(28ab) The Potential of Tetrahydrobiopterin As an Early Biomarker of Cardiovascular Diseases: A Computational Analysis

Cardiovascular diseases (CVDs) are one of the key contributors to morbidity and mortality in both developed and developing countries. Additionally, the high incidence of CVDs globally poses a significant economic burden on the healthcare system of countries. Current diagnostic tools for CVDs (e.g. blood-based biochemical tests, electrocardiogram, echocardiography, coronary angiography, and stress testing) are in many cases unable to detect CVDs in their early stages. Limitations associated with current CVD-based diagnostic tools can be resolved by (i) identification of a suitable biomarker that can be detected under normal physiological conditions and early/intermediate/advanced stages of CVD, and (ii) quantitative estimation of plasma-based concentrations of the identified biomarker at different stages of CVD.

Several studies have reported that endothelial dysfunction (ED) is the initiating event in the pathogenesis of CVDs. ED can be described as a condition where endothelial cells lining blood vessel walls lose their ability to regulate vascular homeostasis. Endothelial cells regulate vascular homeostasis by releasing anti-inflammatory signaling molecules including nitric oxide (NO) and prostacyclin. The NO released by endothelial cells acts to regulate vascular homeostasis by (i) maintaining physiological levels of vascular tone and (ii) inhibiting pro-inflammatory mechanisms including platelet aggregation, leukocyte adhesion, and leukocyte activation. However, prolonged exposure of endothelial cells to cardiovascular risk factors (e.g., smoking, hyperglycemia, and hypercholesterolemia) promotes ED by (i) increasing expression of pro-inflammatory signaling molecules (e.g., reactive oxygen species (ROS), reactive nitrogen species (RNS), endothelin-I and tumor necrosis factor-alpha (TNF-α)) in endothelial and other vascular cells, (ii) reducing the synthesis rate of NO in endothelial cells and (iii) reducing the bioavailability of NO in the vasculature. Endothelial dysfunction has also been reported in patients with no exposure to potential cardiovascular risk factors but with a family history of CVD.

The primary source of NO production in endothelial cells is the constitutive enzyme, endothelial nitric oxide synthase (eNOS). Under normal physiological conditions, eNOS catalyzes the oxidation of the amino acid, L-Arginine to produce NO. However, eNOS uncouples and transforms from a NO-producing enzyme into a ROS-producing enzyme during ED. Several studies have reported that the extent of eNOS uncoupling depends on the availability of the eNOS co-factor tetrahydrobiopterin (BH₄). Hence, the eNOS co-factor tetrahydrobiopterin (BH₄), can be used as a potential biomarker for the early detection of ED/CVDs. It has been reported that the synthesis rates of BH₄ in endothelial cells increase during endothelial dysfunction. However, this does not manifest in the form of improved endothelial cell functionality. Additionally, clinical studies for testing the efficacy of BH₄, during ED, have yielded mixed results. These ambiguous results demonstrate that the molecular mechanisms modulating BH₄ levels in the vasculature remain poorly understood.

Increased vascular ROS and RNS levels during ED can reduce the availability of BH₄ in endothelial cells by oxidizing it to trihydrobiopterin radical (BH₃) and dihydrobiopterin (BH₂). The BH₂ formed competes with BH₄ for binding to eNOS. Binding of BH₂ to eNOS uncouples eNOS. Hence, a quantitative understanding of the complex biochemical interactions between NO, ROS, RNS, and biopterin (BH₄, BH_3, and BH₂) is necessary to examine the feasibility of using BH₄ as a biomarker for ED/CVD. It is to be noted that ED primarily occurs at the microcirculation level where the blood vessels are extremely small in size (50-100 μm in diameter). There are several ROS/RNS-mediated localized events occurring inside the microcirculation-based blood vessels and in the endothelial cells lining these blood vessel walls. These events are often difficult to analyze experimentally due to their reductionist approach. To address the experimental limitations, computational biotransport models can be used to develop a quantitative understanding of the mechanisms regulating BH₄ levels in the vasculature. These biotransport models are developed using the principles of mass conservation and reaction kinetics.

In this study, we have developed a computational biotransport model at the microcirculation level to investigate the complex biochemical interactions between NO, primary ROS and RNS (superoxide and peroxynitrite, respectively), and biopterin (BH₄, BH_3, and BH₂). The different levels of endothelial cell functionality are modeled by changing the endothelial NO and superoxide production levels in accordance with values reported in the literature. The model also accounts for the endothelial cell-based generation of BH₄ and the BH₄ consumption routes due to oxidation by oxygen, superoxide and peroxynitrite. Our model results showed that (i) the transition from normal physiological condition to moderate endothelial cell oxidative stress condition reduced luminal BH₄ levels by an order of magnitude (140 nM to 45 nM) and (ii) the transition from moderate to severe endothelial cell oxidative stress condition reduced luminal BH₄ levels by another one order of magnitude (45 nM to 5 nM). The results clearly indicate that BH₄ levels in the blood is an effective biomarker of cardiovascular health. In future, we want to increase the robustness of our model by taking into account the transport and chemical interactions of other ROS and RNS, which are reported to interact with biopterin (BH₄ and BH₃).