C-reactive protein (CRP) is a short pentraxin mainly found as a pentamer in the circulation, or as non-soluble monomers CRP (mCRP) in tissues, exerting different functions. This review is focused on discussing the role of CRP in cardiovascular disease, including recent advances on the implication of CRP and its forms specifically on the pathogenesis of atherothrombosis and angiogenesis. Besides its role in the humoral innate immune response, CRP contributes to cardiovascular disease progression by recognizing and binding multiple intrinsic ligands. mCRP is not present in the healthy vessel wall but it becomes detectable in the early stages of atherogenesis and accumulates during the progression of atherosclerosis. CRP inhibits endothelial nitric oxide production and contributes to plaque instability by increasing endothelial cell adhesion molecules expression, by promoting monocyte recruitment into the atheromatous plaque and by enzymatically binding to modified low-density lipoprotein. CRP also contributes to thrombosis, but depending on its form it elicits different actions. Pentameric CRP has no involvement in thrombogenesis, whereas mCRP induces platelet activation and thrombus growth. In addition, mCRP has apparently contradictory pro-angiogenic and anti-angiogenic effects determining tissue remodeling in the atherosclerotic plaque and in infarcted tissues. Overall, CRP contributes to cardiovascular disease by several mechanisms that deserve an in-depth analysis.

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Objective-We studied the impact of native (natCRP) and modified CRP (mCRP) isoforms on platelet adhesion and thrombus growth under arterial flow. Methods and Results-Blood was perfused over type I collagen at a wall shear rate of 1500 s Ϫ1 , and platelet deposition and thrombus growth were evaluated by confocal microscopy. natCRP and mCRP were either incubated with blood before perfusion experiments or immobilized in the collagen surface and exposed to flowing blood. mCRP significantly increased platelet adhesion and thrombus growth when directly incubated with blood and when immobilized on a collagen surface (PϽ0.05). In contrast, natCRP did not exert any effect. Confocal immunohistochemistry revealed the presence of CRP on the surface of adhered platelets and within the thrombus and showed an upregulation of P-selectin and CD36 in effluent platelets preincubated with mCRP (PϽ0.05). Flow cytometry analysis of agonist-induced platelet activation demonstrated that mCRP, but not natCRP, significantly increased platelet surface P-selectin (PϽ0.05) without modifying CD63 and PAC-1. Conclusions-Our data indicate that whereas serum natCRP may not affect thrombus growth, mCRP displays a prothrombotic phenotype enhancing not only platelet deposition, but also thrombus growth under arterial flow conditions. (Arterioscler Thromb Vasc Biol. 2008;28:2239-2246.)Key Words: C-reactive protein Ⅲ isoforms Ⅲ thrombosis Ⅲ platelets I n recent years, C-reactive protein (CRP), long associated with inflammation, has emerged as a clinical marker of future cardiovascular events among apparently healthy subjects and of worse prognosis in acute coronary patients. [1][2][3] Thrombus formation on rupture of an atherosclerotic plaque is believed to be the responsible event for most of the coronary syndromes, in a process mainly mediated by platelet adhesion, activation, and aggregation. The first response to vascular injury consists of platelet adhesion to the damaged vessel wall or to exposed tissue components, and is mediated by flow-regulated interactions that have a key influence on subsequent thrombus growth, often culminating in lifethreatening complications. 4,5 Long considered merely a bystander in vascular disease, new evidence indicates that CRP may be not only a marker, but also an active player in the development of cardiovascular pathology. 6 The role of CRP as a modulator of inflammation and thrombosis is controversial, because both proinflammatory and antiinflammatory properties have been ascribed to the molecule. 7-9 For instance, CRP inhibits neutrophil activation and adhesion, 9 and blocks platelet aggregation in vitro, 10,11 whereas arterial injury in CRP-transgenic mice is associated with increased thrombosis. 12 Overexpression of the human CRP gene in atherosclerosis-prone mice has also shown contradictory effects on the development of atherosclerosis. 13,14 To explain these apparently contradictory actions, it was proposed that distinct isoforms of CRP were formed during inflammation. The classically studied serum CRP ...

PurposeIschemic stroke has shown to induce platelet and endothelial microparticle shedding, but whether stroke induces microparticle shedding from additional blood and vascular compartment cells is unclear. Neural precursor cells have been shown to replace dying neurons at sites of brain injury; however, if neural precursor cell activation is associated to microparticle shedding, and whether this activation is maintained at long term and associates to stroke type and severity remains unknown. We analyzed neural precursor cells and blood and vascular compartment cells microparticle shedding after an acute ischemic stroke.MethodsForty-four patients were included in the study within the first 48h after the onset of stroke. The cerebral lesion size was evaluated at 3–7 days of the stroke. Circulating microparticles from neural precursor cells and blood and vascular compartment cells (platelets, endothelial cells, erythrocytes, leukocytes, lymphocytes, monocytes and smooth muscle cells) were analyzed by flow cytometry at the onset of stroke and at 7 and 90 days. Forty-four age-matched high cardiovascular risk subjects without documented vascular disease were used as controls.ResultsCompared to high cardiovascular risk controls, patients showed higher number of neural precursor cell- and all blood and vascular compartment cell-derived microparticles at the onset of stroke, and after 7 and 90 days. At 90 days, neural precursor cell-derived microparticles decreased and smooth muscle cell-derived microparticles increased compared to levels at the onset of stroke, but only in those patients with the highest stroke-induced cerebral lesions.ConclusionsStroke increases blood and vascular compartment cell and neural precursor cell microparticle shedding, an effect that is chronically maintained up to 90 days after the ischemic event. These results show that stroke induces a generalized blood and vascular cell activation and the initiation of neuronal cell repair process after stroke. Larger cerebral lesions associate with deeper vessel injury affecting vascular smooth muscle cells.

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...

Aims Atherosclerosis, the leading cause of cardiovascular diseases, is driven by high blood cholesterol levels and chronic inflammation. Low-Density Lipoprotein Receptor (LDLR) play a critical role in regulating blood cholesterol levels by binding to and clearing LDLs from the circulation. The disruption of the interaction between Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) and LDLR reduces blood cholesterol levels. It is not well known whether other members of the LDLR superfamily may be targets of PCSK9. The aim of this work was to determine if LDLR-related protein 5 (LRP5) is a PCSK9 target, and to study the role of PCSK9 and LRP5 in foam cell formation and lipid accumulation. Methods and Results Primary cultures of human inflammatory cells (monocytes and macrophages) were silenced for LRP5 or PCSK9 and challenged with LDLs. We first show that LRP5 is needed for macrophage lipid uptake since LRP5-silenced macrophages show less intracellular CE accumulation. In macrophages, internalization of LRP5-bound LDL is already highly evident after 5 hours of LDL incubation and lasts up to 24hours; however in the absence of both LRP5 and PCSK9 there is a strong reduction of CE accumulation indicating a role for both proteins in lipid uptake. Immunoprecipitation experiments show that LRP5 forms a complex with PCSK9 in lipid-loaded macrophages. Finally PCSK9 participates in TLR4/NFkB signaling; a decreased TLR4 protein expression levels and a decreased nuclear translocation of NFκB was detected in PCSK9 silenced cells after lipid loading, indicating a down-regulation of the TLR4/NFκB pathway. Conclusion Our results show that both LRP5 and PCSK9 participate in lipid uptake in macrophages. In the absence of LRP5 there is a reduced release of PCSK9 indicating that LRP5 also participates in the mechanism of release of soluble PCSK9. Furthermore, PCSK9 up-regulates TLR4/NFκB favoring inflammation. Translational Perspective We demonstrate that PCSK9 and LRP5 contribute to lipid uptake. We also show that LRP5 participates in PCSK9 transport to the plasma membrane and that PCSK9 inhibition protects against agLDL-induced inflammation associated to the TLR4/NFκB pathway. These results offer new targets to prevent the progression of inflammation and hypercholesterolemia and their increased risk of cardiovascular events.

Our results indicate that LDLs impair human VSMC migration and wound repair after injury. agLDL, and to a lesser extent nLDL, induce dephosphorylation of MRLC and striking changes in the subcellular localization of P-MRLC, a cytoskeleton protein involved in VSMC migration kinetics.

Objective-Therapeutic angiogenesis is a promising strategy for treating ischemia. Our previous work showed that endogenous endothelial tissue factor (TF) expression induces intracrine signaling and switches-on angiogenesis in microvascular endothelial cells (mECs). We have hypothesized that activated mECs could exert a further paracrine regulation through the release of TF-rich microvascular endothelial microparticles (mEMPs) and induce neovascularization of ischemic tissues. Approach and Results-Here, we describe for the first time that activated mECs are able to induce reparative neovascularization in ischemic zones by releasing TF-rich microparticles. We show in vitro and in vivo that mEMPs released by both wildtype and TF-upregulated-mECs induce angiogenesis and collateral vessel formation, whereas TF-poor mEMPs derived from TF-silenced mECs are not able to trigger angiogenesis. Isolated TF-bearing mEMPs delivered to nonperfused adductor muscles in a murine hindlimb ischemia model enhance collateral flow and capillary formation evidenced by MRI. TF-bearing mEMPs increase angiogenesis operating via paracrine regulation of neighboring endothelial cells, signaling through the β1-integrin pathway Rac1-ERK1/2-ETS1 and triggering CCL2 (chemokine [C-C motif] ligand 2) production to form new and competent mature neovessels. Conclusions-These