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.

Isolation of Splenic Microvesicles in a Murine Model of Intraperitoneal Bacterial Infection.

Microvesicles (MVs) are submicron fragments released from the plasma membrane of activated cells that act as proinflammatory and procoagulant cellular effectors. In rats, spleen MVs (SMVs) are surrogate markers of pathophysiological conditions. Previous in vitro studies demonstrated that Porphyromonas gingivalis (P. gingivalis), a major periodontal pathogen, enables the endothelial shedding and apoptosis while lipopolysaccharide (LPS) favors the shedding of splenocyte-derived microvesicles (SMVs). In vivo studies showed the feasibility of pharmacological control of SMV shedding. The present protocol establishes a standardized procedure for isolating splenic SMVs from the P. gingivalis acute murine infection model. P. gingivalis infection was induced in young C57BL/6 mice by intraperitoneal injection (three injections of 5 x 107 bacteria/week). After two weeks, the spleens were collected, weighed, and the splenocytes were counted. SMVs were isolated and quantified by protein, RNA, and prothrombinase assays. Cell viability was assessed by either propidium iodide or trypan blue exclusion dyes. Following splenocyte extraction, neutrophil counts were obtained by flow cytometry after 24 h of splenocyte culture. In P. gingivalis-injected mice, a 2.5-fold increase in spleen weight and a 2.3-fold rise in the splenocyte count were observed, while the neutrophils count was enhanced by 40-folds. The cell viability of splenocytes from P. gingivalis-injected mice ranged from 75%-96% and was decreased by 50% after 24 h of culture without any significant difference compared to unexposed controls. However, splenocytes from injected mice shed higher amounts of MVs by prothrombinase assay or protein measurements. The data demonstrate that the procoagulant SMVs are reliable tools to assess an early spleen response to intraperitoneal P. gingivalis infection.

Significance of neutrophil microparticles in ischaemia-reperfusion: Pro-inflammatory effectors of endothelial senescence and vascular dysfunction.

Endothelial senescence is an emerging cause of vascular dysfunction. Because microparticles are effectors of endothelial inflammation and vascular injury after ischaemia-reperfusion, we examined leucocyte-derived microparticles of spleen origin as possible contributors. Microparticles were generated from primary rat splenocytes by either lipopolysaccharide or phorbol-myristate-acetate/calcium ionophore, under conditions mimicking innate and adaptive immune responses. Incubation of primary porcine coronary endothelial cells with either type of microparticles, but not with those from unstimulated splenocytes, leads to a similar threefold raise in senescence-associated ß-galactosidase activity within 48 hours, indicating accelerated senescence, to endothelial oxidative stress, and a fivefold and threefold increase in p21 and p16 senescence markers after 24 hours. After 12-hour incubation, the endothelial-dependent relaxation of coronary artery rings was reduced by 50%, at distinct optimal microparticle concentration. In vitro, microparticles were pro-thrombotic by up-regulating the local angiotensin system, by prompting tissue factor activity and a secondary generation of pro-coagulant endothelial microparticles. They initiated an early pro-inflammatory response by inducing phosphorylation of NF-κB, MAP kinases and Akt after 1 hour, and up-regulated VCAM-1 and ICAM-1 at 24 hours. Accordingly, VCAM-1 and COX-2 were also up-regulated in the coronary artery endothelium and eNOS down-regulated. Lipopolysaccharide specifically favoured the shedding of neutrophil- and monocyte-derived microparticles. A 80% immuno-depletion of neutrophil microparticles reduced endothelial senescence by 55%, indicating a key role. Altogether, data suggest that microparticles from activated splenocytes prompt early pro-inflammatory, pro-coagulant and pro-senescent responses in endothelial cells through redox-sensitive pathways. The control of neutrophil shedding could preserve the endothelium at site of ischaemia-reperfusion-driven inflammation and delay its dysfunction.

Empagliflozin improved systolic blood pressure, endothelial dysfunction and heart remodeling in the metabolic syndrome ZSF1 rat.

BACKGROUND: Empagliflozin (empa), a selective sodium-glucose cotransporter (SGLT)2 inhibitor, reduced cardiovascular mortality and hospitalization for heart failure in patients with type 2 diabetes at high cardiovascular risk independent of glycemic control. The cardiovascular protective effect of empa was evaluated in an experimental model of metabolic syndrome, the obese ZSF1 rat, and its' lean control. METHODS: Lean and obese ZSF1 rats were either non-treated or treated with empa (30 mg/kg/day) for 6 weeks. Vascular reactivity was assessed using mesenteric artery rings, systolic blood pressure by tail-cuff sphygmomanometry, heart function and structural changes by echocardiography, and protein expression levels by Western blot analysis. RESULTS: Empa treatment reduced blood glucose levels from 275 to 196 mg/dl in obese ZSF1 rats whereas normoglycemia (134 mg/dl) was present in control lean ZSF1 rats and was unaffected by empa. Obese ZSF1 rats showed increased systolic blood pressure, and blunted endothelium-dependent relaxations associated with the appearance of endothelium-dependent contractile responses (EDCFs) compared to control lean rats. These effects were prevented by the empa treatment. Obese ZSF1 rats showed increased weight of the heart and of the left ventricle volume without the presence of diastolic or systolic dysfunction, which were improved by the empa treatment. An increased expression level of senescence markers (p53, p21, p16), tissue factor, VCAM-1, SGLT1 and SGLT2 and a down-regulation of eNOS were observed in the aortic inner curvature compared to the outer one in the control lean rats, which were prevented by the empa treatment. In the obese ZSF1 rats, no such effects were observed. The empa treatment reduced the increased body weight and weight of lungs, spleen, liver and perirenal fat, hyperglycemia and the increased levels of total cholesterol and triglycerides in obese ZSF1 rats, and increased blood ketone levels and urinary glucose excretion in control lean and obese ZSF1 rats. CONCLUSION: Empa reduced glucose levels by 28% and improved both endothelial function and cardiac remodeling in the obese ZSF1 rat. Empa also reduced the increased expression level of senescence, and atherothrombotic markers at arterial sites at risk in the control lean, but not obese, ZSF1 rat.

Thrombin Induces Angiotensin II-Mediated Senescence in Atrial Endothelial Cells: Impact on Pro-Remodeling Patterns.

BACKGROUND: Besides its well-known functions in hemostasis, thrombin plays a role in various non-hemostatic biological and pathophysiologic processes. We examined the potential of thrombin to promote premature atrial endothelial cells (ECs) senescence. METHODS AND RESULTS: Primary ECs were isolated from porcine atrial tissue. Endothelial senescence was assessed by measuring beta-galactosidase (SA-ß-gal) activity using flow cytometry, oxidative stress using the redox-sensitive probe dihydroethidium, protein level by Western blot, and matrix metalloproteinases (MMPs) activity using zymography. Atrial endothelial senescence was induced by thrombin at clinically relevant concentrations. Thrombin induced the up-regulation of p53, a key regulator in cellular senescence and of p21 and p16, two cyclin-dependent kinase inhibitors. Nicotinamide adenine dinucleotide phosphate NADPH oxidase, cyclooxygenases and the mitochondrial respiration complex contributed to oxidative stress and senescence. Enhanced expression levels of vascular cell adhesion molecule (VCAM)-1, tissue factor, transforming growth factor (TGF)-ß and MMP-2 and 9 characterized the senescence-associated secretory phenotype of atrial ECs. In addition, the pro-senescence endothelial response to thrombin was associated with an overexpression of both angiotensin converting enzyme and AT1 receptors and was inhibited by perindoprilat and losartan. CONCLUSIONS: Thrombin promotes premature ageing and senescence of atrial ECs and may pave the way to deleterious remodeling of atrial tissue by a local up-regulation of the angiotensin system and by promoting pro-inflammatory, pro-thrombotic, pro-fibrotic and pro-remodeling responses. Hence, targeting thrombin and/or angiotensin systems may efficiently prevent atrial endothelial senescence.

Immunostimulant activities of the aqueous methanolic extract of Leptadenia pyrotechnica, a plant from Cholistan desert.

ETHNOPHARMACOLOGICAL RELEVANCE: Leptadenia pyrotechnica (Forssk.) Decne (Asclepiadaceae) is a well-renowned medicinal shrub, used by herbal practitioners for various ailments including allergic rhinitis, productive cough, abortion, diabetes, stomach disorders, fever, kidney disorders, stones and cancer AIM OF THE STUDY: On the basis of folkloric uses, L. pyrotechnica was selected from the wide medicinal flora of the Cholistan desert of Pakistan, for the exploration of immunomodulatory potential. MATERIALS AND METHODS: Aqueous methanolic (30:70) extract of aerial parts of L. pyrotechnica (Lp. Cr) was prepared by 3 days thrice maceration and subsequent evaporation under reduced pressure. In-vivo experiments were performed in Wistar albino rats including neutrophil adhesion test, haemagglutinating antibody (HA) titre, delayed-type hypersensitivity response, phagocytic activity and cyclophosphamide-induced myelosuppression. RESULTS: Lp. Cr produced a significant increase in phagocytic index in dose-dependent fashion (3.56, 4.18 and 5.42 at 30, 100 and 300mg/kg, respectively) as well as an augmented response in the delayed-type hypersensitivity reaction and HA titre induced by sheep erythrocytes. Lp. Cr also showed improved adhesion of neutrophils with nylon pellets (15.28, 27.85 and 38.42% at the doses of 30, 100 and 300mg/kg) and prevented myelosuppression in cyclophosphamide-treated rats as evidenced from the hematological parameters. The results of treatment were compared with normal and standard control groups throughout the study and the effects by Lp. Cr (300mg/kg) were found to be comparable with Levamisole. CONCLUSIONS: The results indicated that L. pyrotechnica possesses immunostimulatory activity and justify its traditional use for the control and management of diseases in which the immune system needs to be stimulated like infectious diseases.