Free Arginase-I in Human Pulmonary Microvascular Endothelial Cell Culture Leads to Endothelial Dysfunction and Increased Reactive Oxygen Species.

Endothelial dysfunction in chronic hemolytic diseases (e.g., sickle cell disease) is believed to lead to hemolysis-associated pulmonary hypertension. Dysregulated arginine metabolism, via free arginase-I released during hemolysis, is one mechanism proposed in prior studies. However, the effects of free arginase-I on endothelial cell function have not previously been reported. We hypothesized that free arginase-I would decrease hPMVEC nitric oxide (NO) production and therefore limit NO bioavailability.

Cultured human pulmonary microvascular endothelial cells (hPMVEC), at passage 6-8 and approximately 80% confluence, with increasing arginase-I concentrations added to the media (CN: 0 ng prot/mL, A30: 30 ng prot/mL, A90: 90 ng prot/mL) were studied. To maximize arginase-I activity, 0.1 mM MnCl₂ (an important cofactor for arginase-I) and 3mM of L-arginine were added to each condition. The hPMVEC were incubated for 24h in normoxia (n=8) or hypoxia (1% O₂; n=4), then media was harvested for urea/nitrite analyses and cell protein was harvested for Western blot analyses. ANOVA analyses were conducted to determine statistical significance.

Urea production significantly increased with increasing media arginase-I in both normoxia (CN: 1.47±0.22, A30: 1.46±0.15, A90: 2.09±0.20 umol/mgPr; p<0.05) and hypoxia (CN: 0.913±0.22, A30: 1.01±0.04, A90: 1.99±0.14 umol/mgPr; p<0.005); interestingly, hypoxia means were significantly lower than those in normoxia (p<0.01). However, the percent change from controls in hypoxia (CN: 100±24, A30: 110±4, A90: 218±15) was significantly higher (p<0.05) than in normoxia conditions (CN: 100±15, A30: 99±10, A90: 142±13). Nitrite production showed no significant change with increasing media arginase-I in normoxia (CN: 17.6±2.4, A30: 20.8±4.6, A90: 18.8±2.1 nmol/mgPr; p=0.79); however, in hypoxia nitrites trended down with increasing media arginase-I (CN: 5.26±0.42, A30: 4.35±0.27, A90: 3.69±0.51 nmol/mgPr; p=0.068). In addition, means for nitrite production in hypoxia were significantly less than in normoxia conditions (p<0.0001). In normoxia, Western blot analyses showed significantly decreased 3-nitrotyrosine (3-NT) levels with increasing media arginase-I (CN: 1.00±0.03, A30: 0.88±0.07, A90: 0.77±0.07 [fold change]; p<0.05). 3-NT expression in hypoxia showed no differences with increasing media arginase-I (CN: 1.90±0.16, A30: 1.88±0.19, A90: 1.75±0.27 [fold change, normalized to normoxia control]; p=0.87); however, hypoxic means were significantly higher than in normoxia conditions (p<0.0001). In normoxia, NAD(P)H oxidase isoform 4 (Nox4) show a decreasing trend in protein levels with increasing media arginase-I (CN: 1.00±0.03, A30: 0.93±0.10, A90: 0.86±0.06 [fold change]), with an increase in Nox4 protein levels in hypoxia with increasing media arginase-I (CN: 1.18±0.02, A30: 1.33±0.08, A90: 1.47±0.01 [fold change, normalized to normoxia control]). The hypoxic levels of Nox4 were also higher than in normoxia conditions.

As expected, increasing free media arginase-I concentrations in hPMVEC culture resulted in significant increases in urea production in both normoxia and hypoxia. Hypoxia resulted in lower nitrite production by endothelial nitric oxide synthase (eNOS). However, increasing amounts of free arginase-I only affected nitrite production in hypoxia, suggesting that increased arginase-I serum concentrations may also become an important regulator of NO production in clinical cases of hypoxia (e.g., acute chest syndrome and vaso-occlusive crises in sickle cell patients). The concurrent increased expression of 3-NT in hypoxia, despite lower nitrites, implies an increase in reactive oxygen species (ROS). Our preliminary data for Nox4 expression, the predominant source of ROS in endothelial cells, indicates that ROS production via this pathway may increase in hypoxia with increasing free arginase-I. Further studies on how free arginase-I in the media affects endothelial function are warranted, particularly in regards to the mechanism by which free arginase-I leads to greater Nox4 expression and ROS production. We speculate that arginase-I inhibitors may represent a potential therapeutic target in patients with chronic hemolysis and acute hypoxic events.

No relevant conflicts of interest to declare.