Characterization of Commercially Available Human Primary Alveolar Epithelial Cells.

In vitro lung research requires appropriate cell culture models that adequately mimic in vivo structure and function. Previously, researchers extensively used commercially available and easily expandable A549 and NCI-H441 cells, which replicate some but not all features of alveolar epithelial cells. Specifically, these cells are often restricted by terminally altered expression while lacking important alveolar epithelial characteristics. Of late, human primary alveolar epithelial cells (hPAEpCs) have become commercially available but are so far poorly specified. Here, we applied a comprehensive set of technologies to characterize their morphology, surface marker expression, transcriptomic profile, and functional properties. At optimized seeding numbers of 7,500 cells per square centimeter and growth at a gas-liquid interface, hPAEpCs formed regular monolayers with tight junctions and amiloride-sensitive transepithelial ion transport. Electron microscopy revealed lamellar body and microvilli formation characteristic for alveolar type II cells. Protein and single-cell transcriptomic analyses revealed expression of alveolar type I and type II cell markers; yet, transcriptomic data failed to detect NKX2-1, an important transcriptional regulator of alveolar cell differentiation. With increasing passage number, hPAEpCs transdifferentiated toward alveolar-basal intermediates characterized as SFTPC-, KRT8high, and KRT5- cells. In spite of marked changes in the transcriptome as a function of passaging, Uniform Manifold Approximation and Projection plots did not reveal major shifts in cell clusters, and epithelial permeability was unaffected. The present work delineates optimized culture conditions, cellular characteristics, and functional properties of commercially available hPAEpCs. hPAEpCs may provide a useful model system for studies on drug delivery, barrier function, and transepithelial ion transport in vitro.

Epicardial adipose tissue defined by initial polytrauma CT of mechanically ventilated trauma patients: retrospective single-center cohort study to predict short-term outcomes.

PURPOSE: Epicardial adipose tissue (EAT) detected by computed tomography (CT) is associated with morbidity and mortality in patients with COVID-19 and other critical care patient cohorts, whereas their prognostic relevance in trauma patients remains unclear. The present study explored associations with four potential short-term outcomes in trauma patients. METHODS: All consecutive trauma patients requiring emergency tracheal intubation and mechanical ventilation before initial whole-body CT imaging at a level-1 trauma center over a 12-year period (2008-2019) were reanalyzed for this study. EAT was measured semiquantitatively in initial CT and analyzed regarding associations with 24-hour and 30-day mortality using Cox proportional hazard models. In survivors, associations of EAT with intensive care unit length of stay (ICU LOS) and mechanical ventilation duration were analyzed using linear regression analyses. RESULTS: Four hundred fifty-five patients (74.7% male) with a median age of 49 years, and a median injury severity score (ISS) of 26 points were analyzed. In univariable analysis, EAT index was significantly associated with 24-hour and 30-day mortality (p = 0.007, and p = 0.013, respectively). After adjustment for significant predictors age, body mass index, and ISS, no significant associations were confirmed (p = 0.622, and p = 0.903, respectively). In a subanalysis of 353 survivors, EAT index was significantly associated with ICU LOS and mechanical ventilation duration in univariable analyses (p = 0.031, and p = 0.014, respectively), but not in multivariable analyses (p = 0.81 and p = 0.46, respectively). CONCLUSION: EAT index was associated with short-term outcomes in severely injured trauma patients, which not remained significant in multivariable analysis, suggesting that its prognostic capability is limited.

Sex and statin-related genetic associations at the PCSK9 gene locus: results of genome-wide association meta-analysis.

BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key player of lipid metabolism with higher plasma levels in women throughout their life. Statin treatment affects PCSK9 levels also showing evidence of sex-differential effects. It remains unclear whether these differences can be explained by genetics. METHODS: We performed genome-wide association meta-analyses (GWAS) of PCSK9 levels stratified for sex and statin treatment in six independent studies of Europeans (8936 women/11,080 men respectively 14,825 statin-free/5191 statin-treated individuals). Loci associated in one of the strata were tested for statin- and sex-interactions considering all independent signals per locus. Independent variants at the PCSK9 gene locus were then used in a stratified Mendelian Randomization analysis (cis-MR) of PCSK9 effects on low-density lipoprotein cholesterol (LDL-C) levels to detect differences of causal effects between the subgroups. RESULTS: We identified 11 loci associated with PCSK9 in at least one stratified subgroup (p < 1.0 × 10-6), including the PCSK9 gene locus and five other lipid loci: APOB, TM6SF2, FADS1/FADS2, JMJD1C, and HP/HPR. The interaction analysis revealed eight loci with sex- and/or statin-interactions. At the PCSK9 gene locus, there were four independent signals, one with a significant sex-interaction showing stronger effects in men (rs693668). Regarding statin treatment, there were two significant interactions in PCSK9 missense mutations: rs11591147 had stronger effects in statin-free individuals, and rs11583680 had stronger effects in statin-treated individuals. Besides replicating known loci, we detected two novel genome-wide significant associations: one for statin-treated individuals at 6q11.1 (within KHDRBS2) and one for males at 12q24.22 (near KSR2/NOS1), both with significant interactions. In the MR of PCSK9 on LDL-C, we observed significant causal estimates within all subgroups, but significantly stronger causal effects in statin-free subjects compared to statin-treated individuals. CONCLUSIONS: We performed the first double-stratified GWAS of PCSK9 levels and identified multiple biologically plausible loci with genetic interaction effects. Our results indicate that the observed sexual dimorphism of PCSK9 and its statin-related interactions have a genetic basis. Significant differences in the causal relationship between PCSK9 and LDL-C suggest sex-specific dosages of PCSK9 inhibitors.

The protein "proprotein convertase subtilisin/kexin type 9" (PCSK9) regulates the levels of low-density lipoprotein cholesterol (LDL-C) in blood, and thus, contributes to the risk of cardio-vascular diseases. Women tend to have higher PCSK9 plasma levels throughout their life, although the difference is smaller in patients under LDL-C lowering medication (e.g., statins). We investigated the interplay of genetics, statin-treatment and sex, using combined data from six European studies. We detected 11 genetic regions associated with PCSK9 levels, of which one was specific for women (at SLCO1B3, a statin-transporter gene), and three were specific for men (e.g., ALOX5, encoding a protein linked to chronic inflammatory diseases such as atherosclerosis). We also tested if statin use changed the genetic effect and found five genes only associated with PCSK9 levels in untreated participants. Variants in the gene encoding PCSK9 were most strongly associated and had heterogeneous effects in dependence on statin treatment and sex: On one hand, there were genetic variants with stronger effects in men than women. Those variants are also linked to sex-differential gene expression of PCSK9. On the other hand, there were also variants with treatment-depending effects, linked to protein structure and functionality of PCSK9. This indicates that the observed sexual and treatment-related effects on PCSK9 levels have a genetic basis. In addition, we compared the causal effects of PCSK9 on LDL-C levels between men and women and found a different response to statin treatment. This highlights the need for sex-sensitive dosages of lipid-lowering medication.

X-chromosome and kidney function: evidence from a multi-trait genetic analysis of 908,697 individuals reveals sex-specific and sex-differential findings in genes regulated by androgen response elements.

X-chromosomal genetic variants are understudied but can yield valuable insights into sexually dimorphic human traits and diseases. We performed a sex-stratified cross-ancestry X-chromosome-wide association meta-analysis of seven kidney-related traits (n = 908,697), identifying 23 loci genome-wide significantly associated with two of the traits: 7 for uric acid and 16 for estimated glomerular filtration rate (eGFR), including four novel eGFR loci containing the functionally plausible prioritized genes ACSL4, CLDN2, TSPAN6 and the female-specific DRP2. Further, we identified five novel sex-interactions, comprising male-specific effects at FAM9B and AR/EDA2R, and three sex-differential findings with larger genetic effect sizes in males at DCAF12L1 and MST4 and larger effect sizes in females at HPRT1. All prioritized genes in loci showing significant sex-interactions were located next to androgen response elements (ARE). Five ARE genes showed sex-differential expressions. This study contributes new insights into sex-dimorphisms of kidney traits along with new prioritized gene targets for further molecular research.

Dyslexia risk variant rs600753 is linked with dyslexia-specific differential allelic expression of DYX1C1

Abstract An increasing number of genetic variants involved in dyslexia development were discovered during the last years, yet little is known about the molecular functional mechanisms of these SNPs. In this study we investigated whether dyslexia candidate SNPs have a direct, disease-specific effect on local expression levels of the assumed target gene by using a differential allelic expression assay. In total, 12 SNPs previously associated with dyslexia and related phenotypes were suitable for analysis. Transcripts corresponding to four SNPs were sufficiently expressed in 28 cell lines originating from controls and a family affected by dyslexia. We observed a significant effect of rs600753 on expression levels of DYX1C1 in forward and reverse sequencing approaches. The expression level of the rs600753 risk allele was increased in the respective seven cell lines from members of the dyslexia family which might be due to a disturbed transcription factor binding sites. When considering our results in the context of neuroanatomical dyslexia-specific findings, we speculate that this mechanism may be part of the pathomechanisms underlying the dyslexia-specific brain phenotype. Our results suggest that allele-specific DYX1C1 expression levels depend on genetic variants of rs600753 and contribute to dyslexia. However, these results are preliminary and need replication.