Asad Zeidan

Emerging importance of chemokine receptor CXCR3 and its ligands in cardiovascular diseases

The CXC chemokines, CXCL4, -9, -10, -11, CXCL4L1, and the CC chemokine CCL21, activate CXC chemokine receptor 3 (CXCR3), a cell-surface G protein-coupled receptor expressed mainly by Th1 cells, cytotoxic T (Tc) cells and NK cells that have a key role in immunity and inflammation. However, CXCR3 is also expressed by vascular smooth muscle and endothelial cells, and appears to be important in controlling physiological vascular function. In the last decade, evidence from pre-clinical and clinical studies has revealed the participation of CXCR3 and its ligands in multiple cardiovascular diseases (CVDs) of different aetiologies including atherosclerosis, hypertension, cardiac hypertrophy and heart failure, as well as in heart transplant rejection and transplant coronary artery disease (CAD). CXCR3 ligands have also proven to be valid biomarkers for the development of heart failure and left ventricular dysfunction, suggesting an underlining pathophysiological relation between levels of these chemokines and the development of adverse cardiac remodelling. The observation that several of the above-mentioned chemokines exert biological actions independent of CXCR3 provides both opportunities and challenges for developing effective drug strategies. In this review, we provide evidence to support our contention that CXCR3 and its ligands actively participate in the development and progression of CVDs, and may additionally have utility as diagnostic and prognostic biomarkers.

Adiponectin Attenuates Angiotensin II-Induced Vascular Smooth Muscle Cell Remodeling through Nitric Oxide and the RhoA/ROCK Pathway

Introduction: Adiponectin (APN), an adipocytokine, exerts protective effects on cardiac remodeling, while angiotensin II (Ang II) induces hypertension and vascular remodeling. The potential protective role of APN on the vasculature during hypertension has not been fully elucidated yet. Here, we evaluate the molecular mechanisms of the protective role of APN in the physiological response of the vascular wall to Ang II. Methods and Results: Rat aortic tissues were used to investigate the effect of APN on Ang II-induced vascular remodeling and hypertrophy. We investigated whether nitric oxide (NO), the RhoA/ROCK pathway, actin cytoskeleton remodeling, and reactive oxygen species (ROS) mediate the anti-hypertrophic effect of APN. Ang II-induced protein synthesis was attenuated by pre-treatment with APN, NO donor S-nitroso-N-acetylpenicillamine (SNAP), or cGMP. The hypertrophic response to Ang II was associated with a significant increase in RhoA activation and vascular force production, which were prevented by APN and SNAP. NO was also associated with inhibition of Ang II-induced phosphorylation of cofilin. In addition, immunohistochemistry revealed that 24 h Ang II treatment increased the F- to G-actin ratio, an effect that was inhibited by SNAP. Ang II-induced ROS formation and upregulation of p22phox mRNA expression were inhibited by APN and NO. Both compounds failed to inhibit Nox1 and p47phox expression. Conclusion: Our results suggest that the anti-hypertrophic effects of APN are due, in part, to NO-dependent inhibition of the RhoA/ROCK pathway and ROS formation.

Cigarette Smoking-Induced Cardiac Hypertrophy, Vascular Inflammation and Injury Are Attenuated by Antioxidant Supplementation in an Animal Model

Background: Cardiovascular diseases are the leading causes of morbidity and mortality worldwide. Cigarette smoking remains a global health epidemic with associated detrimental effects on the cardiovascular system. In this work, we investigated the effects of cigarette smoke exposure on cardiovascular system in an animal model. The study then evaluated the effects of antioxidants (AO), represented by pomegranate juice, on cigarette smoke induced cardiovascular injury. This study aims at evaluating the effect of pomegranate juice supplementation on the cardiovascular system of an experimental rat model of smoke exposure. Methods: Adult rats were divided into four different groups: Control, Cigarette smoking (CS), AO, and CS + AO. Cigarette smoke exposure was for 4 weeks (5 days of exposure/week) and AO group received pomegranate juice while other groups received placebo. Assessment of cardiovascular injury was documented by assessing different parameters of cardiovascular injury mediators including: (1) cardiac hypertrophy, (2) oxidative stress, (3) expression of inflammatory markers, (4) expression of Bradykinin receptor 1 (Bdkrb1), Bradykinin receptor 2 (Bdkrb2), and (5) altered expression of fibrotic/atherogenic markers [(Fibronectin (Fn1) and leptin receptor (ObR))]. Results: Data from this work demonstrated that cigarette smoke exposure induced cardiac hypertrophy, which was reduced upon administration of pomegranate in CS + AO group. Cigarette smoke exposure was associated with elevation in oxidative stress, significant increase in the expression of IL-1β, TNFα, Fn1, and ObR in rats aorta. In addition, an increase in aortic calcification was observed after 1 month of cigarette smoke exposure. Furthermore, cigarette smoke induced a significant up regulation in Bdkrb1 expression level. Finally, pomegranate supplementation exhibited cardiovascular protection assessed by the above findings and partly contributed to ameliorating cardiac hypertrophy in cigarette smoke exposed animals. Conclusion: Findings from this work showed that cigarette smoking exposure is associated with significant cardiovascular pathology such as cardiac hypertrophy, inflammation, pro-fibrotic, and atherogenic markers and aortic calcification in an animal model as assessed 1 month post exposure. Antioxidant supplementation prevented cardiac hypertrophy and attenuated indicators of atherosclerosis markers associated with cigarette smoke exposure.

ROS mediates interferon gamma induced phosphorylation of Src, through the Raf/ERK pathway, in MCF-7 human breast cancer cell line.

Interferon gamma (IFN-ɣ) is a pleiotropic cytokine which plays dual contrasting roles in cancer. Although IFN-ɣ has been clinically used to treat various malignancies, it was recently shown to have protumorigenic activities. Reactive oxygen species (ROS) are overproduced in cancer cells, mainly due to NADPH oxidase activity, which results into several changes in signaling pathways. In this study, we examined IFN-ɣ effect on the phosphorylation levels of key signaling proteins, through ROS production, in the human breast cancer cell line MCF-7. After treatment by IFN-ɣ, results showed a significant increase in the phosphorylation of STAT1, Src, raf, AKT, ERK1/2 and p38 signaling molecules, in a time specific manner. Src and Raf were found to be involved in early stages of IFN-ɣ signaling since their phosphorylation increased very rapidly. Selective inhibition of Src-family kinases resulted in an immediate significant decrease in the phosphorylation status of Raf and ERK1/2, but not p38 and AKT. On the other hand, IFN-ɣ resulted in ROS generation, through H2O2 production, whereas pre-treatment with the ROS inhibitor NAC caused ROS inhibition and a significant decrease in the phosphorylation levels of AKT, ERK1/2, p38 and STAT1. Moreover, pretreatment with a selective NOX1 inhibitor resulted in a significant decrease of AKT phosphorylation. Finally, no direct relationship was found between ROS production and calcium mobilization. In summary, IFN-ɣ signaling in MCF-7 cell line is ROS-dependent and follows the Src/Raf/ERK pathway whereas its signaling through the AKT pathway is highly dependent on NOX1.

Mechanical stretch-induced vascular hypertrophy occurs through modulation of leptin synthesis-mediated ROS formation and GATA-4 nuclear translocation.

Background: Obesity and hypertension are associated with increased leptin production contributing to cardiovascular remodeling. Mechanisms involving mechanical stretch-induced leptin production and the cross talk between signaling pathways leading to vascular remodeling have not been fully elucidated. Methods and Results: Rat portal vein (RPV) organ culture was used to investigate the effect of mechanical stretch on leptin protein expression in vascular smooth muscle cells (VSMCs). Moreover, the involvement of reactive oxygen species (ROS), the RhoA/ROCK pathway, actin cytoskeleton dynamics and the transcriptional factor GATA-4 activation in mechanical stretch-induced vascular remodeling were investigated. Stretching the RPV for 1 or 24 h significantly increased leptin protein level and ROS formation in VSMCs, which was prevented by 1 h pretreatment with the ROCK inhibitor Y-27632 and the actin cytoskeleton depolymerization agent cytochalasin D. Moreover, Western blotting and immunohistochemistry revealed that mechanical stretch or treatment with 3.1 nmol/L leptin for 24 h significantly increased actin polymerization, as reflected by an increase in the F-actin to G-actin ratio. Increases in blood vessels’ wet weight and [3H]-leucine incorporation following a 24 h treatment with conditioned media from cultured stretched RPVs indicated RPV hypertrophy. This effect was prevented by 1 h pretreatment with anti-leptin antibody, indicating leptin’s crucial role in promoting VSMC hypertrophy. As an index of GATA-4 activation, GATA-4 nuclear translocation was assessed by immunohistochemistry method. Pretreating VSMC with leptin for 1 h significantly activated GATA-4 nuclear translocation, which was potently attenuated by the NADPH oxidase inhibitor apocynin, Y-27632, and cytochalasin D. Conclusion: Our results demonstrate that ROS formation, RhoA/ROCK pathway, and GATA-4 activation play a pivotal role in mechanical stretch-induced leptin synthesis leading to VSMC remodeling.

Nanotheragnostic Applications for Ischemic and Hemorrhagic Strokes: Improved Delivery for a Better Prognosis

Stroke is the second leading cause of death worldwide and a major cause of long-term severe disability representing a global health burden and one of the highly researched medical conditions. Nanostructured material synthesis and engineering have been recently developed and have been largely integrated into many fields including medicine. Recent studies have shown that nanoparticles might be a valuable tool in stroke. Different types, shapes, and sizes of nanoparticles have been used for molecular/biomarker profiling and imaging to help in early diagnosis and prevention of stroke and for drug/RNA delivery for improved treatment and neuroprotection. However, these promising applications have limitations, including cytotoxicity, which hindered their adoption into clinical use. Future research is warranted to fully develop and effectively and safely translate nanoparticles for stroke diagnosis and treatment into the clinic. This work will discuss the emerging role of nanotheragnostics in stroke diagnosis and treatment applications.

Neuroproteomics and microRNAs studies in multiple sclerosis: transforming research and clinical knowledge in biomarker research

Multiple sclerosis (MS) is a complex disease characterized by extensive phenotypic variability. Biomarkers to capture the different aspects of MS heterogeneity, and to help make a diagnosis and monitor disease progression, while providing insights into etiopathogenesis and response to treatment, are urgently needed. Omics technologies and research efforts with microRNAs have provide unparalleled opportunities for exploring altered protein profiles associated with molecular mechanisms of disease, substantially expanding the list of candidate biomarkers for MS. This review presents evidence from proteomic studies that have focused on identification of biomarkers released in biofluids as a result of the different pathophysiological processes of MS. Also discussed is the emerging role of miRNAs as complementary biomarkers related to cellular processes occurring in MS patients. Also provided is an overview of candidate biomarkers that have been proposed for elucidating pathophysiological processes and disease activity and for guiding clinical diagnosis and/or therapeutic interventions in MS.

Myocardial proteases and cardiac remodeling

Cardiac hypertrophy (CH), characterized by the enlargement of cardiomyocytes, fibrosis and apoptosis, is one of the leading causes of death worldwide. Despite the advances in cardiovascular research, there remains a need to further investigate the signaling pathways that mediate CH in order to identify novel therapeutic targets. One of the hallmarks of CH is the remodeling of the extracellular matrix (ECM). Multiple studies have shown an important role of cysteine proteases and matrix metalloproteinases (MMPs) in the remodeled heart. This review focuses on the role of cysteine cathepins and MMPs in cardiac remodeling.

Characterization and assessment of potential microRNAs involved in phosphate-induced aortic calcification.

Medial artery calcification, a hallmark of type 2 diabetes mellitus and chronic kidney disease (CKD), is known as an independent risk factor for cardiovascular mortality and morbidity. Hyperphosphatemia associated with CKD is a strong stimulator of vascular calcification but the molecular mechanisms regulating this process remain not fully understood. We showed that calcification was induced after exposing Sprague‐Dawley rat aortic explants to high inorganic phosphate level (Pi, 6 mM) as examined by Alizarin red and Von Kossa staining. This calcification was associated with high Tissue‐Nonspecific Alkaline Phosphatase (TNAP) activity, vascular smooth muscle cells de‐differentiation, manifested by downregulation of smooth muscle 22 alpha (SM22α) protein expression which was assessed by immunoblot analysis, immunofluorescence, and trans‐differentiation into osteo‐chondrocyte‐like cells revealed by upregulation of Runt related transcription factor 2 (Runx2), TNAP, osteocalcin, and osteopontin mRNA levels which were determined by quantitative real‐time PCR. To unravel the possible mechanism(s) involved in this process, microRNA (miR) expression profile, which was assessed using TLDA technique and thereafter confirmed by individual qRT‐PCR, revealed differential expression 10 miRs, five at day 3 and 5 at day 6 post Pi treatment versus control untreated aortas. At day 3, miR‐200c, ‐155, 322 were upregulated and miR‐708 and 331 were downregulated. After 6 days of treatment, miR‐328, ‐546, ‐301a were upregulated while miR‐409 and miR‐542 were downregulated. Our results indicate that high Pi levels trigger aortic calcification and modulation of certain miRs. These observations suggest that mechanisms regulating aortic calcification might involve miRs, which warrant further investigations in future studies.

Acute Exposure to Cigarette Smoking Followed by Myocardial Infarction Aggravates Renal Damage in an In Vivo Mouse Model

Cigarette smoking (S) is a risk factor for progressive chronic kidney disease, renal dysfunction, and renal failure. In this study, the effect of smoking on kidney function was investigated in a mouse model of myocardial infarction (MI) using 4 groups: control (C), smoking (S), MI, and S+MI. Histological analysis of S+MI group showed alterations in kidney structure including swelling of the proximal convoluted tubules (PCTs), thinning of the epithelial lining, focal loss of the brush border of PCTs, and patchy glomerular retraction. Molecular analysis revealed that nephrin expression was significantly reduced in the S+MI group, whereas sodium-hydrogen exchanger-1 (NHE-1) was significantly increased, suggesting altered glomerular filtration and kidney functions. Moreover, S+MI group, but not S alone, showed a significant increase in the expression of connective tissue growth factor (CTGF) and fibrotic proteins fibronectin (FN) and α-smooth muscle actin (SMA), in comparison to controls, in addition to a significant increase in mRNA levels of IL-6 and TNF-α inflammatory markers. Finally, reactive oxygen species (ROS) production was significantly accentuated in S+MI group concomitant with a significant increase in NOX-4 protein levels. In conclusion, smoking aggravates murine acute renal damage caused by MI at the structural and molecular levels by exacerbating renal dysfunction.