Heme biosynthesis modulation via δ-aminolevulinic acid administration attenuates chronic hypoxia-induced pulmonary hypertension

Abstract

This study examines how heme biosynthesis modulation with δ-aminolevulinic acid (ALA) potentially functions to prevent 21-day hypoxia (10% oxygen)-induced pulmonary hypertension in mice and the effects of 24-h organoid culture with bovine pulmonary arteries (BPA) with the hypoxia and pulmonary hypertension mediator endothelin-1 (ET-1), with a focus on changes in superoxide and regulation of micro-RNA 204 (miR204) expression by src kinase phosphorylation of signal transducer and activator of transcription-3 (STAT3). The treatment of mice with ALA attenuated pulmonary hypertension (assessed through echo Doppler flow of the pulmonary valve, and direct measurements of right ventricular systolic pressure and right ventricular hypertrophy), increases in pulmonary arterial superoxide (detected by lucigenin), and decreases in lung miR204 and mitochondrial superoxide dismutase (SOD2) expression. ALA treatment of BPA attenuated ET-1-induced increases in mitochondrial superoxide (detected by MitoSox), STAT3 phosphorylation, and decreases in miR204 and SOD2 expression. Because ALA increases BPA protoporphyrin IX (a stimulator of guanylate cyclase) and cGMP-mediated protein kinase G (PKG) activity, the effects of the PKG activator 8-bromo-cGMP were examined and found to also attenuate the ET-1-induced increase in superoxide. ET-1 increased superoxide production and the detection of protoporphyrin IX fluorescence, suggesting oxidant conditions might impair heme biosynthesis by ferrochelatase. However, chronic hypoxia actually increased ferrochelatase activity in mouse pulmonary arteries. Thus, a reversal of factors increasing mitochondrial superoxide and oxidant effects that potentially influence remodeling signaling related to miR204 expression and perhaps iron availability needed for the biosynthesis of heme by the ferrochelatase reaction could be factors in the beneficial actions of ALA in pulmonary hypertension.

Keywords: endothelin; ferrochelatase; guanylate cyclase; micro-RNA 204; superoxide.

Fig. 1.

Effects of treating mice with δ-aminolevulinic acid (ALA) for 21 days in the absence and presence of hypoxia on indicators of chronic hypoxia-induced pulmonary hypertension. A: right ventricle systolic pressure (RVSP) measured through right heart catheterization at the end of 21 days of treatment (n ≥ 5/group). B: pulmonary arterial pressure (PAP) monitored at the end of the 21-day treatment through assessing the pulmonary valve Doppler flow time intervals [pulmonary acceleration time/%ejection time (PAT/%ET)]. Note that a decrease in PAT/%ET is consistent with an increase in PAP (n = 10). C: velocity-time integral (VTI) of the area under the measured pulmonary artery Doppler flow. D: right ventricle normalized to left ventricle (including septum) reported as %weight ratios (n = 12). *P < 0.05 vs. control and #P < 0.05 vs. hypoxia (ANOVA). Note that the modest effect of ALA on lowering RVSP in the normoxic control mice was significant by a t-test analysis.

Fig. 2.

Effects of treating mice with ALA for 21 days in the absence and presence of chronic hypoxia on superoxide generation in isolated pulmonary arteries detected by 5 μM lucigenin chemiluminescence (A) and lung micro-RNA 204 (miR204) expression as measured through quantitative real-time polymerase chain reaction (qRT-PCR) (B). *P < 0.05 vs. hypoxia (t-test, n = 5) (A). *P < 0.05 vs. normoxia control and #P < 0.05 vs. hypoxia (t-test, n = 5) (B).

Fig. 3.

Effects of ALA, an activator of protein kinase G (PKG) and an inhibitor of NADPH oxidase-2 (Nox2), on endothelin (ET)-1-induced increases in superoxide production in 24-h organoid-cultured endothelium-removed bovine pulmonary artery (BPA) segments. A: effects of the absence and presence of ALA during organoid culture on superoxide measured through the chemiluminescence of 5 μM lucigenin (counts/g) in the presence and absence of 10 nM ET (n = 10). B: comparison of the influence of ALA, 8-bromo-cGMP, and gp91ds-tat in 24-h organoid culture on lucigenin-detectible superoxide in the presence of ET, reported as a percent of the non-ET-treated BPA ring for each experimental group of BPA segments that are nontreated, ALA treated, 8-bromo-cGMP treated, and gp91ds-tat treated for 24 h in organoid culture (n = 7).

Fig. 4.

Effects of ALA on ET-1-induced increases in phosphorylation of signal transducer and activator of transcription (STAT-3) and decreases in miR204 expression observed in 24-h organoid-cultured BPA segments. A: STAT3 activation as measured through the phosphorylation of tyrosine-705 residue (79 kDa) via Western blot analysis of the endothelium-removed BPA segments in both the nontreated (Control) and ALA-treated segments with the presence or absence of 10 nM ET-1 in 24-h organoid culture (n = 6, ANOVA). B: miR204 expression measured through qRT-PCR in endothelium-removed BPA 24-h organoid cultured with or without the presence of ET-1 in the absence and presence of exposure to ALA (n = 8, t-test). *P < 0.05 vs. Control.

Fig. 5.

Effects of ALA on ET-1-elicited increases on mitochondrial (A) and extramitochondrial (B) superoxide and decreases in mitochondrial superoxide dismutase (SOD) 2 (C) in 24-h organoid-cultured BPA. D: effects of ET on protoporphyrin (PPIX) accumulation in 24-h organoid-cultured BPA segments. Changes in BPA mitochondrial (n = 10, A) and extramitochondrial (n = 8, B) superoxide generation were quantified in BPA segments using HPLC measurements of the superoxide-specific oxidation product of 5 μM MitoSox and dihydroethidine, respectively, that accumulated during a 1-h incubation after a 24-h organoid culture with 10 nM ET-1 (*P < 0.05 vs. control; #P < 0.05 vs. ET). C: changes in lung SOD2 (25 kDa) expression were detected by Western analysis (n = 5) after 24-h organoid culture of BPA with or without 10 nM ET in the presence and absence of ALA (*P < 0.05 vs. control; #P < 0.05 vs. ET). D: BPA segments were exposed to 24-h organ culture without or with 10 nM ET-1, after which they were subjected to surface fluorescence measurement of PPIX (n = 10). *P < 0.05 vs. Control.

Fig. 6.

Effects of treating mice with ALA for 21 days in the absence and presence of hypoxia on measurements of changes in mouse lung mitochondrial SOD2 expression (A) and pulmonary artery ferrochelatase activity (B). Changes in lung SOD2 (25 kDa) expression were detected by Western analysis (n = 7) (A), and ferrochelatase (FECH) activity in homogenates of isolated pulmonary arteries was detected by the conversion of PPIX to ZnPPIX, followed by HPLC analysis (n = 3–5/group from 5–8 mice) (B). *P < 0.05 vs. control (t-test).

Fig. 7.

Hypothesized processes potentially contributing to the ability of ALA to attenuate the development of hypoxia-induced pulmonary hypertension. The model begins with the initial effects of ALA on attenuating increases in mitochondrial superoxide and decreases in SOD2, which could potentially occur through PPIX stimulating cGMP signaling and by decreased mitochondrial superoxide. Decreased mitochondrial reactive oxygen species (ROS) could then help restore the biosynthesis of heme. A combination of decreased ROS and increased cGMP generation by soluble guanylate cyclase (sGC) could function to prevent increased vascular reactivity and pulmonary arterial smooth muscle remodeling processes that contribute to the progression of pulmonary hypertension development.