooxygenase-2 (P<0.005 versus F and versus UC), IL-6 (P<0.005 versus F and versus UC) and monocyte chemoattractant protein-1 (P<0.01 versus F and versus UC). Plaque CRP mRNA levels correlated with immunohistochemical findings but were independent of plasma high-sensitivity CRP. In UNC plaques endothelial cells and inflammatory cells were strongly positive for CRP around areas of newly formed microvessels. Conclusions— In human high-risk carotid artery plaques (UNC) CRP expression reflects an active proinflammatory stage. Local synthesis of CRP could be involved in plaque neovascularization and increased risk of hemorrhagic transformation.

HMG-CoA reductase inhibitors reduce vascular monocyte chemotactic protein-1 expression in early lesions from hypercholesterolemic swine independently of their effect on plasma cholesterol levels

Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase are widely used in the treatment of dyslipemias and have shown beneficial effects in primary and secondary prevention of cardiovascular diseases. There is new information that seems to suggest that the beneficial effects observed may not be solely attributable to plasma cholesterol reduction. Our objective has been to evaluate the effect of two statins at similar dose, although unequivalent plasma lipid lowering potential, on vessel wall expression of two proteins involved in atherosclerotic lesion progression. We have studied the effects of treatment on vessel wall expression of monocyte chemotactic protein-1 (MCP-1) and the inducible form of nitric oxide synthase (NOS II). Atherosclerosis was induced in pigs by feeding a high cholesterol and saturated fatty acid diet for 50 days. Mild atherosclerotic lesions were found at this early stage of induction. Animals were simultaneously treated with atorvastatin (3 mg/kg/day), pravastatin (3 mg/kg/day) or placebo. Non-HDL-cholesterol levels induced by diet were reduced in the atorvastatin-treated group (63+/-8%, P=0.03) and not as much in the pravastatin treated group (44+/-3, P=0.08). The average MCP-1 expression in carotid, femoral and thoracic aorta was significantly reduced with both statins by 37% (P<0.05), while NOS II expression was unaffected. Therefore, vascular MCP-1 expression was downregulated by statins regardless of their lipid lowering potential and lipo/hydrophilic characteristics. Early downregulation of MCP-1 could attenuate the inflammation within the vascular wall and prevent the development of atherosclerotic lesions.

Involvement of Neuron-Derived Orphan Receptor-1 (NOR-1) in LDL-induced Mitogenic Stimulus in Vascular Smooth Muscle Cells: Role of CREB

Objective—Low density lipoproteins (LDLs) modulate the expression of key genes involved in atherogenesis. Recently, we have shown that the transcription factor neuron-derived orphan receptor-1 (NOR-1) is involved in vascular smooth muscle cell (VSMC) proliferation. Our aim was to analyze whether NOR-1 is involved in LDL-induced mitogenic effects in VSMC. Methods and Results—LDL induced NOR-1 expression in a time- and dose-dependent manner. Antisense oligonucleotides against NOR-1 inhibit DNA synthesis induced by LDL in VSMCs as efficiently as antisense against the protooncogene c-fos. The upregulation of NOR-1 mRNA levels by LDL involves pertusis-sensitive G protein–coupled receptors, Ca2+ mobilization, protein kinases A (PKA) and C (PKC) activation, and mitogen-activated protein kinase pathways (MAPK) (p44/p42 and p38). LDL promotes cAMP response element binding protein (CREB) activation (phosphorylation in Ser133). In transfection assays a dominant-negative of CREB inhibits NOR-1 promoter activity, while mutation of specific (cAMP response element) CRE sites in the NOR-1 promoter abolishes LDL-induced NOR-1 promoter activity. Conclusions—In VSMCs, LDL-induced mitogenesis involves NOR-1 upregulation through a CREB-dependent mechanism. CREB could play a role in the modulation by LDL of key genes (containing CRE sites) involved in atherogenesis.

Sustained long-term improvement of arterial endothelial function in heterozygous familial hypercholesterolemia patients treated with simvastatin

Patients with heterozygous familial hypercholesterolemia (hFH) are at very high risk for premature coronary heart disease. In the last decade, treatment with statins has reduced cardiovascular mortality in these patients. The aim of this study was to analyze arterial endothelial function assessed as flow-mediated dilatation (FMD) and soluble E-selectin (sE-selectin) levels in patients with hFH under a long-term lipid-lowering treatment. Twenty-five patients who completed the study received a dose of simvastatin to achieve a treatment goal of at least 30% reduction in serum low-density lipoprotein (LDL)-cholesterol (LDL-C) for 52 weeks. Functional and biochemical measurements were taken at entry, and at week 12 and 52 of treatment. FMD was measured by vascular ultrasound of the brachial artery. sE-selectin, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 were determined by enzyme linked immunosorbent assay (ELISA). LDL-C levels were significantly reduced by treatment at week 12 and maintained at week 52 (reduction vs. baseline, 44+/-12 and 43+/-11%, respectively, P<0.0001). A significant improvement in endothelial function, measured as FMD (baseline, 4.7+/-6.2%; 12 weeks, 12.3+/-5.9%; 52 weeks, 9.7+/-4.7%; P<0.005) and a reduction in sE-selectin levels (baseline, 16.2+/-3.4 ng/ml; 12 weeks, 11.0+/-3.2 ng/ml; 52 weeks, 12.3+/-4.2 ng/ml; P<0.01) were observed. Endothelial-independent relaxation induced by nitroglycerin was not modified during the study. Our results indicate that a long-term treatment with simvastatin produced a sustained beneficial effect in endothelial function in hFH patients.

C-reactive protein exerts angiogenic effects on vascular endothelial cells and modulates associated signalling pathways and gene expression

BackgroundFormation of haemorrhagic neovessels in the intima of developing atherosclerotic plaques is thought to significantly contribute to plaque instability resulting in thrombosis. C-reactive protein (CRP) is an acute phase reactant whose expression in the vascular wall, in particular, in reactive plaque regions, and circulating levels increase in patients at high risk of cardiovascular events. Although CRP is known to induce a pro-inflammatory phenotype in endothelial cells (EC) a direct role on modulation of angiogenesis has not been established.ResultsHere, we show that CRP is a powerful inducer of angiogenesis in bovine aortic EC (BAEC) and human coronary artery EC (HCAEC). CRP, at concentrations corresponding to moderate/high risk (1–5 μg/ml), induced a significant increase in proliferation, migration and tube-like structure formation in vitro and stimulated blood vessel formation in the chick chorioallantoic membrane assay (CAM). CRP treated with detoxi-gel columns retained such effects. Western blotting showed that CRP increased activation of early response kinase-1/2 (ERK1/2), a key protein involved in EC mitogenesis. Furthermore, using TaqMan Low-density Arrays we identified key pro-angiogenic genes induced by CRP among them were vascular endothelial cell growth factor receptor-2 (VEGFR2/KDR), platelet-derived growth factor (PDGF-BB), notch family transcription factors (Notch1 and Notch3), cysteine-rich angiogenic inducer 61 (CYR61/CCN1) and inhibitor of DNA binding/differentiation-1 (ID1).ConclusionThis data suggests a role for CRP in direct stimulation of angiogenesis and therefore may be a mediator of neovessel formation in the intima of vulnerable plaques.

Mevalonate deprivation impairs IGF-I/insulin signaling in human vascular smooth muscle cells.

3-Hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) reductase inhibitors (statins) are therapeutically used to lower plasma cholesterol levels. In addition, these drugs can block vascular smooth muscle cell (VSMC) proliferation. The present study addressed the question whether the inhibitory effect of lovastatin on premitotic DNA synthesis correlates with a downregulation of c-fos mRNA levels, a marker of signaling efficiency, in human SMC. Here we show that in human SMC exposed to individual growth factors (platelet-derived growth factor, epidermal growth factor, alpha-thrombin, insulin, insulin-like growth factor I (IGF-I)) and human serum, the maximal [3H]thymidine incorporation and c-fos mRNA expression are closely correlated. Only alpha-thrombin elicited overexpression of c-fos as compared with its effect on [3H]thymidine incorporation. Lovastatin efficiently inhibited [3H]thymidine uptake promoted by all mitogens tested (76-87%); however, it significantly inhibited upregulation of c-fos mRNA levels induced only by insulin (33-67%, P < 0.05) and IGF-I (31 57%, P < 0.05). This inhibition was overcome by mevalonate and geranylgeraniol, and partially by farnesol. c-fos mRNA expression induced by 4-beta-phorbol-12-myristate-13-acetate, an activator of protein kinase C, was insensitive to lovastatin treatment. Thus, in human vascular SMC, lovastatin impairs premitotic DNA synthesis induced by growth factors, but only c-fos expression promoted by insulin and IGF-I. These data indicate that statin-sensitive and -insensitive pathways seem to be involved in the regulation of c-fos in the response of human SMC to proliferative stimuli, and suggest a prominent role of isoprenylated proteins in the activation of VSMC through the IGF-I/insulin dependent pathways.

Regulatory Effects of HDL on Smooth Muscle Cell Prostacyclin Release

One mechanism by which high density lipoproteins (HDLs) exert their protective effect against coronary artery disease could be related to the induction of prostacyclin (PGI(2)) release in the vessel wall. We have recently shown that HDL increases PGI(2) production in rabbit smooth muscle cells (RSMCs) and that this increase is dependent on cyclooxygenase-2 (Cox-2). Here we analyze the mechanism by which rabbit HDL induces PGI(2) release in RSMCs. Our results show that although HDL(2) and HDL(3) share a similar capacity to induce Cox-2 protein levels, HDL(3) stimulates a higher PGI(2) release than does HDL(2), probably because of their relative arachidonate contents. Acetylsalicylic acid pretreatment (300 micromol/L, 30 minutes) significantly reduced the HDL-induced PGI(2) release, suggesting that both preexisting and induced Cox-2 activities were involved in the HDL effect. Ca(2+)-dependent cytosolic phospholipase A(2) (cPLA(2)) and Cox-1 protein levels were not altered by HDL. Dexamethasone (2 micromol/L), which also inhibited the HDL-induced PGI(2) release, reduced significantly both Cox-2 mRNA and protein levels without affecting cPLA(2) and Cox-1 protein levels. In addition, methylarachidonyl fluorophosphonate, a potent inhibitor of cPLA(2), did not produce any effect on HDL-induced PGI(2) release. In the presence of cycloheximide, Cox-2 mRNA levels were induced by HDL and inhibited by dexamethasone, suggesting that HDL and dexamethasone work in the absence of de novo protein synthesis. These results indicate an early effect of HDL on PGI(2) biosynthesis, specifically increasing Cox-2. PD98059, an inhibitor of mitogen-activated protein kinase kinase, completely inhibited HDL-induced PGI(2) release, whereas GF109203X, a protein kinase C inhibitor, had no effect. Thus, HDL induces PGI(2) synthesis by a mechanism dependent on the mitogen-activated protein kinase pathway but independent of protein kinase C.