Smooth Muscle Type Research Articles

Platelet-activating Factor (PAF) in Urinary Bladder Smooth Muscle (UBSM): Its Novel Role as a Potential Inducer of Detrusor Overactivity and the Mechanisms Underlying Its Effects in Enhancing UBSM Mechanical Activities

Platelet-activating factor (PAF), a phospholipid mediator, was discovered in 1972 as an inducer of platelet aggregation. Subsequent studies have revealed that PAF has a variety of biological functions, such as its role as a potent proinflammatory mediator. Additionally, PAF regulates the contractile functions of various types of smooth muscle (SM), such as the (1) endothelium-dependent relaxation of vascular SM; (2) contraction and epithelium-dependent relaxation of airway SM; (3) contraction of gastrointestinal SM; and (4) contraction of uterine SM, which occurs more strongly in pregnant females. PAF is produced in platelets, monocytes, neutrophils, and macrophages, which are cells related to thrombus formation and inflammation/immune responses. Furthermore, PAF is produced in various other cells throughout the body. Interestingly, recent studies have focused on the urinary bladder (UB) as a PAF-producing organ since the accumulation of this phospholipid is enhanced in patients with bladder cancer and interstitial cystitis/bladder pain syndrome, especially those who smoke. Therefore, in UB tissue, PAF may play a substantial role as an inducer or enhancer of cancers and inflammatory diseases. However, the effects of PAF on the immediate mechanical activities of UBSM have not been investigated to date. In this regard, we recently discovered that PAF strongly enhances mechanical activities (muscle tone and spontaneous contractile activity) in UBSM tissues isolated from guinea pigs and mice. In this review article, we present our data on these PAF effects together with the possible underlying mechanisms. We also discuss the potential pathophysiological roles of this phospholipid in UB diseases and disorders.

407 The OGT/O-GlcNAc Axis Regulates Fibrosis Resolution in Idiopathic Pulmonary Fibrosis

OBJECTIVES/GOALS: Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by dysregulated collagen accumulation in the lung parenchyma. Our goal is to investigate the role of O-linked N-Acetylglucosamine (O-GlcNAc) transferase (OGT) in pulmonary fibrosis to ultimately discover novel therapies for fibrosis resolution. METHODS/STUDY POPULATION: Lung tissue from IPF and non-IPF donors was subjected to immunohistochemistry (IHC) to assess O-GlcNAc levels. Primary human lung fibroblasts were treated with OGT or O-GlcNAcase (OGA) inhibitors followed by transforming growth factor-beta 1 (TGF-β1) stimulation to assess O-GlcNAc regulation of fibroblast-to-myofibroblast transition (FMT) markers [alpha smooth muscle actin (α-SMA) and type 1 and type 3 collagen (COL1α1, COL3α1)] In Drosophila melanogaster, OGT knockdown (KD)/overexpression (OE) was conditionally induced to assess pericardin, a type IV collagen-like protein, regulation by immunofluorescence. Lastly, a mouse model of bleomycin-induced pulmonary fibrosis was examined following OGT KD and assessed for fibrosis resolution via histology, hydroxyproline assay, and western blotting. RESULTS/ANTICIPATED RESULTS: O-GlcNAc staining was increased in IPF lung tissue compared to non-IPF control lungs. In primary human lung fibroblasts, TGF-α1 administration resulted in increased FMT markers (α-SMA, COL1α1, and COL3α1), which were reduced or increased by OGT or OGA inhibition, respectively. Genetic manipulation in the Drosophila models showed decreased pericardin expression with OGT KD compared to the wild-type, whereas OGT OE increased pericardin compared to control. Additionally, OGT KD in bleomycin treated aged mice resulted in reduced collagen levels at the transcript and protein level and concurrent fibrosis resolution as assessed by Masson’s trichrome staining and total hydroxyproline analysis. Collectively, showing OGT/O-GlcNAc regulating collagen in fibrosis resolution. DISCUSSION/SIGNIFICANCE: These data suggest that the OGT/O-GlcNAc axis regulates collagen deposition in pulmonary fibrosis, and we show that O-GlcNAc is implicated in the pathogenesis of IPF. We identified OGT as a therapeutic target to overcome current drug limitations, opening new horizons for biomedical treatment.

Icariin alleviates diabetic renal interstitial fibrosis aggravation by inhibiting miR-320a-3p targeting BMP6

Diabetic nephropathy is a common complication of diabetes, accumulating evidence underscores the pivotal role of tubulointerstitial fibrosis in the progression of diabetic nephropathy. However, the underlying mechanisms remain incompletely understood. Although the mechanisms in diabetic nephropathy fibrosis have been the focus of many studies, only limited information is currently available concerning microRNA regulation in tubulointerstitial fibrosis. In this study, we aimed to investigate the roles of miR-320a-3p and bone morphogenetic protein-6 (BMP6) in tubulointerstitial fibrosis. After inducing fibrosis with high glucose in HK-2 cells, we found that miR-320a-3p is significantly up-regulated, whereas BMP6 is markedly down-regulated. These changes suggest close link between miR-320a-3p and BMP6 in tubulointerstitial fibrosis. To elucidate this phenomenon, miR-320a-3p mimic, inhibitor and siBMP6 were employed. We observed in miR-320a-3p mimic group the fibrosis marker include alpha smooth muscle actin and type I collagen was significantly up-regulated, whereas BMP6 exhibited the opposite trend. Additionally, we found icariin could alleviate tubulointerstitial fibrosis by downregulation the miR-320a-3p expression. In conclusion, miR-320a-3p promotes tubulointerstitial fibrosis during the development of DN by suppressing BMP signal pathway activity via inhibiting BMP6 expression. Suggesting that miR-320a-3p represents a potential therapeutic target for tubulointerstitial fibrosis induced by diabetic nephropathy.

Insights and perspectives on the enigmatic alary muscles of arthropods.

Three types of muscles, cardiac, smooth and skeletal muscles are classically distinguished in eubilaterian animals. The skeletal, striated muscles are innervated multinucleated syncytia, which, together with bones and tendons, carry out voluntary and reflex body movements. Alary muscles (AMs) are another type of striated syncytial muscles, which connect the exoskeleton to the heart in adult arthropods and were proposed to control hemolymph flux. Developmental studies in Drosophila showed that larval AMs are specified in embryos under control of conserved myogenic transcription factors and interact with excretory, respiratory and hematopoietic tissues in addition to the heart. They also revealed the existence of thoracic AMs (TARMs) connecting to specific gut regions. Their asymmetric attachment sites, deformation properties in crawling larvae and ablation-induced phenotypes, suggest that AMs and TARMs could play both architectural and signalling functions. During metamorphosis, and heart remodelling, some AMs trans-differentiate into another type of muscles. Remaining critical questions include the enigmatic modes and roles of AM innervation, mechanical properties of AMs and TARMS and their evolutionary origin. The purpose of this review is to consolidate facts and hypotheses surrounding AMs/TARMs and underscore the need for further detailed investigation into these atypical muscles.

Identifying peristaltic pacemaker cells in the upper urinary tract.

Urine expulsion from the upper urinary tract is a necessary process that eliminates waste, promotes renal filtration and prevents nephron damage. To facilitate the movement of urine boluses throughout the upper urinary tract, smooth muscle cells that line the renal pelvis contract in a coordinated effort to form peristaltic waves. Resident pacemaker cells in the renal pelvis are critical to this process and spontaneously evoke transient depolarizations that initiate each peristaltic wave and establish rhythmic contractions. Renal pacemakers have been termed atypical smooth muscle cells due to their low expression of smooth muscle myosin and poor organization of myofilaments compared to typical (or contractile) smooth muscle cells that perform peristalsis. Recent findings discovered that pacemaker cells also express the tyrosine kinase receptor PDGFRα, enabling their identification and purification amongst other renal pelvis cell types. Improved identification methods have determined that the calcium-activated chloride channel, ANO1, is expressed by pacemaker cells and may contribute to spontaneous depolarization. A greater understanding of pacemaker and peristaltic mechanisms is warranted since aberrant contractile function may underlie diseases such as hydronephrosis, a deleterious condition that can cause significant and irreversible nephron injury.

Investigating the role of CFTR in human and mouse myometrium

BackgroundAbnormal cystic fibrosis transmembrane conductance regulator (CFTR) function in cystic fibrosis (CF) has been linked to airway smooth muscle abnormalities including bronchial hyperresponsiveness. However, a role for CFTR in other types of smooth muscle, including myometrium, remains largely unexplored. As CF life expectancy and the number of pregnancies increases, there is a need for an understanding of the potential role of CFTR in myometrial function. MethodsWe investigated the role of CFTR in human and mouse myometrium. We used immunofluorescence to identify CFTR expression, and carried out contractility studies on spontaneously contracting term pregnant and non-pregnant mouse myometrium and term pregnant human myometrial biopsies from caesarean sections. ResultsCFTR was found to be expressed in term pregnant mouse myometrium. Inhibition of CFTR, with the selective inhibitor CFTRinh-172, significantly reduced contractility in pregnant mouse and human myometrium in a concentration-dependent manner (44.89 ± 11.02 term pregnant mouse, 9.23 ± 4.75 term-pregnant human; maximal effect at 60 μM expressed as a percentage of the pre-treatment control period). However, there was no effect of CFTRinh-172 in non-pregnant myometrium. ConclusionThese results demonstrate decreased myometrial function when CFTR is inhibited, which may have implications on pregnancy and labour outcome and therapeutic decisions for labour in CF patients.

Transcriptional profiles of non-neuronal and immune cells in mouse trigeminal ganglia.

Non-neuronal cells constitute 90%-95% of sensory ganglia. These cells, especially glial and immune cells, play critical roles in the modulation of sensory neurons. This study aimed to identify, profile, and summarize the types of trigeminal ganglion (TG) non-neuronal cells in naïve male mice using published and our own data generated by single-cell RNA sequencing, flow cytometry, and immunohistochemistry. TG has five types of non-neuronal cells, namely, glial, fibroblasts, smooth muscle, endothelial, and immune cells. There is an agreement among publications for glial, fibroblasts, smooth muscle, and endothelial cells. Based on gene profiles, glial cells were classified as myelinated and non-myelinated Schwann cells and satellite glial cells. Mpz has dominant expression in Schwann cells, and Fabp7 is specific for SCG. Two types of Col1a2+ fibroblasts located throughout TG were distinguished. TG smooth muscle and endothelial cells in the blood vessels were detected using well-defined markers. Our study reported three types of macrophages (Mph) and four types of neutrophils (Neu) in TG. Mph were located in the neuronal bodies and nerve fibers and were sub-grouped by unique transcriptomic profiles with Ccr2, Cx3cr1, and Iba1 as markers. A comparison of databases showed that type 1 Mph is similar to choroid plexus-low (CPlo) border-associated Mph (BAMs). Type 2 Mph has the highest prediction score with CPhi BAMs, while type 3 Mph is distinct. S100a8+ Neu were located in the dura surrounding TG and were sub-grouped by clustering and expressions of Csf3r, Ly6G, Ngp, Elane, and Mpo. Integrative analysis of published datasets indicated that Neu-1, Neu-2, and Neu-3 are similar to the brain Neu-1 group, while Neu-4 has a resemblance to the monocyte-derived cells. Overall, the generated and summarized datasets on non-neuronal TG cells showed a unique composition of myeloid cell types in TG and could provide essential and fundamental information for studies on cell plasticity, interactomic networks between neurons and non-neuronal cells, and function during a variety of pain conditions in the head and neck regions.

Molecular insights of a CBP/β-catenin-signaling inhibitor on nonalcoholic steatohepatitis-induced liver fibrosis and disorder

Nonalcoholic steatohepatitis (NASH) is a progressive fibrotic disease associated with an increased risk of developing hepatocellular carcinoma; at present, no efficient therapeutic strategy has been established. Herein, we examined the efficacy of PRI-724, a potent inhibitor of CBP/β-catenin signaling, for treating NASH-related liver fibrosis and disorder and characterized its mechanism.Choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD)-fed mice exhibited NASH-induced liver fibrosis that is characterized by steatosis, lobular inflammation, hepatocellular injury and collagen fibrils. To examine the therapeutic effect, CDAHFD-fed mice were administered PRI-724.Serum levels of ALT and pro-fibrotic molecule, i.e. Mac-2 bp, alpha smooth muscle actin, type I and type III collagens, decreased significantly. mRNA levels of the matrix metalloproteinases Mmp8 and Mmp9 in the liver were significantly increased, and increases in the abundance of MMP9-producing neutrophils and macrophages were observed. Marco+Mmp9+Cd68+ Kupffer cells were only observed in the livers of mice treated with PRI-724, and Mmp9 expression in Marco+Cd68+ Kupffer cells increased 4.3-fold. Moreover, hepatic expression of the lipid metabolism regulator, pyruvate dehydrogenase kinase 4 and liver lipid droplets also decreased significantly.PRI-724-treated NASH mice not only recovered from NASH-related liver fibrosis through the effect of PRI-724 down-regulating the expression of pro-fibrotic genes and up-regulating the expression of anti-fibrotic genes, but they also recovered from NASH-induced liver disorder. PRI-724, a selective CBP/β-catenin inhibitor, thus shows a potent therapeutic effect for NASH-related liver fibrosis and for decreasing adipose tissue in the liver.

Selective inhibition of neurogenic, but not agonist-induced contractions by phospholipase A2 inhibitors points to presynaptic phospholipase A2 functions in contractile neurotransmission to human prostate smooth muscle.

Phospholipases A2 (PLA2 ) may be involved in α1 -adrenergic contraction by formation of thromboxane A2 in different smooth muscle types. However, whether this mechanism occurs with α1 -adrenergic contractions of the prostate, is still unknown. While α1 -adrenoceptor antagonists are the first line option for medical treatment of voiding symptoms in benign prostatic hyperplasia (BPH), improvements are limited, probably by nonadrenergic contractions including thromboxane A2 . Here, we examined effects of PLA2 inhibitors on contractions of human prostate tissues. Prostate tissues were obtained from radical prostatectomy. Contractions were induced by electric field stimulation (EFS) and by α1 -adrenergic agonists in an organ bath, after application of the cytosolic PLA2 inhibitors ASB14780 and AACOCF3, the secretory PLA2 inhibitor YM26734, the leukotriene receptor antagonist montelukast, or of solvent to controls. Frequency-dependent contractions of human prostate tissues induced by EFS were inhibited by 25% at 8 Hz, 38% at 16 Hz and 37% at 32 Hz by ASB14780 (1 µM), and by 32% at 16 Hz and 22% at 32 Hz by AACOCF3 (10 µM). None of both inhibitors affected contractions induced by noradrenaline, phenylephrine or methoxamine. YM26734 (3 µM) and montelukast (0.3and 1 µM) neither affected EFS-induced contractions, nor contractions by α1 -adrenergic agonists, while all contractions were substantially inhibited by silodosin (100 nM). Our findings suggest presynaptic PLA2 functions in prostate smooth muscle contraction, while contractions induced by α1 -adrenergic agonists occur PLA2 -independent. Lacking sensitivity to montelukast excludes an involvement of PLA2 -derived leukotrienes in promotion of contractile neurotransmission.

Effects of Sprayable, Highly Adhesive Hydrophobized Gelatin Microparticles on Endoscopic Submucosal Dissection: A Swine Model

Introduction: Sprayable wound dressings containing hydrophobized microparticles (hMPs) are characterized by strong adhesiveness. We examined the effect of hMPs derived from Alaska pollock gelatin on endoscopic submucosal dissection (ESD) ulcers. Methods: (1) In an in vivo model of miniature swine gastric ESD, gastric ulcers were created by ESD and then sprayed with hMPs or untreated followed by microscopic examination. (2) In an ex vivo ESD model of resected stomach, a pinhole-shaped perforation was created on the ESD ulcer of resected stomach; hMPs were then sprayed on the perforation; and air leakage and intragastric pressure were measured. (3) In an in vivo duodenal ESD model of miniature swine, duodenal artificial ESD ulcers with pinhole-shaped perforation were examined; ulcers were classified into hMPs-sprayed and nonsprayed groups, and inflammation in the intrinsic muscle layer and serosa were compared between the groups. Results: (1) Histological observation of submucosal tissues showed a decreased number of invading inflammatory cells in hMP-sprayed tissues compared with the control in miniature swine gastric ESD (p < 0.05). In addition, the rates of anti-alpha smooth muscle actin and type I collagen positivity were significantly lower in the hMPs group than in the control group (p < 0.05). (2) Intragastric pressure could not be measured in the nonsprayed group, whereas no air leakage was observed in the sprayed group when pressurized up to 26 mm Hg in the resected stomach model. (3) The sprayed group showed suppressed inflammation of the intrinsic muscular layer and serosa in both cases compared with the nonsprayed group in miniature swine duodenal ESD (p < 0.05). Conclusions: Sprayable, tissue-adhesive hMPs are a promising medical material for intraoperative and postoperative treatment of ESD-induced wound via anti-inflammation and strong adhesiveness.

Prolactin-Induced Protein facilitates corneal wound healing.

The purpose of the study was to investigate the role of Prolactin-Induced Protein (PIP) in corneal wound healing, in vivo and in vitro. In C57BL/6J mice, corneal epithelia was removed using an ocular burr. Phosphate buffered saline (PBS) or PIP (0.5 and 1.0μg/mL) was applied topically or subconjunctivally injected. PIP accelerated wound closure as early as 24h. PIP treatment promoted corneal wound healing and epithelial integrity and thickness. Integrin α6, integrin β4, Thrombospondin-1, and TGF-β1 expressions were all downregulated by PIP after wound closure. In vitro, scratch assays were performed using primary human epithelial cells (HCECs) and human corneal fibroblasts (HCFs), stimulated with PIP at various dosages. PIP treatment promoted both HCECs and HCFs migration. PIP upregulated expression of integrin α6, integrin β4, and Thrombospondin-1 in HCECs. Expression of TGF-β1 in HCECs and expression of smooth muscle actin (SMA) and Type III Collagen (Col III) in HCFs were significantly downregulated at 150ng/mL PIP. PIP exhibits noteworthy anti-fibrotic potentiality. While the mechanism of how PIP is impactful on the corneal wound healing cascade is unknown, our findings are novel and further studies are warranted in order to unravel any therapeutic potential.

Abstract 13708: Il11 Causes Pulmonary Tissue Remodeling and Emphysematous Lung Disease in the Fbn1 C1041G/+ Mouse Model of Marfan Syndrome

Introduction: Marfan Syndrome (MFS) is an inherited connective tissue disorder caused by fibrillin-1 (FBN1) mutations that is associated with aortic disease. Lung abnormalities are common in MFS, but their pathogenesis is poorly understood. Here we studied IL11 in a mouse model of MFS (mMFS). Methods: We studied histological and molecular phenotypes in the lungs of Fbn1 C1041G/+ (mMFS) mice, an established model of MFS. To identify IL11-expressing lung cell types, we studied 4- and 16-week-old Fbn1 C1041G/+ : Il11 EGFP/+ reporter mice lungs by immunohistochemistry. We studied the protective effects of IL11 inhibition in the lung by RT-qPCR, immunoblots and histopathology in two models: 1) 16-week-old IL11 receptor (IL11RA) knockout mMFS mice ( Fbn1 C1041G/+ : Il11ra1 -/- ) and 2) administered IgG isotype control or anti-IL11RA (X209; 20mg/kg) antibodies twice weekly in mMFS mice between 4 to 24 weeks of age. Results: IL11 was upregulated in the lungs of mMFS mice, which exhibited progressive loss and enlargement of distal airspaces and increased extracellular matrix production. In the distal airways of young mMFS mice (4 weeks old), IL11 was specifically expressed in smooth muscle cells. In the older (16 weeks old) mMFS lungs, IL11 was expressed in smooth muscle cells, fibroblasts and type II alveolar epithelial cells. In mMFS mice, genetic ( Fbn1 C1041G/+ : Il11ra1 -/- mice) or pharmacologic (anti-IL11RA) inhibition of IL11 protected against emphysematous changes, pulmonary fibrosis and inflammation and was associated with reduced pulmonary ERK1/2 signaling and matrix metalloproteinase (MMP2 and MMP9) expression. Conclusions: IL11 causes pulmonary disease in mMFS. This adds to the recent finding that IL11 underlies aortic pathology in mMFS and provides a common disease mechanism across affected tissues in MFS. This has potential implications for treating multi-organ disease in MFS.

Foxc1 and Foxc2 are indispensable for the maintenance of nephron and stromal progenitors in the developing kidney

Nephron development proceeds with reciprocal interactions among three layers: nephron progenitors (NPs), ureteric buds and stromal progenitors (SPs). We found that Foxc1 and Foxc2 (Foxc1/2) are expressed in NPs and SPs. Systemic deletion of Foxc1/2 2 days after the onset of metanephros development (embryonic day 13.5) resulted in the epithelialization of NPs and ectopic formation of renal vesicles. NP-specific deletion did not cause these phenotypes, indicating that Foxc1/2 in other cells (likely in SPs) contributed to the maintenance of NPs. Single-cell RNA-sequencing analysis revealed the existence of NP and SP subpopulations, the border between committed NPs and renewing NPs, and similarity between the cortical interstitium and vascular smooth muscle type cells. Integrated analysis of the control and Foxc1/2 knockout data indicated transformation of some NPs to strange cells expressing markers of the vascular endothelium, reduced numbers of self-renewing NP and SP populations, and downregulation of crucial genes for kidney development, such as Fgf20 and Frem1 in NPs, and Foxd1 and Sall1 in SPs. It also revealed upregulation of genes that were not usually expressed in NPs and SPs. Thus, Foxc1/2 maintain NPs and SPs by regulating the expression of multiple genes.

Two novel bombesin-like neuropeptides from the skin secretion of Pelophylax kl. esculentus: Ex vivo pharmacological characterization on rat smooth muscle types.

Mammalian bombesin-like neuropeptides (BLPs) play an important role in regulation of physiological and pathophysiological processes. Frog skin-derived BLPs, of smaller size and diverse lengths and sequences at their N-terminus, have attracted the attention of many researchers. However, these N-terminal variants and the receptors modulating their pharmacological actions are poorly studied and less understood. In this study, two BLPs, namely, [Asn3, Lys6, Thr10, Phe13]3–14-bombesin and [Asn3, Lys6, Phe13]3–14-bombesin with primary structures NLGKQWATGHFM and NLGKQWAVGHFM were isolated from the skin secretion of hybrid Pelophylax kl. esculentus. Both BLPs share a similar primary structure with only a single amino acid substitution at the eighth position (threonine to valine), while they have quite different myotropic potencies with EC50 values in the range of 22.64 ± 9.7 nM (N = 8) to 83.93 ± 46.9 nM (N = 8). The potency of [Asn3, Lys6, Thr10, Phe13]3–14-bombesin was approximately 3-fold higher than that of [Asn3, Lys6, Phe13]3–14-bombesin. Through the investigation of receptor selectivity using a canonical bombesin receptor antagonist, it was found that [Asn3, Lys6, Thr10, Phe13]3–14-bombesin and [Asn3, Lys6, Phe13]3–14-bombesin had an affinity to both BB1 and BB2 receptors. Their contractile functions are mainly modulated by both BB1 and BB2 receptors on rat urinary bladder and BB2 alone on rat uterus smooth muscle preparations. These data may provide new insights into the design of potent and selective ligands for bombesin receptors. Moreover, [Asn3, Lys6, Thr10, Phe13]3–14-bombesin and [Asn3, Lys6, Phe13]3–14-bombesin did not induce significant hemolysis and toxicity in normal human cells, suggesting that these two natural novel BLPs have great potential for development into new drug candidates.

MicroRNA expression signature in the striated and smooth adductor muscles of Yesso scallop Patinopecten yessoensis

Increasing evidences point to the potential role of microRNAs (miRNAs) in muscle growth and development in animals. However, knowledge on the identity of miRNAs and their targets in molluscs remains largely unknown. Scallops have one large adductor muscle, composed of fast (striated) and slow (smooth) muscle types, which display great differences in muscle fibers, meat quality, cell types and molecular components. In the present study, we performed a comprehensive investigation of miRNA transcriptomes in fast and slow adductor muscles of Yesso scallop Patinopecten yessoensis. As a result, 47 differentially expressed miRNAs representing ten miRNA families were identified between the striated and smooth adductor muscles. The KEGG enrichment analysis of their target genes were mainly associated with amino acid metabolism, energy metabolism and glycan biosynthesis. The target genes of miR-133 and miR-71 were validated by the dual-luciferase reporter assays and miRNA antagomir treatment in vivo. The identification and functional validation of these different miRNAs in scallops will greatly help our understanding of miRNA regulatory mechanism that achieves the unique muscle phenotypes in scallops. The present findings provide the direct evidences for muscle-specific miRNAs involved in muscle growth and differentiation in molluscs.

Shapeshifters in Pathology: Paratesticular Solitary Fibrous Tumor Mimicking Leiomyoma.

Solitary fibrous tumors are mesenchymal fibroblastic tumors that were originally described as intrathoracic lesions but have since been found to occur in many other locations. They may rarely occur as paratesticular masses. Here we present a peculiar case of a solitary fibrous tumor arising in the paratesticular region and exhibiting leiomyoma-like morphology. Confirmation of the tumor as a solitary fibrous tumor was achieved by RNA sequencing showing NAB2::STAT6 fusion. Possible explanations for the unusual tumor morphology include entrapment of normal smooth muscle elements and tumor differentiation into smooth muscle type cells.

Mechanosensitive modulation of peristaltic contractions in the mouse renal pelvis

The renal pelvis develops spontaneous phasic contractions (SPCs) that underlie pyeloureteric peristalsis. Increased urine flow into the renal pelvis mechanically stimulates the contractile machinery within the renal pelvis to facilitate the propagation of peristalsis. Here, the effects of mechanostimulation of the pelvicalyceal junction (PCJ), where SPCs originate from, on the properties of SPCs were investigated. Using the wire myograph, isometric tension changes in tubular preparations of mouse renal pelvis with calyces were circumferentially measured, while mechanostimuli were applied to the PCJ. Immunohistochemistry and intracellular Ca2+ imaging were performed, respectively, to investigate the distribution and functional roles of mechanosensitive TRPV4 channels in the renal pelvis. SPCs periodically originated from PCJ and propagated distally. Mechanostimulation of the PCJ reduced the frequency of SPCs by about 60%, while almost quadrupling their amplitude. Capsaicin (100 nM), an agonist of TRPV1 channels, or calcitonin gene-related peptide (CGRP) (30 nM) also slowed and enlarged SPCs. A prolonged pre-exposure to capsaicin or BIBN4096 (1 μM), a CGRP receptor antagonist, inhibited the mechanostimulation-induced reduction in the SPC frequency, but did not block the increase in SPC amplitude. TRPV4 immunoreactivity was expressed in both atypical (ASMCs) and typical smooth muscle cells (TSMCs). GSK1016790A (100 nM), a TRPV4 agonist, enlarged SPCs independently of TRPV1 or CGRP without increasing the amplitude of spontaneous Ca2+ transients in TSMCs. Thus, mechanostimulation of PCJ appears to activate TRPV1-expressing sensory nerves, releasing CGRP that predominantly reduce the SPC frequency. Activation of TRPV4 may be involved in the mechanosensitive enlargement of SPCs. (247 words).

ADP Ribosylation Factor 6 Promotes Contraction and Proliferation, Suppresses Apoptosis and Is Specifically Inhibited by NAV2729 in Prostate Stromal Cells.

The presumed ADP ribosylation factor (ARF) 6 inhibitor NAV2729 inhibits human prostate smooth muscle contraction and proliferation of stromal cells, which are driving factors of voiding symptoms in benign prostatic hyperplasia (BPH). However, its specificity and a confirmed role of ARF6 for smooth muscle contraction are still pending. Here, we generated monoclonal ARF6 knockouts in human prostate stromal cells (WPMY-1), and characterized phenotypes of contractility, growth-related functions, and susceptibility to NAV2729 in knockout and control clones. ARF6 knockout was verified by Western blot. Knockout clones showed impaired contraction and actin organization, reduced proliferation and viability, and increased apoptosis and cell death. In ARF6-expressing control clones, NAV2729 (5 µM) strongly inhibited contraction (67% inhibition across all three control clones), actin organization (72%), proliferation (97%), and viability (up to 82%), and increased apoptosis (5-fold) and cell death (6-fold). In ARF6 knockouts, effects of NAV2729 (5 µM) were widely reduced, including lacking or minor effects on contractions (0% inhibition across all three knockout clones), actin (18%) and proliferation (13%), and lacking increases of apoptosis and cell death. Viability was reduced by NAV2729 with an IC50 of 3.3 µM across all three ARF6 control clones, but of 4.5-8.2 µM in ARF6 knockouts. In conclusion, ARF6 promotes prostate smooth muscle contraction and proliferation of stromal cells. Both are inhibited by NAV2729, which showed high specificity for ARF6 up to 5 µM and represents an attractive compound in the context of BPH. Considering the relevance of smooth muscle-based diseases, shared roles of ARF6 in other smooth muscle types merit further investigation. SIGNIFICANCE STATEMENT: By knockout of ARF6 in prostate stromal cells, this study demonstrates the involvement of ARF6 in promotion of prostate smooth muscle contraction and stromal growth, and defines concentration ranges for their ARF6-specific inhibition by NAV2729. Besides the context of benign prostatic hyperplasia and lower urinary tract symptoms, analog ARF6 functions in contraction and growth appear possible in other smooth muscle-rich organs, which merits further attention considering the high clinical relevance of smooth muscle-based diseases.

IMMUNOHISTOPATHOLOGIC VARIATION OF ORAL MYOPERICYTOMA

<h3>Objective</h3> Myopericytoma (MP) is a rare soft tissue neoplasm infrequently arising in the oral cavity. We aimed to clarify the clinicopathologic characteristics of oral MP based on histologic and immunohistochemical findings. <h3>Methods</h3> We reviewed myogenic neoplasms in our archives and selected 6 cases of MP. Immunostainings were performed using antibodies for smooth muscle actin (SMA), calponin, h-Caldesmon, muscle-specific actin (MSA), desmin, and type IV collagen antibody. <h3>Results</h3> Our patients were predominantly female, and the mean age and size were 57.5 years and 10.8 mm, respectively. Histologically, 3 of 6 cases were well circumscribed but others showed ill-defined borders. Three cases were classic but others were angioleiomatous and myofibromatous cases with a mostly fascicular pattern. Myxoid change appeared in 2 cases. Hyalinized nodules (vascular balls) were found in both classic and nonclassic types. Only one case partially comprised small round cells mimicking glomus cells, but these lacked peculiar type IV collagen expression. Two angioleiomatous cases harbored dilated blood vessels. Two cases partially showed a staghorn and indistinct biphasic pattern but a myofibromatous type demonstrated neither. Immunohistochemically, all cases that tested were positive or focally positive for SMA, MSA, and calponin but negative tumor cells were noted in part. However, h-Caldesmon was only detected in 2 out of 5 cases examined. Desmin was constant-negative except for 1 classic case with focally positive reaction. <h3>Conclusion</h3> Oral MPs are morphologically and phenotypically heterogeneous neoplasms that can sometimes mimic angioleiomyoma or glomangioma. It is necessary to recognize the presence of cases showing varied myogenic natures.

Smooth Muscle Contraction Is Regulated by Chloride Channels: Functional Evidence for TMEM16A in Porcine Coronary Arteries

Contraction of coronary smooth muscle is influenced by ion channels that control membrane potential and, thus, Ca2+ influx. A great deal of attention has been focused on K+ channels, as their opening makes membrane potential more negative, reduces Ca2+ influx, and causes relaxation. Our objective was to investigate ion channels in coronary smooth muscle whose opening would depolarize membrane potential, increase Ca2+ influx, and promote contraction. One potential candidate for study is the Ca2+-activated Cl− channel TMEM16A, which is expressed in a variety of smooth muscle types. We tested the hypothesis that drugs which influence TMEM16A activity would alter contraction. Specifically, we tested the idea that coronary smooth muscle contraction would be enhanced by a TMEM16A activator (Eact), whereas coronary smooth muscle contraction would be attenuated by a TMEM16A inhibitor (T16Ainh-A01). We used isometric tension recording methods on epicardial coronary artery segments (3 mm length) from domestic swine to test the predictions. Contractions in response to elevated extracellular K+ (5-30 mM in 5 mM increments) were recorded before and after treatment with 5 μM Eact or 5 μM T16Ainh-01. Extracellular K+ was varied by adding potassium gluconate, rather than potassium chloride, in order to keep the Cl− concentration constant. Concentration-response curves were fit by nonlinear regression and compared with two-way repeated measures ANOVA. K+ contracted coronary artery rings with an EC50 of 19.1 ± 0.6 mM and a maximum of 11.8 ± 1.4 g. Drug vehicle (DMSO; 0.05%) had no effect on contraction (EC50 or maximum). EACT shifted K+-induced contraction to the left (17.8 ± 0.9 mM; P < 0.05), but did not affect the maximum (105 ± 3% of control). T16Ainh-A01 shifted K+-induced contraction to the right (20.4 ± 0.6 mM; P < 0.05), but did not affect the maximum (90 ± 1% of control). These data suggest that TMEM16A is expressed in the porcine coronary artery and can influence function through electromechanical coupling.