Nonmuscle Research Articles

Aging Related Changes in Smooth Muscle Contractile and Regulatory Proteins

Aging is a significant risk factor for cardiovascular disease and is known to be associated with an increase in vascular stiffness and decrease in sensitivity to NO mediated vasodilatation. However, the underlying mechanism for these changes in vascular function is unknown. Smooth muscle tone is known to be regulated by the expression and phosphorylation of both nonmuscle (NM) and smooth muscle (SM) myosin. NM and SM phosphorylation is regulated by the activity of myosin light chain kinase (MLCK) and MLC phosphatase. MLC phosphatase is a trimeric enzyme consisting of enzymatic, myosin targeting (MYPT1) and a 20kDa subunit of unknown function. Two isoforms of MYPT1 are produced by alternative mRNA splicing, which differ by the presence or absence of a COOH-terminal leucine zipper (LZ+/-), and the relative expression of LZ+/LZ- MYPT1 determines the sensitivity to NO mediated vasodilatation. We defined NM myosin expression using two-dimensional SDS PAGE and LZ+/LZ- MYPT1 expression using immunoblotting in aortic and tertiary mesenteric smooth muscle from 6mo and 24mo old Fischer344 rats. The data demonstrate that aging is associated with a significant decrease in the expression of both LZ+ MYPT1 and NM myosin. The reduction in LZ+ MYPT1 expression would both decrease the sensitivity to NO mediated smooth muscle dilatation and increase vascular tone, which is a hallmark of the vasculature in aging. On the other hand, the reduction in NM myosin would result in a decrease in vascular tone and could represent a compensatory mechanism to normalize blood pressure during aging. These data demonstrate that there are changes in the expression of smooth muscle regulatory and contractile proteins during aging and these changes in protein expression contribute to molecular mechanism for the decrease in sensitivity to NO mediated vasodilatation as well as the increase in vascular tone or the vascular dysfunction that occurs with aging. Supported by a Mayo CV Prospective Grant (FVB). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Aging related decreases in NM myosin expression and contractility in a resistance vessel.

Introduction: Vasodilatation in response to NO is a fundamental response of the vasculature, and during aging, the vasculature is characterized by an increase in stiffness and decrease in sensitivity to NO mediated vasodilatation. Vascular tone is regulated by the activation of smooth muscle and nonmuscle (NM) myosin, which are regulated by the activities of myosin light chain kinase (MLCK) and MLC phosphatase. MLC phosphatase is a trimeric enzyme with a catalytic subunit, myosin targeting subunit (MYPT1) and 20kDa subunit of unknown function. Alternative mRNA splicing produces LZ+/LZ- MYPT1 isoforms and the relative expression of LZ+/LZ- MYPT1 determines the sensitivity to NO mediated vasodilatation. This study tested the hypothesis that aging is associated with changes in LZ+ MYPT1 and NM myosin expression, which alter vascular reactivity. Methods: We determined MYPT1 and NM myosin expression, force and the sensitivity of both endothelial dependent and endothelial independent relaxation in tertiary mesenteric arteries of young (6mo) and elderly (24mo) Fischer344 rats. Results: The data demonstrate that aging is associated with a decrease in both the expression of NM myosin and force, but LZ+ MYPT expression and the sensitivity to both endothelial dependent and independent vasodilatation did not change. Further, smooth muscle cell hypertrophy increases the thickness of the medial layer of smooth muscle with aging. Discussion: The reduction of NM myosin may represent an aging associated compensatory mechanism to normalize the stiffness of resistance vessels in response to the increase in media thickness observed during aging.

MP16-07 CAN WE PREDICT OF THE RESPONSE OF HIGH RISK NON MUSCLE INVASIVE BLADDER CANCER PATIENTS TO INTRAVESICAL BACILLUS CALMETTE-GUERIN? THE ROLE OF IMMUNOLOGICAL MARKERS

You have accessJournal of UrologyBladder Cancer: Non-invasive II (MP16)1 Sep 2021MP16-07 CAN WE PREDICT OF THE RESPONSE OF HIGH RISK NON MUSCLE INVASIVE BLADDER CANCER PATIENTS TO INTRAVESICAL BACILLUS CALMETTE-GUERIN? THE ROLE OF IMMUNOLOGICAL MARKERS Ahmed Elbatta, Amr Elsawy, Hassan abol-Enein, Abdel-Aziz Zeidan, Mohamed Zahran, Mohamed shehata, Ahmed A. Shokeir, and Amira Awadalla Ahmed ElbattaAhmed Elbatta More articles by this author , Amr ElsawyAmr Elsawy More articles by this author , Hassan abol-Enein Hassan abol-Enein More articles by this author , Abdel-Aziz ZeidanAbdel-Aziz Zeidan More articles by this author , Mohamed ZahranMohamed Zahran More articles by this author , Mohamed shehataMohamed shehata More articles by this author , Ahmed A. ShokeirAhmed A. Shokeir More articles by this author , and Amira AwadallaAmira Awadalla More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002001.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: To assess the predictive performance of different immunological markers on BCG-response in high risk NMIBC BCG-naïve patients. METHODS: Patients who underwent TURBT for NMIBC were assessed for study eligibility. Urine and blood samples were retrieved from study participants at baseline (immediately before 1st dose of induction) and after induction (4 hours after last 6th dose). Urine samples were evaluated for IL-2 and IL-10 by solid phase ELISA. Blood samples were evaluated for TNF-α, CTLA4 and transcription factors (TFs) (GATA3+, T-bet+ and FoxP3+) using quantitative RT-PCR analysis. Change pattern and fold change of each evaluable marker was assessed in relation to different oncological outcomes of BCG-response (Initial complete response -ICR-/Recurrence /Progression). RESULTS: Between July 2013 and May 2019, 204 patients were included. Among evaluable markers, IL-2 and TNF-α absolutely increased all patients, CTLA4 and FoxP3+ showed a predominant decrease pattern in 188 (92.2%) and 192 (94.1%) patients, respectively, while, IL-10, GATA3+ and T-bet+ did not show any predominant change pattern. ICR was achieved in 186 (91.2%) patients. TNF-α fold change, IL-10 change pattern were significantly associated with ICR (p= 0.001, 0.03, respectively). At a median (range) follow-up 37 (20-88) months, 104 (56%) patients developed recurrence. IL-10, CTLA4, T-bet+, FoxP3+ change patterns and GATA3+/ T-bet+ ratio were significantly associated with tumor recurrence (p=0.001, 0.001, 0.02, 0.009 and 0.001, respectively). Tumor progression was encountered in 34 (18.3%) patients. IL-10, CTLA4, T-bet+ change patterns and GATA3+/ T-bet+ ratio were independent predictors of tumor progression (p=0.001, 0.001, 0.02 and 0.001, respectively). CONCLUSIONS: Urinary IL-10 and blood TNF-α can significantly predict ICR. Moreover, change pattern of IL-10, CTLA4, TFs (GATA3+, T-bet+ and FoxP3+) and GATA3+/T-bet+ ratio after BCG induction can independently predict further BCG-response. These markers might be implanted in clinical practice when management options are discussed or in systems with severe BCG shortage. Source of Funding: Self © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e298-e298 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ahmed Elbatta More articles by this author Amr Elsawy More articles by this author Hassan abol-Enein More articles by this author Abdel-Aziz Zeidan More articles by this author Mohamed Zahran More articles by this author Mohamed shehata More articles by this author Ahmed A. Shokeir More articles by this author Amira Awadalla More articles by this author Expand All Advertisement Loading ...

Distinct Roles of Smooth Muscle and Non-muscle Myosin Light Chain-Mediated Smooth Muscle Contraction.

Both smooth muscle (SM) and non-muscle (NM) myosin II are expressed in hollow organs such as the bladder and uterus, but their respective roles in contraction and corresponding physiological functions remain to be determined. In this report, we assessed their roles by analyzing mice deficient of Myl9, a gene encoding the SM myosin regulatory light chain (SM RLC). We find that global Myl9-deficient bladders contracted with an apparent sustained phase, despite no initial phase. This sustained contraction was mediated by NM myosin RLC (NM RLC) phosphorylation by myosin light chain kinase (MLCK). NM myosin II was expressed abundantly in the uterus and young mice bladders, of which the force was accordingly sensitive to NM myosin inhibition. Our findings reveal distinct roles of SM RLC and NM RLC in SM contraction.

S100A4 is activated by RhoA and catalyses the polymerization of non-muscle myosin, adhesion complex assembly and contraction in airway smooth muscle.

S100A4 is expressed in many tissues, including smooth muscle (SM), but its physiologic function is unknown. S100A4 regulates the motility of metastatic cancer cells by binding to non-muscle (NM) myosin II. Contractile stimulation causes the polymerization of NM myosin in airway SM, which is necessary for tension development. NM myosin regulates the assembly of adhesion junction signalling complexes (adhesomes) that catalyse actin polymerization. In airway SM, ACh (acetylcholine) stimulated the binding of S100A4 to the NM myosin heavy chain, which was catalysed by RhoA GTPase via the RhoA-binding protein, rhotekin. The binding of S100A4 to NM myosin was required for NM myosin polymerization, adhesome assembly and actin polymerization. S100A4 plays a critical function in the regulation of airway SM contraction by catalysing NM myosin filament assembly. The interaction of S100A4 with NM myosin may also play an important role in the physiologic function of other tissues. S100A4 binds to the heavy chain of non-muscle (NM) myosin II and can regulate the motility of crawling cells. S100A4 is widely expressed in many tissues including smooth muscle (SM), although its role in the regulation of their physiologic function is not known. We hypothesized that S100A4 contributes to the regulation of contraction in airway SM by regulating a pool of NM myosin II at the cell cortex. NM myosin II undergoes polymerization in airway SM and regulates contraction by catalysing the assembly of integrin-associated adhesome complexes that activate pathways that catalyse actin polymerization. ACh stimulated the interaction of S100A4 with NM myosin II in airway SM at the cell cortex and catalysed NM myosin filament assembly. RhoA GTPase regulated the activation of S100A4 via rhotekin, which facilitated the formation of a complex between RhoA, S100A4 and NM myosin II. The depletion of S100A4, RhoA or rhotekin from airway SM tissues using short hairpin RNA or small interfering RNA prevented NM myosin II polymerization as well as the recruitment of vinculin and paxillin to adhesome signalling complexes in response to ACh, and inhibited actin polymerization and tension development. S100A4 depletion did not affect ACh-stimulated SM myosin regulatory light chain phosphorylation. The results show that S100A4 plays a critical role in tension development in airway SM tissue by catalysing NM myosin filament assembly, and that the interaction of S100A4 with NM myosin in response to contractile stimulation is activated by RhoA GTPase. These results may be broadly relevant to the physiologic function of S100A4 in other cell and tissue types.

The vasculature in HFpEF vs HFrEF: differences in contractile protein expression produce distinct phenotypes

Both heart failure with reduced (HFrEF) and preserved (HFpEF) ejection fraction are associated with abnormalities of the vasculature, including a resting vasoconstriction and a decrease in sensitivity to nitric oxide (NO) mediated vasodilation. Vascular tone is controlled by the expression and activation of both smooth muscle (SM) and nonmuscle (NM) myosin, and NO mediated vasodilation is regulated by the expression of the leucine zipper positive (LZ+) isoform of the myosin targeting subunit (MYPT1) of myosin light chain phosphatase (MLCP). This study was designed to determine the expression of these contractile proteins in humans with HFrEF and HFpEF vs normal controls. We isolated tertiary mesenteric vessels from remnant biospecimens of patients undergoing partial or total colectomy at Mayo Clinic Rochester from August 2017 to December 2018, and examined the expression of MYPT1 and the LZ + MYPT1 isoform with immunoblots, while 2D SDS-PAGE was used to resolve the phosphorylated and nonphosphorylated regulatory light chains of NM and SM myosin. Our data show that NM myosin expression, as a percentage of total myosin, was 12 ± 3% (controls, n = 6), 7 ± 5% (HFpEF, n = 4) and 37 ± 18% (HFrEF, n = 5, p < 0.05). Total MYPT1 expression was significantly reduced (p < 0.05) in both HFpEF (70 ± 11%) and HFrEF (48 ± 6%); and in HFrEF, LZ + MYPT1 was also depressed (62 ± 19%, <0.05). These results demonstrate that HFrEF and HFpEF are distinct vascular entities, and the changes in protein expression contribute to the vascular abnormalities associated with these diseases. Further in HFpEF, the decrease in MYPT1 would explain why pharmacologic therapies that are designed to activate the NO/cGMP/PKG signaling pathway do not produce a clinical benefit.

Blebbistatin modulates prostatic cell growth and contrapctility through myosin II signaling.

To investigate the effect of blebbistatin (BLEB, a selective myosin inhibitor) on regulating contractility and growth of prostate cells and to provide insight into possible mechanisms associated with these actions. BLEB was incubated with cell lines of BPH-1 and WPMY-1, and intraprostatically injected into rats. Cell growth was determined by flow cytometry, and in vitro organ bath studies were performed to explore muscle contractility. Smooth muscle (SM) myosin isoform (SM1/2, SM-A/B, and LC17a/b) expression was determined via competitive reverse transcriptase PCR. SM myosin heavy chain (MHC), non-muscle (NM) MHC isoforms (NMMHC-A and NMMHC-B), and proteins related to cell apoptosis were further analyzed via Western blotting. Masson's trichrome staining was applied to tissue sections. BLEB could dose-dependently trigger apoptosis and retard the growth of BPH-1 and WPMY-1. Consistent with in vitro effect, administration of BLEB to the prostate could decrease rat prostatic epithelial and SM cells via increased apoptosis. Western blotting confirmed the effects of BLEB on inducing apoptosis through a mechanism involving MLC20 dephosphorylation with down-regulation of Bcl-2 and up-regulation of BAX and cleaved caspase 3. Meanwhile, NMMHC-A and NMMHC-B, the downstream proteins of MLC20, were found significantly attenuated in BPH-1 and WPMY-1 cells, as well as rat prostate tissues. Additionally, BLEB decreased SM cell number and SM MHC expression, along with attenuated phenylephrine-induced contraction and altered prostate SMM isoform composition with up-regulation of SM-B and down-regulation of LC17a, favoring a faster contraction. Our novel data demonstrate BLEB regulated myosin expression and functional activity. The mechanism involved MLC20 dephosphorylation and altered SMM isoform composition.

Non-muscle (NM) myosin heavy chain phosphorylation regulates the formation of NM myosin filaments, adhesome assembly and smooth muscle contraction.

Non-muscle (NM) and smooth muscle (SM) myosin II are both expressed in smooth muscle tissues, however the role of NM myosin in SM contraction is unknown. Contractile stimulation of tracheal smooth muscle tissues stimulates phosphorylation of the NM myosin heavy chain on Ser1943 and causes NM myosin filament assembly at the SM cell cortex. Expression of a non-phosphorylatable NM myosin mutant, NM myosin S1943A, in SM tissues inhibits ACh-induced NM myosin filament assembly and SM contraction, and also inhibits the assembly of membrane adhesome complexes during contractile stimulation. NM myosin regulatory light chain (RLC) phosphorylation but not SM myosin RLC phosphorylation is regulated by RhoA GTPase during ACh stimulation, and NM RLC phosphorylation is required for NM myosin filament assembly and SM contraction. NM myosin II plays a critical role in airway SM contraction that is independent and distinct from the function of SM myosin. The molecular function of non-muscle (NM) isoforms of myosin II in smooth muscle (SM) tissues and their possible role in contraction are largely unknown. We evaluated the function of NM myosin during contractile stimulation of canine tracheal SM tissues. Stimulation with ACh caused NM myosin filament assembly, as assessed by a Triton solubility assay and a proximity ligation assay aiming to measure interactions between NM myosin monomers. ACh stimulated the phosphorylation of NM myosin heavy chain on Ser1943 in tracheal SM tissues, which can regulate NM myosin IIA filament assembly in vitro. Expression of the non-phosphorylatable mutant NM myosin S1943A in SM tissues inhibited ACh-induced endogenous NM myosin Ser1943 phosphorylation, NM myosin filament formation, the assembly of membrane adhesome complexes and tension development. The NM myosin cross-bridge cycling inhibitor blebbistatin suppressed adhesome complex assembly and SM contraction without inhibiting NM myosin Ser1943 phosphorylation or NM myosin filament assembly. RhoA inactivation selectively inhibited phosphorylation of the NM myosin regulatory light chain (RLC), NM myosin filament assembly and contraction, although it did not inhibit SM RLC phosphorylation. We conclude that the assembly and activation of NM myosin II is regulated during contractile stimulation of airway SM tissues by RhoA-mediated NM myosin RLC phosphorylation and by NM myosin heavy chain Ser1943 phosphorylation. NM myosin II actomyosin cross-bridge cycling regulates the assembly of membrane adhesome complexes that mediate the cytoskeletal processes required for tension generation. NM myosin II plays a critical role in airway SM contraction that is independent and distinct from the function of SM myosin.

MP13-06 CAN INTRAVESICAL ADMINISTRATION OF ANTIFIBRINOLYTIC AGENT POTENTIATE THE ACTION OF BACILLUS CALMETTE- GUERIN AFTER TRANSURETHRAL RESECTION OF NON–MUSCLE INVASIVE BLADDER CANCER?: MULTICENTER PROSPECTIVE RANDOMIZED CONTROLLED STUDY.

You have accessJournal of UrologyBladder Cancer: Non-Invasive I1 Apr 2016MP13-06 CAN INTRAVESICAL ADMINISTRATION OF ANTIFIBRINOLYTIC AGENT POTENTIATE THE ACTION OF BACILLUS CALMETTE- GUERIN AFTER TRANSURETHRAL RESECTION OF NON–MUSCLE INVASIVE BLADDER CANCER?: MULTICENTER PROSPECTIVE RANDOMIZED CONTROLLED STUDY. Mohamed Soliman, Hussein Aldaqadossi, Ahmed El-Abd, Ahmed Abou- Ramadan, Mohamed El-Gharabawy, Abd-Elhamid El-Bahnasy, and Mohamed Abd-Eltawab Mohamed SolimanMohamed Soliman More articles by this author , Hussein AldaqadossiHussein Aldaqadossi More articles by this author , Ahmed El-AbdAhmed El-Abd More articles by this author , Ahmed Abou- RamadanAhmed Abou- Ramadan More articles by this author , Mohamed El-GharabawyMohamed El-Gharabawy More articles by this author , Abd-Elhamid El-BahnasyAbd-Elhamid El-Bahnasy More articles by this author , and Mohamed Abd-EltawabMohamed Abd-Eltawab More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2487AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Intravesical instillation of bacillus Calmette-Guerin (BCG) as an adjuvant therapy after transurethral resection of non–muscle invasive bladder cancer (TURBT) is considered to be the most effective treatment to prevent the recurrence and progression of the disease. The objective from this study is to evaluate the effect of intravesical instillation of antifibrinolytic agents with BCG in the management of superficial bladder tumors after TURBT. METHODS This study was conducted on cohort of patients with primary non–muscle invasive bladder cancer in whom the management decision included TURBT with intravesical BCG instillation postoperatively. Patients were managed by TURBT, two weeks later; patients were randomized into two groups. Patients in group A subjected to intravesical instillation of BCG plus 2 gm of tranexamic acid while patients in group B were subjected to intravesical instillation of BCG alone. Prothrombin time was determined at 2 hours after instillation. Complications in each group were reported. Follow-up cystoscopy was conducted three months after TURBT and then every three months during the first year followed by by annual cystoscopy in the following years. RESULTS This study included 96 patients with mean age of 52.4 ± 6.2. They were prospectively randomized into 2 groups (48 each). Follow-up ranged from 6 to 24 months (mean of 15.2 ± 9 months). Four patients were lost to follow up (2 in each group). Another 15 patients (8 in group A and 7 in group B) developed serious BCG complications and excluded from the study. Postoperative prothrombin time showed no significant difference between both groups (P= 0.3). In the remaining 77 patients (38 patients in group A and 39 patients in group B), recurrence was reported in 3 out of 38 patients in group A (7.9%) and 11 out of 39 patients in group B (28%) with a statistically significant difference between both groups (P=0.036). Progression was reported in only one patient in group A (2.6%) and 8 patients in group B (20.5%) with a statistically significant difference (P=0.0286). CONCLUSIONS Intravesical instillation of antifibrinolytic agents with BCG has improved the antitumor efficacy of BCG in the management of non–muscle invasive bladder cancer following TURBT. Further studies including large number of cases are required to support these findings. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e136 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Mohamed Soliman More articles by this author Hussein Aldaqadossi More articles by this author Ahmed El-Abd More articles by this author Ahmed Abou- Ramadan More articles by this author Mohamed El-Gharabawy More articles by this author Abd-Elhamid El-Bahnasy More articles by this author Mohamed Abd-Eltawab More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Arterial Smooth Muscle Proliferative Responses to ET‐1 are Subject to Hypoxic Pre‐conditioning in Term Fetal Lamb Arteries

This study explores the hypothesis that the vascular proliferative response to ET-1 is subject to hypoxic pre-conditioning in term fetal arteries. To test this idea, pregnant ewes were maintained at sea level or at an altitude of 3,820 m for the final 110 days of gestation. Endothelium-denuded carotid arteries harvested from full-term fetuses were cultured in serum-starved media with 3, 10 and 30 nM of ET-1 in normoxic (21% O2) and hypoxic (12% O2) chambers for 48 hours. Proliferative responses were quantified as changes in the colocalization of Non-Muscle (NM) and Smooth Muscle (SM) Myosin Heavy Chain with smooth muscle alpha-actin (SMaA). In normoxic fetal arteries cultured under normoxic conditions (FN-N), ET-1 produced a modest concentration-dependent increase in NM-SMaA colocalization and this effect was depressed 65% in arteries cultured under hypoxic conditions (FN-H). In hypoxic fetal arteries cultured under normoxic conditions (FH-N), responses to normoxic culture were not different than observed in the FN-N group. In hypoxic fetal arteries cultured under hypoxic conditions (FH-H), however, responses to ET-1 were attenuated only 22%. Corresponding numbers for SM-SMaA colocalization reflected a 46% depression in FN arteries, but 25% depression in FH arteries. Together, these results demonstrate that hypoxic acclimatization enhances the ability of smooth muscle to regulate contractile protein organization in response to ET-1-induced proliferation, suggesting that hypoxic preconditioning involves intracellular changes in fetal arterial smooth muscle. This work was supported by National Institutes of Health Grants HL-54120, HD-31266, HL-64867, and NS-076945 and the Loma Linda University School of Medicine.

Non‐muscle (NM) Myosin II Mediates the Formation of Adhesome Signaling Complexes in Response to Contractile Stimulation of Airway Smooth Muscle (ASM)

Non‐muscle (NM) Myosin II Mediates the Formation of Adhesome Signaling Complexes in Response to Contractile Stimulation of Airway Smooth Muscle (ASM)

Chronic Hypoxia Modulates Endothelial Influences on Smooth Muscle Phenotype in Fetal Cerebral Arteries

This study explores the hypothesis that chronic hypoxia modulates endothelial regulation of smooth muscle phenotype and function in fetal cerebral arteries. Endothelium denuded and intact middle cerebral arteries harvested from term fetuses from ewes kept at sea level (Normoxic) or 3, 820 m (Hypoxic) altitudes for the last 110 days of gestation were assessed for contractile function and smooth muscle phenotype as quantified by changes in the colocalization of Non‐Muscle (NM) and Smooth Muscle (SM) Myosin Heavy Chain (MHC) with Smooth Muscle alpha‐actin (SM‐αA). Chronic hypoxia reduced maximum myogenic stress, but increased maximum K+‐induced stress. Organ culture of arteries from normoxic animals with VEGF reproduced this increase in K+‐induced stress but only in the presence of an intact endothelium. For NM‐MHC/AA colocalization, an index of synthetic smooth muscle, organ culture for normoxic arteries with VEGF had little effect in the absence of endothelium but in the presence of endothelium enhanced colocalization. This effect of the endothelium on NM‐MHC/AA colocalization was enhanced in hypoxic arteries. For SM‐MHC/AA colocalization, a marker for contractile smooth muscle, organ culture of normoxic arteries with VEGF had little effect in the presence or the absence of endothelium, but in hypoxic arteries endothelium dramatically amplified VEGF‐induced increases in colocalization. These results demonstrate that chronic hypoxia modulates VEGF‐dependent endothelial regulation of smooth muscle phenotype in fetal cerebral arteries.This work was supported by National Institutes of Health Grants HL‐54120, HD‐31266, HL‐64867, and NS‐076945 and the Loma Linda University School of Medicine.

MP26-16 NEUTROPHIL TO LYMPHOCYTE RATIO, A NEW PROGNOSTIC FACTOR IN NON MUSCLE INVASIVE BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Non-invasive I1 Apr 2015MP26-16 NEUTROPHIL TO LYMPHOCYTE RATIO, A NEW PROGNOSTIC FACTOR IN NON MUSCLE INVASIVE BLADDER CANCER Vincenzo Favilla, Tommaso Castelli, Daniele Urzì, Sebastiano Cimino, Salvatore Privitera, Eugenia Fragalà, Giorgio Ivan Russo, and Giuseppe Morgia Vincenzo FavillaVincenzo Favilla More articles by this author , Tommaso CastelliTommaso Castelli More articles by this author , Daniele UrzìDaniele Urzì More articles by this author , Sebastiano CiminoSebastiano Cimino More articles by this author , Salvatore PriviteraSalvatore Privitera More articles by this author , Eugenia FragalàEugenia Fragalà More articles by this author , Giorgio Ivan RussoGiorgio Ivan Russo More articles by this author , and Giuseppe MorgiaGiuseppe Morgia More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.1138AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES An elevated neutrophil to lymphocyte ratio (NLR) was associated with a worse overall and disease free survival in a variety of different malignancies, including muscle invasive bladder cancer. The predictive value of NLR in non muscle invasive bladder cancer (NMIBC) has not been well prospectively studied. The aim of our study was to evaluated whether NLR predict disease recurrence and progression in NMIBC. METHODS The NLR, defined as the absolute neutrophil count divided by the absolute lymphocyte count, was prospectively evaluated in 178 consecutive, newly diagnosed, patients with NMIBC treated with transurethral resection of the bladder tumor (TURB), between the years 2009 and 2013. Patients with hematologic disorders or conditions that may have influenced blood cell lines such as autoimmune disease, presence of an active infection and/or immunodeficiency virus infection at the time of surgical intervention, prior or concomitant intravescical therapy with BacilleCalmette– Guérin (BCG), prior blood transfusion, and the presence of other cancer types or prior chemotherapy were excluded. Cutoff points for NLR were tested separately for recurrence and progression using the standardized cutoff-finder algorithm. Univariate and multivariate Cox regression analyses were used to evaluated the association between NLR and disease recurrence and progression RESULTS The study cohort comprised 148 male and 30 female patients. The median age of all 178 patients enrolled in the study was 69.27, with a median follow-up of 53 months (IQR: 33.0-76.25). Median NLR was 2.55 (IQR: 1.90-3.62) Patients with NLR <3 were older (74.45 vs. 67.94; p=0.02) and exhibited significant differences in term of pathological stage (p=0.03) and no. of tumours (p=0.04) if compared with those with NLR ≥3. During the follow-up study, 14 (23.3%) and 44 (37.9%) (p=0.04) patients respectively with NLR <3 and ≥3 experienced recurrence and 2 (3.3%) and 14 (11.9%) experienced progression (p=0.06). At the multivariate Cox regression analysis, NLR < 3 was associated with low disease recurrence (HR: 0.34 [IQR: 0.17-0.68]; p<0.01). Pathological stage pT1 (p<0.01), high grade (p<0.01), no. of tumours (p<0.01) and smoking status (p<0.01) were independently predictors of disease recurrence. No association was found between NLR ≥ 3 and disease progression at the multivariate Cox regression analysis. The 5 years recurrence free survival was 49% and 62% in patients with NLR ≥ 3 and < 3 (p=0.04). The 5 year progression free survival was 77% and 93% in patients with NLR ≥ 3 and < 3 (p=0.69). CONCLUSIONS NLR is an independent predictor of disease recurrence but no of progression in patients with newly diagnosed NMIBC. These findings could offer a new contribution to find new biomarkers of recurrence in NMIBC patients. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e299-e300 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Vincenzo Favilla More articles by this author Tommaso Castelli More articles by this author Daniele Urzì More articles by this author Sebastiano Cimino More articles by this author Salvatore Privitera More articles by this author Eugenia Fragalà More articles by this author Giorgio Ivan Russo More articles by this author Giuseppe Morgia More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Counting Molecules in Non-Muscle Myosin II Filaments

Non-muscle (NM) myosin II is involved in many important cellular processes, including migration, adhesion and cytokinesis. NM myosin II assembles into bipolar filaments; this multimerization is thought to be essential for acto-myosin contractility. Since each head in a myosin filament can bind F-actin, the number of heads per filament will affect the balance among its bundling, crosslinking and sliding activities, as well as the load-dependent feedback on these activities. Electron microscopy studies of interphase cells from different species have shown NM myosin II filaments contain 16-56 heads, but the size of filaments has never been determined in cytokinesis.We first studied the number of NM-myosin II in meiotic and mitotic C. elegans zygotes. GFP tagged myosin was imaged by TIRF microscopy until the fluorescence signal was completely bleached. We developed a novel, yet very simple molecule counting method CoMPaS (counting molecules with photobleaching and subtraction) to measure the number of bleaching events in the fluorescence decay curves from cells with different level of GFP expression. We found that the total number of myosin molecules in a single filament is 100 - 150. As an independent assessment of myosin filament size, we also measured filament length in cultured Drosophila cells using super-resolution microscopy. We measured the distance between diffraction-limited foci containing GFP-tagged regulatory light chain (RLC) at the two ends of myosin filaments, with nm accuracy. On average, myosin filaments are approximately 380 nm long. This method also allowed us to determine the orientation and size of the RLC myosin head foci. Interestingly, increased myosin filament length correlated with more parallel filament head foci. We will combine these measurement techniques with genetic perturbations to understand how NM myosin II filament assembly is controlled and how interaction with actin influences myosin filament structure.

Maternal food restriction modulates cerebrovascular structure and contractility in adult rat offspring: effects of metyrapone

Although the effects of prenatal undernutrition on adult cardiovascular health have been well studied, its effects on the cerebrovascular structure and function remain unknown. We used a pair-fed rat model of 50% caloric restriction from day 11 of gestation to term, with ad libitum feeding after birth. We validated that maternal food restriction (MFR) stress is mediated by glucocorticoids by administering metyrapone, a corticosterone synthesis inhibitor, to MFR mothers at day 11 of gestation. At age 8 mo, offspring from Control, MFR, and MFR + Metyrapone groups were killed, and middle cerebral artery (MCA) segments were studied using vessel-bath myography and confocal microscopy. Colocalization of smooth muscle α-actin (SMαA) with nonmuscle (NM), SM1 and SM2 myosin heavy-chain (MHC) isoforms was used to assess smooth muscle phenotype. Our results indicate that artery stiffness and wall thickness were increased, pressure-evoked myogenic reactivity was depressed, and myofilament Ca(2+) sensitivity was decreased in offspring of MFR compared with Control rats. MCA from MFR offspring exhibited a significantly greater SMαA/NM colocalization, suggesting that the smooth muscle cells had been altered toward a noncontractile phenotype. MET significantly reversed the effects of MFR on stiffness but not myogenic reactivity, lowered SMαA/NM colocalization, and increased SMαA/SM2 colocalization. Together, our data suggest that MFR alters cerebrovascular contractility via both glucocorticoid-dependent and glucocorticoid-independent mechanisms.

The role of pulmonary vascular contractile protein expression in pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is associated with refractory vasoconstriction and impaired NO-mediated vasodilatation of the pulmonary vasculature. Vascular tone is regulated by light chain (LC) phosphorylation of both nonmuscle (NM) and smooth muscle (SM) myosins, which are determined by the activities of MLC kinase and MLC phosphatase. Further, NO mediated vasodilatation requires the expression of a leucine zipper positive (LZ+) isoform of the myosin targeting subunit (MYPT1) of MLC phosphatase. The objective of this study was to define contractile protein expression in the pulmonary arterial vasculature and vascular reactivity in PAH. In severe PAH, compared to controls, relative LZ+MYPT1 expression was decreased (100±14% vs. 60±6%, p<0.05, n=7–8), and NM myosin expression was increased (15±4% vs. 53±5% of total myosin, p<0.05, n=4–6). These changes in contractile protein expression should alter vascular reactivity; following activation with Ang II, force activation and relaxation were slowed, and sustained force was increased. Further, the sensitivity to ACh-mediated relaxation was reduced. These results demonstrate that changes in the pulmonary arterial SM contractile protein expression may participate in the molecular mechanism producing both the resting vasoconstriction and the decreased sensitivity to NO-mediated vasodilatation associated with PAH.

Non‐muscle (NM) and Smooth Muscle (SM) Myosin II Activation are Regulated by Different Mechanisms During Contractile Stimulation of Airway Smooth Muscle (ASM)

Myosin II is the major motor protein in SM, and is expressed as both SM and NM isoforms. However, the function of non‐muscle myosin II in SM is not understood. We hypothesized that NM and SM myosin II may serve different functions during ASM contraction and be regulated by different mechanisms. We detected both NM myosin IIA and IIB heavy chain isoforms in canine ASM tissues. The 20 kD myosin light chain (MLC) is expressed in both NM and SM isoforms, but these isoforms cannot be distinguished immunologically. We used 2‐D gel electrophoresis to distinguish NM and SM MLC isoforms and analyze their phosphorylation in response to acetylcholine (ACh). NM MLC represented a relatively small proportion of the total cellular MLC (20–30%). Both SM and NM MLCs were phosphorylated in similar proportions in response to ACh stimulation (about 50%). When RhoA was inactivated by expressing the inactive RhoA mutant, RhoA T19N, in muscle tissues, NM MLC phosphorylation was substantially inhibited but there was little effect on SM MLC phosphorylation. The MLC kinase inhibitor, ML‐7, suppressed ACh induced SM myosin II LC phosphorylation, but had little or no effect on NM myosin II MLC phosphorylation. We conclude that MLC kinase primarily regulates the phosphorylation of SM MLC, whereas NM MLC phosphorylation depends on RhoA activation. This suggests SM and NM myosin II play distinct roles in the regulation of SM contraction.HL29289, HL074099.

Altered Reactivity of Tertiary Mesenteric Arteries following Acute Myocardial Ischemia

Background: It is unknown if cardiac ischemia has any deleterious effect on the contractile properties of nonischemic, peripheral vascular beds. Thus, the objective of the present study was to determine whether acute myocardial ischemia results in peripheral vascular dysfunction. Methods and Results: This study characterized force maintenance and the sensitivity to acetylcholine (ACh)-mediated smooth muscle (SM) relaxation of tertiary (3rd) mesenteric arteries from Sprague-Dawley rats following 30 min of myocardial ischemia. Both the phosphorylation of nonmuscle (NM) light chain (LC) and SM-LCs as well as the expression of myosin phosphatase targeting subunit 1 (MYPT1) were also determined. Our data demonstrate that acute myocardial ischemia resulted in vascular dysfunction of 3rd mesenteric vessels, characterized by decreases in force maintenance, ACh- and cGMP-mediated SM relaxation, the phosphorylation of NM-LCs and SM-LCs, and MYPT1 expression. Ischemia was also associated with an increase in protein polyubiquitination, suggesting that during ischemia MYPT1 is targeted for degradation or proteolysis. Conclusion: Acute myocardial ischemia produces peripheral vascular dysfunction; the changes in LC phosphorylation and MYPT1 expression result in a decrease in both tone and the sensitivity to NO-mediated SM relaxation of the peripheral vasculature.

Acute Myocardial Ischemia Alters Vascular Reactivity of Tertiary Mesenteric Arteries

Ischemia produces a reduction in vascular tone and the sensitivity to NO mediated vasodilatation of the ischemic vascular bed. However, whether cardiac ischemia has any deleterious effect on the contractile properties of nonischemic, peripheral vascular beds is unknown. Thus, the aim of the current study was to determine whether acute myocardial ischemia results in peripheral vascular dysfunction. We determined force and the sensitivity to ACh mediated smooth muscle relaxation of tertiary (3rd) mesenteric arteries of rats following 30 min of myocardial ischemia (ischemia) compared to sham (perfused). During KCl depolarization, myocardial ischemia did not affect peak force, but significantly depressed force maintenance. For perfused vessels, KCl depolarization resulted in a significant increase in both smooth muscle (SM) and nonmuscle (NM) light chain (LC) phosphorylation. However, in ischemic vessels, there was not only a decrease in the expression of the LCs, but also SM-LC phosphorylation was depressed whilst there was no detectable NM-LC phosphorylation. Compared to perfused vessels, there was a decrease in the relaxation in response to both ACh and 8-Br-cGMP in ischemic 3rd mesenteric arteries, and in the expression of MYPT1. Further, ischemia was associated with an increase in protein poly-ubiquitination, suggesting that during ischemia both the LCs and MYPT1 are targeted for degradation by the proteasome. These results suggest that NM myosin contributes to the contractile properties of 3rd mesenteric vessels, and ischemia inhibits NM myosin to depress vascular tone. Additionally, ischemia results in a decrease in MYPT1 expression, which contributes to the decrease in the sensitivity to NO mediated vasodilatation. In conclusion, acute myocardial ischemia alters the peripheral vascular phenotype, which contributes to the decrease in both force maintenance and the sensitivity to NO mediated smooth muscle relaxation.

Gelsolin and Non-muscle Myosin IIA Interact to Mediate Calcium-regulated Collagen Phagocytosis

The formation of adhesion complexes is the rate-limiting step for collagen phagocytosis by fibroblasts, but the role of Ca(2+) and the potential interactions of actin-binding proteins in regulating collagen phagocytosis are not well defined. We found that the binding of collagen beads to fibroblasts was temporally and spatially associated with actin assembly at nascent phagosomes, which was absent in gelsolin null cells. Analysis of tryptic digests isolated from gelsolin immunoprecipitates indicated that non-muscle (NM) myosin IIA may bind to gelsolin. Immunostaining and immunoprecipitation showed that gelsolin and NM myosin IIA associated at collagen adhesion sites. Gelsolin and NM myosin IIA were both required for collagen binding and internalization. Collagen binding to cells initiated a prolonged increase of [Ca(2+)](i), which was absent in cells null for gelsolin or NM myosin IIA. Collagen bead-induced increases of [Ca(2+)](i) were associated with phosphorylation of the myosin light chain, which was dependent on gelsolin. NM myosin IIA filament assembly, which was dependent on myosin light chain phosphorylation and increased [Ca(2+)](i), also required gelsolin. Ionomycin-induced increases of [Ca(2+)](i) overcame the block of myosin filament assembly in gelsolin null cells. We conclude that gelsolin and NM myosin IIA interact at collagen adhesion sites to enable NM myosin IIA filament assembly and localized, Ca(2+)-dependent remodeling of actin at the nascent phagosome and that these steps are required for collagen phagocytosis.