Glycolaldehyde-derived advanced glycation end products (glycol-AGEs)-induced vascular smooth muscle cell dysfunction is regulated by the AGES-receptor (RAGE) axis in endothelium.

Background: Adipokine resistin induces vascular smooth muscle cell (VSMC) dysfunction, implying a vital role in cardiovascular disease. In this study, we examined the in vivo and in vitro effects of resisin and investigated the underlying mechanisms involving protein kinase C epsilon (PKCε) and reactive oxygen species (ROS). Methods: After guide-wire injury of distal left common carotid artery, ApoE-/- mice fed with a western diet were treated with or without subcutaneous infusion of resistin in the presence or absence of PKCε inhibitor for 4 weeks. The progress of intimal hyperplasia was monitored using an ultrasound micro-image system. NADPH oxidase (Nox) activity in the mouse coronary arteries was measured using a chemiluminescence assay. Additionally, we treated human coronary artery smooth muscle with resistin in the presence or absence of PKCε and Nox-specific inhibitors to determine mechanistic and functional effects of resistin in VSMCs functions. Reactive oxygen species (ROS) production was analyzed using confocal microscopy. Results: Resistin significantly enhanced injury-induced intimal hyperplasia in our murine model, which was mitigated by the PKCε inhibitor. Nox activity was also substantially augmented in the injured artery in a resistin-PKCε dependent manner. On the cellular level, resistin-induced ROS production was time-dependent and mitochondrial ROS seemed to be the trigger for cytosolic Nox-induced ROS. Inhibition of Nox completely abolished resistin-exaggerated VSMC proliferation, migration and dedifferentiation. Consistently, inhibition of PKCε significantly reduced resistin-induced cytosolic ROS production, Nox activity, and VSMC dysfunction. Conclusion: This is the first study showing that resistin-induced VSMC dysfunction and intimal hyperplasia is mediated through PKCε-dependent Nox activation pathway. These findings suggest potential molecular targets for cardiovascular disease.

Angiotensin II (Ang II)‐mediated vascular smooth muscle cell (VSMC) dysfunction plays a major role in the pathogenesis of hypertension. Long non‐coding RNAs (lncRNAs) have recently elicited much interest, but their roles in Ang II actions and hypertension are not well understood. We aim to investigate the regulation and functions of a novel lncRNA “Growth factor‐ and pro‐Inflammatory cytokine‐induced Vascular cell‐Expressed lncRNA (Giver)”, in Ang II‐mediated VSMC dysfunction. RNA‐seq and RT‐qPCR analyses revealed that Ang II stimulation of rat VSMC increased expression of Giver, and Nr4a3, an adjacent gene which encodes a nuclear receptor. Similar changes were observed in rat and mouse aortas treated ex vivo with Ang II. Giver and Nr4a3 were also significantly induced by key mitogenic and pro‐inflammatory stimuli. RNA‐FISH and nuclear fractionations showed that Giver is localized predominantly in the nucleus. Regulation of Giver promoter in response to Ang II occurs through the binding of Nr4a3 to a consensus NBRE binding site. In vitro RNA pull down assays using biotinylated Giver combined with mass spectrometry in VSMC showed that Giver interacts with nuclear and chromatin remodeling related factors like ruvB‐like1, WD repeat‐containing protein 82, histone‐binding protein RBBP7, non‐POU domain‐containing octamer‐binding protein and other RNA binding proteins involved in splicing and RNA processing. Furthermore, microarray profiling combined with RT‐qPCR validation revealed that Giver knockdown attenuates the expression of genes involved in oxidative stress (Nox1) and inflammatation (Il6, Ccl2, and Tnf), but increases Nr4a3, in VSMC. Conversely, Giver overexpression enhanced the expression of these genes. Accordingly, Giver knockdown also inhibited Ang II‐induced oxidative stress and proliferation. Moreover, Giver overexpression in VSMC enhanced enrichment of Pol lI but decreased repressive histone modification H3K27me3 at oxidative stress and inflammatory gene promoters. Human orthologous genes GIVER and NR4A3 were also induced by Ang II in human VSMC and endothelial cells. Interestingly, GIVER and NR4A3 were also increased in arteries from hypertensive patients, but attenuated in hypertensive patients treated with Angiotensin Converting Enzyme Inhibitors or Angiotensin Receptor Blockers. Thus, LncRNA Giver and its regulator Nr4a3 are important players in Ang II‐mediated VSMC dysfunction, and can serve as novel targets for anti‐hypertensive therapy.Support or Funding InformationNIH, NHLBIThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Angiotensin II (Ang II)-mediated vascular smooth muscle cell (VSMC) dysfunction plays a critical role in the pathogenesis of Cardiovascular Diseases (CVDs). However, the role of Ang II-induced transcription factors in the diverse effects of Ang II remains unclear. We profiled Ang II induced gene expression by microarray analysis of RNA isolated from Ang II-treated and control VSMC. Our results identified numerous differentially regulated genes including several key transcription factors in Ang II-stimulated VSMC compared with controls. Ingenuity Pathway Analysis indicated that Ang II-regulated genes are involved in VSMC dysfunction highly relevant to CVDs. We validated the expression of several genes by RT-qPCR and further characterized the functions of the most differentially regulated gene, KLF4, known to regulate growth factor induced VSMC phenotypic switching. We demonstrated that Ang II induced the expression of KLF4 in cultured VSMC in vitro , in mice aortas cultured ex vivo , and in aortas isolated from Ang II-infused mice in vivo . Ang II-induced KLF4 expression was inhibited by Losartan, demonstrating regulation via the AT1 receptor. Transfection experiments using WT and mutant KLF4 promoter constructs revealed the key role of cis -elements with consensus binding sites for p53, SP1 and YY1 in Ang II-induced KLF4 promoter activation. Next, we performed gene expression profiling by Affymetrix gene arrays after siRNA mediated KLF4 knockdown in VSMC. The differentially expressed genes were subsequently analyzed by DAVID to obtain enriched biological processes and potential pathways relevant to cardiovascular functions. Results showed that KLF4 knockdown upregulated the expression of several genes related to cell proliferation and hypertrophy. Interestingly, KLF4 knockdown also enhanced the expression of multiple pro-inflammatory genes including IL-6 and downregulated several anti-inflammatory genes including Thrombomodulin, suggesting an anti-inflammatory role for KLF4 in VSMC. Together, these results suggest that KLF4 may act as a novel molecular brake to modulate Ang II actions that, when disrupted, can further augment Ang II mediated VSMC dysfunction associated with vascular diseases.

Chronic kidney disease (CKD) is associated with increased cardiovascular mortality, and vascular smooth muscle cell (VSMC) dysfunction plays a pivotal role in uremic atherosclerosis. Axl signaling is involved in vascular injury and is highly expressed in VSMCs. Recent reports have shown that cilostazol, a phosphodiesterase type 3 inhibitor (PDE3), can regulate various stages of the atherosclerotic process. However, the role of cilostazol in uremic vasculopathy remains unclear. This study aimed to identify the effect of cilostazol in VSMCs in the experimental CKD and to investigate whether the regulatory mechanism occurs through Axl signaling. We investigated the effect of P-cresol and cilostazol on Axl signaling in A7r5 rat VSMCs and the rat and human CKD models. From the in vivo CKD rats and patients, aortic tissue exhibited significantly decreased Axl expression after cilostazol treatment. P-cresol increased Axl, proliferating of cell nuclear antigen (PCNA), focal adhesion kinase (FAK), and matrix metalloproteinase-2 (MMP-2) expressions, decreased caspase-3 expression, and was accompanied by increased cell viability and migration. Cilostazol significantly reversed P-cresol-induced Axl, downstream gene expressions, and cell functions. Along with the increased Axl expression, P-cresol activated PLCγ, Akt, and ERK phosphorylation and cilostazol significantly suppressed the effect of P-cresol. Axl knockdown significantly reversed the expressions of P-cresol-induced Axl-related gene expression and cell functions. Cilostazol with Axl knockdown have additive changes in downstream gene expression and cell functions in P-cresol culture. Both in vitro and in vivo experimental CKD models elucidate a new signal transduction of cilostazol-mediated protection against uremic toxin-related VSMCs dysfunction and highlight the involvement of the Axl signaling and downstream pathways.

Chlamydia pneumoniae invades into vascular smooth muscle cells through the CXCR4-β-arrestin 2 pathway via TLR2/CXCR4 crosstalk.

BackgroundAdvanced type 2 diabetes mellitus (T2DM) accelerates vascular smooth muscle cell (VSMC) dysfunction which contributes to the development of vasculopathy, associated with the highest degree of morbidity of T2DM. Lysine acetylation, a post-translational modification (PTM), has been associated with metabolic diseases and its complications. Whether levels of global lysine acetylation are altered in vasculature from advanced T2DM remains undetermined. We hypothesized that VSMC undergoes dysregulation in advanced T2DM which is associated with vascular hyperacetylation.MethodsAged male Goto Kakizaki (GK) rats, a non-obese murine model of T2DM, and age-matched male Wistar rats (control group) were used in this study. Thoracic aortas were isolated and examined for measurement of global levels of lysine acetylation, and vascular reactivity studies were conducted using a wire myograph. Direct arterial blood pressure was assessed by carotid catheterization. Cultured human VSMCs were used to investigate whether lysine acetylation participates in high glucose-induced reactive oxygen species (ROS), a crucial factor triggering diabetic vascular dysfunction.ResultsThe GK rats exhibited marked glucose intolerance as well as insulin resistance. Cardiovascular complications in GK rats were confirmed by elevated arterial blood pressure and reduced VSMC-dependent vasorelaxation. These complications were correlated with high levels of vascular global lysine acetylation. Human VSMC cultures incubated under high glucose conditions displayed elevated ROS levels and increased global lysine acetylation. Inhibition of hyperacetylation by garcinol, a lysine acetyltransferase and p300/CBP association factor (PCAF) inhibitor, reduced high glucose-induced ROS production in VSMC.ConclusionThis study provides evidence that vascular hyperacetylation is associated with VSMC dysfunction in advanced T2DM. Understanding lysine acetylation regulation in blood vessels from diabetics may provide insight into the mechanisms of diabetic vascular dysfunction, and opportunities for novel therapeutic approaches to treat diabetic vascular complications.

Vascular smooth muscle cell (VSMC) dysfunction is a critical pathological process in postinjury intima hyperplasia. This process is driven by the adherence and accumulation of platelet-derived extracellular vesicles (PEVs) released from activated platelets to VSMCs at the site of injured intima. However, the precise mechanism remains unclear. Thus, the present study aimed to investigate how PEVs adhere to VSMCs and facilitate VSMC dysfunction in postinjury intimal hyperplasia. Morphological results confirmed that PEVs led to VSMC dysfunction and intimal hyperplasia. Integrated single-cell and proteomic analysis revealed that increased secreted phosphoprotein 1 (SPP1) expression in VSMCs played a central role in this process, possibly by mediating PEV adhesion to VSMCs and activating the focal adhesion kinase (FAK)/phosphoinositide 3 kinase (PI3K)/protein kinase B (Akt) axis. In addition, integrin beta 3 (ITGβ3, CD61) on PEVs, with increased expression under pathological conditions, was predicted to interact with SPP1. Co-immunoprecipitation (Co-IP) analysis further confirmed that ITGβ3 interacted with SPP1, thereby activating the FAK/PI3K/AKT phosphorylation and promoting PEV adhesion. Of note, blocking ITGβ3 expression on PEVs reduced PEV adhesion and intimal hyperplasia. Thus, ITGβ3-SPP1-mediated PEV adhesion to VSMCs may be a novel mechanism in intimal hyperplasia, which proposed to be critical for vascular homeostasis.

Transmembrane protein 98 (TMEM98) is a novel gene, and its function has not been well investigated. In a prior study, we have shown that siRNA-mediated knockdown of TMEM98 inhibited interleukin-8 (IL-8) promoted endothelial cell (EC) adhesion, as well as vascular smooth muscle cell (VSMC) proliferation and migration in the vascular endothelial and smooth muscle cell dysfunction. Herein, we used gain- and loss-of-function approaches combined with biochemical techniques to further explore the role of TMEM98 in the vascular wall cell. The expression and secretion of TMEM98 was increased in cultured human umbilical vein endothelial cells (HUVECs) and VSMCs treated with IL-8 and platelet-derived growth factor-BB (PDGF-BB). Also, PDGF-BB secretion was increased in TMEM98-treated HUVECs and VSMCs. Thus, it appears that TMEM98 and PDGF-BB form a positive feedback loop in potentiation of EC adhesion, as well as VSMC proliferation and migration. Knockdown of TMEM98 mediated by siRNA inhibited PDGF-BB-promoted EC adhesion by downregulating the expression of ICAM-1 and VCAM-1, as well as impaired the proliferation and migration of VSMCs by suppressing the AKT/GSK3β/cyclinD1 signaling pathway and reducing the expression of β-catenin. Hence, TMEM98 promoted EC adhesion by inducing the expression of ICAM-1/VCAM-1 and triggered VSMC proliferation and migration by activating the ERK and AKT/GSK3β signaling pathways. Taken together, TMEM98 may serve as a potential therapeutic target for the clinical treatment of vascular endothelial and smooth muscle cell dysfunction.

Introduction and Aims: Endothelial dysfunction is recognized as a cardiovascular aging hallmark. Administration of nitric oxide synthase blocker N-Ω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) constitutes a well-known small animal model of cardiovascular aging. Despite extensive phenotypic characterization, the exact aortic function changes in L-NAME treated mice are largely unknown. Therefore, this study presents a longitudinal characterization of the aortic reactivity and biomechanical alterations in L-NAME treated C57Bl/6 mice. Methods and Results: Male C57Bl/6 mice were treated with L-NAME (0.5 mg/ml drinking water) for 1, 2, 4, 8, or 16 weeks. Peripheral blood pressure measurement (tail-cuff) and transthoracic echocardiograms were recorded, showing progressive hypertension after 4 weeks of treatment and progressive cardiac hypertrophy after 8–16 weeks of treatment. Aortic stiffness was measured in vivo as aortic pulse wave velocity (aPWV, ultrasound) and ex vivo as Peterson modulus (Ep). Aortic reactivity and biomechanics were investigated ex vivo in thoracic aortic rings, mounted isometrically or dynamically-stretched in organ bath set-ups. Aortic stiffening was heightened in L-NAME treated mice after all treatment durations, thereby preceding the development of hypertension and cardiac aging. L-NAME treatment doubled the rate of arterial stiffening compared to control mice, and displayed an attenuation of the elevated aortic stiffness at high distending pressure, possibly due to late-term reduction of medial collagen types I, III, and IV content. Remarkably, endothelial dysfunction, measured by acetylcholine concentration-response stimulation in precontracted aortic rings, was only observed after short-term (1–4 weeks) treatment, followed by restoration of endothelial function which coincided with increased phosphorylation of endothelial nitric oxide synthase (S1177). In the late-disease phase (8–16 weeks), vascular smooth muscle cell (VSMC) dysfunction developed, including increased contribution of voltage-dependent calcium channels (assessed by inhibition with diltiazem), basal VSMC cytoplasmic calcium loading (assessed by removal of extracellular calcium), and heightened intracellular contractile calcium handling (assessed by measurement of sarcoplasmic reticulum-mediated transient contractions). Conclusion: Arterial stiffness precedes peripheral hypertension and cardiac hypertrophy in chronic L-NAME treated male C57Bl/6 mice. The underlying aortic disease mechanisms underwent a distinct shift from early endothelial dysfunction to late-term VSMC dysfunction, with continued disease progression.

Background and aims:Coronary microvascular dysfunction (CMD) is associated with adverse cardiovascular outcomes. CMD is driven by endothelial and vascular smooth muscle cell (VSMC) dysfunction. We aimed to test whether CMD could be mitigated by a plant-based diet (PBD) in an animal model of hypertension.Methods:We compared 28- and 40-week-old female normotensive Wistar-Kyoto and spontaneously hypertensive (SHR) rats, maintained, from age 4 weeks, on a control refined diet or a PBD, comprised of 28% fruits, vegetables, nuts and legumes. A subset of control SHRs were switched to the PBD at 28 weeks. CMD was assessed by coronary flow reserve via echocardiogram. Cardiac microvascular endothelial function was assessed via cMRI. Endothelial and VSMC function were assessed in the left ventricle (LV) or in isolated VSMCs. The role of gut microbiota was probed via 16S sequencing and antibiotics. Cardiac inflammation, oxidative stress, and fibrosis were also explored.Results:SHRs exhibited endothelial dysfunction and likely VSMC dysfunction. PBD did not ameliorate their hypertension but, nonetheless, prevented and reversed CMD. PBD’s mitigation of CMD was associated with improved endothelial nitric oxide synthase function and NO-mediated VSMC signaling, as well as reductions in LV oxidative stress, inflammatory signaling, and fibrosis. PBD altered the gut microbiota, although antibiotic studies failed to establish its importance in ameliorating CMD.Conclusions:A PBD prevented CMD development and reversed established CMD in SHRs. Such benefits of PBD, which occurred without alleviating hypertension, were possibly due to improved endothelial function and likely improved VSMC function. These results support clinical trials to test PBDs in human CMD.

Many natural angiotensin-converting enzyme inhibitory (ACEI) peptides have been widely studied. However, their stability in vivo is poor in most cases. In this study, peptides were initially digested from broccoli in vitro, and absorption was simulated by Caco2 cells transport and then analyzed by Peptideomics and molecular docking. Subsequently, the mechanisms were verified using a high glucose-induced vascular smooth muscle cells (VSMCs) dysfunction model. Results showed that ACEI activity of broccoli crude peptide increased by 70.73 ± 1.42% after digestion. The enzymatic hydrolysates of crude broccoli peptides before and after digestion were detected by HPLC. The digested crude peptides were highly stable (with a stability level > 90%) in the intestine and possessed a strong absorptive potential. Five peptides with high stability and strong permeability were first identified, including HLEVR, LTEVR, LEHGF, HLVNK, and LLDGR, which exhibited high activity with IC50 values of 3.19 ± 0.23 mM, 17.07 ± 1.37 mM, 0.64 ± 0.02 mM, 0.06 ± 0.01 mM, and 2.81 ± 0.12 mM, respectively. When the VSMCs model was exposed to Ang II, the expressions of PCNA, MMP2, and Bcl2 were increased, while the expression of BAX was inhibited. When the VSMCs was exposed to high glucose (HG), the Ang II concentration significantly increased. This indicates that HG elevated Ang II levels. Finally, five peptides significantly attenuated Ang II-induced VSMCs proliferation and migration by down-regulating AT1R expression and inhibiting ERK and p38 MAPK phosphorylation. Notably, in exploring VSMCs dysfunction on a high glucose-induced model, ACEI peptides resulted in down-regulation of ACE and up-regulation of ACE2 expression. Therefore, it can be further referenced for the functional food against hypertension and cardiovascular diseases.

Coronary microvascular dysfunction (CMD) is associated with adverse cardiovascular outcomes. CMD is driven by endothelial and vascular smooth muscle cell (VSMC) dysfunction. We aimed to test whether CMD could be mitigated by a plant-based diet (PBD) in an animal model of hypertension. We compared 28- and 40-week-old female normotensive Wistar-Kyoto and spontaneously hypertensive rats (SHRs), maintained, from age 4 weeks, on a control refined diet or a PBD, composed of 28% fruits, vegetables, nuts, and legumes. A subset of control SHRs was switched to the PBD at 28 weeks. CMD was assessed by coronary flow reserve via echocardiogram. Cardiac microvascular endothelial function was assessed via cardiac magnetic resonance imaging. Endothelial and VSMC function were assessed in the left ventricle or in isolated VSMCs. The role of gut microbiota was probed via 16S sequencing and antibiotics. Cardiac inflammation, oxidative stress, and fibrosis were also explored. SHRs exhibited endothelial dysfunction and likely VSMC dysfunction. A PBD did not ameliorate their hypertension but, nonetheless, prevented and reversed CMD. The PBD's mitigation of CMD was associated with improved endothelial nitric oxide synthase function and nitric oxide-mediated VSMC signaling, as well as reductions in left ventricular oxidative stress, inflammatory signaling, and fibrosis. A PBD altered the gut microbiota, although antibiotic studies failed to establish its importance in ameliorating CMD. A PBD prevented CMD development and reversed established CMD in SHRs. Such benefits of PBD, which occurred without alleviating hypertension, were possibly due to improved endothelial function and likely improved VSMC function. These results support clinical trials to test PBDs in human CMD.

Vascular Smooth Muscle Cells (VSMC) dysfunction is a major contributor to Type 1 diabetes (T1D)-associated vascular complications. Ca2+ is a key messenger responsible for maintaining VSMC tone and function, and alterations in its cytosolic levels are central to diabetes-related vasculopathy. Heat Shock Protein 70 (HSP70), a multifaceted chaperone present intracellularly (iHSP70), regulates vascular reactivity by supporting Ca2+ handling, and extracellularly (eHSP70) activates immune signaling. Disruption of eHSP70/iHSP70 balance has been implicated in T1D-associated VSMC dysfunction. Curcumin, a phytochemical found in turmeric, is an emerging therapeutic adjuvant for treating a wide range of pathologies, including diabetes. However, whether curcumin modulates Ca2+ dynamics and HSP70 expression, thereby improving VSMC function, in diabetic aorta remains unclear. To investigate this, Streptozotocin-induced diabetic rats (i.p. 65 mg/kg) were treated with curcumin (300 mg/kg) for 28 days. Vascular function was evaluated using wire myography to assess changes in biphasic contraction curve and Ca2+ dynamics, while HSP70 was quantified using Western blotting and ELISA. Structural alterations were analyzed by assessing collagen and elastin using Picrosirius staining and fluorescence microscopy. Chronic curcumin treatment improved vascular function by normalizing Ca2+ mishandling, restoring the eHSP70/iHSP70 ratio, reducing hypercontractility, and mitigating arterial structural alterations. These findings indicate that curcumin could potentially ameliorate diabetes-related VSMC dysfunction by restoring Ca2+ homeostasis and modulating HSP70.

Chronic kidney disease (CKD) is associated with profound vascular remodeling, which accelerates the progression of cardiovascular disease. This remodeling is characterized by intimal hyperplasia, accelerated atherosclerosis, excessive vascular calcification, and vascular stiffness. Vascular smooth muscle cell (VSMC) dysfunction has a key role in the remodeling process. Under uremic conditions, VSMCs can switch from a contractile phenotype to a synthetic phenotype, and undergo abnormal proliferation, migration, senescence, apoptosis, and calcification. A growing body of data from experiments in vitro and animal models suggests that uremic toxins (such as inorganic phosphate, indoxyl sulfate and advanced-glycation end products) may directly impact the VSMCs’ physiological functions. Chronic, low-grade inflammation and oxidative stress—hallmarks of CKD—are also strong inducers of VSMC dysfunction. Here, we review current knowledge about the impact of uremic toxins on VSMC function in CKD, and the consequences for pathological vascular remodeling.

Individuals with diabetes are more susceptible to cerebral vascular aging. However, the underlying mechanisms are not well elucidated. The present study examined whether the myogenic response of the middle cerebral artery (MCA) is impaired in diabetic rats due to high glucose (HG)-induced cerebral vascular smooth muscle cell (CVSMC) dysfunction, and whether this is associated with ATP depletion and changes in mitochondrial dynamics and membrane potential. The diameters of the MCA of diabetic rats increased to 135.3 ± 11.3% when perfusion pressure was increased from 40 to 180mmHg, while it fell to 85.1 ± 3.1% in non-diabetic controls. The production of ROS and mitochondrial-derived superoxide were enhanced in cerebral arteries of diabetic rats. Levels of mitochondrial superoxide were significantly elevated in HG-treated primary CVSMCs, which was associated with decreased ATP production, mitochondrial respiration, and membrane potential. The expression of OPA1 was reduced, and MFF was elevated in HG-treated CVSMCs in association with fragmented mitochondria. Moreover, HG-treated CVSMCs displayed lower contractile and proliferation capabilities. These results demonstrate that imbalanced mitochondrial dynamics (increased fission and decreased fusion) and membrane depolarization contribute to ATP depletion in HG-treated CVSMCs, which promotes CVSMC dysfunction and may play an essential role in exacerbating the impaired myogenic response in the cerebral circulation in diabetes and accelerating vascular aging.