Thoracic Aortic Smooth Muscle Cells Research Articles

Fibronectin/α5 Integrin Contribute to Hypertension-Associated Arterial Ageing and Calcification through Affecting BMP2/MGP Imbalance and Enhancing Vascular Smooth Muscle Cell Phenotypic Transformation.

Hypertension can accelerate and aggravate the process of arterial ageing and calcification. However, the mechanism behind has yet to be well elucidated. Here, we monitored the dynamic changes of fibronectin (FN)/α5 integrin, bone morphogenetic protein 2/matrix Gla protein (BMP2/MGP), and Runx2 in the aorta of spontaneously hypertensive rats (SHRs) and thoracic aortic vascular smooth muscle cells (VSMCs), also the phenotypic transformation of VSMCs during the process of arterial ageing and calcification. Further, study on arterial ageing and calcification through antagonist experiments at the molecular level was explored. We found extracellular FN and its α5 integrin receptor expressions were positively associated with arterial ageing and calcification in SHR during ageing, as well in VSMCs from SHR in vitro. Integrin receptor inhibitor of GRGDSP would delay this arterial ageing and calcification process. Moreover, the elevated FN and α5 integrin receptor expression evoked the disequilibrium of BMP2/MGP, where the expression of BMP2, a potent osteogenic inducer, increased while MGP, a calcification inhibitor, decreased. Furthermore, it was followed by the upregulation of Runx2 and the phenotypic transformation of VSMCs from the contractile phenotype into the osteoblast-like cells. Notably, BMP2 antagonist of rmNoggin was sufficient to ameliorate the ageing and calcification process of VSMCs and exogenous BMP2-adding accelerate and aggregate the process. Our study revealed that hypertension-associated arterial ageing and calcification might be a consequence that hypertension up-regulated FN and its high binding affinity integrin α5 receptor in the aortic wall, which in turn aggravated the imbalance of BMP2/MGP, promoted the transcription of Runx2, and induced the phenotypic transformation of VSMCs from the contractile phenotype into the osteoblast-like cells. Our study would provide insights into hypertension-associated arterial ageing and calcification and shed new light on the control of arterial calcification, especially for those with hypertension.

Effect of Angiotensin II-Mediated Janus Kinase/Signal Transducers and Activators of Transcription Pathways on Thoracic Aortic Smooth Muscle Cells

Effect of Angiotensin II-Mediated Janus Kinase/Signal Transducers and Activators of Transcription Pathways on Thoracic Aortic Smooth Muscle Cells

Molecular Mechanism of Calycosin Inhibited Vascular Calcification.

Vascular calcification (VC) is a pathological condition frequently observed in cardiovascular diseases. Primary factors contributing to VC are osteogenic differentiation of vascular smooth muscle and hydroxyapatite deposition. Targeted autophagy (a lysosome-mediated mechanism for degradation/recycling of unnecessary cellular components) is a useful approach for inhibiting VC and promoting vascular cell health. Calycosin has been shown to alleviate atherosclerosis by enhancing macrophage autophagy, but its therapeutic effect on VC has not been demonstrated. Using an in vitro model (rat thoracic aortic smooth muscle cell line A7r5), we demonstrated effective inhibition of VC using calycosin (the primary flavonoid component of astragalus), based on the enhancement of autophagic flux. Calycosin treatment activated AMPK/mTOR signaling to induce initiation of autophagy and restored mTORC1-dependent autophagosome-lysosome fusion in late-stage autophagy by promoting soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation, thereby preventing stoppage of autophagy in calcified cells. Calycosin substantially reduced degrees of both osteogenic differentiation and calcium deposition in our VC cell model by enhancing autophagy. The present findings clarify the mechanism whereby calycosin mitigates autophagy stoppage in calcified smooth muscle cells and provide a basis for effective VC treatment via autophagy enhancement.

DNA methylation inhibitor regulates guanylyl cyclase/natriuretic peptide receptor-A gene expression via 5'-untranslated region in mesangial and smooth muscle cells

Guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) is activated by cardiac hormones atrial and brain natriuretic peptides (ANP, BNP) and produces intracellular second messenger cGMP. The physiological actions of ANP/NPRA/cGMP signaling cascade largely include diuresis, natriuresis, vasorelaxation, and organ protection, which maintains blood pressure and opposed renal, cardiac, and vascular injury and remodeling. However, the mechanisms that contribute to the activation of Npr1 (coding for GC-A/NPRA) expression is not well understood. The present study was aimed at understanding the epigenetic mechanisms of DNA methyltransferase (DNMT) inhibitor, 5-Azacytidine (5-Aza)-mediated Npr1 gene transcription. The studies were carried out in mouse mesangial cells (MMCs) and rat thoracic aortic smooth muscle (RTASM) cells, cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and ITS (insulin, transferrin, and sodium selenite) and treated with 5-Aza for 24 h. Western blot analysis showed that 5-Aza enhanced NPRA protein levels by 5-fold and 7-fold in a dose-dependent manner in MMCs and RTASM cells, respectively, and greatly stimulated intracellular accumulation of cGMP in ANP-treated cells. Treatment with 5-Aza attenuated DNMT activity and repressed DNMT1 protein levels in cells. The MethPrimer search result showed the presence of three cytosine-phosphate-guanine (CpG) islands in the Npr1 full length promoter and 5'-untranslated region (UTR), namely island 1 (-886 to -752, 134 bp), island 2 (-310 to -158, 152 bp), and island 3 (-154 to +310, 464 bp) from the transcription start site. Addition of 5'-UTR encompassing +56 to +359 bp region to the Npr1 promoter constructs, (-1982/+55) and proximal promoter (-356/+55), was inhibitory to Npr1 transcriptional activity, independent of the cell type. Furthermore, treatment with 5-Aza distinctly induced Npr1 promoter activity of the construct -356/5'-UTR by 10-fold in MMCs suggesting that the repressive effect of DNA methylation on Npr1 gene transcription might be via 5'-UTR. The results demonstrate that 5-Aza acts as a potent inducer of Npr1 expression and signaling and provides new insights in the epigenetic control of Npr1, an important player in the control of hypertension and cardiorenal axis. Supported by the NIH grants (HL062147 and DK133833). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Estrogen ameliorates sepsis-induced vascular hyporeactivity in thoracic aorta of female rats via permissive effect of GRα expression

ObjectiveEstrogen is correlated to the lower mortality and disease severity of female than that of male, which indicates the potential therapeutic role of estrogen supplement therapy in sepsis. The structure of Daidzein is similar to that of 17β estradiol (E2), an estrogen in human body, causing the exogenous Daidzein can interact with estrogen receptor as well as E2 in the body. We aim to explore the therapeutic role of estrogen in sepsis-induced vascular dysfunction. Also, we wonder if estrogen regulates blood pressure via glucocorticoid-mediated vascular reactivity. MethodsFemale SD rats received ovariectomy (OVX) to induce estrogen deficiency. After 12 weeks of administration, cecal ligation and puncture (CLP) was used to establish the in vivo model of sepsis. Lipopolysaccharide (LPS) was used to construct the in vitro model of sepsis in vascular smooth muscle cells (VSMCs). E2 and Daidzein were used for estrogen supplement therapy. ResultsE2 and Daidzein significantly inhibited inflammation infiltration and histopathological injury in thoracic aorta in the rat model with CLP. E2 and Daidzein improved carotid pressure and vascular hyporeactivity in sepsis rats with OVX. Importantly, E2 and Daidzein promoted glucocorticoid permissive action and increased glucocorticoid receptor α (GRα) expression in thoracic aorta smooth muscle cells. E2 and Daidzein upregulated GRα, and inhibits cytokine production, proliferative phenotype and cell migration in LPS-induced VSMCs. ConclusionEstrogen improved vascular hyporeactivity in thoracic aorta induced by sepsis via permissive effect of GRα expression.

Adrenomedullin Improves Hypertension and Vascular Remodeling partly through the Receptor-Mediated AMPK Pathway in Rats with Obesity-Related Hypertension.

Adrenomedullin (ADM) is a novel cardiovascular peptide with anti-inflammatory and antioxidant properties. Chronic inflammation, oxidative stress and calcification play pivotal roles in the pathogenesis of vascular dysfunction in obesity-related hypertension (OH). Our study aimed to explore the effects of ADM on the vascular inflammation, oxidative stress and calcification in rats with OH. Eight-week-old Sprague Dawley male rats were fed with either a Control diet or a high fat diet (HFD) for 28 weeks. Next, the OH rats were randomly subdivided into two groups as follows: (1) HFD control group, and (2) HFD with ADM. A 4-week treatment with ADM (7.2 μg/kg/day, ip) not only improved hypertension and vascular remodeling, but also inhibited vascular inflammation, oxidative stress and calcification in aorta of rats with OH. In vitro experiments, ADM (10 nM) in A7r5 cells (rat thoracic aorta smooth muscle cells) attenuated palmitic acid (PA, 200 μM) or angiotensin II (Ang II, 10 nM) alone or their combination treatment-induced inflammation, oxidative stress and calcification, which were effectively inhibited by the ADM receptor antagonist ADM22-52 and AMP-activated protein kinase (AMPK) inhibitor Compound C, respectively. Moreover, ADM treatment significantly inhibited Ang II type 1 receptor (AT1R) protein expression in aorta of rats with OH or in PA-treated A7r5 cells. ADM improved hypertension, vascular remodeling and arterial stiffness, and attenuated inflammation, oxidative stress and calcification in OH state partially via receptor-mediated AMPK pathway. The results also raise the possibility that ADM will be considered for improving hypertension and vascular damage in patients with OH.

IL-1β knockdown inhibits cigarette smoke extract-induced inflammation and apoptosis in vascular smooth muscle cells.

This study was aimed to investigate the role of interleukin-1β (IL-1β) in cigarette smoke extract (CSE)-induced apoptosis in vascular smooth muscle cells and the underlying mechanism in a rat derived cell line. Rat thoracic aortic smooth muscle cells (A7r5) were divided into six groups including control, CSE (model), CSE+ overexpression empty vector (OvExp-EV), CSE+IL-1β knockdown (KD), and CSE+ IL-1β knockdown empty vector (KD-EV). The mRNA expression levels of IL-1β and pregnancy-associated plasma protein A (PAPP-A) were detected by quantitative polymerase chain reaction (qPCR). The apoptosis of A7r5 cells was detected by flow cytometry. The expression levels of inflammatory mediators (TNFα, IL-6 and IL-8) and apoptotic proteins (Bax and Bcl-2) were determined by western blot. CSE induced significant apoptosis in vascular smooth muscle cells (P < 0.01) and elevated the mRNA levels of IL-1β and PAPP-A (P < 0.01). CSE administration increased protein expression of Bax, TNF-α, IL-6, and IL-8, with significantly reduced Bcl-2 expression (P < 0.01). IL-1β knockdown significantly decreased cell apoptosis via regulating the expression of these proteins (P < 0.05 or P < 0.01). IL-1β is involved in CSE-induced PAPP-A expression and apoptosis in vascular smooth muscle cells, which might be considered as a target for preventing of cardiovascular diseases caused by cigarette smoking.

Aortic Stress Activates an Adaptive Program in Thoracic Aortic Smooth Muscle Cells That Maintains Aortic Strength and Protects Against Aneurysm and Dissection in Mice.

When aortic cells are under stress, such as increased hemodynamic pressure, they adapt to the environment by modifying their functions, allowing the aorta to maintain its strength. To understand the regulation of this adaptive response, we examined transcriptomic and epigenomic programs in aortic smooth muscle cells (SMCs) during the adaptive response to AngII (angiotensin II) infusion and determined its importance in protecting against aortic aneurysm and dissection (AAD). We performed single-cell RNA sequencing and single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq) analyses in a mouse model of sporadic AAD induced by AngII infusion. We also examined the direct effects of YAP (yes-associated protein) on the SMC adaptive response in vitro. The role of YAP in AAD development was further evaluated in AngII-infused mice with SMC-specific Yap deletion. In wild-type mice, AngII infusion increased medial thickness in the thoracic aorta. Single-cell RNA sequencing analysis revealed an adaptive response in thoracic SMCs characterized by upregulated genes with roles in wound healing, elastin and collagen production, proliferation, migration, cytoskeleton organization, cell-matrix focal adhesion, and PI3K-PKB/Akt (phosphoinositide-3-kinase-protein kinase B/Akt) and TGF-β (transforming growth factor beta) signaling. ScATAC-seq analysis showed increased chromatin accessibility at regulatory regions of adaptive genes and revealed the mechanical sensor YAP/transcriptional enhanced associate domains as a top candidate transcription complex driving the expression of these genes (eg, Lox, Col5a2, Tgfb2). In cultured human aortic SMCs, cyclic stretch activated YAP, which directly bound to adaptive gene regulatory regions (eg, Lox) and increased their transcript abundance. SMC-specific Yap deletion in mice compromised this adaptive response in SMCs, leading to an increased AAD incidence. Aortic stress triggers the systemic epigenetic induction of an adaptive response (eg, wound healing, proliferation, matrix organization) in thoracic aortic SMCs that depends on functional biomechanical signal transduction (eg, YAP signaling). Our study highlights the importance of the adaptive response in maintaining aortic homeostasis and preventing AAD in mice.

Vitamin D Regulates Natriuretic Peptide Receptor‐A Gene Expression via Modulation of Histone Deacetylases and DNA Methyltransferase Activity

Cardiac hormones atrial and brain natriuretic peptides (ANP and BNP) activate guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) and produces the second messenger cGMP. cGMP activates downstream signaling and biological consequences of ANP/NPRA such as natriuresis, diuresis, vasorelaxation, antimitogenic responses, and cardiac and renal anti‐hypertrophic effects. The activity and expression of Npr1 gene (coding for GC‐A/NPRA) assessed primarily through ANP‐stimulated cGMP production, are mediated by agents, including autoregulation involving natriuretic peptides, osmotic sensors, and other external and internal stimuli, but the hormonal and epigenetic mechanisms mediating Npr1 regulation are not well understood. The objective of present study was to study the effect of Vitamin D (vitD) in the regulation of Npr1 gene transcription and expression via modulation of epigenetic factors. Our bioinformatics study with the murine Npr1promoter has shown presence of 4 vitD response elements (VDRE) in the region ‐583 to ‐495 from the transcription start site having perfect and VDRE‐like consensus sequence. Npr1promoter activity was determined by transient transfection of Npr1 promoter deletion constructs in cultured rat thoracic aortic smooth muscle cells (RTASMCs) and mouse mesangial cells (MMCs) treated with Vitamin D₃ (1α, 25‐dihydroxy; VD3) using dual luciferase assay kit. Luciferase assay demonstrated that VD3 enhances Npr1 promoter activity by more than 5‐6‐fold in RTASMCs and MMCs in a dose‐dependent manner. Western blot and densitometry analysis showed increasing concentrations of VD3 significantly induced NPRA protein levels by 3.5‐fold in MMCs and 4.5‐fold in RTASMCs and maximum effect was observed at 100 nM. There was 45%‐50% inhibition of histone deacetylase (HDAC) activity in presence of VD3 in both cell lines as measured by HDAC activity/inhibition ELISA kit. Moreover, treatment with VD3 reduced class I HDAC enzymes, HDAC1 and HDAC3 protein levels and dose‐dependently enhanced acetylation level of histones, H3 at lysine residues 9 and 14 (H3‐K9/14 ac) and H4 at lysine residue 12 (H4‐K14ac). Treatment of RTASMCs and MMCs with VD3 attenuated DNA methyltransferase (DNMT) activity by 40%‐55% as measured by DNMT activity/inhibition ELISA kit. The results demonstrate that VD3 regulate Npr1 gene expression by modulation of histone deacetylases and DNMT activity. The identification of epigenetic targets of vitamin D signaling as a regulator of Npr1gene transcription and protein expression will have important implications in hypertension and cardiovascular regulation.

Changes of BKCA current and vascular reactivity in vascular smooth muscle of thoracic aorta and mesenteric artery in type 2 diabetic rats.

The relationship between large conductance calcium activated potassium channel (BKCa) and vascular lesions in type 2 diabetes mellitus (T2DM) was investigated by observing vascular reactivity of thoracic aorta and mesenteric artery and the current changes of BKCa in vascular smooth muscle cells (VSMCs). The thoracic aorta and mesenteric artery of T2DM rats were isolated, the whole cell perforated patch clamp experiment and single channel patch clamp experiment of acute enzyme separation of thoracic aorta and mesenteric artery smooth muscle cells were performed to measure the membrane capacitance and the amplitude of macro current. And the vascular ring experiment was performed to observe the change of relaxation percentage. The results showed that the amplitude of BKCa current in vascular smooth muscle cells of diabetic rats was higher than that of the control group. The channel current had outward rectifying characteristics. BKCa is related to vascular reactivity and smooth muscle relaxation in T2DM rats. The opening probability of BKCa in VSMCs of diabetic rats was significantly increased. This study suggests that BKCa may be a new target for diabetic vascular disease.

USP10 exacerbates neointima formation by stabilizing Skp2 protein in vascular smooth muscle cells

The underlying mechanism of neointima formation remains unclear. Ubiquitin-specific peptidase 10 (USP10) is a deubiquitinase that plays a major role in cancer development and progression. However, the function of USP10 in arterial restenosis is unknown. Herein, USP10 expression was detected in mouse arteries and increased after carotid ligation. The inhibition of USP10 exhibited thinner neointima in the model of mouse carotid ligation. In vitro data showed that USP10 deficiency reduced proliferation and migration of rat thoracic aorta smooth muscle cells (A7r5) and human aortic smooth muscle cells (HASMCs). Mechanically, USP10 can bind to Skp2 and stabilize its protein level by removing polyubiquitin on Skp2 in the cytoplasm. The overexpression of Skp2 abrogated cell cycle arrest induced by USP10 inhibition. Overall, the current study demonstrated that USP10 is involved in vascular remodeling by directly promoting VSMC proliferation and migration via stabilization of Skp2 protein expression.

Green Synthesis of Bioactive CuO@Fe3O4@Walnut Shell Nanocomposite Using Crataegus azarolus Var. aronia L. Extract and its Antivasoconstrictive Action on Rat’s Aortic Smooth Muscle

The antioxidant potential of Crataegus azarolus Var. aronia aqueous extract was used as reducing and stabilizing media to the green synthesis of bioactive CuO@Fe3O4@Walnut shell nanocomposite (NCs). The ability of the plant extract as bioreducing medium was proved using Fourier-transform infrared spectroscopy and ultraviolet–visible spectroscopy. While, the structural identification of synthesized CuO@Fe3O4@Walnut shell NCs was proved using the scanning electron microscopy, electron dispersive spectroscopy, elemental mapping, and point analysis. Then, the antivasoconstriction effects of CuO@Fe3O4@Walnut shell NCs (5*10−2 mg/ml) and (1*10−1 mg/ml) on rats isolated thoracic aortic smooth muscle cells were studied. Green synthesized CuO@Fe3O4@Walnut shell NCs showed effective antivasoconstriction activity against norepinephrine (NE) (1*10−9–10−4 M) induced contraction in endothelium-intact and endothelium-denuded rats aortic smooth muscle. In conclusion, the aqueous extract of C. aronia acts as potential reducing and stabilizing media to the green synthesis of bioactive CuO@Fe3O4@Walnut shell NCs and the NCs play an important role against contractile effects of NE on rats’ aortic smooth muscle.

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.

MiR-22-3p Suppresses Vascular Remodeling and Oxidative Stress by Targeting CHD9 during the Development of Hypertension

Hypertension is considered a risk factor for a series of systematic diseases. Known factors including genetic predisposition, age, and diet habits are strongly associated with the initiation of hypertension. The current study aimed to investigate the role of miR-22-3p in hypertension. In this study, we discovered that the miR-22-3p level was significantly decreased in the thoracic aortic vascular tissues and aortic smooth muscle cells (ASMCs) of spontaneously hypertensive rats. Functionally, the overexpression of miR-22-3p facilitated the switch of ASMCs from the synthetic to contractile phenotype. To investigate the underlying mechanism, we predicted 11 potential target mRNAs for miR-22-3p. After screening, chromodomain helicase DNA-binding 9 (CHD9) was validated to bind with miR-22-3p. Rescue assays showed that the co-overexpression of miR-22-3p and CHD9 reversed the inhibitory effect of miR-22-3p mimics on cell proliferation, migration, and oxidative stress in ASMCs. Finally, miR-22-3p suppressed vascular remodeling and oxidative stress in vivo. Overall, miR-22-3p regulated ASMC phenotype switch by targeting CHD9. This new discovery provides a potential insight into hypertension treatment.

Comparison of the inhibition of high phosphate-induced smooth muscle cell calcification by Porphyra yezoensis and Astragalus polysaccharides

Abstract This study aimed to investigate the inhibitory effects of Porphyra yezoensis polysaccharides (PYP) and Astragalus polysaccharides (APS) with similar molecular weight (approximately 4 KDa) on high phosphate (Pi)-induced vascular calcification (VC) in rat thoracic aortic smooth muscle cells (A7r5) and to develop drugs that can inhibit VC. After co-incubation with 3.0 mmol/L Pi and different concentrations of PYP or APS, calcium deposition and corresponding biochemical indicators of A7r5 cells were detected. PYP and APS remarkably restricted reactive oxygen species generation, restored mitochondrial membrane potential, reduced excessive apoptosis and autophagy, up-regulated expression of α-smooth muscle actin, suppressed alkaline phosphatase activity, and decreased calcium deposition on the surface of A7r5 cells. At the same concentration, PYP with stronger antioxidant capacity exhibited better inhibitory effect on calcification of A7r5 cells. These results suggest that polysaccharide can inhibit calcification by protecting A7r5 cells from high-Pi induced oxidative damage.

The protease inhibition with Ritonavir impairs endothelial function and promotes vascular smooth cell proliferation via RANTES/C‐C chemokine receptor type 5 and Nox1‐derived reactive oxygen species pathway.

Thanks to the advent of highly active antiretroviral therapy (HAART), the life of people living with HIV has been extended to close to that of healthy people. However, patients with HIV are now presenting with accelerated development of cardiovascular disease (CVD) and CVD is currently the leading cause of death in patients with HIV on HAART. Herein, we tested the hypothesis that the protease inhibitor ritonavir contributes to HAART‐associated CVD by impairing vascular function via C‐C chemokine receptor type 5 (CCR5)‐ and reactive oxygen species‐mediated mechanisms. We treated 8–10‐week‐old male C57BL6/J with ritonavir for 4 consecutive weeks (5mg/kg/day, i.p). At the end of the treatment, blood was collected and aortas excised to measure H2O2 via Amplex Red and vascular function via wire myography. Aorta from ritonavir treated animals exhibited higher H2O2 levels, impaired relaxation to acetylcholine (Ach) and increased contractility to phenylephrine (Phe). Nox1 inhibition with GKT771, (10μM) fully restored endothelial function in rings from ritonavir treated animals. Ritonavir treatment also elevated RANTES (CCR5 ligand) plasma levels, as well as CCR5, NADPH oxidase 1 (Nox1) and NoxA1 transcript levels in the aorta. No difference was observed for others oxidases, such as Nox2 and Nox4. Repetition of the ritonavir treatment in CCR5 deficient mice revealed that CCR5 deficiency protects vessels from ritonavir induced‐endothelial dysfunction and upregulation of Nox1 and NoxA1 enzymes. To analyze the direct effects of RANTES on the vasculature, thoracic aorta, endothelial, and vascular smooth muscle cells were exposed to RANTES in different times (20ng/mL). RANTES elevated endothelial NOX1 expression and impaired ACh‐mediated relaxation. Concomitantly, Nox1 inhibition prevented RANTES‐mediated endothelial dysfunction. In addition, RANTES time‐dependently stimulated rat aortic smooth muscle cell proliferation, as measured by Ki67 expression, which was partially reverted by Nox1 inhibition. Our data suggest that the protease inhibitor ritonavir contributes to vascular dysfunction and cardiovascular disease by increasing RANTES‐mediated CCR5 activation ultimately leading to increases in Nox1 expression, ROS production, endothelial dysfunction and vascular smooth muscle cell proliferation. Taken together, these findings provide potential explanation for the increased risk of cardiovascular disease developed by patients living with HIV and present CCR5 as a potential target for treatment.Support or Funding Information1R01HL130301‐01 to EBC; K99: 1K99HL140139‐01A1; R00: 4R00HL140139‐03 to TBN

TSPO ligands prevent the proliferation of vascular smooth muscle cells and attenuate neointima formation through AMPK activation.

Abnormal growth of the intimal layer of blood vessels (neointima formation) contributes to the progression of atherosclerosis and in-stent restenosis. Recent evidence shows that the 18-kDa translocator protein (TSPO), a mitochondrial membrane protein, is involved in diverse cardiovascular diseases. In this study we investigated the role of endogenous TSPO in neointima formation after angioplasty in vitro and in vivo. We established a vascular injury model in vitro by using platelet-derived growth factor-BB (PDGF-BB) to stimulate rat thoracic aortic smooth muscle cells (A10 cells). We found that treatment with PDGF-BB (1–20 ng/mL) dose-dependently increased TSPO expression in A10 cells, which was blocked in the presence of PKC inhibitor or MAPK inhibitor. Overexpression of TSPO significantly promoted the proliferation and migration in A10 cells, whereas downregulation of TSPO expression by siRNA or treatment with TSPO ligands PK11195 or Ro5-4864 (104 nM) produced the opposite effects. Furthermore, we found that PK11195 (10−104 nM) dose-dependently activated AMPK in A10 cells. PK11195-induced inhibition on the proliferation and migration of PDGF-BB-treated A10 cells were abolished by compound C (an AMPK-specific inhibitor, 103 nM). In rats with balloon-injured carotid arteries, TSPO expression was markedly upregulated in the carotid arteries. Administration of PK11195 (3 mg/kg every 3 days, ip), starting from the initial balloon injury and lasting for 2 weeks, greatly attenuated carotid neointima formation by suppressing balloon injury-induced phenotype switching of VSMCs (increased α-SMA expression). These results suggest that TSPO is a vascular injury-response molecule that promotes VSMC proliferation and migration and is responsible for the neointima formation after vascular injury, which provides a novel therapeutic target for various cardiovascular diseases including atherosclerosis and restenosis.

Inhibition of DNA Methylation Regulates Guanylyl Cyclase/Natriuretic Peptide Receptor‐A Gene Expression

Cardiac hormone atrial natriuretic peptide binds to guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) and produces second messenger cGMP, which plays a pivotal role in maintaining blood pressure and cardiovascular homeostasis. The molecular and epigenetic mechanisms that contribute to the activation of Npr1 (coding for GC‐A/NPRA) gene expression are not well understood. The present study was aimed at understanding the mechanisms involved in DNA methylation inhibitor, 5‐Azacytidine (5‐aza)‐mediated Npr1 (coding for GC‐A/NPRA) gene transcription. The studies were carried out in rat thoracic aortic vascular smooth muscle cells (RTASMC), cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and treated with 5‐aza for 24 h. The MethPrimer search result showed the presence of three cytosine‐phosphate‐guanine (CpG) islands on the Npr1 full length promoter, namely island 1 (−886 to −752, 134 bp), island 2 (−310 to −58, 152 bp), and island 3 (−514 to +310, 464 bp) from the transcription start site. 5‐azacytidine significantly induced mRNA levels of Npr1 gene by 4‐fold and NPRA protein expression by 3‐fold in RTASMCs in a dose‐dependent manner. Treatment of cells with 5‐aza significantly (p&lt;0.05) attenuated DNA methyltransferase 1 (DNMT1) protein expression in a dose‐dependent manner. However, there was no noticeable change in the DNMT3a and DNMT3b protein levels. There was a significant reduction in the DNMT activity in 5‐aza‐treated RTASMCs compared with vehicle–treated cells as quantified by EpiQuik DNMT activity assay. The present findings provide a novel regulatory mechanism for Npr1 gene transcription, an important player in the control of hypertension and cardiovascular homeostasis.Support or Funding InformationThis work was supported by an Institutional Development Award (IDeA) from the NIGMS and a partial support from Tulane Carol Lavin Bernick grant award.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Antiapoptotic Effect of β1 Blockers in Ascending Thoracic Aortic Smooth Muscle Cells: The Role of HSP70 Expression.

Heat shock proteins (HSPs) play an important role in the cellular adaptation to stress, a requisite for cell survival. The aortic wall appears to be a target for increased expression of HSPs during surgical stress. We aimed to define the expression and function of aortic HSP70 in 31 patients with normal ascending thoracic aortic diameter who underwent aortic valve replacement due to aortic valve stenosis and in 35 patients with dilated ascending thoracic aorta who underwent replacement of an ascending thoracic aortic aneurysm. To elucidate responsible signaling mechanisms we used an in vitro model of rat hypoxic aortic vascular smooth muscle cell (AVSMC) cultures. We demonstrated an increase in AVSMC HSP70 and an attenuation of the apoptotic markers (TUNEL-positive nuclei, caspase-3 activity, Bax/Bcl2 ratio) in aortic wall tissue specimens from both aortic valve stenosis and ascending thoracic aortic aneurysm patients on β1 blockade with metoprolol. In vitro, metoprolol treatment of hypoxic rat AVSMCs increased nitric oxide (NO) production, induced heat shock factor 1 transport to the nucleus, upregulated HSP70, decreased p53 phosphorylation and attenuated apoptosis. Blockade of NO production, resulted in decreased HSP70 and prevented the metoprolol-induced anti-apoptotic response of hypoxic AVSMCs. We demonstrate an anti-apoptotic effect of metoprolol dependent on NO-induced HSP70 expression, and thus augmentation of HSP70 expression should be considered as a therapeutic approach to limit apoptosis in the human ascending thoracic aorta of patients undergoing cardiac surgery.

Inhibitory effects of solid lipid nanoparticles of rhynchophylline on proliferation of rat vasculars mooth muscle cells induced by TGF-β1

Objective To observe the effect of Rhy-SLN on the proliferation of rat vascular smooth muscle cells (VSMC) induced by TGF-β1, and explore the mechanism. Methods The primary culture of rat thoracic aortic vascular smooth muscle cells was studied by tissue block culture method. The cells were divided into the control group, TGF-β1 group, TGF-β1+ the high, medium and low dosage groups of Rhy-SLN. In addition to the control group, the cells of the other groups were involved in the intervention of TGF-β1 of 20 g/L, and the high, medium and low dosage groups of Rhy-SLN cells were involved in the intervention of 25, 50, 100 mg/L of the hook teng solid lipid nanoparticles. After 24 hours of culture, MTT assay was used to detect cell proliferation inhibition rate in each group, and the cell cycle was detected by flow cytometry. The expression of c-myc and c-Fos protein in each group was detected by Western blot method. Results Compared with the TGF-β1 group, the absorbance value (0.457 ± 0.046 vs. 0.975 ± 0.049) of TGF-β1+ rhy-sln high dose group significantly decreased (P<0.01); the number of S phase cells (15.87% ± 2.47%, 15.23% ± 1.69%, 17.02% ± 2.87% vs. 38.58% ± 2.68%) of TGF-β1+ rhy-sln in each dose group significantly decreased (P<0.01); The c-myc (48.65 ± 3.65, 50.69 ± 4.16, 55.29 ± 3.67 vs. 68.21 ± 3.25) and c-Fos (38.78 ± 4.25, 43.56 ± 3.69, 46.58 ± 3.57 vs. 66.54 ± 4.09) of the TGF-β1+ rhy-sln each dose group significantly decreased (P<0.01). Conclusions The Rhy-SLN can inhibit the proliferation of VSMC in rats induced by TGF-β1.Its mechanism is related to the conversion of G0/G1 phase to the S phase and the expression of the reduction of c-myc and c-fos protein. Key words: Rhynchophylline; Solid lipid nanoparticles; Transforming growth factor beta; Proto-oncogene proteins c-fos; Proto-oncogene proteins c-myc; Muscle, smooth, vascular; Cell proliferation; Rat