Enhancement of Vascular Smooth Muscle Contractility by Alterations of Membranous Architecture.

Plasma membrane (PM) of smooth muscle cells hosts channel molecules regulating the flow of various ions. An intact architecture of PM is essential to orchestrate proper channel functions in order to complete agonist-mediated contraction, which includes Ca2+ release from the sarcoplasmic reticulum (SR) to initiate contraction, and subsequent Ca2+ refilling into SR through PM to sustain muscle contraction. The Junctional Complex (JC), comprising of junctional SR, and its apposing PM and neighboring caveolae, provides a quasi-enclosed microdomain housing receptors as well as ion channels and also restricting ion diffusions into the cytosol so the cell achieves optimal performance. The spatial arrangement of the JC is believed to ensure an uninterrupted Ca2+ cycling route. Full understanding of the functional role of the JC is the key to elucidating the contractile mechanisms of vascular smooth muscle and the physiological function of vessel contraction. The JC can be further divided into two sub-divisions, namely the PM-SR and caveolar regions. Previously, we demonstrated the role of the PM-SR region in the initiation of muscle contraction using pharmacological tools on the inferior vena cava (IVC) of rabbit. In the current study, we further dissected the caveolar region using a cholesterol-disrupting agent to investigate the role of the caveolar region. We conclude that disruption of the caveolar region in rabbit IVC smooth muscle results in augmented muscle contraction in response to adrenergic stimulation and the altered Ca2+ signaling may underlie the augmented contractility. Anat Rec, 302:186-192, 2019. © 2018 Wiley Periodicals, Inc.

Optical Versus Virtual Microscope for Medical Education: A Systematic Review.

Many technological innovations have changed the traditional practice of medical education and clinical practice. Whole slide imaging (WSI) technology provided an unique way of viewing conventional glass slides in histology and pathology laboratories. The WSI technology digitalized glass slide images and made them readily accessible via the Internet using tablets or computers. Users utilized the pan-and-zoom function to view digital images of slides, also referred to as the virtual microscope (VM), simulating use of an optical microscope (OM). Several articles have reported various outcomes on the utility of VM in teaching laboratories. Recently, the Royal College of Physicians and Surgeons of Canada certification examinations for anatomical pathologists ha completely adopted VM for the national licensing examination. To better inform medical educators, there is an urgent need for more structured reviews to draw evidence-based conclusions on the effectiveness of VM and learner's perceptions, in comparison to OM. The current study provides a descriptive summary of published outcomes using the systematic review approach. In conclusion, medical students' performance was improved with adoption of VM into the curriculum and recognized as a preferred learning modality, compared to OM. On the contrary, resident learners' performance was comparable between using OM and VM, with OM being the favored slide-viewing modality.

A Comparison of GATA3, TTF1, CD10, and Calretinin in Identifying Mesonephric and Mesonephric-like Carcinomas of the Gynecologic Tract.

Mesonephric carcinomas of the gynecologic tract are neoplasms that are often under-recognized due to their varied morphologic appearances. Recently, GATA3 and TTF1 have been reported to be useful immunohistochemical markers for distinguishing mesonephric carcinomas from its morphologic mimics. Herein, we compared the performance of GATA3 and TTF1 to the traditional markers used for mesonephric carcinomas, CD10 and calretinin. We studied 694 cases: 8 mesonephric carcinomas (7 cervical [includes 3 mesonephric carcinosarcomas], 1 vaginal), 5 mesonephric-like carcinomas (4 uterine corpus, 1 ovarian), 585 endometrial adenocarcinomas, and 96 cervical adenocarcinomas. Mesonephric-like carcinomas were defined as tumors exhibiting the classic morphologic features of mesonephric carcinoma, but occurring outside of the cervix and without convincing mesonephric remnants. GATA3 had the highest sensitivity and specificity (91% and 94%) compared with TTF1 (45% and 99%), CD10 (73% and 83%), and calretinin (36% and 89%). GATA3, however, also stained a substantial number of uterine carcinosarcomas (23/113, 20%). TTF1 was positive in 5/5 (100%) mesonephric-like carcinomas and only 1/8 (13%) mesonephric carcinomas. In 4/6 (67%) TTF1 positive cases, GATA3 exhibited an inverse staining pattern with TTF1. In summary, GATA3 was the best overall marker for mesonephric and mesonephric-like carcinomas, but cannot be used to distinguish mesonephric carcinosarcomas from Müllerian carcinosarcomas. The inverse staining pattern between GATA3 and TTF1, suggests that TTF1 may be useful when GATA3 is negative in small biopsies where mesonephric or mesonephric-like carcinoma is suspected. The greater TTF1 positivity in mesonephric-like carcinomas suggests they may be biologically different from prototypical mesonephric carcinomas.

Interobserver Agreement in Endometrial Carcinoma Histotype Diagnosis Varies Depending on The Cancer Genome Atlas (TCGA)-based Molecular Subgroup.

The Cancer Genome Atlas recently identified a genomic-based molecular classification of endometrial carcinomas, with 4 molecular categories: (1) ultramutated (polymerase epsilon [POLE] mutated), (2) hypermutated (microsatellite instability), (3) copy number abnormalities-low, and (4) copy number abnormalities-high. Two studies have since proposed models to classify endometrial carcinomas into 4 molecular subgroups, modeled after The Cancer Genome Atlas, using simplified and more clinically applicable surrogate methodologies. In our study, 151 endometrial carcinomas were molecularly categorized using sequencing for the exonuclease domain mutations (EDM) of POLE, and immunohistochemistry for p53 and mismatch repair (MMR) proteins. This separated cases into 1 of 4 groups: (1) POLE EDM, (2) MMR-D, (3) p53 wildtype (p53 wt), or (4) p53 abnormal (p53 abn). Seven gynecologic pathologists were asked to assign each case to one of the following categories: grade 1 to 2 endometrioid carcinoma (EC), grade 3 EC, mucinous, serous carcinoma (SC), clear cell, dedifferentiated, carcinosarcoma, mixed, and other. Consensus diagnosis among all 7 pathologists was highest in the p53 wt group (37/41, 90%), lowest in the p53 abn group (14/36, 39%), and intermediate in the POLE EDM (22/34, 65%) and MMR-D groups (23/40, 58%). Although the majority of p53 wt endometrial carcinomas are grade 1 to 2 EC (sensitivity: 90%), fewer than half of grade 1 to 2 EC fell into the p53 wt category (positive predictive value: 42%). Pure SC almost always resided in the p53 abn group (positive predictive value: 96%), but it was insensitive as a marker of p53 abn (sensitivity 64%) and the reproducibility of diagnosing SC was suboptimal. The limitations in the precise histologic classification of endometrial carcinomas highlights the importance of an ancillary molecular-based classification scheme.

Immunophenotyping of ampullary carcinomata allows for stratification of treatment specific subgroups.

BACKGROUND: Ampullary carcinomata (AC) can be separated into intestinal (IT) or pancreatobiliary (PB) subtypes. Although morphological, immunohistochemical and molecular differentiation of IT and PB have been well documented; the prognostic significance of histological subtype and whether patients with either subtype benefit from differential chemotherapeutic regimens remains unclear. METHODS: As part of a larger cohort study, patients who underwent resection for AC or pancreatic ductal adenocarcinoma (PDAC) were retrospectively identified. Clinicopathological covariates and outcome were obtained and MUC1, MUC2, CDX2 and CK20 were assessed with immunohistochemistry. RESULTS: Of 99 ACs, the resultant immunophenotypes indicated 48% and 22% were IT and PB, respectively. Thirty (30%) cases were quadruple negative (QN). Within the PDAC cohort (N = 257), the most prevalent immunophenotype was QN (53%). Subsequently, all QN ACs were classified as PB immunohistochemically yielding 47.5% and 52.5% classified as IT and PB, respectively. Involved regional lymph nodes and elevated T-stage were significantly associated with PB compared with IT AC (p = 0.0032 and 0.0396, respectively). Progression-free survival revealed inferior survival for PB versus IT AC (p = 0.0156). CONCLUSIONS: AC can be classified into prognostic groups with unique clinicopathological characteristics using immunohistochemistry. Immunophenotypical similarity of PB and PDAC suggests that treatment regimens similar to those used in PDAC should be explored.

Acetylcholine-induced asynchronous calcium waves in intact human bronchial muscle bundle.

Calcium (Ca2+) is an important activator of the contractile machinery in airway smooth muscle (ASM). While agonist-induced Ca2+ signals are well characterized in animal ASM, little is known about what occurs in adult human ASM. In this study, we examined the Ca2+ signal elicited by acetylcholine (ACh) in smooth muscle cells of the intact human bronchial muscle strips obtained from fresh surgical specimens in relation to muscle contraction. We found that ACh induces repetitive Ca2+ waves that spread along the longitudinal axis of individual cells in the intact human bronchial smooth muscle strips. These Ca2+ waves display no apparent synchronization between neighboring cells, and their generation precedes force development. Comparison of the ACh concentration dependence of tissue contraction and selected parameters of the asynchronous Ca2+ waves (ACW) reveals that the graded force generation by ACh-stimulated human bronchial muscle strips is achieved by differential recruitment of cells to initiate Ca2+ waves and by enhancement of the frequency of ACW once the cells are recruited. Furthermore, pharmacologic characterization shows that the ACW are produced by repetitive cycles of SR Ca2+ release via ryanodine-sensitive channels followed by SR Ca2+ reuptake by sarco(endo)plasmic reticulum Ca2+ ATPase. Extracellular Ca2+ entry involving receptor-operated channels/store-operated channels, reverse-mode Na+/Ca2+ exchange, and to a lesser extent L-type voltage-gated Ca2+ channels is required to maintain the ACW. These findings for the first time demonstrate the occurrence and the role of ACW in excitation-contraction coupling in adult human ASM.

Mechanism of ACh-induced asynchronous calcium waves and tonic contraction in porcine tracheal muscle bundle.

Stimulation of the tracheal muscle bundle by acetylcholine (ACh) results in the generation of asynchronous repetitive Ca2+ waves (ACW) in intact tracheal smooth muscle (TSM) cells. We showed previously that ACW underlie cholinergic excitation-contraction coupling in porcine TSM and that Ca2+ entry through the L-type voltage-gated Ca2+ channel (VGCC) contributes partially to maintenance of the ACW. However, the mechanism of the ACW remains undefined. In this study, we pharmacologically characterized the mechanism of ACh-induced ACW in the intact porcine tracheal muscle bundle. We found that inhibition of receptor-operated channels/store-operated channels (ROC/SOC) by SKF-96365 completely abolished the nifedipine-insensitive component of ACh-mediated ACW and tonic contraction. Blockade of Na+/Ca2+ exchange with KB-R7943 or 2',4'-dichlorobenzamil or removal of extracellular Na+ resulted in nearly complete inhibition of the nifedipine-insensitive component of ACh-mediated ACW and tonic contraction. Inhibition of the sarco(endo)plasmic reticulum Ca2+-ATPase by cyclopiazonic acid abolished the ongoing ACW. Application of 2-aminoethoxydiphenyl borate (2-APB) or xestospongin C to inhibit the inositol 1,4,5-trisphosphate-sensitive sarcoplasmic reticulum (SR) Ca2+ release channels produced no effect on ACh-mediated ACW and tonic contraction. However, pretreatment with caffeine or ryanodine inhibited ACh-induced ACW. Furthermore, application of procaine or tetracaine prevented the generation and abolished the ongoing ACh-mediated ACW and tonic contraction. Collectively, these results indicate that the ACh-stimulated ACW in porcine TSM are produced by repetitive cycles of Ca2+ release from SR through 2-APB- and xestospongin C-insensitive Ca2+ release channels, and plasmalemmal Ca2+ entry involving reverse-mode Na+/Ca2+ exchange, ROC/SOC, and L-type VGCC is required to refill the SR via SERCA to support the ongoing ACW.

Critical role of actin in modulating BBB permeability.

A major obstacle in the treatment of degenerative manifestations and debilitating diseases in the central nervous system (CNS) lies in the impediment of drug delivery into these tissues. The impediment is due to a membrane barrier referred to as the blood-brain barrier (BBB). It is known that the BBB is a unique membranous structure in brain capillaries that tightly segregates the brain from systemic blood circulation. It is imperative to have a thorough understanding of the molecular components and their integrated function of this barrier to develop effective therapeutics for CNS disorders and diseases. Although there are other cell and biochemical properties that underlie this barrier function, it is well established that the barrier is mainly made up of the physical elements of tight junction (TJ) complex. The major constituents of TJ, such as occludin, claudins, zonula occludens (ZOs) and junctional adhesion molecule (JAM) have been subjects of intensive studies and reviews. However, after examining currently proposed models, we have come to believe that a cytoskeletal component-actin may play a critical role in interacting TJ molecular constituents and modulating functional TJ complex. In this review, we will discuss the correlation of temporal and spatial distribution and remodeling of actin filaments with altering integrity of TJ complexes in various systems and present a hypothesis to depict its potential role in modulating BBB permeability.

The effect of N-acetylcysteine on cardiac contractility to dobutamine in rats with streptozotocin-induced diabetes.

We examined if myocardial depression at the acute phase of diabetes (3 weeks after injection of streptozotocin, 60 mg/kg i.v.) is due to activation of inducible nitric oxide synthase and production of peroxynitrite, and if treatment with N-acetylcysteine (1.2 g/day/kg for 3 weeks, antioxidant) improves cardiac function. Four groups of rats were used: control, N-acetylcysteine-treated control, diabetic and N-acetylcysteine-treated diabetic. Pentobarbital-anaesthetized diabetic rats, relative to the controls, had reduced left ventricular contractility to dobutamine (1-57 microg/min/kg). The diabetic rats also had increased myocardial levels of thiobarbituric acid reactive substances, immunostaining of inducible nitric oxide synthase and nitrotyrosine, and similar baseline 15-F2t-isoprostane. N-acetylcysteine did not affect responses in the control rats; but increased cardiac contractility to dobutamine, reduced myocardial immunostaining of inducible nitric oxide synthase and nitrotyrosine and level of 15-F2t-isoprostane, and increased cardiac contractility to dobutamine in the diabetic rats. Antioxidant supplementation in diabetes reduces oxidative stress and improves cardiac function.

Rearrangement of the close contact between the mitochondria and the sarcoplasmic reticulum in airway smooth muscle.

The mitochondria and the sarcoplasmic reticulum (SR) are two major intracellular calcium-storing organelles that exhibit close functional interaction with each other. Close spatial association is believed to be important for their functional interaction. In this study, we have characterized the spatial relationship between the SR and the mitochondria in porcine tracheal smooth muscle cells (TSMC) under different conditions. By examining the cross-section of unstimulated TSMC with electron microscopy, we found that 99.4 +/- 0.5% of the mitochondria seen on random cross-sections were situated within 30 nm of the SR and that 82.2 +/- 6.7% of the mitochondria were completely enveloped by the SR network. Overall, 48.0 +/- 3.5% of the mitochondrial outer membrane was within 30 nm with the SR. After stimulation of the TSMC with acetylcholine (ACh) or 80 mM [K(+)] solution 97.0 +/- 2.1% and 98.6 +/- 1.4% of the mitochondria observed were situated within 30 nm of the SR, respectively. However, the proportion of the mitochondria that was completely enveloped by the SR was significantly reduced to 12.2 +/- 5.9% in ACh-stimulated cells and 9.7 +/- 6.6% in 80 mM [K(+)] stimulated cells. The percentage of mitochondrial membrane closely associated with the SR was correspondingly lower at 10.1 +/- 1.0% during ACh stimulation and 10.8 +/- 0.9% during 80 mM [K(+)] stimulation. During smooth muscle cell stimulation, the SR appears to unwrap from the mitochondria and extend into the cytoplasm while maintaining close contact with the mitochondria over a smaller area. Such static and dynamic components of the close spatial association between the mitochondria and the SR may serve as a structural basis for the selective and efficient Ca(2+) trafficking between the two organelles in TSMC.

Calyculin-A disrupts subplasmalemmal junction and recurring Ca2+ waves in vascular smooth muscle.

Excitation-contraction coupling (E-C coupling) in phenylephrine(PE)-stimulated rabbit inferior vena cava (IVC) depends on the generation of asynchronous recurring Ca2+ waves in the in situ vascular smooth muscle cells (VSMC). Previous studies by our group have implicated a putative non-selective cationic store-operated channel and the reverse-mode Na+-Ca2+ exchange in refilling of the intracellular Ca2+ store via the sarco/endoplasmic reticulum ATPase (SERCA) and the maintenance of the recurring Ca2+ waves. We hypothesize that for the proper functioning of these three Ca2+ translocators in the process of SR refilling, the plasma membrane (PM) and the underlying superficial sarcoplasmic reticulum (SR) form specialized PM-SR junctions, which are essential for the maintenance of the recurring Ca2+ waves. In order to test this hypothesis, calyculin-A, a serine/threonine phosphatase inhibitor that has been demonstrated to result in the disruption of the PM-SR junctions was used. In the control rabbit IVC, electron microscopy of the in situ VSMC indicates that 14.2+/-0.7% of the PM is closely apposed by the prominent superficial SR network, forming numerous flattened PM-SR junctional cytoplasmic spaces. In the control IVC stimulation with 5 microM PE resulted in sustained recurring Ca2+ waves with a frequency of 0.42+/-0.02 Hz. In calyculin-A treated rabbit IVC, a concentration-dependent dissociation of the superficial SR and loss of PM-SR junctions was observed. This progressive loss of the PM-SR junctions occurs over the same concentration range as the inhibition of PE-induced recurring Ca2+ waves. These findings offer support for the hypothesis that the presence of the PM-SR junctions is required for the generation of asynchronous recurring Ca2+ waves, which underlie excitation-contraction coupling in the VSMC of the rabbit IVC.