Definitions

It is important to set forth the authors' working definitions before proceeding with this review.

ATHEROSCLEROSIS is a systemic dysfunctional endothelial, focal occurring, chronic inflammatory, fibroprolifertive, angiogenic, prothrombotic, multifactorial disease of the arterial intima caused by the retention of modified low density lipoproteins, hemodynamic stress, and accelerated by redox stress [5, 8–11].

T2DM is a heterogeneous, multifactorial, polygenic disease characterized by a defect in insulin's secretion (the beta cell secretory defect) and action (insulin resistance) which results in elevated glucose [8, 12].

T2DM may be viewed as a dynamic continuum of five stages (table 9) [12].

THE L-ARGININE ARM

The substrate L-arginine is an essential amino acid and is usually not thought to be rate limiting (with the exception of high demands in a dysfunctional endothelial state such as atherosclerosis or the atheroscleropathy associated with T2DM).

Arginine deficiency is a rare occurrence, however, there can be competitive inhibition by the endogenously produced asymmetrical dimethylarginine (ADMA) and nitroarginine. ADMA is emerging as an important cause of endothelial cell dysfunction.

It is an endogenously produced competive inhibitor of L-arginine which results in a relative deficiency of the natural substrate for the eNOS enzyme to produce eNO. ADMA accumulates by either a process of increased synthesis by the protein arginine N-methyltransferases (PRMTs) or decreased elimination by the enzyme dimethylarginine dimethylaminohydrolose (DDAH) [14–17].

DDAH selectively hydrolyzes ADMA to L-citrulline and dimethylamine, so that the higher the DDAH activity, the lower the ADMA levels. Importantly, inhibition of DDAH activity results in increased ADMA levels. Both native and oxidized LDL-cholesterol have been shown to up-regulate PRMT while causing a decrease in the activity of DDAH [18]. This results in an elevation of ADMA levels contributing to endothelial cell dysfunction and increased oxidative stress to the intima with subsequent accelerated atherosclerosis.

There are multiple causes for the elevation in ADMA levels (figure 2). Each of these toxicities relate to the A-FLIGHT acronym responsible for the production of ROS (table 6). Recently, Faldetta CM, Laurenti O, and Desideri G [19] were able to demonstrate in both normal human subjects and T2DM human patients that acute L-arginine infusion decreased plasma total homocysteine concentrations, counteract oxidative stress, and increased the availability of nitric oxide.

In summary, the relative deficiency of L-arginine due to elevation of ADMA levels contribute to oxidative stress and result in the atheroscleropathy associated with insulin resistance, metabolic syndrome, prediabetes, and overt T2DM.

THE eNOS ARM

If there is a complete loss of eNOS enzyme as in the eNOS -/- knockout mouse model of Duplain and Sherrer [20] insulin resistance, hypertension, and hyperlipidemia develop. Their findings indicate that eNOS plays a major role in the regulation of insulin sensitivity. To have an effect on atheroscleropathy and T2DM it would not require a complete knockout of the gene but just one or two changes in the 1203 amino acid protein structure in the eNOS enzyme.

Such a gene polymorphism does exist and may become a major player in the polygenic causes of T2DM and atheroscleropathy:

Glu298 → Asp Gene Polymorphism (Glu298 → Asp GP)

eNOS may be impaired by abnormal structure and or function that occurs in gene polymorphisms.

One of the first significant eNOS gene polymorphisms described to date is Glu 298 → Asp. This amino acid substitution is responsible for a statistically significant increased frequency of hypertension and spastic angina [21]. Also, described in a different set of patients is the occurrence of increased abnormal coronary angiographic findings of atherosclerosis and an association of increased events [22–24].

The Glu298 → Asp GP is responsible for a decrease in basal NO production in healthy subjects according to the publication by Veldman BA et al [25].

This gene polymorphism then could interact with other gene polymorphisms and especially environmental conditions such as smoking, obesity, and the A-FLIGHT toxicities of IR, MS, PD, and overt T2DM. Or as in the hypothesis, could be responsible for the development of IR and MS which then would progress to PD or overt T2DM. We now have a Japanese population, a UK population, and an Italian population that correlates Glu298 → Asp GP to atherosclerosis and CAD events [23–25].

These findings indicate that the eNOS gene is important to the development of accelerated atherosclerosis → atheroscleropathy. Furthermore, these three distinct patient populations indicate that this gene polymorphism is widely distributed throughout diverse patient populations and according to Veldman [25] is present in approximately 10 % of patient populations.

Recently, Noiri E and colleagues have shown that there is a significant association of the Glu298 → Asp GP and end stage renal disease in T2DM which once again points to the importance of the endothelial cell and the eNOS reaction [26].

As the human genome unfolds there may be other related important gene polymorphisms identified.

The BH4 ARM

Tetrahydrobiopterin (BH4) is the necessary cofactor for coupling L-arginine to the NAD(P)H oxidase enzyme in order for L-arginine to be oxidized to NO and L-citrulline. The importance of this cofactor and its mechanism is just starting to emerge.

If this cofactor is not functioning properly the entire eNOS reaction uncouples and the endothelium becomes a net producer of superoxide [O2'] through the uncoupled NAD(P)H oxidase reaction. In addition to BH4 being the necessary cofactor it also serves as a naturally occurring antioxidant capable of scavenging ROS such as superoxide O2' and perioxynitrite [ONOO']. If BH4 is deficient in supply or function the eNOS reaction not only is capable of uncoupling with subsequent excess [O2'] and [ONOO'] but also is at a disadvantage in that there would be less BH4 to scavenge these local occurring reactive oxygen species being produced in excess amounts [27].

It is important to note that a direct effect of folic acid is to keep this important cofactor BH4 intact so that it can maintain coupling of the eNOS reaction.

The lowering of homocysteine is an indirect effect of folic acid aiding in the decreased production of ADMA due to the effect of Hcy on DDAH. Folic acid given to patients with T2DM have an improvement of endothelial relaxation before Hcy has time to be lowered. It works almost immediately before the Hcy is lowered [28, 29].

When you look at the three arms of importance for the eNOS reaction there are two possible changes in the treatment paradigm (in addition to lowering the substrates of the A-FLIGHT toxicities (table 6) and the RAAS acronym [8]) for these patients who suffer from endothelial dysfunction associated with atheroscleropathy, IR, MS, PD, and T2DM.

First, we need to take a global risk reduction approach (by treating each risk factor in the A-FLIGHT toxicities acronym in order to decrease the clinical substrates of lipids, hypertension, and glucotoxicity without elevating endogenous insulin or amylin) in order to decrease the progression of the disease. Second, we should now include in the treatment paradigm the use of folic acid and arginine supplementation. T2DM is now considered to be a coronary risk equivalent and therefore all patients with T2DM should be on folate supplementation, as well as, the non-diabetic atherosclerotic patient.

Those patients with persistent endothelial cell dysfunction after the standard goals have been reached should consider arginine supplementation and possibly should be considered even sooner in the treatment paradigm if there is a history or evidence of symptomatic peripheral arterial disease, congestive heart failure and / or diastolic dysfunction [17, 30, 31].

Folic acid – 5MTHF

This water soluble B vitamin has been recently gaining considerable attention due to its positive role in cardiovascular disease, neurodegenerative disorders, neural tube defects, and cancer. Folic acid plays an important role in the remethylation of homocysteine and is thus capable of lowering elevated levels of homocysteine (figure 3) [32].

Deficiency or impairment of folate metabolism is associated with hyperhomocysteinemia (hHcy), hypomethylation (the decreased one carbon unit transfer to purines and pyrimidines for DNA repair and biosynthesis), DNA damage, and impaired cell proliferation, malignancies, and impaired eNO production [33–37]. 5-MTHF is an electron donor, a hydrogen donor, and a methyl donor.

Each of these attributes confers a unique role to its function. In regards to the eNOS reaction it supplies both hydrogen and electrons to BH2 and BH3 to restore BH4 (the necessary cofactor for the eNOS reaction). This attribute allows the eNOS reaction to re-couple and restores the capability of this reaction to be once again a net producer of eNO and restores endothelial dependent vasodilatation in T2DM and hyperlipidemia [38]. Recently, the combination of folic acid, vitamin B12, and vitamin B6 was able to demonstrate a reduction of in-stent restenosis and recurrent events in up to one year follow up in the Swiss heart study [39–41].