Extracellular matrix components indicate remodelling activity in different fibrosis stages of human non-alcoholic fatty liver disease

AIMS: The composition of several important extracellular matrix components (ECM) has not yet been elucidated in human non-alcoholic fatty liver disease (NAFLD). We aim to investigate the proportion of hepatic stellate cells (HSCs) and activity of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) in human NAFLD liver tissue with respect to severity of inflammation and fibrosis. METHODS AND RESULTS: Histopathological features were quantified by NAFLD activity score and grading assignment. The collagen proportionate area (CPA) was measured. Slides were stained with alpha-smooth muscle actin (alpha-SMA), as a marker of activated HSCs, and alpha-SMA was quantified digitally. Zymography was performed to measure the proteolytic activity of MMP-2 and MMP-9. TIMP-1 and TIMP-2 protein concentration was measured with enzyme-linked immunosorbent assay (ELISA). alpha-SMA was higher in severe fibrosis (6.3%, interquartile range 2.9-13.1) than mild and no fibrosis (median 1.1 and 0.9%, P < 0.001) and correlated strongly with CPA (Rs = 0.870, P < 0.001). ProMMP-2 activity in severe (4.1%, IQR 2.6-16.2) and mild fibrosis (2.7%, IQR 1.9-3.9) was higher than in no fibrosis (1.5%, (IQR 0.95-2.1); P = 0.001 and P = 0.046) and showed a moderate positive correlation with CPA (Rs = 0.495, P = 0.001). TIMP-1 and TIMP-2 were significantly higher in severe fibrosis than mild or no fibrosis. Both showed moderate correlation with CPA (TIMP-1: Rs = 0.471, P = 0.002 and TIMP-2: Rs = 0.325, P = 0.036). MMP-9 correlated as the only ECM component to inflammation severity. CONCLUSIONS: Advanced human NAFLD-fibrosis has a distinct ECM composition with increased HSCs and increased TIMP inhibition, but there is also ongoing remodelling activity of MMP-2

Diabetes Causes Bone Marrow Autonomic Neuropathy and Impairs Stem Cell Mobilization via Dysregulated p66Shc and Sirt1

Diabetes compromises the bone marrow (BM) microenvironment and reduces circulating CD34 + cells. Diabetic autonomic neuropathy (DAN) may impact the BM, because the sympathetic nervous system (SNS) is prominently involved in BM stem cell trafficking. We hypothesize that neuropathy of the BM affects stem cell mobilization and vascular recovery after ischemia in diabetes. We report that, in patients, cardiovascular DAN was associated with fewer circulating CD34 + cells. Experimental diabetes (STZ and Ob/Ob ) or chemical sympathectomy in mice resulted in BM autonomic neuropathy, impaired Lin - cKit + Sca1 + (LKS) cell and endothelial progenitor cells (EPC, CD34 + Flk1 + ) mobilization and vascular recovery after ischemia. DAN increased expression of p66Shc and reduced expression of Sirt1 in mice and humans. p66Shc KO in diabetic mice prevented DAN in the BM, and rescued defective LKS cell and EPC mobilization. Hematopoietic Sirt1 KO mimicked the diabetic mobilization defect, while hematopoietic Sirt1 overexpression in diabetes rescued defective mobilization and vascular repair. Through p66Shc and Sirt1 , diabetes and sympathectomy elevated the expression of various adhesion molecules, including CD62L . CD62L KO partially rescued the defective stem/progenitor cell mobilization. In conclusion, autonomic neuropathy in the BM impairs stem cell mobilization in diabetes with dysregulation of the lifespan regulators p66Shc and Sirt1

Reduced ratio of protective versus proinflammatory cytokine responses to commensal bacteria in HLA-B27 transgenic rats

Germ-free HLA-B27 transgenic (TG) rats do not develop colitis, but colonization with specific pathogen-free (SPF) bacteria induces colitis accompanied by immune activation. To study host-dependent immune responses to commensal caecal bacteria we investigated cytokine profiles in mesenteric lymph node (MLN) cells from HLA-B27 TG versus nontransgenic (non-TG) littermates after in vitro stimulation with caecal bacterial lysates (CBL). Supernatants from CBL-stimulated unseparated T- or B- cell-depleted MLN cells from HLA-B27 TG and non-TG littermates were analysed for IFN-γ, IL-12, TNF, IL-10 and TGF-β production. Our results show that unfractionated TG MLN cells stimulated with CBL produced more IFN-γ, IL-12 and TNF than did non-TG MLN cells. In contrast, CBL-stimulated non-TG MLN cells produced more IL-10 and TGF-β. T cell depletion abolished IFN-γ and decreased IL-12 production, but did not affect IL-10 and TGF-β production. Conversely, neither IL-10 nor TGF-β was produced in cultures of B cell-depleted MLN. In addition, CD4+ T cells enriched from MLN of HLA-B27 TG but not from non-TG rats produced IFN-γ when cocultured with CBL-pulsed antigen presenting cells from non-TG rats. Interestingly, IL-10 and TGF-β, but not IFN-γ, IL-12 and TNF were produced by MLN cells from germ-free TG rats. These results indicate that the colitis that develops in SPF HLA-B27 TG rats is accompanied by activation of IFN-γ-producing CD4+ T cells that respond to commensal bacteria. However, B cell cytokine production in response to components of commensal intestinal microorganisms occurs in the absence of intestinal inflammation

Reevaluation of the role of VEGF-B suggests a restricted role in the revascularization of the ischemic myocardium .

peer reviewedOBJECTIVE: The endogenous role of the VEGF family member vascular endothelial growth factor-B (VEGF-B) in pathological angiogenesis remains unclear. METHODS AND RESULTS: We studied the role of VEGF-B in various models of pathological angiogenesis using mice lacking VEGF-B (VEGF-B(-/-)) or overexpressing VEGF-B(167). After occlusion of the left coronary artery, VEGF-B deficiency impaired vessel growth in the ischemic myocardium whereas, in wild-type mice, VEGF-B(167) overexpression enhanced revascularization of the infarct and ischemic border zone. By contrast, VEGF-B deficiency did not affect vessel growth in the wounded skin, hypoxic lung, ischemic retina, or ischemic limb. Moreover, VEGF-B(167) overexpression failed to enhance vascular growth in the skin or ischemic limb. CONCLUSIONS: VEGF-B appears to have a relatively restricted angiogenic activity in the ischemic heart. These insights might offer novel therapeutic opportunitie

MicroRNAs targeting oncogenes are down-regulated in pancreatic malignant transformation from benign tumors

BACKGROUND MicroRNA (miRNA) expression profiles have been described in pancreatic ductal adenocarcinoma (PDAC), but these have not been compared with pre-malignant pancreatic tumors. We wished to compare the miRNA expression signatures in pancreatic benign cystic tumors (BCT) of low and high malignant potential with PDAC, in order to identify miRNAs deregulated during PDAC development. The mechanistic consequences of miRNA dysregulation were further evaluated. METHODS Tissue samples were obtained at a tertiary pancreatic unit from individuals with BCT and PDAC. MiRNA profiling was performed using a custom microarray and results were validated using RT-qPCR prior to evaluation of miRNA targets. RESULTS Widespread miRNA down-regulation was observed in PDAC compared to low malignant potential BCT. We show that amongst those miRNAs down-regulated, miR-16, miR-126 and let-7d regulate known PDAC oncogenes (targeting BCL2, CRK and KRAS respectively). Notably, miR-126 also directly targets the KRAS transcript at a "seedless" binding site within its 3'UTR. In clinical specimens, miR-126 was strongly down-regulated in PDAC tissues, with an associated elevation in KRAS and CRK proteins. Furthermore, miR-21, a known oncogenic miRNA in pancreatic and other cancers, was not elevated in PDAC compared to serous microcystic adenoma (SMCA), but in both groups it was up-regulated compared to normal pancreas, implicating early up-regulation during malignant change. CONCLUSIONS Expression profiling revealed 21 miRNAs down-regulated in PDAC compared to SMCA, the most benign lesion that rarely progresses to invasive carcinoma. It appears that miR-21 up-regulation is an early event in the transformation from normal pancreatic tissue. MiRNA expression has the potential to distinguish PDAC from normal pancreas and BCT. Mechanistically the down-regulation of miR-16, miR-126 and let-7d promotes PDAC transformation by post-transcriptional up-regulation of crucial PDAC oncogenes. We show that miR-126 is able to directly target KRAS; re-expression has the potential as a therapeutic strategy against PDAC and other KRAS-driven cancers

Hypoxia induces dilated cardiomyopathy in the chick embryo: mechanism, intervention, and long-term consequences

Background: Intrauterine growth restriction is associated with an increased future risk for developing cardiovascular diseases. Hypoxia in utero is a common clinical cause of fetal growth restriction. We have previously shown that chronic hypoxia alters cardiovascular development in chick embryos. The aim of this study was to further characterize cardiac disease in hypoxic chick embryos. Methods: Chick embryos were exposed to hypoxia and cardiac structure was examined by histological methods one day prior to hatching (E20) and at adulthood. Cardiac function was assessed in vivo by echocardiography and ex vivo by contractility measurements in isolated heart muscle bundles and isolated cardiomyocytes. Chick embryos were exposed to vascular endothelial growth factor (VEGF) and its scavenger soluble VEGF receptor-1 (sFlt-1) to investigate the potential role of this hypoxia-regulated cytokine. Principal Findings: Growth restricted hypoxic chick embryos showed cardiomyopathy as evidenced by left ventricular (LV) dilatation, reduced ventricular wall mass and increased apoptosis. Hypoxic hearts displayed pump dysfunction with decreased LV ejection fractions, accompanied by signs of diastolic dysfunction. Cardiomyopathy caused by hypoxia persisted into adulthood. Hypoxic embryonic hearts showed increases in VEGF expression. Systemic administration of rhVEGF165 to normoxic chick embryos resulted in LV dilatation and a dose-dependent loss of LV wall mass. Lowering VEGF levels in hypoxic embryonic chick hearts by systemic administration of sFlt-1 yielded an almost complete normalization of the phenotype. Conclusions/Significance: Our data show that hypoxia causes a decreased cardiac performance and cardiomyopathy in chick embryos, involving a significant VEGF-mediated component. This cardiomyopathy persists into adulthood

Distinct Characteristics of Circulating Vascular Endothelial Growth Factor-A and C Levels in Human Subjects

The mechanisms that lead from obesity to atherosclerotic disease are not fully understood. Obesity involves angiogenesis in which vascular endothelial growth factor-A (VEGF-A) plays a key role. On the other hand, vascular endothelial growth factor-C (VEGF-C) plays a pivotal role in lymphangiogenesis. Circulating levels of VEGF-A and VEGF-C are elevated in sera from obese subjects. However, relationships of VEGF-C with atherosclerotic risk factors and atherosclerosis are unknown. We determined circulating levels of VEGF-A and VEGF-C in 423 consecutive subjects not receiving any drugs at the Health Evaluation Center. After adjusting for age and gender, VEGF-A levels were significantly and more strongly correlated with the body mass index (BMI) and waist circumference than VEGF-C. Conversely, VEGF-C levels were significantly and more closely correlated with metabolic (e.g., fasting plasma glucose, hemoglobin A1c, immunoreactive insulin, and the homeostasis model assessment of insulin resistance) and lipid parameters (e.g., triglycerides, total cholesterol (TC), low-density-lipoprotein cholesterol (LDL-C), and non-high-density-lipoprotein cholesterol (non-HDL-C)) than VEGF-A. Stepwise regression analyses revealed that independent determinants of VEGF-A were the BMI and age, whereas strong independent determinants of VEGF-C were age, triglycerides, and non-HDL-C. In apolipoprotein E-deficient mice fed a high-fat-diet (HFD) or normal chow (NC) for 16 weeks, levels of VEGF-A were not significantly different between the two groups. However, levels of VEGF-C were significantly higher in HFD mice with advanced atherosclerosis and marked hypercholesterolemia than NC mice. Furthermore, immunohistochemistry revealed that the expression of VEGF-C in atheromatous plaque of the aortic sinus was significantly intensified by feeding HFD compared to NC, while that of VEGF-A was not. In conclusion, these findings demonstrate that VEGF-C, rather than VEGF-A, is closely related to dyslipidemia and atherosclerosis

A 50% Reduction of Excitability but Not of Intercellular Coupling Affects Conduction Velocity Restitution and Activation Delay in the Mouse Heart

Computer simulations suggest that intercellular coupling is more robust than membrane excitability with regard to changes in and safety of conduction. Clinical studies indicate that SCN5A (excitability) and/or Connexin43 (Cx43, intercellular coupling) expression in heart disease is reduced by approximately 50%. In this retrospective study we assessed the effect of reduced membrane excitability or intercellular coupling on conduction in mouse models of reduced excitability or intercellular coupling. Epicardial activation mapping of LV and RV was performed on Langendorff-perfused mouse hearts having the following: 1) Reduced excitability: Scn5a haploinsufficient mice; and 2) reduced intercellular coupling: Cx43(CreER(T)/fl) mice, uninduced (50% Cx43) or induced (10% Cx43) with Tamoxifen. Wild type (WT) littermates were used as control. Conduction velocity (CV) restitution and activation delay were determined longitudinal and transversal to fiber direction during S(1)S(1) pacing and S(1)S(2) premature stimulation until the effective refractory period. In both animal models, CV restitution and activation delay in LV were not changed compared to WT. In contrast, CV restitution decreased and activation delay increased in RV during conduction longitudinal but not transverse to fiber direction in Scn5a heterozygous animals compared to WT. In contrast, a 50% reduction of intercellular coupling did not affect either CV restitution or activation delay. A decrease of 90% Cx43, however, resulted in decreased CV restitution and increased activation delay in RV, but not LV. Reducing excitability but not intercellular coupling by 50% affects CV restitution and activation delay in RV, indicating a higher safety factor for intercellular coupling than excitability in R

The clinical spectrum of Fontan-associated liver disease: results from a prospective multimodality screening cohort

AIMS: Liver fibrosis and cirrhosis are a consequence of a Fontan physiology, and determine prognosis. It is unclear whether non-invasive assessment of liver pathology is helpful to provide clinically relevant information. The aims of this study were to assess the spectrum of Fontan-associated liver disease (FALD) and usefulness of non-invasive methods to assess biopsy confirmed liver fibrosis. METHODS AND RESULTS: Hepatic screening of consecutive patients consisted of a blood panel, ultrasonography, elastography, contrast-enhanced magnetic resonance imaging (MRI)/computed tomography (CT) scan, and liver biopsy (scored with Fontan specific fibrosis scores and collagen proportionate area; CPA). Fibrosis parameters, varices, ascites, and splenomegaly were measured on imaging. Thirty-eight of 49 referred patients (27 +/- 6.6 years, 73.7% male) underwent the complete screening protocol. Liver fibrosis on biopsy was present in all patients, and classified as severe (Stages 3-4) in 68%. Median CPA was 22.5% (16.9-29.5) and correlated with individual fibrosis scores. ELF(R) and liver stiffness were elevated, but MELD-XI scores were low in all patients. Fibrosis severity neither correlated to ELF(R) and liver stiffness, nor to (semi-) quantitative fibrosis parameters on MRI/CT. Varices were present in 50% and hyperenhancing nodules in 25% of patients, both independent of fibrosis stage, but varices were associated with higher CPA values. CONCLUSION: The FALD spectrum includes both hepatic congestion and severe fibrosis, with signs of portal hypertension and hyperenhancing nodules as significant manifestations. Routine imaging, transient elastography, and serum biomarkers are unable to accurately assess severity of liver fibrosis in this cohort. Future research should focus on validating new diagnostic tools with biopsy as the reference standard