The Yin and Yang of endothelium-derived vasodilator factors

Pulmonary hypertension (PH) is clinically defined as an elevated mean pulmonary arterial pressure of >25 mmHg. This rise in pressure predominantly occurs in response to increased vascular resistance due to vascular remodeling and enhanced constriction or blunted dilation at multiple levels of the pulmonary arterial tree. Such pathological vascular changes can arise from damage to the lungs, the heart, or the pulmonary arteries directly. Whatever the predisposing event, central to the downstream pathways that drive disease progression, and the target of the main pharmacological agents used to treat PH, is an altered balance of vasoactive agents released from the vascular endothelium. The primary known phenomena include loss of the antiproliferative vasodilators nitric oxide (NO) (treated with sildenafil) and prostacyclin (replaced with epoprostenol) and elevations in the vasoconstrictor endothelin-1 (blocked by bosentan or amrisentan). Despite the relative clinical success of these drugs, morbidity and mortality remain high in all forms of PH, and experts have suggested that new pharmacological agents targeting other mechanisms are needed (21).

Two strategies may aid clinicians and researchers in the development of new endothelium-targeted therapies. As is the case with past research into vascular diseases including PH, most studies exploring these underlying vasoactive mechanisms have examined the endothelium of large arteries with relatively little emphasis on the microvascular endothelium (15). In addition, although NO, prostacyclin, and endothelin-1 are well characterized in the pulmonary and systemic circulation, an additional, provocative, and underexplored target is the vasodilator pathway mediated by endothelium-dependent hyperpolarizing factor (EDHF). While NO is the primary vasodilator in large vessels, one EDHF (H₂O₂) increasingly participates in the control of vascular tone as vessel size decreases in the systemic circulation (30). EDHF often compensates for loss of other endothelium-derived dilator factors, but some levels are necessary for vascular homeostasis (12). However, a role for EDHF in the pulmonary (micro)circulation as a physiological or pathophysiological factor is largely unexplored.

In an article recently published in the American Journal of Physiology-Heart and Circulatory Physiology, Tanaka et al. (27a) addressed this gap in knowledge to expand our understanding of the pathogenesis of PH. This group reported that EDHF/H₂O₂ is responsible for approximately half of endothelium-dependent vasodilation to bradykinin in the murine pulmonary microcirculation under normoxic conditions using an isolated perfused lung, with NO contributing the remainder of the microvascular dilation; in contrast, NO is the sole mediator of dilation to acetylcholine in larger pulmonary arteries. Interestingly, despite a universal emphasis on loss of NO bioavailability as a key contributor to cardiovascular diseases, including PH, data from this group revealed that the contribution of NO to dilation increased, whereas EDHF-mediated dilation decreased, in the microcirculation during the development of PH. This reversal of roles, where NO compensates for loss of EDHF contrasts with the more commonly described loss of NO and compensatory rise in H₂O₂ (10, 16, 19) in other vascular beds, is remarkable. Of note, Tanaka et al. identified a decline in EDHF in the microcirculation as early as day 2 of hypoxia, before any vascular remodeling, suggesting that loss of this vasodilator mechanism may serve as an early marker, if not mechanism, of impending PH. Although yet untested, loss of EDHF/H₂O₂ may also be a key microvascular factor driving increased vascular resistance and microvascular complications in PH. That two established mouse models of chronic hypoxia demonstrated similar findings across a prespecified time course lends rigor to their findings.

The implications of this research are far reaching. First, this study advances the notion that NO and EHDF/H₂O₂ can act as synergistic vasodilators in the microcirculation, in line with recent findings from others (4, 12, 18, 29). This concept sharply contrasts with the prevailing theory that NO and H₂O₂ are mutually exclusive and functionally antagonistic, with NO preventing, and H₂O₂ promoting, (micro)vascular damage (8, 16, 26). The latter theory is weakened by the physiological role played by H₂O₂ in this study, its prosurvival signaling effects (14), and multiple reports suggesting that activation of H₂O₂-mediated dilation alongside NO is a key event in the microvascular adaptations to exercise (9, 23, 28, 31), an indisputably protective physiological stimulus. A possible explanation for this discrepancy is that many of the harmful effects of H₂O₂ result from the exogenous administration of supraphysiological concentrations (8, 11, 25, 27). In addition, subcellular localization of endogenous H₂O₂ may shape its influence on the vasculature, with most reports linking pathological effects of H₂O₂ to selected NADPH oxidases (NOX1 and NOX2) or the mitochondria (7). Second, that NO only plays a partial role, and prostacyclin does not participate at all, during microvascular dilation either before or after the onset of PH supports the call to identify new targets. Since prostacyclin’s contribution is minimal, use of epoprostenol to activate prostacyclin-mediated pathways is unlikely to be sufficient in addressing the underlying microvascular dysfunction in PH despite its potent vasorelaxing effect. Third, the data suggest that simply increasing NO bioavailability, the main focus of therapeutic interventions for many cardiovascular disorders, may not restore a healthy microvascular phenotype, and attention should instead shift to maintaining both NO and EDHF/H₂O₂ in the microcirculation.

Why might release of NO alone be insufficient to maintain a healthy vascular environment at baseline and during the development of PH? Stated another way, why is H₂O₂ useful to the pulmonary circulation at baseline, and how might loss of H₂O₂ contribute to early pathological changes in the pulmonary circulation? For one, NO is susceptible to scavenging by the highly reactive superoxide, and relying on NO alone may hamper the vasculature’s capacity to maintain dilation in the setting of elevated oxidative stress. In contrast, H₂O₂ is less susceptible to scavenging by reactive oxygen species. Moreover, it is known that NO exhibits hormesis, because increasing NO above a certain threshold switches its effects from physiological to pathophysiological (12), possibly through increased formation of the damaging NO-derived molecule peroxynitrite (22). In physiological amounts, H₂O₂ can produce dilation (19) and even blunt pathological vascular remodeling and inflammation (13), two functions that would allow it to counteract the increased vascular resistance and remodeling observed in PH, in concert with the antiproliferative actions of NO.

Aside from individual contributions of NO and EDHF/H₂O₂ to vascular regulation, the present study also raises two additional questions about the complementary nature of these two vasodilators. How might NO and EDHF/H₂O₂ be released and operate together, and what is the physiological benefit of that interplay? Previous research has suggested that in some circumstances NO and EDHF/H₂O₂ share endothelial NO synthase (eNOS) as a common intracellular source (20). In theory, partially uncoupled eNOS, or the simultaneous presence of uncoupled and coupled eNOS at different subcellular locations, could generate both NO and H₂O₂ (via one electron reduction of superoxide). On a mechanistic level, multiple cellular pathways have been determined to enable NO- and H₂O₂-mediated signaling, including caveolin-1, peroxisome proliferator-activated receptor-γ coactivator 1α, PKG, and calmodulin (1, 5, 18, 24). From a functional standpoint, dual release may confer an increased ability to maintain dilation in the presence of an acute vascular stressor that compromises individual NO- or H₂O₂-dependent pathways. For example, dual release of NO and H₂O₂ protects against the damage caused by acute increases in intraluminal pressure (18), a protection not observed in arterioles relying on a single vasodilator (2, 18). In this way, the dual dilator pathway serves as a built-in backup system to preserve endothelium-dependent dilation (17, 18, 23). Since the microcirculation plays a critical role in tissue perfusion, this vasodilatory robustness is likely beneficial to the organism. Another effect of this simultaneous release may be multipronged activation of PKG and large-conductance Ca²⁺-activated K⁺ channels for vasodilation. Endothelium-derived H₂O₂ and NO signal through a common pathway by activating PKG (via cGMP for NO and by PKG dimerization for H₂O₂), thus opening K⁺ channels and inhibiting the contractile process (3, 4, 6, 32). These two effector molecules might be expected to act more potently together than separately on this critical dilator pathway.

Several future research directions are unraveled by Tanaka et al.’s report. Additional investigation of the dual release of NO/H₂O₂ in the microcirculation in other physiological contexts and in other vascular beds, and the implications of this ability to recruit more than one vasodilator, is needed. Restoration of EDHF/H₂O₂ signaling via direct hyperpolarization, inhibition of H₂O₂-targeting antioxidants like catalase or glutathione peroxidase, or exogenous administration of H₂O₂ in the mouse models of PH (i.e., a rescue study) would also add support that targeting this pathway will improve disease parameters, such as vascular resistance and survival. It would be helpful to identify intracellular mechanisms responsible for the dual release of NO/H₂O₂ at baseline and the loss of EDHF/H₂O₂ during the onset of PH, similar to what is already available for the NO-mediated pathway (direct NO donors, guanylate cyclase activators, and phosphodiesterase inhibitors). Finally, evaluating the role of this pathway in humans in physiological and pathophysiological settings is essential. Together, these efforts may identify novel adjunct treatments to add to the armamentarium of tools used to combat the microvascular dysfunction in PH and other cardiovascular diseases.

GRANTS

This work was supported by National Institutes of Health Grants R01-HL-135901-01 (to D. D. Gutterman) and T32-GM-080202 (to the Medical College of Wisconsin Medical Scientist Training Program), an endowment from Northwestern Mutual Foundation, and American Heart Association Predoctoral Fellowship Grant 16PRE29130003 (to A. O. Kadlec).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

A.O.K. and D.D.G. drafted manuscript; A.O.K. and D.D.G. edited and revised manuscript; A.O.K. and D.D.G. approved final version of manuscript.