Sinusoidal Endothelial Dysfunction Precedes Inflammation and Fibrosis in a Model of NAFLD

Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome. Most morbidity associated with the metabolic syndrome is related to vascular complications, in which endothelial dysfunction is a major pathogenic factor. However, whether NAFLD is associated with endothelial dysfunction within the hepatic vasculature is unknown. The aims of this study were to explore, in a model of diet-induced overweight that expresses most features of the metabolic syndrome, whether early NAFLD is associated with liver endothelial dysfunction. Wistar Kyoto rats were fed a cafeteria diet (CafD; 65% of fat, mostly saturated) or a control diet (CD) for 1 month. CafD rats developed features of the metabolic syndrome (overweight, arterial hypertension, hypertryglyceridemia, hyperglucemia and insulin resistance) and liver steatosis without inflammation or fibrosis. CafD rats had a significantly higher in vivo hepatic vascular resistance than CD. In liver perfusion livers from CafD rats had an increased portal perfusion pressure and decreased endothelium-dependent vasodilation. This was associated with a decreased Akt-dependent eNOS phosphorylation and NOS activity. In summary, we demonstrate in a rat model of the metabolic syndrome that shows features of NAFLD, that liver endothelial dysfunction occurs before the development of fibrosis or inflammation.

Entamoeba lysyl-tRNA synthetase contains a cytokine-like fomain with chemokine activity towards human endothelial cells

Immunological pressure encountered by protozoan parasites drives the selection of strategies to modulate or avoid the immune responses of their hosts. Here we show that the parasite Entamoeba histolytica has evolved a chemokine that mimics the sequence, structure, and function of the human cytokine HsEMAPII (Homo sapiens endothelial monocyte activating polypeptide II). This Entamoeba EMAPII-like polypeptide (EELP) is translated as a domain attached to two different aminoacyl-tRNA synthetases (aaRS) that are overexpressed when parasites are exposed to inflammatory signals. EELP is dispensable for the tRNA aminoacylation activity of the enzymes that harbor it, and it is cleaved from them by Entamoeba proteases to generate a standalone cytokine. Isolated EELP acts as a chemoattractant for human cells, but its cell specificity is different from that of HsEMAPII. We show that cell specificity differences between HsEMAPII and EELP can be swapped by site directed mutagenesis of only two residues in the cytokines' signal sequence. Thus, Entamoeba has evolved a functional mimic of an aaRS-associated human cytokine with modified cell specificity.

Obstructive apneas induce early activation of mesenchymal stem cells and enhancement of endothelial wound healing

Background: The aim was to test the hypothesis that the blood serum of rats subjected to recurrent airway obstructions mimicking obstructive sleep apnea (OSA) induces early activation of bone marrow-derived mesenchymal stem cells (MSC) and enhancement of endothelial wound healing. Methods: We studied 30 control rats and 30 rats subjected to recurrent obstructive apneas (60 per hour, lasting 15 s each, for 5 h). The migration induced in MSC by apneic serum was measured by transwell assays. MSC-endothelial adhesion induced by apneic serum was assessed by incubating fluorescent-labelled MSC on monolayers of cultured endothelial cells from rat aorta. A wound healing assay was used to investigate the effect of apneic serum on endothelial repair. Results: Apneic serum showed significant increase in chemotaxis in MSC when compared with control serum: the normalized chemotaxis indices were 2.20 +- 0.58 (m +- SE) and 1.00 +- 0.26, respectively (p < 0.05). MSC adhesion to endothelial cells was greater (1.75 +- 0.14 -fold; p < 0.01) in apneic serum than in control serum. When compared with control serum, apneic serum significantly increased endothelial wound healing (2.01 +- 0.24 -fold; p < 0.05). Conclusions: The early increases induced by recurrent obstructive apneas in MSC migration, adhesion and endothelial repair suggest that these mechanisms play a role in the physiological response to the challenges associated to OSA.

Micropatterning of single endothelial cell shape reveals a tight coupling between nuclear volume in G1 and proliferation

Shape-dependent local differentials in cell proliferation are considered to be a major driving mechanism of structuring processes in vivo, such as embryogenesis, wound healing, and angiogenesis. However, the specific biophysical signaling by which changes in cell shape contribute to cell cycle regulation remains poorly understood. Here, we describe our study of the roles of nuclear volume and cytoskeletal mechanics in mediating shape control of proliferation in single endothelial cells. Micropatterned adhesive islands were used to independently control cell spreading and elongation. We show that, irrespective of elongation, nuclear volume and apparent chromatin decondensation of cells in G1 systematically increased with cell spreading and highly correlated with DNA synthesis (percent of cells in the S phase). In contrast, cell elongation dramatically affected the organization of the actin cytoskeleton, markedly reduced both cytoskeletal stiffness (measured dorsally with atomic force microscopy) and contractility (measured ventrally with traction microscopy), and increased mechanical anisotropy, without affecting either DNA synthesis or nuclear volume. Our results reveal that the nuclear volume in G1 is predictive of the proliferative status of single endothelial cells within a population, whereas cell stiffness and contractility are not. These findings show that the effects of cell mechanics in shape control of proliferation are far more complex than a linear or straightforward relationship. Our data are consistent with a mechanism by which spreading of cells in G1 partially enhances proliferation by inducing nuclear swelling and decreasing chromatin condensation, thereby rendering DNA more accessible to the replication machinery.

Growth factor- and cytokine-driven pathways governing liver stemness and differentiation.

Liver is unique in its capacity to regenerate in response to injury or tissue loss. Hepatocytes and other liver cells are able to proliferate and repopulate the liver. However, when this response is impaired, the contribution of hepatic progenitors becomes very relevant. Here, we present an update of recent studies on growth factors and cytokine-driven intracellular pathways that govern liver stem/progenitor cell expansion and differentiation, and the relevance of these signals in liver development, regeneration and carcinogenesis. Tyrosine kinase receptor signaling, in particular, c-Met, epidermal growth factor receptors or fibroblast growth factor receptors, contribute to proliferation, survival and differentiation of liver stem/progenitor cells. Different evidence suggests a dual role for the transforming growth factor (TGF)-β signaling pathway in liver stemness and differentiation. On the one hand, TGF-β mediates progression of differentiation from a progenitor stage, but on the other hand, it contributes to the expansion of liver stem cells. Hedgehog family ligands are necessary to promote hepatoblast proliferation but need to be shut off to permit subsequent hepatoblast differentiation. In the same line, the Wnt family and β-catenin/T-cell factor pathway is clearly involved in the maintenance of liver stemness phenotype, and its repression is necessary for liver differentiation during development. Collectively, data indicate that liver stem/progenitor cells follow their own rules and regulations. The same signals that are essential for their activation, expansion and differentiation are good candidates to contribute, under adequate conditions, to the paradigm of transformation from a pro-regenerative to a pro-tumorigenic role. From a clinical perspective, this is a fundamental issue for liver stem/progenitor cell-based therapies.

Propietats protectores de la lipoproteïna de alta densitat (HDL) sobre l'acció inflamatòria de la lipoproteïna de baixa densitat electronegativa (LDL(-)) en monòcits humans

Electronegative low-density lipoprotein (LDL(-)) is a modified fraction of LDL present in peripheral blood whose proportion is elevated in subjects with increased cardiovascular risk. LDL(-) has been shown to have an inflammatory effect on human endothelial cells and mononuclear blood cells. On the other hand, high-density lipoprotein (HDL) is known to have a protective effect against cardiovascular disease, partly mediated by its anti-inflammatory properties. The objective of the current work is to study the putative protective properties of HDL towards the inflammatory effect of LDL(-) in human monocytes, in order to elucidate the mechanisms behind their interaction. Total LDL and HDL were isolated by ultracentrifugation and LDL(-) was obtained from total LDL by anion exchange chromatography. HDL and LDL(-) were incubated together and then re-isolated, and their characteristics were compared to those of untreated lipoproteins. The inflammatory activity of the lipoproteins was determined by incubating monocytes with lipoproteins and measuring cytokine release from the cultured monocytes. The biochemical composition and electrophoretic mobility of the lipoproteins were also determined before and after their interaction. Incubation of HDL with LDL(-) reduced the inflammatory effect of LDL(-) and, in turn, HDL gained inflammatory properties. This indicates a transfer of inflammatory potential taking place during the interaction of LDL(-) and HDL. Additionally, LDL(-) lost non-esterified fatty acids (NEFAs) while HDL gained the same. We conclude that a transfer of NEFAs takes place between LDL(-) and HDL. These observations suggest that NEFAs play a role in the inflammatory effect mediated by LDL(-).

Vitamin E but not 17B-estradiol protect against vascular toxicity induced by B-amyloid wild type and the Dutch amyploid variant

Amyloid β-peptide (Aβ) fibril deposition on cerebral vessels produces cerebral amyloid angiopathy that appears in the majority of Alzheimer's disease patients. An early onset of a cerebral amyloid angiopathy variant called hereditary cerebral hemorrhage with amyloidosis of the Dutch type is caused by a point mutation in Aβ yielding AβGlu22→Gln. The present study addresses the effect of amyloid fibrils from both wild-type and mutated Aβ on vascular cells, as well as the putative protective role of antioxidants on amyloid angiopathy. For this purpose, we studied the cytotoxicity induced by Aβ1–40 Glu22→Gln and Aβ1–40 wild-type fibrils on human venule endothelial cells and rat aorta smooth muscle cells. We observed that AβGlu22→Gln fibrils are more toxic for vascular cells than the wild-type fibrils. We also evaluated the cytotoxicity of Aβ fibrils bound with acetylcholinesterase (AChE), a common component of amyloid deposits. Aβ1–40 wild-type–AChE fibrillar complexes, similar to neuronal cells, resulted in an increased toxicity on vascular cells. Previous reports showing that antioxidants are able to reduce the toxicity of Aβ fibrils on neuronal cells prompted us to test the effect of vitamin E, vitamin C, and 17β-estradiol on vascular damage induced by Aβwild-type and AβGlu22→Gln. Our data indicate that vitamin E attenuated significantly the Aβ-mediated cytotoxicity on vascular cells, although 17β-estradiol and vitamin C failed to inhibit the cytotoxicity induced by Aβ fibrils.

Anti-inflammatory effects of pancreatitis associated protein in inflammatory bowel disease

Background and aims: Increased pancreatitis associated protein (PAP) mRNA has been reported in active inflammatory bowel disease (IBD). The aims of the current study were to characterise PAP production in IBD and the effects of PAP on inflammation. Patients and methods: Serum PAP levels were determined in healthy controls (n¿=¿29), inflammatory controls (n¿=¿14), and IBD patients (n¿=¿171). Ex vivo PAP secretion in intestinal tissue was measured in 56 IBD patients and 13 healthy controls. Cellular origin of PAP was determined by immunohistochemistry. The effects of exogenous PAP on nuclear factor ¿B (NF¿B) activation, proinflammatory cytokine production, and endothelial adhesion molecule expression were also analysed ex vivo. Results: Patients with active IBD had increased serum PAP levels compared with controls, and these levels correlated with clinical and endoscopic disease severity. Ex vivo intestinal PAP synthesis was increased in active IBD and correlated with endoscopic and histological severity of inflammatory lesions. PAP localised to colonic Paneth cells. Incubation of mucosa from active Crohn¿s disease with PAP dose dependently reduced proinflammatory cytokines secretion. PAP prevented TNF-¿ induced NF¿B activation in monocytic, epithelial, and endothelial cells and reduced proinflammatory cytokine mRNA levels and adhesion molecule expression. Conclusions: PAP is synthesised by Paneth cells and is overexpressed in colonic tissue of active IBD. PAP inhibits NF¿B activation and downregulates cytokine production and adhesion molecule expression in inflamed tissue. It may represent an anti-inflammatory mechanism and new therapeutic strategy in IBD.

Identification of haptoglobin as an angiogenic factor in sera from patients with systemic vasculitis.

Angiogenesis is an important process in chronic inflammatory diseases. We observed that sera from patients with systemic vasculitis stimulated angiogenesis in an in vitro model using human umbilical vein endothelial cells cultured on a basement membrane (Matrigel) substrate. After 40% ammonium sulfate precipitation, angiogenic activity remained in the low molecular weight fraction and could be inactivated by heat. SDS-page of serum FPLC fractions exhibiting maximal angiogenic activity demonstrated two prominent species of 45 and 16-20 kD in patients' sera. These bands were much less apparent in sera obtained from control subjects. Amino-terminal sequencing of the 45-kD protein demonstrated that it was haptoglobin. Purified haptoglobin stimulated angiogenesis in a dose-dependent manner. The angiogenic activity of vasculitis patients' sera was partially inhibited by an antihaptoglobin antibody. Furthermore, serum haptoglobin levels in vasculitis patients correlated both with disease and angiogenic activity. Haptoglobin angiogenic activity was confirmed in two in vivo models using an implanted disc and a subcutaneous injection of basement membrane. Stimulation of angiogenesis is a newly recognized biological function of haptoglobin. The increased levels of haptoglobin found in chronic inflammatory conditions may play an important role in tissue repair. In systemic vasculitis, haptoglobin might also compensate for ischemia by promoting development of collateral vessels.

The embryonic blood-CSF barrier has molecular elements for specific glucose transport and for the general transport of molecules via transcellular routes.

In vertebrates, early brain development takes place at the expanded anterior end of the neural tube, which is filled with embryonic cerebrospinal fluid (E-CSF). We have recently identified a transient blood-CSF barrier that forms between embryonic days E3 and E4 in chick embryos and that is responsible for the transport of proteins and control of E-CSF homeostasis, including osmolarity. Here we examined the presence of glucose transporter GLUT-1 as well the presence of caveolae-structural protein Caveolin1 (CAV-1) in the embryonic blood-CSF barrier which may be involved in the transport of glucose and of proteins, water and ions respectively across the neuroectoderm. In this paper we demonstrate the presence of GLUT-1 and CAV-1 in endothelial cells of blood vessels as well as in adjacent neuroectodermal cells, located in the embryonic blood-CSF barrier. In blood vessels, these proteins were detected as early as E4 in chick embryos and E12.7 in rat embryos, i.e. the point at which the embryonic blood-CSF barrier acquires this function. In the neuroectoderm of the embryonic blood-CSF barrier, GLUT-1 was also detected at E4 and E12.7 respectively, and CAV-1 was detected shortly thereafter in both experimental models. These experiments contribute to delineating the extent to which the blood-CSF embryonic barrier controls E-CSF composition and homeostasis during early stages of brain development in avians and mammals. Our results suggest the regulation of glucose transport to the E-CSF by means of GLUT-1 and also suggest a mechanism by which proteins are transported via transcellular routes across the neuroectoderm, thus reinforcing the crucial role of E-CSF in brain development.