Acute Hypoxic Pulmonary Vasoconstriction Research Articles

Pannexin 1: a novel regulator of acute hypoxic pulmonary vasoconstriction.

Hypoxic pulmonary vasoconstriction (HPV) is a physiological response to alveolar hypoxia that diverts blood flow from poorly ventilated to better aerated lung areas to optimize ventilation-perfusion matching. Yet, the exact sensory and signalling mechanisms by which hypoxia triggers pulmonary vasoconstriction remain incompletely understood. Recently, ATP release via pannexin 1 (Panx1) and subsequent signalling via purinergic P2Y receptors has been identified as regulator of vasoconstriction in systemic arterioles. Here, we probed for the role of Panx1-mediated ATP release in HPV and chronic hypoxic pulmonary hypertension (PH). Pharmacological inhibition of Panx1 by probenecid, spironolactone, the Panx1 specific inhibitory peptide (10Panx1), and genetic deletion of Panx1 specifically in smooth muscle attenuated HPV in isolated perfused mouse lungs. In pulmonary artery smooth muscle cells (PASMCs), both spironolactone and 10Panx1 attenuated the increase in intracellular Ca2+ concentration ([Ca2+]i) in response to hypoxia. Yet, genetic deletion of Panx1 in either endothelial or smooth muscle cells did not prevent the development of PH in mice. Unexpectedly, ATP release in response to hypoxia was not detectable in PASMC, and inhibition of purinergic receptors or ATP degradation by ATPase failed to attenuate HPV. Rather, transient receptor potential vanilloid 4 (TRPV4) antagonism and Panx1 inhibition inhibited the hypoxia-induced [Ca2+]i increase in PASMC in an additive manner, suggesting that Panx1 regulates [Ca2+]i independently of the ATP-P2Y-TRPV4 pathway. In line with this notion, Panx1 overexpression increased the [Ca2+]i response to hypoxia in HeLa cells. In the present study, we identify Panx1 as novel regulator of HPV. Yet, the role of Panx1 in HPV was not attributable to ATP release and downstream signalling via P2Y receptors or TRPV4 activation, but relates to a role of Panx1 as direct or indirect modulator of the PASMC Ca2+ response to hypoxia. Panx1 did not affect the development of chronic hypoxic PH.

Effects of Recombinant Human Angiotensin-Converting Enzyme 2 on Response to Acute Hypoxia and Exercise: A Randomised, Placebo-Controlled Study

IntroductionAngiotensin-converting enzyme 2 (ACE2) is a key enzyme of the renin-angiotensin system (RAS) that has been implicated in the pathogenesis of acute respiratory distress syndrome (ARDS). Enhancing ACE2 activity using GSK2586881, a recombinant form of human ACE2, could be beneficial in diseases such as ARDS but may blunt the hypoxic pulmonary vasoconstriction (HPV) response and potentially impact systemic and tissue oxygenation. This study aimed to evaluate the effect of GSK2586881 0.8 mg/kg on HPV response in healthy adult volunteers during exercise under hypoxic conditions.MethodsIn this phase I, randomised, double-blind (sponsor open) study, GSK2586881 or placebo was administered as a single intravenous (IV) dose in a two-period crossover design. Treatment periods were separated by a washout period of 3–14 days. The primary endpoint was change from baseline in pulmonary artery systolic pressure (PASP) measured by echocardiography. Secondary endpoints included RAS peptides and oxygen saturation.ResultsSeventeen adults aged 18–40 years were randomised to treatment. There were no clinically relevant differences (defined as a reduction of ≥ 5 mmHg) in change from baseline in PASP between GSK2586881 and placebo. GSK2586881 was well tolerated, with no serious adverse events, no worsening of hypoxaemia and no evidence of immunogenicity. The study was terminated early after review of the data, which showed that the predefined success criteria had not been met. Following GSK2586881 administration, levels of the RAS peptide angiotensin II decreased while angiotensin (1-7) increased, as expected, indicating that GSK2586881 was pharmacologically active.ConclusionsA single IV dose of GSK2586881 0.8 mg/kg was well tolerated but did not impact the acute HPV response in healthy volunteers.Supplementary InformationThe online version contains supplementary material available at 10.1007/s41030-021-00164-7.

Genetic deletion of p66shc and/or cyclophilin D results in decreased pulmonary vascular tone.

AimsThe pulmonary vascular tone and hypoxia-induced alterations of the pulmonary vasculature may be regulated by the mitochondrial membrane permeability transition pore (mPTP) that controls mitochondrial calcium load and apoptosis. We thus investigated, if the mitochondrial proteins p66shc and cyclophilin D (CypD) that regulate mPTP opening affect the pulmonary vascular tone.Methods and resultsMice deficient for p66shc (p66shc−/−), CypD (CypD−/−), or both proteins (p66shc/CypD−/−) exhibited decreased pulmonary vascular resistance (PVR) compared to wild-type mice determined in isolated lungs and in vivo. In contrast, systemic arterial pressure was only lower in CypD−/− mice. As cardiac function and pulmonary vascular remodelling did not differ between genotypes, we determined alterations of vascular contractility in isolated lungs and calcium handling in pulmonary arterial smooth muscle cells (PASMC) as underlying reason for decreased PVR. Potassium chloride (KCl)-induced pulmonary vasoconstriction and KCl-induced cytosolic calcium increase determined by Fura-2 were attenuated in all gene-deficient mice. In contrast, KCl-induced mitochondrial calcium increase determined by the genetically encoded Mito-Car-GECO and calcium retention capacity were increased only in CypD−/− and p66shc/CypD−/− mitochondria indicating that decreased mPTP opening affected KCl-induced intracellular calcium peaks in these cells. All mouse strains showed a similar pulmonary vascular response to chronic hypoxia, while acute hypoxic pulmonary vasoconstriction was decreased in gene-deficient mice indicating that CypD and p66shc regulate vascular contractility but not remodelling.ConclusionsWe conclude that p66shc specifically regulates the pulmonary vascular tone, while CypD also affects systemic pressure. However, only CypD acts via regulation of mPTP opening and mitochondrial calcium regulation.

Hypoxic Pulmonary Vasoconstriction in Isolated Mouse Lungs

BackgroundHypoxic pulmonary vasoconstriction (HPV) is a physiological mechanism of lung to ensure maximal gas exchange and blood oxygenation. Persistent and global alveolar hypoxia leads to sustained vasoconstriction and vascular remodeling causing pulmonary hypertension. It has been demonstrated that acute HPV is initiated by an increase in cytosolic [Ca2+] ([Ca2+]cyt) in pulmonary arterial smooth muscle cells (PASMC) and modulated by factors released from endothelium. Acute HPV is well reserved in isolated perfused/ventilated lungs, whereas HPV in isolated pulmonary arterial rings shows different pattern in comparison to HPV in intact animals and humans. In this study, we aimed to use isolated perfused/ventilated mouse lungs to further study potential mechanisms involved in development of HPV.MethodsC57BL/6 mice (male, 8–10 weeks old) were anesthetized by pentobarbital sodium (120 mg/kg). After tracheostomy, isolated lungs were immediately ventilated with normoxic gas (21% O2/5% CO2) using a rodent ventilator (80 breaths/min with a tidal volume of 250 μl). The mice were then placed in an isolated lung system chamber with a heated water jacket at 37°C. After tracheal intubation, the chest was opened and a catheter was inserted into the main PA via the right ventricle to measure PAP. The lung vasculature was consistently superfused with physiological salt solution (PSS) via a pump while lung airway and alveoli were ventilated with normoxic or hypoxic gas. Before experimentation, the isolated lung was first superfused with the 40 mM K+‐containing PSS (40K) to stabilize the basal PAP. The lung was then repetitively challenged by ventilation of hypoxic gas (1% O2 in N2, for 4 min) to induce an increase in PAP (due to HPV). Pharmacological effects of various inhibitors on HPV were examined by superfusion of PSS containing the inhibitors while the lung was ventilated with hypoxic gas.ResultsOur results show that HPV was dependent on Ca2+ influx through a) nifedipine‐sensitive L‐type voltage‐dependent Ca2+ channels; b) SKF‐sensitive non‐selective cation channels; and c) TRP channels like TRPC6, TRPV1 and TRPP. HPV in isolated perfused lung was also significantly inhibited by blocking Ca2+‐sensing receptors (CaSR) and Notch receptors and by extracellular Mg2+ and Mn2+. Interestingly, blockade of Ca2+‐activated Cl− channels and inhibition of muscarinic receptors and endothelin receptors had little effect on HPV. Furthermore, inhibition of nitric oxide synthase (eNOS) potentiated HPV.ConclusionAn increase in [Ca2+]cyt due to Ca2+ influx through various Ca2+‐permeable cation channels in PASMC influx is required for the initiation of acute HPV. Both voltage‐gated Ca2+ channels (e.g., CaV1.2) and receptor‐operated Ca2+ channels (TRPC6/V1/P) are involved in mediating Ca2+ influx to induce HPV. G protein‐coupled receptor/CaSR‐mediated synthesis of intracellular signaling molecules (e.g., diacylglycerol and inositol 1, 4, 5‐trisphosphate) and Notch‐associated cell‐to‐cell interaction also play important roles in HPV. These data indicate that multiple mechanisms are involved in the initiation and regulation of HPV.Support or Funding InformationNIH NIHLBI HL135807

P-189 - The role of mitochondrial reactive oxygen species in the response of the pulmonary vasculature to hypoxia and right heart remodeling

Increased release of mitochondrial superoxide has been suggested to mediate acute hypoxic pulmonary vasoconstriction (HPV) as well as chronic hypoxia-induced pulmonary hypertension (PH) and right heart remodeling. Thus, we investigated the superoxide release during HPV, chronic hypoxia-induced PH and after pulmonary arterial banding (PAB), as well as the effect of the mitochondria-targeted antioxidant MitoQ on these processes. Superoxide levels were increased in PASMC during acute hypoxia and decreased after 5 days of hypoxia. In parallel MitoQ, but not its inactive carrier substance, TPP+, significantly inhibited acute HPV and the rise in superoxide concentration induced by acute hypoxia. However, MitoQ application did not affect the hypoxia-induced proliferation of PASMC or the development of chronic hypoxia-induced PH. In contrast, MitoQ application attenuated right ventricular remodeling after chronic hypoxic exposure as well as after PAB with regard to development of right heart hypertrophy and dilatation. Increased superoxide concentration mediates acute HPV, while decreased superoxide levels were detected in chronic hypoxia-induced PH. MitoQ may be beneficial under conditions of exaggerated acute HPV and to prevent the development of right heart remodeling.

EETs exacerbate chronic hypoxia‐induced pulmonary hypertension

We have demonstrated that epoxyeicosatrienoic acids (EETs), metabolites of arachidonic acids that are degraded by soluble epoxide hydrolase (sEH), initiate increases in right ventricular systolic pressure (RVSP) by perhaps, increasing pulmonary vascular resistance. Also, EETs enhance acute hypoxic pulmonary vasoconstriction (HPV) by shifting the balance between constrictor and dilator prostaglandins towards constrictor prostanoids. Prolonged HPV promotes hypoxia‐induced pulmonary hypertension (HPH), we therefore, tested the hypothesis that deletion of sEH gene (sEH‐KO) promotes EET‐mediated potentiation of HPH.MethodsMale sEH‐KO (12‐weel old) and age matched C57BL/6J (WT) mice were exposed to 10% O2 for 21 days. Cardiac hemodynamics were assessed by echocardiography and right heart catheterization. Right ventricular hypertrophy (RVH) was expressed as the weight ratio of right ventricle (RV) to left ventricle (LV) with septum (RV/LV+S). sEH expression in pulmonary tissue was assessed by western blot. Pulmonary EET levels were measured by LC‐MS/MS analysis.ResultsPulmonary doppler flow echocardiography showed that hypoxia elicited decreases in pulmonary arterial acceleration time (PAAT), ejection time (ET), and PAAT/ET ratio. The reduction is significantly greater in sEH‐KO mice compared to that of WT mice. Baseline RVSP was comparable between WT and sEH‐KO mice. Hypoxia exposure significantly increased RVSP in both strains of mice, the increment however, was significantly greater, associated with greater RVH in sEH‐KO mice compared to their WT controls. This indicates that sEH‐KO mice developed more severe HPH. Although chronic hypoxia also downregulated pulmonary sEH expression in WT mice, sEH‐KO mice exhibited highest level of EETs that correlated to their greater increases in RVSP and more severe HPH. In conclusion, the deficiency of sEH promotes EET‐mediated exacerbation of HPH.Support or Funding Information(This work was supported by HL070653, HL115124 and HL129797).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impact of the mitochondria-targeted antioxidant MitoQ on hypoxia-induced pulmonary hypertension.

Increased mitochondrial reactive oxygen species (ROS), particularly superoxide have been suggested to mediate hypoxic pulmonary vasoconstriction (HPV), chronic hypoxia-induced pulmonary hypertension (PH) and right ventricular (RV) remodelling.We determined ROS in acute, chronic hypoxia and investigated the effect of the mitochondria-targeted antioxidant MitoQ under these conditions.The effect of MitoQ or its inactive carrier substance, decyltriphenylphosphonium (TPP+), on acute HPV (1% O2 for 10 minutes) was investigated in isolated blood-free perfused mouse lungs. Mice exposed for 4 weeks to chronic hypoxia (10% O2) or after banding of the main pulmonary artery (PAB) were treated with MitoQ or TPP+ (50 mg/kg/day).Total cellular superoxide and mitochondrial ROS levels were increased in pulmonary artery smooth muscle cells (PASMC), but decreased in pulmonary fibroblasts in acute hypoxia. MitoQ significantly inhibited HPV and acute hypoxia-induced rise in superoxide concentration. ROS was decreased in PASMC, while it increased in the RV after chronic hypoxia. Correspondingly, MitoQ did not affect the development of chronic hypoxia-induced PH, but attenuated RV remodelling after chronic hypoxia as well as after PAB.Increased mitochondrial ROS of PASMC mediate acute HPV, but not chronic hypoxia-induced PH. MitoQ may be beneficial under conditions of exaggerated acute HPV.

P 165 - The role of mitochondrial reactive oxygen species in the response of the pulmonary vasculature to hypoxia and right heart remodeling

Introduction Increased release of mitochondrial superoxide has been suggested to mediate acute hypoxic pulmonary vasoconstriction (HPV) as well as chronic hypoxia-induced pulmonary hypertension (PH) and right heart remodeling. Thus, we investigated the superoxide release during HPV, chronic hypoxia-induced PH and after pulmonary arterial banding (PAB), as well as the effect of the mitochondria-targeted antioxidant MitoQ on these processes. Results Superoxide levels were increased in PASMC during acute hypoxia, and decreased after 5 days of hypoxia. In parallel MitoQ, but not its inactive carrier substance, TPP+, significantly inhibited acute HPV and the rise in superoxide concentration induced by acute hypoxia. However, MitoQ application did not affect the hypoxia-induced proliferation of PASMC or the development of chronic hypoxia-induced PH. In contrast, MitoQ application attenuated right ventricular remodeling after chronic hypoxic exposure as well as after PAB with regard to development of right heart hypertrophy and dilatation. Accordingly, superoxide levels were increased in the RV after PAB. Conclusion Increased superoxide concentration mediates acute HPV, while decreased superoxide levels were detected in chronic hypoxia-induced PH. MitoQ may be beneficial under conditions of exaggerated acute HPV and to prevent the development of right heart remodeling.

The Role of Transient Receptor Potential Channel 6 Channels in the Pulmonary Vasculature.

Canonical or classical transient receptor potential channel 6 (TRPC6) is a Ca2+-permeable non-selective cation channel that is widely expressed in the heart, lung, and vascular tissues. The use of TRPC6-deficient (“knockout”) mice has provided important insights into the role of TRPC6 in normal physiology and disease states of the pulmonary vasculature. Evidence indicates that TRPC6 is a key regulator of acute hypoxic pulmonary vasoconstriction. Moreover, several studies implicated TRPC6 in the pathogenesis of pulmonary hypertension. Furthermore, a unique genetic variation in the TRPC6 gene promoter has been identified, which might link the inflammatory response to the upregulation of TRPC6 expression and ultimate development of pulmonary vascular abnormalities in idiopathic pulmonary arterial hypertension. Additionally, TRPC6 is critically involved in the regulation of pulmonary vascular permeability and lung edema formation during endotoxin or ischemia/reperfusion-induced acute lung injury. In this review, we will summarize latest findings on the role of TRPC6 in the pulmonary vasculature.

Mitochondrial Complex IV Subunit 4 Isoform 2 Is Essential for Acute Pulmonary Oxygen Sensing.

Acute pulmonary oxygen sensing is essential to avoid life-threatening hypoxemia via hypoxic pulmonary vasoconstriction (HPV) which matches perfusion to ventilation. Hypoxia-induced mitochondrial superoxide release has been suggested as a critical step in the signaling pathway underlying HPV. However, the identity of the primary oxygen sensor and the mechanism of superoxide release in acute hypoxia, as well as its relevance for chronic pulmonary oxygen sensing, remain unresolved. To investigate the role of the pulmonary-specific isoform 2 of subunit 4 of the mitochondrial complex IV (Cox4i2) and the subsequent mediators superoxide and hydrogen peroxide for pulmonary oxygen sensing and signaling. Isolated ventilated and perfused lungs from Cox4i2-/- mice lacked acute HPV. In parallel, pulmonary arterial smooth muscle cells (PASMCs) from Cox4i2-/- mice showed no hypoxia-induced increase of intracellular calcium. Hypoxia-induced superoxide release which was detected by electron spin resonance spectroscopy in wild-type PASMCs was absent in Cox4i2-/- PASMCs and was dependent on cysteine residues of Cox4i2. HPV could be inhibited by mitochondrial superoxide inhibitors proving the functional relevance of superoxide release for HPV. Mitochondrial hyperpolarization, which can promote mitochondrial superoxide release, was detected during acute hypoxia in wild-type but not Cox4i2-/- PASMCs. Downstream signaling determined by patch-clamp measurements showed decreased hypoxia-induced cellular membrane depolarization in Cox4i2-/- PASMCs compared with wild-type PASMCs, which could be normalized by the application of hydrogen peroxide. In contrast, chronic hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling were not or only slightly affected by Cox4i2 deficiency, respectively. Cox4i2 is essential for acute but not chronic pulmonary oxygen sensing by triggering mitochondrial hyperpolarization and release of mitochondrial superoxide which, after conversion to hydrogen peroxide, contributes to cellular membrane depolarization and HPV. These findings provide a new model for oxygen-sensing processes in the lung and possibly also in other organs.

Sympathetic hyper-excitation in obesity and pulmonary hypertension: physiological relevance to the 'obesity paradox'.

Within the lung, sympathetic nerve activity (SNA) has an important role in facilitating pulmonary vasodilation. As SNA is elevated in obesity, we aimed to assess the impact of sympathetic hyper-excitation on pulmonary vascular homeostasis in obesity, and its potential role in ameliorating the severity of pulmonary hypertension (PH); the well-documented 'obesity paradox' phenomenon. Zucker obese and lean rats were exposed to normoxia or chronic hypoxia (CH-10% O2) for 2 weeks. Subsequently, pulmonary SNA (pSNA) was recorded (electrophysiology), or the pulmonary microcirculation was visualized using Synchrotron microangiography. Acute hypoxic pulmonary vasoconstriction (HPV) was assessed before and after blockade of β1-adrenergic receptors (ARs) (atenolol, 3 mg kg(-1)) and β1+β2-adrenergic (propranolol, 2 mg kg(-1)). pSNA of normoxic obese rats was higher than lean counterparts (2.4 and 0.5 μV s, respectively). SNA was enhanced following the development of PH in lean rats, but more so in obese rats (1.7 and 6.8 μV s, respectively). The magnitude of HPV was similar for all groups (for example, ~20% constriction of the 200-300 μm vessels). Although β-blockade did not modify HPV in lean rats, it significantly augmented the HPV in normoxic obese rats (β1 and β2 blockade), and more so in obese rats with PH (β2-blockade alone). Western blots showed, while the expression of pulmonary β1-ARs was similar for all rats, the expression of β2-ARs was downregulated in obesity and PH. This study suggests that sympathetic hyper-excitation in obesity may have an important role in constraining the severity of PH and, thus, contribute in part to the 'obesity paradox' in PH.

Effect of oral nitrate supplementation on pulmonary hemodynamics during exercise and time trial performance in normoxia and hypoxia: a randomized controlled trial.

Background: Hypoxia-induced pulmonary vasoconstriction increases pulmonary arterial pressure (PAP) and may impede right heart function and exercise performance. This study examined the effects of oral nitrate supplementation on right heart function and performance during exercise in normoxia and hypoxia. We tested the hypothesis that nitrate supplementation would attenuate the increase in PAP at rest and during exercise in hypoxia, thereby improving exercise performance.Methods: Twelve trained male cyclists [age: 31 ± 7 year (mean ± SD)] performed 15 km time-trial cycling (TT) and steady-state submaximal cycling (50, 100, and 150 W) in normoxia and hypoxia (11% inspired O2) following 3-day oral supplementation with either placebo or sodium nitrate (0.1 mmol/kg/day). We measured TT time-to-completion, muscle tissue oxygenation during TT and systolic right ventricle to right atrium pressure gradient (RV-RA gradient: index of PAP) during steady state cycling.Results: During steady state exercise, hypoxia elevated RV-RA gradient (p > 0.05), while oral nitrate supplementation did not alter RV-RA gradient (p > 0.05). During 15 km TT, hypoxia lowered muscle tissue oxygenation (p < 0.05). Nitrate supplementation further decreased muscle tissue oxygenation during 15 km TT in hypoxia (p < 0.05). Hypoxia impaired time-to-completion during TT (p < 0.05), while no improvements were observed with nitrate supplementation in normoxia or hypoxia (p > 0.05).Conclusion: Our findings indicate that oral nitrate supplementation does not attenuate acute hypoxic pulmonary vasoconstriction nor improve performance during time trial cycling in normoxia and hypoxia.

Na+/H+ Exchange and Hypoxic Pulmonary Hypertension

Intracellular pH (pHi) homeostasis is key to the functioning of vascular smooth muscle cells, including pulmonary artery smooth muscle cells (PASMCs). Sodium-hydrogen exchange (NHE) is an important contributor to pHi control in PASMCs. In this review, we examine the role of NHE in PASMC function, in both physiologic and pathologic conditions. In particular, we focus on the contribution of NHE to the PASMC response to hypoxia, considering both acute hypoxic pulmonary vasoconstriction and the development of pulmonary vascular remodeling and pulmonary hypertension in response to chronic hypoxia. Hypoxic pulmonary hypertension remains a disease with limited therapeutic options. Thus, this review explores past efforts at disrupting NHE signaling and discusses the therapeutic potential that such efforts may have in the field of pulmonary hypertension.

Discovery and pharmacological characterization of a novel potent inhibitor of diacylglycerol-sensitive TRPC cation channels.

The cation channel transient receptor potential canonical (TRPC) 6 has been associated with several pathologies including focal segmental glomerulosclerosis, pulmonary hypertension and ischaemia reperfusion-induced lung oedema. We set out to discover novel inhibitors of TRPC6 channels and investigate the therapeutic potential of these agents. A library of potential TRPC channel inhibitors was designed and synthesized. Activity of the compounds was assessed by measuring intracellular Ca(2+) levels. The lead compound SAR7334 was further characterized by whole-cell patch-clamp techniques. The effects of SAR7334 on acute hypoxic pulmonary vasoconstriction (HPV) and systemic BP were investigated. SAR7334 inhibited TRPC6, TRPC3 and TRPC7-mediated Ca(2+) influx into cells with IC50 s of 9.5, 282 and 226 nM, whereas TRPC4 and TRPC5-mediated Ca(2+) entry was not affected. Patch-clamp experiments confirmed that the compound blocked TRPC6 currents with an IC50 of 7.9 nM. Furthermore, SAR7334 suppressed TRPC6-dependent acute HPV in isolated perfused lungs from mice. Pharmacokinetic studies of SAR7334 demonstrated that the compound was suitable for chronic oral administration. In an initial short-term study, SAR7334 did not change mean arterial pressure in spontaneously hypertensive rats (SHR). Our results confirm the role of TRPC6 channels in hypoxic pulmonary vasoregulation and indicate that these channels are unlikely to play a major role in BP regulation in SHR. SAR7334 is a novel, highly potent and bioavailable inhibitor of TRPC6 channels that opens new opportunities for the investigation of TRPC channel function in vivo.

Abstract 13284: A Novel Formulation of Vasoactive Intestinal Peptide Attenuates both Acute Hypoxic Pulmonary Vasoconstriction and the Development of Pulmonary Hypertension

Background: Vasoactive intestinal peptide (VIP) is an endogenous hormone that is known to relax vascular smooth muscle and has established anti-proliferative and immunomodulatory effects in the pulmonary circulation making it an attractive therapeutic target in pulmonary arterial hypertension (PAH). In the current study, a polymer-based nanocarrier (protected graft copolymer - PGC) formulation of VIP, which has been shown to increase the potency and duration of action of VIP, is used to show both acute vasodilatory effects and chronic therapeutic effects in experimental animal models of pulmonary hypertension. Methods: The isolated perfused mouse lung preparation is utilized to test acute hypoxic pulmonary vasoconstriction (HPV) in mice. Two animal models of pulmonary hypertension are used in preventative experiments, chronic hypoxic pulmonary hypertension in mice and monocrotaline-induced pulmonary hypertension in rats. Right ventricular systolic pressure and Fulton’s index (weight ratio of RV/[LV+Septum]) are used for measures of pulmonary hemodynamics and RV hypertrophy respectively. Results: PGC-VIP decreased resting pulmonary artery pressure and attenuated acute HPV elicited by 1% inhaled oxygen tension in a dose dependent manner from 0.1 μM to 1.0 μM. After four weeks of chronic hypoxia, both RVSP measurements and Fulton’s index were significantly decreased in mice receiving 100 mg/kg intraperitoneal PGC-VIP every other day compared to vehicle control. Higher doses were associated with mortality in the treatment group. MCT-PH rats receiving subcutaneous PGC-VIP at a dose of 250 mg/kg failed to show improvement in RVSP or Fulton’s index compared to vehicle control. Conclusion: This novel formulation of VIP demonstrates both acute and chronic vasodilatory effects in the pulmonary circulation. Treatment with PGC-VIP can attenuate the development of hypoxic pulmonary hypertension, yet significant mortality is seen at higher doses. Subcutaneous injection failed to attenuate the development of experimental PH in rats, possibly due to an ineffective dose or route of administration. Further studies are underway to identify the ideal dosing strategy necessary to attenuate and potentially reverse experimental PH in animal models.

Hypoxic pulmonary hypertension: the paradigm is changing.

Hypoxic pulmonary hypertension: the paradigm is changing.

Abstract 18495: Lung Inflammation Impairs Hypoxic Pulmonary Vasoconstriction Through Activation of Nuclear Factor of Activated T-Cells

Hypoxic pulmonary vasoconstriction (HPV) is an important physiologic mechanism that directs blood flow away from poorly ventilated regions of the lung toward well-ventilated regions in order to maintain optimal ventilation (V) and perfusion (Q) matching which supports adequate gas exchange and oxygenation. Impaired HPV has been implicated in multiple disease processes that are associated with both hypoxemia and inflammation. Nuclear factor of activated T-cells (NFAT) is a calcium-dependent transcription factor with emerging evidence to suggest an important role in inflammatory as well as cardiovascular disorders. We hypothesize that lipopolysaccharide (LPS)-induced lung inflammation leads to impaired HPV as a result of NFAT activation. In the current study we have utilized the isolated perfused mouse lung (IPL) preparation to test acute hypoxic pulmonary vasoconstriction. Murine lung inflammation is induced by intratracheal instillation of LPS (1 mcg/gm body weight) and acute hypoxic pulmonary vasoconstriction is elicited by five minute challenges of 1% inspired oxygen tension. LPS-induced lung inflammation significantly impairs HPV at 24 hours compared to sham (saline) treatment. In protein isolates from whole mouse lung lysates, LPS-induced lung inflammation is associated with significant increases in both calcineurin and NFATc3 protein expression at 24 hours. Finally, pretreatment with a calcineurin inhibitor, cyclosporin A, one hour prior to LPS treatment prevents the impaired HPV seen in LPS-induced lung inflammation in a dose dependent manner at 30 and 100 mg/kg. We conclude that LPS-induced lung inflammation impairs hypoxic pulmonary vasoconstriction in mice. This effect is mediated by NFAT activation and we show that inhibition of calcineurin, leading to decreased NFAT activation, can attenuate the impaired HPV seen in murine lung inflammation. Impaired HPV results in V/Q mismatching and worsening hypoxemia, a detrimental effect in both acute and chronic cardiopulmonary disease. These results implicate NFAT as a potential novel therapeutic target in diseases characterized by hypoxemia and impaired HPV. We plan to continue this work to determine the downstream effects of NFAT activation that lead to impaired HPV.

Role of ASIC1 in the development of chronic hypoxia-induced pulmonary hypertension.

Chronic hypoxia (CH) associated with respiratory disease results in elevated pulmonary vascular intracellular Ca(2+) concentration, which elicits enhanced vasoconstriction and promotes vascular arterial remodeling and thus has important implications in the development of pulmonary hypertension (PH). Store-operated Ca(2+) entry (SOCE) contributes to this elevated intracellular Ca(2+) concentration and has also been linked to acute hypoxic pulmonary vasoconstriction (HPV). Since our laboratory has recently demonstrated an important role for acid-sensing ion channel 1 (ASIC1) in mediating SOCE, we hypothesized that ASIC1 contributes to both HPV and the development of CH-induced PH. To test this hypothesis, we examined responses to acute hypoxia in isolated lungs and assessed the effects of CH on indexes of PH, arterial remodeling, and vasoconstrictor reactivity in wild-type (ASIC1(+/+)) and ASIC1 knockout (ASIC1(-/-)) mice. Restoration of ASIC1 expression in pulmonary arterial smooth muscle cells from ASIC1(-/-) mice rescued SOCE, confirming the requirement for ASIC1 in this response. HPV responses were blunted in lungs from ASIC1(-/-) mice. Both SOCE and receptor-mediated Ca(2+) entry, along with agonist-dependent vasoconstrictor responses, were diminished in small pulmonary arteries from control ASIC(-/-) mice compared with ASIC(+/+) mice. The effects of CH to augment receptor-mediated vasoconstrictor and SOCE responses in vessels from ASIC1(+/+) mice were not observed after CH in ASIC1(-/-) mice. In addition, ASIC1(-/-) mice exhibited diminished right ventricular systolic pressure, right ventricular hypertrophy, and arterial remodeling in response to CH compared with ASIC1(+/+) mice. Taken together, these data demonstrate an important role for ASIC1 in both HPV and the development of CH-induced PH.

Effects of Dimethylarginine Dimethylaminohydrolase–1 Overexpression on the Response of the Pulmonary Vasculature to Hypoxia

Acute and sustained hypoxic pulmonary vasoconstriction (HPV), as well as chronic pulmonary hypertension (PH), is modulated by nitric oxide (NO). NO synthesis can be decreased by asymmetric dimethylarginine (ADMA), which is degraded by dimethylarginine dimethylaminohydrolase-1 (DDAH1). We investigated the effects of DDAH1 overexpression (DDAH1(tg)) on HPV and chronic hypoxia-induced PH. HPV was measured during acute (10 min) and sustained (3 h) hypoxia in isolated mouse lungs. Chronic PH was induced by the exposure of mice to 4 weeks of hypoxia. ADMA and cyclic 3',5'-guanosine monophosphate (cGMP) were determined by ELISA, and NO generation was determined by chemiluminescence. DDAH1 overexpression exerted no effects on acute HPV. However, DDAH1(tg) mice showed decreased sustained HPV compared with wild-type (WT) mice. Concomitantly, ADMA was decreased, and concentrations of NO and cGMP were significantly increased in DDAH1(tg). The administration of either Nω-nitro-l-arginine or 1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one potentiated sustained HPV and partly abolished the differences in sustained HPV between WT and DDAH1(tg) mice. The overexpression of DDAH1 exerted no effect on the development of chronic hypoxia-induced PH. DDAH1 overexpression selectively decreased the sustained phase of HPV, partly via activation of the NO-cGMP pathway. Thus, increased ADMA concentrations modulate sustained HPV, but not acute HPV or chronic hypoxia-induced PH.

Predominant role of vasoconstrictors over dilatators derived from arachidonic acid in hypoxic pulmonary vasoconstriction

Prostanoids derived from arachidonic acid (AA) have been shown to play a permissive role in the regulation of vascular tone and wall tension. Conventionally, epoxyeicosatrienoic acids (EETs) and prostacyclin have been considered as dilatators, whereas thromboxane (TX) and hydroxyeicosatetraenoic acid (HETE) were considered as vasoconstrictors. However, the role of these prostanoids in the mediation of acute hypoxic pulmonary vasoconstriction is not yet clearly understood. In the present study, the role of prostanoids in the acute hypoxic response in rat isolated intrapulmonary arteries (IPAs) was investigated. Exogenous AA directly caused vasoconstriction, but exerted a significant inhibition on hypoxic vasoconstriction. The vasoconstriction by AA was mediated by the endothelium. AA metabolites from lipoxygenase (LOX) had no effect on vascular tone or hypoxic vasoconstriction. Consistent results from the blockage of cytochrome P450 (CYP) or CYP epoxide hydrolase showed that HETE contributed to endothelium‑independent hypoxic vasoconstriction. EET via epoxygenase exerted no effect on 80 mM KPSS‑induced vessel contraction or hypoxic vasoconstriction. In addition, prostacyclin also failed to inhibit hypoxic pulmonary vasoconstriction (HPV). However, blockage of thromboxane A2/prostanoid (TP) receptors almost eliminated hypoxic vasoconstriction, suggesting the primary role of TP receptors in the regulation of the hypoxic response in rat IPAs. In conclusion, the current data indicate the predominant role of vasoconstrictors instead of dilatators in mediating HPV. These data also highlight a pivotal role for voltage‑independent Ca2+ entry in pulmonary hypoxic response and suggest that modulation of these channels by prostanoids underlies their regulatory mechanisms.