A lipidomic screen of hyperglycemia-treated HRECs links 12/15-Lipoxygenase to microvascular dysfunction during diabetic retinopathy via NADPH oxidase

Retinal hyperpermeability and subsequent macular edema is a cardinal feature of early diabetic retinopathy (DR). Here, we investigated the role of bioactive lipid metabolites, in particular 12/15-lipoxygenase (LOX)-derived metabolites, in this process. LC/MS lipidomic screen of human retinal endothelial cells (HRECs) demonstrated that 15-HETE was the only significantly increased metabolite (2.4 ± 0.4-fold, P = 0.0004) by high glucose (30 mM) treatment. In the presence of arachidonic acid, additional eicosanoids generated by 12/15-LOX, including 12- and 11-HETEs, were significantly increased. Fluorescein angiography and retinal albumin leakage showed a significant decrease in retinal hyperpermeability in streptozotocin-induced diabetic mice lacking 12/15-LOX compared with diabetic WT mice. Our previous studies demonstrated the potential role of NADPH oxidase in mediating the permeability effect of 12- and 15-HETEs, therefore we tested the impact of intraocular injection of 12-HETE in mice lacking the catalytic subunit of NADPH oxidase (NOX2). The permeability effect of 12-HETE was significantly reduced in NOX2−/− mice compared with the WT mice. In vitro experiments also showed that 15-HETE induced HREC migration and tube formation in a NOX-dependent manner. Taken together our data suggest that 12/15-LOX is implicated in DR via a NOX-dependent mechanism.National Institutes of Health Grant 5R01EY023315 and National Priorities Research Program Grant 4-1046-3-284 from the Qatar National Research Fund (a member of Qatar Foundation). This study was also supported in part by the National Center for Research Resources, National Institutes of Health Grant S10RR027926

Global overview of the management of acute cholecystitis during the COVID-19 pandemic (CHOLECOVID study)

Background: This study provides a global overview of the management of patients with acute cholecystitis during the initial phase of the COVID-19 pandemic. Methods: CHOLECOVID is an international, multicentre, observational comparative study of patients admitted to hospital with acute cholecystitis during the COVID-19 pandemic. Data on management were collected for a 2-month study interval coincident with the WHO declaration of the SARS-CoV-2 pandemic and compared with an equivalent pre-pandemic time interval. Mediation analysis examined the influence of SARS-COV-2 infection on 30-day mortality. Results: This study collected data on 9783 patients with acute cholecystitis admitted to 247 hospitals across the world. The pandemic was associated with reduced availability of surgical workforce and operating facilities globally, a significant shift to worse severity of disease, and increased use of conservative management. There was a reduction (both absolute and proportionate) in the number of patients undergoing cholecystectomy from 3095 patients (56.2 per cent) pre-pandemic to 1998 patients (46.2 per cent) during the pandemic but there was no difference in 30-day all-cause mortality after cholecystectomy comparing the pre-pandemic interval with the pandemic (13 patients (0.4 per cent) pre-pandemic to 13 patients (0.6 per cent) pandemic; P = 0.355). In mediation analysis, an admission with acute cholecystitis during the pandemic was associated with a non-significant increased risk of death (OR 1.29, 95 per cent c.i. 0.93 to 1.79, P = 0.121). Conclusion: CHOLECOVID provides a unique overview of the treatment of patients with cholecystitis across the globe during the first months of the SARS-CoV-2 pandemic. The study highlights the need for system resilience in retention of elective surgical activity. Cholecystectomy was associated with a low risk of mortality and deferral of treatment results in an increase in avoidable morbidity that represents the non-COVID cost of this pandemic

Deletion of Thioredoxin Interacting Protein (TXNIP) Augments Hyperoxia-Induced Vaso-Obliteration in a Mouse Model of Oxygen Induced-Retinopathy

<div><p>We have recently shown that thioredoxin interacting protein (TXNIP) is required for VEGF-mediated VEGFR2 receptor activation and angiogenic signal. Retinas from TXNIP knockout mice (TKO) exhibited higher cellular antioxidant defense compared to wild type (WT). This study aimed to examine the impact of TXNIP deletion on hyperoxia-induced vaso-obliteration in ischemic retinopathy. TKO and WT pups were subjected to oxygen-induced retinopathy model. Retinal central capillary dropout was measured at p12. Retinal redox and nitrative state were assessed by reduced-glutathione (GSH), thioredoxin reductase activity and nitrotyrosine formation. Western blot and QT-PCR were used to assess VEGF, VEGFR-2, Akt, iNOS and eNOS, thioredoxin expression, ASK-1 activation and downstream cleaved caspase-3 and PARP in retinal lysates. Retinas from TKO mice exposed to hyperoxia showed significant increases (1.5-fold) in vaso-obliteration as indicated by central capillary drop out area compared to WT. Retinas from TKO showed minimal nitrotyrosine levels (10% of WT) with no change in eNOS or iNOS mRNA expression. There was no change in levels of VEGF or activation of VEGFR2 and its downstream Akt in retinas from TKO and WT. In comparison to WT, retinas from TKO showed significantly higher level of GSH and thioredoxin reductase activity in normoxia but comparable levels under hyperoxia. Exposure of TKO to hyperoxia significantly decreased the anti-apoptotic thioredoxin protein (∼50%) level compared with WT. This effect was associated with a significant increase in activation of the apoptotic ASK-1, PARP and caspase-3 pathway. Our results showed that despite comparable VEGF level and signal in TKO, exposure to hyperoxia significantly decreased Trx expression compared to WT. This effect resulted in liberation and activation of the apoptotic ASK-1 signal. These findings suggest that TXNIP is required for endothelial cell survival and homeostasis especially under stress conditions including hyperoxia.</p></div

Deletion of TXNIP does not alter VEGF levels under normoxia or hyperoxia.

<p>(A) VEGF mRNA levels were detected from various groups using rt-PCR. (B) VEGF protein expression was examined using heparin-bound beads from p12 WT and TKO retinas. There was no change in levels of VEGF mRNA or VEGF expression between WT and TKO under normoxia. Hyperoxia caused significant decrease in VEGF mRNA compared to normoxia. Hyperoxia did not alter VEGF protein levels from normoxia in WT and TKO. (#P<0.05 Hyperoxia vs Normoxia, n = 4–6).</p

Deletion of TXNIP increases antioxidant defense level under normoxia and hyperoxia.

<p>In comparison to WT, retinas from TKO showed significantly higher level of reduced-GSH (<b>A</b>) and thioredoxin reductase activity (<b>B</b>) under normoxia. A 2×2 analysis showed a significant interaction between gene (TKO) and oxygen levels (hyperoxia) in both reduced-GSH and thioredoxin reductase activity measurements. (#P<0.05 Hyperoxia vs Normoxia, *P<0.05, TKO vs WT, n = 6–8).</p

Deletion of TXNIP augments hyperoxia-induced vaso-obliteration compared to WT.

<p>Wild type (WT) and TXNIP knockout (TKO) mice were subjected to hyperoxia (75% O2, p7–p12). Retinas were fixed and stained with iso-lectin B4 to quantify oxygen induced vaso-obliteration. <b>A</b>–<b>C</b>) Retinas from TKO mice exposed to hyperoxia showed significant increases in vaso-obliteration compared to WT. (*P<0.05 vs WT, n = 12). <b>D</b>) Hyperoxia stimulates TXNIP expression mRNA in WT but not in TKO mice. (*P<0.05 vs WT normoxia, n = 4)</p

Deletion of TXNIP augments hyperoxia-induced ASK-1 activation and apoptotic markers.

<p>Wild type (WT) and TXNIP knockout (TKO) mice were subjected to 5 days hyperoxia (p7–12). Retinas were collected for protein ASK-1 (<b>A</b>) and apoptotic markers (Cleaved Caspase-3 and PARP) (<b>B</b>). A 2×2 analysis showed a significant interaction between gene (TKO) and oxygen levels (Hyperoxia) in activation of ASK-1. In parallen, Hyperoxia caused significant increase in cleaved caspase-3 and PARP expression compared to normoxia in WT and TKO. Reduced Trx levels were associated with a significant increase in activation of the apoptotic ASK-1, PARP and caspase-3 pathway. (#P<0.05 Hyperoxia vs Normoxia, *P<0.05, TKO vs WT, n = 4–6).</p

Deletion of TXNIP decreases nitrative stress under normoxia and hyperoxia.

<p><b>A</b>) Retinas of TKO showed significantly less nitrotyrosine levels at normoxia or hyperoxia compared to WT. <b>B</b>) TKO showed higher eNOS mRNA level in normoxia. Hyperoxia significantly reduced eNOS mRNA levels. A 2×2 analysis showed a significant interaction between the gene (TKO) and the oxygen level (hyperoxia) in both nitrotyrosine and eNOS levels. C) We did not detect difference between TKO and WT in the expression of iNOS. Hyperoxia caused significant reduction of iNOS compared to normoxia. (#P<0.05 Hyperoxia vs Normoxia, *P<0.05, TKO vs WT, n = 4–6).</p

Deletion of TXNIP does not alter VEGFR2/Akt activation under hyperoxia.

<p>Wild type (WT) and TXNIP knockout (TKO) mice were subjected to hyperoxia (75% O2, p7–p12). Activation of VEGFR2 (<b>A</b>) and Akt (<b>B</b>) were examined as downstream signal of VEGF in p12 WT and TKO retinas. TKO showed significant decrease in phosphorylation of VEGFR-2 and Akt compared to WT under normoxic condition. We did not detect significant change in the activation of VEGFR2 and its downstream Akt in retinas from TKO and WT in response to hyperoxia. (*P<0.05, TKO vs WT, n = 4–6).</p

Representative diagram shows the impact of TXNIP deletion on retina vasculature under both normoxia and hyperoxia.

<p>Under normoxia, retinas from TXNIP-deficient mice showed similar VEGF levels, less peroxynitrite (ONOO-) levels, less VEGF receptor-2 (pVEGFR2) activation and upregulated thioredoxin (Trx) that collectively lead to normal vascular development in comparison to WT mice. Under hyperoxia, retinas from WT mice showed higher peroxynitrite formation, less survival Akt activation (pAkt) and upregulated proapoptotic signal of ASK-1 resulting in vaso-obliteration. Retinas from TKO although showed less peroxynitrite levels and maintained Akt activation, retinas experienced significant decreases in thioredoxin (Trx) that shift the balance of the ASK-1-Trx inhibitory complex and increases the activation of the proapoptotic ASK-1 pathway leading to exacerbated vasoobliteration compared to WT.</p