No abstract

Aging is associated with reduced tissue remodeling efficiency and increased fibrosis, characterized by excess collagen accumulation and altered matrix degradation. Ovulation, the process by which an egg is released from the ovary, is one of the most dynamic cycles of tissue wounding and repair. Because the ovary is one of the first organs to age, ovulation and ovarian wound healing is impaired with advanced reproductive age. To test this hypothesis, we induced superovulation in reproductively young and old mice and determined the numbers of eggs ovulated and corpora lutea (CLs), the progesterone producing glands formed post-ovulation. Reproductively old mice ovulated fewer eggs and had fewer CLs relative to young controls. Moreover, reproductively old mice exhibited a greater number of oocytes trapped within CLs and expanded cumulus oocyte complexes within unruptured antral follicles, indicative of failed ovulation. In addition, post-ovulatory tissue remodeling was compromised with age as evidenced by reduced CL vasculature, increased collagen, decreased hyaluronan, decreased cell proliferation and apoptosis, impaired wound healing capacity, and aberrant morphology of the ovarian surface epithelium (OSE). These findings demonstrate that ovulatory dysfunction is an additional mechanism underlying the age-related loss of fertility beyond the reduction of egg quantity and quality.

Summary Voltage-gated sodium channels are targets for many analgesic and antiepileptic drugs whose therapeutic mechanisms and binding sites have been well characterized. We describe the identification of a previously unidentified receptor site within the NavMs voltage-gated sodium channel. Tamoxifen, an estrogen receptor modulator, and its primary and secondary metabolic products bind at the intracellular exit of the channel, which is a site that is distinct from other previously characterized sodium channel drug sites. These compounds inhibit NavMs and human sodium channels with similar potencies and prevent sodium conductance by delaying channel recovery from the inactivated state. This study therefore not only describes the structure and pharmacology of a site that could be leveraged for the development of new drugs for the treatment of sodium channelopathies but may also have important implications for off-target health effects of this widely used therapeutic drug.

Eosinophils are implicated as effector cells in asthma but the functional implications of the precise location of eosinophils in the airway wall is poorly understood. We aimed to quantify eosinophils in the different compartments of the airway wall and associate these findings with clinical features of asthma and markers of airway inflammation.In this cross-sectional study, we utilised design-based stereology to accurately partition the numerical density of eosinophils in both the epithelial compartment and the subepithelial space (airway wall area below the basal lamina including the submucosa) in individuals with and without asthma and related these findings to airway hyperresponsiveness (AHR) and features of airway inflammation.Intraepithelial eosinophils were linked to the presence of asthma and endogenous AHR, the type of AHR that is most specific for asthma. In contrast, both intraepithelial and subepithelial eosinophils were associated with type-2 (T2) inflammation, with the strongest association between IL5 expression and intraepithelial eosinophils. Eosinophil infiltration of the airway wall was linked to a specific mast cell phenotype that has been described in asthma. We found that IL-33 and IL-5 additively increased cysteinyl leukotriene (CysLT) production by eosinophils and that the CysLT LTC4 along with IL-33 increased IL13 expression in mast cells and altered their protease profile.We conclude that intraepithelial eosinophils are associated with endogenous AHR and T2 inflammation and may interact with intraepithelial mast cells via CysLTs to regulate airway inflammation.

Aims/hypothesis Substantial deposition of the extracellular matrix component hyaluronan (HA) is characteristic of insulitis in overt type 1 diabetes. We investigated whether HA accumulation is detectable in islets early in disease pathogenesis and how this affects the development of insulitis and beta cell mass. Methods Pancreas tissue from 15 non-diabetic organ donors who were positive for islet autoantibodies (aAbs) and from 14 similarly aged aAbcontrol donors were examined for the amount of islet HA staining and the presence of insulitis. The kinetics of HA deposition in islets, along with the onset and progression of insulitis and changes in beta cell mass, were investigated in BioBreeding DRLyp/Lyp rats (a model of spontaneous autoimmune diabetes) from 40 days of age until diabetes onset. Results Abundant islet HA deposits were observed in pancreas tissues from n = 3 single-and n = 4 double-aAb + donors (aAb + HA high). In these seven tissues, the HA-stained areas in islets measured 1000 ± 240 μm 2 (mean ± SEM) and were fourfold larger than those from aAbcontrol tissues. The aAb + HA high tissues also had a greater prevalence of islets that were highly rich in HA (21% of the islets in these tissues contained the largest HA-stained areas [>2000 μm 2 ] vs less than 1% in tissues from aAbcontrol donors). The amount of HA staining in islets was associated with the number of aAbs (i.e. single-or double-aAb positivity) but not with HLA genotype or changes in beta cell mass. Among the seven aAb + HA high tissues, three from single-and one from double-aAb + donors did not show any islet immune-cell infiltrates, indicating that HA accumulates in aAb + donors independently of insulitis. The three aAb + HA high tissues that exhibited insulitis had the largest HA-stained areas and, in these tissues, islet-infiltrating immune cells co-localised with the most prominent HA deposits (i.e. with HA-stained areas >2000 μm 2). Accumulation of HA in islets was evident prior to insulitis in 7-8-week-old presymptomatic DRLyp/Lyp rats, in which the islet HA-stained area measured 2370 ± 170 μm 2 (mean ± SEM), which was threefold larger than in 6-week-old rats. This initial islet HA deposition was not concurrent with beta cell loss. Insulitis was first detected in 9-10-week-old rats, in which the HA-stained areas were 4980 ± 500 μm 2. At this age, the rats also exhibited a 44% reduction in beta cell mass. Further enlargement of the HA-positive areas (mean ± SEM: 7220 ± 880 μm 2) was associated with invasive insulitis. HA deposits remained abundant in the islets of rats with destructive insulitis, which had lost 85% of their beta cells. Conclusions/interpretation This study indicates that HA deposition in islets occurs early in type 1 diabetes and prior to insulitis, and points to a potential role of HA in triggering islet immune-cell infiltration and the promotion of insulitis.

The female reproductive system ages before any other organ system in the body. This phenomenon can have tangible clinical implications leading to infertility, miscarriages, birth defects and systemic deterioration due to estrogen loss. “Fibroinflammation” is a hallmark of aging tissues; there is an increase in inflammatory cytokines and fibrotic tissue in the aging ovarian stroma. We systematically evaluated immunomodulatory factors in human follicular fluid, which, like the stroma, is a critical ovarian microenvironment directly influencing the oocyte. Using a cytokine antibody array, we identified a unique fibroinflammatory cytokine signature in follicular fluid across an aging series of women (27.7–44.8 years). This signature (IL-3, IL-7, IL-15, TGFβ1, TGFβ3 and MIP-1) increased with chronologic age, was inversely correlated to anti-Müllerian hormone (AMH) levels, and was independent of body mass index (BMI). We focused on one specific protein, TGFβ3, for further validation. By investigating this cytokine in human cumulus cells and ovarian tissue, we found that the age-dependent increase in TGFβ3 expression was unique to the ovarian stroma but not other ovarian sub-compartments. This study broadens our understanding of inflammaging in the female reproductive system and provides a defined fibroinflammatory aging signature in follicular fluid and molecular targets in the ovary with potential clinical utility.

Type 2 diabetes is associated with upregulation of neprilysin, a peptidase capable of cleaving glucoregulatory peptides such as glucagon-like peptide-1 (GLP-1). In humans, use of the neprilysin inhibitor sacubitril in combination with an angiotensin II receptor blocker was associated with increased plasma GLP-1 levels and improved glycemic control. Whether neprilysin inhibition per se is mediating these effects remains unknown. We sought to determine whether pharmacological neprilysin inhibition on its own confers beneficial effects on glycemic status and beta-cell function in a mouse model of reduced insulin secretion, and whether any such effects are dependent on GLP-1 receptor (GLP-1R) signaling. High-fat-fed male wild-type (Glp1r+/+) and GLP-1R knockout (Glp1r−/−) mice were treated with low-dose streptozotocin (STZ) to recapitulate type 2 diabetes-associated beta-cell dysfunction, or vehicle as control. Mice were continued on high fat diet alone or supplemented with the neprilysin inhibitor sacubitril for 8 weeks. At the end of the study period, beta-cell function was assessed by oral or intravenous glucose tolerance test. Fasting and fed glucose were significantly lower in wild-type mice treated with sacubitril, although active GLP-1 levels and insulin secretion during oral glucose challenge were unchanged. In contrast, insulin secretion in response to intravenous glucose was significantly enhanced in sacubitril-treated wild-type mice, and this effect was blunted in Glp1r−/− mice. Similarly, sacubitril enhanced insulin secretion in vitro in islets from STZ-treated Glp1r+/+ but not Glp1r−/− mice. Together, our data suggest the insulinotropic effects of pharmacological neprilysin inhibition in a mouse model of beta-cell dysfunction are mediated via intra-islet GLP-1R signaling.

Islet endothelial cells produce paracrine factors important for islet beta-cell function and survival. Under conditions of type 2 diabetes, islet endothelial cells exhibit a dysfunctional phenotype including increased expression of genes involved in cellular adhesion and inflammation. We sought to determine whether treatment of hyperglycemia with the sodium glucose co-transporter 2 inhibitor empagliflozin, either alone or in combination with metformin, would improve markers of endothelial cell function in islets, assessed ex vivo, and if such an improvement is associated with improved insulin secretion in a mouse model of diabetes in vivo. For these studies, db/db diabetic mice and non-diabetic littermate controls were treated for 6 weeks with empagliflozin or metformin, either alone or in combination. For each treatment group, expression of genes indicative of islet endothelial dysfunction was quantified. Islet endothelial and beta-cell area was assessed by morphometry of immunochemically stained pancreas sections. Measurements of plasma glucose and insulin secretion during an intravenous glucose tolerance test were performed on vehicle and drug treated diabetic animals. We found that expression of endothelial dysfunction marker genes is markedly increased in diabetic mice. Treatment with either empagliflozin or metformin lowered expression of the dysfunction marker genes ex vivo, which correlated with improved glycemic control, and increased insulin release in vivo. Empagliflozin treatment was more effective than metformin alone, with a combination of the two drugs demonstrating the greatest effects. Improving islet endothelial function through strategies such as empagliflozin/metformin treatment may provide an effective approach for improving insulin release in human type 2 diabetes.