Establishing 3-nitrotyrosine as a biomarker for the vasculopathy of Fabry disease

Abstract

The endothelial dysfunction of Fabry disease results from α-galactosidase A deficiency leading to the accumulation of globotriaosylceramide. Vasculopathy in the α-galactosidase A null mouse is manifested as oxidant-induced thrombosis, accelerated atherogenesis, and impaired arterial reactivity. To better understand the pathogenesis of Fabry disease in humans, we generated a human cell model by using RNA interference. Hybrid endothelial cells were transiently transfected with small interfering RNA (siRNA) specifically directed against α-galactosidase A. Knockdown of α-galactosidase A was confirmed using immunoblotting and globotriaosylceramide accumulation. Endothelial nitric oxide synthase (eNOS) activity was correspondingly decreased by >60%. Levels of 3-nitrotyrosine (3NT), a specific marker for reactive nitrogen species and quantified using mass spectrometry, increased by 40- to 120-fold without corresponding changes in other oxidized amino acids, consistent with eNOS-derived reactive nitrogen species as the source of the reactive oxygen species. eNOS uncoupling was confirmed by the observed increase in free plasma and protein-bound aortic 3NT levels in the α-galactosidase A knockout mice. Finally, 3NT levels, assayed in biobanked plasma samples from patients with classical Fabry disease, were over sixfold elevated compared with age- and gender-matched controls. Thus, 3NT may serve as a biomarker for the vascular involvement in Fabry disease. The endothelial dysfunction of Fabry disease results from α-galactosidase A deficiency leading to the accumulation of globotriaosylceramide. Vasculopathy in the α-galactosidase A null mouse is manifested as oxidant-induced thrombosis, accelerated atherogenesis, and impaired arterial reactivity. To better understand the pathogenesis of Fabry disease in humans, we generated a human cell model by using RNA interference. Hybrid endothelial cells were transiently transfected with small interfering RNA (siRNA) specifically directed against α-galactosidase A. Knockdown of α-galactosidase A was confirmed using immunoblotting and globotriaosylceramide accumulation. Endothelial nitric oxide synthase (eNOS) activity was correspondingly decreased by >60%. Levels of 3-nitrotyrosine (3NT), a specific marker for reactive nitrogen species and quantified using mass spectrometry, increased by 40- to 120-fold without corresponding changes in other oxidized amino acids, consistent with eNOS-derived reactive nitrogen species as the source of the reactive oxygen species. eNOS uncoupling was confirmed by the observed increase in free plasma and protein-bound aortic 3NT levels in the α-galactosidase A knockout mice. Finally, 3NT levels, assayed in biobanked plasma samples from patients with classical Fabry disease, were over sixfold elevated compared with age- and gender-matched controls. Thus, 3NT may serve as a biomarker for the vascular involvement in Fabry disease. Fabry disease (FD) is an X-linked lysosomal storage disease arising from a deficiency in α-galactosidase A (GLA). A loss of GLA activity results in the accumulation of glycosphingolipids with galα-1,4 gal linkages including globotriaosylceramide (Gb3), galabiosylceramide, and globotriaosylsphingosine (lyso-Gb3). FD has a pleiotropic phenotype that includes renal disease, cardiomyopathy, and vasculopathy. The vasculopathy is the basis for the life-threatening complications of FD, including stroke, hypertrophic cardiomyopathy, and renal failure. Whereas enzyme replacement therapy with recombinant GLA has emerged as a therapeutic option for FD, its effectiveness in preventing the long-term cardiovascular morbidities has been questioned.1.El Dib R.P. Nascimento P. Pastores G.M. Enzyme replacement therapy for Anderson-Fabry disease.Cochrane Database Syst Rev. 2013; 2: CD006663PubMed Google Scholar The ability to evaluate and deliver effective therapy is limited by a poor understanding of the pathogenesis of the vasculopathy and by the absence of a biomarker that corresponds directly with the presence and degree of vascular dysfunction. The Gla null mouse has been an useful model from exploring the vascular pathophysiology of FD. Although this mouse does not exhibit a spontaneous vascular phenotype, several inducible models of vascular disease have been reported. These include oxidant-induced thrombosis,2.Eitzman D.T. Bodary P.F. Shen Y. et al.Fabry disease in mice is associated with age-dependent susceptibility to vascular thrombosis.J Am Soc Nephrol. 2003; 14: 298-302Crossref PubMed Scopus (64) Google Scholar accelerated atherogenesis,3.Bodary P.F. Shen Y. Vargas F.B. et al.Alpha-galactosidase A deficiency accelerates atherosclerosis in mice with apolipoprotein E deficiency.Circulation. 2005; 111: 629-632Crossref PubMed Scopus (74) Google Scholar and impaired vasorelaxation.4.Park J.L. Whitesall S.E. D'Alecy L.G. et al.Vascular dysfunction in the alpha-galactosidase A-knockout mouse is an endothelial cell-, plasma membrane-based defect.Clin Exp Pharmacol Physiol. 2008; 35: 1156-1163Crossref PubMed Scopus (45) Google Scholar A common mechanism that could potentially link these experimentally observed abnormalities is endothelial nitric oxide synthase (eNOS) dysfunction.5.Shu L. Park J.L. Byun J. et al.Decreased nitric oxide bioavailability in a mouse model of Fabry disease.J Am Soc Nephrol. 2009; 20: 1975-1985Crossref PubMed Scopus (50) Google Scholar eNOS dysfunction may result in either decreased nitric oxide (NO) bioavailability or enzyme uncoupling, which generates a potent oxidant, peroxynitrite, a reactive nitrogen species.6.Shaul P.W. Regulation of endothelial nitric oxide synthase: location, location, location.Annu Rev Physiol. 2002; 64: 749-774Crossref PubMed Scopus (471) Google Scholar,7.Kietadisorn R. Juni R.P. Moens A.L. Tackling endothelial dysfunction by modulating NOS uncoupling: new insights into its pathogenesis and therapeutic possibilities.Am J Physiol Endocrinol Metab. 2012; 302: E481-E495Crossref PubMed Scopus (172) Google Scholar The relationship between GLA and eNOS was explored by determining whether these changes could be recapitulated in a human endothelial cell line. We report that when the Gb3 content of EA.hy926 cells is increased with GLA knockdown, there is an associated uncoupling of eNOS with the formation of 3-nitrotyrosine (3NT), a specific marker for reactive nitrogen species. The eNOS dysfunction was specifically associated with the loss of GLA activity in that comparable changes were not observed with β-glucocerebrosidase (GBA) knockdown. High circulating levels of 3NT were measured in the plasma and aortic extracts of Gla knockout mice. Finally, the concentrations of protein-bound oxidized amino acids were measured in plasma samples from classic FD patients and compared with age- and gender-matched controls. A more than fivefold elevation in 3NT was observed in the FD samples compared with controls, raising the possibility that 3NT represents an useful biomarker for vasculopathy in FD. Previous work in GLA-deficient mice demonstrated endothelial dysfunction associated with decreased NO bioavailability and eNOS uncoupling.5.Shu L. Park J.L. Byun J. et al.Decreased nitric oxide bioavailability in a mouse model of Fabry disease.J Am Soc Nephrol. 2009; 20: 1975-1985Crossref PubMed Scopus (50) Google Scholar To determine whether similar abnormalities could be documented in a human vascular endothelial cell line, immortalized EA.hy926 cells, derived from fused HUVEC and A549 cells, were employed. EA.hy926 cells retain endothelial characteristics, including Factor VIII-related antigen expression, eNOS gene expression, and Weibel–Palade bodies.8.Edgell C.J. Haizlip J.E. Bagnell C.R. et al.Endothelium specific Weibel-Palade bodies in a continuous human cell line, EA.hy926.In Vitro Cell Dev Biol. 1990; 26: 1167-1172Crossref PubMed Scopus (116) Google Scholar,9.Edgell C.J. McDonald C.C. Graham J.B. Permanent cell line expressing human factor VIII-related antigen established by hybridization.Proc Natl Acad Sci USA. 1983; 80: 3734-3737Crossref PubMed Scopus (1358) Google Scholar Importantly, EA.hy926 cells also are characterized by measureable Gb3 and GLA (Figure 1a and b). The silencing efficacies of Dicer-substrate RNAs (dsiRNA) (27-mer) and traditional small interfering RNA (siRNA) (21-mer) were assessed to determine whether it would be possible to fully suppress the expression and activity of GLA in cultured EA.hy926 cells. The conventional 21-mer siRNA against human GLA demonstrated low efficiency and short blocking times (data not shown). By contrast, anti-human GLA-dsiRNA completely knocked down GLA in cultured EA.hy926 cells for up to 3 days at a relatively low siRNA concentration (10nM) as confirmed using immunoblotting (Figure 1a). As both siRNA and dsiRNA interference are transient, a second transfection was performed 3 days later. Under these conditions, GLA silencing was observed up to day 6. The lower expression of GLA was associated with the accumulation of Gb3 (Figure 1b). A correlation between increased Gb3 and the degree of GLA knockdown was observed. Compared with control-dsiRNA–transfected cells, the Gb3 levels in GLA-dsiRNA–transfected cells increased with the duration of the incubation with GLA-dsiRNA, ranging from 210% of control on day 2 after a single transfection up to 275% on day 6 with a double transfection (Figure 1c). The specificity of the Gb3 changes was evaluated by use of GBA-dsiRNA. GBA encodes β-glucocerebrosidase, the lysosomal glycosidase that degrades glucosylceramide (GlcCer) to ceramide. GBA expression in cultured EA.hy926 cells was suppressed to undetectable levels. The silencing effect lasted until day 6 as measured using immunoblotting (Figure 2a). This silencing effect was observed following both single transfection and double transfection with the 27-mer anti-human GBA-dsiRNA. The corresponding loss of GBA activity resulted in the accumulation of GlcCer (Figure 2b). The specificity of this effect was demonstrated by the absence of any corresponding change in galactosylceramide, a cerebroside that is not a substrate for GlcCerase. GlcCer mass increased fivefold in GBA-dsiRNA–transfected cells relative to the levels in control-dsiRNA cells (Figure 2c). A small but reproducible reduction in GlcCer from its peak level occurred on day 4 after first transfection likely due to transient effect of siRNA. When a double GBA-dsiRNA transfection was performed, the cellular GlcCer levels on day 6 were as high as those observed on day 3 with a single transfection (Figure 2c). Although GlcCer is a precursor in Gb3 synthesis, no increase in Gb3 levels were observed in cells subjected to GBA-dsiRNA transfection (Figure 2d and e). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was silenced with GAPDH-dsiRNA to evaluate the effects of transfection on endothelial cell phenotype, morphology, oxidant stress, and viability because of either nonspecific or specific gene knockdown. GAPDH expression in EA.hy926 cells was lowered >98% by GAPDH-dsiRNA as measured using immunoblotting. A double transfection enhanced and prolonged the interference of GAPDH expression up to 6 days (Figure 3a). Cell numbers and viability following transfections were measured daily. The growth curve showed a similar recovery and growth pattern in cells transfected with control, GBA, and GAPDH-dsiRNAs. However, GLA-dsiRNA transfection was associated with lower growth and increased cell detachment from cultured dishes (Figure 3b). Forty percent of the detached cells remained viable and metabolically active as measured by trypan blue exclusion. Previous investigations uncovered the high levels of 3NT and ortho-tyrosine formation in mouse aortic endothelial cells isolated from Fabry mice, demonstrating that oxidant stress is characteristic of the Fabry mouse aortic endothelial cell phenotype.5.Shu L. Park J.L. Byun J. et al.Decreased nitric oxide bioavailability in a mouse model of Fabry disease.J Am Soc Nephrol. 2009; 20: 1975-1985Crossref PubMed Scopus (50) Google Scholar The levels of protein-bound oxidatively modified amino acids were quantified using tandem mass spectrometry in cultured EA.hy926 cells transfected with GLA-dsiRNA (Gene 1), GBA-dsiRNA (Gene 2), and GAPDH-dsiRNA (Gene 3), respectively. Low levels were detectable in control-dsiRNA–transfected EA.hy926 cells and served as basal levels. 3NT, a specific marker for reactive nitrogen species, was markedly elevated (40- to 120-fold) in GLA-dsiRNA–transfected cells (Figure 4a) without corresponding changes in other oxidized tyrosines, including 3-chlorotyrosine, ortho-tyrosine, meta-tyrosine, and o,o′-dityrosine (Figure 4b), consistent with the mouse model of FD.5.Shu L. Park J.L. Byun J. et al.Decreased nitric oxide bioavailability in a mouse model of Fabry disease.J Am Soc Nephrol. 2009; 20: 1975-1985Crossref PubMed Scopus (50) Google Scholar Double GBA-dsiRNA transfection modestly increased 3NT formation. However, this change was not associated with Gb3 accumulation in that GBA-dsiRNA transfection did not raise Gb3 under any conditions (Figure 2d and e), and also not related with repeated siRNA reagents as double GAPDH-dsiRNA transfection did not increase compared with that in single EA.hy926 cells (Figure eNOS activity was measured in GLA-dsiRNA–transfected cells. As in eNOS activity correspondingly decreased in the EA.hy926 cells, and the reduction was on day 3 following the first GLA-dsiRNA transfection and on day 6 following the second GLA-dsiRNA was in and cells with cells (Figure consistent with a specific effect of Gb3 accumulation on eNOS 3NT levels in plasma samples were measured using mass spectrometry in the GLA knockout A increase in 3NT formation was observed between and mice (Figure A more elevation of in mice was measured compared with mice at the consistent with the age-dependent accumulation of Gb3 in the mice a level of in the plasma than by of to 3NT has been with elevated a of nitric oxide and reactive oxygen PubMed Scopus Google Scholar of aortic demonstrated levels of 3NT in from Gla null compared with mice at 2 and of (Figure A sixfold increase in 3NT formation in the Gla null mouse compared with was confirmed using (Figure plasma samples from Fabry patients were for oxidized amino acids including 3NT and compared with control samples that were age- and gender-matched from the patients classical of FD including and in with low or GLA increased levels of plasma and high concentrations of plasma disease and control disease or or activity of involvement enzyme alpha-galactosidase A activity to the in in alpha-galactosidase not transient and were not between Fabry and control on a was in a new enzyme alpha-galactosidase A activity to the in in alpha-galactosidase not transient and were not between Fabry and control on a was A more than sixfold elevation in 3NT concentration was observed in the Fabry compared with the controls. in the concentrations of or or were measured between the (Figure and correlation was observed between the 3NT levels and the plasma or plasma levels of the Fabry These are consistent with eNOS uncoupling and elevated reactive nitrogen species in patients with classical oxidized amino disease oxidized concentrations were assayed as in the and The are as of and of performed in a new with The oxidized concentrations were assayed as in the and The are as of and of performed in FD the of the by and R. of Rev Physiol. PubMed Scopus Google Scholar the loss of GLA activity by alpha-galactosidase PubMed Scopus Google Scholar and the of lysosomal in endothelial cells and cells of the by et and of the PubMed Scopus Google Scholar work by and C.C. as a and of a PubMed Google Scholar and et et in disease. J PubMed Scopus Google Scholar documented that the disease was due to a deficiency of GLA with the accumulation of The use of recombinant GLA as enzyme replacement therapy is the of for the of FD. several the of enzyme replacement therapy for FD have been to et and of enzyme replacement therapy for Fabry J PubMed Scopus Google et long-term renal after of therapy in patients with Fabry disease.J Am Soc Nephrol. PubMed Scopus Google Scholar the from to a for FD is from the of as or renal have been including patients in disease Finally, there are a limited of of FD and are to be directly related to the pathogenesis of the vasculopathy. a mechanism for the vascular disease and a between a biomarker and the of the vasculopathy would be a in the of these have been for the pathogenesis of the vasculopathy of FD. The first that circulating in the of the arterial cell and of the A increase in stress results in increased expression of with formation of reactive oxygen species and These changes to a in NO and increase in The basis for this has been et in patients with Fabry and Metab. 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Whitesall S.E. D'Alecy L.G. et al.Vascular dysfunction in the alpha-galactosidase A-knockout mouse is an endothelial cell-, plasma membrane-based defect.Clin Exp Pharmacol Physiol. 2008; 35: 1156-1163Crossref PubMed Scopus (45) Google Scholar The was with endothelial consistent with an in the plasma of the of aortic endothelial cells were to that increased plasma Gb3 was the basis for the L. et in model of Fabry disease.J Am Soc Nephrol. 2005; PubMed Scopus Google L. accumulation of globotriaosylceramide in the vascular of alpha-galactosidase A null PubMed Scopus Google Scholar The Gb3 content of Gla knockout endothelial cells was markedly elevated in the plasma as as the This increase was associated with decreased eNOS expression, eNOS and levels of high L. 2012; Scholar These were consistent with a for Gb3 in the of the including These models of thrombosis, atherogenesis, and impaired eNOS as a likely basis for the inducible models of vasculopathy. to the of NO have been consistent but in a for eNOS of the in Fabry patients and controls, have documented decreased arterial in the Fabry et and changes in of Fabry PubMed Scopus Google et of endothelial in patients with Anderson-Fabry 2005; Google Scholar This was confirmed using and using in the presence of dysfunction in Fabry disease.J 2003; PubMed Scopus Google Y. et in patients with J. 1983; PubMed Scopus Google Scholar other decreased has been measured in Fabry J. et but endothelial in patients with Fabry disease.J 2005; PubMed Scopus Google A. in Anderson-Fabry disease measured by 2005; PubMed Scopus Google Scholar However, has demonstrated increased in FD with that when eNOS was by R. et in Fabry PubMed Scopus Google Scholar on have also results either et and impaired in patients with Fabry disease.J PubMed Scopus Google Scholar or enhanced et in Fabry disease with after enzyme 2002; PubMed Scopus Google Scholar These may potentially be by eNOS uncoupling with stress, in A for eNOS uncoupling is by a of endothelial cells in which Gb3 resulted in an increase in reactive oxygen species. Gb3 was also associated with an increase in the of vascular cellular and et stress and cell expression in Fabry disease endothelial Metab. 2008; PubMed Scopus Google Scholar The whether changes in Gb3 content in an endothelial cell line would be to for eNOS EA.hy926 cells were employed. for the reproducible loss of GLA activity by siRNA were and the accumulation of Gb3 was Under these conditions, there was a in eNOS activity consistent with the impaired formation of eNOS uncoupling with reactive nitrogen species formation was observed as measured by the formation of 3NT but not of other These changes were not observed when GBA activity was Previous have a for plasma 3NT as a marker for established cardiovascular disease in et and of patients with established disease high by reactive nitrogen J PubMed Scopus Google et of levels with cardiovascular disease and by 2003; PubMed Scopus Google Scholar To whether 3NT be a marker of vasculopathy in FD, mouse plasma from GLA knockout and mice were assayed and 3NT levels were markedly increased and were elevated in knockout mice. changes in 3NT were measured in the mouse The absence of in ortho-tyrosine, meta-tyrosine, and in mouse plasma or aortic samples from the Gla null mice that eNOS uncoupling is to for the reactive oxygen species amino in human atherosclerosis and of 2002; PubMed Scopus Google Scholar Finally, 3NT levels were measured in biobanked plasma samples from patients with classic FD. A and increase in 3NT concentrations was measured in the absence of comparable changes in other oxidized amino These that eNOS uncoupling is likely in human GLA deficiency and is a biomarker that directly with a vascular This with numbers of including those with of the disease. elevated plasma 3NT levels the degree of vascular involvement in FD, may better the for 3NT in the of the the to patients for vascular and the of to Importantly, the ability to eNOS uncoupling directly by the 3NT in an in endothelial cell the Gla knockout and in patients with FD also a for the of more effective and Fabry mice were and as L. Park J.L. Byun J. et al.Decreased nitric oxide bioavailability in a mouse model of Fabry disease.J Am Soc Nephrol. 2009; 20: 1975-1985Crossref PubMed Scopus (50) Google Scholar were in with the of on the and of EA.hy926 cells were from EA.hy926 cells are a human cell line established by the of human cells with a of A549 C.J. McDonald C.C. Graham J.B. Permanent cell line expressing human factor VIII-related antigen established by hybridization.Proc Natl Acad Sci USA. 1983; 80: 3734-3737Crossref PubMed Scopus (1358) Google Scholar EA.hy926 cells were in growth of and and at a of siRNA were and by The numbers for GBA, and GAPDH were and respectively. siRNA Dicer-substrate 27-mer concentrations of the were at in of and were at for and to storage at day siRNA EA.hy926 cells were into a of growth The transfection was was into of to the and the siRNA was with of at The were and at for to the The was with of without and and the siRNA was into the cell an transfection was to a concentration of the second day of the was by growth A second siRNA transfection was performed on day 4 following the first transfection as The transfected EA.hy926 cells were at the days for amino or cell viability L. accumulation of globotriaosylceramide in the vascular of alpha-galactosidase A null PubMed Scopus Google Scholar of EA.hy926 cells L. Park J.L. Byun J. et al.Decreased nitric oxide bioavailability in a mouse model of Fabry disease.J Am Soc Nephrol. 2009; 20: 1975-1985Crossref PubMed Scopus (50) Google Scholar of GLA anti-human GLA was at a of 3NT levels in mouse aortic were with mouse 3NT at a of or samples were and in A of in cellular were with and with concentrations of including and ortho-tyrosine, 3NT and were and samples were for in with The was subjected to with amino acids were quantified using tandem mass spectrometry in the as L. Park J.L. Byun J. et al.Decreased nitric oxide bioavailability in a mouse model of Fabry disease.J Am Soc Nephrol. 2009; 20: 1975-1985Crossref PubMed Scopus (50) Google Scholar The of the peak of the with corresponding were to levels of the were to the content of the precursor of 3NT, and or to the precursor for and A. Byun J. of amino by tandem mass PubMed Scopus Google Scholar eNOS in and EA.hy926 cells were measured following an as L. Park J.L. Byun J. et al.Decreased nitric oxide bioavailability in a mouse model of Fabry disease.J Am Soc Nephrol. 2009; 20: 1975-1985Crossref PubMed Scopus (50) Google Scholar was from the using a The was at for at of plasma was and at for of oxidized amino from and GLA null mice transfections were in A at mouse were with a and in The was with the of immunoblotting after levels were assayed from biobanked plasma samples from the in that those from classical FD FD was by GLA deficiency as GLA activity relative to the in and by GLA J.B. et for lysosomal in human and its to PubMed Scopus Google Scholar and the presence of elevated J.E. et globotriaosylsphingosine is a of Fabry Natl Acad Sci USA. 2008; PubMed Scopus Google Scholar FD was as in L. et on for Fabry of with of plasma and plasma Gb3 were as in et of globotriaosylsphingosine in plasma and of patients by mass 2013; PubMed Scopus Google Scholar using for of and for plasma Gb3 of was as an in of The was by samples from patients with classical FD were However, Fabry the measured was from the that at the of the was from with renal failure. The FD patients were age- and gender-matched to controls, derived from an that with an of FD on GLA of the or by et al.Vascular of Fabry disease in to and in plasma 2012; PubMed Scopus Google Scholar FD and control The to the of and was under at the of and the of Cell were using the using or of and are as between control and samples were at a on samples were performed using are as between control and FD were compared using between 3NT levels and were compared using was This work was by the of spectrometry were performed in the of of and of 3-nitrotyrosine levels and in the plasma of classical Fabry patients and of plasma 3-nitrotyrosine levels and of classical Fabry patients and of plasma 3-nitrotyrosine levels and concentration in Fabry plasma is to the of the at with 2 with 3 with