C-reactive protein decreases endothelial nitric oxide synthase activity via uncoupling

Abstract

C-reactive protein (CRP), a cardiovascular risk marker, induces endothelial dysfunction. We have previously shown that CRP decreases endothelial nitric oxide synthase (eNOS) expression and bioactivity in human aortic endothelial cells (HAECs). In this study, we examined the mechanisms by which CRP decreases eNOS activity in HAECs. To this end, we explored different strategies such as availability of tetrahydrobiopterin (BH4)-a critical cofactor for eNOS, superoxide (O(2)(-)) production resulting in uncoupling of eNOS and phosphorylation/dephosphorylation of eNOS. CRP treatment significantly decreased levels of BH4 thereby promoting eNOS uncoupling. Pretreatment with sepiapterin, a BH4 precursor, prevented CRP-mediated effects on BH(4) levels, superoxide production as well as eNOS activity. The gene expression and enzymatic activity of GTPCH1, the first enzyme in the de novo biosynthesis of BH(4), were significantly inhibited by CRP. Importantly, GTPCH1 is known to be regulated by cAMP-mediated pathway. In the present study, CRP-mediated inhibition of GTPCH1 activity was reversed by pretreatment with cAMP analogues. Furthermore, CRP-induced O(2)(-) production was reversed by pharmacologic inhibition and siRNAs to p47 phox and p22 phox. Additionally, CRP treatment significantly decreased the eNOS dimer: monomer ratio confirming CRP-mediated eNOS uncoupling. The pretreatment of cells with NO synthase inhibitor (N-nitro-l-arginine methyl ester [l-NAME]) also prevented CRP-mediated O(2)(-) production further strengthening CRP-mediated eNOS uncoupling. Additionally, CRP decreased eNOS phosphorylation at Ser1177 as well as increased phosphorylation at Thr495. CRP appears to mediate these effects through the Fcgamma receptors, CD32 and CD64. To conclude, CRP uncouples eNOS resulting in increased superoxide production, decreased NO production and altered eNOS phosphorylation.

Fig 1

Effect of CRP on eNOS mRNA expression (a), NOS activity (b), and cGMP release (c). HAECs were incubated with CRP (0, 25 and 50 μg/ml) for 12 h. RT-PCR (a) for eNOS mRNA or GAPDH mRNA (as loading control) was performed as described under Methods. 1-C, 2-CRP 25ug/ml, 3- CRP50 ug/ml. The determination of NOS activity (b) and cGMP levels (c) was done as described under Methods.*p<0.05 as compared to control. Data are mean +SEM of 5-7 experiments in duplicate.

Fig 2

a&b) Effect of CRP and supplementation with sepiapterin on BH₄ levels in HAECs. BH₄ measurement was done by HPLC as described in methods section. *p<0.05 as compared to control. The results are mean ± SEM of 5 different experiments done in duplicate. c & d) Effect of supplementation with sepiapterin on CRP-mediated eNOS inhibition as measured by (c) eNOS activity and (d) cGMP release. The results are mean ± SEM of 4 different experiments done in duplicate. *p<0.05 as compared to control, # p<0.05 as compared to CRP.

Fig 3

a & b) Effect of CRP on GTP CH1 mRNA expression (a) and activity (b) in HAECs. The cells were treated with CRP (0, 25 and 50 μg/ml) for 12 hrs. RNA was isolated for RT-PCR for GTPCH1 mRNA or GAPDH mRNA (as loading control) as detailed in methods. 1-C, 2-CRP 25ug/ml, 3- CRP50 ug/ml. GTPCH1 enzyme activity (Neopterin generated) was assayed as detailed in methods. The results are mean ± SD of 3 different experiments done in duplicate and are presented as pmoles/min/mg protein.* p<0.05 as compared to control. c) Role of cAMP-mediated pathway in GTPCH 1 inhibition and eNOS downregulation (d and e) by CRP. Control, CRP (25 ug/ml), cAMP analogues treated cells [(8-Br-cAMP and Db-cAMP 0.5mM each) 1 hr prior to CRP treatment] were used for the measurement of GTPCH1 activity, eNOS activity and NO bioactivity as described in methods. * p<0.05 as compared to control. # p<0.05 as compared to CRP.

Fig 3

a & b) Effect of CRP on GTP CH1 mRNA expression (a) and activity (b) in HAECs. The cells were treated with CRP (0, 25 and 50 μg/ml) for 12 hrs. RNA was isolated for RT-PCR for GTPCH1 mRNA or GAPDH mRNA (as loading control) as detailed in methods. 1-C, 2-CRP 25ug/ml, 3- CRP50 ug/ml. GTPCH1 enzyme activity (Neopterin generated) was assayed as detailed in methods. The results are mean ± SD of 3 different experiments done in duplicate and are presented as pmoles/min/mg protein.* p<0.05 as compared to control. c) Role of cAMP-mediated pathway in GTPCH 1 inhibition and eNOS downregulation (d and e) by CRP. Control, CRP (25 ug/ml), cAMP analogues treated cells [(8-Br-cAMP and Db-cAMP 0.5mM each) 1 hr prior to CRP treatment] were used for the measurement of GTPCH1 activity, eNOS activity and NO bioactivity as described in methods. * p<0.05 as compared to control. # p<0.05 as compared to CRP.

Fig 4

Effect of CRP on eNOS dimer and monomer forms. HAECs were treated with CRP for 12 hrs as reported in methods. The lysates were run on low temperature SDS-PAGE at 70 V on 6% mini-gels. The proteins were transferred on PVDF membrane and blotted for eNOS forms using rabbit anti-human eNOS Ab (1:1000) and anti-rabbit IgG (1:2000). Actin was used as house keeping gene. The upper panel shows the blot which is representative of 5 different experiments. The lower panel shows the densitometric ratio of eNOS dimer:monomer. * p<0.02 compared to control.

Fig 5

(a) Role of NADPH oxidase inhibition on CRP-mediated superoxide production. The cells were transfected with siRNA to p47 phox or control siRNA as detailed in methods section. The cells were incubated with DPI (5 μmol/L) 1 hour prior to CRP treatment (25 ug/ml for 6 hrs). O₂^- production was measured as described in methods. The results are mean ± SEM of 4 different experiments. *p <0.01 as compared to control, #p<0.05 as compared to CRP, **p<0.05 as compared to control, ## p<0.05 as compared to CRP. (b) The cells were transfected with siRNA to p22 phox/CSiRNA or control siRNA as detailed in methods section. At the end of incubation with CRP treatment, western blotting was done for p22 phox and actin as described in methods. Density ratio of p22phox/actin is provided. * p<0.05 as compared to control. (c). Role of p22 phox inhibition on CRP-mediated superoxide production. The cells were transfected with siRNA to p22 phox or control siRNA and superoxide release was measured as detailed in methods section. *p <0.01 as compared to control, #p<0.05 as compared to CRP. The results are mean ± SEM of 4 different experiments. (d) Role of PKC in CRP-mediated superoxide release. The cells were pretreated with PKC inhibitors prior to CRP treatment and superoxide release was measured as detailed in methods. *p <0.01 as compared to control, #p<0.05 as compared to CRP. The results are mean ±SEM of 3 different experiments. (e) Effect of p47 and p22 phox siRNA on CRP-mediated superoxide release as measured by DHE fluorescence as detailed in methods- representative figure of n=3 experiments. (f). Role of NADPH oxidase inhibition on CRP- mediated eNOS inhibition. The cells were incubated with NADPH-oxidase inhibitors (apocynin [0.6 mM] and DPI [5 μM]) 1 hour prior to CRP treatment (25 μg/ml for 12 hrs). The cells were transfected with siRNA to p47 phox or control siRNA as detailed in methods section. At the end of incubation with CRP treatment, eNOS activity and cGMP release were measured as described in methods. The results are mean ± SEM of 4 different experiments done in duplicate. * p<0.05 as compared to control, # p<0.05 as compared to CRP.

Fig 5

(a) Role of NADPH oxidase inhibition on CRP-mediated superoxide production. The cells were transfected with siRNA to p47 phox or control siRNA as detailed in methods section. The cells were incubated with DPI (5 μmol/L) 1 hour prior to CRP treatment (25 ug/ml for 6 hrs). O₂^- production was measured as described in methods. The results are mean ± SEM of 4 different experiments. *p <0.01 as compared to control, #p<0.05 as compared to CRP, **p<0.05 as compared to control, ## p<0.05 as compared to CRP. (b) The cells were transfected with siRNA to p22 phox/CSiRNA or control siRNA as detailed in methods section. At the end of incubation with CRP treatment, western blotting was done for p22 phox and actin as described in methods. Density ratio of p22phox/actin is provided. * p<0.05 as compared to control. (c). Role of p22 phox inhibition on CRP-mediated superoxide production. The cells were transfected with siRNA to p22 phox or control siRNA and superoxide release was measured as detailed in methods section. *p <0.01 as compared to control, #p<0.05 as compared to CRP. The results are mean ± SEM of 4 different experiments. (d) Role of PKC in CRP-mediated superoxide release. The cells were pretreated with PKC inhibitors prior to CRP treatment and superoxide release was measured as detailed in methods. *p <0.01 as compared to control, #p<0.05 as compared to CRP. The results are mean ±SEM of 3 different experiments. (e) Effect of p47 and p22 phox siRNA on CRP-mediated superoxide release as measured by DHE fluorescence as detailed in methods- representative figure of n=3 experiments. (f). Role of NADPH oxidase inhibition on CRP- mediated eNOS inhibition. The cells were incubated with NADPH-oxidase inhibitors (apocynin [0.6 mM] and DPI [5 μM]) 1 hour prior to CRP treatment (25 μg/ml for 12 hrs). The cells were transfected with siRNA to p47 phox or control siRNA as detailed in methods section. At the end of incubation with CRP treatment, eNOS activity and cGMP release were measured as described in methods. The results are mean ± SEM of 4 different experiments done in duplicate. * p<0.05 as compared to control, # p<0.05 as compared to CRP.

Fig 6

a) Western blots showing time course of VEGF (100 ng/ml for 0-15 min)-induced phosphorylation of Ser1177 and dephosphorylation of Thr495 in control and CRP treated cells. The cell lysates were immunoprecipitated for eNOS and subjected to SDS-PAGE and blotted using specific Abs as described in methods section. bi & ii) Phosphorylation was quantified densitometrically as the ratio of phospho-eNOS/eNOS for blot in Fig 6a. Results are presented as mean ± SEM of 3 different experiments. *p<0.05 as compared to control.

Fig 6

a) Western blots showing time course of VEGF (100 ng/ml for 0-15 min)-induced phosphorylation of Ser1177 and dephosphorylation of Thr495 in control and CRP treated cells. The cell lysates were immunoprecipitated for eNOS and subjected to SDS-PAGE and blotted using specific Abs as described in methods section. bi & ii) Phosphorylation was quantified densitometrically as the ratio of phospho-eNOS/eNOS for blot in Fig 6a. Results are presented as mean ± SEM of 3 different experiments. *p<0.05 as compared to control.

Fig 7

Effect of Fc γ R neutralizing antibodies on CRP-mediated eNOS inhibition. HAECs were incubated with respective antibodies for 1 hour prior to CRP treatment as described in methods followed by the measurement of (a) NOS activity and (b) cGMP. * p<0.05 compared with control, # p <0.05 compared with CRP. The results are mean ± SEM of 3 experiments in duplicate. (c) Effect of Fc γ R neutralizing antibodies and syk kinase inhibitor on CRP-mediated GTPCH1 activity inhibition. *p<0.05 compared with control, # p<0.05 compared with CRP. The results are mean ± SEM of 3 experiments in duplicate.

Fig 7

Effect of Fc γ R neutralizing antibodies on CRP-mediated eNOS inhibition. HAECs were incubated with respective antibodies for 1 hour prior to CRP treatment as described in methods followed by the measurement of (a) NOS activity and (b) cGMP. * p<0.05 compared with control, # p <0.05 compared with CRP. The results are mean ± SEM of 3 experiments in duplicate. (c) Effect of Fc γ R neutralizing antibodies and syk kinase inhibitor on CRP-mediated GTPCH1 activity inhibition. *p<0.05 compared with control, # p<0.05 compared with CRP. The results are mean ± SEM of 3 experiments in duplicate.

Fig 8

Hypothetical schema illustrating the possibility of divergent effects for CRP- mediated eNOS inhibition. CRP uncouples eNOS via 2 different ways: stimulation of NADPH oxidase leading to generation of oxidative stress which causes the formation of superoxide rather than NO from eNOS; CRP also inhibits GTPCH1, the rate limiting enzyme for de novo synthesis of BH4, resulting in decreased BH4 levels. The uncoupled eNOS (as evidenced by decreased dimer:monomer ratio) results in altered phosphorylation of Ser1177 and Thr 495 finally leading to decreased functional activity of eNOS.