Alhawaj R, Patel D, Kelly MR, Sun D, Wolin MS. Heme biosynthesis modulation via ␦-aminolevulinic acid administration attenuates chronic hypoxia-induced pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 308: L719 -L728, 2015. First published February 6, 2015 doi:10.1152/ajplung.00155.2014.-This study examines how heme biosynthesis modulation with ␦-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. endothelin; ferrochelatase; guanylate cyclase; micro-RNA 204; superoxide CHRONIC HYPOXIA IS AN IMPORTANT factor in the development of pulmonary hypertension in diseases such as chronic obstructive pulmonary disease (COPD), sleep apnea, and mountain sickness (10,20). It is thought to promote pulmonary hypertension development through persistent pulmonary vasoconstriction and vascular remodeling (31). There is substantial evidence for increases in the generation of reactive oxygen species from sources including Nox oxidases and mitochondria having important roles in the development and/or progression of pulmonary hypertension caused by chronic hypoxia and other stimuli of this disease process (7,15,18,27,30). Pulmonary arterial generation of nitric oxide (NO) is normally thought to oppose this process of vasoconstriction ...
Oxidation of the soluble guanylate cyclase (sGC) heme promotes loss of regulation by nitric oxide (NO) and depletion of sGC. We hypothesized that angiotensin II (ANG II) stimulation of mitochondrial superoxide by its type 1 receptor could function as a potential inhibitor of heme biosynthesis by ferrochelatase, and this could decrease vascular responsiveness to NO by depleting sGC. These processes were investigated in a 24-h organoid culture model of bovine coronary arteries (BCA) with 0.1 μM ANG II. Treatment of BCA with ANG II increased mitochondrial superoxide, depleted mitochondrial superoxide dismutase (SOD2), ferrochelatase, and cytochrome oxidase subunit 4, and sGC, associated with impairment of relaxation to NO. These processes were attenuated by organoid culture with 8-bromo-cGMP and/or δ-aminolevulinic acid (a stimulator of sGC by protoporphyrin IX generation and heme biosynthesis). Organoid culture with Mito-TEMPOL, a scavenger of mitochondrial matrix superoxide, also attenuated ANG II-elicited ferrochelatase depletion and loss of relaxation to NO, whereas organoid culture with Tempol, an extramitochondrial scavenger of superoxide, attenuated the loss of relaxation to NO by ANG II, but not ferrochelatase depletion, suggesting cytosolic superoxide could be an initiating factor in the loss of sGC regulation by NO. The depletion of cytochrome oxidase subunit 4 and sGC (but not catalase) suggests that sGC expression may be very sensitive to depletion of heme caused by ANG II disrupting ferrochelatase activity by increasing mitochondrial superoxide. In addition, cGMP-dependent activation of protein kinase G appears to attenuate these ANG II-stimulated processes through both preventing SOD2 depletion and increases in mitochondrial and extramitochondrial superoxide.
Patel D, Alhawaj R, Wolin MS. Exposure of mice to chronic hypoxia attenuates pulmonary arterial contractile responses to acute hypoxia by increases in extracellular hydrogen peroxide. Am J Physiol Regul Integr Comp Physiol 307: R426 -R433, 2014. First published June 15, 2014 doi:10.1152/ajpregu.00257.2013.-Exposing mice to a chronic hypoxic treatment (10% oxygen, 21 days) that promotes pulmonary hypertension was observed to attenuate the pulmonary vasoconstriction response to acute hypoxia (HPV) both in vivo and in isolated pulmonary arteries. Since catalase restored the HPV response in isolated arteries, it appeared to be attenuated by extracellular hydrogen peroxide. Chronic hypoxia promoted the detection of elevated lung superoxide, extracellular peroxide, extracellular SOD expression, and protein kinase G (PKG) activation [based on PKG dimerization and vasodilator-stimulated phosphoprotein (VASP) phosphorylation], suggesting increased generation of extracellular peroxide and PKG activation may contribute to the suppression of HPV. Aorta from mice exposed to 21 days of hypoxia also showed evidence for extracellular hydrogen peroxide, suppressing the relaxation response to acute hypoxia. Peroxide appeared to partially suppress contractions to phenylephrine used in the study of in vitro hypoxic responses. Treatment of mice with the heme precursor ␦-aminolevulinic acid (ALA; 50 mg·kg Ϫ1 ·day Ϫ1 ) during exposure to chronic hypoxia was examined as a pulmonary hypertension therapy because it could potentially activate beneficial cGMP-mediated effects through promoting a prolonged protoporphyrin IX (PpIX)-elicited activation of soluble guanylate cyclase. ALA attenuated pulmonary hypertension, increases in both superoxide and peroxide, and the suppression of in vitro and in vivo HPV responses. ALA generated prolonged detectible increases in PpIX and PKG-associated phosphorylation of VASP, suggesting PKG activation may contribute to suppression of pulmonary hypertension and prevention of alterations in extracellular peroxide that appear to be attenuating HPV responses caused by chronic hypoxia. aminolevulinic acid; hypoxic pulmonary vasoconstriction; protein kinase G; pulmonary hypertension CHRONIC HYPOXIA IS KNOWN to contribute to the development of pulmonary hypertension in humans with chronic obstructive pulmonary disease (7), and intermittent hypoxia associated with sleep apnea promotes both pulmonary and systemic hypertension (9). A loss of the hypoxic pulmonary vasoconstriction (HPV) response which maintains ventilation to perfusion ratios is seen in humans with chronic obstructive pulmonary disease (COPD) and in pulmonary arteries isolated from these individuals (25). Studies in animal models of chronic hypoxia have provided evidence for processes that are associated with increased generation of reactive oxygen species (ROS) from sources, including various Nox oxidases participating in the progression of hypoxia-induced pulmonary hypertension development (10, 12, 13, 15, 19, 21, 23). Previous studies in pulm...
Under conditions that lead to pulmonary arterial hypertension (PAH), the pro-proliferative oncoprotein Src kinase activity is elevated, contributing to pulmonary arterial smooth muscle cell remodeling, increasing vascular resistance, and leading to PAH. In addition, reactive oxygen species (ROS), including Hydrogen peroxide (H2O2), are elevated in the pulmonary vasculature under pro-PAH conditions. Therefore, we hypothesized that Src kinase is subject to redox modulation by elevated hydrogen peroxide (H2O2) under pro-PAH conditions. We intended to explore whether increased H2O2 can cause the oxidation of Src kinase thiol groups resulting in an inter-molecular disulfide bond forming Src kinase homodimer, increasing its catalytic activity. The results of this study detected evidence that Src kinase undergoes redox-induced homodimer formation as illustrated by western blot analysis under non-reducing conditions of rat MCT-induced PAH lung lysates. Furthermore, the redox-mediated Src kinase homodimer formation was associated with the increased catalytic activity of Src kinase, as seen in its intra-molecular auto-phosphorylation of its tyrosine 416 residue. This process was accompanied by activating one of Src kinase downstream substrates, STAT3, via the phosphorylation of its tyrosine 705 residue. Moreover, these mechanistic changes were associated with a PAH disease state confirmed with elevated right ventricle systolic pressure and right ventricle hypertrophy through right ventricle catheterization and the Fulton index, respectively. From the results above, we can conclude that in rat MCT-PAH lung lysates, Src kinase undergoes a redox-induced modulation that results in a structural modification associated with increased Src kinase activity, as observed in its autophosphorylation and the phosphorylation of its downstream substrate STAT3. In conclusion, under conditions that lead to PAH, Src kinase undergoes a post-translational structural modification. This modification occurs in redox-induced oxidation of Src kinase thiol groups. This process results in an inter-molecular disulfide bond formation yielding a homodimer associated with elevated Src kinase catalytic activity, further adding a mechanistic layer to Src kinase regulation in PAH. Kuwait University Research Sector This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Pulmonary arterial hypertension (PAH) is driven by pulmonary arterial smooth muscle cell (PASMC) vasoconstriction and remodeling. One of the many drivers of PAH‐PASMCs remodeling is increased transferrin receptor‐1 (TFR‐1) expression. Under pro‐PAH conditions, PASMCs exhibit elevated reactive oxygen species (ROS) production which may disrupt ferrochelatase normal heme production. We evaluated the role played by ROS‐deregulated ferrochelatase in TFR‐1‐mediated PAH‐PASMCs remodeling. Intra‐lobar pulmonary arteries (PA) isolated from monocrotaline‐induced PAH model in Sprague‐Dawley rats have shown an up‐regulation of ferrochelatase expression, that can be a result of hypoxia‐inducible factor (HIF) mediated up‐regulation to compensate for decreased ferrochelatase catalytic activity. Decreased ferrochelatase activity was evident in the decreased levels of hemin (oxidized form of heme) in isolated PAH‐PAs. Endothelin‐1 (ET‐1), which is an inducer of mitochondrial ROS and is one of the mediators of PAH; was used in a 24‐hour organ culture of isolated endothelium‐removed bovine pulmonary arteries (BPA). ET‐1 cultured BPAs have shown decreased hemin levels and increased TFR‐1 expression. Thus, ROS‐deregulated ferrochelatase may promote vascular remodeling in PAH through the impairment of hemin‐mediated downregulation of TFR‐1 receptors in PASMCs.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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