Abstract:Atherosclerosis (As) is a chronic inflammatory arterial wall injury process, and vessel wall cells play an important role in the occurrence and development of As. Vascular endothelial cell (VEC) act as a semi-permeable barrier between vascular smooth muscle cell (VSMC) and vascular lumen, and its injury is the initial stage of As. In addition, Through phenotypic transformation, VSMC could transform into many cell phenotype of the plaques, including macrophage, foam cell, mesenchymal stem cell and so on, and these cells further involved in the occurrence of As. Fibroblast is the main component of vascular adventitia,in pathological conditions, fibroblast differentiate into myofibroblast and participate in the occurrence of As. In this article, we will review the involvement of vascular wall cells in the mechanism of As and its potential therapeutic targets for the treatment of As, which provide new therapeutic ideas for As.

Abstract:Aim To explore the mechanism of oxidized lipoprotein(a) (oxLp(a) inducing pyroptosis of vascular endothelial cells. Methods After incubating human umbilical vein endothelial cells (HUVEC) with 100 mg/L oxLp(a) for 24 hours, Western blot and RT-qPCR was used to detect pyroptosis related proteins, pro-inflammatory cytokines, mitochondrial related proteins NRF1, NRF2, PGC-1α and mitochondrial gene cytochrome b (CYTB), ELISA was used to detect the levels of inflammatory factors, scanning electron microscopy was used to detect cell membrane rupture, transmission electron microscopy was used to detect mitochondrial morphology, Hoechst33342/PI staining was used to detect cell apoptosis, MitoSOX probe was used to detect mitochondrial reactive oxygen species (mtROS), Flu-4AM probe was used to detect calcium ions, JC-1 probe was used to detect mitochondrial membrane potential (MMP), and Calcein AM staining was used to detect mitochondrial permeability transition pore (mPTP). Transfecting HUVEC with CYTB overexpressing lentivirus and analyzing its effects on oxLp(a) induced pyroptosis and mitochondrial function. ResultsAfter treatment with oxLp(a), the expression of NLRP3, pro-Caspase-1, Caspase-1, GSDMD and GSDMD-N proteins related to pyroptosis were significantly increased (P＜0.05); the protein and mRNA levels of CYTB and pro-inflammatory cytokine IL-1β, IL-18 were significantly increased (P＜0.05). Small pores appeared on the cell membrane, the percentage of PI stained positive cells significantly increased (P＜0.05). OxLp(a) significantly inhibited the expression of mitochondrial related proteins NRF1, NRF2 and PGC-1α, and the expression of mitochondrial gene CYTB, promoted an increase in mtROS generation, Ca2＋ overload, a decrease in ATP levels, a decrease in MMP, an increase in mPTP values, and abnormal mitochondrial morphology. After transfection with pHelper 2.0 lentivirus vector overexpressing CYTB, it was found that oxLp(a) induced HUVEC pyroptosis and mitochondrial morphological and functional abnormalities were partially reversed by overexpression of CYTB. Conclusion oxLp(a) promotes mitochondrial morphological and functional abnormalities and induces HUVEC pyroptosis by downregulating CYTB.

Abstract:Atherosclerosis (As) is the pathological basis of various vascular diseases, which affects the function of important organs. Recent studies have found that N6-methyladenosine (m6A) modification plays a vital role in As. This paper summarizes the regulatory effects of m6A modification on vascular endothelial cell (VEC) injury, vascular smooth muscle cell (VSMC) transformation, macrophage (M) differentiation, foam cell (FC) formation, pyroptosis, and lipid regulation, and summarizes the regulation function of some m6A regulatory proteins in As.

Abstract:Aim To establish an efficient and stable isolation method of primary mouse brain vascular smooth muscle cells and endothelial cells, and provide experimental materials for the investigation of pathogenesis and treatment of brain vascular diseases. Methods Brain vascular smooth muscle cells were isolated by dextran gradient centrifugation with enzymatic digestion, and endothelial cells were isolated by immunomagnetic beads sorting. Morphology and growth characteristics of two types of cells were observed with an inverted phase contrast microscope, their purity were identified by immunofluorescence, and their proliferation characteristics were observed by CCK-8 assay. At the level of cellular function, angiogenic capacity of endothelial cell was assessed by angiogenesis assay and smooth muscle cell responsiveness to platelet-derived growth factor (PDGF) was assessed by migration assay. Results The two types of cells isolated using this method grew vigorously and were in good condition. Smooth muscle cells exhibited typical “peak valley” growth, and immunofluorescence results showed cytoplasmic specific smooth muscle α-SMA and SM22α expression was positive. Endothelial cells exhibited typical “cobblestone like” growth, with positive expression of platelet endothelial cell adhesion molecule CD31 and atresia zone protein 1. Conclusion This study established a reliable and efficient method for simultaneously isolating two types of cerebrovascular cells, the isolated cells have high purity, good activity, and stable characteristics after passage, which were sufficient to meet the needs of subsequent experiments.

Abstract:Piezo1 protein is a non-selective mechanically gated cation channel. Under the action of mechanical stimulation, it can cause the influx of cations such as calcium, sodium, potassium, etc., and then convert the mechanical signals into bioelectrical signals, and integrate the signals into cells to participate in a variety of physiological and pathological processes. Piezo1 protein widely exists in the cardiovascular system, and plays an important role in many cardiovascular activities, such as blood flow shear stress and vascular tension sensing, vascular development and angiogenesis, and vascular remodeling. Piezo1 can sense the changes of blood flow shear stress and vascular tension, and its expression is also affected by them, thus affecting the morphology, arrangement, synthesis, secretion, inflammation and adhesion of vascular endothelial cells. Piezo1 can also regulate the migration, inflammatory reaction and lipid phagocytosis of macrophages, and participate in regulating the occurrence and development of atherosclerosis. The proliferation of vascular smooth muscle cells is also regulated by Piezo1, which then affects the remodeling of vascular wall. This article mainly reviews the discovery, structure and function of Piezo1 channel, and its research progress in atherosclerosis, in order to provide new ideas for the prevention and treatment of atherosclerosis.

Abstract:Aim To investigate the influences of ginkgetin on the ferroptosis of vascular endothelial cells induced by oxidized low density lipoprotein (ox-LDL) by regulating the nuclear factor erythroid-2 related factor 2 (Nrf2)/solute carrier protein 7 family member 11 (SLC7A11)/glutathione peroxidase 4 (GPX4) signaling pathway. Methods Human umbilical vein cell fusion cells EA.hy926 were grouped into normal control group (normal culture), ox-LDL group (50 mg/L ox-LDL), and ginkgetin low dose group (50 mg/L ox-LDL+10 μmol/L ginkgetin), middle dose group (50 mg/L ox-LDL+20 μmol/L ginkgetin), high dose group (50 mg/L ox-LDL+40 μmol/L ginkgetin), ML385 group (50 mg/L ox-LDL+40 μmol/L ginkgetin+1 μmol/L Nrf2 inhibitor ML385), Erastin group (50 mg/L ox-LDL+40 μmol/L ginkgetin+5 μmol/L SLC7A11 inhibitor Erastin), RSL3 group (50 mg/L ox-LDL+40 μmol/L ginkgetin+0.5 μmol/L GPX4 inhibitor RSL3). Cell viability was detected by tetramethylazolium salt (MTT) method. The kits were applied to detect the levels of cellular superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH). The intracellular iron content was detected by specific fluorescent probe method. The levels of intracellular reactive oxygen species (ROS) and lipid ROS were detected by 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe method and boron dipyrrole (BODIPYTM) method; Western blot was applied to detect the protein expressions of cellular Nrf2, SLC7A11, GPX4,4-hydroxynonenoic acid (4-HNE), cyclooxygenase 2 (COX2), and p53. Results Compared with the normal control group, the cell viability, SOD content, GSH content, expressions of Nrf2, SLC7A11 and GPX4 were obviously decreased in the ox-LDL group (P＜0.05); the MDA content, Fe2＋ content, ROS, lipid ROS, expressions of 4-HNE, COX2, p53 were obviously increased (P＜0.05). Compared with the ox-LDL group, the cell viability, SOD content, GSH content, expressions of Nrf2, SLC7A11 and GPX4 were obviously increased in low, middle and high dose groups of ginkgetin (P＜0.05); the MDA content, Fe2＋ content, ROS, lipid ROS, expressions of 4-HNE, COX2, p53 were obviously decreased (P＜0.05). Compared with the high dose ginkgetin group, the ML385 group, Erastin group and RSL3 group attenuated the inhibitory effect of ginkgetin on ox-LDL-induced vascular endothelial cell ferroptosis. Conclusion Ginkgetin inhibits ox-LDL-induced vascular endothelial cell ferroptosis by activating the Nrf2/SLC7A11/GPX4 pathway.

Abstract:Aim To observe the effect of TSB2 inhibiting the combination of heat shock protein 90 (HSP90) and endothelial nitric oxide synthase (eNOS) on the formation of atherosclerosis. Methods Human umbilical vein endothelial cells (HUVEC) were treated with TSB2 and the combination between HSP90 and eNOS was detected by co-immunoprecipitation. C57BL/6 mice and low density lipoprotein receptor knockout (LDLR－/－) mice were fed with normal diet (ND) or high fat diet (HFD) for 12 weeks while injected with phosphate buffered saline (PBS) or TSB2 intraperitoneally. The mice were divided into four groups:C57BL/6＋ ND＋PBS group, LDLR－/－＋ND＋PBS group, LDLR－/－＋HFD＋PBS group, LDLR－/－＋HFD ＋TSB2 group. Then the aorta was isolated. The combination between HSP90 and eNOS in aorta was measured. The atherosclerotic plaque in aorta and aortic sinus were determined. The production of nitric oxide (NO) and superoxide anion (O2·－) were also detected. At the same time, L-monomethyl-arginine (L-NMMA), a competitive substrate of L-arginine, was used to determine the production of NO, and L-nitroarginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, was used to determine the production of O2·－. Results Compared with control group, the combination between HSP90 and eNOS was decreased by 41.06% (P＜0.05) in cultured HUVEC treated with TBS2. Compared with LDLR－/－＋HFD＋PBS group,the combination between HSP90 and eNOS in the mouse aortas was decreased by 40.95% (P＜0.05) in LDLR－/－ ＋HFD＋TSB2 group, and the production of O2·－ was decreased by 63.73% (P＜0.05) (L-NAME significantly inhibited the production of O2·－ in LDLR－/－＋HFD＋PBS group), while the production of NO had no significant change in the mouse aortic endothelial cells (L-NMMA inhibited NO production in all groups), and the formation of atherosclerotic lesions in aortas and aortic sinus were significantly decreased by 59.39% and 68.86% (P＜0.05) respectively in LDLR－/－＋HFD＋TSB2 group. Conclusion TSB2 can reduce the O2·－ production of uncoupled eNOS in vascular endothelial cells by inhibiting the combination of HSP90 and eNOS in aortic endothelial cells, and finally inhibits the formation of atherosclerosis.

Abstract:Cardiovascular disease is the leading cause of death. Moreover, the incidence of cardiovascular disease in young/middle-aged people (18~45 years old) is still on the rise. Early diagnosis, early warning and treatment of cardiovascular disease are important topics in the current medical research field. Atherosclerosis is the pathophysiological basis of many cardiovascular diseases. Recent studies have shown that the tricarboxylic acid (TCA) cycle intermediate is involved in the occurrence and development of atherosclerosis, and considered as the early diagnosis and warning bio-markers for atherosclerosis. Here, the roles and underlying mechanisms of succinate in the initiation and progression in atherosclerosis were reviewed.

Abstract:Aim To study the regulatory effect of N-acetylcysteine (NAC) on hypoxia-induced injury of cerebrovascular endothelial cells and its molecular mechanism. Methods Healthy male SD rats were selected, cerebrovascular endothelial cells were isolated and cultured. Cells were divided into normal oxygen group, hypoxia group, 0.5 NAC group(hypoxia+0.5 mol/L NAC), 1.0 NAC group(hypoxia+1.0 mol/L NAC), NAC+8-bAMP group((hypoxia+1.0 mol/L NAC+1.0mol/L 8-bAMP). Cell proliferation activity was detected by MTS assay, apoptotic rate was detected by TUNEL assay, oxidative stress index was detected by kit, apoptotic gene and AMPK/SIRT1 pathway molecule expression was detected by western blot. Results OD490 value, T-AOC and expression of Bcl-2, p-AMPK, SIRT1 in hypoxia group were significantly lower than those in normoxia group, while apoptotic rate, contents of ROS, MDA, 8-OHDG and expression of Caspase-3, Cyt-C, Bax of hypoxia group were significantly higher than those in normoxia group. OD490 value, content of T-AOC and expression of Bcl-2, p-AMPK, SIRT1 in 0.5 NAC group, 1.0 NAC group were significantly higher than those in hypoxia group, while apoptotic rate, contents of ROS, MDA, 8-OHDG and expression of Caspase-3, Cyt-C, Bax of 0.5 NAC group, 1.0 NAC group were significantly higher than those in hypoxia group. OD490 value, content of T-AOC and expression of Bcl-2, p-AMPK, SIRT1 in NAC+8-bAMP group were significantly lower than those in 1.0 NAC group, while apoptotic rate, contents of ROS, MDA, 8-OHDG and expression of Caspase-3, Cyt-C, Bax of NAC+8-bAMP group were significantly higher than those in 1.0 NAC group. Conclusion NAC can alleviate oxidative stress and mitochondrial apoptosis-mediated injury of cerebrovascular endothelial cells by activating AMPK/SIRT1 pathway.

Abstract:The vascular endothelial cell (VEC) is a single layer of flat squamous epithelium covering the intima of the blood vessel. It constitutes a biological barrier to the blood vessel wall. It is not only a protective barrier but also produces some autocrine secretion. The substance is used to regulate homeostasis and vascular tone and has a variety of biological functions. VECsenescence can lead to vascular dysfunction, which is a major risk factor for cardiovascular system (CVS) and has a close relationship with cardiovascular disease (CVD). However, themechanism of VEC senescence and the effects of VEC senescence on vascular function are not fully understood. This review summarizes the characteristics of VEC senescence and its related molecular mechanisms, and describes age-related CVD.