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 μmol/L, 24 h) treatment in endothelial cells. Tetrahydrofolate (THF) is formed after incubation of purified DHFR or cellular extracts with 50 μmol/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 H₄B and NO bioavailability while decreasing superoxide (O₂⁻) 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 H₄B bioavailability, and this was highly effective in reducing Ang II infusion (0.7 mg/kg/day, 14 days)-stimulated aortic O₂⁻ 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.

Introduction

The most widely known therapeutic function of folic acid (FA) is to prevent birth defect (primarily neural tube defects), based on its role in neonatal development [1], [2]. Its other major therapeutic function is to treat patients with hyperhomocysteinemia [3]. FA is required for the remethylation of homocysteine to form methionine, thus reducing the homocysteine level in the plasma. Oral supplementation of FA effectively decreases plasma homocysteine levels [3]. Both hyperhomocysteinemia and hyperhomocysteinuria have been shown to be independent risk factors for atherosclerotic vascular diseases [4], [5], [6], [7], [8], [9], [10]. One of the molecular mechanisms responsible for hyperhomocysteinemia-induced disease is increased production of reactive oxygen species and consequent endothelial dysfunction [11], [12]. It is however unclear whether FA, besides reducing homocysteine levels metabolically, modulates endothelial function directly in subjects with or without hyperhomocysteinemia. It is also unclear whether FA treatment of hyperhomocysteinemic patients is beneficial in disease progression and prognosis.

As a FDA-approved agent, FA or combined vitamin therapy has been investigated for its role in cardiovascular therapeutics in various clinical conditions, and the outcomes (myocardial infarction, stroke, thromboembolic events and mortality) have remained controversial [5], [13], [14], [15]. However, it is worth noting that the larger trials (i.e. HOST [14], VISP [16], HOPE-2 [17]) were conducted in subjects with advanced atherosclerotic vascular diseases where disease regression might be more difficult. Even though, a significant reduction was observed in stroke morbidity with FA treatment after removal of the HOST trial [14], [18] (subjects with renal failure) based on meta-analysis [5], [19], [20], [21]. Therefore, FA supplementation can potentially benefit patients with vascular diseases, although the role of homocysteine during the course of interventions is unclear [22]. More interestingly, FA supplementation was demonstrated to improve endothelial function in subjects without elevated homocysteine levels, indicating an independent effect [22]. Whether this is attributed to direct nitric oxide (NO) production and/or reduced oxidant stress, as well as the potential underlying mechanisms, remains to be fully elucidated.

Accumulating evidence has established that a deficiency in endothelial nitric oxide synthase (eNOS) cofactor tetrahydrobiopterin (H₄B) causes eNOS to produce superoxide (O₂⁻) rather than NO, resulting in eNOS uncoupling and increased oxidant stress [23], [24]. This change in eNOS enzymatic function is independent of gene regulation. Under pathological conditions such as diabetes and ischemic renal dysfunction where angiotensin II (Ang II) levels are elevated [23], we and others have shown that a deficiency in H₄B salvage enzyme dihydrofolate reductase (DHFR) is responsible for reduced H₄B and NO bioavailability [23], [25], [26], [27]. Therefore in the present study we examined FA regulation of DHFR protein expression and activity, and DHFR-mediated changes in NO and O₂⁻ productions in cultured endothelial cells and Ang II-infused mice aortas.

A sensitive HPLC-based activity assay for DHFR was first established. Endothelial cells were exposed to Ang II in the presence or absence of FA, followed by detection of NO and O₂⁻ production specifically and quantitatively using electron spin resonance (ESR). Basal FA regulation of DHFR expression and activity were also analyzed. In Ang II-infused mice, oral administration of FA was examined for its effects on aortic productions of NO and O₂⁻. Overall FA potently improved NO bioavailability, intracellular H₄B content while reducing oxidant stress both in vitro and in vivo. These responses were found dependent on an upregulation in DHFR. These data demonstrate an innovative mechanism whereby FA may protect against cardiovascular disorders in a general population.