Localization In Smooth Muscle Cells Research Articles

ImmunoSERS microscopy for the detection of smooth muscle cells in atherosclerotic plaques

We investigated the suitability of immuno-SERS (iSERS) microscopy for imaging of smooth muscle cells (SMCs) in atherosclerotic plaques. Localization of SMCs is achieved by using SERS-labelled antibodies direct against alpha-smooth muscle actin (SMA). The staining quality of the false-colour iSERS images obtained by confocal Raman microscopy with point mapping is compared with wide-field immunofluorescence images. Both direct (labelled primary antibody) and indirect iSERS staining (unlabelled primary and labelled secondary antibody) techniques were employed. Direct iSERS staining yields results comparable to indirect IF staining, demonstrating the suitability of iSERS in research on atherosclerosis and paving the way for future multiplexed imaging experiments.

Apelin Reduces Nitric Oxide-Induced Relaxation of Cerebral Arteries by Inhibiting Activation of Large-Conductance, Calcium-Activated K Channels.

Activation of the apelin/APJ receptor signaling system causes endothelium-dependent and nitric oxide (NO)-dependent relaxation in several peripheral arteries. The effects of apelin in cerebral arteries are unknown; however, apelin inhibits voltage-dependent increases in large-conductance, calcium-activated K channel (BKCa) currents in cerebral artery smooth muscle cells. Because NO-induced relaxation of cerebral arteries is mediated, in part, by activation of BKCa channels, the goals of this study were to determine the net effect of apelin in cerebral arteries, as well as test the hypothesis that the actions of apelin in cerebral arteries are secondary to stimulation of APJ receptors. Immunoblot and quantitative reverse transcription polymerase chain reaction analyses detected APJ receptors in cerebral arteries of male Sprague-Dawley rats, and immunofluorescence studies using confocal microscopy confirmed APJ receptor localization in smooth muscle cells. In myograph studies, apelin itself had no direct vasomotor effect but inhibited relaxations to the NO-donor, diethylamine NONOate, and to the endothelium-dependent vasodilator, bradykinin. These effects of apelin were mimicked by the selective BKCa-channel blocker, iberiotoxin, and suppressed by the APJ receptor antagonist, F13A. Apelin also inhibited relaxations evoked by the BKCa-channel openers, NS1619 and BMS 191011, but had no effect on relaxation to levcromakalim, a selective KATP-channel opener. Apelin had no effect on diethylamine NONOate-induced or bradykinin-induced increases in cyclic guanosine monophosphate levels. Patch clamp recordings demonstrated that apelin and iberiotoxin each suppressed the increase in BKCa currents induced by DEA and NS1619 in freshly isolated cerebral artery smooth muscle cells. The results demonstrate that apelin inhibits NO-induced relaxation of cerebral arteries through a mechanism involving activation of APJ receptors and inhibition of BKCa channels in cerebral arterial smooth muscle cells.

MP19-09 NSC23766, A RAC GTPASE INHIBITOR, INHIBITS SMOOTH MUSCLE CONTRACTION IN THE HYPERPLASTIC HUMAN PROSTATE

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research1 Apr 2014MP19-09 NSC23766, A RAC GTPASE INHIBITOR, INHIBITS SMOOTH MUSCLE CONTRACTION IN THE HYPERPLASTIC HUMAN PROSTATE Thomas Kunit, Andrea Schreiber, Christian Gratzke, Beata Rutz, Raphaela Waidelich, Frank Strittmatter, Christian G. Stief, and Martin Hennenberg Thomas KunitThomas Kunit More articles by this author , Andrea SchreiberAndrea Schreiber More articles by this author , Christian GratzkeChristian Gratzke More articles by this author , Beata RutzBeata Rutz More articles by this author , Raphaela WaidelichRaphaela Waidelich More articles by this author , Frank StrittmatterFrank Strittmatter More articles by this author , Christian G. StiefChristian G. Stief More articles by this author , and Martin HennenbergMartin Hennenberg More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.710AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Prostate smooth muscle contraction may be critical for pathophysiology and therapy of lower urinary tract symptoms (LUTS) in patients with benign prostate hyperplasia (BPH). Although several options are available, medical therapy still remains a challenge, in particular with respect to the imminent demographic transition. Consequently, new strategies for inhibition of smooth muscle contraction in the lower urinary tract are of interest. Rac, a small monomeric GTPase, is an important regulator of smooth muscle tone outside the lower urinary tract. Here, we studied Rac inhibition and expression in the human prostate. METHODS Periurethral prostate tissues were obtained from patients undergoing radical prostatectomy (n=27). Effects of the Rac inhibitor, NSC23766 on adrenergic contraction were studied in the organ bath. Rac expression was examined by Western blot analysis, and immunofluorescence staining. RESULTS The α1-adrenoceptor agonist, phenylephrine (0.1-100 μM), induced concentration-dependent contractions of human prostate strips. NSC23766 (50 μM) inhibited phenylephrine-induced contractions (Fig.). This was significant at 0.3 μM phenylephrine (p<0.05 for NSC23766 vs. control), 1 μM (p<0.003), 3 μM (p<0.002), 10 μM (p<0.0002), and 30 μM (p<0.03). Similarly, NSC23766 (50 μM) inhibited noradrenaline-induced contractions (Fig.). This was significant at 0.3 μM (p<0.05) noreadrenaline, 1 μM (p<0.02), 3 μM (p<0.03), 10 μM (p<0.02), 30 μM (p<0.02), and 100 μM (p<0.03). In Western blot analysis revealed bands with the expected size for Rac1 in prostate tissue from each patient being included in this analysis. After immunofluorescence staining, immunoreactivity for Rac1 was observed in the prostate stroma, and in glands. Double labeling with calponin and pan-cytokeratin indicated localization in smooth muscle cells and in epithelial cells. CONCLUSIONS Rac is critical for prostate smooth muscle contraction. Regulation of smooth muscle tone in the lower urinary tract by Rac may be involved in pathophysiology of LUTS, and may represent a putative new target for therapy. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e192 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Thomas Kunit More articles by this author Andrea Schreiber More articles by this author Christian Gratzke More articles by this author Beata Rutz More articles by this author Raphaela Waidelich More articles by this author Frank Strittmatter More articles by this author Christian G. Stief More articles by this author Martin Hennenberg More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Correction: Phenotypic Switching Induced by Damaged Matrix Is Associated with DNA Methyltransferase 3A (DNMT3A) Activity and Nuclear Localization in Smooth Muscle Cells (SMC)

Correction: Phenotypic Switching Induced by Damaged Matrix Is Associated with DNA Methyltransferase 3A (DNMT3A) Activity and Nuclear Localization in Smooth Muscle Cells (SMC)

Phenotypic Switching Induced by Damaged Matrix Is Associated with DNA Methyltransferase 3A (DNMT3A) Activity and Nuclear Localization in Smooth Muscle Cells (SMC)

Extracellular matrix changes are often crucial inciting events for fibroproliferative disease. Epigenetic changes, specifically DNA methylation, are critical factors underlying differentiated phenotypes. We examined the dependency of matrix-induced fibroproliferation and SMC phenotype on DNA methyltransferases. The cooperativity of matrix with growth factors, cell density and hypoxia was also examined. Primary rat visceral SMC of early passage (0–2) were plated on native collagen or damaged/heat-denatured collagen. Hypoxia was induced with 3% O2 (balanced 5% CO2 and 95% N2) over 48 hours. Inhibitors were applied 2–3 hours after cells were plated on matrix, or immediately before hypoxia. Cells were fixed and stained for DNMT3A and smooth muscle actin (SMA) or smooth muscle myosin heavy chain. Illumina 450 K array of CpG sites was performed on bisulfite-converted DNA from smooth muscle cells on damaged matrix vs native collagen. Matrix exquisitely regulates DNMT3A localization and expression, and influences differentiation in SMCs exposed to denatured matrix +/− hypoxia. Analysis of DNA methylation signatures showed that Matrix caused significant DNA methylation alterations in a discrete number of CpG sites proximal to genes related to SMC differentiation. Matrix has a profound effect on the regulation of SMC phenotype, which is associated with altered expression, localization of DNMTs and discrete changes DNA methylation.

Differential expression of proteins related to smooth muscle cells and myofibroblasts in human thoracic aortic aneurysm.

Increasing knowledge is required for a better comprehension of the etiology of thoracic aortic aneurysm (TAA). The aim of this study was to highlight the modulations in vascular cell phenotypes, including myofibroblasts (MFs), in human TAA specimens compared to healthy aortas. histology, RT-PCR and immunohistochemical analysis of a panel of molecules, including ED-A Fibronectin (Fn), smoothelin, CD34 and alpha-smooth muscle actin (alpha-SMA), selected on the basis of their informative potential as markers of smooth muscle cells (SMCs) and MF phenotypic modulation, were performed on all samples. The media of TAAs was characterized by the absence of smoothelin, the unaltered expression of alpha-SMA accompanied by an alteration of its distribution pattern, and by the activated expression of the ED-A isoform of Fn. We found a concentration of round-shaped cells exclusively in the adventitia and in the perivascular tissue of TAAs, also rich in vasa vasorum, largely expressing alpha-SMA, while a sub-population also expressed ED-A Fn and CD34. CD34 was expressed by several cells in the intima of TAAs, together with cells expressing cytoplasmatic ED-A Fn and alpha-SMA in comparison to healthy aortas. TAA specimens show an altered expression and localization of SMC and MF differentiation markers in comparison to healthy aortas, with possible implications on remodeling.

Expression pattern of endothelin system components and localization of smooth muscle cells in the human pre-ovulatory follicle

Whether ovarian follicular rupture involves contractile activity or not has been debated for decades. Recently, study in the rodents has indicated that an endogenously produced potent vasoconstrictive peptide, endothelin-2 (EDN2), may induce follicular constriction immediately prior to ovulation. This study was aimed to systematically characterize the human ovarian endothelin system and localize smooth muscle cells to assess the possible involvement of contractile activity in human ovulation. This is a prospective experimental study. Study subjects were 20 women aged 20-38 years who underwent IVF owing to tubal or male factors. Expression patterns of messenger RNAs (mRNAs) for EDN1, EDN2, EDN3, endothelin-converting enzyme-1 (ECE1 and ECE2), endothelin receptor A (ET(A)) and ET(B) in the granulosa cells (GCs) and cumulus cells and endothelin peptide concentration in the pre-ovulatory follicles were measured at 36 h after hCG injection. In addition, localization of ovarian smooth muscle cells and endothelin receptor expression were determined in normal (non-IVF patient) ovaries. Pre-ovulatory follicles express mRNA for EDN1 and EDN2, ECE1, ECE2, ET(A) and ET(B), but not EDN3, contain highly concentrated endothelin peptides (105.9 pg/ml) and are surrounded by theca externa that are made mostly of multicell layer non-vascular smooth muscle cells. ET(A) expression is localized in the smooth muscle cells of theca externa, theca interna and GC, whereas ET(B) expression is confined to theca interna. Pre-ovulatory follicles contain highly concentrated endothelins and are surrounded by non-vascular smooth muscle cells that express endothelin receptor, suggesting involvement of endothelin-induced contractile action in ovulation in the human ovary.

Ontogenetic Profile of the Expression of Thyroid Hormone Receptors in Rat and Human Corpora Cavernosa of the Penis

IntroductionIn the last few years, various studies have underlined a correlation between thyroid function and male sexual function, hypothesizing a direct action of thyroid hormones on the penis.AimTo study the spatiotemporal distribution of mRNA for the thyroid hormone nuclear receptors (TR) α1, α2 and β in the penis and smooth muscle cells (SMCs) of the corpora cavernosa of rats and humans during development.MethodsWe used several molecular biology techniques to study the TR expression in whole tissues or primary cultures from human and rodent penile tissues of different ages.Main Outcome MeasureWe measured our data by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) amplification, Northern blot and immunohistochemistry.ResultsWe found that TRα1 and TRα2 are both expressed in the penis and in SMCs during ontogenesis without development-dependent changes. However, in the rodent model, TRβ shows an increase from 3 to 6 days post natum (dpn) to 20 dpn, remaining high in adulthood. The same expression profile was observed in humans. While the expression of TRβ is strictly regulated by development, TRα1 is the principal isoform present in corpora cavernosa, suggesting its importance in SMC function. These results have been confirmed by immunohistochemistry localization in SMCs and endothelial cells of the corpora cavernosa.ConclusionsThe presence of TRs in the penis provides the biological basis for the direct action of thyroid hormones on this organ. Given this evidence, physicians would be advised to investigate sexual function in men with thyroid disorders. Carosa E, Di Sante S, Rossi S, Castri A, D'Adamo F, Gravina GL, Ronchi P, Kostrouch Z, Dolci S, Lenzi A, and Jannini EA. Ontogenetic profile of the expression of thyroid hormone receptors in rat and human corpora cavernosa of the penis. J Sex Med 2010;7:1381–1390.

Aldolase A is present in smooth muscle cell nuclei.

Previously we have shown that aldolase (ALD; EC 4.1.2.13) is present in cardiomyocyte nuclei. Now, we focused our attention on ALD localization in smooth muscle cells. Immunocytochemical methods were used to study the subcellular localization of ALD. Aldolase was localized in the cytoplasm as well as in the nuclei. Within the nuclei ALD was located in the heterochromatin region. Native polyacrylamide gel electrophoresis followed by aldolase activity staining in gel was used to study the ALD isoenzyme pattern in porcine smooth muscle cells. Two ALD isoenzymes, A and C, were found in these cells but in the nuclei only the muscle isoenzyme was detected. To support the nuclear localization of ALD, measurement of aldolase activity in the smooth muscle cell nuclei isolated from porcine stomach was performed. The ALD activity in the isolated nuclei was detectable only after preincubation of the nuclear fraction with Triton X-100 and high concentration of KCl.

LR11, an LDL receptor gene family member, is a novel regulator of smooth muscle cell migration.

LR11, a member of the LDL receptor family, is highly expressed in vascular smooth muscle cells (SMCs) of the hyperplastic intima, and induces enhanced migration of SMCs in vitro via its upregulation of urokinase-type plasminogen activator receptor (uPAR) expression. In this study, we have delineated the mechanism by which LR11 elevates the expression levels of uPAR in SMCs. Secretion of soluble LR11 is induced in SMCs during the rapidly proliferating phase, and the secreted LR11 induces the migration activities of SMCs. Both the cell-anchored and secreted forms of LR11 have the capacity to bind to and form complexes with uPAR. LR11-overexpressing cells show significantly enhanced uPAR binding, but decreased uPAR internalization. LR11 colocalizes with uPAR on the cell surface and inhibits the LDL receptor-related protein (LRP)-mediated binding and internalization of uPAR. Thus, LR11 mediates the uPAR localization to the plasma membrane. LR11 is highly expressed in the atheromatous plaque areas of apoE knockout mice, particularly in the intimal SMCs at the border between intima and media. The neutralization of LR11 function with anti-LR11 antibody reduced cuff-induced intimal thickness in mice. The novel mechanism of regulation of uPAR localization in SMCs accompanied with enhanced migration activity possibly constitutes an important factor in the process of atherosclerosis and arterial remodeling.

Soluble guanylate cyclase and cGMP-dependent protein kinase I expression in the human corpus cavernosum.

Nitric oxide (NO) as a mediator in smooth muscle cells causes rapid and robust increases in cGMP levels. The cGMP-dependent protein kinase I has emerged as an important signal transduction mediator for smooth muscle relaxation. The purpose of this study was to examine the existence and distribution of two key enzymes of the NO/cGMP pathway, the cGMP-dependent kinase I (cGK I) and the soluble guanylate cyclase (sGC) in human cavernosal tissue. The expression of the enzymes were examined in corpus cavernosum specimens of 23 patients. Eleven potent patients suffered from penile deviations and were treated via Nesbit's surgical method. Nine long-term impotent patients underwent implantation of flexible hydraulic prothesis. Three potent patients underwent trans-sexual operations. Expression of the sGC and cGK I were examined immunohistochemically using specific antibodies. In all specimens of cavernosal tissue a distinct immunoreactivity was observed in different parts and structures. We found a high expression of sGC and cGK I in smooth muscle cells of vessels and in the fibromuscular stroma. The endothelium of the cavernosal sinus, of the cavernosal arteries, and the cavernosal nerve fibers showed an immunoreactivity against sGC. The distribution analysis of cGK I revealed a predominately vesicular localization in smooth muscle cells. The examination of the endothelium showed no clear immunoreactivity against cGK I. There was no distinct difference in immunoreactivity and cellular distribution between potent and impotent patients.

An Electron Microscope Study of Calcium and Magnesium in Smooth Muscle.

Recent investigations of electrolytes in Thyone muscle by Steinbach have led him to the conclusion that Ca is present in cells in lower concentration than that of the surrounding medium. Since these results are at variance with our own in a number of respects we have studied again the localization of Ca and Mg in mammalian and frog smooth muscle by the electron microscope as described by Scott and Packer. Naturally, the results obtained cannot be compared directly with those of Steinbach as the same form was not used. Furthermore, our interest lay in determining the localization in smooth muscle cells which had been preserved as nearly as possible in their normal physiological state. Thin strips of smooth muscle were taken from the duodenum and stomach wall of cats and frogs. These pieces were frozen in liquid air while still actively contracting from the mechanical stimulation of the manipulation. The frozen tissues were dehydrated in vacuo at -63°C and infiltrated in water-free paraffin without breaking the vacuum. This procedure precludes any salt shift during dehydration and the addition of water from outside sources at any time and thus maintains the spatial relations of the element in question. Thin sections (8μ) of muscle were prepared and examined by heating on a surfaced cathode in the electron microscope. The technic of preparation of tissues for examination and the method of localizing Ca and Mg are believed to preserve the exact topical cellular relationships. Unfortunately, it is impossible by the method used to differentiate Ca and Mg, since both activate the cathode surface. Quantities of these elements in less than 1 × 10-12 g are easily detected. In no instance was either Ca or Mg found outside the cell wall.