Alzheimer’s disease (AD) increases dramatically in patients with ischaemic stroke. Monomeric C-reactive protein (mCRP) appears in the ECM of ischaemic tissue after stroke, associating with microvasculature, neurons and AD-plaques, Aβ, also, being able to dissociate native-CRP into inflammatory, mCRP in vivo. Here, mCRP injected into the hippocampal region of mice was retained within the retrosplenial tract of the dorsal 3rd ventrical and surrounding major vessels. Mice developed behavioural/cognitive deficits within 1 month, concomitant with mCRP staining within abnormal looking neurons expressing p-tau and in beta-amyloid 1-42-plaque positive regions. mCRP co-localised with CD105 in microvessels suggesting angiogenesis. Phospho-arrays/Western blotting identified signalling activation in endothelial cells and neurons through p-IRS-1, p-Tau and p-ERK1/2-which was blocked following pre-incubation with mCRP-antibody. mCRP increased vascular monolayer permeability and gap junctions, increased NCAM expression and produced haemorrhagic angiogenesis in mouse matrigel implants. mCRP induced tau244–372 aggregation and assembly in vitro. IHC study of human AD/stroke patients revealed co-localization of mCRP with Aβ plaques, tau-like fibrils and IRS-1/P-Tau positive neurons and high mCRP-levels spreading from infarcted core regions matched reduced expression of Aβ/Tau. mCRP may be responsible for promoting dementia after ischaemia and mCRP clearance could inform therapeutic avenues to reduce the risk of future dementia.
C-reactive protein (CRP) is the most acute-phase reactant serum protein of inflammation and a strong predictor of cardiovascular disease. Its expression is associated with atherosclerotic plaque instability and the formation of immature micro-vessels. We have previously shown that CRP upregulates endothelial-derived Notch-3, a key receptor involved in vascular development, remodelling and maturation. In this study, we investigated the links between the bioactive monomeric CRP (mCRP) and Notch-3 signalling in angiogenesis. We used in vitro (cell counting, wound-healing and tubulogenesis assays) and in vivo (chorioallantoic membrane) angiogenic assays and Western blotting to study the angiogenic signalling pathways induced by mCRP and Notch-3 activator chimera protein (Notch-3/Fc). Our results showed an additive effect on angiogenesis of mCRP stimulatory effect combined with Notch-3/Fc promoting bovine aortic endothelial cell (BAEC) proliferation, migration, tube formation in Matrigel(TM) with up-regulation of phospho-Akt expression. The pharmacological blockade of PI3K/Akt survival pathway by LY294002 fully inhibited in vitro and in vivo angiogenesis induced by mCRP/Notch-3/Fc combination while blocking Notch signalling by gamma-secretase inhibitor (DAPT) partially inhibited mCRP/Notch-3/Fc-induced angiogenesis. Using a BAEC vascular smooth muscle cell co-culture sprouting angiogenesis assay and transmission electron microscopy, we showed that activation of both mCRP and Notch-3 signalling induced the formation of thicker sprouts which were shown later by Western blotting to be associated with an up-regulation of N-cadherin expression and a down-regulation of VE-cadherin expression. Thus, mCRP combined with Notch-3 activator promote angiogenesis through the PI3K/Akt pathway and their therapeutic combination has potential to promote and stabilize vessel formation whilst reducing the risk of haemorrhage from unstable plaques.
Excessive angiogenesis (i.e. neovascularization) in atherosclerotic lesions, sites of dissociation of the inflammatory biomarker pentameric C-reactive protein (pCRP) into monomeric CRP (mCRP), represents a focus of plaque instability with haemorrhagic complications. We previously demonstrated mCRP pro-angiogenic effects on cultured aortic endothelial cells. However, mCRP effects in combination with FGF-2, pro-angiogenic factor released by activated macrophages infiltrating developing lesions, have not yet been described. Here, we examined in vitro the angiogenic capabilities of mCRP combined with FGF-2 by performing endothelial cell proliferation, migration, and differentiation including tube formation and spheroid sprouting assays. The signaling pathways were also investigated by Western blotting and all the cell-based assays were used with or without pharmacological inhibitors of mitogen-activated protein kinase (MAPK), phosphatidylinositol-3 kinase (PI3K) and γ-secretase, considered as key regulators of angiogenesis. We showed that mCRP-induced endothelial cell proliferation and migration required activation of PI3K pathway. MAPK pathway was essential in mCRP-induced endothelial cell proliferation and spheroid sprouting while γ-secretase activity was indispensable for mCRP-induced tube formation only. MAPK pathway was required in all FGF-2-stimulated angiogenic assays whereas γ-secretase slightly inhibited FGF-2 angiogenic effects. PI3K pathway was necessary for FGF-2 angiogenic activities except for cell differentiation. In most of the assays, the additive pro-angiogenic effects of mCRP combined to FGF-2 were mainly attenuated by PI3K and MAPK inhibitors. Altogether, mCRP and FGF-2 have common angiogenic signaling pathways through PI3K and MAPK. Thus, the therapeutic use of PI3K and MAPK inhibitors may inhibit this increased vascularization whilst reducing the haemorrhagic complications from unstable plaques.
Health care workers (HCWs) who have occupational exposure to blood are at increased risk for acquiring blood-borne infections. Aims of the study: To determine the prevalence of HIV among HCWs in five major hospitals in the eastern part of Libya. Material and Methods: A cross-sectional study was conducted in a random sample; this study was done on 601 of HCWs healthcare workers in five major healthcare settings in the eastern part of Libya, during the period between July 2008 to June 2009 rapid methods for HIV, and enzyme-linked immunosorbent assay (ELISA) techniques method and polymerase chain reaction (PCR). A structured and anonymous questionnaire sheet was filled by each of the HCWs through direct personal interviews. After verbal consent 5-10 ml, a blood sample was extracted from each HCW. Results: The majority of HCWs (89.0%) were able to identify that HIV is caused by viruses and not by other microorganisms like bacteria or parasites. Although the majority of HCWs know that contamination with blood or blood products, unsterilized needles or surgical instruments, unsafe sex, folk behaviour like tattooing and piercing, and sharing personal items (e.g. toothbrush, razor, and nail scissors) are essential modes of transmission of these viruses, however many of them were falsely reported that sharing clothes, usage of same toilet and skin contact are also an important mode of transmission of these viruses. Moreover, most HCWs did not believe that the infected persons with these viruses may remain asymptomatic for a long time and always persist for one‘s whole life. More than one-third of HCWs were unaware of any available policy or procedure for reporting sharp injuries in their place of work. Moreover, the majority of HCWs (88.7%) do not use needle removers or needle cutters before disposing of injection pieces of equipment. Furthermore, half of the HCWs do not have sufficient quantities of sharps boxes to dispose of sharps safely in their workplaces. More surprisingly, more than half of the studied HCWs never attend any education or training programs on infection control and prevention during their previous work careers. As for HIV infection, it is almost non-existent (0.0%), Conclusion: The present study showed that no infection by HIV and the lack of educational programs and the lack of post-exposure documentation are concerns. A clear, thoughtful, well-planned and carefully structured risk reduction approach to hospital infection control is needed for this high-risk population.
Angiogenesis is the process of new blood vessel growth from pre-existing vascular structures. The new vessels from atherosclerotic lesions may be a focus of instability, since they facilitate the infiltration of inflammatory cells and due to their tendency to leak, they may produce haemorrhagic complications. Pentameric C-reactive protein (CRP), a strong marker of inflammation, is a risk factor for cardiovascular diseases with a direct role in the development of atherosclerotic lesions. In hypoxic tissue damage, CRP dissociates irreversibly into monomeric CRP (mCRP), which was previously demonstrated to be pro-angiogenic on bovine aortic endothelial cells (BAEC). Our main study was to examine the vessel forming capability of CRP in the presence of other angiogenic factors known to be present in the micro-environment of unstable plaques with immature vasculature. Here we studied the effects of mCRP in presence or absence of FGF-2 on BAEC proliferation, migration, tube formation in Matrigel and on the vascular remodelling using spheroids, a tri-dimensional system of endothelial cell culture embedded in collagen gel. A significant synergic effect of mCRP combined with FGF-2 was observed in all angiogenesis assays used, compared to the effect of mCRP or FGF-2 alone. Using the spheroids, both mCRP and FGF-2 stimulated the length of sprouts with endothelial cells more dispersed giving an aspect of thin structures. For a better understanding of the molecular mechanisms involved, the signalling pathways were investigated by Western blotting and all the assays were performed in the presence or absence of pharmacological inhibitors of MAPK (PD98059), γ-secretase (DAPT inhibitor) and phosphatidylinositol 3-kinase (PI3K) pathways (LY294002). We showed mCRP-induced endothelial cell proliferation, migration and tube formation required activation of the PI3K pathway. MAPK activation was essential in mCRP-induced cell proliferation and differentiation (tube formation and sprouting from the core of spheroids) and γ-secretase activity was required for mCRP-induced tube formation only. For its pro-angiogenic activity, FGF-2 required all of these key pathways with the exception that γ-secretase activity was not associated with FGF-2-induced cell migration. In all assays including the over-expression of phospho-ERK, the synergistic pro-angiogenic effect of mCRP added to FGF-2 was completely inhibited by LY294002. Thus, mCRP and FGF-2 have a common signalling pathway through PI3K and an eventual deregulation of their pro-angiogenic effects due to an excessive inflammation inducing a hyper-vascularisation which could contribute to formation of unstable plaque with haemorrhagic risk, and therefore, might be prevented by targeting the key proteins of the PI3K pathway.
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