Apoptosis Of Vascular Smooth Muscle Cells Research Articles

Hsa_circ_0087352 promotes the inflammatory response of macrophages in abdominal aortic aneurysm by adsorbing hsa-miR-149-5p

Abdominal aortic aneurysms (AAA) is a common cardiovascular disease with the risk of rupture. Macrophage depletion can significantly limit the formation of experimental AAA. However, how macrophages in the arterial wall affect the focal distribution and progression of AAA remains unclear. Here, we aimed to evaluate whether circRNAs characterized by stable structure and high tissue specific expression can regulate the inflammatory response of macrophages in AAA. First, we applied bioinformatics to analyze circRNA expression profile in human AAA specimens, and screened out hsa_circ_0087352, which is up-regulated in human AAA specimens and related to inflammatory response of THP-1 macrophages induced by LPS. Besides, hsa_circ_0087352 is stably expressed in THP-1 and mainly distributed in the nucleus. Then, we constructed ceRNA network of circRNA-miRNA-mRNA (IL-6/CCL2/NF-κB) in AAA and found that hsa_circ_0087352 promotes IL-6 transcription and the secretion of inflammatory cytokines by sponging endogenous hsa-miR-149-5p in macrophages. Dual luciferase reporter gene and RNA pull-down suggested hsa_circ_0087352 directly binds to hsa-miR-149-5p. Fluorescence in situ hybridization assay showed the localization of hsa_circ_0087352 and hsa-miR-149-5p in the nucleus of macrophages. Further, western blot demonstrated hsa_circ_0087352 expands the signal transduction of ERK/NF-κB pathway, then IκB phosphorylation promotes NF-κB p65 phosphorylation and nuclear translocation. In addition, hsa_circ_0087352 overexpression in macrophages induces human vascular smooth muscle cells (VSMC) apoptosis in macrophage-VSMC coculture system via the release of proapoptotic cytokines, such as IL-6, TNF-α and IL-1β. Overall, this study provides experimental evidence that hsa_circ_0087352 can be used as a new biomarker and therapeutic target for abdominal aortic aneurysm.

LncRNA OIP5-AS1 promotes the proliferation and migration of vascular smooth muscle cells via regulating miR-141-3p/HMGB1 pathway

BackgroundLong non-coding RNAs (lncRNAs) have been reported to play critical roles in the pathogenesis of cardiovascular diseases. However, whether lncRNA opa-interacting protein 5 antisense RNA 1 (OIP5-AS1) regulates the pathogenesis of atherosclerosis (AS) is still unknown. MethodsHuman vascular smooth muscle cells (VSMCs) were treated with oxidized low-density lipoprotein (ox-LDL). OIP5-AS1, miR-141-3p and HMGB1 mRNA expressions were determined using quantitative real-time polymerase chain reaction (qRT-PCR). The proliferation, migration, and apoptosis of VSMCs were measured using MTT method, Transwell assay and TUNEL assay, respectively. Dual-luciferase reporter gene assay, qRT-PCR, and Western blot were conducted to investigate the interactions among OIP5-AS1, miR-141-3p and high mobility group box 1 (HMGB1). ResultsOIP5-AS1 expression was markedly increased in serum samples of AS patients and VSMCs treated with ox-LDL. OIP5-AS1 over-expression remarkably promoted proliferation, migration and inhibited apoptosis of VSMCs while miR-141-3p exerted the opposite effects. Furthermore, the binding sites between OIP5-AS1 and miR-141-3p were identified. OIP5-AS1 indirectly increased HMGB1 expression in VSMCs by targeting miR-141-3p. ConclusionsOIP5-AS1 promotes the proliferation, migration and suppresses apoptosis of VSMCs through regulating miR-141-3p/HMGB1 axis.

MiR-140-5p upregulation suppressed β-glycerophosphate-induced vascular smooth muscle cell calcification via targeting TLR4

Background The role and function of microRNA (miRNA, miR)-140-5p in the calcification of vascular smooth muscle cells (VSMCs) have been explored in this study. Methods The calcium nodules formed in transfected and β-glycerophosphate (β-GP)-treated VSMCs were observed using Alizarin Red S staining, and alkaline phosphatase (ALP) activity was determined. VSMC apoptosis was detected with flow cytometry assay. The target gene of miR-140-5p was predicted and confirmed with dual-luciferase reporter assay. Relative expressions of miR-140-5p, toll like receptor 4 (TLR4) and vascular calcification-related proteins (α-smooth muscle actin, α-SMA; Msh Homeobox 2, MSX2; bone morphogenetic protein 2, BMP2; Kruppel-like factor 4, KLF4; Runt-related transcription factor 2, RUNX2) were measured through quantitative real time polymerase chain reaction (qRT-PCR) and western blot. Results MiR-140-5p upregulation reversed the effects of β-GP on downregulating miR-140-5p and α-SMA expressions, enhancing ALP activity, calcium nodule formation and cell apoptosis, and upregulating levels of MSX2, BMP2, KLF4 and RUNX2. TLR4 was the target of miR-140-5p, and offset the effects of miR-140-5p on β-GP-induced VSMCs. Conclusions MiR-140-5p upregulation represses β-GP-induced calcification of VSMCs via targeting TLR4, providing a potential therapeutic method for vascular calcification.

Circular RNAs: regulators of vascular smooth muscle cells in cardiovascular diseases.

The aberrant expansion and dysfunction of vascular smooth muscle cells (VSMCs) contribute to the occurrence and development of many cardiovascular diseases. Circular RNAs, a new class of non-coding RNAs with the 3' and 5' ends covalently linked together due to back-splicing, have recently been revealed to function as new regulators of VSMCs. These circular RNAs mainly act as RNA sponge to downregulate other regulatory non-coding RNAs such as microRNAs, influencing the overgrowth and transformation of VSMCs under pathogenic conditions. The purpose of this review is to summarize how circular RNAs fluctuate their own expression in response to multiple stimuli in vitro and in vivo and how they modulate the phenotypic adaptation, proliferation, migration, apoptosis, and senescence of VSMCs, which in turn affects the progression of cardiovascular diseases. Finally, we highlight the potential of circular RNAs as the biomarkers and therapeutic targets for abnormal VSMCs and cardiovascular diseases.

Clock Gene Bmal1 Disruption in Vascular Smooth Muscle Cells Worsens Carotid Atherosclerotic Lesions.

Clock system disruptions are associated with cardiovascular diseases. We previously demonstrated Bmal1 (brain muscle aryl nuclear translocase like-1) expression is significantly attenuated in plaque-derived vascular smooth muscle cells (VSMCs). However, the influence of Bmal1 disruption in VSMCs and its molecular targets are still unclear. Here, we aim to define how Bmal1 disruption in VSMCs influences the atherosclerosis lesions. The relationship among Bmal1, neurological symptoms, and plaque stability was investigated. VSMC Bmal1-/- and VSMC Bmal1+/+mice were generated and injected with adeno associated virus encoding mutant proprotein convertase subtilisin/kexin type 9 to induce atherosclerosis. Carotid artery ligation and cuff placement were performed in these mice to confirm the role of Bmal1 in atherosclerosis progression. The relevant molecular mechanisms were then explored. Bmal1 expression in the carotid plague was significantly lower in symptomatic patients as well as in unstable plaques. Moreover, Bmal1 reduction is an independent risk factor for neurological symptoms and plaque instability. Besides, VSMC Bmal1-/- mice exhibit aggravated atherosclerotic lesions. Further study demonstrated that Bmal1 downregulation in VSMCs increased VSMC migration, monocyte transmigration, reactive oxygen species levels, and VSMCs apoptosis. As for the mechanism, we revealed that Bmal1 suppresses VSMCs migration by inhibiting RAC1 activity in 2 ways: by activating the transcription of RhoGDIα and by interacting with RAC1. Besides, Bmal1 was shown to preserve antioxidant function in VSMCs by activating Nrf2 (nuclear factor erythroid 2-related factor 2) and Bcl-2 transcription. Bmal1 disruption in VSMCs worsens atherosclerosis by promoting VSMC migration and monocyte transmigration and impairing antioxidant function. Therefore, Bmal1 may be a potential therapeutic target and biomarker of atherosclerosis in the future.

Contribution of vascular smooth muscle cell apoptosis to spiral artery remodeling in early human pregnancy.

Uterine spiral artery remodeling is one of the key maternal adaptations of pregnancy, allowing delivery of the large volumes of maternal blood required for both placental and fetal growth. Failure of this process is associated with obstetric complications including preeclampsia, fetal growth restriction and miscarriage. Spiral artery remodeling is characterized by loss of the musculoelastic wall which is replaced by fibrinoid and intramural extravillous trophoblast cells (EVT). In recent years attention has focused on the initial stages of spiral artery remodeling which include separation of the vascular smooth muscle cells (VSMCs) and their phenotypic switch to a more synthetic phenotype, facilitating their migration away from the vessel wall. However, less is known about the final fate of the VSMCs. In vitro studies suggested that EVT could induce VSMC apoptosis, though VSMC apoptosis is not seen within the wall of the spiral arteries undergoing remodeling. However, apoptotic VSMCs have been observed amongst those cells which had migrated away from the vessel wall. This review article gives a brief overview of the current state of knowledge of the processes involved in spiral artery remodeling, and then concentrates on the mechanisms involved in VSMC apoptosis, drawing on knowledge from other vascular beds.

Inhibition of XIST attenuates abdominal aortic aneurysm in mice by regulating apoptosis of vascular smooth muscle cells through miR-762/MAP2K4 axis.

Abdominal aortic aneurysm (AAA) is a common chronic aortic degenerative disease. Long non-coding RNA X-inactive specific transcript (XIST) is associated with the progression of AAA, while the underlying mechanism is still unclear. We investigated the functional role of XIST in AAA. AAA mouse model was established by administration of Angiotensin II (Ang II). Primary mouse vascular smooth muscle cells (VSMCs) were separated from the abdominal aorta of Ang II-induced AAA mice, and then treated with Ang II. XIST was highly expressed in Ang II-treated VSMCs. Cell proliferation ability was decreased and apoptosis was increased in VSMCs following Ang II treatment. XIST knockdown reversed the impact of Ang II on cell proliferation and apoptosis in VSMCs. XIST promoted mitogen-activated protein kinase kinase 4 (MAP2K4) expression by sponging miR-762. XIST overexpression suppressed cell proliferation and apoptosis of Ang II-treated VSMCs by regulating miR-762/MAP2K4 axis. Finally, Ang II-induced AAA mouse model was established to verify the function of XIST in AAA. Inhibition of XIST significantly attenuated the pathological changes of abdominal aorta tissues in Ang II-induced mice. The expression of miR-762 was inhibited, and MAP2K4 expression was enhanced by XIST knockdown in the abdominal aorta tissues of AAA mice. In conclusion, these data demonstrate that inhibition of XIST attenuates AAA in mice, which attributes to inhibit apoptosis of VSMCs by regulating miR-762/MAP2K4 axis. Thus, this study highlights a novel ceRNA circuitry involving key regulators in the pathogenesis of AAA.

Curcumin alleviates lipopolysaccharides-induced inflammation and apoptosis in vascular smooth muscle cells via inhibition of the NF-κB and JNK signaling pathways.

Curcumin plays an important role in inflammation regulation. This study aimed to investigate the effect of curcumin on vascular smooth muscle cells (VSMCs) inflammation induced by lipopolysaccharide (LPS) and its mechanism. VSMCs were treated with different concentrations of curcumin (0, 50, 100 and 150μg/mL). MTT assay and flow cytometry were used to analyze the effects of curcumin on LPS-induced VSMCs viability and apoptosis. The expression and release of inflammatory cytokines in VSMCs were detected by real-time quantitative polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). Moreover, the proteins expressions of NF-κB and JNK signaling pathways were analyzed by western blot. Interestingly, the results showed that curcumin could reduce LPS induced inflammatory injury by increasing VSMC's viability, reducing apoptosis and inhibiting the release of inflammatory cytokines. In addition, curcumin increased the expression of Toll-like receptor 4 (TLR4) in LPS treated VSMCs. Mechanistically, we found that curcumin attenuated LPS-induced cell damage in VSMCs via inhibition of NF-κB and the JNK signal pathway. Curcumin can protect VSMCs from LPS induced inflammatory damage, which may be related to the blocking of NF-κB and the JNK signaling pathway. Herewith, curcumin could be potential therapeutics for the treatment of atherosclerosis.

The role of long noncoding RNA Nron in atherosclerosis development and plaque stability

SummaryThe major clinical consequences of atherosclerosis such as myocardial infarction or stroke are because of thrombotic events associated with acute rupture or erosion of an unstable plaque. Here, we identify an lncRNA Noncoding Repressor of NFAT (Nron) as a critical regulator of atherosclerotic plaque stability. Nron overexpression (OE) in vascular smooth muscle cells (VSMC) induces a highly characteristic architecture of more-vulnerable plaques, while Nron knockdown (KD) suppresses the development of atherosclerosis and favors plaque stability. Mechanistically, Nron specifically binds to and negatively regulates NFATc3, thus inhibiting the proliferation and promoting the apoptosis of VSMCs. Moreover, we also provide evidence that Nron increases the production and secretion of VEGFA from VSMCs, which functions as a paracrine factor to enhance intra-plaque angiogenesis. All of these effects contribute to plaque instability. Genetic or pharmacological inhibition of Nron may have potential for future therapy of atherosclerosis.

Radiation-induced C-reactive protein triggers apoptosis of vascular smooth muscle cells through ROS interfering with the STAT3/Ref-1 complex.

Damage to normal tissue can occur over a long period after cancer radiotherapy. Free radical by radiation can initiate or accelerate chronic inflammation, which can lead to atherosclerosis. However, the underlying mechanisms remain unclear. Vascular smooth muscle cells (VSMCs) proliferate in response to JAK/STAT3 signalling. C‐reactive protein (CRP) can induce VSMCs apoptosis via triggering NADPH oxidase (NOX). Apoptotic VSMCs promote instability and inflammation of atherosclerotic lesions. Herein, we identified a VSMCs that switched from proliferation to apoptosis through was enhanced by radiation‐induced CRP. NOX inhibition using lentiviral sh‐p22phox prevented apoptosis upon radiation‐induced CRP. CRP overexpression reduced the amount of STAT3/Ref‐1 complex, decreased JAK/STAT phosphorylation and formed a new complex of Ref‐1/CRP in VSMC. Apoptosis of VSMCs was further increased by CRP co‐overexpressed with Ref‐1. Functional inhibition of NOX or p53 also prevented apoptotic activity of the CRP‐Ref‐1 complex. Immunofluorescence showed co‐localization of CRP, Ref‐1 and p53 with α‐actin‐positive VSMC in human atherosclerotic plaques. In conclusion, radiation‐induced CRP increased the VSMCs apoptosis through Ref‐1, which dissociated the STAT3/Ref‐1 complex, interfered with JAK/STAT3 activity, and interacted with CRP‐Ref‐1, thus resulting in transcription‐independent cell death via p53. Targeting CRP as a vascular side effect of radiotherapy could be exploited to improve curability.

Endoplasmic reticulum stress mediates parathyroid hormone-induced apoptosis in vascular smooth muscle cells

Vascular calcification is one of the most common complications of chronic kidney disease (CKD), which is closely associated with increased mortality and morbidity rates of CKD patients. It has been reported that increased parathyroid hormone (PTH) aggravates vascular calcification in CKD patients. However, the direct role of PTH in vascular smooth muscle cells (VSMCs) is less elucidated. Here, we present evidence that PTH promotes apoptosis of VSMCs and endoplasmic reticulum (ER) stress participates in this process. Human aorta vascular smooth muscle cells (HASMCs) were treated with different concentrations of PTH for various time. HASMC apoptosis was detected by flow cytometry. Expression of phosphorylated (p)-PERK, CHOP, IRE1, p-JNK, and cleaved caspase 3 was determined by Western blotting. We found that PTH induced HASMC apoptosis and increased the expression of cleaved caspase 3. Furthermore, PTH activated PERK-CHOP and IRE1-JNK ER stress pathways. Either inhibition of JNK by SP600125 or CHOP by siRNA ameliorated PTH-induced apoptosis in HASMCs. We therefore suggest that ER stress participates in PTH-induced apoptosis of VSMCs, which may be a possible mechanism of PTH-promoted vascular calcification in CKD patients.

6-Shogaol Inhibits Oxidative Stress-Induced Rat Vascular Smooth Muscle Cell Apoptosis by Regulating OXR1-p53 Axis.

Apoptosis of vascular smooth muscle cells (VSMCs) is closely related to the pathogenesis of cardiovascular diseases, and oxidative stress is an important cause of VSMCs’ death. Inhibiting VSMCs apoptosis is an effective preventive strategy in slowing down the development of cardiovascular disease, especially for atherosclerosis. In this study, we found that oxidation resistance protein 1 (OXR1), a crucial participator for responding to oxidative stress, could modulate the expression of p53, the key regulator of cell apoptosis. Our results revealed that oxidative stress promoted VSMCs apoptosis by overexpression of the OXR1-p53 axis, and 6-shogaol (6S), a major biologically active compound in ginger, could effectively attenuate cell death by preventing the upregulated expression of the OXR1-p53 axis. Quantitative proteomics analysis revealed that the degradation of p53 mediated by OXR1 might be related to the enhanced assembly of SCF ubiquitin ligase complexes, which is reported to closely relate to the modification of ubiquitination or neddylation and subsequent degradation of p53.

Vascular smooth muscle cell-derived hydrogen sulfide promotes atherosclerotic plaque stability via TFEB (transcription factor EB)-mediated autophagy

ABSTRACT Vascular smooth muscle cells (VSMCs) contribute to plaque stability. VSMCs are also a major source of CTH (cystathionine gamma-lyase)-hydrogen sulfide (H2S), a protective gasotransmitter in atherosclerosis. However, the role of VSMC endogenous CTH-H2S in pathogenesis of plaque stability and the mechanism are unknown. In human carotid plaques, CTH expression in ACTA2+ cells was dramatically downregulated in lesion areas in comparison to non-lesion areas. Intraplaque CTH expression was positively correlated with collagen content, whereas there was a negative correlation with CD68+ and necrotic core area, resulting in a rigorous correlation with vulnerability index (r = −0.9033). Deletion of Cth in VSMCs exacerbated plaque vulnerability, and were associated with VSMC autophagy decline, all of which were rescued by H2S donor. In ox-LDL treated VSMCs, cth deletion reduced collagen and heightened apoptosis association with autophagy reduction, and vice versa. For the mechanism, CTH-H2S mediated VSMC autophagosome formation, autolysosome formation and lysosome function, in part by activation of TFEB, a master regulator for autophagy. Interference with TFEB blocked CTH-H2S effects on VSMCs collagen and apoptosis. Next, we demonstrated that CTH-H2S sulfhydrated TFEB at Cys212 site, facilitating its nuclear translocation, and then promoting transcription of its target genes such as ATG9A, LAPTM5 or LDLRAP1. Conclusively, CTH-H2S increases VSMC autophagy by sulfhydration and activation of TFEB, promotes collagen secretion and inhibits apoptosis, thereby attenuating atherogenesis and plaque vulnerability. CTH-H2S may act as a warning biomarker for vulnerable plaque.

Improved Quantification of Cell Density in the Arterial Wall-A Novel Nucleus Splitting Approach Applied to 3D Two-Photon Laser-Scanning Microscopy.

Accurate information on vascular smooth muscle cell (VSMC) content, orientation, and distribution in blood vessels is indispensable to increase understanding of arterial remodeling and to improve modeling of vascular biomechanics. We have previously proposed an analysis method to automatically characterize VSMC orientation and transmural distribution in murine carotid arteries under well-controlled biomechanical conditions. However, coincident nuclei, erroneously detected as one large nucleus, were excluded from the analysis, hampering accurate VSMC content characterization and distorting transmural distributions. In the present study, therefore, we aim to (1) improve the previous method by adding a “nucleus splitting” procedure to split coinciding nuclei, (2) evaluate the accuracy of this novel method, and (3) test this method in a mouse model of VSMC apoptosis. After euthanasia, carotid arteries from SM22α-hDTR Apoe–/– and control Apoe–/– mice were bluntly dissected, excised, mounted in a biaxial biomechanical tester and brought to in vivo axial stretch and a pressure of 100 mmHg. Nuclei and elastin fibers were then stained using Syto-41 and Eosin-Y, respectively, and imaged using 3D two-photon laser scanning microscopy. Nuclei were segmented from images and coincident nuclei were split. The nucleus splitting procedure determines the likelihood that voxel pairs within coincident nuclei belong to the same nucleus and utilizes these likelihoods to identify individual nuclei using spectral clustering. Manual nucleus counts were used as a reference to assess the performance of our splitting procedure. Before and after splitting, automatic nucleus counts differed −26.6 ± 9.90% (p < 0.001) and −1.44 ± 7.05% (p = 0.467) from the manual reference, respectively. Whereas the slope of the relative difference between the manual and automated counts as a function of the manual count was significantly negative before splitting (p = 0.008), this slope became insignificant after splitting (p = 0.653). Smooth muscle apoptosis led to a 33.7% decrease in VSMC density (p = 0.008). Nucleus splitting improves the accuracy of automated cell content quantification in murine carotid arteries and overcomes the progressively worsening problem of coincident nuclei with increasing cell content in vessels. The presented image analysis framework provides a robust tool to quantify cell content, orientation, shape, and distribution in vessels to inform experimental and advanced computational studies on vascular structure and function.

Yes-associated protein reacts differently in vascular smooth muscle cells under different intensities of mechanical stretch.

Vascular smooth muscle cells (VSMCs) are stromal cells of the vascular wall and are continually exposed to mechanical signals. The loss of VSMCs is closely related to the occurrence of many vascular diseases, such as aortic aneurysms and aortic dissection. The proliferation and apoptosis of VSMCs are mechanically stimulated. Yes-associated protein (YAP), one of the core components of the Hippo pathway, plays a key role in the response of VSMCs to mechanical signals. In this study, we tested the impact of different intensities of mechanical stretch on the proliferation and apoptosis of VSMCs, as well as YAP. We tested VSMCs’ proliferation and apoptosis and YAP reaction via immunocytochemistry, western blotting, CCK-8 and flow cytometric analysis. We found that 10% elongation could increase the phosphorylation of YAP and prevent it from entering the nucleus, as well as inhibit cell proliferation and promote apoptosis. However, 15% elongation reduced YAP phosphorylation and promoted its nuclear entry, thereby promoting cell proliferation and inhibiting apoptosis. Accordingly, YAP knockdown suppressed the phenotype of VMSCs induced by 15% elongation. Taken together, YAP regulates proliferation and apoptosis of VSMCs differently under different intensity of mechanical stretch. Mechanical stretch with appropriate intensity can promote the proliferation and inhibit apoptosis of VSMCs by activating YAP.

Protective effect of dihydromyricetin on vascular smooth muscle cell apoptosis induced by hydrogen peroxide in rats.

Dihydromyricetin (DMY), also called Ampelopsin, which was extracted from Ampelopsis grossedentata, has been demonstrated to have a protective effect against cell oxidative injury and cell apoptosis in vitro. In the present study, we tried to study the role of DMY on apoptosis of vascular smooth muscle cells (VSMCs) induced by hydrogen peroxide (H2O2) and explore the underlying mechanisms. Apoptotic cells were detected by Hematoxylin and Eosin (H.E.) staining, Hoechst 33342 staining, and Annexin V-fluorescein isothiocyanate binding assay. The intracellular reactive oxygen species (ROS) level was estimated through fluorescence assay. The mRNA and protein expression of Caspase-3, Caspase-9, Bcl-2, and Bax were determined by reverse transcription-polymerase chain reaction (RT-PCR) and western blot. The results showed that the pretreatment of VSMCs with DMY not only significantly increased cell viability, reduced intracellular ROS release, alleviated the morphological changes of apoptosis, and decreased the apoptosis rate, but also upregulated Bcl-2 expression and downregulated Caspase-3, Caspase-9, Bax expression, and ultimately attenuated the H2O2-stimulated apoptosis. The inhibition of DMY on VSMC apoptosis may be mediated by ROS scavenging and the activation of the mitochondrial apoptotic signaling pathway.

Investigation of Mitochondrial Bioenergetic Profile and Dysfunction in Atherosclerosis.

Mitochondrial function and activity are key indicators of overall cell health and mitochondrial dysfunction is closely associated with disruptions in normal cellular function. Altered mitochondrial function and cellular metabolism has been implicated in processes involved in ageing and associated pathologies. In atherosclerosis, compromised mitochondrial respiration can promote plaque instability and other processes that encourage pathogenesis and dysfunction. For example, increasing respiration promotes vascular smooth muscle cell (VSMC) proliferation and attenuates macrophage and VSMC apoptosis. Use of Agilent Seahorse technology to study mitochondrial bioenergetics has largely replaced previous outdated methods which provided limited insight into mitochondrial function and were associated with various issues. This chapter describes the use of Seahorse Agilent technology (Mito Stress Test) to study key parameters of mitochondrial respiration on cultured cells relevant to atherosclerosis.

Pharmacologic IRE1α kinase inhibition alleviates aortic dissection by decreasing vascular smooth muscle cells apoptosis.

Aortic dissection (AD) is a rare but catastrophic disorder, and associated with significant morbidity among survivors. This study aimed to target IRE1α-XBP1s pathway pharmacologically, and evaluate its therapeutic potential in the occurrence and progression of AD. Western Blot and immunohistochemistry results showed that expression of XBP1s was significantly increased in the human aorta samples of AD group in compared with the control group, and exclusively in aortic vascular smooth muscle cells (VSMCs). Further in vitro study revealed that Angiotensin II (Ang II) could increase the expression of XBP1s and promote its nuclear translocation in cultured VSMCs, which leads to numerous gene transcription, including gp91phox, Chop, Cleaved-caspase 3, Bax, and Bcl-2. These genes contribute to the production of reactive oxygen species (ROS), VMSCs phenotypic switch and apoptosis. Whereas an IRE1α endoribonuclease domain inhibitor MKC-3946 could reverse it. Finally, the efficacy of MKC-3946 was tested in a mouse AD model. As shown in vitro, MKC-3946 could reduce the expression of XBP1s and protect against AD by suppressing XBP1s associated ROS production and apoptosis in VSMCs in vivo. The current study revealed the relevant role of IRE1α-XBP1s signaling pathway in AD occurrence and progression. MKC-3946 could be of great potential in clinical application.

Cytokines in Abdominal Aortic Aneurysm: Master Regulators With Clinical Application.

Abdominal aortic aneurysm (AAA) is a potentially life-threatening disorder with a mostly asymptomatic course where the abdominal aorta is weakened and bulged. Cytokines play especially important roles (both positive and negative) among the molecular actors of AAA development. All the inflammatory cascades, extracellular matrix degradation and vascular smooth muscle cell apoptosis are driven by cytokines. Previous studies emphasize an altered expression and a changed epigenetic regulation of key cytokines in AAA tissue samples. Such cytokines as IL-6, IL-10, IL-12, IL-17, IL-33, IL-1β, TGF-β, TNF-α, IFN-γ, and CXCL10 seem to be crucial in AAA pathogenesis. Some data obtained in animal studies show a protective function of IL-10, IL-33, and canonical TGF-β signaling, as well as a dual role of IL-4, IFN-γ and CXCL10, while TNF-α, IL-1β, IL-6, IL-12/IL-23, IL-17, CCR2, CXCR2, CXCR4 and the TGF-β noncanonical pathway are believed to aggravate the disease. Altogether data highlight significance of cytokines as informative markers and predictors of AAA. Pathologic serum/plasma concentrations of IL-1β, IL-2, IL-6, TNF-α, IL-10, IL-8, IL-17, IFN-γ, and PDGF have been already found in AAA patients. Some of the changes correlate with the size of aneurysms. Moreover, the risk of AAA is associated with polymorphic variants of genes encoding cytokines and their receptors: CCR2 (rs1799864), CCR5 (Delta-32), IL6 (rs1800796 and rs1800795), IL6R (rs12133641), IL10 (rs1800896), TGFB1 (rs1800469), TGFBR1 (rs1626340), TGFBR2 (rs1036095, rs4522809, rs1078985), and TNFA (rs1800629). Finally, 5 single-nucleotide polymorphisms in gene coding latent TGF-β-binding protein (LTBP4) and an allelic variant of TGFB3 are related to a significantly slower AAA annual growth rate.

NRF2-suppressed vascular calcification by regulating the antioxidant pathway in chronic kidney disease.

Vascular calcification (VC), in which vascular smooth muscle cells (VSMCs) undergo differentiation and osteogenic transition, is a common complication of chronic kidney disease (CKD). Recent findings show that nuclear factor-erythroid-2-related factor 2 (NRF2) is an evolutionarily conserved antioxidant and beneficial in preventing vascular senescence and calcification. The roles of NRF2 in the initiation and progression of VC in CKD still need further investigation. CKD-associated VC model rats exhibited significant upregulation of NRF2, NAD(P)H: quinone oxidoreductase-1 (NQO1), osteogenic markers such as alkaline phosphatase (ALP), runt-related transcription factor-2 (RUNX2) and osteopontin (OPN), and β-catenin compared to CKD rats. Immunohistochemistry further verified these results. In addition, rat aortic VSMCs were isolated and subjected to four treatments: normal control, phosphorus-induced (Pi), Pi+NRF2 activator DMF, and Pi+NRF2 inhibitor ML385. The reactive oxygen species (ROS) generation, malondialdehyde (MDA) content, and calcium deposition of the four treatments were determined. The mRNA and protein expression levels of NRF2, NQO1, and haem oxygenase 1 (HO1) and the osteogenic markers ALP, Runx1, OPN, bone morphogenetic protein 2 (BMP2), and β-catenin were quantified by RT-PCR and western blotting. VSMC apoptosis was calculated by flow cytometry. The in vitro results suggested that intracellular oxidative stress and calcification were closely associated with NRF2 activity and that the activation of NRF2 could significantly suppress osteogenic transition and apoptosis in VSMCs. Thus, this study indicated that the NRF2-related antioxidant pathway can positively respond to and protect against the initiation and progression of VC in CKD by reducing oxidative stress. This study may contribute insights facilitating the application of the NRF2 antioxidative system as a therapeutic treatment for vascular diseases such as CKD.