scholarly journals Mice expressing BMPR2R899X transgene in smooth muscle develop pulmonary vascular lesions

2008 ◽  
Vol 295 (5) ◽  
pp. L744-L755 ◽  
Author(s):  
James West ◽  
Julie Harral ◽  
Kirk Lane ◽  
Yupu Deng ◽  
Brian Ickes ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Gene Array ◽  
Vascular Lesions ◽  
Tail Domain ◽  
Small Vessels ◽  
Morphogenetic Protein ◽  
Large Numbers ◽  
Proliferation And Apoptosis ◽  
And Function

Familial pulmonary arterial hypertension (PAH) is associated with mutations in bone morphogenetic protein type II receptor (BMPR2). Many of these mutations occur in the BMPR2 tail domain, leaving the SMAD functions intact. To determine the in vivo consequences of BMPR2 tail domain mutation, we created a smooth muscle-specific doxycycline-inducible BMPR2 mutation with an arginine to termination mutation at amino acid 899. When these SM22-rtTA x TetO7-BMPR2R899X mice had transgene induced for 9 wk, starting at 4 wk of age, they universally developed pulmonary vascular pruning as assessed by fluorescent microangiography. Approximately one-third of the time, the induced animals developed elevated right ventricular systolic pressures (RVSP), associated with extensive pruning, muscularization of small pulmonary vessels, and development of large structural pulmonary vascular changes. These lesions included large numbers of macrophages and T cells in their adventitial compartment as well as CD133-positive cells in the lumen. Small vessels filled with CD45-positive and sometimes CD3-positive cells were a common feature in all SM22-rtTA x TetO7-BMPR2R899X mice. Gene array experiments show changes in stress response, muscle organization and function, proliferation, and apoptosis and developmental pathways before RVSP increases. Our results show that the primary phenotypic result of BMPR2 tail domain mutation in smooth muscle is pulmonary vascular pruning leading to elevated RVSP, associated with early dysregulation in multiple pathways with clear relevance to PAH. This model should be useful to the research community in examining early molecular and physical events in the development of PAH and as a platform to validate potential treatments.

MIC-Drop: A platform for large-scale in vivo CRISPR screens

Science ◽  
2021 ◽  
pp. eabi8870
Author(s):  
Saba Parvez ◽  
Chelsea Herdman ◽  
Manu Beerens ◽  
Korak Chakraborti ◽  
Zachary P. Harmer ◽  
...  
Keyword(s):  
Large Scale ◽  
Cultured Cells ◽  
Cardiac Development ◽  
Droplet Microfluidics ◽  
Model Organisms ◽  
Genetic Screens ◽  
Large Numbers ◽  
And Function ◽  
Genome Scale

CRISPR-Cas9 can be scaled up for large-scale screens in cultured cells, but CRISPR screens in animals have been challenging because generating, validating, and keeping track of large numbers of mutant animals is prohibitive. Here, we report Multiplexed Intermixed CRISPR Droplets (MIC-Drop), a platform combining droplet microfluidics, single-needle en masse CRISPR ribonucleoprotein injections, and DNA barcoding to enable large-scale functional genetic screens in zebrafish. The platform can efficiently identify genes responsible for morphological or behavioral phenotypes. In one application, we show MIC-Drop can identify small molecule targets. Furthermore, in a MIC-Drop screen of 188 poorly characterized genes, we discover several genes important for cardiac development and function. With the potential to scale to thousands of genes, MIC-Drop enables genome-scale reverse-genetic screens in model organisms.

scholarly journals Protective Effects of Antimuscarinics on the Bladder Remodeling After Bladder Outlet Obstruction

2017 ◽  
Vol 44 (3) ◽  
pp. 907-919 ◽  
Author(s):  
Qiang Liu ◽  
Deyi Luo ◽  
Tongxin Yang ◽  
Banghua Liao ◽  
Hong Li ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Bladder Outlet Obstruction ◽  
Outlet Obstruction ◽  
Structure And Function ◽  
Bladder Function ◽  
Bladder Smooth Muscle ◽  
M3 Receptors ◽  
And Function

Background/Aims: Overactive bladder associated with bladder outlet obstruction (BOO) is a highly prevalent condition, which is usually treated with antimuscarinics. However, the potential effects of antimuscarinics on the structure and function of bladder have not been investigated thus far. Methods: Sprague-Dawley(R) rats accepted bladder neck obstruction surgery or sham surgery, and then received treatment of three different antimuscarinics (Solifenacin, Darifenacin, and Tolterodine) or vehicle. After 3, 6 and 12 weeks, the bladder function and structure were measured. The effect of antimuscarinics on cellular alteration in vitro was observed under mechanical stimulation. Bladder morphology were examined by immunohistochemistry, and the bladder function were investigated by cystometry and strip contractility test. The expression of muscarinic receptors and inflammatory cytokines were measured by PCR and Western blotting. Results: Here we demonstrate, both in vitro and in vivo, that antimuscarinics are protective regulators for the bladder structure and function. Antimuscarinics decrease the weight of bladders with BOO. Antimuscarinics improve the voiding parameter and enhance the contraction of bladder smooth muscle. The results also show that antimuscarinics inhibit the proliferation of bladder smooth muscle cells both in vivo and in vitro, it can reduce the collagen deposition and inflammatory cytokines in bladders with BOO. During this process, the expression of M2 and M3 receptors was altered by antimuscarinics. Conclusion: Antimuscarinics could reverse the structural and functional changes of BOO bladder wall at cellular and tissue level, and the alteration of M2 and M3 receptors may be involved in this biological process.

scholarly journals Bone morphogenetic protein 2 induces pulmonary angiogenesis via Wnt–β-catenin and Wnt–RhoA–Rac1 pathways

2009 ◽  
Vol 184 (1) ◽  
pp. 83-99 ◽  
Author(s):  
Vinicio A. de Jesus Perez ◽  
Tero-Pekka Alastalo ◽  
Jenny C. Wu ◽  
Jeffrey D. Axelrod ◽  
John P. Cooke ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Bone Morphogenetic Protein ◽  
Bone Morphogenetic Protein 2 ◽  
Immunodeficient Mice ◽  
Vascular Growth ◽  
Small Vessels ◽  
Angiogenesis Assay ◽  
Morphogenetic Protein ◽  
Pulmonary Arterial

Mutations in bone morphogenetic protein (BMP) receptor II (BMPRII) are associated with pulmonary artery endothelial cell (PAEC) apoptosis and the loss of small vessels seen in idiopathic pulmonary arterial hypertension. Given the low penetrance of BMPRII mutations, abnormalities in other converging signaling pathways may be necessary for disease development. We hypothesized that BMPRII supports normal PAEC function by recruiting Wingless (Wnt) signaling pathways to promote proliferation, survival, and motility. In this study, we report that BMP-2, via BMPRII-mediated inhibition of GSK3-β, induces β-catenin (β-C) accumulation and transcriptional activity necessary for PAEC survival and proliferation. At the same time, BMP-2 mediates phosphorylated Smad1 (pSmad1) or, with loss of BMPRII, pSmad3-dependent recruitment of Disheveled (Dvl) to promote RhoA–Rac1 signaling necessary for motility. Finally, using an angiogenesis assay in severe combined immunodeficient mice, we demonstrate that both β-C– and Dvl-mediated RhoA–Rac1 activation are necessary for vascular growth in vivo. These findings suggest that the recruitment of both canonical and noncanonical Wnt pathways is required in BMP-2–mediated angiogenesis.

scholarly journals Homodimerization of RBPMS2 through a new RRM-interaction motif is necessary to control smooth muscle plasticity

2014 ◽  
Vol 42 (15) ◽  
pp. 10173-10184 ◽  
Author(s):  
Sébastien Sagnol ◽  
Yinshan Yang ◽  
Yannick Bessin ◽  
Fréderic Allemand ◽  
Ilona Hapkova ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Sequence Motif ◽  
Rna Recognition Motif ◽  
Muscle Plasticity ◽  
Rrm Domain ◽  
Bone Morphogenetic Protein Pathway ◽  
Morphogenetic Protein

Abstract In vertebrates, smooth muscle cells (SMCs) can reversibly switch between contractile and proliferative phenotypes. This involves various molecular mechanisms to reactivate developmental signaling pathways and induce cell dedifferentiation. The protein RBPMS2 regulates early development and plasticity of digestive SMCs by inhibiting the bone morphogenetic protein pathway through its interaction with NOGGIN mRNA. RBPMS2 contains only one RNA recognition motif (RRM) while this motif is often repeated in tandem or associated with other functional domains in RRM-containing proteins. Herein, we show using an extensive combination of structure/function analyses that RBPMS2 homodimerizes through a particular sequence motif (D-x-K-x-R-E-L-Y-L-L-F: residues 39–51) located in its RRM domain. We also show that this specific motif is conserved among its homologs and paralogs in vertebrates and in its insect and worm orthologs (CPO and MEC-8, respectively) suggesting a conserved molecular mechanism of action. Inhibition of the dimerization process through targeting a conserved leucine inside of this motif abolishes the capacity of RBPMS2 to interact with the translational elongation eEF2 protein, to upregulate NOGGIN mRNA in vivo and to drive SMC dedifferentiation. Our study demonstrates that RBPMS2 possesses an RRM domain harboring both RNA-binding and protein-binding properties and that the newly identified RRM-homodimerization motif is crucial for the function of RBPMS2 at the cell and tissue levels.

scholarly journals Birth and delineation of the intestinal stem cell niche

2021 ◽  
Author(s):  
Neil McCarthy ◽  
Guodong Tie ◽  
Shariq Madha ◽  
Adrianna Maglieri ◽  
Judith Kraiczy ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Critical Period ◽  
Stem Cell Niche ◽  
De Novo ◽  
Cell Types ◽  
Muscle Layer ◽  
Intestinal Stem Cells ◽  
Morphogenetic Protein ◽  
Cell Niche

Wnt and Rspondin (RSPO) signaling triggers proliferation, and bone morphogenetic protein inhibitors (BMPi) impede differentiation, of intestinal stem cells (ISCs). Here we report that the functional ISC niche is a complex, multi-layered mesenchymal structure that includes distinct smooth muscle populations and describe how that niche organizes early in mouse life. Diverse sub-cryptal cells provide redundant supportive factors, with distinct BMPi and the most potent Wnt agonist, RSPO2, restricted to single cell types. Two functionally opposing elements arise in tandem during a critical period of crypt morphogenesis: a prominent shelf of BMP+ sub-epithelial myofibroblasts that promote epithelial differentiation and the muscularis mucosae, a specialized muscle layer generated de novo to supplement other RSPO and BMPi sources. In vivo ablation of smooth muscle, while preserving trophocytes, raises crypt BMP activity and potently limits crypt expansion. Thus, distinct and progressively refined mesenchymal components together create the milieu necessary to propagate crypts during rapid organ growth and to sustain ISCs in the adult niche.

scholarly journals Increased Adhesion and Aggregation of Platelets Lacking Cyclic Guanosine 3′,5′-Monophosphate Kinase I

1999 ◽  
Vol 189 (8) ◽  
pp. 1255-1264 ◽  
Author(s):  
Steffen Massberg ◽  
Matthias Sausbier ◽  
Peter Klatt ◽  
Markus Bauer ◽  
Alexander Pfeifer ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Platelet Adhesion ◽  
Ischemia Reperfusion ◽  
Vascular Lesions ◽  
Downstream Target ◽  
Platelet Adhesion And Aggregation ◽  
Smooth Muscle Proliferation ◽  
Aggregation Of Platelets

Atherosclerotic vascular lesions are considered to be a major cause of ischemic diseases, including myocardial infarction and stroke. Platelet adhesion and aggregation during ischemia–reperfusion are thought to be the initial steps leading to remodeling and reocclusion of the postischemic vasculature. Nitric oxide (NO) inhibits platelet aggregation and smooth muscle proliferation. A major downstream target of NO is cyclic guanosine 3′,5′-monophosphate kinase I (cGKI). To test the intravascular significance of the NO/cGKI signaling pathway in vivo, we have studied platelet–endothelial cell and platelet–platelet interactions during ischemia/reperfusion using cGKI-deficient (cGKI−/−) mice. Platelet cGKI but not endothelial or smooth muscle cGKI is essential to prevent intravascular adhesion and aggregation of platelets after ischemia. The defect in platelet cGKI is not compensated by the cAMP/cAMP kinase pathway supporting the essential role of cGKI in prevention of ischemia-induced platelet adhesion and aggregation.

scholarly journals Role of the Cldn6 Cytoplasmic Tail Domain in Membrane Targeting and Epidermal Differentiation In Vivo

2006 ◽  
Vol 26 (15) ◽  
pp. 5876-5887 ◽  
Author(s):  
Azadeh Arabzadeh ◽  
Tammy-Claire Troy ◽  
Kursad Turksen
Keyword(s):  
Protein Unfolding ◽  
Cytoplasmic Tail ◽  
Epidermal Differentiation ◽  
Permeability Barrier ◽  
Membrane Targeting ◽  
Tail Domain ◽  
Truncation Mutant ◽  
And Function

ABSTRACT It is widely recognized that the claudin (Cldn) family of four tetraspan transmembrane proteins is crucial for tight junction assembly and permeability barrier function; however, the precise role of the tail and loop domains in Cldn function is not understood. We hypothesized that the cytoplasmic tail domain of Cldn6 is crucial for membrane targeting and hence epidermal permeability barrier (EPB) formation. To test this hypothesis via a structure-function approach, we generated a tail deletion of Cldn6 (CΔ187) and evaluated its role in epidermal differentiation and EPB formation through its forced expression via the involucrin (Inv) promoter in the suprabasal compartment of the transgenic mouse epidermis. Even though a functional barrier formed, Inv-CΔ187 mice displayed histological and biochemical abnormalities in the epidermal differentiation program and stimulation of epidermal cell proliferation in both the basal and suprabasal compartments of the interfolliclar epidermis, leading to a thickening of the epidermis after 1 week of age that persisted throughout life. Although some membrane localization was evident, our studies also revealed a significant amount of not only Cldn6 but also Cldn10, Cldn11, and Cldn18 in the cytoplasm of transgenic epidermal cells as well as the activation of a protein-unfolding pathway. These findings demonstrate that the overexpression of a tail truncation mutant of Cldn6 mislocalizes Cldn6 and other Cldn proteins to the cytoplasm and triggers a postnatal increase in proliferation and aberrant differentiation of the epidermis, emphasizing the importance of the Cldn tail domain in membrane targeting and function in vivo.

scholarly journals Regulation and function of bone morphogenetic protein signaling in colonic injury and inflammation

2017 ◽  
Vol 312 (1) ◽  
pp. G24-G33 ◽  
Author(s):  
Tuo Ji ◽  
Hidehiko Takabayashi ◽  
Maria Mao ◽  
Xu Han ◽  
Xiang Xue ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Actin ◽  
Bmp Signaling ◽  
Muscle Actin ◽  
Colonic Injury ◽  
Colonic Inflammation ◽  
Morphogenetic Protein ◽  
Tnf Α ◽  
Ifn Γ ◽  
And Function

The bone morphogenetic proteins (BMPs) regulate gastrointestinal homeostasis. We investigated the expression of BMP-4 and the localization and function of BMP signaling during colonic injury and inflammation. Mice expressing the β-galactosidase ( β-gal) gene under the control of a BMP-responsive element (BRE), BMP-4-β-gal/ mice, and animals generated by crossing villin-Cre mice to mice with floxed alleles of BMP receptor 1A ( villin-Cre;Bmpr1a flox/flox) were treated with dextran sodium sulfate (DSS) to induce colonic injury and inflammation. Expression of BMP-4, β-gal, BMPR1A, IL-8, α-smooth muscle actin, and phosphorylated Smad1, -5, and -8 was assessed by X-Gal staining, quantitative RT-PCR, and immunohistochemistry. Morphology of the colonic mucosa was examined by staining with hematoxylin and eosin. The effect of IFN-γ, TNF-α, IL-1β, and IL-6 on BMP-4 mRNA expression was investigated in human intestinal fibroblasts, whereas that of BMP-4 on IL-8 was assessed in human colonic organoids. BMP-4 was localized in α-smooth muscle actin-positive mesenchymal cells while the majority of BMP-generated signals targeted the epithelium. DSS caused injury and inflammation leading to reduced expression of BMP-4 and of BMPR1A mRNAs, and to decreased BMP signaling. Deletion of BMPR1A enhanced colonic inflammation and damage. Administration of anti-TNF-α antibodies to DSS-treated mice ameliorated colonic inflammation and increased the expression of BMP-4 and BMPR1A mRNAs. TNF-α and IL-1β inhibited both basal and IFN-γ-stimulated BMP-4 expression, whereas IL-6 had no effect. BMP-4 reduced TNF-α-stimulated IL-8 mRNA expressor IL-8 mRNA expression in the organoids. Inflammation and injury inhibit BMP-4 expression and signaling, leading to enhanced colonic damage and inflammation. These observations underscore the importance of BMP signaling in the regulation of intestinal inflammation and homeostasis. NEW & NOTEWORTHY In this study we report a series of novel observations that underscore the importance of bone morphogenetic protein (BMP) signaling in the regulation of colonic homeostasis during the development of injury and inflammation. In particular, we present evidence that BMP signaling mitigates the response of the colonic epithelium to injury and inflammation and that cytokines, such as TNF-α and IL-1β, inhibit the expression of BMP-4.

Behavior and Cell-to-Cell Interaction of Cultured Endothelia and Smooth Muscle Cells Seeded on Nucleopore Filter

1990 ◽  
Vol 48 (3) ◽  
pp. 160-161
Author(s):  
Shams Ghoneim ◽  
Zsuzsa Fabry ◽  
Judy Keiner ◽  
Michael Hart
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
In Vitro Study ◽  
Muscle Cells ◽  
Cell Interaction ◽  
Small Vessels ◽  
Close Relationship ◽  
Endothelial Basement Membrane

Vascular endothelia and smooth muscle cultures, derived from small vessels may retain some of their in vivo characteristics. It has been demonstrated by other investigators that there exists in-vivo a close relationship between endothelia cells and smooth muscle cells of most vascular beds. The myo-endothelial junction has functional implications which could explain a humoral transmition of substances between the two cell population. Rhodin demonstrated that arteriolar endothelium exhibited foot-like processes which penetrated the endothelial basement membrane and extended into smooth muscle cells of the media(1). Fawcett, believed that a pathway exists between endothelia and smooth muscle cells for the exchange of metabolites (2).The objective of this in-vitro study was to examine the possible relationship between smooth muscle and endothelia cells in culture. Nucleopore filters with both 0.4μ and 3.0μ pores were used. They were seeded for 6 days with mouse brain endothelia cells on the top side of the filter and for 7 days with smooth muscle cells on the bottom side.

Biomolecule-Impregnated Nanocomposite With Spatiotemporal Control Over Release and Degradation Kinetic for Vascular Engineering

2010 ◽  
Author(s):  
Walter Bonani ◽  
Antonella Motta ◽  
Claudio Migliaresi ◽  
Wei Tan
Keyword(s):  
Degradation Kinetics ◽  
Small Diameter ◽  
Degradation Kinetic ◽  
Small Vessels ◽  
Vascular Bypass ◽  
Synthetic Materials ◽  
Vascular Engineering ◽  
And Function

Autologous vessels are the gold standard for small-diameter (<6 mm) vascular bypass; however, many patients lack suitable autologous tissues due to diseases or prior vein harvest. As an alternative, synthetic vascular grafts made from bioinert synthetic materials such as polytetrafluoroethylene (PTFE) are currently used in the medical field. The high long-term failure rate of these materials in the replacement of small vessels is known to be associated with the lack of proper signalling events by PTFE to vascular cells causing adverse hemodynamic, inflammatory or coagulatory conditions. Therefore, constant and pressing is the demand for a more biocompatible conduit with structure and function similar to native vessels. For this reason, bioresorbable scaffold constructs which can provide not only proper mechanical support, but also precise molecular cues, are desired (1). In particular, proper degradation kinetics and molecule release profiles are needed to facilitate remodeling and integration process in vivo over the time for long-term patency (2).

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