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The blood–retinal barrier (BRB) is strongly compromised in diabetic retinopathy (DR) due to the detachment of pericytes (PCs) from retinal microvessels, resulting in increased permeability and impairment of the BRB. Western blots, immunofluorescence and ELISA were performed on adipose mesenchymal stem cells (ASCs) and pericyte-like (P)-ASCs by co-cultured human retinal endothelial cells (HRECs) under hyperglycemic conditions (HG), as a model of DR. Our results demonstrated that: (a) platelet-derived growth factor receptor (PDGFR) and its activated form were more highly expressed in monocultured P-ASCs than in ASCs, and this expression increased when co-cultured with HRECs under high glucose conditions (HG); (b) the transcription factor Nrf2 was more expressed in the cytoplasmic fraction of ASCs and in the P-ASC nuclear fraction, under normal glucose and, even more, under HG conditions; (c) cytosolic phospholipase A2 activity and prostaglandin E2 release, stimulated by HG, were significantly reduced in P-ASCs co-cultured with HRECs; (d) HO-1 protein content was significantly higher in HG-P-ASCs/HRECs than P-ASCs/HRECs; and (e) VEGF-A levels in media from HG-co-cultures were reduced in P-ASCs/HRECs with respect to ASCs/HRECs. The data obtained highlighted the potential of autologous differentiated ASCs in future clinical applications based on cell therapy to counteract the damage induced by DR. [ABSTRACT FROM AUTHOR]

The SARS-CoV-2 virus that is the cause of coronavirus disease 2019 (COVID-19) affects not only peripheral organs such as the lungs and blood vessels, but also the central nervous system (CNS)—as seen by effects on smell, taste, seizures, stroke, neuropathological findings and possibly, loss of control of respiration resulting in silent hypoxemia. COVID-19 induces an inflammatory response and, in severe cases, a cytokine storm that can damage the CNS. Antimalarials have unique properties that distinguish them from other anti-inflammatory drugs. (A) They are very lipophilic, which enhances their ability to cross the blood-brain barrier (BBB). Hence, they have the potential to act not only in the periphery but also in the CNS, and could be a useful addition to our limited armamentarium against the SARS-CoV-2 virus. (B) They are non-selective inhibitors of phospholipase A2 isoforms, including cytosolic phospholipase A2 (cPLA2). The latter is not only activated by cytokines but itself generates arachidonic acid, which is metabolized by cyclooxygenase (COX) to pro-inflammatory eicosanoids. Free radicals are produced in this process, which can lead to oxidative damage to the CNS. There are at least 4 ways that antimalarials could be useful in combating COVID-19. (1) They inhibit PLA2. (2) They are basic molecules capable of affecting the pH of lysosomes and inhibiting the activity of lysosomal enzymes. (3) They may affect the expression and Fe2+/H+ symporter activity of iron transporters such as divalent metal transporter 1 (DMT1), hence reducing iron accumulation in tissues and iron-catalysed free radical formation. (4) They could affect viral replication. The latter may be related to their effect on inhibition of PLA2 isoforms. Inhibition of cPLA2 impairs an early step of coronavirus replication in cell culture. In addition, a secretory PLA2 (sPLA2) isoform, PLA2G2D, has been shown to be essential for the lethality of SARS-CoV in mice. It is important to take note of what ongoing clinical trials on chloroquine and hydroxychloroquine can eventually tell us about the use of antimalarials and other anti-inflammatory agents, not only for the treatment of COVID-19, but also for neurovascular disorders such as stroke and vascular dementia. [ABSTRACT FROM AUTHOR]

Kaempferol, a phytochemical found in many edible plants, is known to alleviate diseases such as cancer, allergy, and inflammation. The objective of this study was to investigate whether kaempferol could reduce omega-6 and ovalbumin-mediated allergic reactions at lung and trachea in BALB/c mice. Mice were allocated into five groups: 1) control group (CON); 2) positive control group with orally administration of omega-6 (POS); 3) bovine serum albumin (BSA) sensitization group (with BSA injection and ovalbumin inhalation); 4) BSA+K10 group: BSA injection, 10 μg/g of kaempferol administration and ovalbumin inhalation; and 5) BSA+K20 group: BSA injection, 20 μg/g of kaempferol administration and ovalbumin inhalation. Results revealed that serum histamine level was the highest (p<0.01) in BSA group. In lung tissue and trachea, cyclooxygenase 2 (Cox2) expression was significantly (p<0.05) higher in the BSA group compared to that in other groups. However, phosphorylated cytosolic phospholipase A2 (p-cPLA2) expression in the trachea was not significantly different among groups. Taken together, results of this study suggest that kaempferol might be useful for alleviating inflammation reaction associated with Cox2 expression. However, the exact mechanism of action involved in the effect of kaempferol on inflammatory response remains unclear. [ABSTRACT FROM AUTHOR]

Cytosolic phospholipase A2α (cPLA2α) is a key enzyme in regulation of inflammation process and neuromembrane homeostasis, both of which are critical in pathogenesis of Alzheimer’s diseases. By hybride APP/PS1 Tg-AD mice with cPLA2α knockout mice, three lines of APP/PS1 Tg-AD mice were produced with genotypes of cPLA2α +/+ , cPLA2α +/− and cPLA2α −/− . Compared to cPLA2α +/+ Tg-AD mice, the amyloid plaque formation was significantly downregulated in the brain of cPLA2α +/− Tg-AD mice, but not in cPLA2α −/− Tg-AD mice. The reactive gliosis were also significantly downregulated in both cPLA2α +/− and cPLA2α −/− Tg-AD mouse lines. The paradoxical effects of cPLA2α on the amyloid plaques reveal a complex role of cPLA2α in pathogenesis of AD and could be a potential target for prevention and treatment of AD. [ABSTRACT FROM AUTHOR]

Background: The lung is exposed to airborne fungal spores, and fungi that colonize the oral cavity such as Candida albicans, but does not develop disease to opportunistic fungal pathogens unless the immune system is compromised. The Group IVA cytosolic phospholipase A2(cPLA2a) is activated in response to Candida albicans infection resulting in the release of arachidonic acid for eicosanoid production. Although eicosanoids such as prostaglandins and leukotrienes modulate inflammation and immune responses, the role of cPLA2α and eicosanoids in regulating C. albicans lung infection is not understood. Methods: The responses of cPLA2α+/+ and cPLA2α-/- Balb/c mice to intratracheal instillation of C. albicans were compared. After challenge, we evaluated weight loss, organ fungal burden, and the recruitment of cells and the levels of cytokines and eicosanoids in bronchoalveolar lavage fluid. The ability of macrophages and neutrophils from cPLA2α+/+ and cPLA2α-/- mice to recognize and kill C. albicans was also compared. Results: After C. albicans instillation, cPLA2α+/+ mice recovered a modest weight loss by 48 h and completely cleared fungi from the lung by 12 h with no dissemination to the kidneys. In cPLA2α-/- mice, weight loss continued for 72 h, C. albicans was not completely cleared from the lung and disseminated to the kidneys. cPLA2α-/- mice exhibited greater signs of inflammation including higher neutrophil influx, and elevated levels of albumin and pro-inflammatory cytokines/chemokines (IL1a, IL1β, TNFa, IL6, CSF2, CXCL1, CCL20) in bronchoalveolar lavage fluid. The amounts of cysteinyl leukotrienes, thromboxane B2 and prostaglandin E2 were significantly lower in bronchoalveolar lavage fluid from C. albicans-infected cPLA2α-/- mice compared to cPLA2α+/+ mice. Alveolar macrophages and neutrophils from uninfected cPLA2α-/- mice exhibited less killing of C. albicans in vitro than cells from cPLA2α+/+ mice. In addition alveolar macrophages from cPLA2α-/- mice isolated 6 h after instillation of GFP-C. albicans contained fewer internalized fungi than cPLA2α+/+ macrophages. Conclusions: The results demonstrate that cPLA2α contributes to immune surveillance and host defense in the lung to prevent infection by the commensal fungus C. albicans and to dampen inflammation. [ABSTRACT FROM AUTHOR]

Highlights: [•] ATPγS induces lung inflammation via a P2 receptors-dependent COX-2/PGE2 pathway. [•] NF-κB plays a key role in cPLA2 activity in ATPγS-stimulated A549 cells. [•] ATPγS-induced PGE2 release is mediated via increase of [Ca2+]i in A549 cells. [•] ATPγS-induced PGE2 release is not mediated via activation of JNK1/2. [Copyright &y& Elsevier]

Background: Nonspecific airway hyperresponsiveness (AHR) is one of the cardinal features of bronchial asthma. Early AHR is caused by chemical mediators released from pulmonary mast cells activated in an IgE-dependent way. However, the mechanism of late AHR remains unclear. Methods: Features of airway allergic inflammation were analyzed, including antigen-induced AHR, using a murine model of asthma. The model was suitable for examining the sequential early molecular events occurring after the initial airway exposure to antigen. Results: AHR increased at 10-12 h after airway challenge, followed by the second-phase response, which was larger and broader in resistance at 18-30 h. Pretreatment of sensitized animals with anti-tumor necrosis factor (TNF) before airway challenge or induction of allergic asthma in TNF-/- mice resulted in abrogation of the first-phase late AHR. Intratracheal instillation of TNF induced a single peak of AHR at 10 h. IgE and IgG immune complexes induced the development of the first-phase late AHR by TNF production. Pretreatment with cytosolic phospholipase inhibitor and 5-lipoxygenase inhibitors abolished the first-phase late AHR as well as the leukotriene B4 levels in the airway. CpG-oligodeoxynucleotide (ODN) pretreatment reduced airway levels of Th2 cytokines, eosinophil infiltration and second-phase late AHR. However, CpG-ODN did not reduce TNF levels or the magnitude of first-phase late AHR. Conclusion: Biphasic late AHR occurs in a murine model of asthma. First- and second-phase late AHR is caused by TNF and Th2 response, respectively. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Azithromycin is a macrolide antibiotic with well-described anti-inflammatory properties which can be attributed, at least partially, to its action on macrophages. We have previously shown, with 18 different macrolide molecules, that IL-6 and PGE2 inhibition correlates with macrolide accumulation, as well as with their binding to phospholipids in J774A.1 cells. The present study was performed in order to substantiate the hypothesis that biological membranes are a target for macrolide anti-inflammatory activity. By analyzing the effect of azithromycin on overall eicosanoid production, we found that in LPS-stimulated J774A.1 cells, azithromycin, like indomethacin, inhibited the synthesis of all eicosanoids produced downstream of COX. Upstream of COX, azithromycin inhibited arachidonic acid release in the same way as a cPLA2 inhibitor, while indomethacin had no effect. Further comparison revealed that in LPS-stimulated J774A.1 cells, the cPLA2 inhibitor showed the same profile of inhibition as azithromycin in inhibiting PGE2, IL-6, IL-12p40 and arachidonic acid release. Therefore, we propose that the anti-inflammatory activity of azithromycin in this model may be due to interactions with cPLA2, causing inadequate translocation of the enzyme or disturbing physical interactions with its substrates. [Copyright &y& Elsevier]

Abstract: Eicosanoids are potent lipid mediators derived from phospholipase (PL)-released arachidonic acid (AA) coupled to subsequent metabolism by cyclooxygenase (COX)-1/2 or lipoxygenases (LO) which are involved in a variety of homeostatic biological functions and inflammation. We have investigated three lupeolic acids (LA) from the gum resin of Boswellia carterii for their ability to interfere with eicosanoid biosynthesis in human blood cells. A novel, yet unknown C(28)-hydroxylated LA, that is, 3α-acetoxy-28-hydroxylup-20(29)-en-4β-oic acid (Ac-OH-LA) was found to inhibit the biosynthesis of COX-, 5-LO- and 12-LO-derived eicosanoids from endogenous AA in activated platelets, neutrophils, and monocytes from human blood with consistent IC50 values of 2.3–6.9μM. In contrast, two other LAs lacking the C(28)-OH moiety were essentially inactive in this respect. Inhibition of eicosanoids by Ac-OH-LA correlated with reduced release of AA in intact cells. When AA was exogenously provided as substrate for cellular eicosanoid biosynthesis the inhibitory effects of Ac-OH-LA were essentially reversed, even though some inhibition of 5-LO and COX-1 product formation still remained. Finally, by means of a cell-free phospholipid hydrolysis assay using human recombinant cytosolic PLA2α, we show that Ac-OH-LA may directly interfere with cPLA2α activity (IC50 =3.6μM). Together, we identified a novel, naturally occuring C(28)-hydroxylated LA which acts as efficient inhibitor of cPLA2α and consequently suppresses eicosanoid biosynthesis in intact cells. [Copyright &y& Elsevier]

Leptin is a hormone secreted by adipocytes in correlation with total body fat mass. In addition to regulating energy homeostasis, leptin modulates immune functions such as macrophage phagocytosis and cytokine synthesis. Previously, we reported defective leukotriene synthesis in macrophages from leptin-deficient mice that could be restored with exogenous leptin. In the present study, we utilized macrophages from normal rodents to explore the mechanism by which leptin could enhance cellular leukotriene synthesis. Leptin pretreatment of either rat alveolar or murine peritoneal macrophages for 16 h dose dependently increased the synthesis of leukotriene B4 and cysteinyl leukotrienes in response to calcium ionophore or the particulate zymosan. Leptin also enhanced calcium ionophore-stimulated release of free arachidonic acid. Calcium-dependent and -independent arachidonoylselective phospholipase activities in macrophage lysates were likewise increased following leptin treatment. Immunoblot analysis of leptin-treated cells revealed that group IVC iPLA2 (cPLA2γ) protein expression increased ∼80%. These data demonstrate for the first time that phospholipase A2 activity and cPLA2γ protein levels in alveolar macrophages represent targets for upregulation by leptin and provide previously unrecognized mechanisms by which this hormone can promote inflammatory responses. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol-specific phospholipase C (PI-PLC) and cytosolic phospholipase A 2 (cPLA 2) regulate both eosinophil degranulation and leukotriene (LT) synthesis via PI-PLC-mediated calcium influx and cPLA 2 activation. Phosphatidylcholine-specific phospholipase C (PC-PLC) likely plays a key role in cellular signaling, including the eosinophilic allergic inflammatory response. This study examined the role of PC-PLC in eosinophil LT synthesis and degranulation using tricyclodecan-9-yl-xanthogenate (D609), a PC-specific PLC inhibitor. D609 inhibited N -formyl-met-leu-phe + cytochalasin B (fMLP/B)-induced arachidonic acid (AA) release and leukotriene C 4 (LTC 4) secretion. However, at concentrations that blocked both AA release and LTC 4 secretion, D609 had no significant inhibitory effect on stimulated cPLA 2 activity. D609 also partially blocked fMLP/B-induced calcium influx, indicating that inhibition of AA release and LTC 4 secretion by D609 is due to inhibition of calcium-mediated cPLA 2 translocation to intracellular membranes, not inhibition of cPLA 2 activity. In addition, D609 inhibited fMLP/B-stimulated eosinophil peroxidase release, indicating that PC-PLC regulates fMLP/B-induced eosinophil degranulation by increasing the intracellular calcium concentration ([Ca2+] i). Overall, our results showed that PC-PLC is critical for fMLP/B-stimulated eosinophil LT synthesis and degranulation. In addition, degranulation requires calcium influx, while PC-PLC regulates LTC 4 synthesis through calcium-mediated cPLA 2 activation. [ABSTRACT FROM AUTHOR]