Effect of superoxide anion on contractility of aortic smooth muscle cells

Celiac plexus neurolysis, although effective in relieving pain associated with upper abdominal malignancy, occasionally results in paraplegia. Diffusion of the neurolytic agent to arteries supplying the spinal cord has been postulated as a cause, and previous studies with isolated lumbar segmental arteries have demonstrated contraction in response to ethanol and phenol. The mechanism of this contractile effect is unknown, but a role for insular free calcium (Ca2+i) is suggested by the known involvement of Ca2+i in both smooth muscle vasoconstriction and toxic cell injury. The authors sought to determine whether nontoxic concentrations of ethanol cause a direct elevation of Ca2+i in arterial smooth muscle and endothelium. Primary cultures of human aortic smooth muscle and endothelial cells were studied to determine the direct effect of ethanol independent of interactions with agonists or contractile proteins. Ca2+i levels were determined in single cells with digitized video fluorescence microscopy, using ratio imaging of the Ca2+i-sensitive fluorophore fura-2. In aortic smooth muscle cells, initial Ca2+i was 98 +/- 41 nM (n = 59 cells). Histamine (10 microM) as a positive control caused an increase in Ca2+i, as expected. Ethanol alone, at doses of 2-5% (v/v) also caused a sustained elevation in Ca2+i of physiologically significant magnitude. Ethanol at doses of 5% or lower did not cause any visibly apparent injury within 30 minutes. In contrast, 10% or higher ethanol doses quickly caused membrane blebbing, a sign of toxic injury, followed by cell death within 20 minutes. Aortic endothelial cells were more resistant to ethanol than smooth muscle cells, in terms of both Ca2+i elevation and cell death. Ethanol, even at nontoxic concentrations, has a direct effect on aortic smooth muscle Ca2+i, large enough to be associated with significant vasoconstriction. The findings suggest a possible role for pharmacologic agents that preserve Ca2+i homeostasis in protecting against neurolysis-induced paraplegia, although additional study is required before clinical application is appropriate.

Introduction: Spontaneous coronary artery dissection (SCAD) is an increasingly recognized cause of myocardial infarction in young and middle-aged women. A common genetic variant, rs11172113, located in LRP1 (low density lipoprotein receptor-related protein 1) first intron, was associated with several vascular diseases, including SCAD, and regulation of LRP1 expression in arterial tissues. However, the biological mechanisms through which rs11172113 influence LRP1 function in the context of non-atherosclerotic arterial lesions are not known. Hypothesis: We hypothesized that rs11172113 genotype affects LRP1 expression through specific mechanisms in contractile smooth muscle cells (SMCs), leading to alterations of their physiological function. Methods: We differentiated 4 human induced pluripotent stem cells (iPSCs) lines (2 males, 2 females) towards either contractile or synthetic SMCs using a recently optimized 24-day protocol. We performed RNA-Seq and ATAC-Seq at 6 time points during differentiation. We silenced LRP1 using siRNAs in differentiated SMCs and used CRISPR-Cas9 to induce deletions of the enhancer region containing rs11172113 or frame-shift indels in exons 2 or 5 of LRP1 in iPSCs. Results: iPSCs derived SMCs presented the expected morphology and expression of SMC markers. Transcriptomic analyses showed that contractile SMCs harbored SMC properties during late differentiation and resembled SMCs of artery tissue, whereas genes involved in cell stress were upregulated in synthetic SMCs at the end of differentiation. In particular, LRP1 expression and DNA accessibility at rs11172113 were higher in contractile SMCs. The deletion of rs11172113 enhancer region led to a decrease in the expression of LRP1 in both contractile and synthetic SMCs. LRP1 inactivation decreased the proliferation and increased the migration capacity of contractile SMCs. Detailed characterization of the influence of LRP1 on extracellular matrix maintenance and cellular contraction is ongoing. Conclusions: We confirmed rs11172113 to regulate LRP1 expression in iPSCs derived contractile SMCs. Our results support LRP1 influence on SMCs phenotype as a causal factor in the genetic susceptibility to SCAD and related vascular diseases.

We studied stimulus-specific alterations of the excitation-contraction coupling pathway in freshly isolated contractile and subcultured non-contractile vascular smooth muscle cells. Using the calcium indicator aequorin, we detected physiological increases in cytoplasmic free calcium [( Ca2+]i) in subcultured smooth muscle cells subjected to angiotensin or 33 mM potassium depolarization. These increases were qualitatively identical to those previously measured in intact vascular strips. Platelet-derived growth factor (PDGF) induced a slow, sustained [Ca2+]i increase when applied to the subcultured smooth muscle cells at low picomolar concentrations. Freshly isolated, contractile vascular smooth muscle cells, prepared by a novel technique, exhibited a slow shortening of 20% of resting length in response to PDGF. PDGF also markedly potentiated smooth muscle cell shortening in response to an ED50 dose of phenylephrine. This effect was PDGF concentration dependent. The time course of shortening induced by PDGF alone was consistent with the time course of the PDGF-induced [Ca2+]i increase in the cultured smooth muscle cells. These data suggest that agonists which induce [Ca2+]i changes in contractile smooth muscle cells may retain this ability with respect to cultured smooth muscle cells. PDGF, a peptide mitogen for proliferative smooth muscle cells, may also serve to modulate vascular tone by modestly raising [Ca2+]i in contractile smooth muscle cell and, therefore, sensitizing the cells to alpha adrenergic agonists.

Epidemiological studies have demonstrated the importance of cardiovascular diseases in Western countries. Among the cell types associated with a dysfunctional vasculature, smooth muscle (SM) cells are believed to play an essential role in the development of these illnesses. Vascular SM cells are key regulators of the vascular tone and also have an important function in the development of atherosclerosis and restenosis. While in the normal vasculature, contractile SM cells are predominant, in atherosclerotic vascular lesions, synthetic cells migrate toward the neointima, proliferate, and synthetize extracellular matrix proteins. In the present study, we have examined the role of caveolin-3 in the regulation of SM cell phenotype. Caveolin-3 is expressed in vivo in normal arterial SM cells, but its expression appears to be lost in cultured SM cells. Our data show that caveolin-3 expression in the A7r5 SM cell line is associated with increased expression of contractility markers such as SM α-actin, SM myosin heavy chain but decreased expression of the synthetic phenotype markers such as p-Elk and Klf4. Moreover, we also show that caveolin-3 expression can reduce proliferation upon treatment with LDL or PDGF. Finally, we show that caveolin-3-expressing SM cells are less sensitive to apoptosis than control cells upon treatment with oxidized LDL. Taken together, our data suggest that caveolin-3 can regulate the phenotypic switch between contractile and synthetic SM cells. A better understanding of the factors regulating caveolin-3 expression and function in this cell type will permit the development of a better comprehension of the factors regulating SM function in atherosclerosis and restenosis.

Introduction: Apoptogenic protein (Apop) is a mitochondrial protein originally identified in the atherosclerotic cells cultured from thoracic aortas of ApoE-deficient mice. Functional analysis revealed that the expression of Apop using an expression vector induces apoptotic death of smooth muscle cells (SMC) in culture through the release of cytochrome c from mitochondria, followed by the activation of the caspase cascade. Here, we show that Apop is expressed in the contractile SMC, and can be a novel phenotypic marker of the SMC. Methods and Results: Human aortic smooth muscle cells (HASMC) and human coronary atery smooth muscle cells (HCASMC) were cultured in HuMedia-SB2 supplanted with hEGF, hFGF, insulin and 5% FBS. Differentiated HASMC were cultured on laminin-coated dishes in HuMedia-SD2 supplemented with heparin and 1% FBS. Gene expression level was analyzed by real time PCR. SM2 and SMemb were used as differentiation marker and synthetic SMC marker, respectively. The differentiated SMC showed a higher level of expression of SM2, and the ratio of SM2/SMemb was higher compared to the synthetic SMC. Apop gene expression was higher in differentiated HASMC compared to the synthetic phase cells. Similarly, HCASMC showed a higher level of Apop and SM2 expression and SM2/SMemb expression ratio in differentiated cells compared to the cells in synthetic phase. These results indicate that Apop is expressed in differentiated SMC at a higher level than synthetic SMC. CArG box, which is often present in phenotypic marker genes expressed in differentiated SMC, exists in the intron1 region of Apop gene. Serum response factor (SRF) binds to CArG box, and regulates the expression of the genes expressed in differentiated SMC. The silencing of SRF using siRNA reduced the expression of Apop in differentiaed HASMC, indicating that SRF regulates the expression of Apop in differentiated cells. Moreover, immunostaining using human atherosclerotic coronary artery specimens revealed that Apop gene is co-expressed with the differentiation marker, SM2 in the coronary artery SMC. Conclusion: These results indicate that Apop is a novel phenotypic marker of the differentiated SMC, and may be a novel therapeutic target for the prevention of the development of atherosclerosis.

Notch and transforming growth factor-beta (TGFbeta) play pivotal roles during vascular development and the pathogenesis of vascular disease. The interaction of these two pathways is not fully understood. The present study utilized primary human smooth muscle cells (SMC) to examine molecular cross-talk between TGFbeta1 and Notch signaling on contractile gene expression. Activation of Notch signaling using Notch intracellular domain or Jagged1 ligand induced smooth muscle alpha-actin (SM actin), smooth muscle myosin heavy chain, and calponin1, and the expression of Notch downstream effectors hairy-related transcription factors. Similarly, TGFbeta1 treatment of human aortic smooth muscle cells induced SM actin, calponin1, and smooth muscle protein 22-alpha (SM22alpha) in a dose- and time-dependent manner. Hairy-related transcription factor proteins, which antagonize Notch activity, also suppressed the TGFbeta1-induced increase in SMC markers, suggesting a general mechanism of inhibition. We found that Notch and TGFbeta1 cooperatively activate SMC marker transcripts and protein through parallel signaling axes. Although the intracellular domain of Notch4 interacted with phosphoSmad2/3 in SMC, this interaction was not observed with Notch1 or Notch2. However, we found that CBF1 co-immunoprecipitated with phosphoSmad2/3, suggesting a mechanism to link canonical Notch signaling to phosphoSmad activity. Indeed, the combination of Notch activation and TGFbeta1 treatment led to synergistic activation of a TGFbeta-responsive promoter. This increase corresponded to increased levels of phosphoSmad2/3 interaction at Smad consensus binding sites within the SM actin, calponin1, and SM22alpha promoters. Thus, Notch and TGFbeta coordinately induce a molecular and functional contractile phenotype by co-regulation of Smad activity at SMC promoters.

Applications involving freeze-thaw in arteries such as cryoplasty and cryopreservation alter the arterial biomechanics significantly [1]. Tissue dehydration or bulk water loss is observed following freeze-thaw in native arteries as well as other artificial tissues [1, 2]. It is hypothesized that tissue dehydration observed during freeze-thaw is an important mechanism underlying the biomechanical changes in arteries. In order to test this hypothesis, dehydration was induced in arteries (without changing temperature or phase) by treating them with different concentrations of hyperosmotic mannitol solutions. Changes to smooth muscle cell (SMC) contractility, collagen matrix structure and overall artery biomechanics were studied following tissue dehydration. SMC contractility and relaxation were measured by studying the response of arteries to norepinephrine (NE) and acetylcholine (AC) respectively. Collagen matrix structure was assessed by studying the thermal denaturation of collagen due to heating using Fourier transform infrared (FTIR) spectroscopy and the overall artery biomechanics through uniaxial tensile tests.

BackgroundHere we investigated Brahma-related gene 1 (BRG1) expression in aortic smooth muscle cells (SMCs) and its role in the regulation of the pathological changes in aortic SMCs of thoracic arotic dissection (TAD).MethodsBRG1, matrix metalloproteinase 2 (MMP2), and MMP9 mRNA and protein expression in human aortic specimens were examined by qPCR and western blot, respectively. The percentage of apoptotic and contractile SMCs in aortic specimens were determined by TUNEL assay and α-SMA immunohistochemical staining, respectively. The role of BRG1 in MMP2 and MMP9 expression, cell apoptosis, and phenotype transition in aortic SMCs were investigated using a human aortic SMC line via adenovirus mediated gene transfer. MMPs mRNA and protein levels were analyzed by qPCR and western blot, respectively. The percentage of apoptotic and contractile cells were determined through flow cytometry analysis.ResultsThe expression level of BRG1 in the aortic walls (adventitia-removed) was significantly higher in the TAD than the normal group. BRG1 expression was positively correlated to expression of MMP2 and MMP9 and SMC apoptosis, but was negatively correlated to the percentage of contractile aortic SMCs in TAD specimens. In human aortic SMC line, BRG1 transfection led to significant upregulation of MMP2 and MMP9 expression and a concomitant increase in SMC apoptosis as well as a decrease in the percentage of contractile phenotype of cells.ConclusionsBRG1 is significantly upregulated in the aortic SMCs of TAD, and its overexpression might promote the development of TAD by increasing MMP2 and MMP9 expression, inducing SMC apoptosis and the transition from contractile to synthetic phenotype.

Introduction: Smooth muscle cells (SMCs) capacity to phenotype switching between proliferative and quiescent (contractile) is a widely studied mechanism in cardiovascular disease. Primary SMCs tend to lose many physiological features in culture, which makes the study of their contractile function challenging. Recently, an optimized protocol of induced pluripotent stem cells (iPSCs) differentiation into contractile SMCs was described. Here we aimed at defining the transcriptomic and open chromatin dynamics during the acquisition of SMCs phenotypes. Methods: We differentiated 4 human iPSC lines (2 males, 2 females) towards either contractile (Repsox induced) or synthetic (PDGF-BB/TGF-β induced) SMC phenotypes using a 24-days protocol. We performed RNA-Seq and assay for transposase accessible chromatin (ATAC)-Seq at 5 time points of differentiation. We analyzed gene expression profiles and compared them to existing dataset of human aorta by principle component analyses (PCA) and gene set enrichment analyses using GO terms. Results: iPSCs derived SMCs showed expected morphology and positive expression of SMC markers. Synthetic SMCs (SSMCs) exhibited greater capacity of proliferation, migration and lower calcium release capacity, compared to contractile SMCs (CSMCs). RNA-Seq results showed that multiple genes involved in the contractile function of arteries, including myosin light chain kinase (MYLK) and angiotensin type 1 receptor ( AGTR1 ) genes were highly expressed in CSMCs compared to SSMCs. Overall, CSMCs conserved SMC properties beyond 24 days and their gene expression profile clustered near human aorta. During late differentiation stages, CSMCs showed an upregulation of genes involved in cardiovascular system development, whereas genes involved in cell stress were upregulated in SSMCs. Conclusions: We describe global genomic profiles of iPSCs derived CSMCs that presented comparable gene expression profiles to mature artery tissue. Combination with upcoming DNA accessibility maps is expected to allow the functional exploration of genetic risk variation involved in several arterial diseases involving the impairment of the SMCs contractile function.

Airway smooth muscle: A modulator of airway remodeling in asthma

Myocardin, a cofactor of serum response factor (SRF), specifically induces the expression of contractile proteins to promote differentiation and contractile phenotype of smooth muscle cells (SMCs). SRF directly induces the transcription of microRNA-1 (miR-1) in cardiac and skeletal muscle precursor cells and miR-1 promotes the skeletal muscle differentiation and modulates cardiac hypertrophy. We aimed to examine whether miR-1 plays a role in the regulation of smooth muscle contractility. We found that miR-1 expression was induced by myocardin overexpression in human aortic SMCs. In a collagen lattice contraction assay using SMCs harboring a doxycycline-inducible expression system for myocardin, we found that myocardin expression increased the contractility of SMCs, which was significantly inhibited by exogenous miR-1. Our further studies revealed that exogenous miR-1, which did not affect myocardin or SRF expression, suppressed the expression of contractile proteins, such as alpha-SMA and SM22, and impaired the actin cytoskeletal organization. Taken together, our results have revealed that myocardin induces miR-1 expression, which represses the expression of contractile proteins and thereby inhibits the contractility of SMCs. Therefore, our findings suggest a role of miR-1 in the negative feedback loop in the regulation of contractility induced by myocardin.

Objective To investigate the inhibition effect and molecular mechanism of microRNA-124 (miR-124) on rat vascular smooth muscle cell proliferation. Methods We applied 10 male 11-week-old spontaneously hypertensive rats (SHR) as experimental group and 10 age matched Wistar Kyoto (WKY) rats as control group. Aortic smooth muscle cells were isolated from the medial layer of thoracic aorta of SHR and WKY rats and cultured in Dulbecco's modified eagles medium (DMEM). Quantitative real-time PCR (RT-PCR) was used to detect the expression of miR-124 in SHR and WKY aortic smooth muscle cells. The SHR aortic smooth muscle cells were transfected with either miR-124 mimics or NC mimics control. MTT assay was used to explore the proliferation of SHR aortic smooth muscle cells in vitro. The targeted gene of miR-124 was predicted by bioinformatics, and verified by dual luciferase reporter assay. The over-expression of miR-124 in SHR aortic smooth muscle cells was analyzed by the mRNA RT-PCR and protein expression of Meox2 by Western blot analyses. Finally, SHR aortic smooth muscle cells proliferation was also detected using MTT assay after treated with siMeox2. Results The expression of miR-124 in SHR aortic smooth muscle cells was 0.22 times of WKY aortic smooth muscle cells (P<0.01). Over-expression of miR-124 could significantly inhibit SHR aortic smooth muscle cells proliferation. Bioinformatics analysis and dual luciferase reporter assay demonstrated that Meox2 was a target gene of miR-124. The mRNA level of Meox2 in SHR aortic smooth muscle cells after treated with miR-124 mimics was 0.29 times of transfected NC mimics control (P<0.01). Western blot showed that Meox2 protein was also decreased after treated with miR-124 mimics. Knock-down Meox2 could also arrest the SHR aortic smooth muscle cells proliferation. Conclusions The expression of miR-124 is down-regulated in SHR, which may be mediated by Meox2 regulation of SHR aortic smooth muscle cells proliferation and inhibit the occurrence of hypertensive disease. Key words: miR-124; Rats, inbred SHR; Aorta; Myocytes, smooth muscle; Proliferation; Meox2

Deoxynivalenol interferes with intestinal motility via injuring the contractility of enteric smooth muscle cells: A novel hazard to the gastrointestinal tract by environmental toxins

Complete transcriptome profiling of contractile smooth muscle cells (SMCs) differentiated from embryonic stem cells is crucial for the characterization of smooth muscle gene expression signatures and will contribute to defining biological and physiological processes in these cells. We have generated a transgenic embryonic stem cell line expressing both the puromycin acetyl transferase and enhanced green fluorescent protein cassettes under the control of the Acta2 promoter. Applying a specific monolayer culture protocol using retinoic acid, a puromycin-resistant and enhanced green fluorescent protein-positive Acta2(+) SMC population of 95% purity was isolated. Acta2(+) SMCs were characterized by semiquantitative and quantitative RT-PCR profiling of SMC markers and by microarray expression profiling, as well as by immunostaining for SMC-specific cytoskeletal proteins. Patch-clamp electrophysiological characterization of these cells identified SMC-specific channels such as the ATP-sensitive potassium channel and the Ca(2+)-activated potassium channel. Culturing of Acta2(+) SMCs in serum-containing medium resulted in a significant number of hypertrophic and binucleated cells failing to complete cell division. Functional characterization of the cells has been proved by stimulation of the cells with vasoactive agents, such as angiotensin II and endothelin. We concluded that our embryonic stem cell-derived SMC population possesses the contractile and hypertrophic phenotype of SMCs incapable of proliferation. This is the first study describing the complete transcriptome of ES-derived SMCs allowing identification of specific biological and physiological processes in the contractile phenotype SMCs and will contribute to the understanding of these processes in early SMCs derived from embryonic stem cells.

Objective To study the influence of high calcium ion environment on the membrane current of denervated aortic smooth muscles in iso-osmotic condition, as well as study the myogenic spontaneous vasomotion of the aortic smooth muscles under different pre-load state and intervention of calcium-channel blockers. Methods Smooth muscle samples were taken from the aortic smooth muscle layer of Kunming mice. The samples were fixed at two-ends in relaxed state and infiltrated in Ringer's solution. After stabilization, a glass microelectrode was adsorbed on the smooth muscle membrane to form gigaohm-seal. The current state of the membrane was observed under the physiological osmotic pressure. Then, the calcium ions concentration in the Ringer's solution was increased from 0.9 mol/L to 1.2 mol/L, and the immediate change of membrane current was observed after changing of osmotic pressure. Finally, the membrane potential change in iso-osmotic condition was observed when the pre-load was increased to 1 g, and the spontaneous vasomotion curves of the samples were recorded. Results In the relaxation state, the membrane current of the smooth muscle was increased significantly, i.e. (10.25±1.34) pA vs. (24.91±3.27) pA (P<0.05), when the calcium ion concentration was increased from 0.9 mol/L to 1.2 mol/L in iso-osmotic condition. When the pre-load was increased, the variation amplitude of the membrane current was increased in iso-osmotic condition, i.e. (10.25±1.34) pA to (15.33±4.33) pA (P<0.05) for the lower calcium ion concentration, and (24.91±3.27) pA to (33.31±7.25) pA (P<0.05) for the higher calcium ion concentration. If only increasing the pre-load, the myogenic spontaneous vasomotion could be increased by 175% for the condition of lower calcium ion concentration (0.9 mol/L). When the calcium ion concentration was increased to 1.2 mol/L, the spontaneous vasomotion frequency of the specimen was increased, and the range of the vasomotion could be further increased by 40%. After pretreatment with the calcium-channel blocker (0.5 g/L nitrendipine), the membrane current and myogenic spontaneous vasomotion were significantly decreased in the higher calcium ion environment, which indicated that the membrane potential and spontaneous vasomotion was dominated by calcium ions. Conclusions The increase of extracellular calcium ion concentration not only can improve the excitability of the aorta smooth muscle cells, but also can cause significant spontaneous vasomotion, and can improve the compliance of the smooth muscle tissue in pre-load variation. The calcium-channel blockers can inhibit calcium-dominated transmembrane currents, reduce myogenic spontaneous vasomotion, and enhance the stiffness of smooth muscle, which may have negative effects on windkessel vessel function. Key words: Denervation; Quick stretching; Overloading; Spontaneous vasomotion; Membrane current