الفهرس 
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Abstract
Cardiovascular diseases are considered one of the main causes of mortality worldwide, whereby their major denominating hallmark, is endothelial dysfunction (ED). Such dysfunction is the earliest event to occur in pathogenesis of many of diseases, as in case of atherosclerosis. In such disorder, the existing dyslipidemia [a modifiable cardiovascular risk factor] will allow lipid-containing lipoproteins to accumulate in the intima, to disrupt the endothelial quiescent state thus modifying it into a prothrombotic phenotype with development of ED that progresses to atherosclerosis.
In defence, endogenous repair mechanisms must operate in parallel to endothelial injury, in order to counter-act the injurious inflicts posed by dyslipidemia or any other cardiovascular risk factor, to maintain vascular integrity. In such context, it was found that endothelial progenitor cells (EPCs) play a major role in orchestrating such vascular repair processes so as to halt disease progression. Owing to their proliferative capacity, their direct homing into site of arterial injury, their potentiality to differentiate into endothelial lineage cells, aside to their indirect paracrine secretory functions, will all permit EPCs vital contribution to neovascularization of ischemic tissue and/or covering and subsequently regeneration of dysfunctional injured endothelium.
Unfortunately, this is not the case in dyslipidemia, where a significant reduction in EPCs circulating numbers and functional profile is reported to reduce their regenerative capacity which could be due to a reduction in their survival and an increase in their senescence. This is being inflicted by the generated oxidative stress that evolves secondary to excess uptake of oxidized-LDL by EPCs. Additionally, it could be also due to alteration in EPCs microenvironment secondary to the dyslipidemia-induced inflammatory reactions, which impair their ability to participate in vascular repair.
In accordance, it has, for long, been known that NADPH oxidases (NOXs) are the major sources of endothelial reactive oxygen species (ROS). And that they have been accused for inflicting injury that affects cellular lifespan. However, in deviation, now ROS are no more solely considered detrimental but in physiological range, they are rather considered as important signals for proper stem-cell differentiation and renewal. This is being achieved through activation of signaling pathways that maintain vascular homeostasis by controlling endothelial cells phenotype. It was declared that the characteristic gene expression pattern within the EPCs, especially in response to the constitutively expressed endothelial NOX-4, unlike rest of NOXs, activates stress responsive genes. These entail the increase in expression of DNA repair and detoxifying genes to enhance resistance to detrimental environmental stressors.
Despite that, other signalling pathways guiding the EPCs reparative function remain still complex and not fully ravelled. Of concern is the canonical Wnt/-catenin signalling pathway which is generally accepted as a master regulator, crucial to cell fate determination and cell migration. Though, it has been recognized as one of the main regulators of EPCs recruitment and differentiation, yet its interaction particularly with NOX-4 in promoting EPCs regenerative capacity remains unravelled.
Based on the aforementioned background, EPCs became considered a surrogate marker for ED and more importantly an attractive candidate for vascular regenerative therapy. When autologous EPCs were assessed by systemic administration, several obstacles hindered their therapeutic application, so that functional enhancement of endogenous EPCs became a plausible alternative strategy to adopt. One approach of which, enrols the administration of mobilization therapy that intends not only to mimic but also to boost endogenous mobilization of bone marrow EPCs in response to ischemic injury.
In line with this, the current study was designed to assess the EPCs-based vascular protective potentials of some nutritional supplements such as, resveratrol (the nonflavonoid polyphenol with anti-oxidant anti-inflammatory, anti-apoptotic activities), vitamin D3 (fat-soluble vitamin with hormonal/skeletal and pleotropic/non-skeletal effects) and vitamin k2 (the menaquinone, acting as a cofactor for c-glutamyl carboxylase). This is being demonstrated by detecting the effect of studied drugs on endothelial reactivity and EPCs quantification. The molecular mechanism underlying such protection, least present, in relevance to some newly raised vascular signaling reparative pathways as Wnt/-catenin and NOX-4 enzyme expression were also checked. .
The study was carried out on sixty male Wistar rats weighing 200-250g. Rats were weighed and randomly assigned to 2 groups according to the fed diet for 8 weeks.A standard chow, 1.29 KJ/g of diet as fat, was served for normal control rats (n=12) and a high-fat diet (HFD), 7.74 KJ/g of diet as fat, was served for induction of dyslipidemia in rats (n=48). These latter dyslipidemia-induced group was further equally divided into 4 subgroups (12 rats each); the first received resveratrol (10mg/kg/day po), the second received vitamin D3 (125 µg/kg/day po), and the third received vitamin k2 (30 mg/kg/day po) against the non-treated control dyslipidemia-induced group.
By the end of the 8th week, fasting anesthetized rats were weighed then subjected for blood samples collection by cardiac puncture on EDTA-containing tube and stored at 4°C for lipid profile estimation. Then, animals underwent euthanization by an overdose of thiopental. Multiple arterial sections from thoracic aortae were gently dissected and one of the sections were put in fresh Krebs solution for the immediate in-vitro isometric tension studies, other sections were kept at -80°C for later relative quantification of NOX-4 gene expression by real time PCR and the rest sections were preserved in 10% formalin for histopathological and immune-histochemical assessments. And finally, visceral fat was dissected and weighed.
The results of the present study revealed that 8-week HFD induced dyslipidemia retrieved worsening in obesity-related biomarkers and plasma lipid parameters. Simultaneously, a reduction in the number of cells positive for EPCs’ composite surface markers; CD133+/VEGFR-2 +cells, was observed in association with under-expression of both endothelial NOX-4 and -catenin. Such pathophysiological changes contributed to vascular functional impairment and structural damage, which was confirmed with a significant reduction in aortic vascular reactivity to cumulative concentrations of ACh and an elevated atherosclerotic score, respectively.
Administration of resveratrol, vitamin D3, and vitamin k2 for 8 weeks, exhibited a variable endothelial protective profile against HFD-induced dyslipidemia. Resveratrol as well as vitamin D3 more than vitamin K2 preserved serum lipid profile, and EPCs repair potentials evidenced by an increase in their number and function, maintaining vascular structural and functional integrity, in alignment with an upregulation of NOX-4 and β-catenin expression.
Present findings were supported by significant correlative analysis which demonstrated that HFD-induced dyslipidemia compromised EPCs regenerative function as detected by reduced aortic sections reactivity to cumulative concentrations of Ach. A crosstalk between NOX-4 and Wnt/-catenin signaling together with their positive correlation with EPCs composite markers, could mediate the fate of EPCs’ phenotype. Resveratrol attained a robust improvement in both vascular structural integrity and functional endothelial reactivity and even in obesity related biomarkers. Alternatively, vitamin D3 showed a superior preservation of the vascular redox balance through a more significant increase in NOX-4 expression than resveratrol and vitamin k2.On the contrary, vitamin K2 did not markedly enhance vascular reactivity, despite a modest improvement of most studied parameters in exception to the atherogenic profile.
In conclusion, resveratrol and vitamin D3 supplements conferred endothelial protection and halted the atherosclerosis progression in HFD-induced Dyslipidemia. Whereas, vitamin k2 supplement was mainly effective in inhibiting the progression of atherosclerosis. Supplements’ action was achieved via interplay between Wnt/-catenin signalling and endothelial NOX-4 expression that assisted an activation of EPCs repair process improving their number and function.