The G6PD a- Variant Does Not Modulate Pulmonary Hypertension Phenotypes in Mice

Glucose-6-phosphate dehydrogenase (G6PD) is the most common human enzyme deficiency, affecting over five hundred million people worldwide. Clinically, this manifests as acute neonatal jaundice, acute hemolysis following exposure to oxidative stress, or severe chronic non-spherocytic hemolytic anemia. G6PD catalyzes the rate-limiting step of the pentose phosphate pathway (PPP) and regenerates reduced NADPH. Antioxidant enzymes such as glutathione reductase require NADPH. Since RBCs do not contain mitochondria, the PPP pathway is their only source of reduced NADPH, and cells deficient for G6PD are highly susceptible to oxidative hemolysis.

Intravascular hemolysis releases the contents of the RBC into the plasma. Cell-free hemoglobin (Hb) reacts with nitric oxide (NO) in a near-diffusion limited reaction to inhibit NO signaling resulting in Vasoconstriction. Additionally, NO is known to play an important role in the immune response and inflammation. These factors lead to pulmonary vascular dysfunction and can result in pulmonary hypertension (PH). PH is a fatal vascular disease with proliferative vascular remodeling, blocking the lumen of the pulmonary arteries and leading to right ventricular (RV) dysfunction.

There has been a recent focus on determining the relationship between G6PD-deficiency and its contribution to PH, with evidence supporting a protective effect while others demonstrate an increased propensity for elevated pulmonary pressures. To help delineate the contribution of G6PD to PH, we generated a novel humanized mouse model G6PD A- carrying the most common African variant (V68M - < 10% residual enzymatic activity). We induced intravascular hemolysis in G6PD A- mice model by repeated injections of low dose phenylhydrazine (PHZ) and found that these mice are susceptible to oxidative hemolytic anemia following PHZ injection and represent good model to study the effects of this variant on physiology and pathophysiology.

WT and G6PD A- mice were exposed to 10% hypoxia for 3 weeks and hemodynamic parameters were assessed compared to normoxic (21% O₂) controls. Right ventricular systolic pressure was determined by right heart catheterization and right heart remodeling was assessed using the Fulton Index (a weight ratio of the right ventricle divided by the sum of left ventricle and septum (RV/[LV + S])). Following hypoxia treatment both WT and G6PD A- mice developed increased pulmonary pressures and had evidence of right heart remodeling, suggesting that G6PD deficiency is not protective against pulmonary vascular remodeling. Plasma Hb concentrations and reticulocytes were not different between genotypes or treatments, indicating a lack of intravascular hemolysis during hypoxia exposure. To determine if repeated hemolysis results in the development of PH, WT and G6PD A- mice were exposed to primaquine, a common antimalarial that induces hemolysis in erythrocytes with limited oxidative capacity. Following 3 or 12 weeks of Primaquine exposure, G6PD A- mice had decreased in Hb levels, red blood cell counts, hematocrit and increased reticulocytes, suggesting the induction of intravascular hemolysis. Pulmonary pressures were unaffected by primaquine treatment and there was no evidence of right ventricular remodeling in either WT or G6PD A- mice.

In summary, we have demonstrated that G6PD A- mice do not develop spontaneous pulmonary hypertension, nor are they protected from phenotype when exposed to hypoxia. Additionally, repeated episodes of intravascular hemolysis did not cause elevated pulmonary pressures or right heart remodeling in G6PD A- mice. These findings suggest that G6PD A- variant does not modulate pulmonary hypertension phenotypes in mice. These findings are in line with a dearth of clinical data associating incidences of PH in G6PD-deficient individuals.