Activated factor X stimulates atrial endothelial cells and tissues to promote remodeling responses through AT1R/NADPH oxidases/SGLT1/2.

AIMS: Atrial fibrillation (AF), the most common cardiac arrhythmia favoring ischemic stroke and heart failure involves left atrial remodeling, fibrosis and a complex interplay between cardiovascular risk factors. This study examined whether activated factor X (FXa) induces pro-remodeling and pro-fibrotic responses in atrial endothelial cells (AECs) and human atrial tissues and determined the underlying mechanisms. METHODS AND RESULTS: AECs were from porcine hearts and human right atrial appendages (RAA) from patients undergoing heart surgery. Protein expression levels were assessed by Western blot and immunofluorescence staining, mRNA levels by RT-qPCR, formation of reactive oxygen species (ROS) and NO using fluorescent probes, thrombin and angiotensin II generation by specific assays, fibrosis by Sirius red staining and senescence by senescence-associated beta-galactosidase (SA-ß-gal) activity.In AECs, FXa increased ROS formation, senescence (SA-ß-gal activity, p53, p21), angiotensin II generation and the expression of pro-inflammatory (VCAM-1, MCP-1), pro-thrombotic (tissue factor), pro-fibrotic (TGF-ß and collagen-1/3a) and pro-remodeling (MMP-2/9) markers whereas eNOS levels and NO formation were reduced. These effects were prevented by inhibitors of FXa but not thrombin, protease-activated receptors antagonists (PAR-1/2) and inhibitors of NADPH oxidases, ACE, AT1R, SGLT1/SGLT2. FXa also increased expression levels of ACE1, AT1R, SGLT1/2 proteins which was prevented by SGLT1/2 inhibitors. Human RAA showed tissue factor mRNA levels that correlated with markers of endothelial activation, pro-remodeling and pro-fibrotic responses and SGLT1/2 mRNA levels. They also showed protein expression levels of ACE1, AT1R, p22phox, SGLT1/2, and immunofluorescence signals of nitrotyrosine and SGLT1/2 colocalized with those of CD31. FXa increased oxidative stress levels which were prevented by inhibitors of the AT1R/NADPH oxidases/SGLT1/2 pathway. CONCLUSIONS: FXa promotes oxidative stress triggering premature endothelial senescence and dysfunction associated with pro-thrombotic, pro-remodeling and pro-fibrotic responses in AECs and in human RAA involving the AT1R/NADPH oxidases/SGLT1/2 pro-oxidant pathway. Targeting this pathway may be of interest to prevent atrial remodeling and the progression of atrial fibrillation substrate.

COVID-19 promotes endothelial dysfunction and thrombogenicity: role of proinflammatory cytokines/SGLT2 prooxidant pathway.

BACKGROUND: COVID-19 is associated with an increased risk of cardiovascular complications. Although cytokines have a predominant role in endothelium damage, the precise molecular mechanisms are far from being elucidated. OBJECTIVES: The present study hypothesized that inflammation in patients with COVID-19 contributes to endothelial dysfunction through redox-sensitive SGLT2 overexpression and investigated the protective effect of SGLT2 inhibition by empagliflozin. METHODS: Human plasma samples were collected from patients with acute, subacute, and long COVID-19 (n = 100), patients with non-COVID-19 and cardiovascular risk factors (n = 50), and healthy volunteers (n = 25). Porcine coronary artery endothelial cells (ECs) were incubated with plasma (10%). Protein expression levels were determined using Western blot analyses and immunofluorescence staining, mRNA expression by quantitative reverse transcription-polymerase chain reaction, and the level of oxidative stress by dihydroethidium staining. Platelet adhesion, aggregation, and thrombin generation were determined. RESULTS: Increased plasma levels of interleukin (IL)-1ß, IL-6, tumor necrosis factor-α, monocyte chemoattractant protein-1, and soluble intercellular adhesion molecule-1 were observed in patients with COVID-19. Exposure of ECs to COVID-19 plasma with high cytokines levels induced redox-sensitive upregulation of SGLT2 expression via proinflammatory cytokines IL-1ß, IL-6, and tumor necrosis factor-α which, in turn, fueled endothelial dysfunction, senescence, NF-κB activation, inflammation, platelet adhesion and aggregation, von Willebrand factor secretion, and thrombin generation. The stimulatory effect of COVID-19 plasma was blunted by neutralizing antibodies against proinflammatory cytokines and empagliflozin. CONCLUSION: In patients with COVID-19, proinflammatory cytokines induced a redox-sensitive upregulation of SGLT2 expression in ECs, which in turn promoted endothelial injury, senescence, platelet adhesion, aggregation, and thrombin generation. SGLT2 inhibition with empagliflozin appeared as an attractive strategy to restore vascular homeostasis in COVID-19.

Taxifolin as a Major Bioactive Compound in the Vasorelaxant Effect of Different Pigmented Rice Bran Extracts.

Cardiovascular disease is one of the leading causes of morbidity and mortality in recent years. The intake of polyphenol rich diets has been associated with improved cardiovascular function and reduced cardiovascular risks. Oryza sativa L. is one of the most common cereals worldwide. Rice bran, a byproduct of the rice milling process, contains many bioactive ingredients, including polyphenols, polysaccharides, proteins, and micronutrients. It is also consumed as a healthy diet in the form of rice bran oil and powder in many Asian countries like Japan, South Korea, and India for its several health benefits as a natural antioxidant. Thus, this study evaluated the vasorelaxant effect of ethanolic extracts of brown, green, red, and black rice bran and investigated its underlying vasorelaxant mechanism. Among the four rice bran extracts (RBEs) examined, the red rice bran extract (RRBE) had a strong endothelium-dependent vasorelaxant effect, which was markedly prevented by N-ω-nitro-L-arginine [endothelial nitric oxide synthase (eNOS) inhibitor], wortmannin [phosphoinositide-3 kinase (PI3K) inhibitor], and 1H-[1,2,4]oxadiazole[4,3-alpha]quinoxalin-1-one (inhibitor of guanylate cyclase). Likewise, RRBE induced the phosphorylation of eNOS and Src in cultured endothelial cells, thereby stimulating NO formation. Altogether, these findings propose that RRBE induces endothelium-dependent relaxation, involving at least in part, NO-mediated signaling through the PI3K/eNOS pathway. Further, LC-PDA analysis conducted on the four RBEs also revealed that RRBE highly contained taxifolin, which is an active flavanonol that induces endothelium-dependent vasorelaxation, compared to other RBEs. Subsequently, the underlying mechanism of taxifolin was assessed through vascular reactivity studies with pharmacological inhibitors similar to that of RRBE. These findings deciphered a distinct difference in vasorelaxant effects between RRBE and the other RBEs. We also observed that RRBE induced a potent endothelium-dependent NO-mediated relaxation in coronary artery rings, which involved the Src/PI3K pathway that activates eNOS. Additionally, taxifolin exhibited, at least in part, similar vasoprotective effects of RRBE. Therefore, we propose that RRBE may serve as natural sources of functional phytochemicals that improve cardiovascular diseases associated with disturbed NO production and endothelial dysfunction.

Endothelium-Dependent Relaxation Effects of Actinidia arguta Extracts in Coronary Artery: Involvement of eNOS/Akt Pathway.

Cardiovascular diseases (CVD) are the major cause of death globally. Bioavailability of nitric oxide, antioxidative activity, and regulation of ionic homeostasis are the key targets for prevention of CVD. Actinidia arguta (AA) has shown promising effect for anticancer, anti-hypercholesterolemia, and antioxidant agents. However, the vascular effect of AA remains unclear. Therefore, we investigated the vascular relaxation of AA extract as well as the underlying mechanisms. Vascular reactivity was assessed in organ baths using porcine coronary arteries and antioxidant properties were assessed using 2,2-diphenyl-1-picrylhydrazyl (DPPH). Methanol extract of AA stem (AASE) induced significantly vasorelaxation of porcine coronary artery and its effects is endothelium-dependent without cytotoxicity effects. In addition, ASSE scavenged reactive oxygen species (ROS) in vitro and strongly inhibited NADPH-oxidase activity, which is major source of ROS in vasculature. AASE strongly and dose-dependently activate endothelial nitric oxide synthase (eNOS), the major vascular protective enzyme, and Akt, the upstream signaling protein of eNOS, in porcine coronary artery endothelial cell. Altogether, these results have demonstrated that AASE is a potent endotheliumdependent vasodilator and this effect was involved in, at least in part, Akt/eNOS/NO pathway with strong anti-oxidant properties. The present findings indicate that AA stem could be a valuable candidate of herbal medicine for cardiovascular diseases associated with endothelial dysfunction and atherosclerosis.

Fine air pollution particles induce endothelial senescence via redox-sensitive activation of local angiotensin system.

Fine dust (FD) is a form of air pollution and is responsible for a wide range of diseases. Specially, FD is associated with several cardiovascular diseases (CVDs); long-term exposure to FD was shown to decrease endothelial function, but the underlying mechanism remains unclear. We investigated whether exposure to FD causes premature senescence-associated endothelial dysfunction in endothelial cells (ECs) isolated from porcine coronary arteries. The cells were treated with different concentrations of FD and senescence associated-beta galactosidase (SA-ß-gal) activity, cell cycle progression, expression of endothelial nitric oxide synthase (eNOS), oxidative stress level, and vascular function were evaluated. We found that FD increased SA-ß-gal activity, caused cell cycle arrest, and increased oxidative stress, suggesting the premature induction of senescence; on the other hand, eNOS expression was downregulated and platelet aggregation was enhanced. FD exposure impaired vasorelaxation in response to bradykinin and activated the local angiotensin system (LAS), which was inhibited by treatment with the antioxidant N-acetyl cysteine (NAC) and angiotensin II receptor type 1 (AT1) antagonist losartan (LOS). NAC and LOS also suppressed FD-induced SA-ß-gal activity, increased EC proliferation and eNOS expression, and improved endothelial function. These results demonstrate that FD induces premature senescence of ECs and is associated with increased oxidative stress and activation of LAS. This study can serve as a pharmacological target for prevention and/or treatment of air pollution-associated CVD.