Original articleMechanistic 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
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.
Section snippets
Materials
Monoclonal antibodies for DHFR and eNOS were purchased from Research Diagnostics (Flanders, NJ) and BD Transduction Laboratories (San Jose, CA) respectively. Tetrahydrofolate, FA, dihydrofolic acid, recombinant DHFR, NADPH and all other reagents were purchased from Sigma-Aldrich in highest purity (St. Louis, USA).
Cell culture, cell treatments and NO detection
Bovine aortic endothelial cells (BAECs, Cell Systems, Kirkland, WA) were grown in media 199 containing 10% fetal bovine serum (FBS) until confluence, and quiescent with 5% FBS media.
Folic acid upregulates endothelial DHFR expression and NO production
Post confluent bovine aortic endothelial cells were treated with folic acid (FA, 50 μmol/L, 24 h) prior to Western analysis of DHFR and eNOS protein expressions. As shown by representative blot and grouped densitometric data, FA supplementation resulted in a significant upregulation of DHFR while not affecting eNOS expression (Figs. 1A and B). Of note, endothelial cell production of NO was also increased (Figs. 1C and D).
Establishment of a novel HPLC-based assay for DHFR activity
In order to determine specifically DHFR activity from cells, a sensitive
Discussion
The most significant finding of the study is that folic acid (FA) directly improves H4B and NO bioavailability while reducing oxidant production both in vitro and vivo, and that these effects are mediated by an upregulation of dihydrofolate reductase (DHFR) expression and activity. FA treatment of control or Ang II stimulated endothelial cells increased NO production, which was attenuated by DHFR siRNA or methotrexate. In Ang II-infused mice, aortic O2− production was attenuated by FA
Acknowledgments
The authors' work has been supported by National Heart, Lung and Blood Institute (NHLBI) Grant HL077440 (HC), HL057244 (PLL and HC), HL080111 (PPP and HC), an American Diabetes Association Award 7-08-RA-23 (HC), and a Start-up Fund from the University of California Los Angeles (HC). The authors would like to thank Dr. Ting Wang for helpful discussion on HPLC calibration.
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2022, Advances in PharmacologyCitation Excerpt :Under normal physiological conditions BH2 can be reduced to BH4 by dihydrofolate reductase (DHFR), but DHFR is downregulated during oxidative stress in endothelial cells. Folic acid supplementation increases DHFR, thereby reducing BP (Gao, Chalupsky, Stefani, & Cai, 2009; Gao et al., 2012). Sepiapterin, a BH4 precursor, acutely improves endothelium-dependent vasodilation in the SHR aorta (Schuhmacher et al., 2010), and abrogation of its synthesis in mice causes hypertension (Sumi-Ichinose et al., 2017) but does not lower BP in dexamethasone-hypertensive rats (Thida et al., 2010).