Frances L. Jourd'heuil

Cytoglobin Is Expressed in the Vasculature and Regulates Cell Respiration and Proliferation via Nitric Oxide Dioxygenation

Disposition of the second messenger nitric oxide (NO) in mammalian tissues occurs through multiple pathways including dioxygenation by erythrocyte hemoglobin and red muscle myoglobin. Metabolism by a putative NO dioxygenase activity in non-striated tissues has also been postulated, but the exact nature of this activity is unknown. In the present study, we tested the hypothesis that cytoglobin, a newly discovered hexacoordinated globin, participates in cell-mediated NO consumption. Stable expression of small hairpin RNA targeting cytoglobin in fibroblasts resulted in decreased NO consumption and intracellular nitrate production. These cells were more sensitive to NO-induced inhibition of cell respiration and proliferation, which could be restored by re-expression of human cytoglobin. We also demonstrated cytoglobin expression in adventitial fibroblasts as well as vascular smooth muscle cells from various species including human and found that cytoglobin was expressed in the adventitia and media of intact rat aorta. These results indicate that cytoglobin contributes to cell-mediated NO dioxygenation and represents an important NO sink in the vascular wall.

Redox control of G1/S cell cycle regulators during nitric oxide‐mediated cell cycle arrest

Redox regulation of cell cycle progression during nitric oxide (NO) mediated cytostasis is not well‐understood. In this study, we investigated the role of the intracellular antioxidant glutathione (GSH) in regulating specific signaling events that are associated with NO‐mediated cell cycle arrest. Manipulation of intracellular GSH content through pharmacological inhibition of glutamate‐cysteine ligase (GCL) indicated that GSH depletion potentiated nitrosative stress, DNA damage, phosphorylation of the tumor suppressor p53 (Ser‐18) and upregulation of p21cip1/waf1 upon NO stimulation. However, we found that neither overexpression of a dominant negative p53 nor pharmacological inhibition of p53 with cyclic pifithrin‐α (cPFT‐α) was sufficient to reverse NO‐mediated cell cycle arrest or hypophosphorylation of retinoblastoma protein (Rb). We found that the decrease in cyclin D1 levels induced by NO was GSH‐sensitive implying that the redox regulation of NO‐mediated cytostasis was a multifaceted process and that both p53/p21cip1/waf1 and p53 independent cyclin D1 pathways were involved. Together, our results demonstrate that GSH serves as an important component of cellular protective mechanisms against NO‐derived nitrosative stress to regulate DNA damage checkpoint. J. Cell. Physiol. 212:827–839, 2007.

Redox-Sensitivity and Site-Specificity of S- and N- Denitrosation in Proteins

Background S-nitrosation – the formation of S-nitrosothiols (RSNOs) at cysteine residues in proteins – is a posttranslational modification involved in signal transduction and nitric oxide (NO) transport. Recent studies would also suggest the formation of N-nitrosamines (RNNOs) in proteins in vivo, although their biological significance remains obscure. In this study, we characterized a redox-based mechanism by which N-nitroso-tryptophan residues in proteins may be denitrosated. Methodology/Principal Findings The denitrosation of N-acetyl-nitroso Trp (NANT) by glutathione (GSH) required molecular oxygen and was inhibited by superoxide dismutase (SOD). Transnitrosation to form S-nitrosoglutathione (GSNO) was observed only in the absence of oxygen or presence of SOD. Protein denitrosation by GSH was studied using a set of mutant recombinant human serum albumin (HSA). Trp-214 and Cys-37 were the only two residues nitrosated by NO under aerobic conditions. Nitroso-Trp-214 in HSA was insensitive to denitrosation by GSH or ascorbate while denitrosation at Cys-37 was evident in the presence of GSH but not ascorbate. GSH-dependent denitrosation of Trp-214 was restored in a peptide fragment of helix II containing Trp-214. Finally, incubation of cell lysates with NANT revealed a pattern of protein nitrosation distinct from that observed with GSNO. Conclusions We propose that the denitrosation of nitrosated Trp by GSH occurs through homolytic cleavage of nitroso Trp to NO and a Trp aminyl radical, driven by the formation of superoxide derived from the oxidation of GSH to GSSG. Overall, the accessibility of Trp residues to redox-active biomolecules determines the stability of protein-associated nitroso species such that in the case of HSA, N-nitroso-Trp-214 is insensitive to denitrosation by low-molecular-weight antioxidants. Moreover, RNNOs can generate free NO and transfer their NO moiety in an oxygen-dependent fashion, albeit site-specificities appear to differ markedly from that of RSNOs.

Dual Function for Mature Vascular Smooth Muscle Cells During Arteriovenous Fistula Remodeling

Background The arteriovenous fistula (AVF) is the preferred form of hemodialysis access for patients with chronic kidney disease. However, AVFs are associated with significant problems including high incidence of both early and late failures, usually attributed to inadequate venous arterialization and neointimal hyperplasia, respectively. Understanding the cellular basis of venous remodeling in the setting of AVF could provide targets for improving AVF patency rates. Methods and Results A novel vascular smooth muscle cell (VSMC) lineage tracing reporter mouse, Myh11‐Cre/ERT2‐mTmG, was used to track mature VSMCs in a clinically relevant AVF mouse model created by a jugular vein branch end to carotid artery side anastomosis. Prior to AVF surgery, differentiated medial layer VSMCs were labeled with membrane green fluorescent protein (GFP) following tamoxifen induction. Four weeks after AVF surgery, we observed medial VSMC layer thickening in the middle region of the arterialized vein branch. This thickened medial VSMC layer was solely composed of differentiated VSMCs that were GFP+/MYH11+/Ki67−. Extensive neointimal hyperplasia occurred in the AVF region proximal to the anastomosis site. Dedifferentiated VSMCs (GFP+/MYH11−) were a major cellular component of the neointima. Examination of failed human AVF samples revealed that the processes of VSMC phenotypic modulation and intimal hyperplasia, as well as medial VSMC layer thickening, also occurred in human AVFs. Conclusions We demonstrated a dual function for mature VSMCs in AVF remodeling, with differentiated VSMCs contributing to medial wall thickening towards venous maturation and dedifferentiated VSMCs contributing to neointimal hyperplasia. These results provide valuable insights into the mechanisms underlying venous adaptations during AVF remodeling.

Vimentin expression is required for the development of EMT-related renal fibrosis following Unilateral Ureteral Obstruction in mice

Most renal transplants ultimately fail secondary to chronic allograft nephropathy (CAN). Vimentin (vim) is a member of the intermediate filament family of proteins and has been shown to be important in the development of CAN. One of the pathways leading to chronic renal fibrosis after transplant is thought to be epithelial to mesenchymal transition (EMT). Even though vim expression is one of the main steps of EMT, it is unknown whether vim expression is required for EMT leading to renal fibrosis and allograft loss. To this end, the role of vim in renal fibrosis was determined via unilateral ureteral obstruction (UUO) in vim knockout mice (129 svs6 vim -/-). Following UUO, kidneys were recovered and analyzed via Western blotting, immunofluorescence, and transcriptomics. Cultured human proximal renal tubular (HK-2) cells were subjected to lentiviral-driven inhibition of vim expression and then treated with transforming growth factor (TGF)-β to undergo EMT. Immunoblotting as well as wound healing assays were used to determine development of EMT. Western blotting analyses of mice undergoing UUO reveal increased levels of vim soon after UUO. As expected, interstitial collagen deposition increased in control mice following UUO but decreased in vim -/- kidneys. Immunofluorescence analyses also revealed altered localization of β-catenin in vim -/- mice undergoing UUO without significant changes in mRNA levels. However, RNA sequencing revealed a decrease in β-catenin-dependent genes in vim -/- kidneys. Finally, vim-silenced HK-2 cell lines undergoing EMT were shown to have decreased cellular migration during wound healing. We conclude that vim inhibition decreases fibrosis following UUO by possibly altering β-catenin localization and downstream signaling.