Browsing by Subject "Smooth muscle"

Now showing 1 - 10 of 13
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    An Evaluation of Peer-Rated Surgical Skill and its Relationship With Detrusor Muscle Sampling in Transurethral Resection of Bladder Tumor
    (Elsevier, 2022) Pham, Minh N.; Ko, Oliver S.; Huang, Reiping; Vo, Amanda X.; Tsai, Kyle P.; Lai, Jeremy D.; Hudnall, Matthew T.; Halpern, Joshua A.; Meeks, Joshua J.; Benson, Jonas; Soares, Ricardo; Kim, Ronald; Bilimoria, Karl Y.; Stulberg, Jonah J.; Auffenberg, Gregory B.; Surgery, School of Medicine
    Objective: To assess the reliability of peer-review of TURBT videos as a means to evaluate surgeon skill and its relationship to detrusor sampling. Methods: Urologists from an academic health system submitted TURBT videos in 2019. Ten blinded peers evaluated each surgeon's performance using a 10-item scoring instrument to quantify surgeon skill. Normalized composite skill scores for each surgeon were calculated using peer ratings. For surgeons submitting videos, we retrospectively reviewed all TURBT pathology results (2018-2019) to assess surgeon-specific detrusor sampling. A hierarchical logistic regression model was fit to evaluate the association between skill and detrusor sampling, adjusting for patient and surgeon factors. Results: Surgeon skill scores and detrusor sampling rates were determined for 13 surgeons performing 245 TURBTs. Skill scores varied from -6.0 to 5.1 [mean: 0; standard deviation (SD): 2.40]. Muscle was sampled in 72% of cases, varying considerably across surgeons (mean: 64.5%; SD: 30.7%). Among 8 surgeons performing >5 TURBTs during the study period, adjusted detrusor sampling rate was associated with sending separate deep specimens (odds ratio [OR]: 1.97; 95% confidence interval [CI]: 1.02-3.81, P = .045) but not skill (OR: 0.81; 95% CI: 0.57-1.17, P = .191). Conclusion: Surgeon skill was not associated with detrusor sampling, suggesting there may be other drivers of variability of detrusor sampling in TURBT.
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    CRISPR-Cas9 Mediated Epitope Tagging Provides Accurate and Versatile Assessment of Myocardin
    (American Heart Association, 2018-09) Lyu, Qing; Dhagia, Vidhi; Han, Yu; Guo, Bing; Wines-Samuelson, Mary E.; Christie, Christine K.; Yin, Qiangzong; Slivano, Orazio J.; Herring, Paul; Long, Xiaochun; Gupte, Sachin A.; Miano, Joseph M.; Cellular and Integrative Physiology, School of Medicine
    Objective- Unreliable antibodies often hinder the accurate detection of an endogenous protein, and this is particularly true for the cardiac and smooth muscle cofactor, MYOCD (myocardin). Accordingly, the mouse Myocd locus was targeted with 2 independent epitope tags for the unambiguous expression, localization, and activity of MYOCD protein. Approach and Results- 3cCRISPR (3-component clustered regularly interspaced short palindromic repeat) was used to engineer a carboxyl-terminal 3×FLAG or 3×HA epitope tag in mouse embryos. Western blotting with antibodies to each tag revealed a MYOCD protein product of ≈150 kDa, a size considerably larger than that reported in virtually all publications. MYOCD protein was most abundant in some adult smooth muscle-containing tissues with surprisingly low-level expression in the heart. Both alleles of Myocd are active in aorta because a 2-fold increase in protein was seen in mice homozygous versus heterozygous for FLAG-tagged Myocd. ChIP (chromatin immunoprecipitation)-quantitative polymerase chain reaction studies provide proof-of-principle data demonstrating the utility of this mouse line in conducting genome-wide ChIP-seq studies to ascertain the full complement of MYOCD-dependent target genes in vivo. Although FLAG-tagged MYOCD protein was undetectable in sections of adult mouse tissues, low-passaged vascular smooth muscle cells exhibited expected nuclear localization. Conclusions- This report validates new mouse models for analyzing MYOCD protein expression, localization, and binding activity in vivo and highlights the need for rigorous authentication of antibodies in biomedical research.
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    Death-Associated Protein Kinase Regulates Vascular Smooth Muscle Cell Signaling and Migration
    (2011-03-16) Blue, Emily Keller; Gallagher, Patricia J.; Elmendorf, Jeffrey S.; Herring, B. Paul; Rhodes, Simon J.; Thurmond, Debbie C.
    Cardiovascular disease is the number one cause of death for Americans. New treatments are needed for serious conditions like atherosclerosis, as it can lead to stroke and heart attack. Many types of cells contribute to the progression of cardiovascular disease, including smooth muscle cells that comprise the middle layers of arteries. Inappropriate growth and migration of smooth muscle cells into the lumen of arteries has been implicated in vascular diseases. Death associated protein kinase (DAPK) is a protein that has been found to regulate the survival and migration of cancer cells, but has not been well characterized in vascular cells. The objective of this work was to determine the signaling pathways that DAPK regulates in smooth muscle cells. These studies have focused on smooth muscle cells isolated from human coronary arteries (HCASM cells). We have determined that HCASM cells depleted of DAPK exhibit more rapid migration, showing that DAPK negatively regulates migration of vascular cells. Results from a focused RT-PCR array identified matrix metalloproteinase 9 (MMP9) as a gene that is increased in cells depleted of DAPK. MMP9 is an important enzyme that degrades collagen, a component of the extracellular matrix through which smooth muscle cells migrate during atherosclerosis. We found that DAPK regulates phosphorylation of the NF-kappa B transcription factor p65 at serine 536, a modification previously found to correlate with increased nuclear levels and activity of p65. In DAPK-depleted HCASM cells, there was more phosphorylation of p65, which causes increased MMP9 promoter activity. Additional experiments were conducted using transgenic mice in which the DAPK gene has been deleted. By studying these mice, we have determined that under some circumstances DAPK augments maximal MMP9 levels in mouse carotid arteries which have been injured by ligation surgery via other signaling pathways. MMP9 has been previously implicated as a protein that promotes vascular diseases such as atherosclerosis. Our research in identifying DAPK as a regulator of MMP9 expression identifies a new target for treatment of vascular diseases like atherosclerosis.
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    Generation of transgenic mice that conditionally express microRNA miR-145
    (Wiley, 2020-09) Sawant, Dwitiya; Klevenow, Emilie; Baeten, Jeremy T.; Thomas, Shelby; Manivannan, Sathiyanarayanan; Conway, Simon J.; Lilly, Brenda; Pediatrics, School of Medicine
    MicroRNAs are modulators of cellular phenotypes and their functions contribute to development, homeostasis, and disease. miR-145 is a conserved microRNA that has been implicated in regulating an array of phenotypes. These include supporting smooth muscle differentiation, repression of stem cell pluripotency, and inhibition of tumor growth and metastasis. Previously, our lab demonstrated that miR-145 acts to suppress cardiac fibrosis through inhibition of the TGF-β signaling pathway. The range of effects that miR-145 has on different cell types makes it an attractive microRNA for further study. Here we describe the generation of transgenic mice that conditionally express miR-145 through Cre recombinase-mediated activation. Characterization of individual founder lines indicates that overexpression of miR-145 in the developing cardiovascular system has detrimental effects, with three independent miR-145 transgenic lines exhibiting Cre-dependent lethality. Expression analysis demonstrates that the transgene is robustly expressed and our analysis reveals a novel downstream target of miR-145, Tnnt2. The miR-145 transgenic mice represent a valuable tool to understand the role of miR-145 in diverse cell types and to address its potential as a therapeutic mediator for the treatment of disease.
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    Identification of a common polymorphism in COQ8B acting as a modifier of thoracic aortic aneurysm severity
    (Elsevier, 2022-01-13) Landis, Benjamin J.; Lai, Dongbing; Guo, Dong-Chuan; Corvera, Joel S.; Idrees, Muhammad T.; Stadler, Henry W.; Cuevas, Christian; Needler, Gavin U.; Vujakovich, Courtney E.; Milewicz, Dianna M.; Hinton, Robert B.; Ware, Stephanie M.; Pediatrics, School of Medicine
    Thoracic 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 patients 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.H174R) was associated with decreased rate of aortic root dilation in young patients with TAA. In addition, the SNP was less frequent in a second cohort of early-onset thoracic aortic dissection cases compared with controls. COQ8B protein levels in aortic SMCs were increased in TAA patients 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 COQ8B level. Genotyping rs3865452 may be useful for clinical risk stratification and tailored aortopathy management.
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    Idiopathic gastroparesis is associated with specific transcriptional changes in the gastric muscularis externa
    (Wiley, 2018-04) Herring, B. Paul; Hoggatt, April M.; Gupta, Anita; Griffith, Sarah; Nakeeb, Attila; Choi, Jennifer N.; Idrees, Muhammad T.; Nowak, Thomas; Morris, David L.; Wo, John M.; Cellular and Integrative Physiology, School of Medicine
    BACKGROUND: The molecular changes that occur in the stomach that are associated with idiopathic gastroparesis are poorly described. The aim of this study was to use quantitative analysis of mRNA expression to identify changes in mRNAs encoding proteins required for the normal motility functions of the stomach. METHODS: Full-thickness stomach biopsy samples were collected from non-diabetic control subjects who exhibited no symptoms of gastroparesis and from patients with idiopathic gastroparesis. mRNA was isolated from the muscularis externa and mRNA expression levels were determined by quantitative reverse transcriptase (RT)-PCR. KEY RESULTS: Smooth muscle tissue from idiopathic gastroparesis patients had decreased expression of mRNAs encoding several contractile proteins, such as MYH11 and MYLK1. Conversely, there was no significant change in mRNAs characteristic of interstitial cells of Cajal (ICCs) such as KIT or ANO1. There was also a significant decrease in mRNA-encoding platelet-derived growth factor receptor α (PDGFRα) and its ligand PDGFB and in Heme oxygenase 1 in idiopathic gastroparesis subjects. In contrast, there was a small increase in mRNA characteristic of neurons. Although there was not an overall change in KIT expression in gastroparesis patients, KIT expression showed a significant correlation with gastric emptying whereas changes in MYLK1, ANO1 and PDGFRα showed weak correlations to the fullness/satiety subscore of patient assessment of upper gastrointestinal disorder-symptom severity index scores. CONCLUSIONS AND INFERENCES: Our findings suggest that idiopathic gastroparesis is associated with altered smooth muscle cell contractile protein expression and loss of PDGFRα+ cells without a significant change in ICCs.
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    Interaction Between Pannexin 1 and Caveolin-1 in Smooth Muscle Can Regulate Blood Pressure
    (American Heart Association, 2018-09) DeLalio, Leon J.; Keller, Alexander S.; Chen, Jiwang; Boyce, Andrew K. J.; Artamonov, Mykhaylo V.; Askew-Page, Henry R.; Keller, T. C. Stevenson; Johnstone, Scott R.; Weaver, Rachel B.; Good, Miranda E.; Murphy, Sara A.; Best, Angela K.; Mintz, Ellen L.; Penuela, Silvia; Greenwood, Iain A.; Machado, Roberto F.; Somlyo, Avril V.; Swayne, Leigh Anne; Minshall, Richard D.; Isakson, Brant E.; Medicine, School of Medicine
    Objective- Sympathetic nerve innervation of vascular smooth muscle cells (VSMCs) is a major regulator of arteriolar vasoconstriction, vascular resistance, and blood pressure. Importantly, α-adrenergic receptor stimulation, which uniquely couples with Panx1 (pannexin 1) channel-mediated ATP release in resistance arteries, also requires localization to membrane caveolae. Here, we test whether localization of Panx1 to Cav1 (caveolin-1) promotes channel function (stimulus-dependent ATP release and adrenergic vasoconstriction) and is important for blood pressure homeostasis. Approach and Results- We use in vitro VSMC culture models, ex vivo resistance arteries, and a novel inducible VSMC-specific Cav1 knockout mouse to probe interactions between Panx1 and Cav1. We report that Panx1 and Cav1 colocalized on the VSMC plasma membrane of resistance arteries near sympathetic nerves in an adrenergic stimulus-dependent manner. Genetic deletion of Cav1 significantly blunts adrenergic-stimulated ATP release and vasoconstriction, with no direct influence on endothelium-dependent vasodilation or cardiac function. A significant reduction in mean arterial pressure (total=4 mm Hg; night=7 mm Hg) occurred in mice deficient for VSMC Cav1. These animals were resistant to further blood pressure lowering using a Panx1 peptide inhibitor Px1IL2P, which targets an intracellular loop region necessary for channel function. Conclusions- Translocalization of Panx1 to Cav1-enriched caveolae in VSMCs augments the release of purinergic stimuli necessary for proper adrenergic-mediated vasoconstriction and blood pressure homeostasis.
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    Last Word on Point: Alterations in airway smooth muscle phenotype do cause airway hyperresponsiveness in asthma
    (American Physiological Society, 2012) Gunst, Susan J.; Panettieri, Reynold A., Jr.; Cellular and Integrative Physiology, School of Medicine
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    Membrane adhesion junctions regulate airway smooth muscle phenotype and function
    (American Physiological Society, 2023) Zhang, Wenwu; Wu, Yidi; Gunst, Susan J.; Anatomy, Cell Biology and Physiology, School of Medicine
    The local environment surrounding airway smooth muscle (ASM) cells has profound effects on the physiological and phenotypic properties of ASM tissues. ASM is continually subjected to the mechanical forces generated during breathing and to the constituents of its surrounding extracellular milieu. The smooth muscle cells within the airways continually modulate their properties to adapt to these changing environmental influences. Smooth muscle cells connect to the extracellular cell matrix (ECM) at membrane adhesion junctions that provide mechanical coupling between smooth muscle cells within the tissue. Membrane adhesion junctions also sense local environmental signals and transduce them to cytoplasmic and nuclear signaling pathways in the ASM cell. Adhesion junctions are composed of clusters of transmembrane integrin proteins that bind to ECM proteins outside the cell and to large multiprotein complexes in the submembranous cytoplasm. Physiological conditions and stimuli from the surrounding ECM are sensed by integrin proteins and transduced by submembranous adhesion complexes to signaling pathways to the cytoskeleton and nucleus. The transmission of information between the local environment of the cells and intracellular processes enables ASM cells to rapidly adapt their physiological properties to modulating influences in their extracellular environment: mechanical and physical forces that impinge on the cell, ECM constituents, local mediators, and metabolites. The structure and molecular organization of adhesion junction complexes and the actin cytoskeleton are dynamic and constantly changing in response to environmental influences. The ability of ASM to rapidly accommodate to the ever-changing conditions and fluctuating physical forces within its local environment is essential for its normal physiological function.