Single-cell transcriptomics reveals skewed cellular communication and phenotypic shift in pulmonary artery remodeling.

A central feature of progressive vascular remodeling is altered smooth muscle cell (SMC) homeostasis; however, the understanding of how different cell populations contribute to this process is limited. Here, we utilized single-cell RNA sequencing to provide insight into cellular composition changes within isolated pulmonary arteries (PAs) from pulmonary arterial hypertension and donor lungs. Our results revealed that remodeling skewed the balanced communication network between immune and structural cells, in particular SMCs. Comparative analysis with murine PAs showed that human PAs harbored heterogeneous SMC populations with an abundant intermediary cluster displaying a gradient transition between SMCs and adventitial fibroblasts. Transcriptionally distinct SMC populations were enriched in specific biological processes and could be differentiated into 4 major clusters: oxygen sensing (enriched in pericytes), contractile, synthetic, and fibroblast-like. End-stage remodeling was associated with phenotypic shift of preexisting SMC populations and accumulation of synthetic SMCs in neointima. Distinctly regulated genes in clusters built nonredundant regulatory hubs encompassing stress response and differentiation regulators. The current study provides a blueprint of cellular and molecular changes on a single-cell level that are defining the pathological vascular remodeling process.

Impairment of the NKT-STAT1-CXCL9 Axis Contributes to Vessel Fibrosis in Pulmonary Hypertension Caused by Lung Fibrosis.

Rationale: Pulmonary hypertension (PH) is a common, severe comorbidity in interstitial lung diseases such as pulmonary fibrosis (PF), and it has limited treatment options. Excessive vascular fibrosis and inflammation are often present in PH, but the underlying mechanisms are still not well understood. Objectives: To identify a novel functional link between natural killer T (NKT) cell activation and vascular fibrosis in PF-PH. Methods: Multicolor flow cytometry, secretome, and immunohistological analyses were complemented by pharmacological NKT cell activation in vivo, in vitro, and ex vivo. Measurements and Main Results: In pulmonary vessels of patients with PF-PH, increased collagen deposition was linked to a local NKT cell deficiency and decreased IL-15 concentrations. In a mouse model of PH caused by lung fibrosis, pharmacological NKT cell activation using a synthetic α-galactosylceramide analog (KRN7000) restored local NKT cell numbers and ameliorated vascular remodeling and right ventricular systolic pressure. Supplementation with activated NKT cells reduced collagen deposition in isolated human pulmonary arterial smooth muscle cells (hPASMCs) and in ex vivo precision-cut lung slices of patients with end-stage PF-PH. Coculture with activated NKT cells induced STAT1 signaling in hPASMCs. Secretome analysis of peripheral blood mononuclear cells identified CXCL9 and CXCL10 as indicators of NKT cell activation. Pharmacologically, CXCL9, but not CXCL10, potently inhibited collagen deposition in hPASMCs via the chemokine receptor CXCR3. Conclusions: Our results indicate that the absence of NKT cells impairs the STAT1-CXCL9-CXCR3 axis in PF-PH and that restoration of this axis by NKT cell activation may unravel a novel therapeutic strategy to target vascular fibrosis in interstitial lung disease.

RGS5 Determines Neutrophil Migration in the Acute Inflammatory Phase of Bleomycin-Induced Lung Injury.

The regulator of G protein signaling (RGS) represents a widespread system of controllers of cellular responses. The activities of the R4 subfamily of RGSs have been elucidated in allergic pulmonary diseases. However, the R4 signaling in other inflammatory lung diseases, with a strong cellular immune response, remained unexplored. Thus, our study aimed to discern the functional relevance of the R4 family member, RGS5, as a potential modulating element in this context. Gene profiling of the R4 subfamily showed increased RGS5 expression in human fibrosing lung disease samples. In line with this, RGS5 was markedly increased in murine lungs following bleomycin injury. RGS knock-out mice (RGS-/-) had preserved lung function while control mice showed significant combined ventilatory disorders three days after bleomycin application as compared to untreated control mice. Loss of RGS5 was associated with a significantly reduced neutrophil influx and tissue myeloperoxidase expression. In the LPS lung injury model, RGS5-/- mice also failed to recruit neutrophils into the lung, which was accompanied by reduced tissue myeloperoxidase levels after 24 h. Our in-vitro assays showed impaired migration of RGS5-/- neutrophils towards chemokines despite preserved Ca2+ signaling. ERK dephosphorylation might play a role in reduced neutrophil migration in our model. As a conclusion, loss of RGS5 preserves lung function and attenuates hyperinflammation in the acute phase of bleomycin-induced pulmonary fibrosis and LPS-induced lung injury. Targeting RGS5 might alleviate the severity of exacerbations in interstitial lung diseases.

Dysbalance of ACE2 levels - a possible cause for severe COVID-19 outcome in COPD.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a serious threat to healthcare systems worldwide. Binding of the virus to angiotensin-converting enzyme 2 (ACE2) is an important step in the infection mechanism. However, it is unknown if ACE2 expression in patients with chronic lung diseases (CLDs), such as chronic obstructive pulmonary disease (COPD), idiopathic pulmonary arterial hypertension (IPAH), or pulmonary fibrosis (PF), is changed as compared to controls. We used lung samples from patients with COPD (n = 28), IPAH (n = 10), and PF (n = 10) as well as healthy control donor (n = 10) tissue samples to investigate the expression of ACE2 and related cofactors that might influence the course of SARS-CoV-2 infection. Expression levels of the ACE2 receptor, the putative receptor CD147/BSG, and the viral entry cofactors TMPRSS2 (transmembrane serine protease 2), EZR, and FURIN were determined by quantitative PCR and in open-access RNA sequencing datasets. Immunohistochemical and single-cell RNA sequencing (scRNAseq) analyses were used for localization and coexpression, respectively. Soluble ACE2 (sACE2) plasma levels were analyzed by enzyme-linked immunosorbent assay. In COPD as compared to donor, IPAH, and PF lung tissue, gene expression of ACE2, TMPRSS2, and EZR was significantly elevated, but circulating sACE2 levels were significantly reduced in COPD and PF plasma compared to healthy control and IPAH plasma samples. Lung tissue expressions of FURIN and CD147/BSG were downregulated in COPD. None of these changes were associated with changes in pulmonary hemodynamics. Histological analysis revealed coexpression of ACE2, TMPRSS2, and Ezrin in bronchial regions and epithelial cells. This was confirmed by scRNAseq analysis. There were no significant expression changes of the analyzed molecules in the lung tissue of IPAH and idiopathic PF as compared to control. In conclusion, we reveal increased ACE2 and TMPRSS2 expression in lung tissue with a concomitant decrease of protective sACE2 in COPD patients. These changes represent the possible risk factors for an increased susceptibility of COPD patients to SARS-CoV-2 infection.

Simple method of thawing cryo-stored samples preserves ultrastructural features in electron microscopy.

Preservation of ultrastructural features in biological samples for electron microscopy (EM) is a challenging task that is routinely accomplished through chemical fixation or high-pressure freezing coupled to automated freeze substitution (AFS) using specialized devices. However, samples from clinical (e.g. "biobanking" of bulk biopsies) and preclinical (e.g. whole mouse tissues) specimens are often not specifically prepared for ultrastructural analyses but simply immersed in liquid nitrogen before long-term cryo-storage. We demonstrate that ultrastructural features of such samples are insufficiently conserved using AFS and developed a simple, rapid, and effective method for thawing that does not require specific instrumentation. This procedure consists of dry ice-cooled pre-trimming of frozen tissue and aldehyde fixation for 3 h at 37 °C followed by standard embedding steps. Herein investigated tissues comprised human term placentae, clinical lung samples, as well as mouse tissues of different composition (brown adipose tissue, white adipose tissue, cardiac muscle, skeletal muscle, liver). For all these tissues, we compared electron micrographs prepared from cryo-stored material with our method to images derived from directly prepared fresh tissues with standard chemical fixation. Our protocol yielded highly conserved ultrastructural features and tissue-specific details, largely matching the quality of fresh tissue samples. Furthermore, morphometric analysis of lipid droplets and mitochondria in livers of fasted mice demonstrated that statistically valid quantifications can be derived from samples prepared with our method. Overall, we provide a simple and effective protocol for accurate ultrastructural and morphometric analyses of cryo-stored bulk tissue samples.

Endothelial Dysfunction Following Enhanced TMEM16A Activity in Human Pulmonary Arteries.

Endothelial dysfunction is one of the hallmarks of different vascular diseases, including pulmonary arterial hypertension (PAH). Ion channelome changes have long been connected to vascular remodeling in PAH, yet only recently has the focus shifted towards Ca2+-activated Cl- channels (CaCC). The most prominent member of the CaCC TMEM16A has been shown to contribute to the pathogenesis of idiopathic PAH (IPAH) in pulmonary arterial smooth muscle cells, however its role in the homeostasis of healthy human pulmonary arterial endothelial cells (PAECs) and in the development of endothelial dysfunction remains underrepresented. Here we report enhanced TMEM16A activity in IPAH PAECs by whole-cell patch-clamp recordings. Using adenoviral-mediated TMEM16A increase in healthy primary human PAECs in vitro and in human pulmonary arteries ex vivo, we demonstrate the functional consequences of the augmented TMEM16A activity: alterations of Ca2+ dynamics and eNOS activity as well as decreased NO production, PAECs proliferation, wound healing, tube formation and acetylcholine-mediated relaxation of human pulmonary arteries. We propose that the ERK1/2 pathway is specifically affected by elevated TMEM16A activity, leading to these pathological changes. With this work we introduce increased TMEM16A activity in the cell membrane of human PAECs for the development of endothelial dysfunction in PAH.

[Modern thyroid surgery - the surgeon's endocrine-surgical understanding and his responsibility for the extent of surgery and complication rate]. / Moderne Schilddrüsenchirurgie ­ das endokrin-chirurgische Verständnis des Operateurs und seine Verantwortung für Resektionsausmaß und Komplikationsrate.

High quality thyroid surgery implies a surgeon with an endocrine-surgical understanding aiming at best possible outcome. This includes an appropriate extent of the resection and a low rate of complications. It is important that the surgeon is involved at an early stage being part of the decision process for or against partial or total thyroidectomy. Furthermore, the surgeon should not only be able to perform thyroid and cervical lymph node sonography, but also to be capable to interpret cross-sectional imaging modalities and nuclear medicine imaging procedures. A thorough knowledge of modern principles of radicality is essential.Benign goiters require individualized surgical strategy: solitary nodules can be treated with a tissue-preserving selective nodular resection. However, a multinodular goiter does not necessarily require total thyroidectomy-prevention of a permanent hypoparathyroidism is of paramount importance. For recurrent goiters, removal of the dominant side and therefore, unilateral procedure is favored. Nowadays, there is an increasing tendency to set the indication for thyroid surgery separately for each lobe. Graves' disease requires thyroidectomy, and occasionally, hypertrophic Hashimoto's thyroiditis may also result in surgery.The principles of radical surgical treatment of malignant goiters have changed significantly over the past few years and, so far, strict indication for postoperative radioiodine treatment is being reconsidered. This is especially relevant for papillary thyroid microcarcinomas and minimally invasive follicular tumors. Even the radical surgical treatment of medullary thyroid carcinoma, especially considering synchronous or metachronous lateral neck dissection, is currently under review.Hypoparathyroidism is the most relevant complication in radical thyroid surgery and has devastating influence on the patients' life quality. Nowadays, permanent recurrent laryngeal nerve injury and postoperative hemorrhage rarely occur due to subtle surgical techniques. Extracervical surgical access to the thyroid is still a matter of clinical trials and should be restricted to centers. Radiofrequency ablation is an alternative method for benign lesions or hyperfunctioning nodules in patients with high surgical risk.

The laryngeal twitch response - Can it avoid unnecessary two-stage thyroidectomy? - A retrospective cohort study.

BACKGROUND: The objective of this study was to determine if the laryngeal twitch response, when compared to neuromonitoring, can predict postoperative vocal cord function and can thus be used in case of technical failure of the EMG-recording electrode. METHODS: A total of 640 nerves at risk were included in this study based on a prospective protocol. The laryngeal twitch response and the EMG-records were compared with the results of the postoperative laryngoscopy. RESULTS: Of the 640 nerves at risk, 582 showed a normal postoperative vocal cord function. A recurrent laryngeal nerve paralysis (no vocal fold movement) was observed in 39 cases and recurrent laryngeal nerve paresis (reduced vocal cord movement) was diagnosed in 19 cases. The overall negative predictive value (NPV) in final vagus nerve stimulation (V2) was 95.0% for the EMG-records and 94.8% for the laryngeal twitch response. When pareses were excluded, the NPV was 96.8% and 96.6% respectively. The positive predictive value (PPV) of vagus nerve stimulation lies between 51.4% and 57.1% excluding the pareses. It rises to values between 60.0% and 65.1% if they are included. CONCLUSIONS: The laryngeal twitch response and the EMG-records show similar results, and the NPV is good in both. Thus, in case of technical failure or displacement of the EMG-recording electrode, the laryngeal twitch can be used in decision-making for or against a two-stage thyroidectomy.

Expression of FGFR1-4 in Malignant Pleural Mesothelioma Tissue and Corresponding Cell Lines and its Relationship to Patient Survival and FGFR Inhibitor Sensitivity.

Malignant pleural mesothelioma (MPM) is a devastating malignancy with limited therapeutic options. Fibroblast growth factor receptors (FGFR) and their ligands were shown to contribute to MPM aggressiveness and it was suggested that subgroups of MPM patients could benefit from FGFR-targeted inhibitors. In the current investigation, we determined the expression of all four FGFRs (FGFR1-FGFR4) by immunohistochemistry in tissue samples from 94 MPM patients. From 13 of these patients, we were able to establish stable cell lines, which were subjected to FGFR1-4 staining, transcript analysis by quantitative RT-PCR, and treatment with the FGFR inhibitor infigratinib. While FGFR1 and FGFR2 were widely expressed in MPM tissue and cell lines, FGFR3 and FGFR4 showed more restricted expression. FGFR1 and FGFR2 showed no correlation with clinicopathologic data or patient survival, but presence of FGFR3 in 42% and of FGFR4 in 7% of patients correlated with shorter overall survival. Immunostaining in cell lines was more homogenous than in the corresponding tissue samples. Neither transcript nor protein expression of FGFR1-4 correlated with response to infigratinib treatment in MPM cell lines. We conclude that FGFR3 and FGFR4, but not FGFR1 or FGFR2, have prognostic significance in MPM and that FGFR expression is not sufficient to predict FGFR inhibitor response in MPM cell lines.

Complete and incomplete recurrent laryngeal nerve injury after thyroid and parathyroid surgery: Characterizing paralysis and paresis.

BACKGROUND: Injury of the recurrent laryngeal nerve and consequent disorder of vocal fold movement is a typical complication in thyroid and parathyroid surgery. During postoperative laryngoscopy we observed not only a complete standstill (vocal fold paralysis), but also a hypomobility (paresis). In this prospective study, we investigated the difference in incidence and prognosis as well as risk-factors, intraoperative neuromonitoring, and symptoms between vocal fold paralysis and vocal fold paresis. METHODS: Data were prospectively collected and analyzed in a single high-volume thyroid center between 2012 and 2016. Vocal fold paresis was defined as hypomobility in abduction or adduction, a reduction in range and speed of vocal fold movement. Vocal fold paralysis was defined as asymmetry and missing purposeful vocal fold movement. RESULTS: The study included 4,707 surgeries and 7,992 at-risk nerves at risk. Vocal fold paralysis was diagnosed in 374 patients (4.68% of 7,992 nerves at risk) and vocal fold paresis in 114 patients (1.43%). Exclusively in the paralysis group, 36 patients (0.45%) developed permanent loss of vocal fold function (P < .001). In follow-up, vocal fold paresis patients regain normal vocal fold function significantly earlier than vocal fold paralysis (mean duration: 6.96 ± 6.506 vs 10.77 ± 7,827 weeks) and presented with significantly less symptoms like hoarseness, diplophonia, dysphagia, and dyspnea (68.8% vs 95.9 %). In intraoperative neuromonitoring, vocal fold paresis showed a significantly higher postresectional N. vagus amplitude than vocal fold paralysis patients (0.349 mV vs 0.114 mV, P < .001). CONCLUSION: After thyroidectomy, vocal fold paresis must be distinguished from vocal fold paralysis and should be implemented as a separate outcome parameter in the postoperative quality assessment.

How many parathyroid glands can be identified during thyroidectomy?: Evidence-based data for medical experts.

BACKGROUND: The purpose of this study is to provide guidance for medical experts regarding malpractice claims on permanent hypoparathyroidism by analyzing the number of parathyroid glands (PGs) identified during thyroidectomy and the clinical outcome. METHODS: Parathyroid findings were documented in a standardized protocol for 357 patients undergoing thyroidectomy and treated by a single specialized surgeon. The resected thyroid was routinely dissected for accidentally removed PGs with consecutive autotransplantation and the pathological report also described unintentionally resected PGs. Follow-up was performed for 6 months. RESULTS: The mean number of identified PGs was 2.28. No PGs were found in 20 (5.6%), one in 56 (15.7%), two in 126 (35.3%), three in 114 (31.9%), and four in 41 (11.5%) cases. One patient (0.28%) had manifest permanent hypoparathyroidism, while ten patients (2.8%) had latent permanent hypoparathyroidism (hypocalcemia and normoparathyroidism). The risk factors identified for postoperative hypoparathyroidism were an increasing number of visualized PGs, autotransplantation, central neck dissection, and PGs in the histopathological work-up. For permanent hypoparathyroidism, PGs in the histology examination and neck dissection were significant, but the number of identified PGs was not. CONCLUSION: Even an experienced surgeon is not always able to find all four PGs during thyroidectomy and occasionally identifies none. Rather than focusing on identifying a minimum number of PGs, it is more important not to miss them in risky positions. A documented awareness of PGs, i. e., knowledge of variable parathyroid positions and their saving, is a prerequisite for surgical quality and to protect surgeons from claims.

Necrotizing fasciitis due to Vibrio cholerae non-O1/non-O139 after exposure to Austrian bathing sites.

We report on two cases of necrotizing fasciitis of the lower leg due to nontoxigenic Vibrio cholerae (V. cholerae). A 73-year-old woman (case 1) and an 80-year-old man (case 2) were hospitalized with symptoms of necrotizing fasciitis on July 18 and August 15, 2015, respectively. In both cases, symptoms started the day after swimming in local ponds. Swabs gained intraoperatively and a blood culture from the male patient, yielded V. cholerae non-O1/non-O139, negative for cholera toxin gene ctx and positive for hemolysin genes hlyA and hlyB. Water samples taken from pond A on August 17, 2015 (32 days after exposure of case 1) and from pond B on August 20, 2015 (7 days after exposure of case 2) yielded non-O1/non-O139 V. cholerae in most-probable numbers of > 11,000 per 100 ml each. The occurrence of two cases of necrotizing fasciitis within a 1 month period related to two Austrian non-saline bathing waters, previously not known to harbor V. cholerae, is probably linked to the prevailing extreme weather conditions (heat wave, drought) this summer in Austria. While case 1 was discharged in good clinical condition after 73 days, case 2 died after four months of hospitalization. Public health authorities are challenged to assess the effects of long-term climate change on pathogen growth and survival in continental bodies of fresh water.

Increase of papillary thyroid microcarcinoma and a plea for restrictive treatment: A retrospective study of 1,391 prospective documented patients.

BACKGROUND: The incidence of papillary thyroid microcarcinomas (PMCs) has increased sharply and therefore the lack of consensus for treatment has become a clinical dilemma. Our aim was to evaluate a less-radical approach. METHODS: This study includes 1,391 patients with PMC treated at a single surgical referral center in the endemic goiter area in Austria. Data, including long-term follow-up examinations with a median follow-up time of 7 years, were collected from the institutional surgery database. RESULTS: Of the 1,391 patients, 947 (68.1%) had a near-total or total thyroidectomy; 1,090 patients (78.3%) had no lymphadenectomy, and 1,136 patients (81.7%) did not receive radioiodine treatment. Twenty-one patients (1.5%) underwent reoperation, 5 because of lymph node recurrence (0.4%), 16 with clinically benign recurrence, including 4 cases of another PMC. There were no recurrences in the thyroid bed and no disease-related deaths. Risk factors for lymph node recurrences were nonincidental finding, nodal metastases at presentation, young age, aggregate tumor size, and subcapsular tumor localization. Multifocality, sex, maximum tumor size, and the extent of surgery were not relevant factors. CONCLUSION: Nodal recurrence is rare and reoperation cured all patients. Micrometastases are not of clinical relevance. The postoperative findings of most PMCs suggest that, even if multifocal, a limited approach without completion thyroidectomy, lymphadenectomy and radioiodine treatment is sufficient. In case of pre- or intraoperative clinically suspected nodal metastases or postoperatively diagnosed risk factors we propose the standard radical procedure. Routine preoperative cervical lymph node sonography is advisable before any thyroid surgery.