Characterization of porcine arterial endothelial cells cultured on amniotic membrane, a potential matrix for vascular tissue engineering

The existing of basement membrane improves the development of endothelium while constructing blood vessel equivalent. The amniotic membrane (AM) provides a natural basement membrane and has been used in ocular surface reconstruction. This study evaluated the molecular and cellular characteristics of porcine vascular endothelial cells (ECs) cultured on AM. ECs cultured on AM expressed the endothelial marker vWF and exhibited normal endothelial morphology. Here, we demonstrated that AM enhanced the expression of intercellular molecules, platelet-endothelial cell adhesion molecule-1 (PECAM-1), and adhesion molecule VE-cadherin at the intercellular junctions. The expression level of integrin was markedly higher in ECs cultured on AM than on plastic dish. Furthermore, the AM downregulated the expression of E-selectin and P-selectin in both LPS-activated and non-activated ECs. Consistently, adhesion of leukocytes to both activated and non-activated cells was decreased in ECs cultured on AM. Our results suggest that AM is an ideal matrix to develop a functional endothelium in blood vessel equivalent construction.     

GroEL1, from Chlamydia pneumoniae, Induces Vascular Adhesion Molecule 1 Expression by p37(AUF1) in Endothelial Cells and Hypercholesterolemic Rabbit

The expression of vascular adhesion molecule-1 (VCAM-1) by endothelial cells may play a major role in atherogenesis. The actual mechanisms of chlamydia pneumoniae (C. pneumoniae) relate to atherogenesis are unclear. We investigate the influence of VCAM-1 expression in the GroEL1 from C. pneumoniae-administered human coronary artery endothelial cells (HCAECs) and hypercholesterolemic rabbits. In this study, we constructed the recombinant GroEL1 from C. pneumoniae. The HCAECs/THP-1 adhesion assay, tube formation assay, western blotting, enzyme-linked immunosorbent assay, actinomycin D chase experiment, luciferase reporter assay, and immunohistochemical stainings were performed. The results show that GroEL1 increased both VCAM-1expression and THP-1 cell adhesives, and impaired tube-formation capacity in the HCAECs. GroEL1 significantly increased the VCAM-1 mRNA stability and cytosolic AU-binding factor 1 (AUF1) level. Overexpression of the p37(AUF1) significantly increased VCAM-1 gene expression in GroEL1-induced bovine aortic endothelial cells (BAECs). GroEL1 prolonged the stability of VCAM-1 mRNA by increasing both p37(AUF1) and the regulation of the 5' untranslated region (UTR) of the VCAM-1 mRNA in BAECs. In hypercholesterolemic rabbits, GroEL1 administration enhanced fatty-streak and macrophage infiltration in atherosclerotic lesions, which may be mediated by elevated VCAM-1 expression. In conclusion, GroEL1 induces VCAM-1 expression by p37(AUF1) in endothelial cells and enhances atherogenesis in hypercholesterolemic rabbits.     

Synergistic contribution of CD14 and HLA loci in the susceptibility to Buerger disease

Buerger disease (BD) is an occulusive vascular disease of unknown etiology. Although cigarette smoking is a well-known risk factor of BD, genetic factors may also play a role in the etiology. Because chronic bacterial infection such as oral periodontitis is suggested to be involved in the pathogenesis of BD, gene polymorphisms involved in the infectious immunity might be associated with BD as the genetic factor(s). We have previously reported that HLA-DRB1*1501 and B54 was associated with BD in Japanese. In this study, polymorphisms in HLA-DPB1, DRB1 and B were analyzed in 131 Japanese BD patients and 227 healthy controls. In addition, we investigated a functional promoter polymorphism, −260 C > T, of CD14 that is a main receptor of bacterial lipopolysaccharide. It was found that the frequencies of CD14 TT genotype [37.4 vs. 24.2%, P = 0.008 OR = 1.87, 95% confidence interval (CI); 1.18, 2.97], DRB1*1501 (34.4 vs. 13.2%, P _c = 4.4 × 10⁻⁵, OR = 3.44, 95%CI; 2.06, 5.73) and DPB1*0501 (79.4 vs. 55.1%, P _c = 4.7 × 10⁻⁵, OR = 3.14, 95%CI; 1.93, 5.11) were significantly higher in the patients than in the controls, demonstrating that at least three genetic markers were associated with BD. Stratification analyses of these associated markers suggested synergistic roles of the genetic factors. Odds ratios ranged from 4.72 to 12.57 in individuals carrying any two of these three markers. These findings suggested that the susceptibility to BD was in part controlled by genes involved in the innate and adaptive immunity.     

Analysis of human skeletal muscle after 48 h immobilization reveals alterations in mRNA and protein for extracellular matrix components.

We examined the effects of 48 h of knee immobilization on alterations in mRNA and protein in human skeletal muscle. We hypothesized that 48 h of immobilization would increase gene expression and respective protein products for ubiquitin-proteasome pathway (UPP) components. Also, we used microarray analysis to identify novel pathways. Biopsies were taken from the vastus muscle of five men (20.4 +/- 0.5 yr) before and after 48-h immobilization. Global changes in gene expression were analyzed by use of Affymetrix GeneChips. Candidate genes were confirmed via quantitative RT-PCR. Western blotting (WB) was used to quantify protein products of candidate genes and to assess Akt pathway activation. Immunohistochemistry was used to localize proteins found to be altered when assessed via WB. The greatest percentage of genes showing altered expression with the GeneChip included genes involved in the UPP, metallothionein function, and extracellular matrix (ECM) integrity. Quantitative RT-PCR analysis confirmed increases in mRNA for UPP components [USP-6, small ubiquitin-related modifier (SUMO-1)] and the metallothioneins (MT2A, MT1F, MT1H, MT1X) and decreases in mRNA content for matrix metalloproteinases (MMP-28, TIMP-1) and ECM structural components [collagen III (COLIII) and IV (COLIV)]. Only phosphorylated Akt (Ser473, Thr308), COLIII and COLIV protein levels were significantly different postimmobilization (25, 10, 88, and 28% decrease, respectively). Immunohistochemistry confirmed WB showing decreased staining for collagens postimmobilization. Our results suggest that 48 h of immobilization increases mRNA content for components of the UPP and metallothionein function while decreasing mRNA and protein for ECM components as well as decreased phosphorylation of Akt.     

Correlation between body composition and risk factors for cardiovascular disease and metabolic syndrome

Body mass index (BMI) is an important diagnostic tool for determining obesity; however, while BMI reflects the influence of body height over body weight, it does not reveal body fat percentage (BF%). We explored whether BF% correlated with risk factors for cardiovascular disease and metabolic syndrome and whether metabolically obese, normal weight people were at risk for these diseases. A total of 2,867 healthy volunteers participated in this study. Blood pressure, height, weight, waist circumference, BMI, BF%, lipid profile, fasting glucose, uric acid, and lifestyle factors were collected from healthy subjects during their annual health examinations. In both males and females, BF% correlated positively with BMI and waist circumference. Participants were divided into three groups according to BF% and data were compared between groups. The results suggest that BF% correlates with risk factors for cardiovascular disease and metabolic syndrome for both men and women, and that BF% may be a useful predictor of risk, particularly in metabolically obese, normal weight individuals. ? 2012 International Union of Biochemistry and Molecular Biology, Inc.     

Development and fibronectin signaling requirements of the zebrafish interrenal vessel

Background

The early morphogenetic steps of zebrafish interrenal tissue, the teleostean counterpart of the mammalian adrenal gland, are modulated by the peri-interrenal angioblasts and blood vessels. While an organized distribution of intra-adrenal vessels and extracellular matrix is essential for the fetal adrenal cortex remodeling, whether and how an intra-interrenal buildup of vasculature and extracellular matrix forms and functions during interrenal organogenesis in teleosts remains unclear.

Methodology and Principal Findings

We characterized the process of interrenal gland vascularization by identifying the interrenal vessel (IRV); which develops from the axial artery through angiogenesis and is associated with highly enriched Fibronectin (Fn) accumulation at its microenvironment. The loss of Fn1 by either antisense morpholino (MO) knockdown or genetic mutation inhibited endothelial invasion and migration of the steroidogenic tissue. The accumulation of peri-IRV Fn requires Integrin α5 (Itga5), with its knockdown leading to interrenal and IRV morphologies phenocopying those in the fn1 morphant and mutant. fn1b, another known fn gene in zebrafish, is however not involved in the IRV formation. The distribution pattern of peri-IRV Fn could be modulated by the blood flow, while a lack of which altered angiogenic direction of the IRV as well as its ability to integrate with the steroidogenic tissue. The administration of Fn antagonist through microangiography exerted reducing effects on both interrenal vessel angiogenesis and steroidogenic cell migration.

Conclusions and Significance

This work is the first to identify the zebrafish IRV and to characterize how its integration into the developing interrenal gland requires the Fn-enriched microenvironment, which leads to the possibility of using the IRV formation as a platform for exploring organ-specific angiogenesis. In the context of other developmental endocrinology studies, our results indicate a highly dynamic interrenal-vessel interaction immediately before the onset of stress response in the zebrafish embryo.     

GABAB-receptor-mediated suppression of sympathetic outflow from the spinal cord of neonatal rats.

Using a splanchnic nerve-spinal cord preparation in vitro that could spontaneously generate sympathetic nerve discharge (SND), we investigated the roles of intraspinal GABA(B) receptors in the regulation of SND. Despite an age-dependent difference in sensitivity, bath applications of baclofen (Bac; GABA(B)-receptor agonist) consistently reduced SND in a concentration-dependent manner. The drug specificity of Bac in activation of GABA(B) receptors was verified by application of its antagonist saclofen (Sac) or CGP-46381 (CGP). Sac or CGP alone did not change SND. However, in the presence of Sac or CGP, the effects of Bac on SND inhibition were reversibly attenuated. The splanchnic sympathetic preganglionic neuron (SPN) was recorded by blind whole cell, patch-clamp techniques. We examined Bac effects on electrical membrane properties of SPNs. Applications of Bac reduced excitatory synaptic events, induced membrane hyperpolarizations, and inhibited SPN firing. In the presence of 12 mM Mg2+ or 0.5 microM TTX to block Ca2+- or action potential-dependent synaptic transmissions, applications of Bac induced an outward baseline current that reversed at -29 +/- 6 mV. Because the K+ equilibrium potential in our experimental conditions was -100 mV, the Bac-induced currents could not simply be attributed to an alteration of K+ conductance. On the other hand, applications of Bac to Cs+-loaded SPNs reduced Cd2+-sensitive and high-voltage-activated inward currents, indicating an inhibition of voltage-gated Ca2+ currents. Our results suggest that the activation of intraspinal GABA(B) receptors suppresses SND via a mixture of ion events that may link to a change in Ca2+ conductance.     

An overview of epigenetics and chemoprevention

It is now appreciated that both genetic alteration, e.g. mutations, and aberrant epigenetic changes, e.g. DNA methylation, cause cancer. Epigenetic dysregulation is potentially reversible which makes it attractive as targets for cancer prevention. Synthetic drugs targeting enzymes, e.g. DNA methyltransferase and histone deacetylase, that regulate epigenetic patterns are active in clinical settings. In addition, dietary factors have been suggested to have potential to reverse aberrant epigenetic patterns. Uncovering the human epigenome can lead us to better understand the dynamics of DNA methylation in disease progression which can further assist in cancer prevention.