Effects of hydrogen peroxide on endothelial function in three-dimensional hydrogel vascular model and regulation mechanism of polar protein Par3.

Three-dimensional (3D) cell cultures better reflect the function of endothelial cells (ECs) than two-dimensional (2D) cultures. In recent years, studies have found that ECs cultured in a 3D luminal structure can mimic the biological characteristics and phenotypes of vascular ECs, thus making it more suitable for endothelial dysfunction research. In this study, we used a 3D model and 2D tissue culture polystyrene (TCP) to investigate the effects of cell polarity on hydrogen peroxide (H2O2)-induced endothelial dysfunction and its related mechanisms. We observed the cell morphology, oxidative stress, and barrier and endothelial function of human umbilical vein ECs (HUVECs) in 3D and 2D cultures. We then used Illumina to detect the differentially expressed genes (DEGs) in the 3D-cultured HUVEC with and without H2O2stimulation, using clusterProfiler for Gene Ontology function enrichment analysis and Kyoto Encyclopaedia of Genes and Genomes pathway enrichment analysis of DEGs. Finally, we explored the role and mechanism of polar protein partitioning defective protein 3 (Par3) in the regulation of ECs. ECs were inoculated into the 3D hydrogel channel; after stimulation with H2O2, the morphology of HUVECs changed, the boundary was blurred, the expression of intercellular junction proteins decreased, and the barrier function of the EC layer was damaged. 3D culture increased the oxidative stress response of cells stimulated by H2O2compared to 2D TCPs. The polarity-related protein Par3 and cell division control protein 42 were screened using bioinformatics analysis, and western blotting was used to verify the results. Par3 knockdown significantly suppressed claudin1 (CLDN1) and vascular endothelial cadherin. These results suggest that the polar protein Par3 can protect H2O2-induced vascular ECs from damage by regulating CLDN1 and VE-cadherin.

SIRT6 inhibits inflammatory response through regulation of NRF2 in vascular endothelial cells.

Emerging evidence suggests that inflammation plays a pivotal role in Atherosclerosis. Sirtuin 6 (SIRT6), a member of NAD+-dependent protein lysine deacylases of the sirtuin family, plays an important role in the regulation of metabolism, aging and stress resistance. However, the role of SIRT6 in vascular inflammation and its molecular mechanism is unknown. The present study showed that TNF-α significantly reduced the expression of SIRT6 protein and mRNA in a concentration- and time-dependent manner and increased the expression of monocyte chemotactic protein 1 (MCP-1), interleukin (IL) -6 and IL-1ß in human umbilical vein endothelial cells (HUVECs). Overexpression of SIRT6 but not its catalytically inactive mutant inhibited TNF-α-induced expression of MCP-1, IL-6 and IL-1ß. Knockdown of SIRT6 significantly enhanced TNF-α-induced expression of MCP-1, IL-6 and IL-1ß. Moreover, knockdown of SIRT6 reduced TNF-α-induced nuclear factor erythroid 2 related factor 2 (NRF2) nucleus protein expression, whereas knockdown of NRF2 significantly enhanced TNF-α-induced expression of MCP-1, IL-6 and IL-1ß. In addition, overexpression of SIRT6 increased NRF2 and its target genes expression, and knockdown of SIRT6 decreased NRF2 and its target genes expression. Meanwhile, knockdown of SIRT6 inhibited NRF2 nucleus protein expression. Further, knockdown of SIRT6 decreased phosphorylation of NRF2, overexpression of SIRT6 increased phosphorylation of NRF2. SIRT6 interacted with NRF2. In vivo, the levels of TNF-α and IL-1ß were increased in the serum of hyperlipidemia mice. Hyperlipidemia-induced production of MCP-1, IL-6 and IL-1ß was significantly augmented in the endothelium specific SIRT6 knockout mice. In contrast, the expression of NRF2 and its target genes was reduced. Taken together, these results indicate that SIRT6 protects against vascular inflammation via its deacetylase activity and the NRF2-dependent signaling pathway.

Joint effect of alcohol drinking and tobacco smoking on all-cause mortality and premature death in China: A cohort study.

BACKGROUND: Tobacco smoking and alcohol drinking are associated with several diseases, and studies on the joint effects of smoking and drinking are rare. OBJECTIVE: This study investigates the joint effects of tobacco smoking and alcohol drinking on all-cause and premature mortality in a contemporary cohort. METHODS: The China Health and Retirement Longitudinal Study (CHARLS) is an ongoing nationally representative survey of subjects aged over 45 years in China that was performed every two years for a total of three waves from 2011 to 2015 in China. We used weighted logistic regression models to estimate the joint effects of tobacco smoking and alcohol drinking on all-cause and premature mortality. RESULTS: After adjusting for prespecified confounders, the odds ratios (ORs) of all-cause mortality were 1.51 (95% CI: 1.09-2.10) and 1.47 (95% CI: 1.03-2.08) in smokers and smokers/drinkers, respectively. Compared with nonsmokers/nondrinkers, the OR of smokers/drinkers for premature death was 3.14 (95% CI: 1.56-6.34). In the female subgroup, there was an approximately 5-fold (OR = 4.95; 95% CI: 2.00-12.27) odds of premature mortality for smokers/drinkers compared to nonsmokers/nondrinkers. CONCLUSION: This study found a joint effect of tobacco smoking and alcohol drinking on all-cause and premature mortality among a contemporary and nationally representative cohort in China. Our results suggested that the joint effects were more pronounced in women, but further research is needed.

Synergistic Effect of Tangeretin and Atorvastatin for Colon Cancer Combination Therapy: Targeted Delivery of These Dual Drugs Using RGD Peptide Decorated Nanocarriers.

PURPOSE: Colorectal cancer (CRC) is the third most frequently diagnosed cancer and the fourth leading cause of cancer death over the world. Nano-sized drug delivery systems are used for the treatment of cancers. The aim of this study was to develop a tangeretin (TAGE) and atorvastatin (ATST) combined nano-system decorated with RGD (RGD-ATST/TAGE CNPs) for colon cancer combination therapy. MATERIALS AND METHODS: In this study, cyclized arginine-glycine-aspartic acid sequences (RGD) contained ligand was synthesized by conjugating cyclo (Arg-Gly-Asp-d-Phe-Lys) (cRGDfK) with D-α-tocopheryl succinate dichloromethane (TOSD) using polyethylene glycol (PEG) as a linker to obtain cRGDfK-PEG-TOSD. ATST and TAGE combined nano-systems: RGD-ATST/TAGE CNPs were prepared. The combination effects as well as antitumor effects of these two agents were evaluated on colon cancer cells and mice bearing cancer models. RESULTS: Drug entrapment efficiencies of nano-systems were high (around 90%), suggesting the good loading capacity. The release profiles of ATST or TAGE from RGD-ATST/TAGE CNPs followed Higuchi model. The RGD-decorated nano-system showed more obvious cytotoxicity on HT-29 cells than the undecorated nano-system, but no obvious difference was found on normal CCD-18 cells. The strongest synergism was observed when the weight ratio of ATST to TAGE was 1:1. In vivo biodistribution of RGD-ATST/TAGE CNPs in the tumor site is high and prominently inhibited the in vivo tumor growth. CONCLUSION: The results demonstrated that RGD-ATST/TAGE CNPs showed the most significant synergistic therapeutic efficacy, exhibited no significant toxicity to major organs and tissues, and body weight of the treated mice was stable. Therefore, the combination nano-system is a promising platform for colon cancer therapy.

Assessment of direct oral anticoagulants administered as potentially inappropriate medications to elderly inpatients.

Background There is evidence that direct oral anticoagulants administered as potentially inappropriate medications increase the risk of bleeding and thromboembolic complications, which represent serious threats to human health. Objective To identify direct oral anticoagulants administered as potentially inappropriate medications for hospitalized patients aged ≥ 65 years in our hospital, and to determine associated factors and the correlation between potentially inappropriate medications and adverse reactions. Setting Xi'an Central Hospital, China. Method A retrospective cross-sectional study of elderly hospitalized patients who received either dabigatran or rivaroxaban at Xi'an Central Hospital between June 1, 2018 and June 1, 2019. The evaluation criteria of direct oral anticoagulants were formulated based on drug labels, disease guidelines and the 2019 American Geriatrics Society Beers Criteria, and any non-compliance with the criteria was considered to be potentially inappropriate medications. The Pearson chi-square test and a binary logistic regression model were used. Main outcome measure Factors associated with potentially inappropriate medications and correlation between potentially inappropriate medications and adverse reactions. Results This study analysed 315 patients aged ≥ 65 years. The application of our evaluation criteria identified 155 (49.2%) instances of potentially inappropriate medications, comprising 5 different types of potentially inappropriate medications. Fifteen adverse drug reactions occurred in the study participants. The Pearson chi-square test revealed significant differences in number of medications (p = 0.021) and creatinine clearance rate (p = 0.002) between potentially inappropriate medications and non-potentially inappropriate medications groups. In the binary logistic regression model, potentially inappropriate medications use was associated with creatinine clearance (creatinine clearance < 30: OR = 3.590, 95% CI = 1.214-10.615, p = 0.021), and there was no significant correlation between potentially inappropriate medications and adverse drug reactions after eliminating the confounding factors (age, length of hospitalization, number of disease combined) with p values of less than 0.25 (adjusted OR = 0.372, 95% CI = 0.117-1.182, p = 0.094). Conclusion This study revealed that the incidence of potentially inappropriate medications was relatively high, number of medications and creatinine clearance differed significantly between potentially inappropriate medications and non-potentially inappropriate medications groups, and potentially inappropriate medications was associated with creatinine clearance (creatinine clearance < 30 mL/min), but there was no significant correlation between potentially inappropriate medications and adverse drug reactions after eliminating the confounding factors.

A comparative study unraveling the effects of TNF-α stimulation on endothelial cells between 2D and 3D culture.

Endothelial cell (EC) dysfunction is an important predictor of and contributor to the pathobiology of cardiovascular diseases. However, most in vitro studies are performed using monolayer cultures of ECs on 2D tissue polystyrene plates (TCPs), which cannot reflect the physiological characteristics of cells in vivo. Here, we used 2D TCPs and a 3D culture model to investigate the effects of dimensionality and cardiovascular risk factors in regulating endothelial dysfunction. Cell morphology, oxidative stress, inflammatory cytokines and endothelial function were investigated in human umbilical vein endothelial cells (HUVECs) cultured in 2D/3D. The differentially expressed genes in 2D/3D-cultured HUVECs were analysed using Enrichr, Cytoscape and STRING services. Finally, we validated the proteins of interest and confirmed their relevance to TNF-α and the culture microenvironment. Compared with 2D TCPs, 3D culture increased TNF-α-stimulated oxidative stress and the inflammatory response and changed the mediators secreted by ECs. In addition, the functional characteristics, important pathways and key proteins were determined by bioinformatics analysis. Furthermore, we found that some key proteins, notably ACE, CD40, Sirt1 and Sirt6, represent a critical link between endothelial dysfunction and dimensionality, and these proteins were screened by bioinformatics analysis and verified by western blotting. Our observations provide insight into the interdependence between endothelial dysfunction and the complex microenvironment, which enhances our understanding of endothelial biology or provides a therapeutic strategy for cardiovascular-related diseases.

Effect of muscarinic receptors agonist in the rat model of coronary heart disease: A potential therapeutic target in cardiovascular diseases.

Cardiac hypertrophy is a one of common type of CHD, responsible for cardiac mortality worldwide. The present study designed to investigate the effect of muscarinic receptors agonist in the rat model of cardiac hypertrophy. A total of 30 male adult Wistar rats having body weight 300-400 gram were equally distributed in two groups (Test group: Rats with Angiotensin II + M3 receptor agonist [acetylcholine]; Reference group: Rats with cardiac hypertrophy induced by Angiotensin II). Rat model of cardiac hypertrophy were induced by Angiotensin II. Effect of M3 receptor agonist on cardiac hypertrophy was evaluated by electrocardiography, hemodynamic and histological assessment. Also, expression of M3 receptor was analyzed using by real-time-PCR and Western blot analysis. Also, vital signs such as pulse rate, and blood pressure were measured. Echocardiographic related variable including ejection fraction were also assessed in both the groups. The results of this study showed acetylcholine attenuates the hypertrophic response triggered by Angiotensin II, by upregulation of M3 receptor. Upregulation of M3 receptor after administration of acetylcholine ameliorates hypertrophic responses induced by angiotensin II. Also acetylcholine treatment prevents Angiotensin II induced increase in level of ANP and ß-myosin, which are responsible for inducing cardiac hypertrophic responses. Moreover, acetylcholine ameliorates Angiotensin II induced cell enlargement by reducing the surface area of cells. Overall finding suggested that acetylcholine improves left ventricle hypertrophy and ejection fraction by activating M3 receptor in heart. The finding of this study gives the new vision to cardiovascular researchers to develop anti- hypertrophy therapy based on M3 receptor.