Targeted Ablation of Periostin-Expressing Activated Fibroblasts Prevents Adverse Cardiac Remodeling in Mice.

RATIONALE: Activated cardiac fibroblasts (CF) are crucial players in the cardiac damage response; excess fibrosis, however, may result in myocardial stiffening and heart failure development. Inhibition of activated CF has been suggested as a therapeutic strategy in cardiac disease, but whether this truly improves cardiac function is unclear. OBJECTIVE: To study the effect of CF ablation on cardiac remodeling. METHODS AND RESULTS: We characterized subgroups of murine CF by single-cell expression analysis and identified periostin as the marker showing the highest correlation to an activated CF phenotype. We generated bacterial artificial chromosome-transgenic mice allowing tamoxifen-inducible Cre expression in periostin-positive cells as well as their diphtheria toxin-mediated ablation. In the healthy heart, periostin expression was restricted to valvular fibroblasts; ablation of this population did not affect cardiac function. After chronic angiotensin II exposure, ablation of activated CF resulted in significantly reduced cardiac fibrosis and improved cardiac function. After myocardial infarction, ablation of periostin-expressing CF resulted in reduced fibrosis without compromising scar stability, and cardiac function was significantly improved. Single-cell transcriptional analysis revealed reduced CF activation but increased expression of prohypertrophic factors in cardiac macrophages and cardiomyocytes, resulting in localized cardiomyocyte hypertrophy. CONCLUSIONS: Modulation of the activated CF population is a promising approach to prevent adverse cardiac remodeling in response to angiotensin II and after myocardial infarction.

Gentamicin submucosal lavage during peroral endoscopic myotomy (POEM): a retrospective analysis.

BACKGROUND AND AIMS: Peroral endoscopic myotomy (POEM) is an evolving therapeutic modality for achalasia. According to the original Inoue's technique, a submucosal lavage with gentamicin has been practiced due to the fear of infection. This single-tertiary center study was intended to assess the clinical significance of the topical antibiotic lavage during POEM. METHODS: A retrospective analysis of prospectively collected data was conducted. The outcomes of patients who received the gentamicin lavage (group A) during POEM were compared to those who did not (group B). The main outcome variables were infectious adverse events, post-POEM fever, and markers of systemic inflammatory response. One day before and after POEM, all patients received systemic antibiotic prophylaxis with ceftriaxone. RESULTS: Of 124 consecutive patients having undergone POEM, 60 patients received a lavage with 80 mg of gentamicin into the submucosal tunnel before starting the myotomy, while 64 patients did not. The overall treatment success at 3 months did not differ between the two groups (group A 94.7 vs. 97.5% group B). We did not experience any significant infectious adverse events in either group. CRP and WBC levels were lower in patients with lavage versus those without [CRP: median 52.7 (IQR 34.9) vs. 69.5 (54.1); p = 0.01; WBCs: median 10.9 (IQR 3.3) vs. 12.6 (3.9); p < 0.01]. Post-procedural fever was present in 10% of patients in either group. CONCLUSIONS: During POEM, the submucosal lavage with gentamicin prior to the myotomy does not play a role in the prevention of clinically significant infectious adverse events, although the systemic inflammatory response may be decreased.

Deficiency in lymphotoxin ß receptor protects from atherosclerosis in apoE-deficient mice.

RATIONALE: Lymphotoxin ß receptor (LTbR) regulates immune cell trafficking and communication in inflammatory diseases. However, the role of LTbR in atherosclerosis is still unclear. OBJECTIVE: The aim of this study was to elucidate the role of LTbR in atherosclerosis. METHODS AND RESULTS: After 15 weeks of feeding a Western-type diet, mice double-deficient in apolipoprotein E and LTbR (apoE(-/-)/LTbR(-/-)) exhibited lower aortic plaque burden than did apoE(-/-) littermates. Macrophage content at the aortic root and in the aorta was reduced, as determined by immunohistochemistry and flow cytometry. In line with a decrease in plaque inflammation, chemokine (C-C motif) ligand 5 (Ccl5) and other chemokines were transcriptionally downregulated in aortic tissue from apoE(-/-)/LTbR(-/-) mice. Moreover, bone marrow chimeras demonstrated that LTbR deficiency in hematopoietic cells mediated the atheroprotection. Furthermore, during atheroprogression, apoE(-/-) mice exhibited increased concentrations of cytokines, for example, Ccl5, whereas apoE(-/-)/LTbR(-/-) mice did not. Despite this decreased plaque macrophage content, flow cytometric analysis showed that the numbers of circulating lymphocyte antigen 6C (Ly6C)(low) monocytes were markedly elevated in apoE(-/-)/LTbR(-/-) mice. The influx of these cells into atherosclerotic lesions was significantly reduced, whereas apoptosis and macrophage proliferation in atherosclerotic lesions were unaffected. Gene array analysis pointed to chemokine (C-C motif) receptor 5 as the most regulated pathway in isolated CD115(+) cells in apoE(-/-)/LTbR(-/-) mice. Furthermore, stimulating monocytes from apoE(-/-) mice with agonistic anti-LTbR antibody or the natural ligand lymphotoxin-α1ß2, increased Ccl5 mRNA expression. CONCLUSIONS: These findings suggest that LTbR plays a role in macrophage-driven inflammation in atherosclerotic lesions, probably by augmenting the Ccl5-mediated recruitment of monocytes.

LimiTT: link miRNAs to targets.

BACKGROUND: MicroRNAs (miRNAs) impact various biological processes within animals and plants. They complementarily bind target mRNAs, effecting a post-transcriptional negative regulation on mRNA level. The investigation of miRNA target interactions (MTIs) by high throughput screenings is challenging, as frequently used in silico target prediction tools are prone to emit false positives. This issue is aggravated for niche model organisms, where validated miRNAs and MTIs both have to be transferred from well described model organisms. Even though DBs exist that contain experimentally validated MTIs, they are limited in their search options and they utilize different miRNA and target identifiers. RESULTS: The implemented pipeline LimiTT integrates four existing DBs containing experimentally validated MTIs. In contrast to other cumulative databases (DBs), LimiTT includes MTI data of 26 species. Additionally, the pipeline enables the identification and enrichment analysis of MTIs with and without species specificity based on dynamic quality criteria. Multiple tabular and graphical outputs are generated to permit the detailed assessment of results. CONCLUSION: Our freely available web-based pipeline LimiTT ( https://bioinformatics.mpi-bn.mpg.de/ ) is optimized to determine MTIs with and without species specification. It links miRNAs and/or putative targets with high granularity. The integrated mapping to homologous target identifiers enables the identification of MTIs not only for standard models, but for niche model organisms as well.

Combination of Sterile Injury and Microbial Contamination to Model Post-surgical Peritoneal Adhesions in Mice.

Abdominal surgeries are frequently associated with the development of post-surgical adhesions. These are irreversible fibrotic scar bands that appear between abdominal organs and the abdominal wall. Patients suffering from adhesions are at risk of severe complications, such as small bowel obstruction, chronic pelvic pain, or infertility. To date, no cure exists, and the understanding of underlying molecular mechanisms of adhesion formation is incomplete. The current paradigm largely relies on sterile injury mouse models. However, abdominal surgeries in human patients are rarely completely sterile procedures. Here, we describe a modular surgical procedure for simultaneous or separate induction of sterile injury and microbial contamination. Combined, these insults synergistically lead to adhesion formation in the mouse peritoneal cavity. Surgical trauma is confined to a localized sterile injury of the peritoneum. Microbial contamination of the peritoneal cavity is induced by a limited perforation of the microbe-rich large intestine or by injection of fecal content. The presented protocol extends previous injury-based adhesion models by an additional insult through microbial contamination, which may more adequately model the clinical context of abdominal surgery. Graphical abstract.

Influence of High Energy Diet and Polygenic Predisposition for Obesity on Postpartum Health in Rat Dams.

It is estimated that 30% of pregnant women worldwide are overweight or obese, leading to adverse health effects for both mother and child. Women with obesity during pregnancy are at higher risk for developing both metabolic and mental disorders, such as diabetes and depression. Numerous studies have used rodent models of maternal obesity to understand its consequences on the offspring, yet characterization of changes in the dams is rare, and most rodent models rely solely on a high fat diet to induce maternal obesity, without regarding genetic propensity for obesity. Here we present the influence of both peripartum high energy diet (HE) and obesity-proneness on maternal health using selectively bred diet-resistant (DR) and diet-induced obese (DIO) rat dams. Outbred Sprague-Dawley rats were challenged with HE diet prior to mating and bred according to their propensity to gain weight. The original outbred breeding dams (F0) were maintained on low-fat chow during pregnancy and lactation. By comparison, the F1 dams consuming HE diet during pregnancy and lactation displayed higher gestational body weight gain (P < 0.01), and HE diet caused increased meal size and reduced meal frequency (P < 0.001). Sensitivity to the hormone amylin was preserved during pregnancy, regardless of diet. After several rounds of selective breeding, DIO and DR dams from generation F3 were provided chow or HE during pregnancy and lactation and assessed for their postpartum physiology and behaviors. We observed strong diet and phenotype effects on gestational weight gain, with DIO-HE dams gaining 119% more weight than DR-chow (P < 0.001). A high-resolution analysis of maternal behaviors did not detect main effects of diet or phenotype, but a subset of DIO dams showed delayed nursing behavior (P < 0.05). In generation F6/F7 dams, effects on gestational weight gain persisted (P < 0.01), and we observed a main effect of phenotype during a sucrose preference test (P < 0.05), with DIO-chow dams showing lower sucrose preference than DR controls (P < 0.05). Both DIO and DR dams consuming HE diet had hepatic steatosis (P < 0.001) and exhibited reduced leptin sensitivity in the arcuate nucleus (P < 0.001). These data demonstrate that both diet and genetic obesity-proneness have consequences on maternal health.

Intraperitoneal microbial contamination drives post-surgical peritoneal adhesions by mesothelial EGFR-signaling.

Abdominal surgeries are lifesaving procedures but can be complicated by the formation of peritoneal adhesions, intra-abdominal scars that cause intestinal obstruction, pain, infertility, and significant health costs. Despite this burden, the mechanisms underlying adhesion formation remain unclear and no cure exists. Here, we show that contamination of gut microbes increases post-surgical adhesion formation. Using genetic lineage tracing we show that adhesion myofibroblasts arise from the mesothelium. This transformation is driven by epidermal growth factor receptor (EGFR) signaling. The EGFR ligands amphiregulin and heparin-binding epidermal growth factor, are sufficient to induce these changes. Correspondingly, EGFR inhibition leads to a significant reduction of adhesion formation in mice. Adhesions isolated from human patients are enriched in EGFR positive cells of mesothelial origin and human mesothelium shows an increase of mesothelial EGFR expression during bacterial peritonitis. In conclusion, bacterial contamination drives adhesion formation through mesothelial EGFR signaling. This mechanism may represent a therapeutic target for the prevention of adhesions after intra-abdominal surgery.

Interoceptive accuracy is related to long-term stress via self-regulation.

Interoception describes the ability to perceive internal bodily signals. Previous research found a relationship between interoceptive accuracy (IAcc) and cardiovascular outcomes during or after acute stress. So far, the association between IAcc and long-term stress has not been investigated, although this would be important to identify a starting point to prevent long-term stress. To address this idea in the current study, we examined the relationship between IAcc and long-term stress, which was assessed with different questionnaires and biological markers, including cortisol and dehydroepiandrosterone (DHEA). Furthermore, we investigated self-regulation as a mechanism linking IAcc to long-term stress. The sample consisted of 98 participants. To measure IAcc, participants completed the heartbeat perception task. Perceived long-term stress and self-regulation were assessed via an online questionnaire. Moreover, hair samples were taken from 65 participants to determine long-term stress with cortisol and DHEA as well as the ratio of both. Results showed that IAcc was positively related to DHEA and weakly negatively related to the other indicators of long-term stress, except for the nonsignificant relationships to the indicators cortisol and stress experiences due to negative events. Furthermore, these relationships were mediated by participants' enhanced self-regulation. Thus, our results suggest that enhanced self-regulation could be a mechanism explaining why IAcc is associated with long-term stress.

Targeting sphingosine-1-phosphate lyase as an anabolic therapy for bone loss.

Sphingosine-1-phosphate (S1P) signaling influences bone metabolism, but its therapeutic potential in bone disorders has remained unexplored. We show that raising S1P levels in adult mice through conditionally deleting or pharmacologically inhibiting S1P lyase, the sole enzyme responsible for irreversibly degrading S1P, markedly increased bone formation, mass and strength and substantially decreased white adipose tissue. S1P signaling through S1P2 potently stimulated osteoblastogenesis at the expense of adipogenesis by inversely regulating osterix and PPAR-γ, and it simultaneously inhibited osteoclastogenesis by inducing osteoprotegerin through newly discovered p38-GSK3ß-ß-catenin and WNT5A-LRP5 pathways. Accordingly, S1P2-deficient mice were osteopenic and obese. In ovariectomy-induced osteopenia, S1P lyase inhibition was as effective as intermittent parathyroid hormone (iPTH) treatment in increasing bone mass and was superior to iPTH in enhancing bone strength. Furthermore, lyase inhibition in mice successfully corrected severe genetic osteoporosis caused by osteoprotegerin deficiency. Human data from 4,091 participants of the SHIP-Trend population-based study revealed a positive association between serum levels of S1P and bone formation markers, but not resorption markers. Furthermore, serum S1P levels were positively associated with serum calcium , negatively with PTH , and curvilinearly with body mass index. Bone stiffness, as determined through quantitative ultrasound, was inversely related to levels of both S1P and the bone formation marker PINP, suggesting that S1P stimulates osteoanabolic activity to counteract decreasing bone quality. S1P-based drugs should be considered as a promising therapeutic avenue for the treatment of osteoporotic diseases.

ADMIRE: analysis and visualization of differential methylation in genomic regions using the Infinium HumanMethylation450 Assay.

BACKGROUND: DNA methylation at cytosine nucleotides constitutes epigenetic gene regulation impacting cellular development and a wide range of diseases. Cytosine bases of the DNA are converted to 5-methylcytosine by the methyltransferase enzyme, acting as a reversible regulator of gene expression. Due to its outstanding importance in the epigenetic field, a number of lab techniques were developed to interrogate DNA methylation on a global range. Besides whole-genome bisulfite sequencing, the Infinium HumanMethylation450 Assay represents a versatile and cost-effective tool to investigate genome-wide changes of methylation patterns. RESULTS: Analysis of DNA Methylation In genomic REgions (ADMIRE) is an open source, semi-automatic analysis pipeline and visualization tool for Infinium HumanMethylation450 Assays with a special focus on ease of use. It features flexible experimental settings, quality control, automatic filtering, normalization, multiple testing, and differential analyses on arbitrary genomic regions. Publication-ready graphics, genome browser tracks, and table outputs include summary data and statistics, permitting instant comparison of methylation profiles between sample groups and the exploration of methylation patterns along the whole genome. ADMIREs statistical approach permits simultaneous large-scale analyses of hundreds of assays with little impact on algorithm runtimes. CONCLUSIONS: The web-based version of ADMIRE provides a simple interface to researchers with limited programming skills, whereas the offline version is suitable for integration into custom pipelines. ADMIRE may be used via our freely available web service at https://bioinformatics.mpi-bn.mpg.de without any limitations concerning the size of a project. An offline version for local execution is available from our website or GitHub (https://github.molgen.mpg.de/loosolab/admire).