Abstract 124: Integrated Omics Approaches to Elucidate Targets of Cyclic AMP-Epac-Rap1A Signaling in Microvascular Smooth Muscle Cells: Identification of Role in Production and Deposition of Extracellular Matrix Fibrillar Collagen

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Maqsood A Chotani
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Cyclic Amp ◽  
Blood Vessels ◽  
Intracellular Signaling ◽  
Small Gtpase ◽  
Cytoskeletal Proteins ◽  
Global Changes ◽  
Fibrillar Collagen ◽  
Healthy Human

Objective: Intracellular signaling by cyclic AMP (cAMP) has an essential role in vascular smooth muscle (VSM) physiology, including relaxation of large blood vessels such as aorta and pulmonary artery. Studies support a mechanism of signaling through e xchange p rotein a ctivated by c AMP (epac) and Ras-related small GTPase Rap1B down-regulation of RhoA activity. The role of cAMP in small blood vessels however was not examined, and remained unknown. This study elucidated the targets of cAMP signaling in peripheral blood vessels, specifically VSM explanted from healthy human (h) cutaneous arterioles and mouse (m) tail artery (microVSM). Results: Global changes in protein expression were assessed by Differential-in-Gel Electrophoresis in quiescent (h)-microVSM treated with the adenylate cyclase activator and cAMP elevating agent forskolin (10 μM, 30 min or 18 hr). Detectable changes were observed in proteins associated with the cytoskeleton, stress-response, protein-synthesis, -folding, -membrane transport, and extracellular matrix. Notably, actin modulating proteins caldesmon, ezrin-moesin, zyxin, gelsolin, and α-adducin, as well as cytoskeletal proteins, tubulin and vimentin were differentially expressed ( P< 0.05; 3-5 replicates). These results agreed with our recent demonstration of cAMP activation of epac-Rap1A, and RhoA-ROCK-F-actin signaling in (h)- and (m)-microVSM to increase expression and cell surface transport of functional α 2C -adrenoceptors (α 2C -ARs) that mediate vasoconstriction. Transcriptome analysis of Rap1A-null (knockout) (m)-microVSM transduced with constitutively active Rap1A showed connections to signaling linked to vessel production and deposition of extracellular matrix fibrillar collagen, compared with control cells ( P< 0.05, 4 replicates). Conclusions: Rap1 subtypes have a discrete role in the vasculature. This study links cAMP-Rap1A signaling to collagen and α 2C -AR expression in arteriole VSM. It suggests over-activation of Rap1A-coupled signaling in the peripheral circulation during chronic inflammation could lead to increased α 2C -AR mediated vessel reactivity, progressive perivascular fibrosis, and dysfunction as seen in human pathologies such as Raynaud’s phenomenon and scleroderma.

scholarly journals Cyclic AMP-Rap1A signaling mediates cell surface translocation of microvascular smooth muscle α2C-adrenoceptors through the actin-binding protein filamin-2

2013 ◽  
Vol 305 (8) ◽  
pp. C829-C845 ◽  
Author(s):  
Hanaa K. B. Motawea ◽  
Selvi C. Jeyaraj ◽  
Ali H. Eid ◽  
Srabani Mitra ◽  
Nicholas T. Unger ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Cyclic Amp ◽  
Cell Surface ◽  
Smooth Muscle Cells ◽  
Blood Vessels ◽  
Muscle Cells ◽  
Small Gtpase ◽  
Actin Binding ◽  
Surface Translocation

The second messenger cyclic AMP (cAMP) plays a vital role in vascular physiology, including vasodilation of large blood vessels. We recently demonstrated cAMP activation of Epac-Rap1A and RhoA-Rho-associated kinase (ROCK)-F-actin signaling in arteriolar-derived smooth muscle cells increases expression and cell surface translocation of functional α2C-adrenoceptors (α2C-ARs) that mediate vasoconstriction in small blood vessels (arterioles). The Ras-related small GTPAse Rap1A increased expression of α2C-ARs and also increased translocation of perinuclear α2C-ARs to intracellular F-actin and to the plasma membrane. This study examined the mechanism of translocation to better understand the role of these newly discovered mediators of blood flow control, potentially activated in peripheral vascular disorders. We utilized a yeast two-hybrid screen with human microvascular smooth muscle cells (microVSM) cDNA library and the α2C-AR COOH terminus to identify a novel interaction with the actin cross-linker filamin-2. Yeast α-galactosidase assays, site-directed mutagenesis, and coimmunoprecipitation experiments in heterologous human embryonic kidney (HEK) 293 cells and in human microVSM demonstrated that α2C-ARs, but not α2A-AR subtype, interacted with filamin. In Rap1-stimulated human microVSM, α2C-ARs colocalized with filamin on intracellular filaments and at the plasma membrane. Small interfering RNA-mediated knockdown of filamin-2 inhibited Rap1-induced redistribution of α2C-ARs to the cell surface and inhibited receptor function. The studies suggest that cAMP-Rap1-Rho-ROCK signaling facilitates receptor translocation and function via phosphorylation of filamin-2 Ser2113. Together, these studies extend our previous findings to show that functional rescue of α2C-ARs is mediated through Rap1-filamin signaling. Perturbation of this signaling pathway may lead to alterations in α2C-AR trafficking and physiological function.

scholarly journals Cyclic AMP-Rap1A signaling activates RhoA to induce α2c-adrenoceptor translocation to the cell surface of microvascular smooth muscle cells

2012 ◽  
Vol 303 (5) ◽  
pp. C499-C511 ◽  
Author(s):  
Selvi C. Jeyaraj ◽  
Nicholas T. Unger ◽  
Ali H. Eid ◽  
Srabani Mitra ◽  
N. Paul El-Dahdah ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Cyclic Amp ◽  
Cell Surface ◽  
Smooth Muscle Cells ◽  
Intracellular Signaling ◽  
Muscle Cells ◽  
Small Gtpase ◽  
Hek293 Cells ◽  
Intracellular Camp

Intracellular signaling by the second messenger cyclic AMP (cAMP) activates the Ras-related small GTPase Rap1 through the guanine exchange factor Epac. This activation leads to effector protein interactions, activation, and biological responses in the vasculature, including vasorelaxation. In vascular smooth muscle cells derived from human dermal arterioles (microVSM), Rap1 selectively regulates expression of G protein-coupled α2C-adrenoceptors (α2C-ARs) through JNK-c-jun nuclear signaling. The α2C-ARs are generally retained in the trans-Golgi compartment and mobilize to the cell surface and elicit vasoconstriction in response to cellular stress. The present study used human microVSM to examine the role of Rap1 in receptor localization. Complementary approaches included murine microVSM derived from tail arteries of C57BL6 mice that express functional α2C-ARs and mice deficient in Rap1A (Rap1A-null). In human microVSM, increasing intracellular cAMP by direct activation of adenylyl cyclase by forskolin (10 μM) or selectively activating Epac-Rap signaling by the cAMP analog 8-pCPT-2′- O-Me-cAMP (100 μM) activated RhoA, increased α2C-AR expression, and reorganized the actin cytoskeleton, increasing F-actin. The α2C-ARs mobilized from the perinuclear region to intracellular filamentous structures and to the plasma membrane. Similar results were obtained in murine wild-type microVSM, coupling Rap1-Rho-actin dynamics to receptor relocalization. This signaling was impaired in Rap1A-null murine microVSM and was rescued by delivery of constitutively active (CA) mutant of Rap1A. When tested in heterologous HEK293 cells, Rap1A-CA or Rho-kinase (ROCK-CA) caused translocation of functional α2C-ARs to the cell surface (∼4- to 6-fold increase, respectively). Together, these studies support vascular bed-specific physiological role of Rap1 and suggest a role in vasoconstriction in microVSM.

scholarly journals Multiple Microvascular and Astroglial 5-Hydroxytryptamine Receptor Subtypes in Human Brain: Molecular and Pharmacologic Characterization

1999 ◽  
Vol 19 (8) ◽  
pp. 908-917 ◽  
Author(s):  
Zvi Cohen ◽  
Isabelle Bouchelet ◽  
André Olivier ◽  
Jean-Guy Villemure ◽  
Rita Ball ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Cyclic Amp ◽  
Human Brain ◽  
Blood Vessels ◽  
Cell Cultures ◽  
Receptor Subtypes ◽  
Reaction Products ◽  
Astroglial Cells ◽  
Complex Interactions ◽  
Capillary Endothelial Cells

Physiologic and anatomic evidence suggest that 5-hydroxytryptamine (5-HT) neurons regulate local cerebral blood flow and blood-brain barrier permeability. To evaluate the possibility that some of these effects occur directly on the blood vessels, molecular and/or pharmacologic approaches were used to assess the presence of 5-HT receptors in human brain microvascular fractions, endothelial and smooth muscle cell cultures, as well as in astroglial cells which intimately associate with intraparenchymal blood vessels. Isolated microvessels and' capillaries consistently expressed messages for the h5-HT1B, h5-HT1D, 5-HT1F, 5-HT2A but not 5-HT7 receptors. When their distribution within the vessel wall was studied in more detail, it was found that capillary endothelial cells exhibited mRNA for the h5-HT1D and for the 5-HT7 receptors whereas microvascular smooth muscle cells, in addition to h5-HT1D and 5-HT7, also showed polymerase chain reaction products for h5-HT1B receptors. Expression of 5-HT1F and 5-HT2A receptor mRNAs was never detected in any of the microvascular cell cultures. In contrast, messages for all 5-HT receptors tested were detected in human brain astrocytes with a predominance of the 5-HT2A and 5-HT7 subtypes. In all cultures, sumatriptan inhibited (35–58%, P < .05) the forskolin-stimulated production of cyclic AMP, an effect blocked by the 5-HT1B/1d receptor antagonists GR127935 and GR55562. In contrast, 5-carboxamidotryptamine induced strong increases (≥ 400%, P < .005) in basal cyclic AMP levels that were abolished by mesulergine, a nonselective 5-HT7 receptor antagonist. Only astroglial cells showed a ketanserin-sensitive increase (177%, P < .05) in IP3 formation when exposed to 5-HT. These results show that specific populations of functional 5-HT receptors are differentially distributed within the various cellular compartments of the human cortical microvascular bed, and that human brain astroglial cells are endowed with multiple 5-HT receptors. These findings emphasize the complex interactions between brain serotonergic pathways and non-neuronal cells within the CNS and, further, they raise the possibility that some of these receptors may be activated by antimigraine compounds such as brain penetrant triptan derivatives.

scholarly journals Cytoskeleton and Adhesion in Myogenesis

2014 ◽  
Vol 2014 ◽  
pp. 1-15 ◽  
Author(s):  
Manoel Luís Costa
Keyword(s):  
Skeletal Muscle ◽  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Muscle Function ◽  
Force Production ◽  
Structure And Function ◽  
Cytoskeletal Proteins ◽  
Skeletal Muscle Differentiation ◽  
And Function ◽  
Structural Adaptations

The function of muscle is to contract, which means to exert force on a substrate. The adaptations required for skeletal muscle differentiation, from a prototypic cell, involve specialization of housekeeping cytoskeletal contracting and supporting systems into crystalline arrays of proteins. Here I discuss the changes that all three cytoskeletal systems (microfilaments, intermediate filaments, and microtubules) undergo through myogenesis. I also discuss their interaction, through the membrane, to extracellular matrix and to other cells, where force will be exerted during contraction. The three cytoskeletal systems are necessary for the muscle cell and must exert complementary roles in the cell. Muscle is a responsive system, where structure and function are integrated: the structural adaptations it undergoes depend on force production. In this way, the muscle cytoskeleton is a portrait of its physiology. I review the cytoskeletal proteins and structures involved in muscle function and focus particularly on their role in myogenesis, the process by which this incredible muscle machine is made. Although the focus is on skeletal muscle, some of the discussion is applicable to cardiac and smooth muscle.

scholarly journals Different DNA methylome, transcriptome and histological features in uterine fibroids with and without MED12 mutations

2021 ◽  
Author(s):  
Ryo Maekawa ◽  
Shun Sato ◽  
Tetsuro Tamehisa ◽  
Takahiro Sakai ◽  
Takuya Kajimura ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Blood Vessels ◽  
Uterine Fibroids ◽  
Muscle Cells ◽  
Histological Features ◽  
Dna Methylome

Abstract Background: Somatic mutations in Mediator complex subunit 12 (MED12m) have been reported as a biomarker of uterine fibroids (UFs). However, the role of MED12m is still unclear in the pathogenesis of UFs. Therefore, we investigated the differences in DNA methylome, transcriptome, and histological features between MED12m-positive and -negative UFs. Methods: DNA methylomes and transcriptomes were obtained from MED12m-positive and -negative UFs and myometrium, and hierarchically clustered. Differentially expressed genes in comparison with the myometrium and co-expressed genes detected by weighted gene co-expression network analysis were subjected to gene ontology enrichment analyses. The amounts of collagen fibers and the number of blood vessels and smooth muscle cells were histologically evaluated. Results: Hierarchical clustering based on DNA methylation clearly separated the myometrium, MED12m-positive, and MED12m-negative UFs. MED12m-positive UFs had the increased activities of extracellular matrix formation, whereas MED12m-negative UFs had the increased angiogenic activities and smooth muscle cell proliferation. Conclusion: The MED12m-positive and -negative UFs had different DNA methylation, gene expression, and histological features. The MED12m-positive UFs form the tumor with a rich extracellular matrix and poor blood vessels and smooth muscle cells compared to the MED12m-negative UFs, suggesting MED12 mutations affect the tissue composition of UFs.

scholarly journals Molecular Mechanisms for the Mechanical Modulation of Airway Responsiveness

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Wenwu Zhang ◽  
Susan J. Gunst
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Actin Polymerization ◽  
Molecular Mechanisms ◽  
Airway Responsiveness ◽  
Small Gtpase ◽  
Cortical Actin

The smooth muscle of the airways is exposed to continuously changing mechanical forces during normal breathing. The mechanical oscillations that occur during breathing have profound effects on airway tone and airway responsiveness both in experimental animals and humans in vivo and in isolated airway tissues in vitro. Experimental evidence suggests that alterations in the contractile and mechanical properties of airway smooth muscle tissues caused by mechanical perturbations result from adaptive changes in the organization of the cytoskeletal architecture of the smooth muscle cell. The cytoskeleton is a dynamic structure that undergoes rapid reorganization in response to external mechanical and pharmacologic stimuli. Contractile stimulation initiates the assembly of cytoskeletal/extracellular matrix adhesion complex proteins into large macromolecular signaling complexes (adhesomes) that undergo activation to mediate the polymerization and reorganization of a submembranous network of actin filaments at the cortex of the cell. Cortical actin polymerization is catalyzed by Neuronal-Wiskott–Aldrich syndrome protein (N-WASP) and the Arp2/3 complex, which are activated by pathways regulated by paxillin and the small GTPase, cdc42. These processes create a strong and rigid cytoskeletal framework that may serve to strengthen the membrane for the transmission of force generated by the contractile apparatus to the extracellular matrix, and to enable the adaptation of smooth muscle cells to mechanical stresses. This model for the regulation of airway smooth muscle function can provide novel perspectives to explain the normal physiologic behavior of the airways and pathophysiologic properties of the airways in asthma.

scholarly journals Characterization of Collagens Fibers (I, III, IV) and Elastin in the Extracellular Matrix of Normal and Neoplastic Canine Prostate

2019 ◽  
Author(s):  
Luis Gabriel Rivera Calderón ◽  
Priscila Emiko Kobayashi ◽  
Rosemeri Oliveira Vasconcelos ◽  
Carlos Eduardo Fonseca-Alves ◽  
Renee Laufer-Amorim
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Blood Vessels ◽  
Basal Membrane ◽  
Elastic Fibers ◽  
Normal Prostate ◽  
Type Iv ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Imagej Software

Collagen (Coll) is the most common protein in the extracellular matrix, responsible for providing tissue structure and support. In some types of cancer, including prostate cancer (PC) abundant collagen was identified and related to tumor progression and metastasis. This study aimed to investigate Coll-I, III, IV and elastin in normal canine prostatic tissue and PC, using the Picrosirius red (PSR) and Immunohistochemical (IHC) analysis. Eight normal prostates and 10 PC from formalin-fixed, paraffin-embedded samples were used. Collagen fibers area was analyzed with ImageJ software. The distribution of Coll-I and Col-III was approximately 80% around prostatic ducts and acini, 15% among smooth muscle and 5% around in blood vessels, in both normal prostate and PC. Immunostaining for type IV collagen was observed in the basal membrane of prostate acini, smooth muscle, blood vessels, and never fibers of normal and PC samples. Elastic fibers were found in the septa dividing the lobules and around the prostatic acini of normal samples. A high amount of elastic fibers was observed around the ducts and the urethra in normal and canine PC. The distribution and area percentage of staining for collagen are similar in normal and neoplastic canine prostate when analyzed with PSR and IHC.

scholarly journals Characterization of Collagens Fibers (I, III, IV) and Elastin in the Extracellular Matrix of Normal and Neoplastic Canine Prostatic Tissues

2019 ◽  
Author(s):  
Luis Gabriel Rivera Calderón ◽  
Priscila Emiko Kobayashi ◽  
Rosemeri Oliveira Vasconcelos ◽  
Carlos Eduardo Fonseca-Alves ◽  
Renee Laufer-Amorim
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Blood Vessels ◽  
Basal Membrane ◽  
Elastic Fibers ◽  
Normal Prostate ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Imagej Software ◽  
Area Percentage

Collagen (Coll) is the most common protein in the extracellular matrix, responsible for providing tissue structure and support. In some types of cancer, including prostate cancer (PC) Coll deregulation was described and related to tumor progression and metastasis. This study aimed to investigate Coll-I, III, IV and elastin in canine normal prostate and PC, using Picrosirius red (PSR) and Immunohistochemical (IHC) analysis. Eight normal prostates and 10 PC from formalin-fixed, paraffin-embedded samples were used. Collagen fibers area was analyzed with ImageJ software. The distribution of Coll-I and Coll-III was approximately 80% around prostatic ducts and acini, 15% among smooth muscle and 5% around in blood vessels, in both normal prostate and PC. Immunostaining for Coll-IV was observed in the basal membrane of prostate acini, smooth muscle, blood vessels, and never fibers of normal and PC samples. Elastic fibers were found in the septa dividing the lobules and around the prostatic acini of normal samples. A high amount of elastic fibers was observed around the ducts and the urethra in normal and PC. The distribution and area percentage of staining for collagen are similar in normal and neoplastic canine prostate when analyzed with PSR and IHC.

scholarly journals Matrix mechanotransduction mediated by thrombospondin-1/integrin/YAP in the vascular remodeling

2020 ◽  
Vol 117 (18) ◽  
pp. 9896-9905 ◽  
Author(s):  
Yoshito Yamashiro ◽  
Bui Quoc Thang ◽  
Karina Ramirez ◽  
Seung Jae Shin ◽  
Tomohiro Kohata ◽  
...  
Keyword(s):  
Blood Vessels ◽  
Intracellular Signaling ◽  
Vascular Remodeling ◽  
Vessel Wall ◽  
Focal Adhesions ◽  
Small Gtpase ◽  
Cyclic Stretch ◽  
Matricellular Protein ◽  
Mechanical Stimuli ◽  
Thrombospondin 1

The extracellular matrix (ECM) initiates mechanical cues that activate intracellular signaling through matrix–cell interactions. In blood vessels, additional mechanical cues derived from the pulsatile blood flow and pressure play a pivotal role in homeostasis and disease development. Currently, the nature of the cues from the ECM and their interaction with the mechanical microenvironment in large blood vessels to maintain the integrity of the vessel wall are not fully understood. Here, we identified the matricellular protein thrombospondin-1 (Thbs1) as an extracellular mediator of matrix mechanotransduction that acts via integrin αvβ1 to establish focal adhesions and promotes nuclear shuttling of Yes-associated protein (YAP) in response to high strain of cyclic stretch. Thbs1-mediated YAP activation depends on the small GTPase Rap2 and Hippo pathway and is not influenced by alteration of actin fibers. Deletion of Thbs1 in mice inhibited Thbs1/integrin β1/YAP signaling, leading to maladaptive remodeling of the aorta in response to pressure overload and inhibition of neointima formation upon carotid artery ligation, exerting context-dependent effects on the vessel wall. We thus propose a mechanism of matrix mechanotransduction centered on Thbs1, connecting mechanical stimuli to YAP signaling during vascular remodeling in vivo.

scholarly journals Collective cell traction force analysis on aligned smooth muscle cell sheet between three-dimensional microwalls

2014 ◽  
Vol 4 (2) ◽  
pp. 20130056 ◽  
Author(s):  
Ying Zhang ◽  
Soon Seng Ng ◽  
Yilei Wang ◽  
Huixing Feng ◽  
Wei Ning Chen ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Smooth Muscle ◽  
Blood Vessels ◽  
Cell Sheet ◽  
Adhesion Formation ◽  
Three Dimensional ◽  
Traction Force ◽  
Cytoskeletal Proteins ◽  
Cell Alignment ◽  
Biomaterial Scaffold

During the past two decades, novel biomaterial scaffold for cell attachment and culture has been developed for applications in tissue engineering, biosensing and regeneration medicine. Tissue engineering of blood vessels remains a challenge owing to the complex three-layer histology involved. In order to engineer functional blood vessels, it is essential to recapitulate the characteristics of vascular smooth muscle cells (SMCs) inside the tunica media, which is known to be critical for vasoconstriction and vasodilation of the circulatory system. Until now, there has been a lack of understanding on the mechanotransduction of the SMC layer during the transformation from viable synthetic to quiescent contractile phenotypes. In this study, microfabricated arrays of discontinuous microwalls coated with fluorescence microbeads were developed to probe the mechanotransduction of the SMC layer. First, the system was exploited for stimulating the formation of a highly aligned orientation of SMCs in native tunica medium. Second, atomic force microscopy in combination with regression analysis was applied to measure the elastic modulus of a polyacrylamide gel layer coated on the discontinuous microwall arrays. Third, the conventional traction force assay for single cell measurement was extended for applications in three-dimensional cell aggregates. Then, the biophysical effects of discontinuous microwalls on the mechanotransduction of the SMC layer undergoing cell alignment were probed. Generally, the cooperative multiple cell–cell and cell–microwall interactions were accessed quantitatively by the newly developed assay with the aid of finite-element modelling. The results show that the traction forces of highly aligned cells lying in the middle region between two opposing microwalls were significantly lower than those lying adjacent to the microwalls. Moreover, the spatial distributions of Von Mises stress during the cell alignment process were dependent on the collective cell layer orientation. Immunostaining of the SMC sheet further demonstrated that the collective mechanotransduction induced by three-dimensional topographic cues was correlated with the reduction of actin and vinculin expression. In addition, the online two-dimensional LC–MS/MS analysis verified the modulation of focal adhesion formation under the influence of microwalls through the regulation in the expression of three key cytoskeletal proteins.

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