The Effects of Cigarette Smoke on Airway Smooth Muscle Function

Ph. D. Thesis.Cigarette smoking is the largest risk factor for developing chronic obstructive pulmonary disease (COPD), which is associated with hyperresponsiveness of airway smooth muscle. While it is well established that chronic exposure to cigarette smoke (CS) leads to dysregulated calcium signalling, airway inflammation and remodelling of the airway smooth muscle, there is limited information about the acute effects of CS or CS extract (CSE) on human airway smooth muscle cell (hASMC) function, particularly Ca2+ homeostasis. Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is also linked to disrupted Ca2+ homeostasis in ASMC, but its role in ASMC function remains poorly understood. This study investigated the following: (1) The role of CFTR in regulating Ca2+ homeostasis in ASMC; (2) The acute effects of aqueous CSE on hASMC Ca2+ homeostasis; and (3) The acute effects of gaseous CS on hASMC Ca2+ homeostasis. The results demonstrate that (1) CFTR inhibition in rat ASMC had minor effects on store-operated Ca2+ entry and the extent of Ca2+ reuptake; (2) Acute exposure to CSE or CS rapidly elevated cytosolic Ca2+ in hASMC through stimulation of plasmalemmal Ca2+ influx, but not through the release of calcium from intracellular sarcoplasmic reticulum stores; (3) Both CSE and CS specifically stimulated Ca2+ influx through the neurogenic pain receptor channel, transient receptor potential ankyrin 1 (TRPA1), but not through L-type or store-operated Ca2+ channels; (4) The CSE/CS-induced calcium influx through TRPA1 led to myosin light-chain phosphorylation, a key process regulating ASMC contractility. Overall, results suggest that CFTR plays a minor role in regulating calcium homeostasis in ASM. However, this study provides the first experimental evidence that TRPA1 is an important target of both CSE and CS in hASMC. The CSE/CS-induced activation of TRPA1 may lead to exacerbated ASMC contraction thus contributing to airway hyperresponsiveness in COPD.Newcastle Universit

Extracellular phosphate enhances the function of F508del-CFTR rescued by CFTR correctors

Background: The clinical response to cystic fibrosis transmembrane conductance regulator (CFTR) modulators varies between people with cystic fibrosis (CF) of the same genotype, in part through the action of solute carriers encoded by modifier genes. Here, we investigate whether phosphate transport by SLC34A2 modulates the function of F508del-CFTR after its rescue by CFTR correctors. Methods: With Fischer rat thyroid (FRT) cells heterologously expressing wild-type and F508del-CFTR and fully-differentiated CF and non-CF human airway epithelial cells, we studied SLC34A2 expression and the effects of phosphate on CFTR-mediated transepithelial ion transport. F508del-CFTR was trafficked to the plasma membrane by incubation with different CFTR correctors (alone or in combination) or by low temperature. Results: Quantitative RT-PCR demonstrated that both FRT and primary airway epithelial cells express SLC34A2 mRNA and no differences were found between cells expressing wild-type and F508del-CFTR. For both heterologously expressed and native F508del-CFTR rescued by either VX-809 or C18, the magnitude of CFTR-mediated Cl(−) currents was dependent on the presence of extracellular phosphate. However, this effect of phosphate was not detected with wild-type and low temperature-rescued F508del-CFTR Cl(−) currents. Importantly, the modulatory effect of phosphate was observed in native CF airway cells exposed to VX-445, VX-661 and VX-770 (Trikafta) and was dependent on the presence of both sodium and phosphate. Conclusions: Extracellular phosphate modulates the magnitude of CFTR-mediated Cl(−) currents after F508del-CFTR rescue by clinically-approved CFTR correctors. This effect likely involves electrogenic phosphate transport by SLC34A2. It might contribute to inter-individual variability in the clinical response to CFTR correctors

Signaling and structures underpinning conducted vasodilation in human and porcine intramyocardial coronary arteries

BACKGROUND: Adequate blood flow into coronary micro-arteries is essential for myocardial function. Here we assess the mechanisms responsible for amplifying blood flow into myogenically-contracting human and porcine intramyocardial micro-arteries ex vivo using endothelium-dependent and -independent vasodilators. METHODS: Human and porcine atrial and ventricular small intramyocardial coronary arteries (IMCAs) were studied with pressure myography and imaged using confocal microscopy and serial section/3-D reconstruction EM. RESULTS: 3D rendered ultrastructure images of human right atrial (RA-) IMCAs revealed extensive homo-and hetero-cellular contacts, including to longitudinally-arranged smooth muscle cells (l-SMCs) found between the endothelial cells (ECs) and radially-arranged medial SMCs (r-SMCs). Local and conducted vasodilatation followed focal application of bradykinin in both human and porcine RA-IMCAs, and relied on hyperpolarization of SMCs, but not nitric oxide. Bradykinin initiated asynchronous oscillations in endothelial cell Ca(2+) in pressurized RA-IMCAs and, as previously shown in human RA-IMCAs, hyperpolarized porcine arteries. Immunolabelling showed small- and intermediate-conductance Ca(2+)-activated K(+) channels (K(Ca)) present in the endothelium of both species, and concentration-dependent vasodilation to bradykinin followed activation of these K(Ca) channels. Extensive electrical coupling was demonstrated between r-SMCs and l-SMCs, providing an additional pathway to facilitate the well-established myoendothelial coupling. Conducted dilation was still evident in a human RA-IMCA with poor myogenic tone, and heterocellular contacts were visible in the 3D reconstructed artery. Hyperpolarization and conducted vasodilation was also observed to adenosine which, in contrast to bradykinin, was sensitive to combined block of ATP-sensitive (K(ATP)) and inwardly rectifying (K(IR)) K(+) channels. CONCLUSIONS: These data extend our understanding of the mechanisms that coordinate human coronary microvascular blood flow and the mechanistic overlap with porcine IMCAs. The unusual presence of l-SMCs provides an additional pathway for rapid intercellular signaling between cells of the coronary artery wall. Local and conducted vasodilation follow hyperpolarization of the ECs or SMCs, and contact-coupling between l-SMCs and r-SMCs likely facilitates this vasodilation

VisorGPT: Learning Visual Prior via Generative Pre-Training

Various stuff and things in visual data possess specific traits, which can be learned by deep neural networks and are implicitly represented as the visual prior, e.g., object location and shape, in the model. Such prior potentially impacts many vision tasks. For example, in conditional image synthesis, spatial conditions failing to adhere to the prior can result in visually inaccurate synthetic results. This work aims to explicitly learn the visual prior and enable the customization of sampling. Inspired by advances in language modeling, we propose to learn Visual prior via Generative Pre-Training, dubbed VisorGPT. By discretizing visual locations of objects, e.g., bounding boxes, human pose, and instance masks, into sequences, VisorGPT can model visual prior through likelihood maximization. Besides, prompt engineering is investigated to unify various visual locations and enable customized sampling of sequential outputs from the learned prior. Experimental results demonstrate that VisorGPT can effectively model the visual prior, which can be employed for many vision tasks, such as customizing accurate human pose for conditional image synthesis models like ControlNet. Code will be released at https://github.com/Sierkinhane/VisorGPT.Comment: Project web-page: https://sierkinhane.github.io/visor-gpt

Pharmacological inhibitors of the cystic fibrosis transmembrane conductance regulator exert off-target effects on epithelial cation channels

The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel and the epithelial Na+ channel (ENaC) play essential roles in transepithelial ion and fluid transport in numerous epithelial tissues. Inhibitors of both channels have been important tools for defining their physiological role in vitro. However, two commonly used CFTR inhibitors, CFTRinh-172 and GlyH-101, also inhibit non-CFTR anion channels, indicating they are not CFTR specific. However, the potential off-target effects of these inhibitors on epithelial cation channels has to date not been addressed. Here, we show that both CFTR blockers, at concentrations routinely employed by many researchers, caused a significant inhibition of store-operated calcium entry (SOCE) that was time-dependent, poorly reversible and independent of CFTR. Patch clamp experiments showed that both CFTRinh-172 and GlyH-101 caused a significant block of Orai1-mediated whole cell currents, establishing that they likely reduce SOCE via modulation of this Ca2+ release-activated Ca2+ (CRAC) channel. In addition to off-target effects on calcium channels, both inhibitors significantly reduced human αβγ-ENaC-mediated currents after heterologous expression in Xenopus oocytes, but had differential effects on δβγ-ENaC function. Molecular docking identified two putative binding sites in the extracellular domain of ENaC for both CFTR blockers. Together, our results indicate that caution is needed when using these two CFTR inhibitors to dissect the role of CFTR, and potentially ENaC, in physiological processes

Global research status and frontiers on microvascular invasion of hepatocellular carcinoma: A bibliometric and visualized analysis

IntroductionOver the past decade, several studies on the microvascular invasion (MVI) of hepatocellular carcinoma (HCC) have been published. However, they have not quantitatively analyzed the remarkable impact of MVI. Therefore, a more comprehensive understanding of the field is now needed. This study aims to analyze the evolution of HCC-MVI research and to systematically evaluate the scientific outputs using bibliometric citation analysis.MethodsA systematic search was conducted on the Web of Science Core Collection on 2 May 2022 to retrieve studies on HCC-MVI published between 2013 and 2022. Then, a bibliometric analysis of the publications was performed using CiteSpace, VOSviewer, and other visualization tools.ResultsA total of 1,208 articles on HCC MVI were identified. Of these, China (n = 518) was the most prolific country, and Fudan University (n = 90) was the most notable institution. Furthermore, we observed that Lau Wan Yee participated in most studies (n = 26), and Frontiers in Oncology (IF2020:6.24) published the highest number of documents (n = 49) on this subject, with 138 publications. The paper “Bray F, 2018, CA-CANCER J CLIN, V68, P394” has the highest number of co-cited references, with 119 citations. In addition, the top three keywords were “survival”, “recurrence”, and “microvascular invasion”. Moreover, the research hot spots and frontiers of HCC-MVI for the last 3 years included imaging characteristics and transarterial chemoembolization (TACE) therapy studies.ConclusionsThis study comprehensively summarized the most significant HCC-MVI documents from past literature and highlighted key contributions made to the advancement of this subject and the advancement of this field over the past decade. The trend of MVI research will gradually shift from risk factors and prognosis studies to imaging characteristics and TACE therapy studies

Asymmetric dimethylarginine enables depolarizing spikes and vasospasm in mesenteric and coronary resistance arteries

BACKGROUND: Increased vasoreactivity due to reduced endothelial NO bioavailability is an underlying feature of cardiovascular disease, including hypertension. In small resistance arteries, declining NO enhances vascular smooth muscle (VSM) reactivity partly by enabling rapid depolarizing Ca2+-based spikes that underlie vasospasm. The endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) is metabolized by DDAH1 (dimethylarginine dimethylaminohydrolase 1) and elevated in cardiovascular disease. We hypothesized ADMA might enable VSM spikes and vasospasm by reducing NO bioavailability, which is opposed by DDAH1 activity and L-arginine. METHODS: Rat isolated small mesenteric arteries and myogenic rat-isolated intraseptal coronary arteries (RCA) were studied using myography, VSM intracellular recording, Ca2+ imaging, and DDAH1 immunolabeling. Exogenous ADMA was used to inhibit NO synthase and a selective DDAH1 inhibitor, NG-(2-methoxyethyl) arginine, to assess the functional impact of ADMA metabolism. RESULTS: ADMA enhanced rat-isolated small mesenteric arteries vasoreactivity to the α1-adrenoceptor agonist, phenylephrine by enabling T-type voltage-gated calcium channel-dependent depolarizing spikes. However, some endothelium-dependent NO-vasorelaxation remained, which was sensitive to DDAH1-inhibition with NG-(2-methoxyethyl) arginine. In myogenically active RCA, ADMA alone stimulated depolarizing Ca2+ spikes and marked vasoconstriction, while NO vasorelaxation was abolished. DDAH1 expression was greater in rat-isolated small mesenteric arteries endothelium compared with RCA, but low in VSM of both arteries. L-arginine prevented depolarizing spikes and protected NO-vasorelaxation in rat-isolated small mesenteric artery and RCA. CONCLUSIONS: ADMA increases VSM electrical excitability enhancing vasoreactivity. Endothelial DDAH1 reduces this effect, and low levels of DDAH1 in RCAs may render them susceptible to endothelial dysfunction contributing to vasospasm, changes opposed by L-arginine

Choice of Differentiation Media Significantly Impacts Cell Lineage and Response to CFTR Modulators in Fully Differentiated Primary Cultures of Cystic Fibrosis Human Airway Epithelial Cells

In vitro cultures of primary human airway epithelial cells (hAECs) grown at air–liquid interface have become a valuable tool to study airway biology under normal and pathologic conditions, and for drug discovery in lung diseases such as cystic fibrosis (CF). An increasing number of different differentiation media, are now available, making comparison of data between studies difficult. Here, we investigated the impact of two common differentiation media on phenotypic, transcriptomic, and physiological features of CF and non-CF epithelia. Cellular architecture and density were strongly impacted by the choice of medium. RNA-sequencing revealed a shift in airway cell lineage; one medium promoting differentiation into club and goblet cells whilst the other enriched the growth of ionocytes and multiciliated cells. Pathway analysis identified differential expression of genes involved in ion and fluid transport. Physiological assays (intracellular/extracellular pH, Ussing chamber) specifically showed that ATP12A and CFTR function were altered, impacting pH and transepithelial ion transport in CF hAECs. Importantly, the two media differentially affected functional responses to CFTR modulators. We argue that the effect of growth conditions should be appropriately determined depending on the scientific question and that our study can act as a guide for choosing the optimal growth medium for specific applications

DataSheet_1_Global research status and frontiers on microvascular invasion of hepatocellular carcinoma: A bibliometric and visualized analysis.zip

IntroductionOver the past decade, several studies on the microvascular invasion (MVI) of hepatocellular carcinoma (HCC) have been published. However, they have not quantitatively analyzed the remarkable impact of MVI. Therefore, a more comprehensive understanding of the field is now needed. This study aims to analyze the evolution of HCC-MVI research and to systematically evaluate the scientific outputs using bibliometric citation analysis.MethodsA systematic search was conducted on the Web of Science Core Collection on 2 May 2022 to retrieve studies on HCC-MVI published between 2013 and 2022. Then, a bibliometric analysis of the publications was performed using CiteSpace, VOSviewer, and other visualization tools.ResultsA total of 1,208 articles on HCC MVI were identified. Of these, China (n = 518) was the most prolific country, and Fudan University (n = 90) was the most notable institution. Furthermore, we observed that Lau Wan Yee participated in most studies (n = 26), and Frontiers in Oncology (IF2020:6.24) published the highest number of documents (n = 49) on this subject, with 138 publications. The paper “Bray F, 2018, CA-CANCER J CLIN, V68, P394” has the highest number of co-cited references, with 119 citations. In addition, the top three keywords were “survival”, “recurrence”, and “microvascular invasion”. Moreover, the research hot spots and frontiers of HCC-MVI for the last 3 years included imaging characteristics and transarterial chemoembolization (TACE) therapy studies.ConclusionsThis study comprehensively summarized the most significant HCC-MVI documents from past literature and highlighted key contributions made to the advancement of this subject and the advancement of this field over the past decade. The trend of MVI research will gradually shift from risk factors and prognosis studies to imaging characteristics and TACE therapy studies.</p