Mechanistic insights into folic acid-dependent vascular protection: dihydrofolate reductase (DHFR)-mediated reduction in oxidant stress in endothelial cells and angiotensin II-infused mice: a novel HPLC-based fluorescent assay for DHFR activity

Abstract

Folate supplementation improves endothelial function in patients with hyperhomocysteinemia. Mechanistic insights into potential benefits of folate on vascular function in general population however, remain mysterious. Expression of dihydrofolate reductase (DHFR) was markedly increased by folic acid (FA, 50 micromol/L, 24 h) treatment in endothelial cells. Tetrahydrofolate (THF) is formed after incubation of purified DHFR or cellular extracts with 50 micromol/L of substrate dihydrofolic acid. THF could then be detected and quantified by high performance liquid chromatography (HPLC) with a fluorescent detector (295/365 nm). Using this novel and sensitive assay, we found that DHFR activity was significantly increased by FA. Furthermore, FA improved redox status of Ang II treated cells by increasing H(4)B and NO() bioavailability while decreasing superoxide (O(2)(-)) production. It however failed to restore NO() levels in DHFR siRNA-transfected or methotrexate pre-treated cells, implicating a specific and intermediate role of DHFR. In mice orally administrated with FA (15 mg/kg/day, 16 days), endothelial upregulation of DHFR expression and activity occurred in correspondence to improved NO() and H(4)B bioavailability, and this was highly effective in reducing Ang II infusion (0.7 mg/kg/day, 14 days)-stimulated aortic O(2)(-) production. 5'-methyltetrahydrofolate (5'-MTHF) levels, GTPCH1 expression and activity remained unchanged in response to FA or Ang II treatment in vitro and in vivo. FA supplementation improves endothelial NO() bioavailability via upregulation of DHFR expression and activity, and protects endothelial cells from Ang II-provoked oxidant stress both in vitro and in vivo. These observations likely represent a novel mechanism (intermediate role of DHFR) whereby FA induces vascular protection.

Figures

Figure 1. Effects of folic acid on endothelial DHFR expression and NO• production

A) Representative Western blot of on DHFR expression in FA (50 µmol/L, 24h)-treated endothelial cells; B) Densitometric analysis of the immunoblots from A; C) Representative spectra of NO• production in FA (50 µmol/L, 24h)-treated endothelial cells; D) Grouped densitometric data of NO• production. *p < 0.05 vs. Ctrl.

Figure 2. Specific tetrahydrofolate (THF) peak in the HPLC based DHFR activity assay

A) 50 nmol/L of tetrahydrofolate in assay buffer; B) 50 µmol/L of dihydrofolate in assay buffer (no enzyme and NADPH); C) recombinant DHFR with 50 µmol/L of dihydrofolate in assay buffer (no NADPH); D) boiled recombinant DHFR with 50 µmol/L of dihydrofolate and 200 µmol/L of NADPH in assay buffer (deactivated enzyme); E) recombinant DHFR with 200 µmol/L of NADPH in assay buffer (no substrate); F) recombinant DHFR with 50 µmol/L of dihydrofolate and 200 µmol/L of NADPH in assay buffer; G) Sample constituents.

Figure 3. Optimization of the assay reaction for DHFR activity

DHFR activity is expressed as amount of tetrahydrofolate (THF) produced after 20 min incubation with 200 µmol/L NADPH, 500 ng/ml of recombinant DHFR and 80 µmol/L of dihydrofolic acid at pH 7.4. A) The THF production was examined after 2, 4, 6, 8, 10, 12, 16 and 20 min incubation while the rest of the assay condition remained the same as above; B) THF production was examined at different concentrations of NADPH; C) THF production was examined at different concentrations of recombinant DHFR; D) THF production was examined at different protein concentration of bovine endothelial cell lysates; E) THF production was examined at different pH; F) Km and Vmax of recombinant DHFR from the optimized assay reaction were calculated using Program Hyper 22 for hyperbolic regression analysis of enzyme kinetic data (http://homepage.ntlworld.com/john.easterby/software.html).

Figure 4. Effects of folic acid pre-incubation on Ang II induced changes in NO•, H4B availability and DHFR expression/activity

A) Effect of FA on Ang II induced changes in DHFR expression; B) Effect of FA on Ang II induced changes in DHFR activity; C) Effect of FA on Ang II induced changes in H4B bioavailability; D) Effects of FA on the ratio of oxidized biopterins over total pterins; E) Effects of FA on Ang II induced NO• production. p < 0.05 vs. control, #p < 0.05 vs. Ang II alone.

Figure 5. Effect of DHFR inhibition on folic acid induced NO• production

A) DHFR siRNA on FA-induced DHFR-upregulation; B) DHFR siRNA on FA induced NO• production; C) Methotrexate on FA induced NO• production; D) FA treatment on GTPCH1 mRNA expression; E) FA treatment on GTPCH1 activity; F) FA treatment on intracellular 5’-MTHF level. *p < 0.05 vs. control, #p < 0.05 vs. AngII alone.

Figure 6. Effect of oral folic acid supplementation on aortic NO• and O2•− production in Ang II-infused mice

A) Kinetic assay of aortic superoxide (O2•−) production in AngII infused mice with or without FA supplementation; B) Group analysis of aortic O2•− production from A; C) Representative spectra of NO• production in Ang II infused mice with or without FA supplementation; D) Group analysis of NO• production from C. *p < 0.05 vs. control; #p < 0.05 vs. AngII alone.

Figure 7. Endothelium regulation of DHFR expression and activity by Ang II and folic acid in Ang II infused mice

A) Differential expression profile of DHFR in endothelium-denuded aortas and endothelial cells of untreated and Ang II infused mice; B) Effects of FA on endothelium regulation of DHFR expression in Ang II infused mice; C) Grouped densitometric data of B; D) Effects of FA on endothelium regulation of DHFR activity E) FA diet on endothelium GTPCH1 activity; F) FA diet on endothelium 5’-MTHF level. *p < 0.05 vs. control; #p < 0.05 vs. AngII alone.