Reference Gene Validation for RT-qPCR in PBMCs from Asthmatic Patients with or without Obesity

Obesity is known to impair the efficacy of glucocorticoid medications for asthma control. Glucocorticoid-induced gene expression studies may be useful to discriminate those obese asthmatic patients who present a poor response to glucocorticoids. The expression of genes of interest is normalized with respect to reference genes (RGs). Ideally, RGs have a stable expression in different samples and are not affected by experimental conditions. The objective of this work was to analyze suitable RGs to study the role of glucocorticoid-induced genes in obese asthmatic patients in further research. The gene expression of eight potential RGs (GUSB, B2M, POLR2A, PPIA, ACTB, GAPDH, HPRT1, and TBP) was assessed with reverse transcription-quantitative polymerase chain reaction in peripheral blood mononuclear cells (PBMCs) from asthmatic, obese asthmatic, and healthy individuals. Their stability was analyzed using four different algorithms-BestKeeper, ?Ct, geNorm, and NormFinder. geNorm analysis recommended the use of a minimum of three genes for normalization. Moreover, intergroup variation due to the treatment was calculated by NormFinder, which found that B2M was the gene that was least affected by different treatments. Comprehensive rankings indicated GUSB and HPRT1 as the best RGs for qPCR in PBMCs from healthy and asthmatic subjects, while B2M and PPIA were the best for obese asthmatic subjects. Finally, our results demonstrated that B2M and HPRT1 were the most stable RGs among all groups, whereas ACTB, TBP, and GAPDH were the worst shared ones

Acetylsalicylic acid prevents intermittent hypoxia-induced vascular remodeling in a murine model of sleep apnea

Study objectives: Chronic intermittent hypoxia (CIH), a hallmark feature of obstructive sleep apnea (OSA), induces accelerated atherogenesis as well as aorta vascular remodeling. Although the cyclooxygenase (COX) pathway has been proposed to contribute to the cardiovascular consequences of OSA, the potential benefits of a widely employed COX-inhibitor such (acetylsalicylic acid, ASA) on CIH-induced vascular pathology are unknown. Therefore, we hypothesized that a common non-selective COX inhibitor such as ASA would attenuate the aortic remodeling induced by CIH in mice. Methods: 40 wild-type C57/BL6malemice were randomly allocated to CIH or normoxic exposures (N) and treated with daily doses of ASA or placebo for 6 weeks. At the end of the experiments, intima-media thickness (IMT), elastin disorganization (ED), elastin fragmentation (EF), length between fragmented fiber endpoints (LFF), aortic wall collagen abundance (AC) and mucoid deposition (MD) were assessed. Results: Compared to N, CIH promoted significant increases in IMT (52.58 ± 2.82μm vs. 46.07 ± 4.18μm, p < 0.003), ED (25.29 ± 14.60% vs. 4.74 ± 5.37%, p < 0.001), EF (5.80 ± 2.04 vs. 3.06 ± 0.58, p < 0.001), LFF (0.65 ± 0.34% vs. 0.14 ± 0.09%, p < 0.001), AC (3.43 ± 1.52% vs. 1.67 ± 0.67%, p < 0.001) and MD (3.40 ± 2.73 μm2 vs. 1.09 ± 0.72 μm2, p < 0.006). ASA treatment mitigated the CIH-induced alterations in IMT: 44.07 ± 2.73μm; ED: 10.57 ± 12.89%; EF: 4.63 ± 0.88; LFF: 0.25 ± 0.17% and AC: 0.90 ± 0.13% (p<0.05 for all comparisons). Conclusions: ASA prevents the CIH-induced aortic vascular remodeling, and should therefore be prospectively evaluated as adjuvant treatment in patients with OSA.This work was supported by the Spanish Respiratory Society (SEPAR), SOCAP, the Associació Lleidatana de Respiratori (ALLER), and the Spanish Fondo de Investigaciones Sanitarias (PI14/00486 and PI14-00004), Instituto de Salud Carlos III (ISCIII), European Regional Development Fund (ERDF) “Una manera de hacer Europa”. DG is supported by National Institutes of Health grant HL130984