Indoxyl Sulfate-Induced Valve Endothelial Cell Endothelial-to-Mesenchymal Transition and Calcification in an Integrin-Linked Kinase-Dependent Manner.

Calcific Aortic Valve Disease (CAVD) is a significant concern for cardiovascular health and is closely associated with chronic kidney disease (CKD). Aortic valve endothelial cells (VECs) play a significant role in the onset and progression of CAVD. Previous research has suggested that uremic toxins, particularly indoxyl sulfate (IS), induce vascular calcification and endothelial dysfunction, but the effect of IS on valve endothelial cells (VECs) and its contribution to CAVD is unclear. Our results show that IS reduced human VEC viability and increased pro-calcific markers RUNX2 and alkaline phosphatase (ALP) expression. Additionally, IS-exposed VECs cultured in pro-osteogenic media showed increased calcification. Mechanistically, IS induced endothelial-to-mesenchymal transition (EndMT), evidenced by the loss of endothelial markers and increased expression of mesenchymal markers. IS triggered VEC inflammation, as revealed by NF-kB activation, and decreased integrin-linked kinase (ILK) expression. ILK overexpression reversed the loss of endothelial phenotype and RUNX2, emphasizing its relevance in the pathogenesis of CAVD in CKD. Conversely, a lower dose of IS intensified some of the effects in EndMT caused by silencing ILK. These findings imply that IS affects valve endothelium directly, contributing to CAVD by inducing EndMT and calcification, with ILK acting as a crucial modulator.

New environmental factors related to diabetes risk in humans: Emerging bisphenols used in synthesis of plastics.

BACKGROUND: Diabetes mellitus (DM) is one of the largest global health emergencies of the 21st century. In recent years, its connection with environmental pollutants, such as bisphenol A (BPA), has been demonstrated; consequently, new structurally similar molecules are used to replace BPA in the plastics industry (BPS, BPF and BPAF). AIM: To carry out a systematic review to allow coherent evaluation of the state of the art. Subsequently, a meta-analysis was performed to unify the existing quantitative data. METHODS: Firstly, a systematic review was carried out, using the terms "(bisphenol) AND (Diabetes OR Hyperglycemia)", to maximize the number of results. Subsequently, three authors analyzed the set of articles. Finally, a meta-analysis was performed for each BP, using RevMan software. In addition, funnel plots were developed to study publication bias. RESULTS: The systematic analysis of the literature revealed 13 recent articles (2017-2023) related to the study paradigm. The qualitative analysis showed interesting data linking diabetes to the three most widely used substitute BPs in the industry: BPS, BPF and BPAF. Finally, the meta-analysis determined a positive relationship with BPS, BPF and BPAF, which was only statistically significant with BPS. CONCLUSION: There is a need to apply the precautionary principle, regulating the use of new BPs. Therefore, replacing BPA with BPS, BPF or BPAF is unlikely to protect the population from potential health risks, such as DM.

Combination of Bisphenol A and Its Emergent Substitute Molecules Is Related to Heart Disease and Exerts a Differential Effect on Vascular Endothelium.

Plastic production, disposal, and recycling systems represent one of the higher challenges for the planet's health. Its direct consequence is the release of endocrine disruptors, such as bisphenol A (BPA), and its emerging substitute molecules, bisphenol F and S (BPF and BPS), into the environment. Consequently, bisphenols are usually present in human biological fluids. Since BPA, BPS, and BPF have structural analogies and similar hormonal activity, their combined study is urgently needed. The present manuscript studied the effect of the mixture of bisphenols (BPmix) in one of the world's largest human cohorts (NHANES cohort). Descriptive and comparative statistics, binomial and multinomial logistic regression, weighted quantile sum regression, quantile g-computation, and Bayesian kernel machine regression analysis determined a positive association between BPmix and heart disease, including confounders age, gender, BMI, ethnicity, Poverty/Income Ratio, and serum cotinine. Endothelial dysfunction is a hallmark of cardiovascular disease; thus, the average ratio of bisphenols found in humans was used to conduct murine aortic endothelial cell studies. The first results showed that BPmix had a higher effect on cell viability than BPA, enhancing its deleterious biological action. However, the flow cytometry, Western blot, and immunofluorescence assays demonstrated that BPmix induces a differential effect on cell death. While BPA exposure induces necroptosis, its combination with the proportion determined in the NHANES cohort induces apoptosis. In conclusion, the evidence suggests the need to reassess research methodologies to study endocrine disruptors more realistically.

Integrin-Linked Kinase Expression in Human Valve Endothelial Cells Plays a Protective Role in Calcific Aortic Valve Disease.

Calcific aortic valve disease (CAVD) is highly prevalent during aging. CAVD initiates with endothelial dysfunction, leading to lipid accumulation, inflammation, and osteogenic transformation. Integrin-linked kinase (ILK) participates in the progression of cardiovascular diseases, such as endothelial dysfunction and atherosclerosis. However, ILK role in CAVD is unknown. First, we determined that ILK expression is downregulated in aortic valves from patients with CAVD compared to non-CAVD, especially at the valve endothelium, and negatively correlated with calcification markers. Silencing ILK expression in human valve endothelial cells (siILK-hVECs) induced endothelial-to-mesenchymal transition (EndMT) and promoted a switch to an osteoblastic phenotype; SiILK-hVECs expressed increased RUNX2 and developed calcified nodules. siILK-hVECs exhibited decreased NO production and increased nitrosative stress, suggesting valvular endothelial dysfunction. NO treatment of siILK-hVECs prevented VEC transdifferentiation, while treatment with an eNOS inhibitor mimicked ILK-silencing induction of EndMT. Accordingly, NO treatment inhibited VEC calcification. Mechanistically, siILK-hVECs showed increased Smad2 phosphorylation, suggesting a TGF-ß-dependent mechanism, and NO treatment decreased Smad2 activation and RUNX2. Experiments performed in eNOS KO mice confirmed the involvement of the ILK-eNOS signaling pathway in valve calcification, since aortic valves from these animals showed decreased ILK expression, increased RUNX2, and calcification. Our study demonstrated that ILK endothelial expression participates in human CAVD development by preventing endothelial osteogenic transformation.

Bisphenol A Induces Accelerated Cell Aging in Murine Endothelium.

Bisphenol A (BPA) is a widespread endocrine disruptor affecting many organs and systems. Previous work in our laboratory demonstrated that BPA could induce death due to necroptosis in murine aortic endothelial cells (MAECs). This work aims to evaluate the possible involvement of BPA-induced senescence mechanisms in endothelial cells. The ß-Gal assays showed interesting differences in cell senescence at relatively low doses (100 nM and 5 µM). Western blots confirmed that proteins involved in senescence mechanisms, p16 and p21, were overexpressed in the presence of BPA. In addition, the UPR (unfolding protein response) system, which is part of the senescent phenotype, was also explored by Western blot and qPCR, confirming the involvement of the PERK-ATF4-CHOP pathway (related to pathological processes). The endothelium of mice treated with BPA showed an evident increase in the expression of the proteins p16, p21, and CHOP, confirming the results observed in cells. Our results demonstrate that oxidative stress induced by BPA leads to UPR activation and senescence since pretreatment with N-acetylcysteine (NAC) in BPA-treated cells reduced the percentage of senescent cells prevented the overexpression of proteins related to BPA-induced senescence and reduced the activation of the UPR system. The results suggest that BPA participates actively in accelerated cell aging mechanisms, affecting the vascular endothelium and promoting cardiovascular diseases.

Bisphenol A impaired cell adhesion by altering the expression of adhesion and cytoskeleton proteins on human podocytes.

Bisphenol A (BPA), a chemical -xenoestrogen- used in food containers is present in the urine of almost the entire population. Recently, several extensive population studies have proven a significant association between urinary excretion of BPA and albuminuria. The alteration of glomerular podocytes or "podocytopathy" is a common event in chronic albuminuric conditions. Since many podocytes recovered from patients' urine are viable, we hypothesized that BPA could impair podocyte adhesion capabilities. Using an in vitro adhesion assay, we observed that BPA impaired podocyte adhesion, an effect that was abrogated by Tamoxifen (an estrogen receptor blocker). Genomic and proteomic analyses revealed that BPA affected the expression of several podocyte cytoskeleton and adhesion proteins. Western blot and immunocytochemistry confirmed the alteration in the protein expression of tubulin, vimentin, podocin, cofilin-1, vinculin, E-cadherin, nephrin, VCAM-1, tenascin-C, and ß-catenin. Moreover, we also found that BPA, while decreased podocyte nitric oxide production, it lead to overproduction of ion superoxide. In conclusion, our data show that BPA induced a novel type of podocytopathy characterizes by an impairment of podocyte adhesion, by altering the expression of adhesion and cytoskeleton proteins. Moreover, BPA diminished production of podocyte nitric oxide and induced the overproduction of oxygen-free metabolites. These data provide a mechanism by which BPA could participate in the pathogenesis and progression of renal diseases.

The BCL-2 family members NOXA and BIM mediate fluorizoline-induced apoptosis in multiple myeloma cells.

Fluorizoline is a new synthetic molecule that induces apoptosis by selectively targeting prohibitins. In this study, we have assessed the pro-apoptotic effect of fluorizoline in 3 different multiple myeloma cell lines and 12 primary samples obtained from treatment-naïve multiple myeloma patients. Fluorizoline induced apoptosis in both multiple myeloma cell lines and primary samples at concentrations in the low micromolar range. All primary samples were sensitive to fluorizoline. Moreover, fluorizoline increased the mRNA and protein levels of the pro-apoptotic BCL-2 family member NOXA both in cell lines and primary samples analyzed. Finally, NOXA-depletion by CRISPR/Cas9 in cells that do not express BIM conferred resistance to fluorizoline-induced apoptosis in multiple myeloma cells. These results suggest that targeting prohibitins could be a new therapeutic strategy for myeloma multiple.