Early-onset preeclampsia, plasma microRNAs, and endothelial cell function

Background: Preeclampsia is a hypertensive pregnancy disorder in which generalized systemic inflammation and maternal endothelial dysfunction are involved in the pathophysiology. MiRNAs are small noncoding RNAs responsible for post-transcriptional regulation of gene expression and involved in many physiological processes. They mainly downregulate translation of their target genes. Objective: We aimed to compare the plasma miRNA concentrations in preeclampsia, healthy pregnant women, and nonpregnant women. Furthermore, we aimed to evaluate the effect of 3 highly increased plasma miRNAs in preeclampsia on endothelial cell function in vitro. Study Design: We compared 3391 (precursor) miRNA concentrations in plasma samples from early-onset preeclamptic women, gestational age–matched healthy pregnant women, and nonpregnant women using miRNA 3.1. arrays (Affymetrix) and validated our findings by real-time quantitative polymerase chain reaction. Subsequently, endothelial cells (human umbilical vein endothelial cells) were transfected with microRNA mimics (we choose the 3 miRNAs with the greatest fold change and lowest false-discovery rate in preeclampsia vs healthy pregnancy). After transfection, functional assays were performed to evaluate whether overexpression of the microRNAs in endothelial cells affected endothelial cell function in vitro. Functional assays were the wound-healing assay (which measures cell migration and proliferation), the proliferation assay, and the tube-formation assay (which assesses formation of endothelial cell tubes during the angiogenic process). To determine whether the miRNAs are able to decrease gene expression of certain genes, RNA was isolated from transfected endothelial cells and gene expression (by measuring RNA expression) was evaluated by gene expression microarray (Genechip Human Gene 2.1 ST arrays; Life Technologies). For the microarray, we used pooled samples, but the differently expressed genes in the microarray were validated by real-time quantitative polymerase chain reaction in individual samples. Results: No significant differences (fold change 1.2 with a false-discovery rate <0.05) were found in miRNA plasma concentrations between healthy pregnant and nonpregnant women. The plasma concentrations of 26 (precursor) miRNAs were different between preeclampsia and healthy pregnancy. The 3 miRNAs that were increased with the greatest fold change and lowest false-discovery rate in preeclampsia vs healthy pregnancy were miR-574-5p, miR-1972, and miR-4793-3p. Transfection of endothelial cells with these miRNAs in showed that miR-574-5p decreased (P<.05) the wound-healing capacity (ie, decreased endothelial cell migration and/or proliferation) and tended (P<.1) to decrease proliferation, miR-1972 decreased tube formation (P<.05), and also tended (P<.1) to decrease proliferation, and miR-4793-3p tended (P<.1) to decrease both the wound-healing capacity and tube formation in vitro. Gene expression analysis of transfected endothelial cells revealed that miR-574-5p tended (P<.1) to decrease the expression of the proliferation marker MKI67. Conclusion: We conclude that in the early-onset preeclampsia group in our study different concentrations of plasma miRNAs are present as compared with healthy pregnancy. Our results suggest that miR-574-5p and miR-1972 decrease the proliferation (probably via decreasing MKI67) and/or migration as well as the tube-formation capacity of endothelial cells. Therefore, these miRNAs may be antiangiogenic factors affecting endothelial cells in preeclampsia

Age-associated Impairment of the Mucus Barrier Function is Associated with Profound Changes in Microbiota and Immunity

Aging significantly increases the vulnerability to gastrointestinal (GI) disorders but there are few studies investigating the key factors in aging that affect the GI tract. To address this knowledge gap, we used 10-week- and 19-month-old litter-mate mice to investigate microbiota and host gene expression changes in association with ageing. In aged mice the thickness of the colonic mucus layer was reduced about 6-fold relative to young mice, and more easily penetrable by luminal bacteria. This was linked to increased apoptosis of goblet cells in the upper part of the crypts. The barrier function of the small intestinal mucus was also compromised and the microbiota were frequently observed in contact with the villus epithelium. Antimicrobial Paneth cell factors Ang4 and lysozyme were expressed in significantly reduced amounts. These barrier defects were accompanied by major changes in the faecal microbiota and significantly decreased abundance of Akkermansia muciniphila which is strongly and negatively affected by old age in humans. Transcriptomics revealed age-associated decreases in the expression of immunity and other genes in intestinal mucosal tissue, including decreased T cell-specific transcripts and T cell signalling pathways. The physiological and immunological changes we observed in the intestine in old age, could have major consequences beyond the gut.</p

The muscle metabolome differs between healthy and frail older adults

Populations around the world are aging rapidly. Age-related loss of physiological functions negatively affects quality of life. A major contributor to the frailty syndrome of aging is loss of skeletal muscle. In this study we assessed the skeletal muscle biopsy metabolome of healthy young, healthy older and frail older subjects to determine the effect of age and frailty on the metabolic signature of skeletal muscle tissue. In addition, the effects of prolonged whole-body resistance-type exercise training on the muscle metabolome of older subjects were examined. The baseline metabolome was measured in muscle biopsies collected from 30 young, 66 healthy older subjects and 43 frail older subjects. Follow-up samples from frail older (24 samples) and healthy older subjects (38 samples) were collected after 6 months of prolonged resistance-type exercise training. Young subjects were included as a reference If thisgroup. Primary differences in skeletal muscle metabolite levels between young and healthy older subjects were related to mitochondrial function, muscle fiber type, and tissue turnover. Similar differences were observed when comparing frail older subjects with healthy older subjects at baseline. Prolonged resistance-type exercise training resulted in an adaptive response of amino acid metabolism, especially reflected in branched chain amino acids and genes related to tissue remodeling. The effect of exercise training on branched-chain amino acid-derived acylcarnitines in older subjects points to a downward shift in branched-chain amino acid catabolism upon training. We observed only modest correlations between muscle and plasma metabolite levels, which pleads against the use of plasma metabolites as a direct read-out of muscle metabolism and stresses the need for direct assessment of metabolites in muscle tissue biopsies

β2→ 1-fructans modulate the immune system in vivo by direct interaction with the mucosa in a microbiota-independent fashion

It has been shown in vitro that only specific dietary fibers contribute to immunity, but studies in vivo are not conclusive. Here, we investigated degree of polymerization (DP) dependent effects of beta2-->1-fructans on immunity via microbiota-dependent and -independent effects. To this end, conventional or germ-free mice received short- or long-chain beta2-->1-fructan for 5 days. Immune cell populations in the spleen, mesenteric lymph nodes (MLNs), and Peyer's patches (PPs) were analyzed with flow cytometry, genome-wide gene expression in the ileum was measured with microarray, and gut microbiota composition was analyzed with 16S rRNA sequencing of fecal samples. We found that beta2-->1-fructans modulated immunity by both microbiota and microbiota-independent effects. Moreover, effects were dependent on the chain-length of the beta2-->1-fructans type polymer. Both short- and long-chain beta2-->1-fructans enhanced T-helper 1 cells in PPs, whereas only short-chain beta2-->1-fructans increased regulatory T cells and CD11b-CD103- dendritic cells (DCs) in the MLN. A common feature after short- and long-chain beta2-->1-fructan treatment was enhanced 2-alpha-l-fucosyltransferase 2 expression and other IL-22-dependent genes in the ileum of conventional mice. These effects were not associated with shifts in gut microbiota composition, or altered production of short-chain fatty acids. Both short- and long-chain beta2-->1-fructans also induced immune effects in germ-free animals, demonstrating direct effect independent from the gut microbiota. Also, these effects were dependent on the chain-length of the beta2-->1-fructans. Short-chain beta2-->1-fructan induced lower CD80 expression by CD11b-CD103- DCs in PPs, whereas long-chain beta2-->1-fructan specifically modulated B cell responses in germ-free mice. In conclusion, support of immunity is determined by the chemical structure of beta2-->1-fructans and is partially microbiota independent

Loss of c-Jun N-terminal Kinase 1 and 2 Function in Liver Epithelial Cells Triggers Biliary Hyperproliferation Resembling Cholangiocarcinoma

Targeted inhibition of the c-Jun N-terminal kinases (JNKs) has shown therapeutic potential in intrahepatic cholangiocarcinoma (CCA)-related tumorigenesis. However, the cell-type-specific role and mechanisms triggered by JNK in liver parenchymal cells during CCA remain largely unknown. Here, we aimed to investigate the relevance of JNK1 and JNK2 function in hepatocytes in two different models of experimental carcinogenesis, the dethylnitrosamine (DEN) model and in nuclear factor kappa B essential modulator (NEMO)(hepatocyte-specific knockout (Deltahepa)) mice, focusing on liver damage, cell death, compensatory proliferation, fibrogenesis, and tumor development. Moreover, regulation of essential genes was assessed by reverse transcription polymerase chain reaction, immunoblottings, and immunostainings. Additionally, specific Jnk2 inhibition in hepatocytes of NEMO(Deltahepa)/JNK1(Deltahepa) mice was performed using small interfering (si) RNA (siJnk2) nanodelivery. Finally, active signaling pathways were blocked using specific inhibitors. Compound deletion of Jnk1 and Jnk2 in hepatocytes diminished hepatocellular carcinoma (HCC) in both the DEN model and in NEMO(Deltahepa) mice but in contrast caused massive proliferation of the biliary ducts. Indeed, Jnk1/2 deficiency in hepatocytes of NEMO(Deltahepa) (NEMO(Deltahepa)/JNK(Deltahepa)) animals caused elevated fibrosis, increased apoptosis, increased compensatory proliferation, and elevated inflammatory cytokines expression but reduced HCC. Furthermore, siJnk2 treatment in NEMO(Deltahepa)/JNK1(Deltahepa) mice recapitulated the phenotype of NEMO(Deltahepa)/JNK(Deltahepa) mice. Next, we sought to investigate the impact of molecular pathways in response to compound JNK deficiency in NEMO(Deltahepa) mice. We found that NEMO(Deltahepa)/JNK(Deltahepa) livers exhibited overexpression of the interleukin-6/signal transducer and activator of transcription 3 pathway in addition to epidermal growth factor receptor (EGFR)-rapidly accelerated fibrosarcoma (Raf)-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) cascade. The functional relevance was tested by administering lapatinib, which is a dual tyrosine kinase inhibitor of erythroblastic oncogene B-2 (ErbB2) and EGFR signaling, to NEMO(Deltahepa)/JNK(Deltahepa) mice. Lapatinib effectively inhibited cystogenesis, improved transaminases, and effectively blocked EGFR-Raf-MEK-ERK signaling. Conclusion: We define a novel function of JNK1/2 in cholangiocyte hyperproliferation. This opens new therapeutic avenues devised to inhibit pathways of cholangiocarcinogenesis

Lactobacillus plantarum Strains Can Enhance Human Mucosal and Systemic Immunity and Prevent Non-steroidal Anti-inflammatory Drug Induced Reduction in T Regulatory Cells

Orally ingested bacteria interact with intestinal mucosa and may impact immunity. However, insights in mechanisms involved are limited. In this randomized placebo-controlled cross-over trial, healthy human subjects were given Lactobacillus plantarum supplementation (strain TIFN101, CIP104448, or WCFS1) or placebo for 7 days. To determine whether L. plantarum can enhance immune response, we compared the effects of three stains on systemic and gut mucosal immunity, by among others assessing memory responses against tetanus toxoid (TT)-antigen, and mucosal gene transcription, in human volunteers during induction of mild immune stressor in the intestine, by giving a commonly used enteropathic drug, indomethacin [non-steroidal anti-inflammatory drug (NSAID)]. Systemic effects of the interventions were studies in peripheral blood samples. NSAID was found to induce a reduction in serum CD4+/Foxp3 regulatory cells, which was prevented by L. plantarum TIFN101. T-cell polarization experiments showed L. plantarum TIFN101 to enhance responses against TT-antigen, which indicates stimulation of memory responses by this strain. Cell extracts of the specific L. plantarum strains provoked responses after WCFS1 and TIFN101 consumption, indicating stimulation of immune responses against the specific bacteria. Mucosal immunomodulatory effects were studied in duodenal biopsies. In small intestinal mucosa, TIFN101 upregulated genes associated with maintenance of T- and B-cell function and antigen presentation. Furthermore, L. plantarum TIFN101 and WCFS1 downregulated immunological pathways involved in antigen presentation and shared downregulation of snoRNAs, which may suggest cellular destabilization, but may also be an indicator of tissue repair. Full sequencing of the L. plantarum strains revealed possible gene clusters that might be responsible for the differential biological effects of the bacteria on host immunity. In conclusion, the impact of oral consumption L. plantarum on host immunity is strain dependent and involves responses against bacterial cell components. Some strains may enhance specific responses against pathogens by enhancing antigen presentation and leukocyte maintenance in mucosa. In future studies and clinical settings, caution should be taken in selecting beneficial bacteria as closely related strains can have different effects. Our data show that specific bacterial strains can prevent immune stress induced by commonly consumed painkillers such as NSAID and can have enhancing beneficial effects on immunity of consumers by stimulating antigen presentation and memory responses

Subanalytic sets and feedback control

AbstractThe theory of subanalytic sets is used to prove: If a real analytic control system is completely controllable, then for every point p in the state space there exists a piecewise analytic feedback control that steers every state into p

Supplementation with Lactobacillus plantarum WCFS1 Prevents Decline of Mucus Barrier in Colon of Accelerated Aging Ercc1−/Δ7 Mice

textabstractAlthough it is clear that probiotics improve intestinal barrier function, little is known about the effects of probiotics on the aging intestine. We investigated effects of 10-week bacterial supplementation of Lactobacillus plantarum WCFS1, Lactobacillus casei BL23, or Bifidobacterium breve DSM20213 on gut barrier and immunity in 16-week-old accelerated aging Ercc1-/Δ7 mice, which have a median lifespan of ~20 weeks, and their wild-type littermates. The colonic barrier in Ercc1-/Δ7 mice was characterized by a thin (< 10 μm) mucus layer. L. plantarum prevented this decline in mucus integrity in Ercc1-/Δ7 mice, whereas B. breve exacerbated it. Bacterial supplementations affected the expression of immune-related genes, including Toll-like receptor 4. Regulatory T cell frequencies were increased in the mesenteric lymph nodes of L. plantarum- and L. casei-treated Ercc1-/Δ7 mice. L. plantarum- and L. casei-treated Ercc1-/Δ7 mice showed increased specific antibody production in a T cell-dependent immune response in vivo. By contrast, the effects of bacterial supplementation on wild-type control mice were negligible. Thus, supplementation with L. plantarum - but not with L. casei and B. breve - prevented the decline in the mucus barrier in Ercc1-/Δ7 mice. Our data indicate that age is an important factor influencing beneficial or detrimental effects of candidate probiotics. These findings also highlight the need for caution in translating beneficial effects of probiotics observed in young animals or humans to the elderly

Adipose Tissue Dysfunction Signals Progression of Hepatic Steatosis Towards Nonalcoholic Steatohepatitis in C57Bl/6 Mice

OBJECTIVE - Nonalcoholic fatty liver disease (NAFLD) is linked to obesity and diabetes, suggesting an important role of adipose tissue in the pathogenesis of NAFLD. Here, we aimed to investigate the interaction between adipose tissue and liver in NAFLD and identify potential early plasma markers that predict nonalcoholic steatohepatitis (NASH). RESEARCH DESIGN AND METHODS - C57Bl/6 mice were chronically fed a high-fat diet to induce NAFLD and compared with mice fed a low-fat diet. Extensive histological and phenotypical analyses coupled with a time course study of plasma proteins using multiplex assay were performed. RESULTS - Mice exhibited pronounced heterogeneity in liver histological scoring, leading to classification into four subgroups: low-fat low (LFL) responders displaying normal liver morphology, low-fat high (LFH) responders showing benign hepatic steatosis, high-fat low (HFL) responders displaying pre-NASH with macrovesicular lipid droplets, and high fat high (HFH) responders exhibiting overt NASH characterized by ballooning of hepatocytes, presence of Mallory bodies, and activated inflammatory cells. Compared with HFL responders, HFH mice gained weight more rapidly and exhibited adipose tissue dysfunction characterized by decreased final fat mass, enhanced macrophage infiltration and inflammation, and adipose tissue remodeling. Plasma haptoglobin, IL-1β, TIMP-1, adiponectin, and leptin were significantly changed in HFH mice. Multivariate analysis indicated that in addition to leptin, plasma CRP, haptoglobin, eotaxin, and MIP-1α early in the intervention were positively associated with liver triglycerides. Intermediate prognostic markers of liver triglycerides included IL-18, IL-1β, MIP-1γ, and MIP-2, whereas insulin, TIMP-1, granulocyte chemotactic protein 2, and myeloperoxidase emerged as late markers. CONCLUSIONS - Our data support the existence of a tight relationship between adipose tissue dysfunction and NASH pathogenesis and point to several novel potential predictive biomarkers for NASH