The endothelial isoform of nitric-oxide synthase (eNOS) is regulated by a complex pattern of post-translational modifications. In these studies, we show that eNOS is dynamically regulated by S-nitrosylation, the covalent adduction of nitric oxide (NO)-derived nitrosyl groups to the cysteine thiols of proteins. We report that eNOS is tonically S-nitrosylated in resting bovine aortic endothelial cells and that the enzyme undergoes rapid transient denitrosylation after addition of the eNOS agonist, vascular endothelial growth factor. eNOS is thereafter progressively renitrosylated to basal levels. The receptor-mediated decrease in eNOS S-nitrosylation is inversely related to enzyme phosphorylation at Ser 1179 , a site associated with eNOS activation. We also document that targeting of eNOS to the cell membrane is required for eNOS S-nitrosylation. Acylation-deficient mutant eNOS, which is targeted to the cytosol, does not undergo S-nitrosylation. Using purified eNOS, we show that eNOS S-nitrosylation by exogenous NO donors inhibits enzyme activity and that eNOS inhibition is reversed by denitrosylation. We determine that the cysteines of the zinc-tetrathiolate that comprise the eNOS dimer interface are the targets of S-nitrosylation. Mutation of the zinc-tetrathiolate cysteines eliminates eNOS S-nitrosylation but does not eliminate NO synthase activity, arguing strongly that disruption of the zinc-tetrathiolate does not necessarily lead to eNOS monomerization in vivo. Taken together, these studies suggest that eNOS S-nitrosylation may represent an important mechanism for regulation of NO signaling pathways in the vascular wall.

Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid that binds to S1P 1 (EDG-1) receptors and activates the endothelial isoform of NO synthase (eNOS). S1P and the polypeptide growth factor vascular endothelial growth factor (VEGF) act independently to modulate angiogenesis and activate eNOS. In these studies, we explored the cross-talk between S1P and VEGF signaling pathways. When cultured bovine aortic endothelial cells were treated with VEGF (10 ng͞ml), the expression of S1P 1 protein and mRNA increased by Ϸ4-fold. S1P1 up-regulation by VEGF was seen within 30 min of VEGF addition and reached a maximum after 1.5 h. By contrast, expression of neither bradykinin B2 receptors nor the scaffolding protein caveolin-1 was altered by VEGF treatment. The EC 50 for VEGF-promoted induction of S1P 1 expression was Ϸ2 ng͞ml, within its physiological concentration range. S1P1 induction by VEGF was attenuated by the tyrosine kinase inhibitor genistein and by the PKC inhibitor calphostin C. Preincubation of bovine aortic endothelial cells with VEGF (10 ng͞ml for 90 min) markedly enhanced subsequent S1P-dependent eNOS activation. VEGF pretreatment of cultured endothelial cells also markedly potentiated S1P-promoted eNOS phosphorylation at Ser-1179, as well as S1P-mediated activation of kinase Akt. In isolated rat arteries, VEGF pretreatment markedly potentiated S1P-mediated vasorelaxation and eNOS Ser-1179 phosphorylation. Taken together, these data indicate that VEGF specifically induces expression of S1P 1 receptors, associated with enhanced intracellular signaling responses to S1P and the potentiation of S1P-mediated vasorelaxation. We suggest that VEGF acts to sensitize the vascular endothelium to the effects of lipid mediators by promoting the induction of S1P 1 receptors, representing a potentially important point of cross-talk between receptor-regulated eNOS signaling pathways in the vasculature.

Endothelial nitric-oxide synthase (eNOS) undergoes a complex pattern of post-translational modifications that regulate its activity. We have recently reported that eNOS is constitutively S-nitrosylated in endothelial cells and that agonists promote eNOS denitrosylation concomitant with enzyme activation (Erwin, P. A., Lin, A. J., Golan, D. E., and Michel, T. (2005) , modifications that are dependent on the prior co-translational and irreversible N-myristoylation of eNOS Gly 2 (4). Upon agonist stimulation, eNOS is rapidly depalmitoylated and translocates from peripheral membrane caveolae to internal membrane structures; over time, the enzyme is retargeted to caveolae and repalmitoylated as eNOS returns to basal activity levels (2, 5, 6). We have previously reported that S-nitrosylation reversibly inhibits eNOS activity, that eNOS is tonically S-nitrosylated in vascular endothelial cells, and that the enzyme undergoes rapid, transient denitrosylation upon agonist stimulation, followed by progressive renitrosylation as the enzyme returns to resting activity levels (7). We also showed that endogenous eNOS S-nitrosylation in transfected cells is abolished when the zinc-tetrathiolate cysteines (residues 96 and 101 in the bovine eNOS sequence) of eNOS are changed to serine by PCR-directed mutagenesis (7). We used mass spectrometry to analyze the S-nitrosylation pattern of purified recombinant eNOS and showed directly that the zinc-tetrathiolate of eNOS is preferentially S-nitrosylated in the intact enzyme. We also report that subcellular targeting is a determinant of eNOS S-nitrosylation and that translocation between cellular compartments is necessary for agonist-modulated eNOS denitrosylation. Finally, we extended our work from cultured bovine aortic endothelial cells (BAEC) to show that dynamic S-nitrosylation occurs in intact mouse blood vessels. Taken together, these data suggest that S-nitrosylation is a dynamic and physiologically relevant regulator of NO signaling pathways in the vascular endothelium.

The inducible isoform of nitric oxide synthase (iNOS) and three zinc tetrathiolate mutants (C104A, C109A, and C104A/C109A) were expressed in Escherichia coli and purified. The mutants were found by ICP-AES and the zinc-specific PAR colorimetric assay to be zinc free, whereas the wild-type iNOS zinc content was 0.38 +/- 0.01 mol of Zn/mol of iNOS dimer. The cysteine mutants (C104A and C109A) had an activity within error of wild-type iNOS (2.24 +/- 0.12 micromol of NO min(-1) mg(-1)), but the double cysteine mutant had a modestly decreased activity (1.75 +/- 0.14 micromol of NO min(-1) mg(-1)). To determine if NO could stimulate release of zinc and dimer dissociation, wild-type protein was allowed to react with an NO donor, DEA/NO, followed by buffer exchange. ICP-AES of samples treated with 10 microM DEA/NO showed a decrease in zinc content (0.23 +/- 0.01 to 0.09 +/- 0.01 mol of Zn/mol of iNOS dimer) with no loss of heme iron. Gel filtration of wild-type iNOS treated similarly resulted in approximately 20% more monomeric iNOS compared to a DEA-treated sample. Only wild-type iNOS had decreased activity (42 +/- 2%) after reaction with 50 microM DEA/NO compared to a control sample. Using the biotin switch method under the same conditions, only wild-type iNOS had increased levels of S-biotinylation. S-Biotinylation was mapped to C104 and C109 on wild-type iNOS using LysC digestion and MALDI-TOF/TOF MS. Immunoprecipitation of iNOS from the mouse macrophage cell line, RAW-264.7, and the biotin switch method were used to confirm endogenous S-nitrosation of iNOS. The data show that S-nitrosation of the zinc tetrathiolate cysteine results in zinc release from the dimer interface and formation of inactive monomers, suggesting that this mode of inhibition might occur in vivo.

Fibromuscular dysplasia (FMD) is a non-atherosclerotic, non-inflammatory disease of medium sized arteries that has been described in multiple anatomic territories with a wide variety of manifestations (e.g. beading, stenosis, occlusion, aneurysm, or dissection). While the first case of FMD is thought to have been described over 75 years ago, the causes, natural history, and epidemiology of FMD in the general population remain incompletely understood. This article reviews important historical and contemporary contributions to the FMD literature that inform our current understanding of the prevalence and epidemiology of this important disorder. A particular focus is given to studies which form the basis for FMD prevalence estimates. Prevalence estimates for renal FMD are derived from renal transplant donor studies and sub-studies of clinical trials of renal artery stenting; however, it is unclear how well these estimates generalize to the overall population as a whole. Newer data are emerging examining the genetic associations and environmental interactions with FMD. Significant contributions to the understanding of FMD have come from the United States Registry for Fibromuscular Dysplasia; however, many unanswered questions remain, and future studies are required to further characterize FMD epidemiology in general populations and advance our understanding of this important disorder.

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Transplant renal artery stenosis (TRAS),R enal transplantation is the definitive therapy for end-stage kidney disease and provides substantial lifesaving and quality-of-life benefits for patients who would otherwise undergo dialysis.1 Posttransplantation stenosis of the renal artery-more commonly called TRAS, or transplant renal artery stenosis-is the most frequent vascular complication of kidney transplantation.2 It is implicated in graft dysfunction, concomitant congestive heart failure, and refractory hypertension. Transplant renal artery stenosis is often detected during routine Doppler-ultrasonographic screening of the transplanted kidney or during the clinical evaluation that occurs when graft function deteriorates.3 Percutaneous revascularization of TRAS is indicated to treat graft dysfunction; however, the conventional use of angiography to guide the intervention can put the graft at substantial risk of contrast nephropathy. 4,5 We present a case of TRAS that was discovered during the evaluation for causes of a patient's failing transplanted kidney. Case ReportIn January 2013, a 70-year-old man with end-stage polycystic kidney disease underwent cadaveric kidney transplantation. He had a history of hypertension, transient ischemic attack, and coronary artery disease. In the 4 months after his kidney transplantation, he had hospital admissions for heart failure, gastrointestinal bleeding, and acute coronary syndrome requiring percutaneous coronary intervention. The patient's serum creatinine levels increased steadily during this time, from 1.47 mg/ dL at the time of transplantation to 6.99 mg/dL 5 months after transplantation. The rise in creatinine was attributed to cumulative kidney damage from intra-arterial contrast medium administered during the above-mentioned percutaneous coronary intervention, to acute tubular necrosis from hypotension, and possibly to cardiorenal syndrome. Five months after the transplantation, he presented with refractory pulmonary edema and acute-on-chronic renal failure (serum creatinine level, 6.05 mg/ dL). He was treated with diuresis, which further elevated the creatinine to a peak of 7.31 mg/dL. Hemodialysis was initiated to treat azotemia and volume overload. Biopsy specimens of the transplanted kidney did not reveal any specific cause for his Case Reports

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