Pulmonary arterial hypertension (PH), high blood pressure within the lung, is a progressive disease characterized by an increase in pulmonary arterial pressure and the formation of muscle around normally non-muscular small pulmonary arteries. Without treatment, PH progresses rapidly to right heart failure and death. One model used to study this disease is chronic exposure to low oxygen levels. This causes PH in humans and animals and has led to the identification of a number of proteins involved in the development of this disease. However, the mechanism(s) sensing the initiating event and transducing this signal into changes in protein expression to alter pulmonary physiology are unclear.

This proposal examines the role of S-nitrosothiols (SNO) in the development of PH. In the pulmonary circulation, red blood cells (RBCs) deliver SNOs to recipient target proteins as a function of oxygen saturation. In this context, RBCs act as a molecular switch, monitoring changes in oxygen saturation to deliver SNOs to the vascular endothelium, the cells lining the blood vessel. In the experiments proposed, the compound N-acetyl cysteine (NAC) will be used as a tracer to (1) monitor SNO formation, transfer and metabolism in vivo, (2) address the physiological and pathological consequences of SNO signaling in the pulmonary vasculature, and (3) identify SNO target proteins in this signaling pathway in vivo.

The formation, transfer, and degradation of SNO will be examined by monitoring the formation of S-nitroso-N-acetyl cysteine (SNOAC), the S-nitrosylated form of NAC in plasma. Using mass spectroscopy and reductive chemiluminescence, the abundance and rate of formation of SNOAC will be determined as a function of exposure time, dose, and red blood cell oxygenation state. Transfer of SNOs to and from hemoglobin present in RBC and albumin in the serum will be measured. Physiological changes in the pulmonary vasculature indicative of PH (right heart weight, right ventricular pressure, and vessel morphology) as a result of unregulated delivery of SNOAC to the pulmonary endothelium can be monitored. The importance of endothelial nitric oxide synthase (eNOS, involved in SNO formation), S-nitrosoglutathione reductase (GSNO-R, involved in SNO breakdown), and hypoxia inducible factor-1 (HIF-1, the downstream effector) in NAC-induced PH will be elucidated.

The mechanism by which NAC activates HIF signaling will be determined. Studies will examine whether the effect of NAC on HIF-1 expression is transcriptional or post-transcriptional and whether the expression of HIF responsive genes is activated. The effects of NAC on the expression and activity of enzymes that target HIF for degradation (specifically HIF prolyl hydroxylases) will be examined.

One risk factor for the development of PH is gender. This may be tied to the differences in the ability of males and female humans to transport oxygen (thus deliver SNOs to the endothelium), degrade SNOs (activity of GSNO-R), and regulate nitric oxide synthase activity (formation of SNOs). Currently, there are no studies examining the relationship between SNO bioavailability, sex hormones, and the development of PH.

In summary, preliminary data indicate that chronic NAC administration resembles long-term exposure to low levels of oxygen in causing PH in a male mouse. The ability of NAC to affect the pulmonary vasculature appears to be mediated through S-nitrosylation mediated through RBC deoxygenation and requiring the expression of eNOS. NAC- and SNOAC-induced increases in HIF expression and activity appear to occur by altered HIF prolyl hydroxylase expression and/or activity. Gender differences can be explained by differential SNO breakdown in male and female mice and an effect of testosterone.

PH causes significant morbidity and is ultimately fatal despite promising new research and therapies. The results produced from the proposed experiments may (1) highlight the importance of SNOs in the pathogenesis of PH, (2) define a mechanism by which S-nitrosothiols can upregulate genes associated with PH in the pulmonary vascular endothelium, and (3) explain the gender imbalance for the development of this disease. Clinically, it will be important to screen individuals who use NAC as a dietary supplement or receive long-term NAC treatment, as the risk to develop PH may be elevated. Lastly, identifying the components within this signaling cascade may provide novel approaches for the treatment of this disease.