Smooth Muscle Function Research Articles

Protective Effects of Inhibition of Mitochondrial Fission on Organ Function After Sepsis.

Sepsis-associated organ dysfunction plays a critical role in its high mortality, mainly in connection with mitochondrial dysfunction. Whether the inhibition of mitochondrial fission is beneficial to sepsis-related organ dysfunction and underlying mechanisms are unknown. Cecal ligation and puncture induced sepsis in rats and dynamic related protein 1 knockout mice, lipopolysaccharide-treated vascular smooth muscle cells and cardiomyocytes, were used to explore the effects of inhibition of mitochondrial fission and specific mechanisms. Our study showed that mitochondrial fission inhibitor Mdivi-1 could antagonize sepsis-induced organ dysfunction including heart, vascular smooth muscle, liver, kidney, and intestinal functions, and prolonged animal survival. The further study showed that mitochondrial functions such as mitochondrial membrane potential, adenosine-triphosphate contents, reactive oxygen species, superoxide dismutase and malonaldehyde were recovered after Mdivi-1 administration via improving mitochondrial morphology. And sepsis-induced inflammation and apoptosis in heart and vascular smooth muscle were alleviated through inhibition of mitochondrial fission and mitochondrial function improvement. The parameter trends in lipopolysaccharide-stimulated cardiomyocytes and vascular smooth muscle cells were similar in vivo. Dynamic related protein 1 knockout preserved sepsis-induced organ dysfunction, and the animal survival was prolonged. Taken together, this finding provides a novel effective candidate therapy for severe sepsis/septic shock and other critical clinical diseases.

Contractile and Structural Properties of Detrusor from Children with Neurogenic Lower Urinary Tract Dysfunction

Simple SummaryDisorders of bladder function can result from congenital spinal cord developmental defects and can remain in a significant number of patients despite surgical improvements to repair the primary defect. We studied the ability of bladder wall muscle from such patients to contract, a function essential to void collected urine and avoid urinary tract infections and potential damage to the kidneys. Tissue was taken when patients were several years old, at the time of surgical operations to improve bladder function. This tissue would otherwise have been discarded and was collected with the full ethical approval and consent of parents or guardians. We found that the ability of the bladder wall samples to contract was impaired and was generally stiffer; both of which would make it more difficult for the bladder to void urine. These functional changes were associated with a replacement of muscle with connective tissue (fibrosis). The experiments provide a pathway to devise strategies that might improve bladder function in these patients through reversal of the intrinsic tissue pathways that increase fibrosis.Neurogenic lower urinary tract (NLUT) dysfunction in paediatric patients can arise after congenital or acquired conditions that affect bladder innervation. With some patients, urinary tract dysfunction remains and is more difficult to treat without understanding the pathophysiology. We measured in vitro detrusor smooth muscle function of samples from such bladders and any association with altered Wnt-signalling pathways that contribute to both foetal development and connective tissue deposition. A comparator group was tissue from children with normally functioning bladders. Nerve-mediated and agonist-induced contractile responses and passive stiffness were measured. Histology measured smooth muscle and connective tissue proportions, and multiplex immunohistochemistry recorded expression of protein targets associated with Wnt-signalling pathways. Detrusor from the NLUT group had reduced contractility and greater stiffness, associated with increased connective tissue content. Immunohistochemistry showed no major changes to Wnt-signalling components except down-regulation of c-Myc, a multifunctional regulator of gene transcription. NLUT is a diverse term for several diagnoses that disrupt bladder innervation. While we cannot speculate about the reasons for these pathophysiological changes, their recognition should guide research to understand their ultimate causes and develop strategies to attenuate and even reverse them. The role of changes to the Wnt-signalling pathways was minor.

Abstract MP14: Endothelial Cullin3 Mutation Causes Decreased Nitric Oxide (NO) Bioavailability And Vascular Dysfunction Through Protein Phosphatase 2A

Mutations in CULLIN3 gene (in-frame deletion of exon 9, termed Cul3Δ9) cause human hypertension (HT) driven by a combination of renal tubular and vascular mechanisms. To test the importance of endothelial Cul3 in vivo , we bred the conditionally activatable Cul3Δ9 mice with tamoxifen-inducible Tie2-CRE ERT2 mice. The resultant mice (E-Cul3Δ9) developed arterial stiffening (pulse wave velocity, 3.7±0.3 vs 2.7±0.1 m/s, n=5-7, p<0.05) and a trend towards elevated nighttime blood pressure (peak systolic BP, E-Cul3Δ9 136±3 vs control 128±3 mmHg, n=9-11) that were not associated with any alterations in locomotion, food/water intake or sleep/wake behaviors. No difference was seen in daytime BP. To determine whether vascular remodeling impairs baroreflex function, we performed power spectral analysis. Heart rate (HR), low frequency/high frequency ratio of HR variability, and baroreflex gain were comparable between control and E-Cul3Δ9 mice, suggesting no change in cardiac sympathetic nerve activity. However, low frequency amplitude of arterial pressure variability (16±4 vs 7±2 mmHg 2 , n=5-9, p<0.05) at night was markedly augmented in E-Cul3Δ9 mice, suggesting increased sympathetic activity in vascular tone regulation. Consistently, E-Cul3Δ9 mice exhibited impaired endothelial-dependent relaxation in carotid artery (max ACh relaxation: 69% vs 84%, n=5-7, p<0.05) and cerebral resistance basilar artery (41% vs 77%, n=4-6, p<0.05). However, no dilatory impairment in mesenteric resistance artery and no difference in smooth muscle function were observed, suggesting that the effects of Cul3Δ9 are arterial bed specific. Expression of Cul3Δ9 in primary mouse aortic endothelial cells markedly decreased wild type Cul3 protein, phosphorylated eNOS and NO production. Protein phosphatase (PP) 2A, a known Cul3 substrate, dephosphorylates eNOS. Therefore, we determined whether impaired eNOS activity was attributable to PP2A. Cul3Δ9-induced impairment of eNOS activity was rescued by a selective PP2A inhibitor okadaic acid (4nM), but not by a PP1 inhibitor tautomycetin (4nM). Thus, CUL3 mutations in the endothelium may contribute to human HT in part through decreased endothelial NO bioavailability, arterial stiffening and secondary sympathoexcitation.

MP08-09 NERVE TRANSFER FOR RESTORATION OF LOWER MOTOR NEURON-LESIONED BLADDER FUNCTION. CORRELATION BETWEEN HISTOLOGICAL CHANGES AND NERVE EVOKED CONTRACTIONS

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology (MP08)1 Sep 2021MP08-09 NERVE TRANSFER FOR RESTORATION OF LOWER MOTOR NEURON-LESIONED BLADDER FUNCTION. CORRELATION BETWEEN HISTOLOGICAL CHANGES AND NERVE EVOKED CONTRACTIONS Mary Barbe, Courtney L. Testa, Geneva E. Cruz, Nagat A. Frara, Ekta Tiwari, Lucas J. Hobson, Brian S McIntyre, Danielle S. Porreca, Dania Giaddui, Alan S. Braverman, Emily P. Day, Mamta Amin, Justin M. Brown, Michael Mazzei, Michel A. Pontari, Ida J. Wagner, and Michael R. Ruggieri Mary BarbeMary Barbe More articles by this author , Courtney L. TestaCourtney L. Testa More articles by this author , Geneva E. CruzGeneva E. Cruz More articles by this author , Nagat A. FraraNagat A. Frara More articles by this author , Ekta TiwariEkta Tiwari More articles by this author , Lucas J. HobsonLucas J. Hobson More articles by this author , Brian S McIntyreBrian S McIntyre More articles by this author , Danielle S. PorrecaDanielle S. Porreca More articles by this author , Dania GiadduiDania Giaddui More articles by this author , Alan S. BravermanAlan S. Braverman More articles by this author , Emily P. DayEmily P. Day More articles by this author , Mamta AminMamta Amin More articles by this author , Justin M. BrownJustin M. Brown More articles by this author , Michael MazzeiMichael Mazzei More articles by this author , Michel A. PontariMichel A. Pontari More articles by this author , Ida J. WagnerIda J. Wagner More articles by this author , and Michael R. RuggieriMichael R. Ruggieri More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000001981.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Our main objective was to determine the effects of long-term decentralization of 9-13 months and then reinnervation (followed by an 8-12 month post-reinnervation recovery period), versus the effects of 11-21 months of decentralization, on the histology of the urinary bladder, compared to sham/unoperated control animals. Our second objective was to correlate the histological findings with functional findings from ex vivo smooth muscle strip contractions in response to KCl and electric field stimulation, and with in vivo increases in bladder pressure evoked by electrical stimulation of intact pelvic nerves, transferred peripheral nerves and spinal roots. METHODS: Twelve dogs underwent decentralization by bilateral transection of coccygeal and sacral (S) spinal roots, dorsal roots of lumbar (L)7, and hypogastric nerves. Eight were reinnervated 9-13 months post-decentralization with obturator-to-pelvic nerve and sciatic-to-pudendal nerve transfers, then euthanized 8-12 months later; the remainder served as long-term decentralized only animals. Controls included 11 sham-operated and 3 unoperated animals. Before euthanasia, pelvic or transferred nerves and L1-S3 spinal roots were stimulated and maximum detrusor pressure (MDP) recorded. Bladder specimens were collected for histological and ex vivo smooth muscle contractility studies. RESULTS: Decentralized and reinnervated animals showed less urothelium or a denuded urothelium, fewer intramural ganglia, and more inflammation and collagen in the bladder wall, than controls, although percent muscle was maintained. In reinnervated animals, pgp9.5+ axon density was higher, compared to decentralized animals. Ex vivo smooth muscle contractions in response to KCl correlated positively with submucosal inflammation, detrusor muscle thickness and pgp9.5+ axon density. In vivo, decentralized and reinnervated animals showed lower MDP than controls after stimulation of transferred or pelvic nerves and L7-S3 roots. Reinnervated animals showed higher MDP after stimulation of L1-L6 roots, compared to L7-S3 roots. MDP correlated negatively with detrusor collagen and inflammation, and positively with pgp9.5+ axon density and intramural ganglia numbers. CONCLUSIONS: Although percent muscle and some bladder smooth muscle function was maintained in reinnervated animals, perhaps due to the innervation change, enhanced collagen deposition and inflammation were associated with decreased contractile function. Source of Funding: NIH 1R01NS070267 © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e153-e153 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Mary Barbe More articles by this author Courtney L. Testa More articles by this author Geneva E. Cruz More articles by this author Nagat A. Frara More articles by this author Ekta Tiwari More articles by this author Lucas J. Hobson More articles by this author Brian S McIntyre More articles by this author Danielle S. Porreca More articles by this author Dania Giaddui More articles by this author Alan S. Braverman More articles by this author Emily P. Day More articles by this author Mamta Amin More articles by this author Justin M. Brown More articles by this author Michael Mazzei More articles by this author Michel A. Pontari More articles by this author Ida J. Wagner More articles by this author Michael R. Ruggieri More articles by this author Expand All Advertisement Loading ...

Retraction Note: The anti-proliferative and anti-inflammatory response of COPD airway smooth muscle cells to hydrogen sulfide

COPD is a common, highly debilitating disease of the airways, primarily caused by smoking. Chronic inflammation and structural remodelling are key pathological features of this disease caused, in part, by the aberrant function of airway smooth muscle (ASM). We have previously demonstrated that hydrogen sulfide (H2S) can inhibit ASM cell proliferation and CXCL8 release, from cells isolated from non-smokers. We examined the effect of H2S upon ASM cells from COPD patients. ASM cells were isolated from non-smokers, smokers and patients with COPD (n = 9). Proliferation and cytokine release (IL-6 and CXCL8) of ASM was induced by FCS, and measured by bromodeoxyuridine incorporation and ELISA, respectively. Exposure of ASM to H2S donors inhibited FCS-induced proliferation and cytokine release, but was less effective upon COPD ASM cells compared to the non-smokers and smokers. The mRNA and protein expression of the enzymes responsible for endogenous H2S production (cystathionine-β-synthase [CBS] and 3-mercaptopyruvate sulphur transferase [MPST]) were inhibited by H2S donors. Finally, we report that exogenous H2S inhibited FCS-stimulated phosphorylation of ERK–1/2 and p38 mitogen activated protein kinases (MAPKs), in the non-smoker and smoker ASM cells, with little effect in COPD cells. H2S production provides a novel mechanism for the repression of ASM proliferation and cytokine release. The ability of COPD ASM cells to respond to H2S is attenuated in COPD ASM cells despite the presence of the enzymes responsible for H2S production.

Acetylation of Abelson interactor 1 at K416 regulates actin cytoskeleton and smooth muscle contraction.

Actin cytoskeletal reorganization plays an important role in regulating smooth muscle contraction, which is essential for the modulation of various physiological functions including airway tone. The adapter protein Abi1 (Abelson interactor 1) participates in the control of smooth muscle contraction. The mechanisms by which Abi1 coordinates smooth muscle function are not fully understood. Here, we found that contractile stimulation elicited Abi1 acetylation in human airway smooth muscle (HASM) cells. Mutagenesis analysis identified lysine‐416 (K416) as a major acetylation site. Replacement of K416 with Q (glutamine) enhanced the interaction of Abi1 with neuronal Wiskott‐Aldrich syndrome protein (N‐WASP), an important actin‐regulatory protein. Moreover, the expression of K416Q Abi1 promoted actin polymerization and smooth muscle contraction without affecting myosin light chain phosphorylation at Ser‐19 and vimentin phosphorylation at Ser‐56. Furthermore, p300 is a lysine acetyltransferase that catalyzes acetylation of histone and non‐histone proteins in various cell types. Here, we discovered that a portion of p300 was localized in the cytoplasm of HASM cells. Knockdown of p300 reduced the agonist‐induced Abi1 acetylation in HASM cells and in mouse airway smooth muscle tissues. Smooth muscle conditional knockout of p300 inhibited actin polymerization and the contraction of airway smooth muscle tissues without affecting myosin light chain phosphorylation and vimentin phosphorylation. Together, our results suggest that contractile stimulation induces Abi1 acetylation via p300 in smooth muscle. Acetylation at K416 promotes the coupling of Abi1 with N‐WASP, which facilitates actin polymerization and smooth muscle contraction. This is a novel acetylation‐dependent regulation of the actin cytoskeleton in smooth muscle.

Human Vascular Smooth Muscle Function and Oxidative Stress Induced by NADPH Oxidase with the Clinical Implications.

Among reactive oxygen species, superoxide mediates the critical vascular redox signaling, resulting in the regulation of the human cardiovascular system. The reduced form of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase, NOX) is the source of superoxide and relates to the crucial intracellular pathology and physiology of vascular smooth muscle cells, including contraction, proliferation, apoptosis, and inflammatory response. Human vascular smooth muscle cells express NOX1, 2, 4, and 5 in physiological and pathological conditions, and those enzymes play roles in most cardiovascular disorders caused by hypertension, diabetes, inflammation, and arteriosclerosis. Various physiologically active substances, including angiotensin II, stimulate NOX via the cytosolic subunits’ translocation toward the vascular smooth muscle cell membrane. As we have shown, some pathological stimuli such as high glucose augment the enzymatic activity mediated by the phosphatidylinositol 3-kinase-Akt pathway, resulting in the membrane translocation of cytosolic subunits of NOXs. This review highlights and details the roles of human vascular smooth muscle NOXs in the pathophysiology and clinical aspects. The regulation of the enzyme expressed in the vascular smooth muscle cells may lead to the prevention and treatment of human cardiovascular diseases.

Stair climbing activity and vascular function in patients with hypertension.

We evaluated the relationship between daily stair climbing activity and vascular function as assessed by flow-mediated vasodilation (FMD) and nitroglycerine-induced vasodilation (NID). This study was a cross-sectional study. A total of 374 patients with hypertension were enrolled. The subjects were divided into three groups based on their daily stair climbing habit: no stairs group, climbing stairs to the 2nd-floor group, and climbing stairs to the ≥3rd-floor group. There was a significant difference in FMD between the ≥3rd-floor group and the other two groups (3.3 ± 2.5% vs. 2.3 ± 2.7% and 2.4 ± 2.7%, p = 0.02, respectively). FMD values were similar in the no stairs group and the 2nd-floor group (p = 0.96). There was a significant difference in NID between the no stairs group and the other two groups (7.4 ± 4.2% vs. 10.9 ± 5.3% and 11.3 ± 5.1%, p < 0.001, respectively). NID values were similar in the second-floor group and the ≥3rd-floor group (p = 0.86). These findings suggest that both endothelial function and vascular smooth muscle function are impaired in individuals who do not climb stairs and that endothelial function but not vascular smooth muscle function is impaired in individuals who climb stairs to the second floor compared with individuals who climb stairs to the ≥3rd floor. Stair climbing activity, a simple method for assessing daily physical activity, may reflect vascular function in patients with hypertension.

Study of serum electrolytes, calcium and magnesium in preeclampsia at a tertiary hospital in central Karnataka

Preeclampsia(PE) is a hypertensive disorder affecting about 5-14% of pregnancies worldwide and around 20% of high-risk pregnancies, resulting in maternal and perinatal morbidity and mortality. Imbalances in milieu of serum sodium and potassium, that are involved in functioning of vascular smooth muscles, leads to vasoconstriction and hypertension in PE. Calcium deficiency stimulates parathyroid hormone, leading to vasoconstriction. Magnesium regulates blood pressure by monitoring vascular tone and structure. There is scarcity of studies on these factors in PE in central Karnataka. To estimate levels of serum calcium, magnesium, sodium and potassium in preeclampsia. Hospital based cross-sectional study conducted from September, 2020 to February, 2021 at a Tertiary Teaching Hospital in Chitradurga. 60 diagnosed cases of preeclampsia and equal number of age, parity and gestational age matched women with singleton normal pregnancies, attending antenatal clinic or admitted and treated in obstetrics wards of the hospital were included in the study. Clinical examination and laboratory investigations to estimate biochemical parameters were conducted. Data was analyzed in SPSS V:20. Statistical tests like chi-square and One-way ANOVA test were applied to find the significance of associations. Hypernatremia, hypokalemia, hypomagnesemia and hypocalcemia were significant in preeclampsia compared to normal pregnancy group. In severe preeclamptia, highest levels of serum sodium and lowest levels of serum potassium, magnesium and calcium were found compared to the mild PE group. Timely estimation of serum electrolytes, calcium and magnesium through the course of pregnancy could help in early detection and prompt treatment of preeclampsia and also can prevent the adverse outcomes of pregnancies. Also, dietary restriction of sodium and dietary supplementation of calcium, magnesium and potassium rich foods to the pregnant women and calcium supplementation could reduce the risk of preeclampsia.

Increased Level of Tumor Necrosis Factor-Alpha (TNF-α) Leads to Downregulation of Nitrergic Neurons Following Bilateral Cavernous Nerve Injury and Modulates Penile Smooth Tone

Erectile dysfunction (ED) after injury to peripheral cavernous nerve (CN) is partly a result of inflammation in pelvic ganglia, suggesting that ED may be prevented by inhibiting neuroinflammation. The aim of this study is to examine temporal changes of TNF-α, after bilateral CN injury (BCNI), to evaluate effect of exogenous TNF-α on neurite outgrowth from major pelvic ganglion (MPG), and to investigate effect of TNF-α signal inhibition to evaluate effects of TNF-α on penile tone with TNF-α receptor knockout mice (TNFRKO). Seventy Sprague-Dawley rats were randomized to undergo BCNI or sham surgery. Sham rats' MPGs were harvested after 48 hours, whereas BCNI groups' MPGs were at 6, 12, 24, 48 hours, 7, or 14 days after surgery. qPCR was used to evaluate gene expression of markers for neuroinflammation in MPGs. Western blot was performed to evaluate TNF-α protein amount in MPGs. MPGs were harvested from healthy rats and cultured in Matrigel with TNF-α. Neurite outgrowth from MPGs was measured after 3 days, and TH and nNOS immunofluorescence was assessed. Wild type (WT) and TNFRKO mice were used to examine effect of TNF-α inhibition on smooth muscle function after BCNI. MPGs were harvested 48 hours after sham or BCNI surgery to evaluate gene expression of nNOS and TH. Gene expression of TNF-α signaling pathway, Schwann cell and macrophage markers, protein expression of TNF-α in MPGs, and penile smooth muscle function to electrical field stimulation (EFS) were evaluated. BCNI increased gene and protein expression of TNF-α in MPGs. Exogenous TNF-α inhibited MPG neurite outgrowth. MPGs cultured with TNF-α had decreased gene expression of nNOS (P < .05). MPGs cultured with TNF-α had shorter nNOS+ neurites than TH+ neurites (P < .01). Gene expression of nNOS was enhanced in TNFRKO mice compared to WT mice (P < .01). WT mice showed enhanced smooth muscle contraction of penises of WT mice was enhanced to EFS, compared to TNFKO (P < .01). Penile smooth-muscle relaxation to EFS was greater in TNFKO mice compared to WT (P < .01). TNF-α inhibition may prevent ED after prostatectomy. TNF-α inhibition might prevent loss of nitrergic nerve apoptosis after BCNI and preserve corporal smooth muscle function but further investigation is required to evaluate protein expression of nNOS in MPGs of TNFKO mice. TNF-α inhibited neurite outgrowth from MPGs by downregulating gene expression of nNOS and TNFRKO mice showed enhanced gene expression of nNOS and enhanced penile smooth-muscle relaxation. Matsui H, Sopko NA, Campbell JD, etal. Increased Level of Tumor Necrosis Factor-Alpha (TNF-α) Leads to Downregulation of Nitrergic Neurons Following Bilateral Cavernous Nerve Injury and Modulates Penile Smooth Tone. J Sex Med 2021;18:1181-1190.

A mechanism linking perinatal 2,3,7,8 tetrachlorodibenzo-p-dioxin exposure to lower urinary tract dysfunction in adulthood.

ABSTRACTBenign prostatic hyperplasia/lower urinary tract dysfunction (LUTD) affects nearly all men. Symptoms typically present in the fifth or sixth decade and progressively worsen over the remainder of life. Here, we identify a surprising origin of this disease that traces back to the intrauterine environment of the developing male, challenging paradigms about when this disease process begins. We delivered a single dose of a widespread environmental contaminant present in the serum of most Americans [2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD), 1 µg/kg], and representative of a broader class of environmental contaminants, to pregnant mice and observed an increase in the abundance of a neurotrophic factor, artemin, in the developing mouse prostate. Artemin is required for noradrenergic axon recruitment across multiple tissues, and TCDD rapidly increases prostatic noradrenergic axon density in the male fetus. The hyperinnervation persists into adulthood, when it is coupled to autonomic hyperactivity of prostatic smooth muscle and abnormal urinary function, including increased urinary frequency. We offer new evidence that prostate neuroanatomical development is malleable and that intrauterine chemical exposures can permanently reprogram prostate neuromuscular function to cause male LUTD in adulthood.

Does and duration of exposure time dependent regulation of proteins in the urinary bladder of hamsters exposed to sodium arsenite

Does and duration of exposure time dependent regulation of proteins in the urinary bladder of hamsters exposed to sodium arsenite

Dystrophin deficiency impairs vascular structure and function in the canine model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a muscle-wasting disease caused by dystrophin deficiency. Vascular dysfunction has been suggested as an underlying pathogenic mechanism in DMD. However, this has not been thoroughly studied in a large animal model. Here we investigated structural and functional changes in the vascular smooth muscle and endothelium of the canine DMD model. The expression of dystrophin and endothelial nitric oxide synthase (eNOS), neuronal NOS (nNOS), and the structure and function of the femoral artery from 15 normal and 16 affected adult dogs were evaluated. Full-length dystrophin was detected in the endothelium and smooth muscle in normal but not affected dog arteries. Normal arteries lacked nNOS but expressed eNOS in the endothelium. NOS activity and eNOS expression were reduced in the endothelium of dystrophic dogs. Dystrophin deficiency resulted in structural remodeling of the artery. In affected dogs, the maximum tension induced by vasoconstrictor phenylephrine and endothelin-1 was significantly reduced. In addition, acetylcholine-mediated vasorelaxation was significantly impaired, whereas exogenous nitric oxide-induced vasorelaxation was significantly enhanced. Our results suggest that dystrophin plays a crucial role in maintaining the structure and function of vascular endothelium and smooth muscle in large mammals. Vascular defects may contribute to DMD pathogenesis. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Caldesmon ablation in mice causes umbilical herniation and alters contractility of fetal urinary bladder smooth muscle.

The actin-, myosin-, and calmodulin-binding protein caldesmon (CaD) is expressed in two splice isoforms: h-CaD, which is an integral part of the actomyosin domain of smooth muscle cells, and l-CaD, which is widely expressed and is involved in many cellular functions. Despite extensive research for many years, CaD's in vivo function has remained elusive. To explore the role of CaD in smooth muscle contraction in vivo, we generated a mutant allele that ablates both isoforms. Heterozygous animals were viable and had a normal life span, but homozygous mutants died perinatally, likely because of a persistent umbilical hernia. The herniation was associated with hypoplastic and dysmorphic abdominal wall muscles. We assessed mechanical parameters in isometrically mounted longitudinal strips of E18.5 urinary bladders and in ring preparations from abdominal aorta using wire myography. Ca2+ sensitivity was higher and relaxation rate was slower in Cald1-/- compared with Cald1+/+ skinned bladder strips. However, we observed no change in the content and phosphorylation of regulatory proteins of the contractile apparatus and myosin isoforms known to affect these contractile parameters. Intact fibers showed no difference in actin and myosin content, regardless of genotype, although KCl-induced force tended to be lower in homozygous and higher in heterozygous mutants than in WTs. Conversely, in skinned fibers, myosin content and maximal force were significantly lower in Cald1-/- than in WTs. In KO abdominal aortas, resting and U46619 elicited force were lower than in WTs. Our results are consistent with the notion that CaD impacts smooth muscle function dually by (1) acting as a molecular brake on contraction and (2) maintaining the structural integrity of the contractile machinery. Most importantly, CaD is essential for resolution of the physiological umbilical hernia and ventral body wall closure.

Impact of human immunodeficiency virus on pulmonary vascular disease.

With the advent of anti-retroviral therapy, non-AIDS-related comorbidities have increased in people living with HIV. Among these comorbidities, pulmonary hypertension (PH) is one of the most common causes of morbidity and mortality. Although chronic HIV-1 infection is independently associated with the development of pulmonary arterial hypertension, PH in people living with HIV may also be the outcome of various co-morbidities commonly observed in these individuals including chronic obstructive pulmonary disease, left heart disease and co-infections. In addition, the association of these co-morbidities and other risk factors, such as illicit drug use, can exacerbate the development of pulmonary vascular disease. This review will focus on these complex interactions contributing to PH development and exacerbation in HIV patients. We also examine the interactions of HIV proteins, including Nef, Tat, and gp120 in the pulmonary vasculature and how these proteins alter the endothelial and smooth muscle function by transforming them into susceptible PH phenotype. The review also discusses the available infectious and non-infectious animal models to study HIV-associated PAH, highlighting the advantages and disadvantages of each model, along with their ability to mimic the clinical manifestations of HIV-PAH.

Essential role of smooth muscle Rac1 in severe asthma associated-airway remodelling

Severe asthma is a chronic lung disease characterized by inflammation, airway hyperresponsiveness (AHR) and airway remodelling with hyperplasia of airway smooth muscle cells (aSMC). Small G proteins of the Rho family (RhoA, Rac1 and Cdc42) are key regulators of smooth muscle functions and we recently demonstrated that Rac1 controls bronchoconstriction. The objective of this study was to assess the role of Rac1 in severe asthma associated airway remodelling. We have studied the level of Rac1 activation by immunofluorescence in bronchial biopsies from severe asthmatic patients and control patients. In order to identify the role of Rac1 in aSMC remodelling, mice with specific deletion of Rac1 in SMC were sensitized to house dust mite to mimic the characteristics of severe allergic asthma in humans. Immunofluorescence analysis in human bronchial biopsies revealed an increased Rac1 activity in aSMC from severe asthmatic patients compared to control subjects. The basal proliferation rate of aSMC from severe asthmatics subjects is increased compared to control subjects. We have also observed that bFGF and PDGF-BB, growth factors described to be implicated in airway remodelling, stimulate the proliferation of aSMC from severe asthmatic subjects by 33% and 37% respectively. Moreover, the inhibition of Rac1 by EHT1864 decreased this proliferation by more than 50%. By a biochemical approach we have demonstrated that Rac1 signalling controlled human aSMC proliferation induced by mitogenic stimuli through the STAT3 signalling pathway. In vivo, we have demonstrated that specific deletion of Rac1 in SMC prevents aSMC hyperplasia and hypertrophy. This study demonstrates that Rac1 is overactive in the airways of patients with severe asthma and is essential for aSMC proliferation and airway remodelling. Inhibition of Rac1 dependant signalling pathway may represent an interesting target for the treatment of severe asthma.

The cold receptor TRPM8 activation leads to attenuation of endothelium-dependent cerebral vascular functions during head cooling

Using the cranial window technique, we investigated acute effects of head cooling on cerebral vascular functions in newborn pigs. Head cooling lowered the rectal and extradural brain temperatures to 34.3 ± 0.6°C and 26.1 ± 0.6°C, respectively. During the 3-h hypothermia period, responses of pial arterioles to endothelium-dependent dilators bradykinin and glutamate were reduced, whereas the responses to hypercapnia and an endothelium-independent dilator sodium nitroprusside (SNP) remained intact. All vasodilator responses were restored after rewarming, suggesting that head cooling did not produce endothelial injury. We tested the hypothesis that the cold-sensitive TRPM8 channel is involved in attenuation of cerebrovascular functions. TRPM8 is immunodetected in cerebral vessels and in the brain parenchyma. During normothermia, the TRPM8 agonist icilin produced constriction of pial arterioles that was antagonized by the channel blocker AMTB. Icilin reduced dilation of pial arterioles to bradykinin and glutamate but not to hypercapnia and SNP, thus mimicking the effects of head cooling on vascular functions. AMTB counteracted the impairment of endothelium-dependent vasodilation caused by hypothermia or icilin. Overall, mild hypothermia produced by head cooling leads to acute reversible reduction of selected endothelium-dependent cerebral vasodilator functions via TRPM8 activation, whereas cerebral arteriolar smooth muscle functions are largely preserved.

Coronary microvascular adaptations distal to epicardial artery stenosis.

Until recently, epicardial coronary stenosis has been considered the primary outcome of coronary heart disease, and clinical interventions have been dedicated primarily to the identification and removal of flow-limiting stenoses. However, a growing body of literature indicates that both epicardial stenosis and microvascular dysfunction contribute to damaging myocardial ischemia. In this review, we discuss the coexistence of macro- and microvascular disease, and how the structure and function of the distal microcirculation is impacted by the hemodynamic consequences of an epicardial, flow-limiting stenosis. Mechanisms of endothelial dysfunction as well as alterations of smooth muscle function in the coronary microcirculation distal to stenosis are discussed. Risk factors including diabetes, metabolic syndrome, and aging exacerbate microvascular dysfunction in the myocardium distal to a stenosis, and our current understanding of the role of these factors in limiting collateralization and angiogenesis of the ischemic myocardium is presented. Importantly, exercise training has been shown to promote collateral growth and improve microvascular function distal to stenosis; thus, the current literature reporting the mechanisms that underlie the beneficial effects of exercise training in the microcirculation distal to epicardial stenosis is reviewed. We also discuss recent studies of therapeutic interventions designed to improve microvascular function and stimulate angiogenesis in clinically relevant animal models of epicardial stenosis and microvascular disease. Finally, microvascular adaptation to removal of epicardial stenosis is considered.

Nerve transfer for restoration of lower motor neuron‐lesioned bladder function: Correlation between histological changes and nerve evoked contractions

We determined the effect of pelvic organ decentralization and reinnervation one year later on urinary bladder histology and function. Twelve dogs underwent decentralization by bilateral transection of coccygeal and sacral (S) spinal roots, dorsal roots of lumbar (L)7, and hypogastric nerves. One was euthanized at 11 and three at 18-21 months post-decentralization. Eight were reinnervated 9-13 months post-decentralization with obturator-to-pelvic nerve and sciatic-to-pudendal nerve transfer, then euthanized 8-12 months later. Controls included 11 sham-operated and 3 unoperated animals. Before euthanasia, pelvic or transferred nerves and L1-S3 spinal roots were stimulated and maximum detrusor pressure (MDP) recorded. Bladder specimens were collected for histological and ex vivo smooth muscle contractility studies. Decentralized and reinnervated animals showed loss of urothelium integrity, fewer intramural ganglia, and more inflammation and collagen, than controls, although percent muscle was maintained. In reinnervated animals, pgp9.5+ axon density was higher, compared to decentralized animals. Ex vivo smooth muscle contractions in response to KCl correlated positively with submucosal inflammation, detrusor muscle thickness and pgp9.5+ axon density. In vivo, decentralized and reinnervated animals showed lower MDP than controls after stimulation of transferred or pelvic nerves and L7-S3 roots. Reinnervated animals showed higher MDP after stimulation of L1-L6 roots, compared to L7-S3 roots. MDP correlated negatively with detrusor collagen and inflammation, and positively with pgp9.5+ axon density and intramural ganglia numbers. Although percent muscle and some bladder smooth muscle function was maintained in reinnervated animals, perhaps due to the innervation change, enhanced collagen deposition and inflammation were associated with decreased contractile function.

The Evaluation of Acute Lung Injury Induced by Nitrogen Mustard in Mouse Precision Cut Lung Slices

Nitrogen Mustard (NM; mechlorethamine hydrochloride) is a cytotoxic alkylating agent known to cause progressive pulmonary injury. Its toxic properties elicit extensive tissue remodeling within the respiratory tract, contributing to the development of chronic lung disease. Precision-Cut Lung Slices (PCLS) represent an ex vivo alternative to in vivo studies, minimizing animal numbers while increasing experimental replicates, which is advantageous for mechanistic toxicology studies and evaluating potential therapeutics. PCLS are three-dimensional excerpts of the lung that include all resident cell types; they remain viable in culture for up to 14 days, allowing for evaluation of functional activity, structural alterations and cytotoxicity. In the present studies, PCLS were used to assess NM-induced lung injury and alterations in pulmonary mechanics. C57BL/6 mice were euthanized, the lungs filled with agarose, isolated after congealment, and sliced (300 mM in thickness); slices were cultured in Dulbecco's Modified Eagle's Medium (DMEM) and Ham's F-12 Nutrient Mixture (DMEM/F-12). Twenty-four hours later, PCLS were treated with NM (1, 10, and 100 mM) for one hour, washed, refed with medium and cultured for 3 days. NM caused a dose-related increase in cytotoxicity and a decrease in mitochondrial activity as measured by lactate dehydrogenase (LDH) and WST-1 assays, respectively. Analysis of histologic sections revealed that NM caused damage to the airway epithelium; this was associated with decreased mRNA expression of collagen1A1 and collagen3A1, markers of tissue repair. To determine if these changes were linked to altered pulmonary function, we measured airway responsiveness using video microscopy. In these studies, changes in airway area after stimulation with increasing concentrations of methacholine were assessed in cross-sectional airways. Consistent with structural alterations, NM caused a ~30% decrease in maximal airway contraction after methacholine, relative to control, indicating a loss in airway smooth muscle function. These data demonstrate that PCLS can be used effectively to mechanistically assess the progression of injury induced by NM. Further understanding of the progression of damage and repair processes following exposure to mustards is essential in order to develop therapeutics for treating pulmonary toxicity.