High-Fat Diet: Bacteria Interactions Promote Intestinal Inflammation Which Precedes and Correlates with Obesity and Insulin Resistance in Mouse

Obesity induced by high fat (HF) diet is associated with inflammation which contributes to development of insulin resistance. Most prior studies have focused on adipose tissue as the source of obesity-associated inflammation. Increasing evidence links intestinal bacteria to development of diet-induced obesity (DIO). This study tested the hypothesis that HF western diet and gut bacteria interact to promote intestinal inflammation, which contributes to the progression of obesity and insulin resistance.Conventionally raised specific-pathogen free (CONV) and germ-free (GF) mice were given HF or low fat (LF) diet for 2-16 weeks. Body weight and adiposity were measured. Intestinal inflammation was assessed by evaluation of TNF-alpha mRNA and activation of a NF-kappaB(EGFP) reporter gene. In CONV but not GF mice, HF diet induced increases in body weight and adiposity. HF diet induced ileal TNF-alpha mRNA in CONV but not GF mice and this increase preceded obesity and strongly and significantly correlated with diet induced weight gain, adiposity, plasma insulin and glucose. In CONV mice HF diet also resulted in activation of NF-kappaB(EGFP) in epithelial cells, immune cells and endothelial cells of small intestine. Further experiments demonstrated that fecal slurries from CONV mice fed HF diet are sufficient to activate NF-kappaB(EGFP) in GF NF-kappaB(EGFP) mice.Bacteria and HF diet interact to promote proinflammatory changes in the small intestine, which precede weight gain and obesity and show strong and significant associations with progression of obesity and development of insulin resistance. To our knowledge, this is the first evidence that intestinal inflammation is an early consequence of HF diet which may contribute to obesity and associated insulin resistance. Interventions which limit intestinal inflammation induced by HF diet and bacteria may protect against obesity and insulin resistance

Capture and 3D culture of colonic crypts and colonoids in a microarray platform

Crypts are the basic structural and functional units of colonic epithelium and can be isolated from the colon and cultured in vitro into multi-cell spheroids termed “colonoids”. Both crypts and colonoids are ideal building blocks for construction of an in vitro tissue model of the colon. Here we proposed and tested a microengineered platform for capture and in vitro 3D culture of colonic crypts and colonoids. An integrated platform was fabricated from polydimethylsiloxane which contained two fluidic layers separated by an array of cylindrical microwells (150-μm diameter, 150-μm depth) with perforated bottoms (30-μm opening, 10-μm depth) termed “microstrainers”. As fluid moved through the array, crypts or colonoids were retained in the microstrainers with a >90% array-filling efficiency. Matrigel as an extracellular matrix was then applied to the microstrainers to generate isolated Matrigel pockets encapsulating the crypts or colonoids. After supplying the essential growth factors, epidermal growth factor, Wnt-3A, R-spondin 2 and noggin, 63±13% of the crypts and 77±8% of the colonoids cultured in the microstrainers over a 48–72 h period formed viable 3D colonoids. Thus colonoid growth on the array was similar to that under standard culture conditions (78±5%). Additionally the colonoids displayed the same morphology and similar numbers of stem and progenitor cells as those under standard culture conditions. Immunofluorescence staining confirmed that the differentiated cell-types of the colon, goblet cells, enteroendocrine cells and absorptive enterocytes, formed on the array. To demonstrating the utility of the array in tracking the colonoid fate, quantitative fluorescence analysis was performed on the arrayed colonoids exposed to reagents such as Wnt-3A and the γ-secretase inhibitor LY-411575. The successful formation of viable, multi-cell type colonic tissue on the microengineered platform represents a first step in the building of a “colon-on-a-chip” with the goal of producing the physiologic structure and organ-level function of the colon for controlled experiments

Distinct SOX9 levels differentially mark stem/progenitor populations and enteroendocrine cells of the small intestine epithelium

SOX transcription factors have the capacity to modulate stem/progenitor cell proliferation and differentiation in a dose-dependent manner. SOX9 is expressed in the small intestine epithelial stem cell zone. Therefore, we hypothesized that differential levels of SOX9 may exist, influencing proliferation and/or differentiation of the small intestine epithelium. Sox9 expression levels in the small intestine were investigated using a Sox9 enhanced green fluorescent protein (Sox9EGFP) transgenic mouse. Sox9EGFP levels correlate with endogenous SOX9 levels, which are expressed at two steady-state levels, termed Sox9EGFPLO and Sox9EGFPHI. Crypt-based columnar cells are Sox9EGFPLO and demonstrate enriched expression of the stem cell marker, Lgr5. Sox9EGFPHI cells express chromogranin A and substance P but do not express Ki67 and neurogenin3, indicating that Sox9EGFPHI cells are postmitotic enteroendocrine cells. Overexpression of SOX9 in a crypt cell line stopped proliferation and induced morphological changes. These data support a bimodal role for SOX9 in the intestinal epithelium, where low SOX9 expression supports proliferative capacity, and high SOX9 expression suppresses proliferation

CD24 and CD44 mark human intestinal epithelial cell populations with characteristics of active and facultative stem cells

Recent seminal studies have rapidly advanced the understanding of intestinal epithelial stem cell (IESC) biology in murine models. However, the lack of techniques suitable for isolation and subsequent downstream analysis of IESCs from human tissue has hindered the application of these findings toward the development of novel diagnostics and therapies with direct clinical relevance. This study demonstrates that the cluster of differentiation genes CD24 and CD44 are differentially expressed across LGR5 positive “active” stem cells as well as HOPX positive “facultative” stem cells. Fluorescence-activated cell sorting enables differential enrichment of LGR5 cells (CD24−/CD44+) and HOPX (CD24+/CD44+) cells for gene expression analysis and culture. These findings provide the fundamental methodology and basic cell surface signature necessary for isolating and studying intestinal stem cell populations in human physiology and disease

Origin of myofibroblasts in liver fibrosis

Most chronic liver diseases of all etiologies result in progressive liver fibrosis. Myofibroblasts produce the extracellular matrix, including type I collagen, which constitutes the fibrous scar in liver fibrosis. Normal liver has little type I collagen and no detectable myofibroblasts, but myofibroblasts appear early in experimental and clinical liver injury. The origin of the myofibroblast in liver fibrosis is still unresolved. The possibilities include activation of endogenous mesenchymal cells including fibroblasts and hepatic stellate cells, recruitment from the bone marrow, and transformation of epithelial or endothelial cells to myofibroblasts. In fact, the origin of myofibroblasts may be different for different types of chronic liver diseases, such as cholestatic liver disease or hepatotoxic liver disease. This review will examine our current understanding of the liver myofibroblast

Polymorphisms of the endothelial nitric oxide synthase (NOS3) gene in preeclampsia: a candidate-gene association study

<p>Abstract</p> <p>Background</p> <p>The endothelial nitric oxide synthase gene (<it>NOS3</it>) has been proposed as a candidate gene for preeclampsia. However, studies so far have produced conflicting results. This study examines the specific role of variants and haplotypes of the <it>NOS3 </it>gene in a population of Caucasian origin.</p> <p>Methods</p> <p>We examined the association of three common variants of the <it>NOS3 </it>gene (4b/a, T-786C and G894T) and their haplotypes in a case-control sample of 102 patients with preeclampsia and 176 women with a history of uncomplicated pregnancies. Genotyping for the <it>NOS3 </it>variants was performed and odds ratios and 95% confidence intervals were obtained to evaluate the association between <it>NOS3 </it>polymorphisms and preeclampsia.</p> <p>Results</p> <p>The single locus analysis for the three variants using various genetic models and a model-free approach revealed no significant association in relation to clinical status. The analysis of haplotypes also showed lack of significant association.</p> <p>Conclusions</p> <p>Given the limitations of the candidate-gene approach in investigating complex traits, the evidence of our study does not support the major contributory role of these common <it>NOS3 </it>variants in preeclampsia. Future larger studies may help in elucidating the genetics of preeclampsia further.</p

Vasodilator factors in the systemic and local adaptations to pregnancy

We postulate that an orchestrated network composed of various vasodilatory systems participates in the systemic and local hemodynamic adaptations in pregnancy. The temporal patterns of increase in the circulating and urinary levels of five vasodilator factors/systems, prostacyclin, nitric oxide, kallikrein, angiotensin-(1–7) and VEGF, in normal pregnant women and animals, as well as the changes observed in preeclamptic pregnancies support their functional role in maintaining normotension by opposing the vasoconstrictor systems. In addition, the expression of these vasodilators in the different trophoblastic subtypes in various species supports their role in the transformation of the uterine arteries. Moreover, their expression in the fetal endothelium and in the syncytiotrophoblast in humans, rats and guinea-pigs, favour their participation in maintaining the uteroplacental circulation. The findings that sustain the functional associations of the various vasodilators, and their participation by endocrine, paracrine and autocrine regulation of the systemic and local vasoactive changes of pregnancy are abundant and compelling. However, further elucidation of the role of the various players is hampered by methodological problems. Among these difficulties is the complexity of the interactions between the different factors, the likelihood that experimental alterations induced in one system may be compensated by the other players of the network, and the possibility that data obtained by manipulating single factors in vitro or in animal studies may be difficult to translate to the human. In addition, the impossibility of sampling the uteroplacental interface along normal pregnancy precludes obtaining longitudinal profiles of the various players. Nevertheless, the possibility of improving maternal blood pressure regulation, trophoblast invasion and uteroplacental flow by enhancing vasodilation (e.g. L-arginine, NO donors, VEGF transfection) deserves unravelling the intricate association of vasoactive factors and the systemic and local adaptations to pregnancy