Porphyria is a group of metabolic disorders due to altered enzyme activities within the heme biosynthetic pathway. It is a systemic disease with multiple potential contributions to mitochondrial ...dysfunction and oxidative stress. Recently, it has become possible to measure mitochondrial function from cells isolated from peripheral blood (cellular bioenergetics) using the XF96 analyzer (Seahorse Bioscience). Mitochondrial respiration in these cells is measured with the addition of activators and inhibitors of respiration. The output is measured as the O2 consumption rate (OCR) at basal conditions, ATP linked, proton leak, maximal, reserve capacity, non-mitochondrial, and oxidative burst. We performed cellular bioenergetics on 22 porphyria (12 porphyria cutanea tarda (PCT), seven acute hepatic porphyria (AHP), and three erythropoietic protoporphyria (EPP)) patients and 18 age and gender matched healthy controls. Of porphyria cases, eight were active (2 PCT, 1 EPP, and 5 AHP) and 14 in biochemical remission. The OCR were decreased in patients compared to healthy controls. The bioenergetic profile was significantly lower when measuring proton leak and the non-mitochondrial associated OCR in the eight active porphyria patients when compared to 18 healthy controls. In conclusion, we demonstrate that the bioenergetic profile and mitochondrial activities assessed in porphyria patients and is different than in healthy control individuals. Further, our novel preliminary findings suggest the existence of a mitochondrial dysfunction in porphyria and this may be used as potential non-invasive biomarker for disease activity. This needs to be assessed with a systematic examination in a larger patient cohort. Studies are also suggested to examine mitochondrial metabolism as basis to understand mechanisms of these findings and deriving mitochondrial based therapies for porphyria.
Hepatopulmonary syndrome (HPS) following rat common bile duct ligation results from pulmonary molecular changes that may be influenced by circulating TNF-alpha and increased vascular shear stress, ...through activation of NF-kappaB or Akt. Increased pulmonary microvascular endothelin B (ET(B)) receptor and endothelial nitric oxide synthase (eNOS) levels contribute to nitric oxide production and the development of experimental HPS. Pentoxifylline (PTX), a phosphodiesterase and nonspecific TNF-alpha inhibitor, ameliorates experimental HPS when begun before hepatic injury. However, how PTX influences the molecular events associated with initiation of experimental HPS after liver injury is established is unknown. We assessed the effects of PTX on the molecular and physiological features of HPS in vivo and on shear stress or TNF-alpha-mediated events in rat pulmonary microvascular endothelial cells in vitro. PTX significantly improved HPS without altering portal or systemic hemodynamics and downregulated pulmonary ET(B) receptor levels and eNOS expression and activation. These changes were associated with a reduction in circulating TNF levels and NF-kappaB activation and complete inhibition of Akt activation. In rat pulmonary microvascular endothelial cells, PTX inhibited shear stress-induced ET(B) receptor and eNOS expression and eNOS activation. These effects were also associated with inhibition of Akt activation and were reproduced by wortmanin. In contrast, TNF-alpha had no effects on endothelial ET(B) and eNOS alterations in vitro. PTX has direct effects in the pulmonary microvasculature, likely mediated through Akt inhibition, that ameliorate experimental HPS.
Oxidative modifications to cellular proteins are critical in mediating redox-sensitive processes such as autophagy, the antioxidant response, and apoptosis. The proteins that become modified by ...reactive species are often compartmentalized to specific organelles or regions of the cell. Here, we detail protocols for identifying the subcellular protein targets of lipid oxidation and for linking protein modifications with biological responses such as autophagy. Fluorophores such as BODIPY-labeled arachidonic acid or BODIPY-conjugated electrophiles can be paired with organelle-specific probes to identify specific biological processes and signaling pathways activated in response to oxidative stress. In particular, we demonstrate “negative” and “positive” labeling methods using BODIPY-tagged reagents for examining oxidative modifications to protein nucleophiles. The protocol describes the use of these probes in slot immunoblotting, quantitative Western blotting, in-gel fluorescence, and confocal microscopy techniques. In particular, the use of the BODIPY fluorophore with organelle- or biological process-specific dyes and chromophores is highlighted. These methods can be used in multiple cell types as well as isolated organelles to interrogate the role of oxidative modifications in regulating biological responses to oxidative stress.
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► A method was developed to quantify oxidative modifications using fluorescent tags. ► BODIPY–electrophile conjugates track endogenous protein thiol modifications. ► Fluorescent lipid peroxidation substrates were created to identify redox-sensitive proteomes. ► Organelle-specific proteomes that react with lipid species can be examined by confocal microscopy. ► BODIPY–arachidonic acid can be used to examine autophagic responses to oxidative stress.
Sepsis is an acute inflammatory disease characterized by dysfunctional blood flow and hypotension. Nitric oxide (NO) is elevated during sepsis and plays an integral role in the associated vascular ...pathology. However, precise mechanisms and functions of NO in sepsis remain unclear. In this study, we show that red blood cells (RBCs) are foci for nitrosative reactions during acute inflammation, resulting in the formation of cells that can promote systemic vascular relaxation in an uncontrolled manner. Specifically, using experimental models of endotoxemia and surgical sepsis, NO adducts were found in the RBCs, including S-nitrosohemoglobin (SNOHb). These RBCs, referred to as septic RBCs, spontaneously stimulated vasodilation in a manner consistent with elevated SNOHb concentrations. Moreover, relaxation was cyclic guanosine monophosphate (cGMP) dependent and was inhibited by RBC lysis and glutathione but not by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5 tetramethylimidazoline 1-oxyl 3-oxide (C-PTIO). The potential mechanism of septic RBC–mediated vasorelaxation is discussed and may involve the intermediate, nitroxyl (HNO). Coupled with data showing that NO adducts in septic RBCs were dependent on the inducible nitric oxide synthase and correlated with plasma nitrite, these findings provide a novel framework to understand mechanisms underlying dysfunctional blood flow responses during sepsis. Specifically, the concept that RBCs directly mediate systemic hypotension through NO-dependent mechanisms is discussed.
The antiatherogenic effects of soy isoflavone consumption have been demonstrated in a variety of studies. However, the mechanisms involved remain poorly defined. Adhesion of monocytes to vascular ...endothelial cells is a key step within the inflammatory cascade that leads to atherogenesis. Many factors, including the physical forces associated with blood flow, regulate this process. Using an in vitro flow assay, we report that genistein, a principal component of most isoflavone preparations, inhibits monocyte adhesion to cytokine (TNF-alpha)-stimulated human vascular endothelial cells at physiologically relevant concentrations (0-1 microM). This effect is absolutely dependent on flow and is not observed under static conditions. Furthermore, this inhibition was dependent on activation of endothelial peroxisomal proliferator-activated receptor-gamma. No significant role for other reported properties of genistein, including antioxidant effects, inhibition of tyrosine kinases, or activation of estrogen receptors, was observed. Furthermore, the antiadhesive effects of genistein did not occur via modulation of the adhesion molecules E-selectin, ICAM-1, VCAM-1, or platelet-endothelial cell adhesion molecule-1. These data reveal a novel anti-inflammatory mechanism for isoflavones and identify the physical forces associated with blood flow and a critical mediator of this function.
The mechanisms by which isoflavones protect against inflammatory vascular disease remain unclear. Our previous observations suggest that one mechanism involves inhibition of monocyte-endothelial cell ...interactions in a process that is absolutely dependent on flow. The molecular mechanisms involved and the effects of structurally distinct isoflavones on this process are not known and are investigated herein. Using static and flow-dependent monocyte adhesion assays, our data show that exposure of endothelial cells to biologically relevant concentrations of isoflavones inhibits subsequent TNF-α induced monocyte adhesion only during flow. This inhibition involved activating endothelial PPARγ by stimulating promoter sequences containing the PPARγ response element by isoflavones and attenuating antiadhesive effects by siRNA targeting of PPARγ. A comparison of structurally distinct isoflavones suggested a critical role for the A-ring. Using chlorinated derivatives of daidzein, a key structural requirement for PPARγ agonist activity appears to be the presence of the 7-OH group and the lack of chlorine at the 6- or 8-positions in the A-ring. Collectively, these data support 1) a novel flow-dependent anti-inflammatory mechanism for PPARγ ligands in vascular endothelial cells and 2) exemplify the current concepts of nutrients modulating disease via regulating specific cell signaling pathways.
Abstract Arteriovenous grafts used for hemodialysis frequently develop intimal hyperplasia (IH), which ultimately leads to graft failure. Although the turbulent jet from the dialysis needle may ...contribute to vessel wall injury, its role in the pathogenesis of IH is relatively unexplored. In the current study, using bovine aortic endothelial cells (BAEC) cultured on the inner surface of a compliant tube, we evaluated the effects of simulated hemodialysis conditions on morphology and nitric oxide (NO) production. The flows via the graft and needle were 500 ml/min (Reynolds number = 819 ) and 100 ml/min (Reynolds number = 954 ), respectively. In the presence of the needle jet for 6 h, 19.3% (±1.53%) of BAEC were sheared off, whereas no loss of BAEC was observed in the presence of graft flow alone ( P < 0.05 ) . In the presence of graft flow alone, assessment of cell orientation by the Saltykov method revealed that BAEC were oriented along the flow direction. This alignment, however, was lost in the presence of needle flow. Finally, NO production was also significantly decreased in the presence of the needle flow compared to the presence of graft flow alone ( 16 ± 3.1 vs 34.7 ± 1.9 nmol / 1 0 6 cells / h , P < 0.05 ). NO is a key player in vascular homeostasis mechanisms modulating vasomotor tone, inhibiting inflammation and smooth muscle cell proliferation. Thus, the loss of NO signaling and the loss of endothelial integrity caused by needle jet turbulence may contribute to the cascade of events leading to IH formation during hemodialysis.
Reducing tidal volume as a part of a protective ventilation strategy may result in hypercapnia. In this study, we focused on the influence of hypercapnia on endothelial–neutrophil responses in models ...of inflammatory-stimulated human pulmonary microvascular endothelial cells (HMVEC) and in an animal model of lipopolysaccharide (LPS)-induced acute lung injury. Neutrophil adhesion and adhesion molecules expression and nuclear factor-κB (NF-κB) were analyzed in TNF-α and LPS-treated HMVEC exposed to either eucapnia or hypercapnia. In the
in vivo limb, bronchoalveolar lavage fluid cell counts and differentials, adhesion molecule and chemokine expression were assessed in LPS-treated rabbits ventilated with either low tidal volume ventilation and eucapnia or hypercapnia. In both the
in vitro and
in vivo models, hypercapnia significantly increased neutrophil adhesion and adhesion molecule expression compared to eucapnia. Activity of NF-κB was significantly enhanced by hypercapnia in the
in vitro experiments. IL-8 expression was greatest both
in vitro and
in vivo under conditions of hypercapnia and concomitant inflammation. CD11a expression was greatest in isolated human neutrophils exposed to hypercapnia
+
LPS. Our results demonstrate that endothelial–neutrophil responses per measurement of fundamental molecules of adhesion are significantly increased during hypercapnia and that hypercapnia mimics conditions of eucapnia
+
inflammation.
Dextran-binding immunoglobulin (DIg) was detected in circulating immune complexes in young healthy blood donors. High molecular weight dextran or non-dialysable polysaccharides of edible sugar added ...to serum markedly increased DIg-containing immune complexes. DIg from plasma was purified by an improved affinity chromatography using Sephadex G-200 to increase yield. Recognition by DIg of α (1→6) glucans in polystyrene-coated dextran was inhibited totally and its less avid recognition of β (1→3) glucans in yeast and barley partially by 50 mM 1-O-methyl-α-D-glucoside. All fractions of non-dialysable edible sugar separated by electrophoresis were recognized by DIg. While purified DIg contained nearly equal amounts of IgM and IgG, DIg in immune complexes isolated after addition of dextran to serum was 72% IgM. Recognition of bovine thyroglobulin, tumour laminin, and peanut-agglutinin-binding bovine heart glycoproteins, all containing terminal α (1→3)-linked galactose epitopes, by DIg was inhibitable by 1-O-methyl-α-D-glucoside. Enzymatic removal or modification of the respective terminal α (1→3)-linked galactose groups in these glycoproteins abolished their recognition by the antibody. Results suggest that DIg recognizes α (1→6) glucans, α (1→3) - linked galactose epitopes and less avidly β (1→3) glucans. Results also point to a new immunomodulatory effect of non-dialysable sugar and dietary β-glucans.
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