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Foam cell formation is an important event in atherosclerosis. Fisetin, a bioflavonoid, has been identified to possess anti-inflammatory, antilipidemic, and anticancerous properties; however, its role as a lipid homeostasis regulator in macrophages, specifically in the presence of metabolic stressors such as oxidized low-density lipoprotein (oxLDL) is not well understood. In this study, we have investigated the role of fisetin in preventing oxLDL-induced macrophage foam cell formation. U937-derived macrophages were stimulated with oxLDL with or without fisetin for varied time points, and various parameters were assessed including cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay; reactive oxygen species (ROS) by dichlorofluorescin diacetate assay; lipid accumulation by Oil Red O staining; and expression of NLR family pyrin domain containing 3 (NLRP3), sterol regulatory element-binding protein (SREBP)-1, and associated downstream proteins 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) and fatty acid synthase (FAS) by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunoblotting. Functionality of FAS enzyme was determined using enzyme activity assay. Docking studies were performed to determine the in silico interaction between NLRP3 and fisetin. The results showed that fisetin up to the dose of 10 µM did not alter cell viability but at the same dose could decrease the accumulation of lipids in macrophages and prevented foam cell formation. Fisetin could also ameliorate and reduce oxLDL-induced upregulation of SREBP-1 and thereby the expression of its downstream lipid synthesis genes HMGCR and FAS and inhibited ROS-induced NLRP3 inflammasome activation. In conclusion, fisetin could inhibit foam cell formation by blocking oxLDL-induced ROS formation and subsequent NLRP3 activation, thereby inhibiting SREBP-1 and its downstream genes including FAS and HMGCR.

During metabolic disorders, the adipocytes increase in number and size along with the alteration in their functions. They secrete various types of chemical factors which contribute to inflammation and various physiological dysregulations. The adipose tissue acts as a reservoir of the immune cells such as macrophages, T-cells, and B-cells that maintain the homeostasis of body’s metabolic processes. However, the dysfunctional adipocytes and overpopulated immune cells secrete various cytokines and chemokines which promote low-grade chronic inflammation in the body causing the development of several metabolic diseases including diabetes, vascular diseases, and cancer.

Extra-cellular signal regulated kinase-5 (Erk-5), a transcriptional activator and regulator of endothelial cells (ECs) homeostasis, has been implicated in shear stress-induced endothelial dysfunction (ED), however its role in oxidized low-density lipoprotein (oxLDL)- induced ED during metabolic stress is not known. Herein, regulation and function of Erk-5 in oxLDL-induced EC death, inflammation and dysfunction has been investigated. Primary Human Umbilical Vein Endothelial Cells (pHUVECs) were stimulated with oxLDL. MTT and Trypan blue exclusion assays to assess cell viability, RT-qPCR and Western blotting assays to determine expression of endothelial and inflammatory markers and ED mediators at mRNA and protein levels, respectively were performed. Monocyte adhesion assay was performed to examine monocytes adherence to oxLDL-stimulated pHUVECs. The exposure of oxLDL induced a dose- and time-dependent decrease in pHUVECs viability, which concurred with decreased Erk-5 expression. Further, oxLDL (100 μg/ml) decreased the expression of endothelial markers eNOS and vWF, and increased the expression of ICAM-1, at both mRNA and protein levels. SiRNA-mediated silencing of Erk-5 or its inhibition showed that changes in eNOS, vWF and ICAM-1 expression could be mediated through Erk-5. Furthermore, oxLDL decreased the levels of Erk-5's upstream regulator MEK5 and downstream regulators Mef2c and KLF2, which were similar to their expressions in Erk-5 silenced cells. Fisetin, a phytochemical and bioflavonoid, could reduce the effect of oxLDL in ECs by upregulating the expression of endothelial markers including Erk-5, and downregulating the expression of inflammation markers. These results suggest that Erk-5 could be a critical regulator of oxLDL-induced EC death, inflammation and dysfunction via downregulation of Erk-5/Mef2c-KLF2 signaling pathway, which can be ameliorated by a bioflavonoid, fisetin.

Macrophage foam cell formation (FCF) has long been known to play a critical role during atherosclerotic plaque development. In the presence of atherogenic molecules such as oxidized low-density lipoprotein (oxLDL) macrophages accumulate massive amounts of lipid through uptake. However, in the presence of oxLDL mechanism of dysregulated lipid homeostasis in the macrophages remains largely unknown. Herein we have investigated the role of Sterol regulatory element binding protein (SREBP)-1 in oxLDL-induced inflammation and altered lipid homeostasis in macrophages. The U937 monocytes and monocyte-derived macrophages (MDMs) were stimulated with different doses of oxLDL. MTT assay to study the effect of oxLDL on cell viability, Oil-Red-O (ORO) staining to observe cytosolic lipid accumulation, semi-quantitative PCR and Western blotting to analyze mRNA and protein expressions, respectively, and spectrophotometric assay to measure the lipid synthesizing enzyme's activity were performed. Our results indicate that oxLDL increased proliferation in monocytes and decreased the viability in MDMs in a time- and dose-dependent manner. The oxLDL (100 μg/ml) enhanced lipid accumulation via increased expressions of SREBP-1 and its downstream proteins such as fatty acid synthase (FAS) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) at both RNA and protein levels in monocytes as well as in MDMs. Inhibiting SREBP-1 by a synthetic inhibitor prevented excessive lipid accumulation by downregulating the expression of its downstream proteins. Further, oxLDL increased reactive oxygen species (ROS) levels, NLRP3 inflammasome activation and active interleukin 1β (IL-1β) release in both the cell types. The oxLDL-induced NLRP3 could be responsible for SREBP-1 and downstream proteins overexpression as siRNA silencing of NLRP3 decreased SERBP-1 levels. In summary, we have demonstrated that SREBP-1 could be a key player in oxLDL-induced excessive lipid accumulation leading to macrophage FCF via ROS-mediated NLRP3/IL-1β/SREBP-1 pathway.

Cardiovascular diseases (CVDs) are the most prevalent cause of morbidity and mortality worldwide. Oxidative stress and chronic inflammation are the major causes of CVDs. In the vasculature, inflammation occurs due to injury, oxidation of lipids, infection, etc. that result in damage to cells of the blood vessels and progression towards plaque formation. Inflammation portrays an important role in all stages of plaque formation which leads to cardiac arrest or heart failure, peripheral vascular diseases and stroke and could be fatal. Unlike plaque formation, the birth defect in cardiovascular system also leads to short and poor quality of life. Among all preventive and therapeutic strategies for CVDs, herbal interventions are known to be effective with little or no side effects. Herbal interventions have been in use for medical treatments from the beginning of civilisation. WHO report states that approximately 80% of the global population depends on the herbal resources, of which many have moved to clinical use in the recent times. Phytochemicals have a wide array of properties especially anti-inflammatory, anti-oxidative and anti-lipidaemic which are effective in preventing and treating CVDs. They increase the expression of antioxidant and anti-inflammatory molecules mainly via nuclear factor erythroid 2-related factor 2 (Nrf2) activation and reduce the levels of cholesterol, low-density lipoprotein (LDL) and inflammatory cytokines, hence effective in treatment of CVDs. In this chapter, herbal derivative-induced alteration in the mechanisms pertaining to pathophysiology of CVDs has been discussed.

Context: Atherosclerosis is a progressive pathological process and a leading cause of mor-tality worldwide. Clinical research and epidemiological studies state that atherosclerosis is caused by an amalgamation of metabolic and inflammatory deregulation involving three important pathological events including Endothelial Dysfunction (ED), Foam Cell Formation (FCF), and Vascular Smooth Muscle Cells (VSMCs) proliferation and migration. Objectives: Research in recent years has identified Metabolic Syndrome (MS), which involves factors such as obesity, insulin resistance, dyslipidemia and diabetes, to be responsible for the pathophysiol-ogy of atherosclerosis. These factors elevate oxidative stress and inflammation-induced key signalling molecules and various microRNAs (miRs). In present study, we have reviewed recently identified molecular targets in the pathophysiology of atherosclerosis. Methods: Scientific literature obtained from databases such as university library, PubMed and Google along with evidences from published experimental work in relevant journals has been sum-marized in this review article. Results: The molecular events and cell signalling implicated in atherogenic processes of ED, FCF and VSMCs hyperplasia are sequential and progressive, and involve cross talks at many levels. Specific molecules such as transcription factors, inflammatory cytokines and chemokines and miRs have been identified playing crucial role in most of the events leading to atherosclerosis. Conclusion: Studies associated with MS induced oxidative stress- and inflammation- mediated sig-nalling pathways along with critical miRs help in better understanding of the pathophysiology of ath-erosclerosis. Several key molecules discussed in this review could be potent target for the prevention and treatment of atherosclerosis.