Contribution Of Individual Isoforms Research Articles

The Role of NADPH Oxidases in the Etiology of Obesity and Metabolic Syndrome: Contribution of Individual Isoforms and Cell Biology.

Significance: Highly prevalent in Western cultures, obesity, metabolic syndrome, and diabetes increase the risk of cardiovascular morbidity and mortality and cost health care systems billions of dollars annually. At the cellular level, obesity, metabolic syndrome, and diabetes are associated with increased production of reactive oxygen species (ROS). Increased levels of ROS production in key organ systems such as adipose tissue, skeletal muscle, and the vasculature cause disruption of tissue homeostasis, leading to increased morbidity and risk of mortality. More specifically, growing evidence implicates the nicotinamide adenine dinucleotide phosphate oxidase (NOX) enzymes in these pathologies through impairment of insulin signaling, inflammation, and vascular dysfunction. The NOX family of enzymes is a major driver of redox signaling through its production of superoxide anion, hydrogen peroxide, and attendant downstream metabolites acting on redox-sensitive signaling molecules. Recent Advances: The primary goal of this review is to highlight recent advances and survey our present understanding of cell-specific NOX enzyme contributions to metabolic diseases. Critical Issues: However, due to the short half-lives of individual ROS and/or cellular defense systems, radii of ROS diffusion are commonly short, often restricting redox signaling and oxidant stress to localized events. Thus, special emphasis should be placed on cell type and subcellular location of NOX enzymes to better understand their role in the pathophysiology of metabolic diseases. Future Directions: We discuss the targeting of NOX enzymes as potential therapy and bring to light potential emerging areas of NOX research, microparticles and epigenetics, in the context of metabolic disease.

Roles of fibronectin isoforms in neonatal vascular development and matrix integrity.

Fibronectin (FN) exists in two forms—plasma FN (pFN) and cellular FN (cFN). Although the role of FN in embryonic blood vessel development is well established, its function and the contribution of individual isoforms in early postnatal vascular development are poorly understood. Here, we employed a tamoxifen-dependent cFN inducible knockout (cFN iKO) mouse model to study the consequences of postnatal cFN deletion in smooth muscle cells (SMCs), the major cell type in the vascular wall. Deletion of cFN influences collagen deposition but does not affect life span. Unexpectedly, pFN translocated to the aortic wall in the cFN iKO and in control mice, possibly rescuing the loss of cFN. Postnatal pFN deletion did not show a histological aortic phenotype. Double knockout (dKO) mice lacking both, cFN in SMCs and pFN, resulted in postnatal lethality. These data demonstrate a safeguard role of pFN in vascular stability and the dispensability of the individual FN isoforms in postnatal vascular development. Complete absence of FNs in the dKOs resulted in a disorganized tunica media of the aortic wall. Matrix analysis revealed common and differential roles of the FN isoforms in guiding the assembly/deposition of elastogenic extracellular matrix (ECM) proteins in the aortic wall. In addition, we determined with two cell culture models that that the two FN isoforms acted similarly in supporting matrix formation with a greater contribution from cFN. Together, these data show that pFN exerts a critical role in safeguarding vascular organization and health, and that the two FN isoforms function in an overlapping as well as distinct manner to maintain postnatal vascular matrix integrity.

Impaired activation of platelets lacking protein kinase C-θ isoform

Protein kinase C (PKC) isoforms have been implicated in several platelet functional responses, but the contribution of individual isoforms has not been thoroughly evaluated. Novel PKC isoform PKC-theta is activated by glycoprotein VI (GPVI) and protease-activated receptor (PAR) agonists, but not by adenosine diphosphate. In human platelets, PKC-theta-selective antagonistic (RACK; receptor for activated C kinase) peptide significantly inhibited GPVI and PAR-induced aggregation, dense and alpha-granule secretion at low agonist concentrations. Consistently, in murine platelets lacking PKC-theta, platelet aggregation and secretion were also impaired. PKC-mediated phosphorylation of tSNARE protein syntaxin-4 was strongly reduced in human platelets pretreated with PKC-theta RACK peptide, which may contribute to the lower levels of granule secretion when PKC-theta function is lost. Furthermore, the level of JON/A binding to activated alpha(IIb)beta(3) receptor was also significantly decreased in PKC-theta(-/-) mice compared with wild-type littermates. PKC-theta(-/-) murine platelets showed significantly lower agonist-induced thromboxane A(2) (TXA(2)) release through reduced extracellular signal-regulated kinase phosphorylation. Finally, PKC-theta(-/-) mice displayed unstable thrombus formation and prolonged arterial occlusion in the FeCl(3) in vivo thrombosis model compared with wild-type mice. In conclusion, PKC-theta isoform plays a significant role in platelet functional responses downstream of PAR and GPVI receptors.

Impaired Activation of Platelets Lacking Protein Kinase C Theta (PKCΘ) Isoform.

Protein kinase C (PKC) has been implicated in platelet functional responses, but the contribution of individual isoforms has not been directly evaluated. PKCΘ is activated by glycoprotein VI (GPVI) and protease-activated receptor (PAR) agonists, but not by ADP. In human platelets, PKCΘ-selective receptor for activated C kinase (RACK) antagonistic peptide inhibited agonist-induced aggregation and secretion. Consistently, in murine platelets lacking PKCΘ, GPVI- or PAR-mediated aggregation and secretion were also impaired. Previously, fibrinogen receptor has been shown to be activated independently by calcium and PKC pathways. In the presence of dimethyl BAPTA, AYPGKF-induced platelet aggregation was inhibited by PKCΘ antagonistic RACK peptides, suggesting a role for this isoform in PKC-dependent fibrinogen receptor activation. In addition, the levels of thromboxane A2 (TXA2) release measured in GPVI and PAR-mediated activation of PKCΘ −/− murine platelets, were significantly lower compared to WT platelets. Moreover, agonist-induced extracellular-signal regulated kinase (ERK) phosphorylation was also significantly decreased in PKCΘ −/− murine platelets, which could be contributing to decreased TXA2 levels. PKCΘ −/− mice displayed unstable thrombus formation and prolonged arterial occlusion in the FeCl3 in vivo thrombosis model versus WT mice. In conclusion, PKCΘ isoform plays a significant role in platelet functional responses downstream of GPVI and PARs.

Characterization of Methimazole Metabolism by Mouse Fmo1, 2 and 3: Determination of Enzyme Co‐expression

Flavin-containing monooxygenases (FMOs) efficiently catalyze oxidation of chemicals such as thioureas and thioethers. Enzyme activities of baculovirus-expressed mouse Fmo isoforms were characterized in order to discriminate FMOs co-expressed in mouse tissues. We hypothesized that if the activity of each isoform could be determined under two sets of conditions and isoforms are uniquely discriminated by the second set of conditions, the contribution of individual isoforms from mouse tissues expressing multiple Fmos could be mathematically determined. Metabolism of 2.0 mM methimazole by expressed mouse Fmo1, 2 and 3 was measured at pH 8.5 to determine the activity (nmol·min-1·nmol-1) of each isoform under standard conditions. Mixtures of expressed Fmos were prepared and predictions of combined activity made and compared with observed activity. Activity of individual Fmos was also monitored with sodium cholate at a concentration of 1%. Under standard conditions, mixtures performed as expected (e.g. observed change in activity was the combined change of Fmos assayed individually). After addition of cholate, Fmo1, Fmo2 and Fmo3 retained 26%, 103% and 45% activity, respectively. Since all Fmos were distinguishable with cholate it was possible to develop mathematical equations for simultaneous co-determination of all three isoforms. We are confirming our model with expressed isoforms and will apply this model to mouse tissues. Supported by PHS grant HL038650.