No abstract

These results demonstrate the involvement of LRP5 in the innate inflammatory reaction to lipid infiltration in atherosclerosis.

Objective-Tissue factor (TF) triggers arterial thrombosis. TF is also able to initiate cellular signaling mechanisms leading to angiogenesis. Because high cardiovascular risk atherosclerotic plaques show significant angiogenesis, our objective was to investigate whether TF is able to trigger and stabilize atherosclerotic plaque neovessel formation. Methods and Results-In this study, we showed, by real-time confocal microscopy in 3-dimensional basement membrane cocultures, that TF in human microvascular endothelial cells (HMEC-1) and in human vascular smooth muscle cells (HVSMCs) plays an important role in the formation of capillary-like networks. TF silencing in endothelial cells and smooth muscle cells inhibits the formation of tube-like structures with stable phenotype. Using an in vivo model, we observed that TF inhibition in either HMEC-1 or HVSMCs reduced their shared ability to form new capillaries. The phenotypic changes induced by TF silencing were linked to reduced chemokine (C-C motif) ligand 2 (CCL2) expression in endothelial cells. Wound healing and chemotactic assays demonstrated that TF-induced release of CCL2 stimulated HVSMC migration to HMEC-1. Key Words: angiogenesis Ⅲ atherosclerosis Ⅲ cytokines Ⅲ endothelial function Ⅲ vascular biology A therosclerotic plaque angiogenesis, the outgrowth of new capillaries from preexisting vascular networks, is a pathological feature of advanced complicated plaques. 1,2 Interestingly, coronary type VI plaques, according to the American Heart Association classification, are those with higher amount of microvessels and those with higher risk of inducing a clinical cardiovascular event. 3 In advanced plaques, inflammatory cell infiltration and concomitant production of proangiogenic cytokines may be responsible for induction of uncontrolled neointimal microvessel proliferation resulting in production of immature and fragile neovessels. The final stage of microvessel formation occurs when maturation requires the formation of tight endothelial cell-tocell contacts, 4 the downregulation of endothelial proliferation, and the deposition of a basal lamina to which the endothelium tightly adheres, as well as the recruitment of supporting cells to the vessel wall, such as pericytes and smooth muscle cells (SMCs). 5,6 During angiogenesis, the communication between endothelial cells (ECs) and SMCs requires a precise temporal and spatial regulation of pro-and antiangiogenic molecules, but the process is not yet fully understood. Conclusion-Endogenous See accompanying article on page 2364In recent years, it has become clear that the angiogenesis process is highly dependent on components of the blood coagulation cascade. One of these proteins is tissue factor (TF). 7-9 TF is the cellular receptor and cofactor for blood coagulation factor VII (FVII). 10,11 In addition to its primary role in blood coagulation, accumulating evidence has transformed our view of TF from the cellular receptor for activated FVII (FVIIa) to a multifaceted transmembrane signaling receptor. The bioch...

Objective-Hypoxia is considered a key factor in the progression of atherosclerotic lesions. Low-density lipoprotein receptor-related protein (LRP1) plays a pivotal role in the vasculature. The aim of this study was to investigate the effect of hypoxia on LRP1 expression and function in vascular smooth muscle cells (VSMC) and the role of hypoxia-inducible factor-␣ (HIF-1␣). Methods and Results-Real-time polymerase chain reaction and Western blot analysis demonstrated that hypoxia (1% O 2 ) time-dependently induced LRP1 mRNA (maximum levels at 1 to 2 hours) and protein expression (maximum levels at 12 to 24 hours). The delayed hypoxic upregulation of LRP1 protein versus mRNA may be explained by the long half-life of LRP1 protein. Luciferase assays demonstrated that hypoxia and HIF-1␣ overaccumulation induced LRP1 promoter activity and that 2 consensus hypoxia response element sites located at Ϫ1072/Ϫ1069 and Ϫ695/Ϫ692 participate in the induction. Chromatin immunoprecipitation showed the in vivo binding of HIF-1␣ to LRP1 promoter in hypoxic VSMC. Hypoxia effects on LRP1 protein expression were functionally translated into an increased cholesteryl ester (CE) accumulation from aggregated low-density lipoprotein (agLDL) uptake. The blockade of HIF-1␣ expression inhibited the upregulatory effect of hypoxia on LRP1 expression and agLDL-derived intracellular CE overaccumulation, suggesting that both LRP1 overexpression and CE overaccumulation in hypoxic vascular cells are dependent on HIF-1␣. Immunohistochemical analysis showed the colocalization of LRP1 and HIF-1␣ in vascular cells of human advanced atherosclerotic plaques. H ypoxia plays a pivotal role in the pathophysiology of cancer, myocardial infarction, and atherosclerosis. Arterial wall thickness associated with atherosclerotic plaque progression reduces the oxygen supply in certain areas of the vascular intima. 1,2 Hypoxia is present in human advanced atherosclerotic lesions and correlates with the presence of angiogenesis and thrombus. 3,4 Most of the cells in hypoxic areas of atherosclerotic plaques respond by altering the expression of genes involved in vasculogenesis, angiogenesis, inflammation, and lipid deposition. [5][6][7][8][9][10][11] These genes are activated through hypoxia-inducible factor-1 complex (HIF-1). The HIF-1␣ subunit is subjected to quick oxygendependent proteasomal degradation through hydroxylation of proline residues by hydroxylases. [12][13][14] Under low oxygen tensions, HIF-1␣ accumulates and heterodimerizes with the constitutively expressed beta subunit, allowing the HIF-1 complex to bind to hypoxia response element (HRE) sequences in target promoters. 15 It has been reported that HIF-1␣ accumulation in macrophages promotes foam cell formation and atherosclerosis. 16 In experimental models of hypercholesterolemia and hypertension, HIF-1␣ is also associated with vascular smooth muscle cells (VSMC). 17 30 -32 It is unknown whether hypoxia may modulate LRP1 expression in human atherosclerotic plaques and, if so, which transcription ...

Coronary thrombi show rapid dynamic changes both in structure and cell composition as a function of elapsed onset-of-pain-to-PCI time. Aged ischaemic thrombi were more likely to have reduced Pfn-1 content releasing Pfn-1 to the circulation. Onset-of-pain-to-PCI elapsed time in STEMI patients and hence age of occlusive thrombus can be profiled by Pfn-1 levels found in the peripheral circulation.

Aggregated LDL (agLDL) is internalized by LDL receptor-related protein (LRP1) in vascular smooth muscle cells (VSMCs) and human monocyte-derived macrophages (HMDMs). AgLDL is, therefore, a potent inducer of massive intracellular cholesteryl ester accumulation in lipid droplets. The adipocyte differentiation-related protein (ADRP) has been found on the surface of lipid droplets. The objectives of this work were to analyze whether agLDL uptake modulates ADRP expression levels and whether the effect of agLDL internalization on ADRP expression depends on LRP1 in human VSMCs and HMDMs. AgLDL strongly upregulates ADRP mRNA (real-time PCR) and protein expression (Western blot) in human VSMCs (mRNA: by 3.06-fold; protein: 8.58-fold) and HMDMs (mRNA: by 3.5-fold; protein: by 3.71-fold). Treatment of VSMCs and HMDMs with small anti-LRP1-interfering RNA (siRNA-LRP1) leads to specific inhibition of LRP1 expression. siRNA-LRP1 treatment significantly reduced agLDL-induced ADRP overexpression in HMDMs (by 69%) and in VSMCs (by 53%). Immunohystochemical studies evidence a colocolocalization between ADRP/macrophages and ADRP/VSMCs in advanced lipid-enriched atherosclerotic plaques. These results demonstrate that agLDL-LRP1 engagement induces ADRP overexpression in both HMDMs and human VSMCs and that ADRP is highly expressed in advanced lipid-enriched human atherosclerotic plaques. Therefore, LRP1-mediated agLDL uptake might play a pivotal role in vascular foam cell formation.-Llorente-Cortés, V., T. Royo, O. Juan-Babot, and L. Badimon. Adipocyte differentiation-related protein is induced by LRP1-mediated aggregated LDL internalization in human vascular smooth muscle cells and macrophages.

Objective-Our aim was to analyze the regulation of CC Chemokine ligand 20 (CCL20) by LDL in human vascular smooth muscle cells (VSMC). Methods and Results-In asymptomatic subjects, circulating CCL20 levels were higher in patients with hypercholesterolemia (18.5Ϯ3.2 versus 9.1Ϯ1.3 pg/mL; PϽ0.01). LDL induced the expression of CCL20 in VSMC in a dose-and time-dependent manner. Increased levels of CCL20 secreted by LDL-treated VSMC significantly induced human lymphocyte migration, an effect reduced by CCL20 silencing. The upregulation of CCL20 by LDL was dependent on the activation of kinase signaling pathways and NF-B. By site-directed mutagenesis, electrophoretic mobility shift assay, and chromatin immunoprecipitation, we identified a NF-B site (Ϫ80/Ϫ71) in CCL20 promoter critical for LDL responsiveness. Lysophosphatidic acid mimicked the upregulation of CCL20 induced by LDL, and minimal oxidation of LDL increased the ability of LDL to induce CCL20 through a mechanism that involves lysophosphatidic acid receptors. CCL20 was overexpressed in atherosclerotic lesions from coronary artery patients, colocalizing with VSMC. CCL20 was detected in conditioned media from healthy human aorta and its levels were significantly higher in secretomes from carotid endarterectomy specimens. A therosclerosis is essentially an inflammatory chronic disease. [1][2][3] Inflammation is a necessary response to injury and infection. Virtually all cardiovascular risk factors are capable of promoting an inflammatory response; among them, however, elevated levels of plasma cholesterol, in particular LDL, are recognized as one of the most important risk factors for atherosclerosis. 4,5 The inflammatory response involves the coordinated regulation of cell adhesion and migration and the establishment of a chemotactic gradient that guides inflammatory cells to damaged tissues. Key elements in this communication network are cytokines and chemokines, which orchestrate the recruitment, survival, expansion, and effector function of inflammatory cells. 6 -8 Chemokines are a superfamily of structurally related small chemotactic cytokines that control leukocyte function through interactions with their cognate 7-transmembranedomain G protein-coupled receptors. Monocytes/macrophages and T lymphocytes are the most abundant inflammatory cells found in atherosclerotic plaques, 9,10 but also B cells, dendritic cells, and neutrophils contribute to the pathogenesis of atherosclerosis. 9,11,12 Native and modified LDL modulate the expression of key genes involved in the recruitment and trafficking of inflammatory cells including cellular adhesion molecules and chemokines such as monocyte chemotactic protein 1. 4,5,[13][14][15] Recent studies have implicated other chemokines in atherosclerosis and have extended the knowledge about the regulation of chemokines/chemokine receptors on vascular cells, 6 -8 but the complete picture of these molecules involved in atherogenesis is not completely understood. Conclusion-ThisIncreasing data involving innate and adapti...

Cyclin‐dependent kinase 5 (Cdk5) is expressed in neurons, and growing evidence suggests that it can promote neuronal death. Little has been published on Cdk5 expression after stroke in man. In this study, RT‐PCR, western blotting and immunohistochemistry have been employed to study Cdk5 expression in human post‐mortem stroke tissue, and in human neurons, astrocytes and brain endothelial cells exposed to oxygen‐glucose deficiency and reperfusion. Since Cdk5 activation results both from binding to its regulatory subunit p35 and from phosphorylation, expression of both p35 and phosphorylated Cdk5 (p‐Cdk5) was investigated. An increase in the number of neurons, astrocytes and microvessels stained for Cdk5, p‐Cdk5 and p35 occurred in the infarct and penumbra of patients after stroke. Staining became irregular and clumped in the cytoplasm and nuclear translocation occurred. Association of Cdk5 with apoptosis after stroke was found, as Cdk5 colocalized with TUNEL positive neurons and p‐Cdk5 was found in the nucleus of active caspase‐3 stained cells. Similar upregulation in human cerebral cortical foetal neurons, astrocytes and human brain microvascular endothelial cells (HBMEC) subjected to oxygen‐glucose deficiency (OGD) was seen. These results provide evidence for a role of Cdk5 in the events associated with response to ischemic injury. Financial support for this study came from HEFCE and RIHSC.