Levels In Smooth Muscle Cells Research Articles

Ameliorative Effect of Coenzyme Q10 on Phenotypic Transformation in Human Smooth Muscle Cells with FBN1 Knockdown.

Mutations of the FBN1 gene lead to Marfan syndrome (MFS), which is an autosomal dominant connective tissue disorder featured by thoracic aortic aneurysm risk. There is currently no effective treatment for MFS. Here, we studied the role of mitochondrial dysfunction in the phenotypic transformation of human smooth muscle cells (SMCs) and whether a mitochondrial boosting strategy can be a potential treatment. We knocked down FBN1 in SMCs to create an MFS cell model and used rotenone to induce mitochondrial dysfunction. Furthermore, we incubated the shFBN1 SMCs with Coenzyme Q10 (CoQ10) to assess whether restoring mitochondrial function can reverse the phenotypic transformation. The results showed that shFBN1 SMCs had decreased TFAM (mitochondrial transcription factor A), mtDNA levels and mitochondrial mass, lost their contractile capacity and had increased synthetic phenotype markers. Inhibiting the mitochondrial function of SMCs can decrease the expression of contractile markers and increase the expression of synthetic genes. Imposing mitochondrial stress causes a double-hit effect on the TFAM level, oxidative phosphorylation and phenotypic transformation of FBN1-knockdown SMCs while restoring mitochondrial metabolism with CoQ10 can rapidly reverse the synthetic phenotype. Our results suggest that mitochondria function is a potential therapeutic target for the phenotypic transformation of SMCs in MFS.

Circulating Extracellular Vesicles in Subarachnoid Hemorrhage Patients: Characterization and Cellular Effects.

Circulating extracellular vesicles (EVs) may play a pathophysiological role in the onset of complications of subarachnoid hemorrhage (SAH), potentially contributing to the development of vasospasm (VP). In this study, we aimed to characterize circulating EVs in SAH patients and examine their effects on endothelial and smooth muscle cells (SMCs). In a total of 18 SAH patients, 10 with VP (VP), 8 without VP (NVP), and 5 healthy controls (HC), clinical variables were recorded at different time points. EVs isolated from plasma samples were characterized and used to stimulate human vascular endothelial cells (HUVECs) and SMCs. We found that EVs from SAH patients expressed markers of T-lymphocytes and platelets and had a larger size and a higher concentration compared to those from HC. Moreover, EVs from VP patients reduced cell viability and mitochondrial membrane potential in HUVECs and increased oxidants and nitric oxide (NO) release. Furthermore, EVs from SAH patients increased intracellular calcium levels in SMCs. Altogether, our findings reveal an altered pattern of circulating EVs in SAH patients, suggesting their pathogenic role in promoting endothelial damage and enhancing smooth muscle reactivity. These results have significant implications for the use of EVs as potential diagnostic/prognostic markers and therapeutic tools in SAH management.

PDGFRα+ Interstitial Cells are Effector Cells of PACAP Signaling in Mouse and Human Colon.

Background & AimsPlatelet-derived growth factor receptor α (PDGFRα)-positive interstitial cells (PIC) are interposed between enteric nerve fibers and smooth muscle cells (SMCs) in the tunica muscularis of the gastrointestinal tract. PIC have robust expression of small conductance Ca2+ activated K+ channels 3 (SK3 channels) and transduce inhibitory inputs from purinergic and sympathetic nerves in mouse and human colon. We investigated whether PIC also express pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, PAC1 (PAC1R), and are involved in mediating inhibitory regulation of colonic contractions by PACAP in mouse and human colons.MethodsGene expression analysis, Ca2+ imaging, and contractile experiments were performed on mouse colonic muscles. Ca2+ imaging, intracellular electrical recordings, and contractile experiments were performed on human colonic muscles.ResultsAdcyap1r1 (encoding PAC1R) is highly expressed in mouse PIC. Interstitial cells of Cajal (ICC) and SMCs expressed far lower levels of Adcyap1r. Vipr1 and Vipr2 were expressed at low levels in PIC, ICC, and SMCs. PACAP elicited Ca2+ transients in mouse PIC and inhibited spontaneous phasic contractions via SK channels. In human colonic muscles, PAC1R agonists elicited Ca2+ transients in PIC, hyperpolarized SMCs through SK channels and inhibited spontaneous phasic contractions.ConclusionsPIC of mouse and human colon utilize PAC1R-SK channel signal pathway to inhibit colonic contractions in response to PACAP. Effects of PACAP are in addition to the previously described purinergic and sympathetic inputs to PIC. Thus, PIC integrate inhibitory inputs from at least 3 neurotransmitters and utilize several types of receptors to activate SK channels and regulate colonic contractile behaviors.

Identification of a common polymorphism in COQ8B acting as a modifier of thoracic aortic aneurysm severity.

SummaryThoracic aortic aneurysm (TAA) predisposes to sudden, life-threatening aortic dissection. The factors that regulate interindividual variability in TAA severity are not well understood. Identifying a molecular basis for this variability has the potential to improve clinical risk stratification and advance mechanistic insight. We previously identified COQ8B, a gene important for biosynthesis of coenzyme Q, as a candidate genetic modifier of TAA severity. Here, we investigated the physiological role of COQ8B in human aortic smooth muscle cells (SMCs) and further tested its genetic association with TAA severity. We find COQ8B protein localizes to mitochondria in SMCs, and loss of mitochondrial COQ8B leads to increased oxidative stress, decreased mitochondrial respiration, and altered expression of SMC contractile genes. Oxidative stress and mitochondrial cristae defects were prevalent in the medial layer of human proximal aortic tissues in individuals with TAA, and COQ8B expression was decreased in TAA SMCs compared with controls. A common single nucleotide polymorphism (SNP) rs3865452 in COQ8B (c.521A>G [p.His174Arg]) was associated with decreased rate of aortic root dilation in young individuals with TAA. In addition, the SNP was less frequent in a second cohort of early-onset thoracic aortic dissection (TAD) cases compared with controls. COQ8B protein levels in aortic SMCs were increased in TAA individuals homozygous for rs3865452 compared with those homozygous for the reference allele. Thus, COQ8B is important for aortic SMC metabolism, which is dysregulated in TAA, and rs3865452 may decrease TAA severity by increasing the COQ8B level. Genotyping rs3865452 may be useful for clinical risk stratification and tailored aortopathy management.

MicroRNA expression profile in Smooth Muscle Cells isolated from thoracic aortic aneurysm samples

PurposeThoracic aortic aneurysm (TAA) is a cardiovascular disease characterized by increased aortic diameter, treated with surgery and endovascular therapy in order to avoid aortic dissection or rupture. The mechanism of TAA formation has not been thoroughly studied and many factors have been proposed to drive its progression; however strong focus is attributed to modification of smooth muscle cells (SMCs). Latest research indicates, that microRNAs (miRNAs) may play a significant role in TAA development – these are multifunctional molecules consisting of 19–24 nucleotides involved in regulation of the gene expression level related to many biological processes, i.e. cardiovascular disease pathophysiology, immunity or inflammation. Materials and methodsPrimary SMCs were isolated from aortic scraps of TAA patients and age- and sex-matched healthy controls. Purity of isolated SMCs was determined by flow cytometry using specific markers: α-SMA, CALP, MHC and VIM. Real-time polymerase chain reaction (RT-PCR) was conducted for miRNA analysis. ResultsWe established an isolation protocol and investigated the miRNA expression level in SMCs isolated from aneurysmal and non-aneurysmal aortic samples. We identified that let-7 g (0.71-fold, p = 0.01), miR-130a (0.40-fold, p = 0.04), and miR-221 (0.49-fold, p = 0.05) significantly differed between TAA patients and healthy controls. ConclusionsFurther studies are required to improve our understanding of the pathophysiology underlying TAA, which may aid the development of novel, targeted therapies. The pivotal role of miRNAs in the cardiovascular system provides a new perspective on the pathophysiology of thoracic aortic aneurysms.

MiR-34a Promotes Vascular Smooth Muscle Cell Calcification by Downregulating SIRT1 (Sirtuin 1) and Axl (AXL Receptor Tyrosine Kinase).

Objective- Vascular calcification (VC) is age dependent and a risk factor for cardiovascular and all-cause mortality. VC involves the senescence-induced transdifferentiation of vascular smooth muscle cells (SMCs) toward an osteochondrogenic lineage resulting in arterial wall mineralization. miR-34a increases with age in aortas and induces vascular SMC senescence through the modulation of its target SIRT1 (sirtuin 1). In this study, we aimed to investigate whether miR-34a regulates VC. Approach and Results- We found that miR-34a and Runx2 (Runt-related transcription factor 2) expression correlates in young and old mice. Mir34a+/+ and Mir34a-/- mice were treated with vitamin D, and calcium quantification revealed that Mir34a deficiency reduces soft tissue and aorta medial calcification and the upregulation of the VC Sox9 (SRY [sex-determining region Y]-box 9) and Runx2 and the senescence p16 and p21 markers. In this model, miR-34a upregulation was transient and preceded aorta mineralization. Mir34a-/- SMCs were less prone to undergo senescence and under osteogenic conditions deposited less calcium compared with Mir34a+/+ cells. Furthermore, unlike in Mir34a+/+ SMC, the known VC inhibitors SIRT1 and Axl (AXL receptor tyrosine kinase) were only partially downregulated in calcifying Mir34a-/- SMC. Strikingly, constitutive miR-34a overexpression to senescence-like levels in human aortic SMCs increased calcium deposition and enhanced Axl and SIRT1 decrease during calcification. Notably, we also showed that miR-34a directly decreased Axl expression in human aortic SMC, and restoration of its levels partially rescued miR-34a-dependent growth arrest. Conclusions- miR-34a promotes VC via vascular SMC mineralization by inhibiting cell proliferation and inducing senescence through direct Axl and SIRT1 downregulation, respectively. This miRNA could be a good therapeutic target for the treatment of VC.

Beta-3 Adrenoceptor Signaling Pathways in Urothelial and Smooth Muscle Cells in the Presence of Succinate.

Succinate, an intermediate metabolite of the Krebs cycle, can alter the metabolomics response to certain drugs and controls an array of molecular responses in the urothelium through activation of its receptor, G-protein coupled receptor 91 (GPR91). Mirabegron, a β3-adrenergic receptor (β3-AR) agonist used to treat overactive bladder syndrome (OAB), increases intracellular cAMP in the detrusor smooth muscle cells (SMC), leading to relaxation. We have previously shown that succinate inhibits forskolin-stimulated cAMP production in urothelium. To determine whether succinate interferes with mirabegron-mediated bladder relaxation, we examined their individual and synergistic effect in urothelial-cell and SMC signaling. We first confirmed β3-AR involvement in the mirabegron response by quantifying receptor abundance by immunoblotting in cultured urothelial cells and SMC and cellular localization by immunohistochemistry in rat bladder tissue. Mirabegron increased cAMP levels in SMC but not in urothelial cells, an increase that was inhibited by succinate, suggesting that it impairs cAMP-mediated bladder relaxation by mirabegron. Succinate and mirabegron increased inducible nitric oxide synthesis and nitric oxide secretion only in urothelial cells, suggesting that its release can indirectly induces SMC relaxation. Succinate exposure decreased the expression of β3-AR protein in whole bladder in vivo and in SMC in vitro, indicating that this metabolite may lead to impaired pharmacodynamics of the bladder. Together, our results demonstrate that increased levels of succinate in settings of metabolic stress (e.g., the metabolic syndrome) may lead to impaired mirabegron and β3-AR interaction, inhibition of cAMP production, and ultimately requiring mirabegron dose adjustment for its treatment of OAB related to these conditions.

Integrin beta3 regulates clonality and fate of smooth muscle-derived atherosclerotic plaque cells

Smooth muscle cells (SMCs) play a key role in atherogenesis. However, mechanisms regulating expansion and fate of pre-existing SMCs in atherosclerotic plaques remain poorly defined. Here we show that multiple SMC progenitors mix to form the aorta during development. In contrast, during atherogenesis, a single SMC gives rise to the smooth muscle-derived cells that initially coat the cap of atherosclerotic plaques. Subsequently, highly proliferative cap cells invade the plaque core, comprising the majority of plaque cells. Reduction of integrin β3 (Itgb3) levels in SMCs induces toll-like receptor 4 expression and thereby enhances Cd36 levels and cholesterol-induced transdifferentiation to a macrophage-like phenotype. Global Itgb3 deletion or transplantation of Itgb3(−/−) bone marrow results in recruitment of multiple pre-existing SMCs into plaques. Conditioned medium from Itgb3-silenced macrophages enhances SMC proliferation and migration. Together, our results suggest SMC contribution to atherogenesis is regulated by integrin β3-mediated pathways in both SMCs and bone marrow-derived cells.

Simvastatin Treatment Upregulates HO-1 in Patients with Abdominal Aortic Aneurysm but Independently of Nrf2.

Heme oxygenase-1 (HO-1), encoded by HMOX1 gene and regulated by Nrf2 transcription factor, is a cytoprotective enzyme. Its deficiency may exacerbate abdominal aortic aneurysm (AAA) development, which is also often associated with hyperlipidemia. Beneficial effects of statins, the broadly used antilipidemic drugs, were attributed to modulation of Nrf2/HO-1 axis. However, the effect of statins on Nrf2/HO-1 pathway in patients with AAA has not been studied yet. We analyzed AAA tissue from patients treated with simvastatin (N = 28) or without statins (N = 14). Simvastatin treatment increased HO-1 protein level in AAA, both in endothelial cells (ECs) and in smooth muscle cells (SMCs), but increased Nrf2 localization was restricted only to vasa vasorum. Nrf2 target genes HMOX1, NQO1, and GCLM expression remained unchanged in AAA. In vitro studies showed that simvastatin raises HO-1 protein level slightly in ECs and to much higher extent in SMCs, which is not related to Nrf2/ARE activation, although HMOX1 expression is upregulated by simvastatin in both cell types. In conclusion, simvastatin-induced modulation of HO-1 level in ECs and SMCs in vitro is not related to Nrf2/ARE activity. Likewise, divergent HO-1 and Nrf2 localization together with stable expression of Nrf2 target genes, including HMOX1, in AAA tissue denotes Nrf2 independency.

Caveolin 2: a facultative marker of unfavourable prognosis in long-term patency rate of internal thoracic artery grafts used in coronary artery bypass grafting. Preliminary report.

Intimal hyperplasia leading to graft failure in patients undergoing coronary artery bypass grafting (CABG) is related to vascular smooth muscle cells (SMCs) proliferation. SMCs respond to a variety of mediators, the most important of which is platelet-derived growth factor (PDGF). The platelet-derived growth factor-induced cellular response has been shown to be mediated by caveolins. The aim of this study was to analyze CAV1-3 expression in internal thoracic artery (ITA) grafts used in CABG and correlate their expression with graft occlusion. Six hundred patients undergoing CABG with the use of ITA grafts between 2008 and 2014 were enrolled into this prospective study. CAV1-3 expression in the ITA grafts was analyzed prior to graft transplantation into the coronary circulation via immunohistochemistry. Estimated caveolins expression pattern was then correlated with the occurrence of ITA graft failure observed within 24-months of surgery. Thirty-four patients developed ITA graft failure (subgroup A) and 566 study participants presented no adverse events (subgroup B). CAV1 and CAV3 expression levels in SMCs of the tunica media of the ITA grafts did not differ between the study subgroups and were not associated with the risk of graft failure. CAV2 was expressed within SMCs of the ITA grafts in 94.1% of the patients from subgroup A and 2.5% from subgroup B, and its expression was associated with ITA graft occlusion observed within 24-months after CABG. CAV2 expression in SMCs of the tunica media in autologous ITA transplants might indicate the risk of graft failure.

Oleic acid activates MMPs up-regulation through SIRT1/PPAR-γ inhibition: a probable linkage between obesity and coronary arterial disease.

Obesity is positively related to the growing prevalence of coronary arterial disease (CAD). It is well established in terms of the plasma concentrations of free fatty acid (FFA) that are up-regulated in cases associating with obesity. Oleic acid (OA) is known as the most abundant monounsaturated fatty acid in the human circulatory system. Several pro-atherosclerotic responses of OA have been established. Sirtuin 1 (SIRT1) acts as a key role in regulating the normal physical function in smooth muscle cells (SMCs). SIRT1 activation is developed as a novel approach to delay the progression of atherosclerotic injuries. However, the mechanism is still unclear as to whether OA affects SIRT1 expression and its activity in SMCs. We confirmed that OA treatment represses SIRT1 and peroxisome proliferator-activated receptors-γ levels in SMCs. Moreover, OA enhances by transforming the growth factor-β1 (TGF-β1) release via activation of NF-κB. OA causes NO production by inducing the inducible nitric oxide synthase overexpression, thereby promoting the secretions of matrix metalloproteinases-1 (MMP-1) and MMP-3. Overall, we suggested that OA enhances MMPs activation through SIRT1 down-regulation. Therefore, our findings might provide a novel route for developing new therapeutic treatments for FFAs-related CADs.

Free fatty acid activates MMP‐1 and MMP‐3 through PPAR‐γ mitigation

Obesity is positively related with the prevalence of coronary arterial disease (CAD). It is well known about the plasma concentrations of free fatty acid (FFA) are up‐regulated in cases with obesity. Oleic acid (OA) is known as the most abundant monounsaturated fatty acid in human circulatory system. Several pro‐atherosclerotic responses of OA had been established. Sirtuin 1 (SIRT1) acts a key role in regulation of the normal physical function in smooth muscle cells (SMCs). SIRT1 activation is thought as a novel approach to delay the progression of atherosclerotic injuries. However, the mechanism is still unclear as to whether OA affects SIRT1 expression and activity in SMCs. This study was designed to validate the hypothesis that OA treatment mitigates the expression level of SITR1 and its down‐stream genes, which is a negative regulator of NF‐κB pathway. We confirmed that OA treatment represses SIRT1 and PPAR‐γ levels in SMCs. Moreover, OA enhances transforming growth factor‐β1 (TGF‐b1) release by activation of NF‐κB. OA causes NO production by inducible nitric oxide synthase (iNOS) overexpression, thereby promoting the secretion of matrix metalloproteinases‐1 (MMP‐1) and matrix metalloproteinases‐3 (MMP‐3). Taken together, we suggested OA enhances MMPs activation by SIRT1 down‐regulation. MMPs activation had been shown to positively link with the atherosclerotic plaque generation, therefore, our findings might provide a novel route for developing new therapeutic treatments for FFAs‐related CAD.Support or Funding InformationMOST‐103‐2320‐B‐006‐050

Distribution of Matrix Metalloproteinases in Human Atherosclerotic Carotid Plaques and Their Production by Smooth Muscle Cells and Macrophage Subsets.

PurposeIn this study, the potential of matrix metalloproteinase (MMP) sense for detection of atherosclerotic plaque instability was explored. Secondly, expression of MMPs by macrophage subtypes and smooth muscle cells (SMCs) was investigated.ProceduresTwenty-three consecutive plaques removed during carotid endarterectomy were incubated in MMPSense™ 680 and imaged with IVIS® Spectrum. mRNA levels of MMPs, macrophage markers, and SMCs were determined in plaque specimens, and in in vitro differentiated M1 and M2 macrophages.ResultsThere was a significant difference between autofluorescence signals and MMPSense signals, both on the intraluminal and extraluminal sides of plaques. MMP-9 and CD68 messenger RNA (mRNA) expression was higher in hot spots, whereas MMP-2 and αSMA expression was higher in cold spots. In vitro M2 macrophages had higher mRNA expression of MMP-1, MMP-9, MMP-12, and TIMP-1 compared to M1 macrophages.ConclusionMMP-9 is most dominantly MMP present in atherosclerotic plaques and is produced by M2 rather than M1 macrophages.

Vascular Smooth Muscle as a Target for Novel Therapeutics.

Cardiovascular disease is the principal cause of death in patients with type 2 diabetes (T2DM). Exposure of the vasculature to metabolic disturbances leaves a persistent imprint on vascular walls, and specifically on smooth muscle cells (SMC) that favours their dysfunction and potentially underlies macrovascular complications of T2DM. Current diabetes therapies and continued development of newer treatments has led to the ability to achieve more efficient glycaemic control. There is also some evidence to suggest that some of these treatments may exert favourable pleiotropic effects, some of which may be at the level of SMC. However, emerging interest in epigenetic markers as determinants of vascular disease, and a putative link with diabetes, opens the possibility for new avenues to develop robust and specific new therapies. These will likely need to target cell-specific epigenetic changes such as effectors of DNA histone modifications that promote or inhibit gene transcription, and/or microRNAs capable of regulating entire cellular pathways through target gene repression. The growing epidemic of T2DM worldwide, and its attendant cardiovascular mortality, dictates a need for novel therapies and personalised approaches to ameliorate vascular complications in this vulnerable population.

Effects of Myoendothelial Interactions on Arteriolar Myogenic and Agonist‐mediated Vascular Control

Local regulation of blood flow to tissue is governed by contraction and relaxation of circumferential vascular smooth muscle in resistance vessels. Interaction between endothelial cells (EC) and smooth muscle cells (SMC) play a critical role in regulating vascular tone. Although ion flux, IP3 and nitric oxide (NO) transport between cells are known to be involved in regulation of SMC activity, little is known about their relative contributions to myogenic and agonist-mediated vascular tone. To quantify their contributions we have assembled an isolated vessel model of a resistance artery by integrating models of vessel wall mechanics, smooth muscle force generation, and SMC and EC Ca2+ dynamics and electrophysiology. Steady-state pressure-diameter and agonist response data from pressure and isovolumic myography studies are analyzed to determine contributions of ionic, IP3 and NO transport between ECs and SMCs on isolated vessel tone. The model predicts levels of SMC and EC Ca2+ at a given vascular tone and can also show how the presence/absence of endothelial layers influences pressure/agonist-induced contraction response, under isobaric and isovolumic conditions. In order to replicate experimentally observed vasoconstriction response to the smooth muscle agonist phenylephrine, the model analysis shows there is a limit to ionic and IP3 coupling allowed between cells. Below these limits, the NO-cGMP pathway has a minimal effect on vasoconstriction response to pressure but yet still has a significant effect on endothelial agonist-induced vasodilatory response. Supported by NIH U01 HL118738, NSF CBET 1133260 and NIH P50-GM094503.

Toll like receptor-2 regulates production of glial-derived neurotrophic factors in murine intestinal smooth muscle cells.

Gut microbiota-innate immunity axis is emerging as a key player to guarantee the structural and functional integrity of the enteric nervous system (ENS). Alterations in the composition of the gut microbiota, derangement in signaling of innate immune receptors such as Toll-like receptors (TLRs), and modifications in the neurochemical coding of the ENS have been associated with a variety of gastrointestinal disorders. Indeed, TLR2 activation by microbial products controls the ENS structure and regulates intestinal neuromuscular function. However, the cellular populations and the molecular mechanisms shaping the plasticity of enteric neurons in response to gut microbes are largely unexplored. In this study, smooth muscle cells (SMCs), enteric glial cells (EGCs) and macrophages/dendritic cells (MΦ/DCs) were isolated and cultured from the ileal longitudinal muscle layer of wild-type (WT) and Toll-like receptor-2 deficient (TLR2(-/-)) mice. Quantification of mRNA levels of neurotrophins at baseline and following stimulation with TLR ligands was performed by RT-PCR. To determine the role of neurotrophins in supporting the neuronal phenotype, we performed co-culture experiments of enteric neurons with the conditioned media of cells isolated from the longitudinal muscle layer of WT or TLR2(-/-) mice. The neuronal phenotype was investigated evaluating the expression of βIII-tubulin, HuC/D, and nNOS by immunocytochemistry. As detected by semi-quantitative RT-PCR, SMCs expressed mRNA coding TLR1-9. Among the tested cell populations, un-stimulated SMCs were the most prominent sources of neurotrophins. Stimulation with TLR2, TLR4, TLR5 and TLR9 ligands further increased Gdnf, Ngf, Bdnf and Lif mRNA levels in SMCs. Enteric neurons isolated from TLR2(-/-) mice exhibited smaller ganglia, fewer HuC/D(+ve) and nNOS(+ve) neurons and shorter βIII-tubulin axonal networks as compared to neurons cultured from WT mice. The co-culture with the conditioned media from WT-SMCs but not with those from WT-EGCs or WT-MΦ/DCs corrected the altered neuronal phenotype of TLR2(-/-) mice. Supplementation of TLR2(-/-) neuronal cultures with GDNF recapitulated the WT-SMC co-culture effect whereas the knockdown of GDNF expression in WT-SMCs using shRNA interference abolished the effect on TLR2(-/-) neurons. These data revealed that by exploiting the repertoire of TLRs to decode gut-microbial signals, intestinal SMCs elaborate a cocktail of neurotrophic factors that in turn supports neuronal phenotype. In this view, the SMCs represent an attractive target for novel therapeutic strategies.

Smooth muscle cells of penis in the rat: noninvasive quantification with shear wave elastography.

Purpose. Smooth muscle cells (SMCs) of cavernosum play an important role in erection. It is of great significance to quantitatively analyze the level of SMCs in penis. In this study, we investigated the feasibility of shear wave elastography (SWE) on evaluating the level of SMCs in penis quantitatively. Materials and Methods. Twenty healthy male rats were selected. The SWE imaging of penis was carried out and then immunohistochemistry analysis of penis was performed to analyze the expression of alpha smooth muscle actin in penis. The measurement index of SWE examination was tissue stiffness (TS). The measurement index of immunohistochemistry analysis was positive area percentage of alpha smooth muscle actin (AP). Results. Sixty sets of data of TS and AP were obtained. The results showed that TS was significantly correlated with AP and the correlation coefficient was −0.618 (p < 0.001). The result of TS had been plotted against the AP measurements. The relation between the two results has been fitted with quadric curve; the goodness-of-fit index was 0.364 (p < 0.001). Conclusions. The level of SMCs in penis was successfully quantified in vivo with SWE. SWE can be used clinically for evaluating the level of SMCs in penis quantitatively.

A20 Regulates Atherogenic Interferon (IFN)-γ Signaling in Vascular Cells by Modulating Basal IFNβ Levels

IFNγ signaling in endothelial (EC) and smooth muscle cells (SMC) is a key culprit of pathologic vascular remodeling. The impact of NF-κB inhibitory protein A20 on IFNγ signaling in vascular cells remains unknown. In gain- and loss-of-function studies, A20 inversely regulated expression of IFNγ-induced atherogenic genes in human EC and SMC by modulating STAT1 transcription. In vivo, inadequate A20 expression in A20 heterozygote mice aggravated intimal hyperplasia following partial carotid artery ligation. This outcome uniquely associated with increased levels of Stat1 and super-induction of Ifnγ-dependent genes. Transcriptome analysis of the aortic media from A20 heterozygote versus wild-type mice revealed increased basal Ifnβ signaling as the likely cause for higher Stat1 transcription. We confirmed higher basal IFNβ levels in A20-silenced human SMC and showed that neutralization or knockdown of IFNβ abrogates heightened STAT1 levels in these cells. Upstream of IFNβ, A20-silenced EC and SMC demonstrated higher levels of phosphorylated/activated TANK-binding kinase-1 (TBK1), a regulator of IFNβ transcription. This suggested that A20 knockdown increased STAT1 transcription by enhancing TBK1 activation and subsequently basal IFNβ levels. Altogether, these results uncover A20 as a key physiologic regulator of atherogenic IFNγ/STAT1 signaling. This novel function of A20 added to its ability to inhibit nuclear factor-κB (NF-κB) activation solidifies its promise as an ideal therapeutic candidate for treatment and prevention of vascular diseases. In light of recently discovered A20/TNFAIP3 (TNFα-induced protein 3) single nucleotide polymorphisms that impart lower A20 expression or function, these results also qualify A20 as a reliable clinical biomarker for vascular risk assessment.

Impact of tetrodotoxin application and lidocaine supplementation on equine jejunal smooth muscle contractility and activity of the enteric nervous system in vitro

By blocking the enteric nervous system (ENS) using tetrodotoxin (TTX), previous studies have documented the contractility-enhancing (CE) effects of lidocaine in equine intestinal smooth muscle (SM) at the level of SM cells and/or interstitial cells of Cajal (ICC). The present study examined the impact of ENS deactivation on CE lidocaine effects, and investigated the effects of lidocaine on ENS activity. TTX application did not affect the CE effects of lidocaine, indicating that these were not mediated by TTX-sensitive sodium channels. Application of TTX or ≥100 mg/L lidocaine reduced ENS activity. Although such concentrations of lidocaine exceed therapeutic blood concentrations, tissue concentrations may be higher with the potential to reduce ENS activity and impair intestinal motility in vivo. Improved understanding of underlying mechanisms is relevant for therapeutic use of lidocaine in horses with postoperative ileus.

Monocytes and smooth muscle cells cross-talk activates STAT3 and induces resistin and reactive oxygen species and production

During the early phase of atherosclerosis, monocytes attach to and migrate through the vessel wall where they activate and communicate with smooth muscle cells (SMC) affecting plaque progression by largely unknown mechanisms. Activation of STAT3 transcription factor is suggested to be critically involved in dedifferentiation, migration, and proliferation of SMC in the neointima formation after vascular injury. Monocytes-SMC cross-talk induces an inflammatory phenotype of the resident SMC, but the involvement of STAT3 in phenotype switching is not known. Resistin is a cytokine found in human atheroma associated to monocytes/macrophages with role in inflammation associated with cardiovascular disease. The aim of this study was to follow the effect of activated monocytes-SMC cross-talk on STAT3 activation and subsequent resistin and reactive oxygen species (ROS) production. Our results showed that the interaction of activated monocytes with SMC determines: (i) phosphorylation of STAT3 and reduction of SOCS3 expression in both cell types; (ii) intracellular ROS production dependent on NADPH oxidase (by increased Nox1 expression) and STAT3 activation in SMC; (iii) up-regulation of resistin expression in monocytes dependent on STAT3 activation. Furthermore, exposure of SMC to resistin induces ROS by increasing NADPH oxidase activity and the p22phox and Nox1 expression. In conclusion, the cross-talk between SMC and monocytes activates STAT3 transcription factor and lead to resistin up-regulation in monocytes and ROS production in SMC. Moreover, resistin increases the ROS levels in SMC. These data indicate that monocyte-SMC communication may represent an important factor for progression of the atherosclerotic lesion. J. Cell. Biochem. 114: 2273–2283, 2013. © 2013 Wiley Periodicals, Inc.