Inflammatory Cell Dynamics after Murine Femoral Artery Wire Injury: A Multi-Parameter Flow Cytometry-Based Analysis.

An acute inflammatory response following arterial surgery for atherosclerosis, such as balloon angioplasty, stenting, and surgical bypass, is an important driver of neointimal hyperplasia after arterial injury, which leads to recurrent ischemia. However, a comprehensive understanding of the dynamics of the inflammatory infiltrate in the remodeling artery is difficult to attain due to the shortcomings of conventional methods such as immunofluorescence. We developed a 15-parameter flow cytometry method to quantitate leukocytes and 13 leukocyte subtypes in murine arteries at 4 time points after femoral artery wire injury. Live leukocyte numbers peaked at 7 days, which preceded the peak neointimal hyperplasia lesion at 28 days. Neutrophils were the most abundant early infiltrate, followed by monocytes and macrophages. Eosinophils were elevated after 1 day, while natural killer and dendritic cells gradually infiltrated over the first 7 days; all decreased between 7 and 14 days. Lymphocytes began accumulating at 3 days and peaked at 7 days. Immunofluorescence of arterial sections demonstrated similar temporal trends of CD45+ and F4/80+ cells. This method allows for the simultaneous quantitation of multiple leukocyte subtypes from small tissue samples of injured murine arteries and identifies the CD64+Tim4+ macrophage phenotype as being potentially important in the first 7 days post-injury.

Ketogenic Metabolic Therapy for Glioma.

PURPOSE: This study describes a retrospective case series of patients with glioma who received ketogenic metabolic therapy through dietary adherence and intermittent fasting. METHODS: A retrospective chart review of a single surgeon's clinic records was performed to identify patients who maintained nutritional ketosis for at least four months between January 2015 and October 2020. RESULTS: Sixteen patients who met the inclusion criteria constituted a heterogeneous population of patients with diagnoses including eight World Health Organization (WHO) grade IV gliomas (seven glioblastoma, one gliosarcoma), seven WHO grade III gliomas (three oligodendroglioma, four astrocytoma), and one WHO grade II oligodendroglioma. IDH1 mutation status was present for 12 patients, and MGMT methylation status was present for eight patients. The mean (standard deviation [SD]) duration of ketogenic metabolic therapy was 20.6 (13.8) months. The Response Assessment in Neuro-oncology Criteria was applied during the ketogenic metabolic therapy interval, indicating a complete response in eight patients and partial response in eight patients. The mean (SD) progression-free survival while patients maintained ketogenic metabolic therapy was 20.0 (14.4) months. CONCLUSION: Ketogenic metabolic therapy appears to convey a survival advantage within this patient series, which highlights the possibility that this therapy, when strictly applied, can augment the standard of care. Further exploration of this modality in a prospective series is warranted to formally explore this therapy.

eNOS controls angiogenic sprouting and retinal neovascularization through the regulation of endothelial cell polarity.

The roles of nitric oxide (NO) and endothelial NO synthase (eNOS) in the regulation of angiogenesis are well documented. However, the involvement of eNOS in the sprouting of endothelial tip-cells at the vascular front during sprouting angiogenesis remains poorly defined. In this study, we show that downregulation of eNOS markedly inhibits VEGF-stimulated migration of endothelial cells but increases their polarization, as evidenced by the reorientation of the Golgi in migrating monolayers and by the fewer filopodia on tip cells at ends of sprouts in endothelial cell spheroids. The effect of eNOS inhibition on EC polarization was prevented in Par3-depleted cells. Importantly, downregulation of eNOS increased the expression of polarity genes, such as PARD3B, PARD6A, PARD6B, PKCΖ, TJP3, and CRB1 in endothelial cells. In retinas of eNOS knockout mice, vascular development is retarded with decreased vessel density and vascular branching. Furthermore, tip cells at the extremities of the vascular front have a marked reduction in the number of filopodia per cell and are more oriented. In a model of oxygen-induced retinopathy (OIR), eNOS deficient mice are protected during the initial vaso-obliterative phase, have reduced pathological neovascularization, and retinal endothelial tip cells have fewer filopodia. Single-cell RNA sequencing of endothelial cells from OIR retinas revealed enrichment of genes related to cell polarity in the endothelial tip-cell subtype of eNOS deficient mice. These results indicate that inhibition of eNOS alters the polarity program of endothelial cells, which increases cell polarization, regulates sprouting angiogenesis and normalizes pathological neovascularization during retinopathy.

Tie2 signalling through Erk1/2 regulates TLR4 driven inflammation.

The inflammatory response is essential for eradication of lipopolysaccharide (LPS) presenting microbial invaders but requires exquisite regulation to prevent detrimental vascular inflammation. Endothelial cells play active roles in both the initiation of inflammation, through the detection of LPS by Toll-like Receptor 4 (TLR4), and the resolution of inflammation, through the actions of the receptor tyrosine kinase, Tie2. The process by which Tie2 attenuates LPS-TLR4 driven inflammation is poorly understood. To investigate the effects of Tie2 on TLR4 signalling, Nf-κB activation was monitored in cells expressing Tie2 mutants harboring tyrosine (Y) to phenylalanine (F) substitutions in the cytoplasmic domain. Tie2 attenuated LPS induced Nf-κB activation in a manner requiring Tie2 kinase activation, the carboxy-terminal tyrosine residue Y1100 and downstream Erk1/2 signalling. Tyrosine 1100 was also required for the Tie2 dependent decrease in expression of the TLR4 signalling proteins, TRAF6 and IRAK1 and stabilization of the Nf-κB inhibitor, IκBα. In contrast, upregulation of known TLR4 antagonist miRNA-146b-5p required all three tyrosine phosphorylation sites in Tie2. Finally, we confirmed in an in vivo model that activation of Tie2 signalling reduces LPS mediated inflammation. Our results show that Y1100 initiated Erk1/2 signalling is essential for the anti-inflammatory effect of Tie2 on TLR4 mediated inflammation.

Tissue-specific sex differences in docosahexaenoic acid and Δ6-desaturase in rats fed a standard chow diet.

Docosahexaenoic acid (DHA, 22:6n-3) is higher in the blood and tissues of females relative to males, but the underlying mechanism is not clear. The present study examined the expression of enzymes involved in the biosynthesis of DHA from short-chain n-3 polyunsaturated fatty acids in male and female rats (n = 6 for each sex). Rats were maintained on an AIN-93G diet and sacrificed at 14 weeks of age after an overnight fast. Plasma, erythrocytes, liver, heart, and brain were collected for fatty acid composition analysis and the determination of enzyme and transcription factor expression by RT-PCR and immunoblotting. Females had higher DHA concentrations in the total lipids of liver, plasma, erythrocyte, and heart (53%, 75%, 36%, and 25% higher, respectively, compared with males) with no sex differences in brain DHA concentrations. The mRNA content of Δ5-desaturase, Δ6-desaturase, and elongase 2 was 1.0-, 1.4-, and 1.1-fold higher, respectively, in the livers of female rats compared with males, with no differences in the hearts or brains. The protein content of Δ6-desaturase was also higher in females. Higher hepatic mRNA of sterol-regulatory element-binding protein 1-c and estrogen receptor α in the females suggests that lipogenic and estrogen signaling mechanisms are involved. The sex difference in DHA concentration is tissue specific and is associated with higher Δ6-desaturase expression in females relative to males, which appears to be limited to the liver.

Effects of diet-induced weight gain on insulin sensitivity and plasma hormone and lipid concentrations in horses.

OBJECTIVE: To determine the effects of diet-induced weight gain on glucose and insulin dynamics and plasma hormone and lipid concentrations in horses. ANIMALS: 13 adult geldings. PROCEDURES: Horses were fed 200% of their digestible energy requirements for maintenance for 16 weeks to induce weight gain. Frequently sampled IV glucose tolerance tests were performed before and after weight gain to evaluate glucose and insulin dynamics. Adiposity (assessed via condition scoring, morphometric measurements, and subcutaneous fat depth) and plasma concentrations of insulin, glucose, nonesterified fatty acids, triglycerides, and leptin were measured on a weekly or biweekly basis. RESULTS: Mean + or - SD body weight increased by 20% from 440 + or - 44 kg to 526 + or - 53 kg, and body condition score (scale, 1 to 9) increased from 6 + or - 1 to 8 + or - 1. Plasma glucose, triglyceride, and nonesterified fatty acid concentrations were similar before and after weight gain. Leptin and insulin concentrations increased with weight gain. Mean + or - SD insulin sensitivity decreased by 71 + or - 28%, accompanied by a 408 + or - 201% increase in acute insulin response to glucose, which resulted in similar disposition index before and after weight gain. CONCLUSIONS AND CLINICAL RELEVANCE: Diet-induced weight gain in horses occurred concurrently with decreased insulin sensitivity that was effectively compensated for by an increase in insulin secretory response. Obesity resulted in hyperinsulinemia and hyperleptinemia, compared with baseline values, but no changes in lipid concentrations were apparent. Preventing obesity is a potential strategy to help avoid insulin resistance, hyperinsulinemia, and hyperleptinemia in horses.