Chronic intermittent hypoxia triggers cardiac fibrosis: Role of epididymal white adipose tissue senescent remodeling?

AIM: Obstructive sleep apnea (OSA) is a growing health problem affecting nearly 1 billion people worldwide. The landmark feature of OSA is chronic intermittent hypoxia (CIH), accounting for multiple organ damage, including heart disease. CIH profoundly alters both visceral white adipose tissue (WAT) and heart structure and function, but little is known regarding inter-organ interaction in the context of CIH. We recently showed that visceral WAT senescence drives myocardial alterations in aged mice without CIH. Here, we aimed at investigating whether CIH induces a premature visceral WAT senescent phenotype, triggering subsequent cardiac remodeling. METHODS: In a first experiment, 10-week-old C57bl6J male mice (n = 10/group) were exposed to 14 days of CIH (8 h daily, 5%-21% cyclic inspired oxygen fraction, 60 s per cycle). In a second series, mice were submitted to either epididymal WAT surgical lipectomy or sham surgery before CIH exposure. Finally, we used p53 deficient mice or Wild-type (WT) littermates, also exposed to the same CIH protocol. Epididymal WAT was assessed for fibrosis, DNA damages, oxidative stress, markers of senescence (p16, p21, and p53), and inflammation by RT-qPCR and histology, and myocardium was assessed for fibrosis and cardiomyocyte hypertrophy. RESULTS: CIH-induced epididymal WAT remodeling characterized by increased fibrosis, oxidative stress, DNA damage response, inflammation, and increased expression of senescent markers. CIH-induced epididymal WAT remodeling was associated with subtle and early myocardial interstitial fibrosis. Both epididymal WAT surgical lipectomy and p53 deletion prevented CIH-induced myocardial fibrosis. CONCLUSION: Short-term exposure to CIH induces epididymal WAT senescent remodeling and cardiac interstitial fibrosis, the latter being prevented by lipectomy. This finding strongly suggests visceral WAT senescence as a new target to mitigate OSA-related cardiac disorders.

Long-Term High-Fat Diet Limits the Protective Effect of Spontaneous Physical Activity on Mammary Carcinogenesis.

Breast cancer is influenced by factors such as diet, a sedentary lifestyle, obesity, and postmenopausal status, which are all linked to prolonged hormonal and inflammatory exposure. Physical activity offers protection against breast cancer by modulating hormones, immune responses, and oxidative defenses. This study aimed to assess how a prolonged high-fat diet (HFD) affects the effectiveness of physical activity in preventing and managing mammary tumorigenesis. Ovariectomised C57BL/6 mice were provided with an enriched environment to induce spontaneous physical activity while being fed HFD. After 44 days (short-term, ST HFD) or 88 days (long-term, LT HFD), syngenic EO771 cells were implanted into mammary glands, and tumour growth was monitored until sacrifice. Despite similar physical activity and food intake, the LT HFD group exhibited higher visceral adipose tissue mass and reduced skeletal muscle mass. In the tumour microenvironment, the LT HFD group showed decreased NK cells and TCD8+ cells, with a trend toward increased T regulatory cells, leading to a collapse of the T8/Treg ratio. Additionally, the LT HFD group displayed decreased tumour triglyceride content and altered enzyme activities indicative of oxidative stress. Prolonged exposure to HFD was associated with tumour growth despite elevated physical activity, promoting a tolerogenic tumour microenvironment. Future studies should explore inter-organ exchanges between tumour and tissues.

25-Hydroxyvitamin D potentializes extracellular cathelicidin release from human PBMC stimulated ex vivo with either bacterial (LPS) or viral (P: IC) mimetics

Human cathelicidin refers to the cationic antimicrobial peptide hCAP18/LL-37. LL-37 is formed by cleavage of the propeptide hCAP18 coded by the CAMP gene. The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)D), has been shown to induce the CAMP gene expression through promoter activation. We previously failed to demonstrate in a clinical trial that supplementation of 25-hydroxyvitamin D (25(OH)D) improves LL-37 serum levels. The aim of this work was to evaluate the impact of 25(OH)D supplementation on intracellular expression of CAMP and secretion of LL-37 in an ex vivo model using the peripheral blood mononuclear cells (PBMC). PBMC collected from healthy donors and incubated with different concentrations of 25(OH)D (0 ng/ml: control (D0); 25 ng/ml: deficient (D25); 75 ng/ml: physiological (D75); 125 ng/ml: supraphysiological (D125)) were stimulated or not with lipopolysaccharide (LPS, 100 ng/ml) or synthetic double-stranded RNA Poly (I: C) (PIC, 10 µg/ml). The intracellular expressions of the CAMP gene and the hCAP18 peptide were measured respectively after 24-h and 48-h incubation periods. The concentration of LL-37 was determined in the culture medium after 48-h incubation. 25(OH)D significantly induced CAMP gene expression at 24 h with a maximum effect at a dose of D125 in either unstimulated (tenfold expression) or stimulated (LPS: 100-fold expression; PIC: 15-fold expression) conditions. Intracellular hCAP18 peptide was overexpressed at 48 h under unstimulated (1.5-fold, D125) and stimulated conditions, LPS (twofold, D125) and PIC (2.5-fold, D125). The secretion of LL-37 in the culture medium was significantly induced by 25(OH)D only in both stimulated (LPS and PIC) conditions in a dose-dependent manner. (AU)

25-Hydroxyvitamin D potentializes extracellular cathelicidin release from human PBMC stimulated ex vivo with either bacterial (LPS) or viral (P: IC) mimetics.

Human cathelicidin refers to the cationic antimicrobial peptide hCAP18/LL-37. LL-37 is formed by cleavage of the propeptide hCAP18 coded by the CAMP gene. The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)D), has been shown to induce the CAMP gene expression through promoter activation. We previously failed to demonstrate in a clinical trial that supplementation of 25-hydroxyvitamin D (25(OH)D) improves LL-37 serum levels. The aim of this work was to evaluate the impact of 25(OH)D supplementation on intracellular expression of CAMP and secretion of LL-37 in an ex vivo model using the peripheral blood mononuclear cells (PBMC). PBMC collected from healthy donors and incubated with different concentrations of 25(OH)D (0 ng/ml: control (D0); 25 ng/ml: deficient (D25); 75 ng/ml: physiological (D75); 125 ng/ml: supraphysiological (D125)) were stimulated or not with lipopolysaccharide (LPS, 100 ng/ml) or synthetic double-stranded RNA Poly (I: C) (PIC, 10 µg/ml). The intracellular expressions of the CAMP gene and the hCAP18 peptide were measured respectively after 24-h and 48-h incubation periods. The concentration of LL-37 was determined in the culture medium after 48-h incubation. 25(OH)D significantly induced CAMP gene expression at 24 h with a maximum effect at a dose of D125 in either unstimulated (tenfold expression) or stimulated (LPS: 100-fold expression; PIC: 15-fold expression) conditions. Intracellular hCAP18 peptide was overexpressed at 48 h under unstimulated (1.5-fold, D125) and stimulated conditions, LPS (twofold, D125) and PIC (2.5-fold, D125). The secretion of LL-37 in the culture medium was significantly induced by 25(OH)D only in both stimulated (LPS and PIC) conditions in a dose-dependent manner. Our results demonstrate that 25(OH)D incubation increases intracellular expression of CAMP and hCAP18, but extracellular secretion of LL-37 antimicrobial peptide is increased by 25(OH)D only when PBMC from healthy donors were stimulated with bacterial or viral immune mimetic.

Vitamin D supplementation associated with physical exercise promotes a tolerogenic immune environment without effect on mammary tumour growth in C57BL/6 mice.

PURPOSE: High plasma vitamin D (VitD) level and regular exercise (Ex) are known to have anti-cancer and immunomodulatory effects. This study aimed to evaluate the impact of VitD supplementation and imposed physical Ex on mammary tumour growth and immune response in ovariectomised mice fed high-fat (HF) diet. METHODS: Ovariectomised 33-week-old mice C57BL/6 (n = 60), housed in enriched environment (EE), were fed HF diet (450 kcal/100 g) supplemented or not with VitD (HF/HF + D: 125/1225 IU/100 g) for 12 weeks and submitted or not to Ex (HF + Ex; HF + D + Ex) on treadmill (45 min/day, 5 days/week). At w8, syngeneic tumour cells EO771 were orthotopically injected into the 4th mammary gland. Spontaneous activity (SPA), maximal speed (MS) and forelimb grip strength (GS) were measured. Tumour immune cells infiltrate was phenotyped by FACS. Data (mean ± SEM) were analysed by two-way ANOVA + Tukey post-test. RESULTS: Ex (p = 0.01) and VitD (p = 0.05) reduced body weight gain. Exercise decreased visceral fat mass [g: 1.5 ± 0.8 (HF); 1.2 ± 0.65 (HF + Ex); 0.9 ± 0.6 (HF + D + Ex); p = 0.03]. SPA (p < 0.0001) and GS (p = 0.01) were higher in HF + D + Ex mice vs others. No effect of Ex or VitD on tumour growth was detected. In tumour, VitD decreased the proportion of NK (p = 0.03), while Ex increased it (p = 0.03). The Th1/Th2 ratio is lowered by VitD (p = 0.05), while Tc/Treg ratio was not affected either by Exercise or VitD. CONCLUSION: In our experimental conditions, VitD supplementation and physical exercise have synergetic effects reducing the weight gain under HF diet and improving the physical capacities of mice. VitD coupled with exercise induces an immunosuppressive response without effect on tumour growth.

EO771, the first luminal B mammary cancer cell line from C57BL/6 mice.

BACKGROUND: Despite decades of therapeutic trials, effective diagnosis, many drugs available and numerous studies on breast cancer, it remains the deadliest cancer in women. In order to choose the most appropriate treatment and to understand the prognosis of the patients, breast cancer is divided into different subtypes using a molecular classification. Just as there remains a need to discover new effective therapies, models to test them are also required. METHODS: The EO771 (also named E0771 or EO 771) murine mammary cancer cell line was originally isolated from a spontaneous tumour in C57BL/6 mouse. Although frequently used, this cell line remains poorly characterized. Therefore, the EO771 phenotype was investigated. The phenotype was compared to that of MCF-7 cells, known to be of luminal A subtype and to express estrogen receptors, as well as MDA-MB-231 cells, which are triple negative. Their sensitivity to hormonal treatment was evaluated by viability tests. RESULTS: The EO771 were estrogen receptor α negative, estrogen receptor ß positive, progesterone receptor positive and ErbB2 positive. This phenotype was associated with a sensitivity to anti-estrogen treatments such as tamoxifen, 4-hydroxy-tamoxifen, endoxifen and fulvestrant. CONCLUSIONS: On account of the numerous results published with the EO771 cell line, it is important to know its classification, to facilitate comparisons with corresponding types of tumours in patients. Transcriptomic and protein analysis of the EO771 cell line classified it within the luminal B subtype. Luminal B cancers correspond to one of the subtypes most frequently encountered in patients and associated with a poor prognosis.