Glycation does not alter LDL-induced secretion of tissue plasminogen activator and plasminogen activator inhibitor-1 from human aortic endothelial cells

Diabetes may induce both quantitative and qualitative changes in lipoproteins, especially low-density lipoprotein (LDL). Effects of LDL glycation on endothelial cell secretion of tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) have not been fully elucidated. Human aortic endothelial cell (HAEC) tPA and PAI-1 production were determined after incubation with LDL (50 to 500 microg/mL protein, 24 h) from three sources: (1) nondiabetic LDL (N-LDL) modified in vitro to form six preparations: native, nonmodified (N); glycated (G); minimally oxidized (MO); minimally oxidized and glycated (MOG); heavily oxidized (HO); and heavily oxidized and glycated (HOG); (2) in vivo glycated and relatively nonglycated LDL subfractions from type 1 diabetic patients; (3) LDL from type 1 diabetic patients and matched controls, which was subfractionated using density gradient ultracentrifugation. In experiments using LDL modified in vitro, the rate of tPA release by HAECs incubated with N-LDL (83 +/- 4 ng/mg cell protein/24 h) did not differ significantly from those incubated with G-LDL (73 +/- 7), MO-LDL (74 +/- 13), or MOG-LDL (66 +/- 15) and was not influenced by LDL concentration. The rate of PAI-1 release was similar in HAECs incubated with N-LDL (5.7 +/- 0.6 mug/mg cell protein/24 h), G-LDL (5.7 +/- 0.7), MO-LDL (5.5 +/- 0.8), or MOG-LDL (5.7 +/- 0.9) and was not influenced by LDL concentration. In contrast, tPA release was significantly decreased in cells incubated with LDL (10 microg/mL) modified extensively by oxidation, and averaged 45.2 +/- 5.0 and 43.7 +/- 9.9 ng/mg/24 h for HO-LDL and HOG-LDL, respectively, and was further decreased with increasing concentrations of the heavily oxidized LDL preparations. PAI-1 release was not significantly decreased relative to N-LDL in cells incubated with low concentrations (5 to 50 microg/mL) of HO-LDL and HOG-LDL, but was decreased to 3.2 +/- 0.5 and 3.1 +/- 0.7 microg/mg/24 h for HO-LDL and HOG-LDL at 200 microg/mL, respectively. Results using in vivo glycated versus nonglycated LDL showed that tPA and PAI-1 release did not differ between subfractions. Release of tPA averaged 5.11 +/- 0.6 and 5.12 +/- 0.7 ng/mg/24 h, whereas release of PAI-1 averaged 666 +/- 27 ng/mg/24 h and 705 +/- 30 ng/mg/24 h for nonglycated and glycated LDL subfractions, respectively. Using LDL of different density subclasses, tPA and PAI-1 release in response to LDL from diabetic patients compared with control subjects did not differ when HAECs were incubated with LDLs of increasing density isolated from each subject pair. We conclude that oxidation of LDL, but not glycation, may contribute to the altered fibrinolysis observed in diabetes.

Misonidazole binding in murine liver tissue: a marker for cellular hypoxia in vivo

The hepatic microcirculation is believed to cause variable cellular oxygenation within the organ. In this study a marker of cellular hypoxia was used to demonstrate liver oxygen tension gradients in vivo. Covalent binding of misonidazole adducts to cellular macromolecules is enhanced by hypoxia. Autoradiographs of liver from mice treated with radiolabeled misonidazole demonstrated enhanced binding of adducts within hepatocytes surrounding hepatic veins. Livers from both hypoxic and normal mice had characteristic autoradiographic grain patterns reflecting regional oxygen tension variation in vivo. Differential binding of misonidazole adducts formed in hypoxic cells could have an application in studies of liver physiology and biochemistry.

Role of nitric oxide in maintenance of basal oral tissue blood flow in anesthetized cats

Experiments were undertaken to determine if nitric oxide (NO) plays a role in regulation of basal blood flow in the oral cavity of pentobarbital anesthetized cats and, if so, to quantify this effect using dose-response relationships. Blood flow was continuously measured from the surface of the tongue and mandibular gingiva (laser-Doppler flowmetry) and from the lingual artery (ultrasonic flowmetry). Cardiovascular parameters also were recorded. Administration of the nonselective inhibitor of nitric oxide synthase (NOS), L-NAME (0.08-20 mg/kg i.v.), produced a dose-related increase of blood pressure associated with decreases of blood flow at all three measurement sites. Maximal blood flow depression of 50-60% was seen 30-60 min after administration of 1.25 mg/kg of L-NAME. D-NAME (1.25 mg/kg i.v.) was inactive at all sites. Subsequent administration of L-arginine partially reversed effects of L-NAME in the lingual artery and tongue, but not in the gingival circulation. The neuronally selective NOS inhibitor, 7-nitroindazole (7-NI, 30 mg/kg i.p.), was devoid of effect on any of the measured parameters. These results suggest that endothelial (but not neuronally derived) NO plays an important role in control of basal blood flow in oral tissues of the cat.

Empirical assessment of non-invasive population genetics in bats:Comparison of DNA quality from faecal and tissue samples

Non-invasive population genetics has become a valuable tool in ecology and conservation biology, allowing genetic studies of wild populations without the need to catch, handle or even observe the study subjects directly. We address some of the concerns regarding the limitations of using non-invasive samples by comparing the quality of population genetic information gained through DNA extracted from faecal samples and biopsy samples of two elusive bat species, Myotis mystacinus and Myotis nattereri. We demonstrate that DNA extracted from faeces and tissue samples gives comparable results for frequency based population genetic analyses, despite the occurrence of genotyping errors when using faecal DNA. We conclude that non-invasive genetic sampling for population genetic analysis in bats is viable, and although more labour-intensive and expensive, it is an alternative to tissue sampling, which is particularly pertinent when specimens are rare, endangered or difficult to capture. © 2012 Museum and Institute of Zoology PAS.

p130Cas, a substrate associated with v-Src and v-Crk, localizes to focal adhesions and binds to focal adhesion kinase

p130(Cas) (crk associated substrate) has the structural characteristics of an adapter protein, containing multiple consensus SH2 binding sites, an SH3 domain, and a proline-rich domain. The structure of p130(Cas) suggests that it may act to provide a framework for protein-protein interactions; however, as yet, its functional role in cells is unknown. In this report we show that p130(Cas) is localized to focal adhesions. We demonstrate that p130(Cas) associates both in vitro and in vivo with pp125(FAK) (focal adhesion kinase), a kinase implicated in signaling by the integrin family of cell adhesion receptors. p130(Cas) also associates with pp41/43(FRNK) (pp125(FAK)-related, non-kinase), an autonomously expressed form of pp125(FAK) composed of only the C-terminal noncatalytic domain. We show that the association of p130(Cas) with pp125(Fak) and pp41/43(FRNK) is direct, and is mediated by the binding of the SH3 domain of p130(Cas) to a proline-rich sequence present in both the C terminus of pp125(FAK) and in pp41/43(FRNK). In agreement with recent studies we show that p130(Cas) is tyrosine-phosphorylated upon integrin mediated cell adhesion. The association of p130(Cas) with pp125(FAK), a kinase which is activated upon cell adhesion, is likely to be functionally important in integrin mediated signal transduction.

Tissue plasminogen activator for hepatic vein thrombosis in paroxysmal nocturnal haemoglobinuria

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired clonal disorder thought to arise in a multipotent haemopoietic stem cell. A distinct clinical feature is a tendency to thrombosis, with a particular predilection for the hepatic veins (Budd-Chiari syndrome). We report here on two patients with PNH who developed hepatic vein thrombosis (HVT) and who were treated with tissue plasminogen activator (t-PA). Both patients had a marked clinical and radiological improvement following the t-PA treatment and remain well over 2 years and 6 years after the treatment. This method of thrombolysis for HVT occurring in PNH has only been reported in two previous patients with limited follow-up. We suggest that this therapy is a useful first-line treatment for PNH patients who develop HVT.

Experimental Autoimmune Uveoretinitis (EAU)-Related Tissue Damage and Angiogenesis Is Reduced in CCL2−/−CX3CR1gfp/gfp Mice

Purpose: To investigate the roles of the CCL2-CCR2 and CX3CL1-CX3CR1 pathways in experimental autoimmune uveoretinitis (EAU)-mediated retinal tissue damage and angiogenesis.

Methods: The C57BL/6J wild-type (WT) and CCL2−/−CX3CR1gfp/gfp (double knockout [DKO]) mice were immunized with IRBP1-20. Retinal inflammation and tissue damage were evaluated clinically and histologically at different days postimmunization (p.i.). Retinal neovascular membranes were evaluated by confocal microscopy of retinal flat mounts, and immune cell infiltration by flow cytometry.

Results: At day 25 p.i., DKO mice had lower clinical and histological scores and fewer CD45highCD11b+ infiltrating cells compared with WT mice. The F4/80+macrophages constitute 40% and 21% and CD11b+Gr-1+Ly6G+ neutrophils constitute 10% and 22% of retinal infiltrating cells in WT and DKO mice, respectively. At the late stages of EAU (day 60–90 p.i.), DKO and WT mice had similar levels of inflammatory score. However, less structural damage and reduced angiogenesis were detected in DKO mice. Neutrophils were rarely detected in the inflamed retina in both WT and DKO mice. Macrophages and myeloid-derived suppressor cells (MDSCs) accounted for 8% and 3% in DKO EAU retina, and 19% and 10% in WT EAU retina; 71% of infiltrating cells were T/B-lymphocytes in DKO EAU retina and 50% in WT EAU retina.

Conclusions: Experimental autoimmune uveoretinitis–mediated retinal tissue damage and angiogenesis is reduced in CCL2−/−CX3CR1gfp/gfp mice. Retinal inflammation is dominated by neutrophils at the acute stage and lymphocytes at the chronic stage in these mice. Our results suggest that CCR2+ and CX3CR1+monocytes are both involved in tissue damage and angiogenesis in EAU.

Quantitative Measurement of [Na+] and [K+] in Postmortem Human Brain Tissue Indicates Disturbances in Subjects with Alzheimer's Disease and Dementia with Lewy Bodies

Alzheimer’s disease (AD) is associated with significant disturbances in the homeostasis of Na+ and K+ ions as well as reduced levels of Na+/K+ ATPase in the brain. This study used ICP-MS to accurately quantify Na+ and K+ concentrations in human postmortem brain tissue. We analyzed parietal cortex (Brodmann area 7) from 28 cognitively normal age-matched controls, 15 cases of moderate AD, 30 severe AD, and 15 dementia with Lewy bodies (DLB). Associations were investigated between [Na+] and [K+] and a number of variables including diagnosis, age, gender, Braak tangle stage, amyloid-β (Aβ) plaque load, tau load, frontal tissue pH, and APOE genotype. Brains from patients with severe AD had significantly higher (26%; p<0.001) [Na+] (mean 65.43 ± standard error 2.91 mmol/kg) than controls, but the concentration was not significantly altered in moderate AD or DLB. [Na+] correlated positively with Braak stage (r=0.45; p<0.0001), indicating association with disease severity. [K+] in tissue was 10% lower (p<0.05) in moderate AD than controls. However, [K+] in severe AD and DLB (40.97±1.31 mmol/kg) was not significantly different from controls. There was a significant positive correlation between [K+] and Aβ plaque load (r=0.46; p=0.035), and frontal tissue pH (r=0.35; p=0.008). [Na+] was not associated with [K+] across the groups, and neither ion was associated with tau load or APOE genotype. We have demonstrated disturbances of both [Na+] and [K+] in relation to the severity of AD and markers of AD pathology, although it is possible that these relate to late-stage secondary manifestations of the disease pathology.