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.

Summary: Thrombin is a multifunctional serine protease generated at the site of vascular injury that transforms fibrinogen into fibrin, activates blood platelets and elicits multiple effects on a variety of cell types including endothelial cells, vascular smooth muscle cells (VSMC), monocytes,T lymphocytes and fibroblasts. Cellular effects of thrombin are mediated by protease-activated receptors (PARs), members of the G protein-coupled receptors that carry their own ligand which remains cryptic until unmasked by proteolytic cleavage. Thrombin signalling in platelets contributes to haemostasis and thrombosis. In normal arteries PARs are mainly expressed in endothelial cells, while their expression in VSMC is limited. Endothelial PARs participate in the regulation of vascular tone, vascular permeability and endothelial secretory activity while in VSMC they mediate contraction, migration, proliferation, hypertrophy and production of extra-cellular matrix. PARs contribute to the pro-inflammatory phenotype observed in endothelial dysfunction and their up-regulation inVSMC seems to be a key element in the pathogenesis of atherosclerosis and restenosis. In the last years a myriad of studies have emphasized the critical role of PAR signalling in thrombin mediated effects in haemostasis, inflammation, cancer and embryonic development. Lately, PARs have become a therapeutic target to inhibit platelet aggregation and thrombosis. Early data from a clinical trial (TRA-PCI) to evaluate safety and efficacy of a potent new oral thrombin receptor antagonist (TRA) have promisingly indicated that overallTRA treatment reduces adverse event rates without an increase in bleeding risk. In this paper we review cellular responses triggered by thrombin and their implication in vascular pathophysiology.

The molecular mechanisms regulating endothelial cell activation and vascular smooth muscle cell proliferation are critical in the pathological processes underlying atherosclerosis. Numerous growth factors and cytokines trigger the complex and redundant signaling pathways that regulate cell cycle entry; however, the genes controlling these processes are not fully known. Applying techniques for differential gene expression analysis, new transcription factors have been identified in these mechanisms, among them the three members of the NR4A subfamily of nuclear receptors (NRs). These transcription factors (NOR-1, Nur77 and Nurr1) are products of immediate-early genes whose expression and activity is regulated in a cell-specific manner by a variety of extracellular mitogenic, apoptotic and differentiation stimuli. Unlike most NRs whose transcriptional activity is regulated by direct modulatory ligands, NR4A genes do not appear to require ligand binding for activation, and in vascular cells they are highly responsive to growth factors, cytokines, lipoproteins and thrombin. In this review, we discuss our present knowledge on the role of this subfamily of NRs in vascular cell function.

—Foam cell formation is a key event in the onset and progression of atherosclerotic lesions. We have previously reported that internalization of aggregated low density lipoproteins (agLDLs) by vascular smooth muscle cells (VSMCs) produces cholesteryl ester (CE) accumulation in these cells. The aim of this study was to analyze whether the low density lipoprotein receptor–related protein (LRP) mediates the uptake of agLDL by VSMCs. First, immunocytochemistry and fluorescence microscopic analysis with the use of anti-LRP antibodies indicated that there was a high expression of LRP in VSMCs. Confocal microscopic analysis with the use of agLDLs labeled with fluorochrome 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine and anti-LRP antibodies showed the colocalization of agLDL and LRP. The second approach was to analyze the effect of LRP ligands on agLDL internalization; lactoferrin strongly inhibited CE accumulation from agLDLs (85.0±5.7% at 25 μg/mL) by impairing agLDL binding. Coincubation of agLDL with anti-LRP antibodies decreased in a dose-dependent manner agLDL-derived CE accumulation (from 20% at 12.5 μg/mL to 80% at 50 μg/mL). The third approach was to evaluate whether antisense LRP oligodeoxynucleotides were able to block agLDL internalization. Treatment of VSMCs with 5 μmol/L antisense LRP oligodeoxynucleotides reduced agLDL-derived CE accumulation by 84±2%. In conclusion, these results from immunologic, biochemical, and molecular interventions demonstrate that LRP mediates the binding and internalization of agLDL in human VSMCs. Because LRP is highly expressed in VSMCs and the uptake of 1 LDL aggregate amounts to the deposition of several hundreds of LDL particles, the uptake of agLDL through LRP could have a crucial role for lipid deposition in VSMCs.

Aims: Vascular stiffness, structural elastin abnormalities, and increased oxidative stress are hallmarks of hypertension. Lysyl oxidase (LOX) is an elastin crosslinking enzyme that produces H ₂ O ₂ as a by-product. We addressed the interplay between LOX, oxidative stress, vessel stiffness, and elastin. Results: Angiotensin II (Ang II)-infused hypertensive mice and spontaneously hypertensive rats (SHR) showed increased vascular LOX expression and stiffness and an abnormal elastin structure. Mice over-expressing LOX in vascular smooth muscle cells (TgLOX) exhibited similar mechanical and elastin alterations to those of hypertensive models. LOX inhibition with β-aminopropionitrile (BAPN) attenuated mechanical and elastin alterations in TgLOX mice, Ang II-infused mice, and SHR. Arteries from TgLOX mice, Ang II-infused mice, and/or SHR exhibited increased vascular H ₂ O ₂ and O ₂ ^.− levels, NADPH oxidase activity, and/or mitochondrial dysfunction. BAPN prevented the higher oxidative stress in hypertensive models. Treatment of TgLOX and Ang II-infused mice and SHR with the mitochondrial-targeted superoxide dismutase mimetic mito-TEMPO, the antioxidant apocynin, or the H ₂ O ₂ scavenger polyethylene glycol-conjugated catalase (PEG-catalase) reduced oxidative stress, vascular stiffness, and elastin alterations. Vascular p38 mitogen-activated protein kinase (p38MAPK) activation was increased in Ang II-infused and TgLOX mice and this effect was prevented by BAPN, mito-TEMPO, or PEG-catalase. SB203580, the p38MAPK inhibitor, normalized vessel stiffness and elastin structure in TgLOX mice. Innovation: We identify LOX as a novel source of vascular reactive oxygen species and a new pathway involved in vascular stiffness and elastin remodeling in hypertension. Conclusion: LOX up-regulation is associated with enhanced oxidative stress that promotes p38MAPK activation, elastin structural alterations, and vascular stiffness. This pathway contributes to vascular abnormalities in hypertension. Antioxid. Redox Signal. 27, 379–397.