Smooth Muscle Gene Expression Research Articles

Serum Response Factor Expression in Excess Permits a Dual Contractile-Proliferative Phenotype of Airway Smooth Muscle.

The transcription factors (TFs) MyoCD (myocardin) and Elk-1 (ETS Like-1 protein) competitively bind to SRF (serum response factor) and control myogenic- and mitogenic-related gene expression in smooth muscle, respectively. Their functions are therefore mutually inhibitory, which results in a contractile-versus-proliferative phenotype dichotomy. Airway smooth muscle cell (ASMC) phenotype alterations occur in various inflammatory airway diseases, promoting pathological remodeling and contributing to airflow obstruction. We characterized MyoCD and Elk-1 interactions and their roles in phenotype determination in human ASMCs. MyoCD overexpression in ASMCs increased smooth muscle gene expression, force generation, and partially restored the loss of smooth muscle protein associated with prolonged culturing while inhibiting Elk-1 transcriptional activities and proliferation induced by EGF (epidermal growth factor). However, MyoCD overexpression failed to suppress these responses induced by FBS, as FBS also upregulated SRF expression to a degree that allowed unopposed function of both TFs. Inhibition of the RhoA pathway reversed said SRF changes, allowing inhibition of Elk-1 by MyoCD overexpression and suppressing FBS-mediated contractile protein gene upregulation. Our study confirmed that MyoCD in increased abundance can competitively inhibit Elk-1 function. However, SRF upregulation permits a dual contractile-proliferative ASMC phenotype that is anticipated to exacerbate pathological alterations, whereas therapies targeting SRF may inhibit pathological ASMC proliferation and contractile protein gene expression.

A Preliminary Study on Factors That Drive Patient Variability in Human Subcutaneous Adipose Tissues.

Adipose tissue is a dynamic regulatory organ that has profound effects on the overall health of patients. Unfortunately, inconsistencies in human adipose tissues are extensive and multifactorial, including large variability in cellular sizes, lipid content, inflammation, extracellular matrix components, mechanics, and cytokines secreted. Given the high human variability, and since much of what is known about adipose tissue is from animal models, we sought to establish correlations and patterns between biological, mechanical, and epidemiological properties of human adipose tissues. To do this, twenty-six independent variables were cataloged for twenty patients, which included patient demographics and factors that drive health, obesity, and fibrosis. A factorial analysis for mixed data (FAMD) was used to analyze patterns in the dataset (with BMI > 25), and a correlation matrix was used to identify interactions between quantitative variables. Vascular endothelial growth factor A (VEGFA) and actin alpha 2, smooth muscle (ACTA2) gene expression were the highest loadings in the first two dimensions of the FAMD. The number of adipocytes was also a key driver of patient-related differences, where a decrease in the density of adipocytes was associated with aging. Aging was also correlated with a decrease in overall lipid percentage of subcutaneous tissue, with lipid deposition being favored extracellularly, an increase in transforming growth factor-β1 (TGFβ1), and an increase in M1 macrophage polarization. An important finding was that self-identified race contributed to variance between patients in this study, where Black patients had significantly lower gene expression levels of TGFβ1 and ACTA2. This finding supports the urgent need to account for patient ancestry in biomedical research to develop better therapeutic strategies for all patients. Another important finding was that TGFβ induced factor homeobox 1 (TGIF1), an understudied signaling molecule, which is highly correlated with leptin signaling, was correlated with metabolic inflammation. Furthermore, this study draws attention to what we define as "extracellular lipid droplets", which were consistently found in collagen-rich regions of the obese adipose tissues evaluated here. Reduced levels of TGIF1 were correlated with higher numbers of extracellular lipid droplets and an inability to suppress fibrotic changes in adipose tissue. Finally, this study indicated that M1 and M2 macrophage markers were correlated with each other and leptin in patients with a BMI > 25. This finding supports growing evidence that macrophage polarization in obesity involves a complex, interconnecting network system rather than a full switch in activation patterns from M2 to M1 with increasing body mass. Overall, this study reinforces key findings in animal studies and identifies important areas for future research, where human and animal studies are divergent. Understanding key drivers of human patient variability is required to unravel the complex metabolic health of unique patients.

Abstract 2141: An IL12/IL23 Positive Feedback Loop Modulates Smooth Muscle Differentiation Via Setd4 During Macrophage-mediated Inflammation

Diabetic patients have increased risk of vascular disease (e.g., atherosclerosis), which is driven by macrophage-mediated inflammation. In diseases such as atherosclerosis, smooth muscle cells (SMC) undergo phenotypic modulation from a mature, contractile to a synthetic phenotype, thus leading to increased neointima formation. We hypothesize that pro-inflammatory macrophages as in the setting of diabetes epigenetically pattern SMC during vascular disease. In this study, we use single cell-RNA sequencing (scRNA-seq) of human arteries, loss of function experiments, and mouse models of disease to identify epigenetic mechanisms that regulate SMC phenotype. We identified the H3K4 and H3K9 histone lysine methyltransferase Setd4, which was increased in aortic SMC from diabetic mice and in our scRNA-seq dataset from human atherosclerotic arteries. siRNA mediated knockdown of Setd4 in mouse aortic SMC increased expression of the early SMC differentiation markers, Acta2 and Tagln (p<0.05). To model the pro-inflammatory environment in vitro, supernatant from LPS-stimulated macrophages increased Setd4 expression and inhibited smooth muscle gene expression ( Acta2, Tagln, Cnn1, Myh11 ), which was reversed by siRNA-mediated knockdown of Setd4 in SMC (p<0.05). IL23 increased Setd4 expression in SMC and enrichment at smooth muscle gene promoters, while deceasing SMC-specific gene expression (p<0.05). Treatment of SMC with an IL23 receptor neutralizing antibody prevented downregulation of SMC gene expression by macrophage supernatant (p<0.05). Intriguingly, ELISA of mouse aortic SMC supernatant revealed that IL23 increased SMC secretion of IL12, which also inhibited SMC gene expression via Setd4 (p<0.05). In conclusion, we demonstrate that pro-inflammatory macrophage IL23 represses smooth muscle gene expression via SETD4, thereby shifting the SMC to a synthetic, diseased phenotype. IL23 also stimulates vascular SMC secretion of IL12, which further increases production of inflammatory cytokines from macrophages and leads to additional repression of smooth muscle gene expression. Thus, targeting the IL23-SETD4 axis in SMC may be a potential therapy for the treatment of atherosclerosis, especially in patients who also have diabetes.

Effect of aging on the human myometrium at single-cell resolution

Age-associated myometrial dysfunction can prompt complications during pregnancy and labor, which is one of the factors contributing to the 7.8-fold increase in maternal mortality in women over 40. Using single-cell/single-nucleus RNA sequencing and spatial transcriptomics, we have constructed a cellular atlas of the aging myometrium from 186,120 cells across twenty perimenopausal and postmenopausal women. We identify 23 myometrial cell subpopulations, including contractile and venous capillary cells as well as immune-modulated fibroblasts. Myometrial aging leads to fewer contractile capillary cells, a reduced level of ion channel expression in smooth muscle cells, and impaired gene expression in endothelial, smooth muscle, fibroblast, perivascular, and immune cells. We observe altered myometrial cell-to-cell communication as an aging hallmark, which associated with the loss of 25 signaling pathways, including those related to angiogenesis, tissue repair, contractility, immunity, and nervous system regulation. These insights may contribute to a better understanding of the complications faced by older individuals during pregnancy and labor.

Identifying target organ location of Radix Achyranthis Bidentatae: a bioinformatics approach on active compounds and genes.

Background: Herbal medicines traditionally target organs for treatment based on medicinal properties, and this theory is widely used for prescriptions. However, the scientific evidence explaining how herbs act on specific organs by biological methods has been still limited. This study used bioinformatic tools to identify the target organ locations of Radix Achyranthis Bidentatae (RAB), a blood-activating herb that nourishes the liver and kidney, strengthens bones, and directs prescription to the lower body. Methods: RAB's active compounds and targets were collected and predicted using databases such as TCMSP, HIT2.0, and BATMAN-TCM. Next, the RAB's target list was analyzed based on two approaches to obtain target organ locations. DAVID and Gene ORGANizer enrichment-based approaches were used to enrich an entire gene list, and the BioGPS and HPA gene expression-based approaches were used to analyze the expression of core genes. Results: RAB's targets were found to be involved in whole blood, blood components, and lymphatic organs across all four tools. Each tool indicated a particular aspect of RAB's target organ locations: DAVID-enriched genes showed a predominance in blood, liver, and kidneys; Gene ORGANizer showed the effect on low body parts as well as bones and joints; BioGPS and HPA showed high gene expression in bone marrow, lymphoid tissue, and smooth muscle. Conclusion: Our bioinformatics-based target organ location prediction can serve as a modern interpretation tool for the target organ location theory of traditional medicine. Future studies should predict therapeutic target organ locations in complex prescriptions rather than single herbs and conduct experiments to verify predictions.

Molecular machines stimulate intercellular calcium waves and cause muscle contraction.

Intercellular calcium waves (ICW) are complex signalling phenomena that control many essential biological activities, including smooth muscle contraction, vesicle secretion, gene expression and changes in neuronal excitability. Accordingly, the remote stimulation of ICW could result in versatile biomodulation and therapeutic strategies. Here we demonstrate that light-activated molecular machines (MM)-molecules that perform mechanical work on the molecular scale-can remotely stimulate ICW. MM consist of a polycyclic rotor and stator that rotate around a central alkene when activated with visible light. Live-cell calcium-tracking and pharmacological experiments reveal that MM-induced ICW are driven by the activation of inositol-triphosphate-mediated signalling pathways by unidirectional, fast-rotating MM. Our data suggest that MM-induced ICW can control muscle contraction in vitro in cardiomyocytes and animal behaviour in vivo in Hydra vulgaris. This work demonstrates a strategy for directly controlling cell signalling and downstream biological function using molecular-scale devices.

458-P: Proinflammatory Macrophage Phenotype Modulates Vascular Smooth Muscle Cell Differentiation via the Histone Demethylase JMJD3

Patients with diabetes are at increased risk of a multitude of vascular diseases, including atherosclerosis, which is driven by macrophage-mediated inflammation. While it is known that atherosclerosis is characterized by a proliferative, synthetic smooth muscle phenotype that underlies disease, the exact role of other cell types, namely myeloid cells and their interaction with smooth muscle cells is less understood. Furthermore, how diabetes modifies atherosclerotic severity is another important question. To address this, we performed an epigenetic superarray to identify key chromatin modifying enzymes in vascular smooth muscle cells (vSMC) from normal diet (ND) and diabetic-induced obese (DIO) aortas. We identified the histone demethylase, JMJD3, which was significantly decreased in DIO compared to ND vSMC (p<0.05). Furthermore, in our single cell RNA-seq data set from human atherosclerotic lesions, JMJD3 was expressed in smooth muscle cells. It is known that macrophages in atherosclerotic lesions exhibit a pro-inflammatory phenotype. To model this pro-inflammatory process, we stimulated bone marrow derived macrophages with LPS and found that supernatant from LPS-stimulated BMDMs significantly inhibited smooth muscle specific gene expression (SMA, SM22, CNN1, SM-MHC) as well as expression of JMJD3 compared to control media (p<0.05). Additionally, siRNA-mediated knockdown of JMJD3 in vSMC reduced SMC-specific gene expression, and treatment of mouse aortic vSMC with the JMJD3 selective inhibitor, GSK4J, decreased smooth muscle gene expression (p<0.05). Our data shows that pro-inflammatory macrophages drive reduced vascular smooth muscle gene expression and that JMJD3 in SMC is a regulator of this process. These results implicate JMJD3 in macrophage-smooth muscle cell crosstalk in the pro-inflammatory state and identify it as a potential therapeutic target in the management of atherosclerosis. Disclosure K.D.Mangum: None. A.Joshi: None. P.Mazumder: None. S.Wolf-fortune: None. E.Barrett: None. B.Moore: None. F.M.Davis: None. K.Gallagher: None. Funding National Institutes of Health (1F32DK131799-01); American College of Surgeons; Vascular & Endovascular Surgery Society

Abstract 158: The Histone Demethylase JMJD3 Regulates Smooth Muscle Differentiation And Is Associated With Blood Pressure

Hypertension is associated with significant morbidity and mortality in the United States, and despite its prevalence an unmet need exists for more personalized targeted therapies. Blood pressure is regulated by environmental and genetic factors, and genome wide association studies (GWAS) have identified numerous genes in the human population associated with hypertension. Recently, one GWAS identified the single nucleotide polymorphism rs62059712 that is associated with systolic blood pressure in humans. rs62059712 is located upstream of the gene JMJD3, a histone demethylase that our lab has studied in a variety of disease contexts, and the rs62059712 major C allele (MAF 0.92) is associated with increased blood pressure in humans. Based on our analysis of data from the UCSC genome browser, we found that rs62059712 is located in a DNase Hypersensitive Site marked by H3K27Ac, suggesting it may regulate JMJD3 transcription. In luciferase assays, the fragment containing rs62059712 displayed transcription activity in smooth muscle cells (SMC), and the major C allele exhibited increased luciferase activity compared to the minor T allele in mouse aortic SMC (p<0.05). Notably, rs62059712 is in linkage disequilibrium with rs74480102, which did not display allele-specific transcription activity in SMC. siRNA knockdown of JMJD3 in mouse aortic SMC significantly decreased SMC-specific gene expression (p<0.05), and SMC marker gene expression was also reduced by treatment of SMC with the JMJD3 selective inhibitor GSK4J (p<0.05). Likewise, SMC contractile gene expression was reduced in aortas from SM22Cre;JMJD3 flox/flox mice harboring SMC-specific deletion of JMJD3. Further, angiotensin II increased JMJD3 expression in human aortic SMC. Finally, in chromatin immunoprecipitation assays in mouse aortic SMC, JMJD3 was significantly enriched at smooth muscle gene promoters (p<0.05). Our results identify the blood pressure-associated gene JMJD3 as a key epigenetic regulator of smooth muscle differentiation. Additionally, we show that the rs62059712 major C allele, which is associated with increased blood pressure, increases JMJD3 transcription activity, and likely expression, to drive increased downstream contractile smooth muscle gene expression.

An organ-on-chip model of pulmonary arterial hypertension identifies a BMPR2-SOX17-prostacyclin signalling axis

Pulmonary arterial hypertension (PAH) is an unmet clinical need. The lack of models of human disease is a key obstacle to drug development. We present a biomimetic model of pulmonary arterial endothelial-smooth muscle cell interactions in PAH, combining natural and induced bone morphogenetic protein receptor 2 (BMPR2) dysfunction with hypoxia to induce smooth muscle activation and proliferation, which is responsive to drug treatment. BMPR2- and oxygenation-specific changes in endothelial and smooth muscle gene expression, consistent with observations made in genomic and biochemical studies of PAH, enable insights into underlying disease pathways and mechanisms of drug response. The model captures key changes in the pulmonary endothelial phenotype that are essential for the induction of SMC remodelling, including a BMPR2-SOX17-prostacyclin signalling axis and offers an easily accessible approach for researchers to study pulmonary vascular remodelling and advance drug development in PAH.

Atypical Expression of Smooth Muscle Markers and Co-activators and Their Regulation in Rheumatic Aortic and Calcified Bicuspid Valves

ObjectiveWe have previously reported that human calcified aortic cusps have abundant expression of smooth muscle (SM) markers and co-activators. We hypothesised that cells in bicuspid aortic valve (BAV) cusps and those affected by rheumatic heart valve (RHV) disease may follow a similar phenotypic transition into smooth muscle cells, a process that could be regulated by transforming growth factors (TGFs).AimsCusps from eight patients with BAV and seven patients with RHV were analysed for early and late SM markers and regulators of SM gene expression by immunocytochemistry and compared to healthy aortic valves from 12 unused heart valve donors. The ability of TGFs to induce these markers in valve endothelial cells (VECs) on two substrates was assessed.ResultsIn total, 7 out of 8 BAVs and all the RHVs showed an increased and atypical expression of early and late SM markers α-SMA, calponin, SM22 and SM-myosin. The SM marker co-activators were aberrantly expressed in six of the BAV and six of the RHV, in a similar regional pattern to the expression of SM markers. Additionally, regions of VECs, and endothelial cells lining the vessels within the cusps were found to be positive for SM markers and co-activators in three BAV and six RHV. Both BAVs and RHVs were significantly thickened and HIF1α expression was prominent in four BAVs and one RHV. The ability of TGFβs to induce the expression of SM markers and myocardin was greater in VECs cultured on fibronectin than on gelatin. Fibronectin was shown to be upregulated in BAVs and RHVs, within the cusps as well as in the basement membrane.ConclusionBicuspid aortic valves and RHVs expressed increased numbers of SM marker-positive VICs and VECs. Concomittantly, these cells expressed MRTF-A and myocardin, key regulators of SM gene expression. TGFβ1 was able to preferentially upregulate SM markers and myocardin in VECs on fibronectin, and fibronectin was found to be upregulated in BAVs and RHVs. These findings suggest a role of VEC as a source of cells that express SM cell markers in BAVs and RHVs. The similarity between SM marker expression in BAVs and RHVs with our previous study with cusps from patients with aortic stenosis suggests the existance of a common pathological pathway between these different pathologies.

Heterogeneous pdgfrb+ cells regulate coronary vessel development and revascularization during heart regeneration.

Endothelial cells emerge from the atrioventricular canal to form coronary blood vessels in juvenile zebrafish hearts. We find that pdgfrb is first expressed in the epicardium around the atrioventricular canal and later becomes localized mainly in the mural cells. pdgfrb mutant fish show severe defects in mural cell recruitment and coronary vessel development. Single-cell RNA sequencing analyses identified pdgfrb+ cells as epicardium-derived cells (EPDCs) and mural cells. Mural cells associated with coronary arteries also express cxcl12b and smooth muscle cell markers. Interestingly, these mural cells remain associated with coronary arteries even in the absence of Pdgfrβ, although smooth muscle gene expression is downregulated. We find that pdgfrb expression dynamically changes in EPDCs of regenerating hearts. Differential gene expression analyses of pdgfrb+ EPDCs and mural cells suggest that they express genes that are important for regeneration after heart injuries. mdka was identified as a highly upregulated gene in pdgfrb+ cells during heart regeneration. However, pdgfrb but not mdka mutants show defects in heart regeneration after amputation. Our results demonstrate that heterogeneous pdgfrb+ cells are essential for coronary development and heart regeneration.

MiRNA-200c-3p promotes endothelial to mesenchymal transition and neointimal hyperplasia in artery bypass grafts.

Increasing evidence has suggested a critical role for endothelial‐to‐mesenchymal transition (EndoMT) in a variety of pathological conditions. MicroRNA‐200c‐3p (miR‐200c‐3p) has been implicated in epithelial‐to‐mesenchymal transition. However, the functional role of miR‐200c‐3p in EndoMT and neointimal hyperplasia in artery bypass grafts remains largely unknown. Here we demonstrated a critical role for miR‐200c‐3p in EndoMT. Proteomics and luciferase activity assays revealed that fermitin family member 2 (FERM2) is the functional target of miR‐200c‐3p during EndoMT. FERMT2 gene inactivation recapitulates the effect of miR‐200c‐3p overexpression on EndoMT, and the inhibitory effect of miR‐200c‐3p inhibition on EndoMT was reversed by FERMT2 knockdown. Further mechanistic studies revealed that FERM2 suppresses smooth muscle gene expression by preventing serum response factor nuclear translocation and preventing endothelial mRNA decay by interacting with Y‐box binding protein 1. In a model of aortic grafting using endothelial lineage tracing, we observed that miR‐200c‐3p expression was dramatically up‐regulated, and that EndoMT contributed to neointimal hyperplasia in grafted arteries. MiR‐200c‐3p inhibition in grafted arteries significantly up‐regulated FERM2 gene expression, thereby preventing EndoMT and reducing neointimal formation. Importantly, we found a high level of EndoMT in human femoral arteries with atherosclerotic lesions, and that miR‐200c‐3p expression was significantly increased, while FERMT2 expression levels were dramatically decreased in diseased human arteries. Collectively, we have documented an unexpected role for miR‐200c‐3p in EndoMT and neointimal hyperplasia in grafted arteries. Our findings offer a novel therapeutic opportunity for treating vascular diseases by specifically targeting the miR‐200c‐3p/FERM2 regulatory axis. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Dexamethasone rescues TGF-\u03b21-mediated \u03b22-adrenergic receptor dysfunction and attenuates phosphodiesterase 4D expression in human airway smooth muscle cells

Glucocorticoids (GCs) and β2-adrenergic receptor (β2AR) agonists improve asthma outcomes in most patients. GCs also modulate gene expression in human airway smooth muscle (HASM), thereby attenuating airway inflammation and airway hyperresponsiveness that define asthma. Our previous studies showed that the pro-fibrotic cytokine, transforming growth factor- β1 (TGF-β1) increases phosphodiesterase 4D (PDE4D) expression that attenuates agonist-induced levels of intracellular cAMP. Decreased cAMP levels then diminishes β2 agonist-induced airway relaxation. In the current study, we investigated whether glucocorticoids reverse TGF-β1-effects on β2-agonist-induced bronchodilation and modulate pde4d gene expression in HASM. Dexamethasone (DEX) reversed TGF-β1 effects on cAMP levels induced by isoproterenol (ISO). TGF-β1 also attenuated G protein-dependent responses to cholera toxin (CTX), a Gαs stimulator downstream from the β2AR receptor. Previously, we demonstrated that TGF-β1 treatment increased β2AR phosphorylation to induce hyporesponsiveness to a β2 agonist. Our current data shows that expression of grk2/3, kinases associated with attenuation of β2AR function, are not altered with TGF-β1 stimulation. Interestingly, DEX also attenuated TGF-β1-induced pde4d gene expression. These data suggest that steroids may be an effective therapy for treatment of asthma patients whose disease is primarily driven by elevated TGF-β1 levels.

The effects of membrane potential and extracellular matrix composition on vascular differentiation of cardiac progenitor cells

Historically, the field of tissue engineering has been adept at modulating the chemical and physical microenvironment. This approach has yielded significant progress, but it is imperative to further integrate our understanding of other fundamental cell signaling paradigms into tissue engineering methods. Bioelectric signaling has been demonstrated to be a vital part of tissue development, regeneration, and function across organ systems and the extracellular matrix is known to alter the bioelectric properties of cells. Thus, there is a need to bolster our understanding of how matrix and bioelectric signals interact to drive cell phenotype. We examine how cardiac progenitor cell differentiation is altered by simultaneous changes in both resting membrane potential and extracellular matrix composition. Pediatric c-kit+ cardiac progenitor cells were differentiated on fetal or adult cardiac extracellular matrix while being treated with drugs that alter resting membrane potential. Smooth muscle gene expression was increased with depolarization and decreased with hyperpolarization while endothelial and cardiac expression were unchanged. Early smooth muscle protein expression is modified by matrix developmental age, with fetal ECM appearing to amplify the effects of resting membrane potential. Thus, combining matrix composition and bioelectric signaling represents a potential alternative for guiding cell behavior in tissue engineering and regenerative medicine.

Runx1 promotes scar deposition and inhibits myocardial proliferation and survival during zebrafish heart regeneration.

ABSTRACTRunx1 is a transcription factor that plays a key role in determining the proliferative and differential state of multiple cell types, during both development and adulthood. Here, we report how Runx1 is specifically upregulated at the injury site during zebrafish heart regeneration, and that absence of runx1 results in increased myocardial survival and proliferation, and overall heart regeneration, accompanied by decreased fibrosis. Using single cell sequencing, we found that the wild-type injury site consists of Runx1-positive endocardial cells and thrombocytes that induce expression of smooth muscle and collagen genes. Both these populations cannot be identified in runx1 mutant wounds that contain less collagen and fibrin. The reduction in fibrin in the mutant is further explained by reduced myofibroblast formation and upregulation of components of the fibrin degradation pathway, including plasminogen receptor annexin 2A as well as downregulation of plasminogen activator inhibitor serpine1 in myocardium and endocardium, resulting in increased levels of plasminogen. Our findings suggest that Runx1 controls the regenerative response of multiple cardiac cell types and that targeting Runx1 is a novel therapeutic strategy for inducing endogenous heart repair.

Loss-of-function variants in myocardin cause congenital megabladder in humans and mice.

Myocardin (MYOCD) is the founding member of a class of transcriptional coactivators that bind the serum-response factor to activate gene expression programs critical in smooth muscle (SM) and cardiac muscle development. Insights into the molecular functions of MYOCD have been obtained from cell culture studies, and to date, knowledge about in vivo roles of MYOCD comes exclusively from experimental animals. Here, we defined an often lethal congenital human disease associated with inheritance of pathogenic MYOCD variants. This disease manifested as a massively dilated urinary bladder, or megabladder, with disrupted SM in its wall. We provided evidence that monoallelic loss-of-function variants in MYOCD caused congenital megabladder in males only, whereas biallelic variants were associated with disease in both sexes, with a phenotype additionally involving the cardiovascular system. These results were supported by cosegregation of MYOCD variants with the phenotype in 4 unrelated families by in vitro transactivation studies in which pathogenic variants resulted in abrogated SM gene expression and by the finding of megabladder in 2 distinct mouse models with reduced Myocd activity. In conclusion, we have demonstrated that variants in MYOCD result in human disease, and the collective findings highlight a vital role for MYOCD in mammalian organogenesis.

Enrichment of Smooth Muscle Signature in Gastric Cancer Is Associated with Low Burden of Genomic Aberrations and Poor Survival

Background: Bormann IV gastric cancer (B-IV GC) is characterized by stealth tumor growth and invasion of cancer cells into the stomach wall. It is also associated with very poor prognosis. The molecular mechanism of Bormann IV gastric cancer is still unclear. Methods: In this study, we compare the mRNA expression profiles between Borrman IV and non-Borrmann IV gastric cancer to find out differences in gene expression with GEO dataset. Then we validated our findings using TCGA gastric cancer dataset. Results: We found that B-IV gastric cancer had a distinct gene expression pattern. The smooth muscle geneset was highly enriched in B-IV gastric cancer, and was associated with low tumor purity. Using this smooth musclegeneset, we classified GC into smooth muscle enriched and non-enriched groups. Notably, enrichment of smooth muscle was associated with both poor disease free survival and overall survival. A B-IV GC signature was derived by comparing gene expression of B-IV and non-B-IV gastric cancers. Analysis of RNA sequencing data from TCGA gastric cancer study revealed a similar subgroup of tumors that showed gene expression patterns B-IV GC. This group of tumors mainly came from the genome stable (GS) molecular subtype. More importantly, these tumors were associated with higher CDH1 mutation rate, but lower overall tumor mutation burden and copy number variation burden. Conclusions: In conclusion, Borrmann IV gastric cancer tissue is characterized by smooth muscle gene expression pattern and low tumor purity, and the seemly stable genome of these tumors may not reveal their true genomic background. Funding Statement: This study was supported by International Program for Ph.D. Candidates from Sun Yat-sen University. This work was also Supported by the Natural Science Foundation of Guangdong Province, China (No. 2016A030310155, 2016A020213002) and National Natural Science Foundation of China (No. 81602049). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: All procedures were in accordance with the institutional ethical standards of the responsible committee on human experimentation and with the Helsinki Declaration of 1964 and later versions. Informed consent was obtained from all patients for being included in the study by the original investigators.

Alterations in phenotype and gene expression of adult human aneurysmal smooth muscle cells by exogenous nitric oxide

Abdominal aortic aneurysms (AAA) are characterized by matrix remodeling, elastin degradation, absence of nitric oxide (NO) signaling, and inflammation, influencing smooth muscle cell (SMC) phenotype and gene expression. Little is known about the biomolecular release and intrinsic biomechanics of human AAA-SMCs. NO delivery could be an attractive therapeutic strategy to restore lost functionality of AAA-SMCs by inhibiting inflammation and cell stiffening. We aim to establish the differences in phenotype and gene expression of adult human AAA-SMCs from healthy SMCs. Based on our previous study which showed benefits of optimal NO dosage delivered via S-Nitrosoglutathione (GSNO) to healthy aortic SMCs, we tested whether such benefits would occur in AAA-SMCs. The mRNA expression of three genes involved in matrix degradation (ACE, ADAMTS5 and ADAMTS8) was significantly downregulated in AAA-SMCs. Total protein and glycosaminoglycans synthesis were higher in AAA-SMCs than healthy-SMCs (p < 0.05 for AAA-vs. healthy- SMC cultures) and was enhanced by GSNO and 3D cultures (p < 0.05 for 3D vs. 2D cultures; p < 0.05 for GSNO vs. non-GSNO cases). Elastin gene expression, synthesis and deposition, desmosine crosslinker levels, and lysyl oxidase (LOX) functional activity were lower, while cell proliferation, iNOS, LOX and fibrillin-1 gene expressions were higher in AAA-SMCs (p < 0.05 between respective cases), with differential benefits from GSNO exposure. GSNO and 3D cultures reduced MMPs −2, −9, and increased TIMP-1 release in AAA-SMC cultures (p < 0.05 for GSNO vs. non-GSNO cultures). AAA-SMCs were inherently stiffer and had smoother surface than healthy SMCs (p < 0.01 in both cases), but GSNO reduced stiffness (~25%; p < 0.01) and increased roughness (p < 0.05) of both cell types. In conclusion, exogenously-delivered NO offers an attractive strategy by providing therapeutic benefits to AAA-SMCs.

MicroRNA-126 regulates the expression of inflammationrelated genes in vascular smooth muscle cells of diabetic rats

Purpose: To investigate the regulatory role of miR-126 in inflammation-associated gene expression in vascular smooth muscle (VSMCs) cells of diabetic rats.Methods: Diabetes was induced in rats by intraperitoneal injection of streptozocin (STZ) at a dose of 50 mg/kg. VSMCs were isolated from the aortic intimal-medial layers of the rats by a standard protocol. Expression of miR-126 was determined by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Transfection was performed with Lipofectamine 2000 reagent.Results: Expression of miR-126 was significantly (p < 0.05) downregulated in the VSMCs of diabetic rats. Similarly, expressions of SOD, APX and CAT were downregulated, while those of COX, LOX and NOS were significantly upregulated in VSMCs. However, transfection-induced miR-126 overexpression in the VSMCs of diabetic rats led to significant (p < 0.05) upregulation of SOD, CAT, APX and downregulation of COX, LOX and NOS. TargetScan analysis revealed that miR-126 exerted these effects by targeting SIRT1 gene. Furthermore, qRT-PCR and western blotting revealed that miR-126 overexpression in diabetic VMSCs caused significant (p < 0.05) upregulation of the expression of SIRT-1.Conclusion: The results indicate that miR-126 regulates the expression of inflammation-related genes by targeting SIRT-1 genes in vascular smooth muscle cells of diabetic rats. Thus, miR-126 may be beneficial in the management of diabetes.Keywords: Diabetes, Inflammation, Genes, microRNA, vascular smooth muscle cells

Substrate topography interacts with substrate stiffness and culture time to regulate mechanical properties and smooth muscle differentiation of mesenchymal stem cells.

Substrate stiffness and topography are two powerful means by which mesenchymal stem cells (MSCs) activities can be modulated. The effects of substrate stiffness on the MSCs mechanical properties were investigated previously, however, the role of substrate topography in this regard is not yet well understood. Moreover, in vessel wall, these two physical cues act simultaneously to regulate cellular function, hence it is important to investigate their cooperative effects on cellular activity. Herein, we investigated the combined effects of substrate stiffness, substrate topography and culture time on the mechanical behavior of MSCs. The MSCs were cultured on the stiff and soft substrates with or without micro-grooved topography for 10 days and their viscoelastic properties and smooth muscle (SM) gene expression were investigated on days 2, 6 and 10. In general, substrate topography significantly interacted with substrate stiffness as well as culture time in the modulation of cell viscoelastic behavior and SM gene expression. The micro-grooved, stiff substrates resulted in the maximum cell stiffness and gene expression of α-actin and h1-calponin, and these values were detected to be minimum in the smooth, soft substrates. The findings can be helpful in the mechano-regulation of MSCs for vascular tissue engineering applications.