Oxidative Stress In Vascular Smooth Muscle Cells Research Articles

4-Octyl itaconate inhibits vascular calcification partially via modulation of HMOX-1 signaling

Vascular calcification frequently occurs in patients with chronic conditions such as chronic kidney disease (CKD), diabetes, and hypertension and represents a significant cause of cardiovascular events. Thus, identifying effective therapeutic targets to inhibit the progression of vascular calcification is essential. 4-Octyl itaconate (4-OI), a derivative of itaconate, exhibits anti-inflammatory and antioxidant activity, both of which play an essential role in the progression of vascular calcification. However, the role and molecular mechanisms of 4-OI in vascular calcification have not yet been elucidated. In this study, we investigated the effects of exogenous 4-OI on vascular calcification using vascular smooth muscle cells (VSMCs), arterial rings, and mice. Alizarin red staining and western blot revealed that 4-OI inhibited calcification and osteogenic differentiation of human VSMCs. Similarly, 4-OI inhibited calcification of rat arterial rings and VitD3-overloaded mouse aortas. Mechanistically, RNA sequencing analysis revealed that 4-OI treatment is most likely to affect heme oxygenase 1 (HMOX-1) mRNA expression. The study demonstrated that 4-OI treatment increased HMOX-1 mRNA and protein levels, but suppressed inflammation and oxidative stress in VSMCs under osteogenic conditions. Moreover, 4-OI treatment. HMOX-1 knockdown by siRNA or treatment with the HMOX-1 inhibitor ZnPP9 significantly reversed the suppression effect on calcification of VSMCs and aortas of VitD3-overloaded mice by 4-OI. Furthermore, HMOX-1 knockdown by siRNA markedly abrogated the inhibitory effect of 4-OI on inflammation in VSMCs. These findings suggest that 4-OI alleviates vascular calcification and inhibits oxidative stress and inflammation through modulation of HMOX-1, indicating its potential as a therapeutic target for vascular calcification.

Phoenixin-14 maintains the contractile type of vascular smooth muscle cells in cerebral aneurysms rats.

The rupture of intracranial aneurysm (IA) is the primary reason contributing to the occurrence of life-threatening subarachnoid hemorrhages. The oxidative stress-induced phenotypic transformation from the contractile phenotype to the synthetic phenotype of vascular smooth muscle cells (VSMCs) plays a pivotal role in IA formation and rupture. Our study aimed to figure out the role of phoenixin-14 in VSMC phenotypic switching during the pathogenesis of IA by using both cellular and animal models. Primary rat VSMCs were isolated from the Willis circle of male Sprague-Dawley rats. VSMCs were stimulated by hydrogen peroxide (H2O2) to establish a cell oxidative damage model. After pretreatment with phoenixin-14 and exposure to H2O2, VSMC viability, migration, and invasion were examined through cell counting kit-8 (CCK-8), wound healing, and Transwell assays. Intracellular reactive oxygen species (ROS) production in VSMCs was evaluated by using 2',7'-Dichlorofluorescin diacetate (DCFH-DA) fluorescence probes and flow cytometry. Rat IA models were established by ligation of the left common carotid arteries and posterior branches of both renal arteries. The histopathological changes of rat intracranial blood vessels were observed through hematoxylin and eosin staining. The levels of contractile phenotype markers (alpha-smooth muscle actin [α-SMA] and smooth muscle 22 alpha [SM22α]) in VSMCs and rat arterial rings were determined through real-time quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. Our results showed that H2O2 stimulated the production of intracellular ROS and induced oxidative stress in VSMCs, while phoenixin-14 pretreatment attenuated intracellular ROS levels in H2O2-exposed VSMCs. H2O2 exposure promoted VSMC migration and invasion, which, however, was reversed by phoenixin-14 pretreatment. Besides, phoenixin-14 administration inhibited IA formation and rupture in rat models. The decrease in α-SMA and SM22α levels in H2O2-exposed VSMCs and IA rat models was antagonized by phoenixin-14. Collectively, phoenixin-14 ameliorates the progression of IA through preventing the loss of the contractile phenotype of VSMCs.

Asprosin contributes to vascular remodeling in hypertensive rats via superoxide signaling.

Proliferation and migration of vascular smooth muscle cells (VSMCs) contribute to vascular remodeling. Asprosin, a newly discovered protein hormone, is involved in metabolic diseases. Little is known about the roles of asprosin in cardiovascular diseases. This study focused on the role and mechanism of asprosin on VSMC proliferation and migration, and vascular remodeling in a rat model of hypertension. VSMCs were obtained from the aortic media of 8-week-old male Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Asprosin was upregulated in the VSMCs of SHR. For in vitro studies, asprosin promoted VSMC proliferation and migration of WKY and SHR, and increased Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activity, NOX1/2/4 protein expressions and superoxide production. Knockdown of asprosin inhibited the proliferation, migration, NOX activity, NOX1/2 expressions and superoxide production in the VSMCs of SHR. The roles of asprosin in promoting VSMC proliferation and migration were not affected by hydrogen peroxide scavenger, but attenuated by superoxide scavenger, selective NOX1 or NOX2 inhibitor. Toll-like receptor 4 (TLR4) was upregulated in SHR, TLR4 knockdown inhibited asprosin overexpression-induced proliferation, migration and oxidative stress in VSMCs of WKY and SHR. Asprosin was upregulated in arteries of SHR, and knockdown of asprosin in vivo not only attenuated oxidative stress and vascular remodeling in aorta and mesentery artery, but also caused a subsequent persistent antihypertensive effect in SHR. Asprosin promotes VSMC proliferation and migration via NOX-mediated superoxide production. Inhibition of endogenous asprosin expression attenuates VSMC proliferation and migration, and vascular remodeling of SHR.

CircCDYL Contributes to Apoptosis, Ferroptosis, and Oxidative Stress of Ang II-Induced Vascular Smooth Muscle Cells in Thoracic Aortic Aneurysm.

Circular RNAs (circRNAs) have important regulation in thoracic aortic aneurysm (TAA). The function and mechanism of circCDYL (circ_0008285) was explored in TAA here. Angiotensin II (Ang II) was used to construct a TAA model. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed for the detection of circCDYL, miR-1270, and a disintegrin and metalloproteinase 10 (ADAM10). Cell viability was examined via cell counting kit-8 (CCK-8) assay and proliferation was analyzed using Ethynyl-2'-deoxyuridine (EdU) assay. Apoptosis rate was assessed via flow cytometry. Western blot was used for protein detection. Oxidative stress was evaluated by commercial kits. CircCDYL was upregulated in TAA tissues and Ang II-induced circCDYL upregulation in vascular smooth muscle cells (VSMCs). Knockdown of circCDYL weakened Ang II-aroused inhibition of viability, proliferation, and promotion of apoptosis, ferroptosis, and oxidative stress in VSMCs. CircCDYL served as a miR-1270 sponge. The mitigated regulation of circCDYL knockdown for Ang II-induced injury was restored after miR-1270 downregulation. CircCDYL positively regulated ADAM10 through interacting with miR-1270. Overexpression of miR-1270 abated Ang II-induced injury by downregulating ADAM10. In conclusion, circCDYL was involved in the Ang II-induced VSMC injury in TAA via the miR-1270/ADAM10 axis.

Disulfiram protects against abdominal aortic aneurysm by ameliorating vascular smooth muscle cells pyroptosis

Recent studies demonstrated that pyroptosis is involved in abdominal aortic aneurysm (AAA) progression, suggesting a potential target for AAA treatment. This study aimed to identify if disulfiram could inhibit angiotensin II (Ang II)-induced vascular smooth muscle cells (VSMCs) damage, thereby exerting protective effects on AAA. The AAA mouse model was established by continuous subcutaneous Ang II infusion for 28 days. Then aortic tissue of the mice was isolated and subjected to RNA sequencing, qRT-PCR, Western blotting, and immunofluorescence staining. To explore the therapeutic effect of disulfiram, mice were orally administered disulfiram (50 mg/kg/day) or vehicle for 28 days accompanied with Ang II infusion. Pathological changes in aortic tissues were measured using microultrasound imaging analysis and histopathological analysis. In addition, inflammatory response, pyroptosis, and oxidative stress damage were examined in mouse aortic vascular smooth muscle (MOVAS) cells stimulated with Ang II in vitro. The RNA sequencing and bioinformatic analysis results suggested that pyroptosis- and inflammation-related genes were significantly upregulated in AAA, consistent with the results of qRT-PCR and Western blotting. Most importantly, the therapeutic effect of disulfiram on AAA was identified in our study. First, disulfiram administration significantly attenuated Ang II-induced inflammation, pyroptosis, and oxidative stress in VSMCs, which is associated with the inhibition of the NF-κB-NLRP3 pathway. Second, in-vivo studies revealed that disulfiram treatment reduced AAA formation and significantly ameliorated collagen deposition and elastin degradation in the aortic wall. Our findings suggest that disulfiram has a novel protective effect against AAA by inhibiting Ang II-induced VSMCs pyroptosis.

Nuclear receptor Nur77 protects against oxidative stress by maintaining mitochondrial homeostasis via regulating mitochondrial fission and mitophagy in smooth muscle cell

Angiotensin II (AngII) induces disruption of mitochondrial homeostasis and oxidative stress. Nuclear receptor NR4A1 (Nur77) plays an important role in vascular smooth muscle cells (VSMCs) function. However, the role of Nur77 in AngII-induced mitochondrial dynamics and oxidative stress in VSMCs remains unknown. In an in vitro model of AngII-treated cells, we discovered that Nur77 knockout aggravated AngII-induced oxidative stress in VSMCs, whereas activation of Nur77 by celastrol diminished them. Concomitantly, disturbance of mitochondrial dynamics induced by AngII was further exacerbated in Nur77 deficient VSMCs compared to wild-type (WT) VSMCs. Interestingly, Nur77 deletion increased mitochondrial fission but not fusion as evidenced by upregulated fission related genes (Fis1 and Drp1) but not fusion (Opa1 and Mfn2) under AngII stimulation in VSMCs. Mechanically, Nur77 could directly bind to the promoter regions of Fis1 and Drp1 and repress their transcription. Furthermore, we observed that Nur77 additionally promoted mitochondrial homeostasis by increasing mitophagic flux in a transcription-independent manner upon AngII challenge. By using an in vivo model of AngII-induced abdominal aortic aneurysm (AAA), we finally validated the protective role of Nur77 involved in the mitochondrial fission process and mitophagic flux in aortas, which was correlated with the occurrence and development of AAA in AngII-infused mice. Our data defines an essential role of Nur77 in regulating oxidative stress by maintaining mitochondrial homeostasis in VSMCs via both transcription-dependent and transcription-independent manner, supporting the therapeutic potential of Nur77 targeting in vascular diseases.

β‑aminoisobutyric acid ameliorates hypertensive vascular remodeling via activating the AMPK/SIRT1 pathway in VSMCs

ABSTRACT Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) play a fundamental role in the pathogenesis of hypertension-related vascular remodeling. β-aminoisobutyric acid (BAIBA) is a nonprotein β-amino acid with multiple pharmacological actions. Recently, BAIBA has been shown to attenuate salt‑sensitive hypertension, but the role of BAIBA in hypertension-related vascular remodeling has yet to be fully clarified. This study examined the potential roles and underlying mechanisms of BAIBA in VSMC proliferation and migration induced by hypertension. Primary VSMCs were cultured from the aortas of Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Our results showed that BAIBA pretreatment obviously alleviated the phenotypic transformation, proliferation, and migration of SHR-derived VSMCs. Exogenous BAIBA significantly inhibited the release of inflammatory cytokines by diminishing phosphorylation and nuclear translocation of p65 NFκB, retarding IκBα phosphorylation and degradation, as well as erasing STAT3 phosphorylation in VSMCs. Supplementation of BAIBA triggered Nrf2 dissociation from Keap1 and inhibited oxidative stress in VSMCs from SHR. Mechanistically, activation of the AMPK/sirtuin 1 (SIRT1) axis was required for BAIBA to cube hypertension-induced VSMC proliferation, migration, oxidative damage and inflammatory response. Most importantly, exogenous BAIBA alleviated hypertension, ameliorated vascular remodeling and fibrosis, abated vascular oxidative burst and inflammation in SHR, an effect that was abolished by deficiency of AMPKα1 and SIRT1. BAIBA might serve as a novel therapeutic agent to prevent vascular remodeling in the context of hypertension.

Off‐target effects of oral anticoagulants – vascular effects of vitamin K antagonist and non‐vitamin K antagonist oral anticoagulant dabigatran etexilate

IntroductionVitamin K antagonists (VKA) and non‐vitamin K oral antagonist anticoagulants (NOAC) are used in the clinic to reduce risk of thrombosis. However, they also exhibit vascular off‐target effects. The aim of this study is to compare VKA and NOAC on atherosclerosis progression and calcification in an experimental setup. Material and methodsFemale Apoe−/− mice (age 12 weeks) were fed Western‐type diet as control or supplemented with dabigatran etexilate or warfarin for 6 or 18 weeks. Vascular calcification was measured in whole aortic arches using µCT and [18F]‐NaF. Atherosclerotic burden was assessed by (immuno)histochemistry. Additionally, in vitro effects of warfarin, thrombin, and dabigatran on primary vascular smooth muscle cells (VSMC) were assessed. ResultsShort‐term treatment with warfarin promoted formation of atherosclerotic lesions with a pro‐inflammatory phenotype, and more rapid plaque progression compared with control and dabigatran. In contrast, dabigatran significantly reduced plaque progression compared with control. Long‐term warfarin treatment significantly increased both presence and activity of plaque calcification compared with control and dabigatran. Calcification induced by warfarin treatment was accompanied by increased presence of uncarboxylated matrix Gla protein. In vitro, both warfarin and thrombin significantly increased VSMC oxidative stress and extracellular vesicle release, which was prevented by dabigatran. ConclusionWarfarin aggravates atherosclerotic disease activity, increasing plaque inflammation, active calcification, and plaque progression. Dabigatran lacks undesired vascular side effects and reveals beneficial effects on atherosclerosis progression and calcification. The choice of anticoagulation impacts atherosclerotic disease by differential off target effect. Future clinical studies should test whether this beneficial effect also applies to patients.

Coenzyme Q10 inhibits intracranial aneurysm formation and progression in a mouse model.

The aim of this study was to investigate the effect of coenzyme Q10 (CoQ10), a commonly used nutritional supplement, on intracranial aneurysm (IA) initiation and progression in a mouse model, as well as the mechanism. Hydrogen peroxide (H2O2) was used to treat mouse-derived vascular smooth muscle cells (VSMCs) to induce oxidative injury, followed by incubation with CoQ10. In the mouse IA model established by elastase injection, CoQ10 was orally administered at 10 mg/kg every other day for 14 days, during which the incidence of IA, rupture rate, symptom-free survival, and systolic blood pressure were recorded. CoQ10 promoted the expression of nuclear factor erythroid 2-related factor 2 and antioxidant enzymes. In H2O2-treated VSMCs, reactive oxygen species and cell apoptosis were reduced by CoQ10. In IA mice, CoQ10 treatment decreased the rupture rate of IA, improved the symptom-free survival, and reduced systolic blood pressure. Macrophage infiltration and expression of pro-inflammatory cytokines in the cerebral arteries were mitigated by CoQ10 treatment. CoQ10 is effective in reducing oxidative stress in VSMCs, thereby attenuating IA formation and rupture in mice. CoQ10 also alleviates inflammation and restores normal phenotypes of VSMCs in the cerebral arteries. Our data suggest that CoQ10 is a potentially effective drug for managing IA. To investigate the effect of CoQ10, a commonly used nutritional supplement, on IA initiation and progression in a mouse model, as well as the mechanism. CoQ10 promoted the expression of Nrf2 and antioxidant enzymes. In H2O2-treated VSMCs, ROS and cell apoptosis were reduced by CoQ10. CoQ10 is effective in reducing oxidative stress in VSMCs, thereby attenuating IA formation and rupture in mice.

Fisetin Alleviates Neointimal Hyperplasia via PPARγ/PON2 Antioxidative Pathway in SHR Rat Artery Injury Model.

The phenotypic transformation of proliferation and migration in vascular smooth muscle cells (VSMCs) from media to intima is the basic pathology of neointimal hyperplasia after angioplasty in hypertensive patients. Angiotensin II (AngII) stimulates oxidative stress in VSMC, inducing VSMC proliferation and migration, which is a critical factor in both developments of hypertension and angioplasty-induced arterial restenosis. Fisetin, a plant flavonoid polyphenol, has been reported to be antioxidative and potent senolytic. It is unknown whether fisetin would inhibit neointimal hyperplasia. Therefore, we investigated the role of fisetin in neointimal formation in vitro and in vivo. The rat thoracic aortic smooth muscle cells (A10 cells) stimulated by AngII were used as the in vitro neointimal hyperplasia model, where AngII significantly induced the proliferation and migration in A10 cells. We found that fisetin could dose-dependently inhibit the effect of AngII via inducing the expression of an antioxidant, paraoxonase-2 (PON2), whose overexpression could inhibit the proliferation and migration of A10 cells and downexpression by siRNA had the opposite effect. Furthermore, we found the mechanism of fisetin's inducing PON2 expression involved PPARγ. Rosiglitazone, a PPARγ agonist, could increase PON2 expression in A10 cells, while the PPARγ inhibitor prevented the effect of fisetin on PON2. The in vivo neointimal hyperplasia model was established 2 weeks after the carotid artery balloon injury in SHR rats. Administration of fisetin (ip 3 mg/kg daily for 2 weeks) right after the injury significantly increased PON2 expression in the artery, inhibiting ROS production, and efficiently reduced carotid neointimal hyperplasia. These results indicate that fisetin increases the expression of antioxidant PON2 via activation of PPARγ, reducing oxidative stress, inhibiting VSMC proliferation and migration, and alleviates neointimal hyperplasia after intimal injury. PON2 may be a potential therapeutic target to reduce arterial remodeling after angioplasty in hypertensive patients.

PDK4 promotes vascular calcification by interfering with autophagic activity and metabolic reprogramming

Pyruvate dehydrogenase kinase 4 (PDK4) is an important mitochondrial matrix enzyme in cellular energy regulation. Previous studies suggested that PDK4 is increased in the calcified vessels of patients with atherosclerosis and is closely associated with mitochondrial function, but the precise regulatory mechanisms remain largely unknown. This study aims to investigate the role of PDK4 in vascular calcification and the molecular mechanisms involved. Using a variety of complementary techniques, we found impaired autophagic activity in the process of vascular smooth muscle cells (VSMCs) calcification, whereas knocking down PDK4 had the opposite effect. PDK4 drives the metabolic reprogramming of VSMCs towards a Warburg effect, and the inhibition of PDK4 abrogates VSMCs calcification. Mechanistically, PDK4 disturbs the integrity of the mitochondria-associated endoplasmic reticulum membrane, concomitantly impairing mitochondrial respiratory capacity, which contributes to a decrease in lysosomal degradation by inhibiting the V-ATPase and lactate dehydrogenase B interaction. PDK4 also inhibits the nuclear translocation of the transcription factor EB, thus inhibiting lysosomal function. These changes result in the interruption of autophagic flux, which accelerates calcium deposition in VSMCs. In addition, glycolysis serves as a metabolic adaptation to improve VSMCs oxidative stress resistance, whereas inhibition of glycolysis by 2-deoxy-D-glucose induces the apoptosis of VSMCs and increases the calcium deposition in VSMCs. Our results suggest that PDK4 plays a key role in vascular calcification through autophagy inhibition and metabolic reprogramming.

MiR155-5p Inhibits Cell Migration and Oxidative Stress in Vascular Smooth Muscle Cells of Spontaneously Hypertensive Rats.

Migration of vascular smooth muscle cells (VSMCs) is essential for vascular reconstruction in hypertension and several vascular diseases. Our recent study showed that extracellular vesicles derived from vascular adventitial fibroblasts of normal rats inhibited VSMC proliferation by delivering miR155-5p to VSMCs. It is unknown whether miR155-5p inhibits cell migration and oxidative stress in VSMCs of spontaneously hypertensive rats (SHR) and in angiotensin II (Ang II)-treated VSMCs. The purpose of this study was to determine the role of miR155-5p in VSMC migration and its underlying mechanisms. Primary VSMCs were isolated from the aortic media of Wistar-Kyoto rats (WKY) and SHR. Wound healing assay and Boyden chamber assay were used to evaluate VSMC migration. A miR155-5p mimic inhibited, and a miR155-5p inhibitor promoted the migration of VSMC of SHR but had no significant effect on the migration of VSMC of WKY. The miR155-5p mimic inhibited angiotensin-converting enzyme (ACE) mRNA and protein expression in VSMCs. It also reduced superoxide anion production, NAD(P)H oxidase (NOX) activity, as well as NOX2, interleukin-1β (IL-1β), and tumor necrosis factor α (TNF-α) expression levels in VSMCs of SHR but not in VSMCs of WKY rats. Overexpression of miR155-5p inhibited VSMC migration and superoxide anion and IL-1β production in VSMCs of SHR but had no impact on exogenous Ang II-induced VSMC migration and on superoxide anion and IL-1β production in WKY rats and SHR. These results indicate that miR155-5p inhibits VSMC migration in SHR by suppressing ACE expression and its downstream production of Ang II, superoxide anion, and inflammatory factors. However, miR155-5p had no effects on exogenous Ang II-induced VSMC migration.

MicroRNA-24 inhibits the oxidative stress induced by vascular injury by activating the Nrf2/Ho-1 signaling pathway

Background and aimsThe process of endothelial repair in diabetic patients after stent implantation was significantly delayed compared with that in non-diabetic patients, and oxidative stress is increasingly considered to be relevant to the pathogenesis of diabetic endothelial repair. However, the mechanisms linking diabetes and reendothelialization after vascular injury have not been fully elucidated. The aim of this study was to evaluate the effect of microRNA-24 (miR-24) up-regulation in delayed endothelial repair caused by oxidative stress after balloon injury in diabetic rats. MethodsIn vitro, vascular smooth muscle cells (VSMCs) isolated from the thoracic aorta were stimulated with high glucose (HG) after miR-24 recombinant adenovirus (Ad-miR-24-GFP) transfection for 3 days. In vivo, diabetic rats induced using high-fat diet (HFD) and low-dose streptozotocin (30 mg/kg) underwent carotid artery balloon injury followed by Ad-miR-24-GFP transfection for 20 min. ResultsThe expression of miR-24 was decreased in HG-stimulated VSMCs and balloon-injured carotid arteries of diabetic rats, which was accompanied by increased expression of Ogt and Keap1 and decreased expression of Nrf2 and Ho-1. Up-regulation of miR-24 suppressed VSMC oxidative stress induced by HG in vitro, and miR-24 up-regulation promoted reendothelialization in balloon-injured diabetic rats. The underlying mechanism was related to the activation of the Nrf2/Ho-1 signaling pathway, which subsequently suppressed intracellular reactive oxidative species (ROS) production and malondialdehyde (MDA) and NADPH oxidase (Nox) activity, and to the restoration of Sod and Gsh-px activation. ConclusionsThe up-regulation of miR-24 significantly promoted endothelial repair after balloon injury through inhibition of oxidative stress by activating the Nrf2/Ho-1 signaling pathway.

Natriuretic peptide receptor-C activation attenuates angiotensin II-induced enhanced oxidative stress and hyperproliferation of aortic vascular smooth muscle cells.

We showed previously that natriuretic peptide receptor-C (NPR-C) agonist, C-ANP4-23, attenuated the enhanced expression of Giα proteins in vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) through the inhibition of enhanced oxidative stress. Since the enhanced levels of endogenous angiotensin II (Ang II) contribute to the overexpression of Giα proteins and augmented oxidative stress in VSMC from SHR, the present study was undertaken to investigate if C-ANP4-23 could also attenuate angiotensin II (Ang II)-induced oxidative stress and associated signaling. Ang II treatment of aortic VSMC augmented the levels of superoxide anion (O2-), NADPH oxidase activity, and the expression of NADPH oxidase subunits and C-ANP4-23 treatment attenuated all these to control levels. In addition, Ang II-induced enhanced levels of thiobarbituric acid-reactive substances (TBARS) and protein carbonyl content were also attenuated toward control levels by C-ANP4-23 treatment. On the other hand, Ang II inhibited the levels of nitric oxide (NO) and augmented the levels of peroxynitrite (OONO-) in VSMC which were restored to control levels by C-ANP4-23 treatment. Furthermore, C-ANP4-23 treatment attenuated Ang II-induced enhanced expression of Giα proteins, phosphorylation of p38, JNK, and ERK 1,2 as well as hyperproliferation of VSMC as determined by DNA synthesis, and metabolic activity. These results indicate that C-ANP4-23, via the activation of NPR-C, attenuates Ang II-induced enhanced nitroxidative stress, overexpression of Giα proteins, increased activation of the p38/JNK/ERK 1,2 signaling pathways, and hyperproliferation of VSMC. It may be suggested that C-ANP4-23 could be used as a therapeutic agent in the treatment of vascular remodeling associated with hypertension and atherosclerosis.

Vasculopathy in the setting of cardiorenal syndrome: roles of protein-bound uremic toxins.

Chronic kidney disease (CKD) often leads to and accelerates the progression of cardiovascular disease (CVD), while CVD also causes kidney dysfunction. This bidirectional interaction leads to the development of a complex syndrome known as cardiorenal syndrome (CRS). CRS not only involves both the heart and the kidney but also the vascular system through a vast array of contributing factors. In addition to hemodynamic, neurohormonal, mechanical, and biochemical factors, nondialyzable protein-bound uremic toxins (PBUTs) are also key contributing factors that have been demonstrated through in vitro, in vivo, and clinical observations. PBUTs are ineffectively removed by hemodialysis because their complexes with albumins are larger than the pores of the dialysis membranes. PBUTs such as indoxyl sulfate and p-cresyl sulfate are key determinate and predictive factors for the progression of CVD in CKD patients. In CRS, both vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) exhibit significant dysfunction that is associated with the progression of CVD. PBUTs influence proliferation, calcification, senescence, migration, inflammation, and oxidative stress in VSMCs and ECs through various mechanisms. These pathological changes lead to arterial remodeling, stiffness, and atherosclerosis and thus reduce heart perfusion and impair left ventricular function, aggravating CRS. There is limited literature about the effect of PBUT on the vascular system and their contribution to CRS. This review summarizes current knowledge on how PBUTs influence vasculature, clarifies the relationship between uremic toxin-related vascular disease and CRS, and highlights the potential therapeutic strategies of uremic vasculopathy in the setting of CRS.

PPARγ Inhibits VSMC Proliferation and Migration via Attenuating Oxidative Stress through Upregulating UCP2.

Increasing evidence showed that abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are common event in the pathophysiology of many vascular diseases, including atherosclerosis and restenosis after angioplasty. Among the underlying mechanisms, oxidative stress is one of the principal contributors to the proliferation and migration of VSMCs. Oxidative stress occurs as a result of persistent production of reactive oxygen species (ROS). Recently, the protective effects of peroxisome proliferator-activated receptor γ (PPARγ) against oxidative stress/ROS in other cell types provide new insights to inhibit the suggests that PPARγ may regulate VSMCs function. However, it remains unclear whether activation of PPARγ can attenuate oxidative stress and further inhibit VSMC proliferation and migration. In this study, we therefore investigated the effect of PPARγ on inhibiting VSMC oxidative stress and the capability of proliferation and migration, and the potential role of mitochondrial uncoupling protein 2 (UCP2) in oxidative stress. It was found that platelet derived growth factor-BB (PDGF-BB) induced VSMC proliferation and migration as well as ROS production; PPARγ inhibited PDGF-BB-induced VSMC proliferation, migration and oxidative stress; PPARγ activation upregulated UCP2 expression in VSMCs; PPARγ inhibited PDGF-BB-induced ROS in VSMCs by upregulating UCP2 expression; PPARγ ameliorated injury-induced oxidative stress and intimal hyperplasia (IH) in UCP2-dependent manner. In conclusion, our study provides evidence that activation of PPARγ can attenuate ROS and VSMC proliferation and migration by upregulating UCP2 expression, and thus inhibit IH following carotid injury. These findings suggest PPARγ may represent a prospective target for the prevention and treatment of IH-associated vascular diseases.

Oxidative stress contributes to the enhanced expression of Gqα/PLCβ1 proteins and hypertrophy of VSMC from SHR: role of growth factor receptor transactivation.

We showed previously that vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHRs) exhibit overexpression of Gqα/PLCβ1 proteins, which contribute to increased protein synthesis through the activation of MAP kinase signaling. Because oxidative stress has been shown to be increased in hypertension, the present study was undertaken to examine the role of oxidative stress and underlying mechanisms in enhanced expression of Gqα/PLCβ1 proteins and VSMC hypertrophy. Protein expression was determined by Western blotting, whereas protein synthesis and cell volume, markers for VSMC hypertrophy, were determined by [(3)H]-leucine incorporation and three-dimensional confocal imaging, respectively. The increased expression of Gqα/PLCβ1 proteins, increased protein synthesis, and augmented cell volume exhibited by VSMCs from SHRs were significantly attenuated by antioxidants N-acetyl-cysteine (NAC), a scavenger of superoxide anion, DPI, an inhibitor of NAD(P)H oxidase. In addition, PP2, AG1024, AG1478, and AG1295, inhibitors of c-Src, insulin-like growth factor receptor (IGFR), epidermal growth factor receptor (EGFR), and platelet-derived growth factor receptor (PDGFR), respectively, also attenuated the enhanced expression of Gqα/PLCβ1 proteins and enhanced protein synthesis in VSMCs from SHRs toward control levels. Furthermore, the levels of IGF-1R and EGFR proteins and not of PDGFR were also enhanced in VSMCs from SHRs, which were attenuated significantly by NAC, DPI, and PP2. In addition, NAC, DPI, and PP2 also attenuated the enhanced phosphorylation of IGF-1R, PDGFR, EGFR, c-Src, and EKR1/2 in VSMCs from SHRs. These data suggest that enhanced oxidative stress in VSMCs from SHRs activates c-Src, which through the transactivation of growth factor receptors and MAPK signaling contributes to enhanced expression of Gqα/PLCβ1 proteins and resultant VSMC hypertrophy.

Ferulic acid inhibits H2O2-induced oxidative stress and inflammation in rat vascular smooth muscle cells via inhibition of the NADPH oxidase and NF-κB pathway

Ferulic acid (FA) is a dietary phenolic acid and has a wide range of therapeutic effects, including anti-aging, antitumor activity and antihypertensive effects. The aim of present study was to evaluate the inhibitory effects of FA on cell inflammation and oxidative stress against hydrogen peroxide (H2O2)-induced injury in rat vascular smooth muscle cells (VSMCs) in vitro. VSMCs were pretreated with FA 2h before H2O2 incubation. The results suggested that FA inhibited H2O2-induced cell injury by reducing the MDA and increasing the SOD activity and GSH content. In rat VSMCs exposed to H2O2, FA increased the cell viability and restored the mitochondrial membrane depolarization. The level of ROS generation was reduced by pretreatment with FA through inhibiting the expression of NADPH oxidase and down-regulating MAPK and AKT pathways. We found that H2O2 stimulated the production of IL-6, IL-1β, TNF-α and NO, which could be reduced by pretreatment with FA through inhibiting the p-NF-κB as well as the iNOS expression. In conclusion, our results show that FA may serve as a novel drug in the treatment of these pathologies by inhibiting NADPH oxidase and NF-κB and subsequently decreasing VSMC oxidative stress and inflammation. These suggest that the inhibitory effect of FA on VSMC inflammation and oxidative stress is partially attributed to depressing NADPH and NF-κB expressions in VSMCs, decreasing the ROS production and reducing apoptosis of VSMCs.

Txnip ablation reduces vascular smooth muscle cell inflammation and ameliorates atherosclerosis in apolipoprotein E knockout mice.

Inflammation of vascular smooth muscle cells (VSMC) is intimately linked to atherosclerosis and other vascular inflammatory disease. Thioredoxin interacting protein (Txnip) is a key regulator of cellular sulfhydryl redox and a mediator of inflammasome activation. The goals of the present study were to examine the impact of Txnip ablation on inflammatory response to oxidative stress in VSMC and to determine the effect of Txnip ablation on atherosclerosis invivo. Using cultured VSMC, we showed that ablation of Txnip reduced cellular oxidative stress and increased protection from oxidative stress when challenged with oxidized phospholipids and hydrogen peroxide. Correspondingly, expression of inflammatory markers and adhesion molecules were diminished in both VSMC and macrophages from Txnip knockout mice. The blunted inflammatory response was associated with a decrease in NF-ĸB nuclear translocation. Loss of Txnip in VSMC also led to a dramatic reduction in macrophage adhesion to VSMC. Invivo data from Txnip-ApoE double knockout mice showed that Txnip ablation led to 49% reduction in atherosclerotic lesion in the aortic root and 71% reduction in the abdominal aorta, compared to control ApoE knockout mice. Our data show that Txnip plays an important role in oxidative inflammatory response and atherosclerotic lesion development in mice. The atheroprotective effect of Txnip ablation implicates that modulation of Txnip expression may serve as a potential target for intervention of atherosclerosis and inflammatory vascular disease.

Roles of the kinase TAK1 in CD40-mediated effects on vascular oxidative stress and neointima formation after vascular injury.

Although TAK1 has been implicated in inflammation and oxidative stress, its roles in vascular smooth muscle cells (VSMCs) and in response to vascular injury have not been investigated. The present study aimed to investigate the role of TAK1 in modulating oxidative stress in VSMCs and its involvement in neointima formation after vascular injury. Double immunostaining reveals that vascular injury induces a robust phosphorylation of TAK1 (Thr187) in the medial VSMCs of injured arteries in wildtype mice, but this effect is blocked in CD40-deficient mice. Upregulation of TAK1 in VSMCs is functionally important, as it is critically involved in pro-oxidative and pro-inflammatory effects on VSMCs and eventual neointima formation. In vivo, pharmacological inhibition of TAK1 with 5Z-7-oxozeaenol blocked the injury-induced phosphorylation of both TAK1 (Thr187) and NF-kB/p65 (Ser536), associated with marked inhibition of superoxide production, 3-nitrotyrosine, and MCP-1 in the injured arteries. Cell culture experiments demonstrated that either siRNA knockdown or 5Z-7-oxozeaenol inhibition of TAK1 significantly attenuated NADPH oxidase activation and superoxide production induced by CD40L/CD40 stimulation. Co-immunoprecipitation experiments indicate that blockade of TAK1 disrupted the CD40L-induced complex formation of p22phox with p47phox, p67phox, or Nox4. Blockade of TAK1 also inhibited CD40L-induced NF-kB activation by modulating IKKα/β and NF-kB p65 phosphorylation and this was related to reduced expression of proinflammatory genes (IL-6, MCP-1 and ICAM-1) in VSMCs. Lastly, treatment with 5Z-7-oxozeaenol attenuated neointimal formation in wire-injured femoral arteries. Our findings demonstrate previously uncharacterized roles of TAK1 in vascular oxidative stress and the contribution to neointima formation after vascular injury.