It has been reported that, in the spontaneously hypertensive rat (SHR) model of hypertension, different components of the G-protein/adenylate cyclase (AC)/Calcium-activated potassium channel of high conductance (BK) channel signaling pathway are altered differently. In the upstream part of the pathway (G-protein/AC), a comparatively low efficacy has been established, whereas downstream BK currents seem to be increased. Thus, the overall performance of this signaling pathway in SHR is elusive. For a better understanding, we focused on one aspect, the direct targeting of the BK channel by the G-protein/AC pathway and tested the hypothesis that the comparatively low AC pathway efficacy in SHR results in a reduced agonist-induced stimulation of BK currents. This hypothesis was investigated using freshly isolated smooth muscle cells from WKY and SHR rat tail artery and the patch-clamp technique. It was observed that: (1) single BK channels have similar current-voltage relationships, voltage-dependence and calcium sensitivity; (2) BK currents in cells with a strong buffering of the BK channel activator calcium have similar current-voltage relationships; (3) the iloprost-induced concentration-dependent increase of the BK current is larger in WKY compared to SHR; (4) the effects of activators of the PKA pathway, the catalytic subunit of PKA and the potent and selective cAMP-analogue Sp-5,6-DCl-cBIMPS on BK currents are similar. Thus, our data suggest that the lower iloprost-induced stimulation of the BK current in freshly isolated rat tail artery smooth muscle cells from SHR compared with WKY is due to the lower efficacy of upstream elements of the G-Protein/AC/BK channel pathway.

OBJECTIVE: Cantu Syndrome (CS), a multisystem disease with a complex cardiovascular phenotype, is caused by GoF variants in the Kir6.1/SUR2 subunits of ATP-sensitive potassium (KATP) channels, and is characterized by low systemic vascular resistance, as well as tortuous, dilated vessels, and decreased pulse-wave velocity. Thus, CS vascular dysfunction is multifactorial, with both hypomyotonic and hyperelastic components. To dissect whether such complexities arise cell-autonomously within vascular smooth muscle cells (VSMCs), or as secondary responses to the pathophysiological milieu, we assessed electrical properties and gene expression in human induced pluripotent stem cell-derived VSMCs (hiPSC-VSMCs), differentiated from control and CS patient-derived hiPSCs, and in native mouse control and CS VSMCs.
RESULTS: Whole-cell voltage-clamp of isolated aortic and mesenteric arterial VSMCs isolated from wild type (WT) and Kir6.1[V65M] (CS) mice revealed no clear differences in voltage-gated K+ (Kv) or Ca2+ currents. Kv and Ca2+ currents were also not different between validated hiPSC-VSMCs differentiated from control and CS patient-derived hiPSCs. While pinacidil-sensitive KATP currents in control hiPSC-VSMCs were consistent with those in WT mouse VSMCs, they were considerably larger in CS hiPSC-VSMCs. Under current-clamp conditions, CS hiPSC-VSMCs were also hyperpolarized, consistent with increased basal K conductance, and providing an explanation for decreased tone and decreased vascular resistance in CS. Increased compliance was observed in isolated CS mouse aortae, and was associated with increased elastin mRNA expression. This was consistent with higher levels of elastin mRNA in CS hiPSC-VSMCs, suggesting that the hyperelastic component of CS vasculopathy is a cell-autonomous consequence of vascular KATP GoF.
CONCLUSIONS: The results show that hiPSC-VSMCs reiterate expression of the same major ion currents as primary VSMCs, validating the use of these cells to study vascular disease. Results in hiPSC-VSMCs derived from CS patient cells suggest that both the hypomyotonic and hyperelastic components of CS vasculopathy are cell-autonomous phenomena driven by KATP overactivity within VSMCs.

Atherosclerosis is rare in internal thoracic arteries (ITA) even in patients with severe atherosclerotic coronary artery (ACA) disease. To explore cellular differences, ITA SMC from 3 distinct donors and ACA SMC from 3 distinct donors were grown to sub-confluence and growth arrested for 48 h. Proliferation and thrombospondin-1 (TSP1) production were determined using standard techniques. ITA SMC were larger, grew more slowly and survived more passages than ACA SMC. ACA SMC had a more pronounced proliferative response to 10% serum than ITA SMC. Both ACA SMC and ITA SMC proliferated in response to exogenous TSP1 (12.5 µg/ml and 25 µg/ml) and platelet derived growth factor-BB (PDGF-BB; 20 ng/ml) but TSP1- and PDGF-BB-induced proliferation were partially inhibited by anti-TSP1 antibody A4.1, microRNA-21(miR-21)-3p inhibitors and miR-21-5p inhibitors in each of the 3 ACA SMC lines, but not in any of the ITA SMC lines. PDGF-BB stimulated TSP1 production in ACA SMC but not in ITA SMC but there was no increase in TSP1 levels in conditioned media in either SMC type. In summary, there are significant differences in morphology, proliferative capacity and in responses to TSP1 and PDGF-BB in SMC derived from ITA compared to SMC derived from ACA.

Bile acids regulate gastrointestinal motility by mechanisms that are poorly understood. Standard isolated tissue bath assays might not recapitulate in vivo physiology if contractile responses to certain bile acids require direct application to the intestinal mucosa. We sought to determine the feasibility of quantifying longitudinal smooth muscle contractile responses to bile acids from intact segments of everted mouse ileum. Ileum from adult female C57BL/6J mice was isolated, gently everted over a notched metal rod, and mounted in tissue baths. Individual bile acids and agonists of bile acid receptors were added to the baths, and longitudinal smooth muscle contractile responses were quantified by isometric force transduction. Ursodeoxycholic acid robustly increased contractile responses in a dose-dependent manner. Deoxycholic acid stimulated contractility at low doses but inhibited contractility at high doses. Chenodeoxycholic acid, glycocholic acid, and lithocholic acid did not alter contractility. The dose-dependent increase in contractility resulting from the application of ursodeoxycholic acid was recapitulated by INT-777, an agonist of the Takeda G protein-coupled receptor 5 (TGR5), and by cevimeline, a muscarinic acetylcholine receptor agonist. Agonists to the nuclear receptors farnesoid X receptor, glucocorticoid receptor, pregnane X receptor, vitamin D receptor, and to the plasma membrane epidermal growth factor receptor did not modify baseline contractile patterns. These results demonstrate that gentle eversion of intact mouse ileum facilitates the quantification of longitudinal smooth muscle contractile responses to individual bile acids. Prokinetic effects of ursodeoxycholic acid and low-dose deoxycholic acid are replicated by agonists to TGR5 and muscarinic acetylcholine receptors.

暂无摘要。

BACKGROUND: The natural history and pathophysiology of diverticular disease (DD) are still uncertain. An ex-vivo human complicated DD (cDD) model has recently shown a predominant transmural oxidative imbalance. The present study aims to evaluate whether the previously described alterations may precede the symptomatic form of the disease.
METHODS: Colonic surgical samples obtained from patients with asymptomatic diverticulosis (DIV), complicated DD, and controls were systematically and detailed morphologically and molecularly analyzed. Therefore, histologic, histomorphometric, immunohistochemical evaluation, and gene and protein expression analysis were performed to characterize colonic muscle changes and evaluate chronic inflammation, oxidative imbalance, and hypoxia. Functional muscle activity was tested on strips and isolated cells in response to contractile and relaxant agents.
RESULTS: Compared with controls, DD showed a marketed increase in muscle layer thickness, smooth muscle cell syncytium disarray, and increased interstitial fibrosis; moreover, the observed features were more evident in the cDD group. These changes mainly affected longitudinal muscle and were associated with altered contraction-relaxation dynamics and fibrogenic switch of smooth muscle cells. Chronic lymphoplasmacytic inflammation was primarily evident in the mucosa and spared the muscle. A transmural increase in carbonylated and nitrated proteins, with loss of antioxidant molecules, characterized both stages of DD, suggesting early oxidative stress probably triggered by recurrent ischemic events, more pronounced in cDD, where HIF-1 was detected in both muscle and mucosa.
CONCLUSIONS: The different DD clinical scenarios are part of a progressive process, with oxidative imbalance representing a new target in the management of DD.

Asthma is characterized by aberrant airway smooth muscle (ASM) proliferation, which increases the thickness of the ASM layer within the airway wall and exacerbates airway obstruction during asthma attacks. The mechanisms that drive ASM proliferation in asthma are not entirely elucidated. Ten-eleven translocation methylcytosine dioxygenase (TET) is an enzyme that participates in the regulation of DNA methylation by catalyzing the hydroxylation of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC). The generation of 5-hmC disinhibits the gene silencing effect of 5-mC. In this study, TET1 activity and protein were enhanced in asthmatic human ASM cell cultures. Moreover, the level of 5-hmC was higher in asthmatic ASM cells as compared to nonasthmatic ASM cells. Knockdown (KD) of TET1, but not TET2, reduced the level of 5-hmC in asthmatic cells. Because the cytoskeletal protein nestin controls cell proliferation by modulating mechanistic target of rapamycin (mTOR), we evaluated the effects of TET1 KD on this pathway. TET1 KD reduced nestin expression in ASM cells. Moreover, TET1 inhibition alleviated the platelet-derived growth factor (PDGF)-induced phosphorylation of p70S6K, 4E-BP, S6, and Akt. TET1 inhibition also attenuated the proliferation of ASM cells. Taken together, these results suggest that TET1 drives ASM proliferation via the nestin-mTOR axis.

暂无摘要。

BACKGROUND: Congenital inguinal hernia, hydrocele and undescended testis (UDT) are associated with patent processus vaginalis. The smooth muscles present in the processus vaginalis aid in the descent of the testis and undergo programmed cell death after testicular descent leading to obliteration. The persisting amount of smooth muscle in the processus vaginalis influences the clinical outcome as inguinal hernia, hydrocele or UDT. Therefore, a study was conducted to evaluate the processus vaginalis in these three conditions to observe the presence and phenotype of smooth muscle cells and the presence of myofibroblasts.
METHODS: The processus vaginalis sacs in patients with inguinal hernia, hydrocele and UDT were examined using light microscopy for the presence and distribution of smooth muscle cells and immunohistochemical staining for vimentin, desmin, and α-smooth muscle actin (SMA) to identify the smooth muscle phenotype. Transmission electron microscopy was also performed in all the sacs to observe the presence of myofibroblasts.
RESULTS: Seventy-eight specimens of processus vaginalis (from seventy-four patients), distributed as 47%, 27%, and 26% as inguinal hernia, hydrocele and UDT respectively, were included in the study. The sacs from inguinal hernia and hydrocele had significantly more presence of smooth muscles distributed as multiple smooth muscle bundles (p < 0.001). Desmin and SMA staining of smooth muscle cells was observed in significantly more sacs from hydrocele, followed by inguinal hernia and UDT (p < 0.001). The sacs from UDT had a significant presence of striated muscles (p = 0.028). The sacs from inguinal hernia had a significant presence of myofibroblasts, followed by hydrocele and UDT (p < 0.001) and this significantly correlated with the light microscopy and immunohistochemical features. The processus vaginalis sacs from four female patients did not differ statistically from the male inguinal hernia sacs in any of the above parameters.
CONCLUSIONS: The processus vaginalis sacs in pediatric inguinal hernia, hydrocele and undescended testis differ in the presence, distribution and phenotype of smooth muscles and the presence of myofibroblasts. The clinical presentations in these entities reflect these differences.

The vagina is an essential component of the female reproductive system and is responsible for providing female sexual satisfaction. Vaginal smooth muscle contraction plays a crucial role in various physiological processes, including sexual arousal, childbirth, and urinary continence. In pathophysiological conditions, such as pelvic floor disorders, aberrations in vaginal smooth muscle function can lead to urinary incontinence and pelvic organ prolapse. A set of cellular and sub-cellular physiological mechanisms regulates the contractile properties of the vaginal smooth muscle cells. Calcium influx is a crucial determinant of smooth muscle contraction, facilitated through voltage-dependent calcium channels and calcium release from intracellular stores. Comprehensive reviews on smooth muscle biophysics are relatively scarce within the scientific literature, likely due to the complexity and specialized nature of the topic. The objective of this review is to provide a comprehensive description of alterations in the cellular physiology of vaginal smooth muscle contraction. The benefit associated with this particular approach is that conducting a comprehensive examination of the cellular mechanisms underlying contractile activation will enable the creation of more targeted therapeutic agents to control vaginal contraction disorders.