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Formation of peroxynitrite in vascular endothelial cells exposed to cyclosporine A¹

JAVIER NAVARRO-ANTOLÍN, MARÍA JOSÉ LÓPEZ-MUÑOZ^*, PETER KLATT, JAVIER SORIA^*, THOMAS MICHEL and SANTIAGO LAMAS²

Centro de Investigaciones Biológicas and Instituto Reina Sofía de Investigaciones Nefrológicas,
^* Instituto de Catálisis y Petroleoquímica, Consejo Superior de Investigaciones Científicas, Madrid, Spain; and
Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts

²Correspondence: Centro de Investigaciones Biológicas, c/ Velázquez 144, 28006 Madrid, Spain. E-mail: slamas{at}cib.csic.es

SPECIFIC AIMS

We evaluate the endogenous formation of specific reactive oxygen species (ROS)—superoxide anion (O₂·^-)—and reactive nitrogen species (RNS)—nitric oxide (NO·)—in bovine aortic endothelial cells (BAEC) upon treatment with cyclosporine A (CsA), and the potential role of this immunosupressant as an inducer of peroxynitrite formation.

PRINCIPAL FINDINGS

1. Generation of superoxide anion in endothelial cells exposed to CsA
In media from BAEC treated with the O₂·^- generator 2,3-dimethoxy-1-naphthoquinone (DMNQ), the superoxide dismutase (SOD)- and dihydroethidium (DHE)-inhibitable paramagnetic signal of the adduct 5,5,-dimethyl-1-pyrroline-N-oxide-hydroxyl (DMPO-OH) was detected, implying the formation of O₂·^- and validating the specificity of DHE to detect O₂·^- in our cell culture conditions, respectively (Fig. 1A ). Using flow cytometry, a dose-dependent intracellular increase in the 2 h accumulated fluorescence of ethidium (oxidized and fluorescent form of DHE) was detected in intact BAEC treated with CsA or DMNQ (Fig. 1B ). A synergic effect was achieved when cells were coincubated in the presence of both CsA and DMNQ (Fig. 1C ). The SOD lipophilic mimetic Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) completely abolished the oxidation of DHE by CsA or DMNQ (Fig. 1D ). No intracellular oxidation of DHE was observed in BAEC treated with other ROS/RNS-generating compounds such as 2-(N,N-diethylamino)-diazenolate-2-oxide sodium salt (DEA/NO), 3-morpholinosydnonimine (SIN-1), authentic peroxynitrite (ONOO^-), and hydrogen peroxide (Fig. 1E ).

View larger version (35K): [in this window] [in a new window]   Figure 1. Detection of O2·- in BAEC upon treatment with CsA. A) Representative experiment of three of DMNQ-mediated superoxide accumulation in supernatants of BAEC measured by electron spin resonance (ESR) using the spin trap DMPO. BAEC were preincubated for 15 min in the absence and presence of SOD (200 units/ml) or DHE (5 µM) before addition of DMNQ (100 µM). B–E) Flow cytometry detection of O2·- with the probe DHE. BAEC were preincubated for 1 h with 5 µM DHE and incubated for 2 h with the indicated doses of CsA or/and DMNQ (n=3–7) (B, C). Where indicated, BAEC were incubated with 10 µM DMNQ or 10 µM CsA in the absence or presence of the lipophilic SOD mimetic MnTMPyP (50 µM, 30 min preincubation) (n=3) (D) or with CsA, DMNQ, DEA/NO, SIN-1, peroxynitrite, or hydrogen peroxide (100 µM, each) (n=4) (E). Data are represented as mean intracellular fluorescence. *P < 0.05 vs. corresponding control. P < 0.05: coincubation of the indicated dose of CsA + DMNQ vs. the same dose of CsA or DMNQ. DPPH: magnetic field marker.

2. Generation of nitric oxide in endothelial cells exposed to CsA
We first tested whether the cell-permeable fluorescent probe diaminofluorescein/diacetate (DAF-2/DA) could be used with the flow cytometry technique to detect intracellular NO·. Endothelial nitric oxide synthase (eNOS) protein was expressed in Sf9 insect cells by baculovirus transfection. eNOS-transfected Sf9 cells showed an increased intracellular accumulation of diaminofluorescein triazol (DAF-2T) (oxidized and fluorescent form of the probe) that was sensitive to the eNOS inhibitor N^G-nitro-L-arginine (L-NNA) (Fig. 2A ). A dose-dependent increase in the intracellular fluorescence of DAF-2 was detected in BAEC treated with CsA (Fig. 2B ). As a positive control, a dose-dependent increase in the intracellular fluorescence of DAF-2 was achieved in intact BAEC treated with DEA/NO, whereas DEA was unable to produce any significant nitration of the probe (Fig. 2B ). The increase in DAF-2 fluorescence induced by CsA was sensitive to N^G-nitro-L-arginine methyl ester (L-NAME) (30 min preincubation) (Fig. 2C ). A dose-dependent increase in the accumulation of extracellular nitrite (a marker for NO· formation) in supernatants from BAEC treated with CsA was detected (Fig. 2D ). No intracellular oxidation of DHE was observed in BAEC treated with other ROS/RNS generating compounds (Fig. 2E ).

View larger version (37K): [in this window] [in a new window]   Figure 2. Detection of intracellular and extracellular RNS in BAEC treated with CsA. A) Effect of expression of eNOS in Sf9 cells on the intracellular fluorescence of the NO·-sensitive dye DAF-2. Sf9 cells infected with recombinant baculovirus expressing eNOS (confirmed by immunoblotting) were incubated with 10 µM DAF-2/DA for 3 h in the absence and presence of the NO· synthesis inhibitor L-NNA (1 mM) for the last 2 h. Intracellular fluorescence of DAF-2T was evaluated by flow cytometry (n=3). B) Mean fluorescence intensity of intracellular DAF-2T detected in BAEC incubated for 2 h with the indicated doses of DEA/NO, its inactive form DEA, or the indicated doses of CsA (n=4). C) BAEC were incubated with 10 µM CsA in the absence and presence of 1 mM L-NAME. Inset: histograms from a representative experiment are shown (n=11). D) Effect of the indicated doses of CsA on the 2 h accumulation of nitrites in supernatants of BAEC detected by chemiluminescence (n=11). (E) Effect of 10 µM of various pro-oxidants on DAF-2/DA (n=4). A, B, C, E) For each condition, the mean intracellular fluorescence is shown. *P < 0.05 vs. corresponding control. P < 0.05: L-NNA vs. no L-NNA.

3. Generation of peroxynitrite in endothelial cells exposed to CsA
We first validated the use of dihydrorhodamine 123 (DHR) as a ONOO^--sensitive sensor in endothelial cells. An increase in the oxidation of DHR in BAEC was observed with either authentic ONOO^- or ONOO^- derived from SIN-1, as well as with CsA (graphics inside Fig. 3 ), but not with pH-inactivated ONOO^-, hydrogen peroxide, DEA/NO, and DMNQ. The oxidation of DHR by CsA, SIN-1, or ONOO^- was sensitive to N-acetyl-cysteine (NAC) (graphics in Fig. 3 ). The 2 h or 4 h oxidation of DHR by CsA (0.01 to 10 µM, including therapeutic concentrations) showed a dose-dependent increase; this process was sensitive to L-NAME.

View larger version (31K): [in this window] [in a new window]   Figure 3. Schematic diagram of the production of intracellular superoxide anion (oxidation of DHE to ethidium), nitric oxide (oxidation of DAF-2 to DAF-2T), and peroxynitrite (oxidation of DHR 123 to rhodamine 123) and of its biomarker 3-nitrotyrosine (immunocytochemistry with an anti-nitrotyrosine antibody) induced by CsA in endothelial cells (upper panel). As CsA was shown to produce superoxide and nitric oxide, peroxynitrite formation was evaluated. For flow cytometry with DHR, BAEC were preincubated for 1 h with the probe and incubated for an additional 2 h (unless otherwise indicated) with CsA (10 µM), SIN-1 (10 µM) or peroxynitrite (ONOO-) (100 µM) in the absence and presence of the antioxidant NAC (30 min preincubation, 3 mM) (middle panel) (n=6). For immunocytochemistry with a polyclonal rabbit anti-nitrotyrosine antibody, BAEC were treated for 2 h with CsA vehicle, 10 µM CsA, or 10 µM SIN-1 in the absence and presence of MnTMPyP (50 µM, 30 min preincubation) or NAC (3 mM, 30 min preincubation) (n=4). Pretreatment of the nitrotyrosine antibody with 10 mM 3-nitrotyrosine was used as a control for the specificity of the antibody (original magnification x16) (lower panel).

4. Formation of nitrotyrosine in BAEC treated with CsA
As shown in the immunocytofluorescence of Fig. 3 , immunodetection with a polyclonal antinitrotyrosine antibody showed an increase of nitrotyrosine formation in BAEC treated with CsA. In the presence of 3-nitrotyrosine, nitrotyrosine immunodetection was completely blocked, indicating that the immunocytochemical signal was specific. Nitrotyrosine generation by CsA in BAEC was significantly attenuated by the presence of the antioxidants NAC and MnTMPyP.

CONCLUSIONS AND SIGNIFICANCE

We show data supporting the early enhanced rate of formation of superoxide anion and NO· in CsA-treated endothelial cells. We provide experimental evidence that the enhanced rate of formation of these species coincides within a common spatial-temporal frame and that they react to form the powerful oxidant peroxynitrite, which finally leads to the tyrosine nitration of endothelial proteins.

Peroxynitrite formation and protein nitration are considered a mediator and marker, respectively, of ROS/RNS-induced vascular damage as observed, for example, in atherosclerotic lesions, suggesting that reactive nitrogen species may promote LDL oxidation in vivo. These observations are of special interest in the context of clinical treatment with CsA, since accelerated atherosclerosis has been described in allograft receptors and CsA has been shown to impair the accelerated atherosclerosis in an animal model. Another CsA-associated process in which the endothelial cell appears to be one of the major targets and where a pathogenetic role for peroxynitrite has been suggested is the hemolytic uremic syndrome and its associated thrombotic microangiopathy, the etiology of which is poorly understood. In keeping with this concept, DNA mutations in the context of peroxynitrite production by CsA could play a role in the reported high incidence (32%) of oncogenesis in transplant patients receiving CsA.

Therapeutic blood levels of CsA in patients receiving this drug (approximate trough levels are 150–300 ng/ml, i.e., 0.125–0.250 µM) are in the range of concentrations able to oxidize the peroxynitrite-sensitive probe DHR 123. Oxidation of DHR was detected in human umbilical vein endothelial cells upon treatment with 1 µM CsA for 24 h (166±13% of control; n=3; P<0.05). Even when the present study does not include in vivo experimental models, we believe that the unprecedented demonstration of peroxynitrite formation and protein nitration by CsA in endothelial cells should be considered as potential mechanisms underlying the deleterious side effects of CsA therapy.

That nitration of tyrosine residues in endothelial cells was abrogated by the presence of the antioxidant NAC could also open new possibilities to explore therapeutic alternatives to prevent side-effects in the context of CsA therapy.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0636fje ; to cite this article, use FASEB J. (March 20, 2001) 10.1096/fj.00-0636fje

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