No abstract

Native C-reactive protein (nCRP) is a pentameric oligo-protein and an acute phase reactant whose serum expression is increased in patients with inflammatory disease. We have identified by immunohistochemistry, significant expression of a tissue-binding insoluble modified version or monomeric form of CRP (mCRP) associated with angiogenic microvessels in peri-infarcted regions of patients studied with acute ischaemic stroke. mCRP, but not nCRP was expressed in the cytoplasm and nucleus of damaged neurons. mCRP co-localized with CD105, a marker of angiogenesis in regions of revascularisation. In vitro investigations demonstrated that mCRP was preferentially expressed in human brain microvessel endothelial cells following oxygen-glucose deprivation and mCRP (but not column purified nCRP) associated with the endothelial cell surface, and was angiogenic to vascular endothelial cells, stimulating migration and tube formation in matrigel more strongly than fibroblast growth factor-2. The mechanism of signal transduction was not through the CD16 receptor. Western blotting showed that mCRP stimulated phosphorylation of the key down-stream mitogenic signalling protein ERK1/2. Pharmacological inhibition of ERK1/2 phosphorylation blocked the angiogenic effects of mCRP. We propose that mCRP may contribute to the neovascularization process and because of its abundant presence, be important in modulating angiogenesis in both acute stroke and later during neuro-recovery.

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, nϭ19), fibrous (F, nϭ12) and ulcerated (complicated/hemorrhagic) plaques (UC, nϭ7). 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 (Pϭ0.001). UNC also exhibit the highest expression levels of other genes involved in the inflammatory responses: cyclooxygenase-2 (PϽ0.005 versus F and versus UC), IL-6 (PϽ0.005 versus F and versus UC) and monocyte chemoattractant protein-1 (PϽ0.01 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. (Stroke.

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.

Rupture-prone unstable arterial plaques develop concomitantly with the appearance of intraplaque hemorrhage and tissue ulceration, in association with deregulation of smooth muscle cell mitogenesis and leakage of newly formed blood vessels. Using microarray technology, we have identified novel protein deregulation associated with unstable carotid plaque regions. Overexpression of proapoptotic proteins caspase-9 and TRAF4 was seen in endothelial cells and smooth muscle cells from unstable hemorrhagic and ulcerated plaque regions. Topoisomerase-II-␣ (TOPO-II-␣), which is associated with DNA repair mechanisms, was also overexpressed by these cells. Cell signaling molecules c-src, G-protein-coupled receptor kinase-interacting protein (GIT1), and c-jun N-terminal kinase (JNK) were up-regulated in endothelial cells from the same areas, whereas an increase in expression of junctional adhesion molecule-1 (JAM-1) in blood vessels and infiltrating macrophages from inflammatory regions might form part of a leukocyte rolling response, increasing the plaque volume. Grb2-like adaptor protein (Gads), responsible for differentiation of monocytes into macrophages, was expressed by macrophages from unstable plaques, suggesting a potential mechanism through which increased scavenging could occur in ruptureprone areas. We conclude that modulation of novel cell signaling intermediates, such as those described here, could be useful in the therapy of angiogenesis and apoptosis, designed to reduce unstable plaque formation.

The physiologic properties of the normal cellular prion protein (PrPC) have not been established fully, although recent evidence showed its upregulation in cerebral ischaemia. Using patients, animal models, and in vitro studies we aimed to identify in detail the expression and localization of PrPC in ischemic stroke. Patients in acute phase of ischaemic stroke had increased plasma levels of circulating PrPC as compared to healthy age‐ and gender‐matched controls (3.1 ± 1.4 vs. 1.9 ± 0.7 ng/ml, P = 0.002). Immunohistochemistry showed increased expression of PrPC in the soma of peri‐infarcted neurones as well as in the endothelial cells (EC) of micro‐vessels and inflammatory cells in peri‐infarcted brain tissue from patients who survived for 2–34 days after an initial stroke. The same pattern was repeated 1–48 hr after MCAO. RT‐PCR showed increased gene expression of PrPC by human foetal neurons (HFN) after 12 hr of oxygen glucose deprivation (OGD), which remained increased after 24 hr reperfusion. Western blotting confirmed that protein expression was similarly upregulated, and fluorescent labeling showed a notable increase in peri‐nuclear and axonal PrPC staining intensity. Increased plasma PrPC seems to reflect endogenous expression in acute stroke‐affected brain tissue. Increased cellular expression in peri‐infarcted regions may influence hypoxia‐induced cell damage, although the effects on EC survival and angiogenesis remain to be elucidated. © 2006 Wiley‐Liss, Inc.

Hypoxia, angiogenesis and inflammation leads to plaque progression and remodelling and may significantly contribute towards plaque rupture and subsequent cerebrovascular events. Our aim was to study, markers of hypoxia and inflammation previously identified by microarray analysis, in atherosclerotic carotid arteries with low to moderate stenosis. We hoped to describe different cellular populations expressing the studied markers. The location of selected inflammatory molecules obtained as vascular transplants from organ donors were analysed by immunohistochemistry with monoclonal and polyclonal antibodies. Paraffin-embedded sections were cut and probed with antibodies recognizing active B and T-lymphocytes (CD30), hypoxia-inducible factor-1alpha, endoglin (CD105), Interleukin-6 and C-reactive protein. We observed a notable overexpression of HIF-1alpha in inflammatory and hypoxic areas of carotid arteries in all types of lesions from type II-V taken from the patients with carotid stenosis less than 50%. This suggests that HIF-1alpha may have a putative role in atherosclerosis progression and angiogenesis. Dynamic changes in the non-occluding plaques may explain some of the clinical events in patients with low to moderate carotid stenosis.

Carotid atherosclerosis is a leading cause of cerebrovascular events. The control of cardiovascular risk factors, i.e. tobacco smoking, alcohol abuse, hypertension, dyslipidemia, diabetes and obesity proved to reduce number of fatal and non-fatal strokes but failed to prevent important number of them. Screening for biomarkers in individuals at high risk of symptomatic vascular disease helped to identify some of them. However, as disease is by its nature multifocal, global testing for biomarkers may have limited practical application. New imaging techniques, including direct visualization of artery metabolism, by 18-FDG-PET, has brought new tools to study local atherosclerosis progression and individual plaque metabolic activity. Advances in molecular biology helped to identify inflammatory genes and its strong link to angiogenesis. The later, is thought to play a key role in the transformation to unstable plaque. Studies of the complex role that plays angiogenesis in plaque development will help in future to design effective therapies addressed at the individual cell level. The purpose of the review is to bring new insights into complicated pathophysiology of carotid atherosclerosis.