Ubiquitination is involved in PKC-mediated degradation of cell surface Kv1.5 channels.

The voltage-gated Kv1.5 potassium channel, conducting the ultra-rapid delayed rectifier K+ current (IKur) in human cells, plays important roles in the repolarization of atrial action potentials and regulation of the vascular tone. We previously reported that activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) induces endocytic degradation of cell-surface Kv1.5 channels, and a point mutation removing the phosphorylation site, T15A, in the N terminus of Kv1.5 abolished the PMA-effect. In the present study, using mutagenesis, patch clamp recording, Western blot analysis and immunocytochemical staining, we demonstrate that ubiquitination is involved in PMA-mediated degradation of mature Kv1.5 channels. Since the expression of Kv1.4 channel is unaffected by PMA treatment, we swapped the N- and/or C-termini between Kv1.5 and Kv1.4. We found that N-terminus alone did not, but both N- and C-termini of Kv1.5 did confer PMA sensitivity to mature Kv1.4 channels, suggesting the involvement of Kv1.5 C-terminus in the channel ubiquitination. Removal of each of the potential ubiquitination residue Lysine at position 536, 565, and 591 by Arginine substitution (K536R, K565R, and K591R) had little effect, but removal of all three Lysine residues with Arginine substitution (3K-R) partially reduced PMA-mediated Kv1.5 degradation. Furthermore, removing the cysteine residue at position 604 by Serine substitution (C604S) drastically reduced PMA-induced channel degradation. Removal of the three Lysines and Cys604 with a quadruple mutation (3K-R/C604S) or a truncation mutation (Δ536) completely abolished the PKC activation-mediated degradation of Kv1.5 channels. These results provide mechanistic insight into PKC activation-mediated Kv1.5 degradation.

Evaluation of the Metered Dose Inhaler Technique: Initial Assessment and Post-counseling Improvements Among the Indian Population.

Objective The objective of this study was to evaluate errors in the use of metered-dose inhalers (MDIs) among patients diagnosed with asthma or chronic obstructive pulmonary disease (COPD). Additionally, we aimed to assess improvements following corrective interventions. Settings and design This cross-sectional study was done by simple random sampling. Methods and materials This study was done at a tertiary care center in South India in an outpatient department and ward for tuberculosis and chest disease to find out the right way to use an MDI and investigate the reasons why people with asthma and COPD don't use it correctly. There were a total of 12 steps. The patient was given an empty canister to try the inhalation technique and was scored one point for every correct step and zero for every incorrect step, for a total of 12 steps. Following the demonstration, an educator used a variety of tools, including verbal communication, pictorial demonstrations, and practical demonstrations, to correct the mistakes. After education was provided, post-interventional data was collected. Results During pre-intervention of the 12 steps out of the 183 participants, step one had 183 correct participants (100%), step two had 104 correct participants (56.83%), step three had 129 correct participants (70.49%), step four had 71 correct participants (38.79%), step five had 167 correct participants (91.25%), step six had 123 correct participants (67.21%), step seven had 132 correct participants (72.13%), step eight had 81 correct participants (43.71%), step nine had 123 correct participants (67.21%), step 10 had 108 correct participants (59.01%), step 11 had 128 correct participants (69.94%), and step 12 had 175 correct participants (95.62%). During the post-intervention of the 12 steps, out of the 183 participants, step one remained at 183 correct participants (100%), step two increased to 149 correct participants (81.42%), step three to step seven increased to 183 correct participants (100%), step eight increased to 142 correct participants (77.59%), step nine increased to 174 correct participants (95.08%), step 10 increased to 177 correct participants (96.72%), step 11 increased to 143 correct participants (78.14%) and step 12 increased to 177 correct participants (96.72%). Conclusion This study highlights the prevalent errors in the use of metered-dose inhalers (MDIs) among patients diagnosed with asthma or chronic obstructive pulmonary disease (COPD). The results demonstrate significant improvements in the MDI technique. Following educational interventions such as verbal communication, pictorial demonstrations, and practical exercises, patients were able to correct their inhaler technique effectively and emphasized the importance of patient education and counseling to ensure the maintenance of correct usage over time.

Role of miRNAs in Brain Development.

Non-coding RNAs that are small in size, called microRNAs (miRNAs), exert a conse-quence in neutralizing gene activity after transcription. The nervous system is a massively ex-pressed organ, and an expanding body of research reveals the vital functions that miRNAs play in the brain's growth and neural activity. The significant benefit of miRNAs on the development of the central nervous system is currently shown through new scientific methods that concentrate on targeting and eradicating vital miRNA biogenesis pathways the elements involving Dicer and DGCR8. Modulation of miRNA has been associated with numerous essential cellular processes on neural progenitors, like differentiation, proliferation, and destiny determination. Current re-search discoveries that emphasize the significance of miRNAs in the complex process of brain development are included in this book. The miRNA pathway plays a major role in brain devel-opment, its operational dynamics, and even diseases. Recent studies on miRNA-mediated gene regulation within neural discrepancy, the circadian period and synaptic remodeling are signs of this. We also discussed how these discoveries may affect our comprehension of the fundamental processes behind brain diseases, highlighting the novel therapeutic opportunities miRNAs pro-vide for treating various human illnesses.

Assessment of dietary polyvinylchloride, polypropylene and polyethylene terephthalate exposure in Nile tilapia, Oreochromis niloticus: Bioaccumulation, and effects on behaviour, growth, hematology and histology.

Microplastics (MPs) have been recognized as emerging aquatic pollutants receiving major concern due to their detrimental effects on aquatic life. Nile Tilapia, Oreochromis niloticus is a model species considered in toxicological studies to address the effects of pollutants in freshwater animals. However, comprehensive knowledge comparing the impacts on fish across various MPs polymers is scarce. Therefore, the overarching aim of the current study was to examine the bioconcentration of MPs polymers: polyvinylchloride (PVC), polypropylene (PP), and polyethylene terephthalate (PET), and their toxic effects on growth, and behavioral responses, hematology, and histology of gills, liver, and intestine in O. niloticus. Fishes were subjected to a 21-day dietary exposure to MPs by assigning them into six treatment groups: T1 (4% of PVC), T2 (4% of PP), T3 (4% of PET), T4 (8% of PVC), T5 (8% of PP), T6 (8% of PET), and control (0% of MPs), to assess the effects on fish across the polymers and dosage. Results showed several abnormalities in anatomical and behavioral parameters, lower growth, and high mortality in MPs-exposed fish, indicating a dose-dependent relationship. The elevated dosage of polymers raised the bioavailability of PVC, PP, and PET in gills and gut tissues. Noteworthy erythrocyte degeneration referred to cytotoxicity and stress imposed by MPs, whereas the alterations in hematological parameters were possibly due to blood cell damage, also indicating mechanisms of defense against MPs toxicity. Histopathological changes in the gills, liver, and intestine confirmed the degree of toxicity and associated dysfunctions in fish. A higher sensitivity of O. niloticus to PET-MPs compared to other polymers is likely due to its chemical properties and species-specific morphological and physiological characteristics. Overall, the present study reveals valuable insights into the emerging threat of MPs toxicity in freshwater species, which could be supportive of future toxicological research.

Does Organizational Messaging Make a Difference? Investigating Themes and Language Style in Twitter Discourse and Engagement by Mental Health Organizations.

The present study investigated the latent topics and language styles present in mental health organizational discourse on Twitter. The researchers sought to analyze identifying the prevalence of and language used in social support messaging in tweets about mental health care, the overarching topics regarding mental health care, and predicted that tweets with higher engagement will have increased frequency of words with positively valenced emotion and cognitive processing. A GSDMM was run to uncover latent themes that emerged in a data set of 326.9k tweets and 7.2 m words about organizational discussions of mental health. A generalized linear model using the Poisson distribution was used to assess the role of engagement, positive emotion, and cognitive processing. The study found support for both positive emotion and cognitive processing as statistically significant predictors of engagement. Directions for research include the development of health message strategies, policy needs, and online interventions.

Unraveling the relationship between the renin-angiotensin system and endometrial cancer: a comprehensive review.

Endometrial cancer (EC), the most common adenocarcinoma, represents 90% of uterine cancer in women with an increased incidence of occurrence attributed to age, obesity, hypertension, and hypoestrogenism. Being the most common gynecological malignancy in women, it shows a relation with the activation of different components of the renin-angiotensin system (RAS), which is predominantly involved in maintaining blood pressure, salt, water, and aldosterone secretion, thereby playing a significant role in the etiology of hypertension. The components of the RAS, i.e., ACE-I, ACE-II, AT1R, AT2R, and Pro(renin) receptor, are widely expressed in both glandular and stromal cells of the endometrium, with varying levels throughout the different phases of the menstrual cycle. This causes the endometrial RAS to implicate angiogenesis, neovascularization, and cell proliferation. Thus, dysfunctioning of the endometrial RAS could predispose the growth and spread of EC. Interestingly, the increased expression of AngII, AGTR1, and AGTR2 showed advancement in the stages and progression of EC via the prorenin/ATP6AP2 and AngII/AGTR1 pathway. Therefore, this review corresponds to unraveling the relationship between the progression and development of endometrial cancer with the dysfunction in the expression of various components associated with RAS in maintaining blood pressure.

Molecularly Imprinted Polymer Interface on Screen-Printed ZnO Nanorod Field Effect Transistors for Serotonin Detection in Clinical Samples.

Ultrasensitive detection of serotonin is crucial for the early diagnosis of several diseases like Parkinson's and Alzheimer's. Most of the existing detection strategies are still not suitable for sensitive point-of-care applications. This study presents direct molecular imprinting of serotonin on the surface of three-dimensional zinc oxide (ZnO) nanorod devices connected in a field effect transistor (FET) configuration to achieve ultrasensitive, real-time, and rapid detection with a convenient and affordable approach, which has significant potential for translation to clinical settings. This strategy has enabled pushing the detection limit to 0.1 fM in a physiological analyte in real time with screen-printed electrodes, thereby resulting in the convenient batch fabrication of sensors for clinical validation. The response of the sensor with the clinical sample has been correlated with that of the gold standard and has been observed to be statistically similar.

Wnt7a deficit is associated with dysfunctional angiogenesis in pulmonary arterial hypertension.

INTRODUCTION: Pulmonary arterial hypertension (PAH) is characterised by loss of microvessels. The Wnt pathways control pulmonary angiogenesis but their role in PAH is incompletely understood. We hypothesised that Wnt activation in pulmonary microvascular endothelial cells (PMVECs) is required for pulmonary angiogenesis, and its loss contributes to PAH. METHODS: Lung tissue and PMVECs from healthy and PAH patients were screened for Wnt production. Global and endothelial-specific Wnt7a -/- mice were generated and exposed to chronic hypoxia and Sugen-hypoxia (SuHx). RESULTS: Healthy PMVECs demonstrated >6-fold Wnt7a expression during angiogenesis that was absent in PAH PMVECs and lungs. Wnt7a expression correlated with the formation of tip cells, a migratory endothelial phenotype critical for angiogenesis. PAH PMVECs demonstrated reduced vascular endothelial growth factor (VEGF)-induced tip cell formation as evidenced by reduced filopodia formation and motility, which was partially rescued by recombinant Wnt7a. We discovered that Wnt7a promotes VEGF signalling by facilitating Y1175 tyrosine phosphorylation in vascular endothelial growth factor receptor 2 (VEGFR2) through receptor tyrosine kinase-like orphan receptor 2 (ROR2), a Wnt-specific receptor. We found that ROR2 knockdown mimics Wnt7a insufficiency and prevents recovery of tip cell formation with Wnt7a stimulation. While there was no difference between wild-type and endothelial-specific Wnt7a -/- mice under either chronic hypoxia or SuHx, global Wnt7a +/- mice in hypoxia demonstrated higher pulmonary pressures and severe right ventricular and lung vascular remodelling. Similar to PAH, Wnt7a +/- PMVECs exhibited an insufficient angiogenic response to VEGF-A that improved with Wnt7a. CONCLUSIONS: Wnt7a promotes VEGF signalling in lung PMVECs and its loss is associated with an insufficient VEGF-A angiogenic response. We propose that Wnt7a deficiency contributes to progressive small vessel loss in PAH.

Utilization of Hypolipidemic Drugs, Patterns, and Factors Affecting Dyslipidemia Among Type 2 Diabetes Mellitus at a Tertiary Care Teaching Hospital in South India.

Background The prevalence of dyslipidemia is higher in type 2 diabetes mellitus (T2DM) and hypolipidemic drugs like statins are effective for the primary and secondary prevention of cardiovascular events. Most of the patients with type 2 diabetes have a mixed type of dyslipidemia. This study aimed to evaluate the utilization of hypolipidemic drugs, patterns, and factors affecting dyslipidemia in T2DM participants. Methods This cross-sectional observational study was approved by the institutional ethics committee (IEC) of the Vydehi Institute of Medical Sciences and Research Center. It was conducted for a period of one year from July 2021 to June 2022. Participants with T2DM visiting the Department of General Medicine and Endocrinology were enrolled after obtaining informed consent. Demographic details, medication history, and laboratory data were recorded in case report form and statistical measures were applied. Results Out of 237 participants enrolled in the study, the predominance (n=133, 56%) was males. The mean age of the study population was 47.92±9.17 years, and the mean duration of diabetes was 6.8±5.3 years. Out of the total participants, 164 (69%) had deranged lipid profiles. Out of them, 129 (78.65%) were on hypolipidemic drugs. Regarding drug utilization, 122 (94.6%) received statins either rosuvastatin (54%) or atorvastatin (40%). In the deranged lipid profiles pattern, 24% (58) participants had one abnormal lipid parameter and the majority 70% (166) had combined lipid profile abnormality. Factors like increased BMI were significantly associated with dyslipidemia (p=0.004). Utilization of hypolipidemic drugs was also significantly associated with the control of dyslipidemia (p<0.001). It was observed that participants who were not on lipid-lowering drugs had 5.38 times more chance of dyslipidemia (OR=5.38; CI=2.82-10.28; p<0.001). Conclusion A high prevalence of dyslipidemia was observed among diabetic patients. Statins were the most prescribed drug in the study. BMI and lack of pharmacotherapy were found to have a statistically significant association with dyslipidemia in diabetic patients.

NADPH oxidase 1 in chronic pancreatitis-activated pancreatic stellate cells facilitates the progression of pancreatic cancer.

Patients suffering from chronic pancreatitis (CP) have a higher risk of pancreatic ductal adenocarcinoma (PDAC) compared to the general population. For instance, the presence of an activated pancreatic stellate cell (PaSC)-rich stroma in CP has facilitated the progression of non-invasive pancreatic intraepithelial neoplasia (PanIN) lesions to invasive PDAC. We have previously found that in a mouse model of CP, NADPH oxidase 1 (Nox1) in activated PaSCs forms fibrotic tissue and up-regulates both matrix metalloproteinase (MMP) 9 and the transcription factor Twist1. Yet, the role and mechanism of Nox1 in activated PaSCs from mice with CP (CP-activated PaSCs) in the progression of PDAC is unknown. For that, we tested the ability of Nox1 in CP-activated PaSCs to facilitate the growth of pancreatic cancer cells, and the mechanisms involved in these effects by identifying proteins in the secretome of CP-activated PaSCs whose production were Nox1-dependent. We found that, in vitro, Nox1 evoked a pro-invasive and cancer-promoting phenotype in CP-activated PaSCs via Twist1/MMP-9 expression, causing changes in the extracellular matrix composition. In vivo, Nox1 in CP-activated PaSCs facilitated tumor growth and stromal expansion. Using mass spectrometry, we identified proteins protecting from endoplasmic reticulum, oxidative and metabolic stresses in the secretome of CP-activated PaSCs whose production was Nox1-dependent, including peroxiredoxins (Prdx1 and Prdx4), and thioredoxin reductase 1. In conclusion, inhibiting the Nox1 signaling in activated PaSCs from patients with CP at early stages can reduce the reorganization of extracellular matrix, and the protection of neoplastic cells from cellular stresses, ameliorating the progression of PDAC.

Potential long-term effects of SARS-CoV-2 infection on the pulmonary vasculature: Multilayered cross-talks in the setting of coinfections and comorbidities.

The Coronavirus Disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its sublineages pose a new challenge to healthcare systems worldwide due to its ability to efficiently spread in immunized populations and its resistance to currently available therapies. COVID-19, although targeting primarily the respiratory system, is also now well established that later affects every organ in the body. Most importantly, despite the available therapy and vaccine-elicited protection, the long-term consequences of viral infection in breakthrough and asymptomatic individuals are areas of concern. In the past two years, investigators accumulated evidence on how the virus triggers our immune system and the molecular signals involved in the cross-talk between immune cells and structural cells in the pulmonary vasculature to drive pathological lung complications such as endothelial dysfunction and thrombosis. In the review, we emphasize recent updates on the pathophysiological inflammatory and immune responses associated with SARS-CoV-2 infection and their potential long-term consequences that may consequently lead to the development of pulmonary vascular diseases.

Association of Gastroesophageal Reflux Disease With Dental Erosion.

Gastroesophageal reflux disorder (GERD) or chronic acid reflux disorder is a condition in which acid-containing contents continuously leak from the stomach and return to the esophagus. Acid reflux disease occurs in nearly every person at some unspecified time. In reality, it is considered as a reoccurrence of acid reflux disease disorder and heartburn, every day. However, when you have acid reflux disorder/heartburn greater than two times every week over numerous weeks, constantly take heartburn medicinal tablets and antacids. However, if your signs and symptoms and symptoms keep returning, you can have superior GERD. Your GERD needs to be handled with the aid of your healthcare employer. Now not simply to alleviate your symptoms, but because of the reality, GERD can result in extra intense issues. Dental erosion (DE) is the shortage of the ground of your tooth because of acids you eat or drink or acids arising from your stomach. Those acids can wash away the tough substance that makes up your enamel, number one to tooth floor loss. Acid also can melt the teeth floor, making it much less complicated to wear away with the beneficial aid of erosion. This is called acid put on or erosive enamel wear. The belly contains many sturdy acids that are used to digest food. Vomiting and reflux can reason those belly acids to enter your mouth. Gastric acids are very sturdy and might purpose considerable harm to the tooth. DE is the lack of the enamel's hard tissues due to the interplay of gastric juice, pepsin, and acid.

Adverse Events Following COVID-19 Vaccination in Selected Apartments in Bangalore, India.

Background Vaccination has provided a ray of hope in combating the coronavirus disease 2019 (COVID-19). Vaccines were rolled out as an emergency measure, with an expedited approval process. The available clinical trial data reveals the fact that vaccines mostly produce mild adverse events following immunization (AEFIs). Since the experiences are relatively new, it is important to monitor safety in a real-world setting. With this background, this survey was conducted. Methods This cross-sectional study was approved by the institutional ethics committee (IEC) of Vydehi Institute of Medical Sciences and Research Centre. This was conducted over a period of four months at select apartment complexes around Whitefield, Bangalore. The participants were invited to fill up data through online Google Forms (Google, Mountain View, CA, USA). They were requested to provide demographic details, information related to vaccination, and AEFIs. Eligibility to participate included recipients of vaccines who received Emergency Use Authorization (EUA) in India. Data were analyzed using SPSS version 20.00 (IBM Corporation, Armonk, NY, USA). Results The total number of participants in the study was 322. Out of this, 37.6% (121) were males and 62.4% (201) were females. The mean age of the participants was 34.9 ± 12.4 (mean ± standard deviation (SD) years. About 30% (96) of the study participants had comorbidities. Overall, 67.4% (217) of the participants suffered from AEFI. Of them, immediate reactions were reported by 18.3% (59) and 10.2% (32) of the participants after the first and second doses, respectively. A total of 0.9% (3) of the participants had immediate allergic reactions. The most common local and systemic AEFIs were pain at the injection site and extreme tiredness. AEFIs were found to be mild and with a probable association with vaccination as per the WHO scale. The number of females experiencing AEFIs was found to be higher when compared with males for both local and systemic reactions. There was a statistically significant increase in the number of individuals experiencing general adverse effects following the first dose of CovishieldTM (Serum Institute of India Private Limited, Pune, India) when compared with CovaxinTM  (Bharat Biotech Limited, Hyderabad, India) (P < 0.05). Of the participants, 5.9% (19) were diagnosed with COVID-19 post-vaccination. Among them, 15.8% (3) required hospitalization, with 10.5% (2) of them requiring an oxygen bed. It was observed that 76.5% (166) of the participants did not report their reactions to concerned authorities. Conclusion Based on our sample, the study reflects that COVID-19 vaccination causes mild AEFI in most vaccine recipients. It also provides an insight that reporting of AEFI is very low. It is, therefore, important to take up more awareness campaigns about reporting of AEFIs through the COVID Vaccine Intelligence Network (CoWIN) portal.

Novel Mechanisms Targeted by Drug Trials in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a rare disease associated with abnormally elevated pulmonary pressures and right heart failure resulting in high morbidity and mortality. Although the prognosis for patients with PAH has improved with the introduction of pulmonary vasodilators, disease progression remains a major problem. Given that available therapies are inadequate for preventing small-vessel loss and obstruction, there is active interest in identifying drugs capable of targeting angiogenesis and mechanisms involved in the regulation of cell growth and fibrosis. Among the mechanisms linked to PAH pathogenesis, preclinical studies have identified promising compounds that are currently being tested in clinical trials. These drugs target seven of the major mechanisms associated with PAH pathogenesis: bone morphogenetic protein signaling, tyrosine kinase receptors, estrogen metabolism, extracellular matrix, angiogenesis, epigenetics, and serotonin metabolism. In this review, we discuss the preclinical studies that led to prioritization of these mechanisms, and discuss completed and ongoing phase 2/3 trials using novel interventions such as sotatercept, anastrozole, rodatristat ethyl, tyrosine kinase inhibitors, and endothelial progenitor cells, among others. We anticipate that the next generation of compounds will build on the success of the current standard of care and improve clinical outcomes and quality of life for patients with PAH.

ADR in Journals: Are They Translated into Regulatory Frameworks?

INTRODUCTION: An adverse drug reaction case report refers to a scientific publication that is written by a health care professional who suspects a casual relationship between a drug and an adverse drug reaction (ADR). ADR case reports help to identify potential risks associated with the use of drug. Most of the case reports do not mention about reporting the ADR to regulatory authorities. With this objective, the aim of this study was to analyze the number of Adverse Drug Reactions (ADR) published as case reports (PubMed indexed journals) from January 2018 to June 2019, and observe if they are translated in regulatory frameworks like Vigibase, and package inserts. MATERIALS AND METHODS: 321 ADRs were obtained with the keywords "Adverse Drug Reaction". Out of those, 158 were independently extracted by two investigators, observed and categorized according to classes of the drugs, geographic location, severity, hospitalization, Completeness of ADR, whether reported to the regulatory authority (Vigibase), or listed in the package insert. Literature review articles were excluded. RESULTS: Out of the 158 ADRs, antibiotics accounted for 12.65%, CNS drugs and monoclonal antibodies11.39%, anticancer drugs 9.49%, CVS drugs 4.43%, anti-viral 3.79%, others 45.56%, respectively. According to geographic region, 26 ADRs published were from USA, Australia 4, Italy 3, India 17, Turkey 9, Singapore and UK 1, China 20, Denmark and Canada 2, Japan 10, France 9, Austria 1, Korea 5, South America 3, Switzerland 2, respectively. Depending upon the severity, causality assessment was done only for 45 ADRs, and not done for 113 ADRs. 41.13% patients (from 65 case reports) were hospitalized. Among the 158 ADRs, 14 ADRs were not found in Vigibase. 32 ADRs were not mentioned in the Drug package inserts. When categorized according to the completeness of case reports, weight accounted for1.89%, lab values and procedure for diagnosis, 96.8%, risk factors, 95.56%, prior exposure, 88.60%, Post ADR status, 60.12%, start-stop medication, route of administration, first dose, last dose, duration of illness accounted for 100%, respectively. CONCLUSION: Depending upon our observation, we have noticed that there is deficiency in reporting of suspected ADRs to regulatory authorities. Reporting can be included as mandatory criteria for ADR case reports. Also, there is an increased need to aware various healthcare workers for reporting ADR.

Lung Pericytes in Pulmonary Vascular Physiology and Pathophysiology.

Pericytes are mesenchymal-derived mural cells localized within the basement membrane of pulmonary and systemic capillaries. Besides structural support, pericytes control vascular tone, produce extracellular matrix components, and cytokines responsible for promoting vascular homeostasis and angiogenesis. However, pericytes can also contribute to vascular pathology through the production of pro-inflammatory and pro-fibrotic cytokines, differentiation into myofibroblast-like cells, destruction of the extracellular matrix, and dissociation from the vessel wall. In the lung, pericytes are responsible for maintaining the integrity of the alveolar-capillary membrane and coordinating vascular repair in response to injury. Loss of pericyte communication with alveolar capillaries and a switch to a pro-inflammatory/pro-fibrotic phenotype are common features of lung disorders associated with vascular remodeling, inflammation, and fibrosis. In this article, we will address how to differentiate pericytes from other cells, discuss the molecular mechanisms that regulate the interactions of pericytes and endothelial cells in the pulmonary circulation, and the experimental tools currently used to study pericyte biology both in vivo and in vitro. We will also discuss evidence that links pericytes to the pathogenesis of clinically relevant lung disorders such as pulmonary hypertension, idiopathic lung fibrosis, sepsis, and SARS-COVID. Future studies dissecting the complex interactions of pericytes with other pulmonary cell populations will likely reveal critical insights into the origin of pulmonary diseases and offer opportunities to develop novel therapeutics to treat patients afflicted with these devastating disorders. © 2021 American Physiological Society. Compr Physiol 11:2227-2247, 2021.

Novel TNIP2 and TRAF2 Variants Are Implicated in the Pathogenesis of Pulmonary Arterial Hypertension.

Background: Pulmonary arterial hypertension (PAH) is a rare disease characterized by pulmonary vascular remodeling and right heart failure. Specific genetic variants increase the incidence of PAH in carriers with a family history of PAH, those who suffer from certain medical conditions, and even those with no apparent risk factors. Inflammation and immune dysregulation are related to vascular remodeling in PAH, but whether genetic susceptibility modifies the PAH immune response is unclear. TNIP2 and TRAF2 encode for immunomodulatory proteins that regulate NF-κB activation, a transcription factor complex associated with inflammation and vascular remodeling in PAH. Methods: Two unrelated families with PAH cases underwent whole-exome sequencing (WES). A custom pipeline for variant prioritization was carried out to obtain candidate variants. To determine the impact of TNIP2 and TRAF2 in cell proliferation, we performed an MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay on healthy lung pericytes transfected with siRNA specific for each gene. To measure the effect of loss of TNIP2 and TRAF2 on NF-kappa-beta (NF-κB) activity, we measured levels of Phospho-p65-NF-κB in siRNA-transfected pericytes using western immunoblotting. Results: We discovered a novel missense variant in the TNIP2 gene in two affected individuals from the same family. The two patients had a complex form of PAH with interatrial communication and scleroderma. In the second family, WES of the proband with PAH and primary biliary cirrhosis revealed a de novo protein-truncating variant in the TRAF2. The knockdown of TNIP2 and TRAF2 increased NF-κB activity in healthy lung pericytes, which correlated with a significant increase in proliferation over 24 h. Conclusions: We have identified two rare novel variants in TNIP2 and TRAF2 using WES. We speculate that loss of function in these genes promotes pulmonary vascular remodeling by allowing overactivation of the NF-κB signaling activity. Our findings support a role for WES in helping identify novel genetic variants associated with dysfunctional immune response in PAH.

From 2D to 3D: Promising Advances in Imaging Lung Structure.

The delicate structure of murine lungs poses many challenges for acquiring high-quality images that truly represent the living lung. Here, we describe several optimized procedures for obtaining and imaging murine lung tissue. Compared to traditional paraffin cross-section and optimal cutting temperature (OCT), agarose-inflated vibratome sections (aka precision-cut lung slices), combines comparable structural preservation with experimental flexibility. In particular, we discuss an optimized procedure to precision-cut lung slices that can be used to visualize three-dimensional cell-cell interactions beyond the limitations of two-dimensional imaging. Super-resolution microscopy can then be used to reveal the fine structure of lung tissue's cellular bodies and processes that regular confocal cannot. Lastly, we evaluate the entire lung vasculature with clearing technology that allows imaging of the entire volume of the lung without sectioning. In this manuscript, we combine the above procedures to create a novel and evolutionary method to study cell behavior ex vivo, trace and reconstruct pulmonary vasculature, address fundamental questions relevant to a wide variety of vascular disorders, and perceive implications to better imaging clinical tissue.