Pulmonary hypertension (PH) is a progressive vascular disease driven by pulmonary arterial remodeling, characterized by cellular hyperproliferation, resistance to apoptosis, and phenotypic plasticity. Our laboratory has shown that the proton-gated cation channel, acid-sensing ion channel 1a (ASIC1a), is essential for the development of chronic hypoxia (CH)-induced PH in rodents. Importantly, ASIC1a activation occurs without changes in total ASIC1a levels but reflects a hypoxia-dependent redistribution to the plasma membrane in pulmonary arterial smooth muscle cells (PASMCs). In neurons, mitochondrial-localized ASIC1a (mtASIC1a) contributes to oxidative stress-induced mitochondrial membrane potential (Δψm) depolarization and apoptosis. Although mtASIC1a has not been described in vascular cells, its role in PASMCs may be relevant to mitochondrial dysfunction and apoptosis resistance in PH. We hypothesize that mtASIC1a is a crucial regulator of PASMC mitochondrial homeostasis, and its loss following CH promotes mitochondrial dysfunction and apoptosis resistance. Consistent with this, mtASIC1a localization was decreased in PASMCs and intrapulmonary arteries from CH rats compared to controls. Functionally, PASMCs from CH rats or Asic1a knockout mice exhibited Δψm hyperpolarization, elevated mitochondrial Ca 2+ ; and superoxide, impaired mitophagy, and reduced cleaved caspase-3. Transmission electron microscopy revealed mitochondrial morphological changes, including increased size and circularity, decreased aspect ratio, and reduced mitochondrial number per cell, while fusion/fission proteins remained largely unchanged. Lentiviral restoration of mtASIC1a prevented Δψm hyperpolarization and restored caspase-3 cleavage. These findings identify mtASIC1a as a novel regulator of mitochondrial function in PASMCs, where its loss following CH promotes Δψm hyperpolarization, impaired mitophagy, and resistance to apoptosis.

Chronic hypoxia (CH) leads to the development of pulmonary hypertension, and pulmonary vascular remodeling contributes to disease pathogenesis. Although the mechanisms underlying CH-induced pulmonary vascular remodeling are not fully understood, they involve proliferation of pulmonary arterial smooth muscle cells (PASMCs). Previous studies suggest that extracellular signal-regulated kinases 1 and 2 (ERK1/2) may be important drivers of PASMC proliferation. Therefore, we hypothesized that CH activates extracellular signal-regulated kinases 1 and 2 to facilitate PASMC proliferation. To test our hypothesis, we performed immunoblotting in both pulmonary arteries and primary cultures of PASMCs from normoxic and CH (1 wk, 0.5 atm) rats to assess 1) ERK1/2 phosphorylation status (Thr202/Tyr204) as a measure of ERK1/2 activation, and 2) PCNA abundance as a marker of cell proliferation. To evaluate the role of ERK1/2 in PASMC proliferation, we treated primary cultures with the mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor, U0126 (10 μM). Hypoxia increased ERK1/2 phosphorylation (phosphorylated ERK1/2 to total ERK1/2) and PCNA abundance (PCNA to β-actin) in pulmonary arterial homogenates, with a significant positive correlation (r=0.75; p< 0.05), suggesting ERK1/2 activation is associated with CH-induced PAMSC proliferation. Similar changes were observed in primary cultures of PASMCs from these groups. Treatment with U0126 abolished hypoxia-induced ERK1/2 phosphorylation in primary PASMC. U0126 significantly reduced PCNA abundance in both cell groups, with levels remaining statistically higher in the hypoxic group than in the normoxic group. In conclusion, these results suggest that 1) hypoxia increases proliferation of PASMCs; 2) ERK1/2 is activated by hypoxia in PASMCs; and 3) ERK1/2 plays a key role in driving PASMC proliferation, but it is not the only contributing pathway under hypoxic conditions. This work is supported by NIH grant R01 HL169945 (to T.C. Resta). This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Acid-sensing ion channel 1a (ASIC1a) is a proton-gated cation channel activated by extracellular acidosis, a condition often associated with ischaemia, inflammation and metabolic stress. Our laboratory has previously demonstrated that ASIC1a contributes to systemic endothelium-dependent vasodilatation; however, the role of ASIC1a in blood pressure regulation remains unclear. The objective of this study was to investigate the role of ASIC1a in blood pressure regulation using a model of angiotensin II-induced hypertension. We further determined the effects of age and sex, as these are non-modifiable risk factors for hypertension. Radiotelemeters were implanted in 6- and 18-month-old male and female wild-type (Asic1a+/+) and ASIC1a knockout (Asic1a-/-) mice to continuously measure blood pressure and heart rate under baseline conditions and following angiotensin II. Blood gases, electrolytes, hormones and indices of end-organ injury were also assessed. Deletion of ASIC1 did not significantly alter blood pressure in 6-month-old male or female mice. However, aged male Asic1a-/- mice develop hypertension, whereas female Asic1a-/- mice remain unaffected. This hypertension was associated with aldosterone excess, increased sympathetic activity, and evidence of aortic fibrosis, left ventricular hypertrophy and renal injury. Importantly, hyperaldosteronism occurred independently of the renin-angiotensin system and was associated with blunting of angiotensin II-induced hypertension. Prior to the onset of hypertension, 6-month-old male Asic1a-/- mice exhibit elevated corticosterone, hypokalaemia, reduced urine osmolality, increased pulse pressure and cardiomyocyte hypertrophy. Together, these findings identify ASIC1a as a novel, sex-specific regulator of cardiovascular function, suggesting that ASIC1a deficiency may be a potential driver of endocrine-related hypertension. KEY POINTS: Extracellular acidosis, which occurs during pathological conditions such as ischaemia, inflammation and metabolic stress, contributes to the pathogenesis of cardiometabolic disease. Acid-sensing ion channels (ASICs) are key sensors of acidosis and important mediators of endothelium-dependent vasodilatation; however, the role of ASIC1a in blood pressure regulation remains poorly understood. Here we report that ASIC1a deficiency causes hypertension in aged male mice that is driven by an excess of aldosterone and increased sympathetic activity, while female mice remain unaffected. Hyperaldosteronism occurs independently of angiotensin II but is associated with elevated corticosterone that precedes the development of hypertension. We further show that male, but not female, ASIC1a knockout mice have a reduced sensitivity to angiotensin II-induced hypertension. These findings identify ASIC1a as a novel, sex-specific regulator of cardiovascular function, suggesting that ASIC1a deficiency contributes to endocrine-related hypertension.

In pulmonary hypertension (PHTN), a metabolic shift to aerobic glycolysis promotes a hyperproliferative, apoptosis-resistant phenotype in pulmonary arterial smooth muscle cells (PASMCs). Enhanced glycolysis induces extracellular acidosis, which can activate proton-sensing membrane receptors and ion channels. We previously reported that activation of the proton-gated cation channel acid-sensing ion channel 1a (ASIC1a) contributes to the development of hypoxic PHTN. Therefore, we hypothesize that enhanced glycolysis and subsequent acidification of the PASMC extracellular microenvironment activate ASIC1a in hypoxic PHTN. We observed decreased oxygen consumption rate and increased extracellular acidification rate in PASMCs from chronic hypoxia (CH)-induced PHTN rats, indicating a shift to aerobic glycolysis. In addition, we found that intracellular alkalization and extracellular acidification occur in PASMCs following CH and in vitro hypoxia, which were prevented by the inhibition of glycolysis with 2-deoxy-d-glucose (2-DG). Inhibiting H+ transport/secretion through carbonic anhydrases, Na+/H+ exchanger 1, or vacuolar-type H+-ATPase did not prevent this pH shift following hypoxia. Although the putative monocarboxylate transporter 1 (MCT1) and -4 (MCT4) inhibitor syrosingopine prevented the pH shift, the specific MCT1 inhibitor AZD3965 and/or the MCT4 inhibitor VB124 were without effect, suggesting that syrosingopine targets the glycolytic pathway independent of H+ export. Furthermore, 2-DG and syrosingopine prevented enhanced ASIC1a-mediated store-operated Ca2+ entry in PASMCs from CH rats. These data suggest that multiple H+ transport mechanisms contribute to extracellular acidosis and that inhibiting glycolysis-rather than specific H+ transporters-more effectively prevents extracellular acidification and ASIC1a activation. Together, these data reveal a novel pathological relationship between glycolysis and ASIC1a activation in hypoxic PHTN. NEW & NOTEWORTHY In pulmonary hypertension, a metabolic shift to aerobic glycolysis drives a hyperproliferative, apoptosis-resistant phenotype in pulmonary arterial smooth muscle cells. We demonstrate that this enhanced glycolysis induces extracellular acidosis and activates the proton-gated ion channel, acid-sensing ion channel 1a (ASIC1a). Although multiple H+ transport/secretion mechanisms are upregulated in PHTN and likely contribute to extracellular acidosis, inhibiting glycolysis with 2-deoxy-d-glucose or syrosingopine effectively prevents extracellular acidification and ASIC1a activation, revealing a promising therapeutic avenue.

Acid-sensing ion channel 1a (ASIC1a) is a proton-gated cation channel that contributes to the development of chronic hypoxia (CH)-induced pulmonary hypertension (PH). Our prior studies demonstrate that ASIC1a is expressed in pulmonary arterial smooth muscle cells (PASMCs) and contributes to plasmalemmal membrane potential ( E m ) depolarization and augmented intracellular Ca 2+ signaling leading to enhanced vasoconstrictor reactivity and vascular remodeling. During PH, mitochondrial dysfunction associated with mitochondrial membrane potential (Δψm) hyperpolarization can inhibit opening of the mitochondrial permeability transition pore (mPTP), thereby repressing apoptosis and promoting the hyperproliferative and apoptosis-resistant PASMC phenotype. Recent evidence suggests that ASIC1a is localized to mitochondria and regulates the mPTP. However, it is unknown if mitochondrial-localized ASIC1a (mtASIC1a) contributes to the pathogenesis of PH. Therefore, the overall objective of this study is to determine the functional role of mtASIC1a in the apoptosis-resistant PASMC phenotype associated with PH. We hypothesized that CH causes Δψm hyperpolarization and apoptosis-resistance in PASMCs due to loss of mtASIC1a localization. To test this hypothesis, we housed Asic1a knockout ( Asic1a -/- ) and wild-type ( Asic1a +/+ ) mice in a hypobaric chamber (barometric pressure: ~380 mmHg) for 4 weeks to induce PH. Following CH, we found increased expression of ASIC1a at the plasma membrane and decreased expression in mitochondria, which were associated with PASMC E m depolarization and Δψm hyperpolarization. Using tetramethylrhodamine (TMRE) fluorescence to measure Δψm, we found that TMRE fluorescence was significantly increased (p=0.02) in PASMCs from normoxic Asic1a -/- mice compared to Asic1a +/+ mice, indicating that deletion of Asic1a leads to Δψm hyperpolarization. We utilized a lentiviral vector encoding mitochondrial-targeted human ASIC1a (mtASIC1a LV) to overexpress mtASIC1a in PASMCs (p=0.003) and found that transduction with mtASIC1a LV in PASMCs from Asic1a -/- mice significantly decreased TMRE fluorescence (p=0.004), further supporting the functional role of mtASIC1a to regulate Δψm. To assess apoptosis, we measured levels of active caspase-3 by western blot analysis in intrapulmonary arteries (PAs) from Asic1a +/+ and Asic1a -/- mice following CH. CH decreased levels of active caspase-3 in PAs from Asic1a +/+ (p=0.032), but not Asic1a -/- mice. Under normoxic conditions, active caspase-3 was lower in PAs from Asic1a -/- compared to Asic1a +/+ mice (p=0.044). These data suggest mtASIC1a plays an important role in the regulation of Δψm and that the loss of mtASIC1a contributes to Δψm hyperpolarization and apoptosis resistance in PASMCs following CH. Future studies will examine the contributions of mtASIC1a to the pathogenesis of PH through the regulation of mitochondrial reactive oxygen species, mitochondrial Ca 2+ levels, mPTP sensitivity, and mitochondrial dynamics. NIH R01 HL111084 to N.L. Jernigan and NIH T32 HL007736 to T.C. Resta 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.

Introduction: Chronic hypoxia (CH) leads to increased pulmonary vascular resistance and resultant pulmonary hypertension (PH) in patients with chronic obstructive pulmonary disease and sleep apnea. We recently demonstrated that CH augments vasoconstrictor reactivity through a switch in pulmonary arterial smooth muscle cell (PASMC) signaling from Ca 2+ -dependent mechanisms towards a novel epidermal growth factor receptor (EGFR)-NADPH oxidase 2 (NOX2)-Rho kinase Ca 2+ sensitization pathway. The overall objective of this study is to understand the mechanism by which CH mediates this switch in signaling. We have recently demonstrated that CH decreases membrane cholesterol, a lipid membrane domain component that can regulate both EGFR and NOX2 activity in other cell types. Since Rac1 is an important regulator of NOX2, we hypothesized that cholesterol depletion following CH unmasks EGFR-signaling through disinhibition of Rac1. Methods: We validated our ability to manipulate membrane cholesterol levels by treating isolated pulmonary arteries from control and CH (4 wk @ P B = 380 mmHg) Sprague Dawley rats with either cholesterol (2 mM), epicholesterol (cholesterol epimer; 2 mM), MβCD (cholesterol chelator; 10 mM), or vehicle. PASMC membrane cholesterol content was quantified by filipin (50 μg/ml) fluorescence using confocal microscopy. NOX2 activity was assessed via epidermal growth factor (EGF)-induced superoxide production +/- gp91ds-tat (selective NOX2 inhibitor; 50 μM) in primary cultures of PASMCs from control and CH rats. Superoxide levels were measured by dihydroethidium fluorescence (5 μM). We additionally assessed Rac1 activity by G-LISA (Cytoskeleton) in these cells. Subsequently, we examined vasoconstrictor responses to increasing concentrations of EGF (10 -10 – 10 -6 M) in pressurized, endothelium-disrupted pulmonary arteries (~150 μm diameter) from control rats. A subset of arteries was treated with gp91ds-tat (50 μM), fasudil (Rho kinase inhibitor; 10 μM), or their respective vehicles. Results: Cholesterol supplementation significantly (P<0.05) increased PASMC membrane cholesterol in both control and CH groups. Conversely, treatment with either MβCD or epicholesterol significantly decreased membrane cholesterol in arteries from control animals only. Additionally, epicholesterol increased NOX2-dependent superoxide production and Rac1 activity in PASMCs from control but not CH rats. In agreement with our hypothesis, the epicholesterol-dependent substitution of membrane cholesterol revealed concentration-dependent vasoconstriction to EGF in pulmonary arteries from control rats. Treatment with either fasudil or gp91ds-tat attenuated this effect. Conclusions: These results suggest decreased PASMC membrane cholesterol following CH augments EGFR-mediated vasoconstriction through disinhibition of NOX2 and Rac1. This work was supported by NIH R01 HL132883 to T. Resta, NIH T32 HL007736 to R. Ahmadian, and NIH F31 HL162502-01 to R. Ahmadian. 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.

Acid-sensing ion channel 1a (ASIC1a) belongs to a novel family of proton-gated cation channels that are permeable to both Na+ and Ca2+. ASIC1a is expressed in vascular smooth muscle and endothelial cells in a variety of vascular beds, yet little is known regarding the potential impact of ASIC1a to regulate local vascular reactivity. Our previous studies in rat mesenteric arteries suggest ASIC1a does not contribute to agonist-induced vasoconstriction but may mediate a vasodilatory response. The objective of the current study is to determine the role of ASIC1a in systemic vasodilatory responses by testing the hypothesis that the activation of endothelial ASIC1a mediates vasodilation of mesenteric resistance arteries through an endothelium-dependent hyperpolarization (EDH)-related pathway. The selective ASIC1a antagonist psalmotoxin 1 (PcTX1) largely attenuated the sustained vasodilatory response to acetylcholine (ACh) in isolated, pressurized mesenteric resistance arteries and ACh-mediated Ca2+ influx in freshly isolated mesenteric endothelial tubes. Similarly, basal tone was enhanced and ACh-induced vasodilation blunted in mesenteric arteries from Asic1a knockout mice. ASIC1a colocalizes with intermediate- and small-conductance Ca2+-activated K+ channels (IKCa and SKCa, respectively), and the IKCa/SKCa-sensitive component of the ACh-mediated vasodilation was blocked by ASIC1a inhibition. To determine the role of ASIC1a to activate IKCa/SKCa channels, we measured whole-cell K+ currents using the perforated-patch clamp technique in freshly isolated mesenteric endothelial cells. Inhibition of ASIC1a prevented ACh-induced activation of IKCa/SKCa channels. The ASIC1 agonist, α/β-MitTx, activated IKCa/SKCa channels and induced an IKCa/SKCa-dependent vasodilation. Together, the present study demonstrates that ASIC1a couples to IKCa/SKCa channels in mesenteric resistance arteries to mediate endothelium-dependent vasodilation.

Acid‐sensing ion channel 1 (ASIC1) is a proton‐gated cation channel that conducts both Na+ and Ca2+. ASIC1 is expressed in multiple cell types including vascular smooth muscle cells (SMCs) and endothelial cells (ECs). Our laboratory has previously shown that ASIC1 contributes to pulmonary arterial constriction and the development of chronic hypoxia (CH)‐induced pulmonary hypertension. However, the contribution of SMC versus EC ASIC1 to CH‐induced pulmonary hypertension is unknown. We tested the hypothesis that SMC expression of ASIC1 contributes to pulmonary hypertension and vascular remodeling by examining the development of CH‐induced pulmonary hypertension (barometric pressure of 380 mmHg for 6 weeks) in inducible SMC‐specific ASIC1 knockout (SMC‐Asic1‐/‐) and EC‐specific ASIC1 knockout (EC‐Asic1‐/‐) mice. The SMC‐Asic1‐/‐animals were used to examine the prevention and reversal of pulmonary hypertension by either inducing Asic1 knockout before CH exposure or following the establishment of pulmonary hypertension (3 weeks post‐CH). Pulmonary hypertension was assessed by direct cardiac puncture in anesthetized mice to measure right ventricular systolic pressure. Similar to global ASIC1 knockout (Asic1‐/‐) mice, SMC‐Asic1‐/‐ mice were protected from the development of CH‐induced pulmonary hypertension. In contrast, EC‐Asic1‐/‐ mice developed pulmonary hypertension comparable to wildtype Asic1+/+ mice (Figure 1). Furthermore, CH‐induced pulmonary hypertension was reversed by induction of SMC‐specific ASIC1 knockout following established pulmonary hypertension. CH‐induced vascular remodeling was assessed using smooth muscle α‐actin immunofluorescence to determine the medial thickness of small pulmonary arteries. The degree of arterial muscularization was significantly attenuated and/or reversed in SMC‐Asic1‐/‐ arteries (P<0.05). These findings were additionally supported by decreased CH‐induced proliferation (P<0.05) and migration (P<0.05) of pulmonary arterial SMC from Asic1‐/‐ mice, assessed by flow cytometry of BrdU positive cells and transwell migration assay, respectively. Together these data demonstrate that SMC, but not EC, ASIC1 contributes to CH‐induced pulmonary hypertension and vascular remodeling. Furthermore, these studies provide evidence for the therapeutic potential of ASIC1 inhibition to reverse pulmonary hypertension.

Acid-sensing ion channel 1a (ASIC1a) is a voltage-independent, non-selective cation channel that conducts both Na + and Ca 2+ . Activation of ASIC1a elicits plasma membrane depolarization and stimulates intracellular Ca 2+ -dependent signaling pathways in multiple cell types, including vascular smooth muscle (SM) and endothelial cells (ECs). Previous studies have shown that increases in pulmonary vascular resistance accompanying chronic hypoxia (CH)-induced pulmonary hypertension requires ASIC1a to elicit enhanced pulmonary vasoconstriction and vascular remodeling. Both SM and EC dysfunction drive these processes; however, the involvement of ASIC1a within these different cell types is unknown. Using the Cre-LoxP system to generate cell-type-specific Asic1a knockout mice, we tested the hypothesis that SM- Asic1a contributes to CH-induced pulmonary hypertension and vascular remodeling, whereas EC- Asic1a opposes the development of CH-induced pulmonary hypertension. The severity of pulmonary hypertension was not altered in mice with specific deletion of EC- Asic1a (Tek Cre - Asic1a fl/fl ). However, similar to global Asic1a knockout ( Asic1a −/- ) mice, mice with specific deletion of SM- Asic1a (MHC CreER - Asic1a fl/fl ) were protected from the development of CH-induced pulmonary hypertension and right heart hypertrophy. Furthermore, pulmonary hypertension was reversed when deletion of SM- Asic1a was initiated in conditional MHC CreER - Asic1a fl/fl mice with established pulmonary hypertension. CH-induced vascular remodeling was also significantly attenuated in pulmonary arteries from MHC CreER - Asic1a fl/fl mice. These findings were additionally supported by decreased CH-induced proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) from Asic1a −/- mice. Together these data demonstrate that SM-, but not EC- Asic1a contributes to CH-induced pulmonary hypertension and vascular remodeling. Furthermore, these studies provide evidence for the therapeutic potential of ASIC1a inhibition to reverse pulmonary hypertension.

Pulmonary hypertension (pHTN) is defined by mean pulmonary arterial pressure at rest exceeding 20 mmHg with mortality often resulting from right ventricular failure that is secondary to increased afterload. During pHTN, increased reliance on aerobic glycolysis and inhibition of mitochondrial oxidative respiration is observed in pulmonary arterial smooth muscle cells (PASMCs). This metabolic shift is associated with hyperproliferation and resistance to apoptosis contributing to the pathogenesis of pHTN. Along with these phenotypic changes, cells undergoing aerobic respiration exhibit increased glucose uptake and subsequent lactate and H efflux, contributing to extracellular acidosis. Acid-sensing ion channel 1 (ASIC1) is a voltage-independent, proton-gated cation channel that contributes to the development of chronic hypoxia (CH)-induced pHTN. While the function of ASIC1 is well characterized in pHTN, it has not been determined whether the metabolic shift observed during pHTN contributes to the activation of ASIC1. Therefore, we hypothesized that enhanced glucose uptake and subsequent acidification of the extracellular microenvironment in PASMCs lead to the activation of ASIC1 in CH-induced pHTN. As an initial step in testing this hypothesis, rats were housed in a hypobaric chamber (barometric pressure: ~380 mmHg) to induce pHTN and compared to age-matched normoxic controls. After 4 weeks, PASMCs were collected and transiently cultured under normoxia for 3-4 days. As expected, PASMCs from CH rats showed a reduced oxygen consumption rate (p<0.0001) and greater extracellular acidification (p<0.0001) compared to those from control animals, indicating increased glycolytic activity. Utilizing the fluorescent pH indicators SNARF-5/AM, we found that PASMCs from CH rats exhibit intracellular alkalization (p<0.0001 vs. control) and extracellular acidosis (p<0.001 vs. control). We further examined the H transporter(s) involved in mediating extracellular acidosis by inhibiting carbonic anhydrase IX (CA-IX), the Na /H exchanger (NHE1), the vacuolar-type H -type ATPase (V-ATPase), or monocarboxylate transporters 1 and -4 (MCT1 and MCT4). The dual MCT1/4 inhibitor, syrosingopine, prevented intracellular alkalization and extracellular acidosis following CH (Figure 1A), which corresponded with an increase in MCT4, but not MCT1, expression (Figure 1B). These data suggest MCT plays an important role in regulating PASMC pH homeostasis. Future studies will assess the role of MCT to activate ASIC1 and contribute to pHTN.

Hypoxia is the reduction of alveolar partial pressure of oxygen ([Formula: see text]). Military members and people who practice recreational activities from moderate to high altitudes are at risk for hypoxic exposure. Hypoxemia’s signs and symptoms vary from asymptomatic to severe responses, such as excessive hypoxic ventilatory responses and residual neurobehavioral impairment. Therefore, it is essential to identify hypoxia-induced biomarkers to indicate people with exposure to hypoxia. Advances have been made in understanding physiological responses to hypoxia, including elevations in circulating levels of endothelin 1 (ET-1) and microRNA 21 (miR-21) and reduction in circulating levels of hydrogen sulfide (H 2 S). Although the levels of these factors change upon exposure to hypoxia, it is unclear if these changes are sustained on return to normoxia. We hypothesize that hypoxia-induced ET-1 and miR-21 remain elevated, whereas hypoxia-reduction in H 2 S sustains after returning to normoxic conditions. To test this hypothesis, we exposed male rats to 6 h of 12% O 2 and measured circulating levels of ET-1 and miR-21, pre, during, and posthypoxia. We found that ET-1 plasma levels increased in response to hypoxia but returned to normal levels within 30 min after the restoration of normoxia. miR-21 plasma levels and transdermal H 2 S emissions decreased in response to hypoxia, remaining decreased on return to normoxia, thus following the biomarker criteria. Therefore, this study supports a unique role for plasma miR21 and transdermal H 2 S as hypoxia biomarkers that could be used to identify individuals after exposure to hypoxia.

The EGFR-NOX2-RhoA signaling cascade is present in arteries from normoxic animals but is normally inhibited by membrane cholesterol. The results of this study will allow for improved mechanistic analysis of how cholesterol regulates this pathway that is central to enhanced vasoconstrictor reactivity in PH.

Pulmonary arterial constriction and remodeling resulting from chronic hypoxia (CH) lead to increased pulmonary vascular resistance and resultant pulmonary hypertension (PH) in patients with chronic obstructive pulmonary diseases, sleep apnea, or in residents at high altitude. Recent studies from our laboratory have demonstrated a novel paradigm of CH-induced vasoconstriction and PH mediated by an epidermal growth factor receptor-NADPH oxidase 2-RhoA Ca2+sensitization pathway in pulmonary arterial smooth muscle cells (PASMCs). We have further demonstrated a central role for decreased PASMC membrane cholesterol in this response. However, the mechanism by which CH lowers cholesterol is unknown. Since CH has an effect to both increase reactive oxygen species (ROS) in PASMC and decrease membrane cholesterol, we hypothesized that elevated ROS production during CH diminishes PASMC membrane cholesterol. To test this hypothesis, PASMC were collected from control and CH Sprague Dawley Rats (4 wk, PB = 0.5 atm, N=7-9/group). After 3 days in cell culture, PASMC were treated with the ROS scavengers Tiron (10 mM), EUK-134 (EUK) (10 mM), or their respective vehicles for 30 minutes. PASMC membrane cholesterol was quantified with the fluorescent cholesterol marker filipin (50 mg/ml) via confocal microscopy. A two-way ANOVA was used to make comparisons and if differences were detected, individual groups were compared using the Tukey's multiple comparisons test. A probability of P < 0.05 was considered significant for comparisons. In agreement with our hypothesis, the CH-dependent reduction in PASMC membrane cholesterol was acutely reversed following treatment with the ROS scavengers Tiron (Figure 1A) and EUK (Figure 1B). We conclude that ROS mediate CH-dependent decreases in PASMC membrane cholesterol following CH exposure. These findings may provide a mechanistic basis by which CH links vasoconstrictor stimuli to EGFR signaling and resultant pulmonary hypertension. Future directions will explore the mechanism by which ROS mediate this response, including cholesterol oxidation or dysregulation of membrane cholesterol trafficking.

Pulmonary hypertension is characterized by sustained vasoconstriction and remodelling of the small pulmonary arteries, which is associated with persistent depolarization of the resting membrane potential (E-m) of pulmonary arterial smooth muscle cells (PASMCs). It is well-known that the underlying mechanism of this depolarization includes inhibition of K+ channels; however, whether other ion channels contribute to this depolarization is unknown. We previously reported that acid-sensing ion channel 1 (ASIC1), a non-selective cation channel (NSCC) that conducts both Na+ and Ca2+, is present in PASMCs and contributes to the development of chronic hypoxia (CH)-induced pulmonary hypertension. Therefore, we tested the hypothesis that ASIC1-mediated Na+ influx contributes to PASMC E-m regulation following CH-induced pulmonary hypertension. Using sharp electrode intracellular recordings in isolated, pressurized small pulmonary arteries from rats and mice, we show that exposure to CH leads to PASMC membrane depolarization compared with control animals, and this is independent of intraluminal pressure-induced depolarization. In addition to a decrease in PASMC whole-cell K+ currents following CH, we demonstrate that whole-cell NSCC currents are increased and essential to the persistent CH-induced E-m depolarization in PASMCs. Both the specific inhibitor of ASIC1, psalmotoxin 1, and global knockout ofASIC1 (Asic1(-/-)) preventsCH-inducedEm depolarization and largely inhibitswhole-cell NSCC currents, without affecting whole-cell K+ currents. Our results show a combination of factors, including inhibition of K+ efflux and augmented Na+ influx, mediate CH-induced PASMC depolarization. Furthermore, this study demonstrates a novel role for ASIC1 in the regulation of E-m in PASMCs during CH-induced pulmonary hypertension.

Chronic hypoxia (CH) augments pulmonary arterial tone through O2−‐dependent stimulation of RhoA, a response that may contribute to the vasoconstrictor component of pulmonary hypertension. Although mitochondria are an important source of reactive oxygen species (ROS) in the pulmonary vasculature, the role of mitochondria‐derived ROS (mitoROS) in enhanced vasoconstrictor reactivity following CH is unknown. We hypothesized that mitoROS contribute to elevated basal pulmonary arterial tone and endothelin‐1 (ET‐1) induced vasoconstriction following CH. To test our hypothesis, we measured basal tone by videomicroscopy in isolated, pressurized small pulmonary arteries [~150 μm inner diameter (i.d.)] from normoxic and CH (4 wk, 0.5 atm) rats following administration of the mitochondria‐targeted antioxidants, MitoQ (1 μM) or MitoTEMPO (200 μM), or their respective vehicles. Parallel studies evaluated effects of mitoROS inhibition on vasoconstrictor responses to increasing concentrations of ET‐1. Experiments were performed either in the presence of the nitric oxide (NO) synthase inhibitor Nω‐nitro‐L‐arginine (300 μM) or after endothelial disruption to limit the influence of endogenous NO. Pulmonary arterial tone was calculated as the percent difference in i.d. between Ca2+‐free and Ca2+‐containing conditions, whereas vasoconstriction to ET‐1 was expressed as a percentage of baseline i.d. Exposure to CH significantly (P < 0.05) increased basal pulmonary arterial tone and vasoconstrictor responsiveness to ET‐1 in both NOS‐inhibited and endothelium‐disrupted arteries. Treatment with either MitoQ or MitoTEMPO prevented these responses to CH and normalized values between groups. We conclude that mitoROS provide a major contribution to CH‐induced increases in pulmonary vasoconstrictor reactivity.Support or Funding InformationThis work was supported by NIH grant R01 HL132883 (to T.C. Resta).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hypoxic pulmonary hypertension (PH) can be caused by lung disease, injury, and chronic hypoxia (CH) exposure. Hallmarks for PH include recruitment of immune cells to the perivascular region of the lungs, thickening of the pulmonary arterial walls, elevated pulmonary vascular resistance and pressure, and right ventricular remodeling. These signature signs of PH are what lead to right heart failure and ultimately death. We have previously reported that 21 days of CH leads to both an upregulation of collagen type V (Col V) as well as natural T helper 17 (nTh17) cell‐reactivity towards Col V in mouse pulmonary arteries without affecting the number of T regulatory cells (Tregs) in the lungs. However, it is unknown if Treg suppressive capacity and/or peripheral tolerance to autoantigens is affected by CH. Col V‐specific Tregs, e.g. peripheral tolerance, can be induced by nasal instillation of Col V without adjuvant. Using right ventricular systolic pressure (RVSP) and pulmonary arterial wall thickening as indicators of PH, we hypothesized that induction of mucosal tolerance to Col V attenuates hypoxic PH in mice. Our data support this hypothesis, as both RVSP and wall thickening are lower in CH‐exposed Col V‐inoculated vs. vehicle‐inoculated mice (Figure). Future studies will characterize the effect of CH on Treg suppressive activity, nTh17 perivascular infiltration, and nTh17‐mediated Col V reactivity. In conclusion, our study suggests that induction of mucosal tolerance to Col V attenuates chronic hypoxia‐induced PH.

Studies from our group and others demonstrate a protective role of estrogen in attenuating the progression of chronic hypoxia (CH)‐induced pulmonary hypertension. Physiological responses to estrogen are mediated by the nuclear estrogen receptors a and b, as well as by the membrane‐bound G protein‐coupled estrogen receptor (GPER). However, the role of GPER in the development of pulmonary hypertension resulting from CH is unknown. We hypothesized that GPER acts to limit the severity of CH‐induced pulmonary hypertension.To test this hypothesis, we evaluated indices of pulmonary hypertension in control and CH (4 wk, PB = 0.5 atm) female rats administered the GPER antagonist G36 via osmotic pumps (1 mg/kg/day, s.c.) during the entire 4 wk exposure period (n=7–8/group). Right ventricular systolic pressure (RVSP) was measured using a fluid‐filled catheter advanced through the right jugular vein into the right ventricle (RV) during isoflurane anesthesia. Additionally, RV hypertrophy was determined from Fulton’s index [RV/(LV+S)], and the polycythemic response to CH was assessed by measuring hematocrit.CH increased RVSP, Fulton’s Index, and hematocrit in vehicle‐treated rats (P<0.0001), indicative of pulmonary hypertension and associated polycythemia. Unexpectedly, RVSP was lower in G36‐treated CH rats compared to control animals (P<0.05), with a downward trending Fulton’s Index (not significant). However, hematocrit was unaltered by GPER inhibition. We conclude that GPER mediates a novel effect to facilitate the development of pulmonary hypertension resulting from CH exposure.Support or Funding InformationNIH Grants R01 HL132883, T32 HL007736, and P20 GM103451. AHA Grant 18AIREA33960020

Chronic hypoxia (CH)-induced pulmonary hypertension (PH) results, in part, from T helper-17 (TH17) cell-mediated perivascular inflammation. However, the antigen(s) involved is unknown. Cellular immunity to collagen type V (col V) develops after ischemia-reperfusion injury during lung transplant and is mediated by naturally occurring (n)TH17 cells. Col5a1 gene codifies for the α1-helix of col V, which is normally hidden from the immune system within type I collagen in the extracellular matrix. COL5A1 promoter analysis revealed nuclear factor of activated T cells, cytoplasmic 3 (NFATc3) binding sites. Therefore, we hypothesized that smooth muscle NFATc3 upregulates col V expression, leading to nTH17 cell-mediated autoimmunity to col V in response to CH, representing an upstream mechanism in PH development. To test our hypothesis, we measured indexes of PH in inducible smooth muscle cell (SMC)-specific NFATc3 knockout (KO) mice exposed to either CH (380 mmHg) or normoxia and compared them with wild-type (WT) mice. KO mice did not develop PH. In addition, COL5A1 was one of the 1,792 genes differentially affected by both CH and SMC NFATc3 in isolated intrapulmonary arteries, which was confirmed by RT-PCR and immunostaining. Cellular immunity to col V was determined using a trans vivo delayed-type hypersensitivity assay (Tv-DTH). Tv-DTH response was evident only when splenocytes were used from control mice exposed to CH but not from KO mice, and mediated by nTH17 cells. Our results suggest that SMC NFATc3 is important for CH-induced PH in adult mice, in part, by regulating the expression of the lung self-antigen COL5A1 protein contributing to col V-reactive nTH17-mediated inflammation and hypertension.

Elevated resistance of pulmonary circulation after chronic hypoxia exposure leads to pulmonary hypertension. Contributing to this pathological process is enhanced pulmonary vasoconstriction through both calcium-dependent and calcium sensitization mechanisms. Reactive oxygen species (ROS), as a result of increased enzymatic production and/or decreased scavenging, participate in augmentation of pulmonary arterial constriction by potentiating calcium influx as well as activation of myofilament sensitization, therefore mediating the development of pulmonary hypertension. Here, we review the effects of chronic hypoxia on sources of ROS within the pulmonary vasculature including NADPH oxidases, mitochondria, uncoupled endothelial nitric oxide synthase, xanthine oxidase, monoamine oxidases and dysfunctional superoxide dismutases. We also summarize the ROS-induced functional alterations of various Ca2+ and K+ channels involved in regulating Ca2+ influx, and of Rho kinase that is responsible for myofilament Ca2+ sensitivity. A variety of antioxidants have been shown to have beneficial therapeutic effects in animal models of pulmonary hypertension, supporting the role of ROS in the development of pulmonary hypertension. A better understanding of the mechanisms by which ROS enhance vasoconstriction will be useful in evaluating the efficacy of antioxidants for the treatment of pulmonary hypertension.

Chronic hypoxia (CH) augments depolarization‐induced pulmonary vasoconstriction through a myofilament Ca2+ sensitization mechanism involving epidermal growth factor receptor (EGFR) and NADPH oxidase‐dependent Rho kinase activation. Although the mechanism that links CH exposure to activation of this pathway is unknown, previous studies support a role for caveolin‐1 and cholesterol to regulate both EGFR and NADPH oxidase. Furthermore, membrane cholesterol content can influence caveolin‐1 interactions with a variety of proteins. Therefore, we hypothesized that derangement of these lipid domain components contributes to increased depolarization‐induced Ca2+ sensitization and pulmonary vasoconstriction following CH.To test this hypothesis, we examined vasoconstrictor responses to depolarizing concentrations of KCl (30–120 mM) in pressurized, endothelium‐disrupted pulmonary arteries (~150 um diameter) from control and CH (4 wk at 0.5 atm) rats under conditions in which vascular smooth muscle Ca2+ was clamped with ionomycin (3 mM). The effects of altered lipid domains on vasomotor responses were tested utilizing a solution of MβCD and cholesterol (10 mM and 2 mM respectively) to increase cholesterol, and the caveolin‐1 scaffolding domain peptide, AP‐Cav (10 mM). We also assessed the incidence of caveolae using transmission electron microscopy and caveolin‐1 levels by western blotting. Membrane cholesterol content was quantified by filipin (50 mg/ml) fluorescence using confocal microscopy.In agreement with our hypothesis, CH reduced membrane cholesterol content in pulmonary arterial smooth muscle. However, CH was without effect on caveolin‐1 expression or the incidence of caveolae. CH augmented vasoconstrictor sensitivity in response to depolarizing concentrations of KCl as previously described. Both cholesterol repletion and AP‐Cav prevented effects of CH to enhance KCl‐dependent constriction and normalized reactivity between groups. Interestingly, AP‐Cav restored smooth muscle membrane cholesterol in arteries from CH rats to the level of controls, consistent with evidence that caveolin‐1 can regulate cholesterol trafficking to the cell membrane. These results support an effect of AP‐Cav to attenuate KCl‐dependent vasoconstriction following CH by normalizing membrane cholesterol levels. We conclude that alterations in the lipid domains resulting from reduced cholesterol facilitate enhanced depolarization induced pulmonary vasoconstriction following CH.Support or Funding InformationNIH Grants R01 HL132883 and T32 HL007736. AHA Grant 13PRE14580015