Restoration of renal allograft function via reduced-contrast percutaneous revascularization of transplant renal artery stenosis.

Transplant renal artery stenosis (TRAS), the most common vascular complication of kidney transplantation, can lead to heart failure, uncontrolled hypertension, and irreversible dysfunction of the transplanted kidney. Percutaneous revascularization can improve outcomes in well-selected patients with symptomatic TRAS, but the intervention itself poses risk to the transplanted kidney because of the quantities of nephrotoxic contrast solution that often are used. We report the case of a patient with TRAS who, 5 months after undergoing a kidney transplant, developed allograft dysfunction and heart failure that required hemodialysis. We performed angioplasty and stenting of the TRAS, using intravascular ultrasonography and fluoroscopy as our primary imaging methods. To minimize further damage to a potentially viable kidney, the volume of intravascular contrast medium used was trivial (a total of 9 cc). Revascularization of the patient's TRAS restored his renal function: within 4 weeks of the procedure, he no longer needed hemodialysis, and his heart failure symptoms had resolved. This case emphasizes the value of early definitive treatment of TRAS and the usefulness of intravascular ultrasonography to minimize the amount of contrast medium used in endovascular procedures.

Evaluation of the patient who presents with critical limb ischemia: diagnosis, prognosis, and medical management.

Patients with critical limb ischemia usually have severe atherosclerotic disease and are at a high risk of limb loss as well as major adverse cardiovascular events. The current article provides a description of the clinical presentation of patients with critical limb ischemia and also discusses the initial evaluation of these patients, including physical examination, use of noninvasive vascular tests, and other imaging modalities. An overview of the general management of these patients is also provided, including the identification of patients who benefit from revascularization or primary amputation, principles of wound care, and therapies for cardiovascular risk reduction.

Contemporary Management of Femoral Popliteal Revascularization.

Symptomatic peripheral artery disease of the femoral popliteal segment can be treated by surgical and endovascular revascularization, but controversy exists about the best approach. Conventional approaches to revascularization have focused on lesion anatomy to decide on bypass versus endovascular treatment, but advances in endovascular therapy make an endovascular-first approach increasingly feasible-either as a single approach or as an adjunct to short-segment bypass (ie, hybrid procedure). In this review, we discuss the medical, endovascular, and surgical treatment of femoral popliteal revascularization with a special emphasis on advances in percutaneous therapy.

Subcellular targeting and differential S-nitrosylation of endothelial nitric-oxide synthase.

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), J. Biol. Chem. 280, 19888-19894). In the present studies, we use mass spectrometry to confirm that the zinc-tetrathiolate cysteines of eNOS are S-nitrosylated. eNOS targeting to the plasma membrane is necessary for enzyme S-nitrosylation, and we report that translocation between cellular compartments is necessary for dynamic eNOS S-nitrosylation. We transfected cells with cDNA encoding wild-type eNOS, which is membrane-targeted, or with acylation-deficient mutant eNOS (Myr-), which is expressed solely in the cytosol. While wild-type eNOS is robustly S-nitrosylated, we found that S-nitrosylation of the Myr- eNOS mutant is nearly abolished. When we transfected cells with a fusion protein in which Myr- eNOS is ligated to the CD8-transmembrane domain (CD8-Myr-), we found that CD8-Myr- eNOS, which does not undergo dynamic subcellular translocation, is hypernitrosylated relative to wild-type eNOS. Furthermore, we found that when endothelial cells transfected with wild-type or CD8-Myr- eNOS are stimulated with eNOS agonist, only wild-type eNOS is denitrosylated; CD8-Myr- eNOS S-nitrosylation is unchanged. These findings indicate that subcellular targeting is a critical determinant of eNOS S-nitrosylation. Finally, we show that eNOS S-nitrosylation can be detected in intact arterial preparations from mouse and that eNOS S-nitrosylation is a dynamic agonist-modulated process in intact blood vessels. These studies suggest that receptor-regulated eNOS S-nitrosylation may represent an important determinant of NO-dependent signaling in the vascular wall.

Receptor-regulated dynamic S-nitrosylation of endothelial nitric-oxide synthase in vascular endothelial cells.

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.

S-Nitrosation and regulation of inducible nitric oxide synthase.

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.

VEGF induces S1P1 receptors in endothelial cells: Implications for cross-talk between sphingolipid and growth factor receptors.

Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid that binds to S1P1 (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 ng/ml), the expression of S1P1 protein and mRNA increased by approximately 4-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 EC50 for VEGF-promoted induction of S1P1 expression was approximately 2 ng/ml, 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 ng/ml 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 S1P1 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 S1P1 receptors, representing a potentially important point of cross-talk between receptor-regulated eNOS signaling pathways in the vasculature.