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Published in final edited form as: Am J Physiol Regul Integr Comp Physiol. 2024 Nov 25;328(1):R75–R80. doi: 10.1152/ajpregu.00201.2024

ADRENAL EPINEPHRINE FACILITATES ERYTHROPOIETIN GENE ACTIVATION BY HYPOXIA THROUGH β2 ADRENERGIC RECEPTOR INTERACTION WITH Hif-2α

Xiaoyu Su 1, Matthew Hildreth 1, Srikar Rapaka 1,*, Ying-Jie Peng 1, Jayasri Nanduri 1, Nanduri R Prabhakar 1,$
PMCID: PMC12288984  NIHMSID: NIHMS2085175  PMID: 39585744

Abstract

Hypobaric hypoxia (HH) occurring at high altitude activates the sympathetic nervous system (SNS) and increases circulating erythropoietin (Epo) levels. Epo stimulates red blood cell production (erythropoiesis), enhancing oxygen transport in arterial blood to counteract hypoxemia. Present study tested the hypothesis that SNS contributes to Epo activation by HH through epinephrine (Epi) release from the adrenal medullae. Adult male C57B6 mice were exposed to 18 hours of HH (0.4 atm), and renal Epo mRNA and plasma Epo levels were measured. HH increased Epo mRNA and plasma Epo levels, and SNS activation, as indicated by elevated plasma NE and Epi levels. In adrenalmedullectomized mice, HH-induced Epo response was reduced, correlating with decreased circulating NE and absence of Epi elevation. Epi, but not NE infusion, mimicked the effects of HH in room air breathing mice. Epo responses to HH were reduced with β-adrenergic receptor (AR) blockade using dl-propranolol and in β2 adrenergic receptor knockout mice. Mice with heterozygous Hif-2α deficiency (Hif-2α+/−), but not Hif-1α+/−, showed attenuated the Epo gene activation and elevated plasma Epo levels in response to HH and Epi infusion. These results demonstrate that adrenal Epi facilitates the Epo gene activation by HH through interaction of β2 AR with HIF-2α.

Keywords: Catecholamines, Sympathetic nervous system, protein kinase A

NEWS AND NOTEWORTHY

Hypobaric hypoxia activates the sympathetic nervous system (SNS) and the erythropoietin (Epo) gene. Whether SNS activation by hypoxia influences the Epo gene activation is an unresolved question. The present study demonstrates that adrenal epinephrine facilitates hypoxia-induced Epo gene activation through interaction of β2 adrenergic receptors (β2 AR) with the transcriptional activator HIF-2α.

Graphical Abstract

graphic file with name nihms-2085175-f0007.jpg

Introduction

Hypobaric hypoxia (HH) occurs at high altitudes, triggering a series of time-dependent physiological responses known as high-altitude adaptation or acclimatization. These adaptive responses ensure adequate oxygenation to maintain homeostasis. Shortly after ascending to high altitude, HH increases breathing, activates the sympathetic nervous system (SNS), and elevates blood pressure (BP). While increased breathing ensures sufficient O2 intake, SNS activation and BP elevation secure the delivery of oxygenated blood to vital organs. HH also increases red blood cell production (erythropoiesis) to enhance oxygen transport in arterial blood. HH induced erythropoiesis is mediated by elevated levels of erythropoietin (Epo), a glycoprotein hormone. Whether SNS activation contribute Epo activation by HH remains an unresolved question.

Activation of the SNS releases norepinephrine (NE) from sympathetic nerve terminals. NE infusion increases plasma Epo levels in anesthetized dogs (1). Bilateral ablation of the splanchnic nerves, which provide sympathetic innervation to the kidneys, reduces the Epo response to anoxia in rats, suggesting a role for NE in the Epo response to hypoxia (2). Besides NE, SNS activation also releases epinephrine (Epi) from the adrenal medulla. However, the role of Epi in Epo activation by hypoxia has not been examined.

HH-induced Epo levels require transcriptional activation of the Epo gene by hypoxia-inducible factors (HIFs). HIF-1 and HIF-2 are the most studied members of the HIF family. They consist of an O2-regulated α subunit and a constitutive β subunit (3). Whether NE or Epi require interaction with Hifs to activate Epo is not known.

This study tested the hypothesis that epinephrine (Epi) released from adrenal medullae contributes to HH-induced Epo through interaction of adrenergic receptors with Hifs. This hypothesis is tested by measuring the Epo mRNA and plasma Epo in hypobaric hypoxia (HH) treated mice for 18 hours, a duration chosen because Epo levels in humans and mice return to control levels after 24 hours of hypoxia (4, 5). The role of HIFs was assessed in mice with heterozygous deficiency of the regulatory α subunit of the HIF-1 and HIF-2 complex (Hif-1α+/− or Hif-2α+/−; heterozygotes; HETs). Our results showed that selective ablation of the adrenal medulla reduced HH-induced Epo gene activation and elevated plasma Epo levels. Infusion of Epi, but not NE, mimicked the effects of HH. Additionally, Epo responses to HH were attenuated in β2 adrenergic receptor null mice as well as in Hif-2α but not Hif-1α heterozygous (HET) mice.

Methods

Preparation of Animals

Experiments were performed on adult, male C57B6 mice weighing between 25–30gm (Charles River). Male mice were chosen to exclude confounding influence of ovarian estrogen (E2) hormone on Epo in females (6). Experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Chicago (Protocol No. ACUP 71811, approved on February 27, 2019). Adrenal medulectomized mice were obtained from Charles River (C57B6 genetic background), β2 adrenergic receptor knockout mice (C57B6 genetic background) were a gift from Dr. Gökhan M Mutlu, University of Chicago; global Hif1α+/– and Hif2α+/– (C57B6/129 genetic background) mice were gifts from Drs. G. L. Semenza (Johns Hopkins University, MD) and Joseph A. Garcia (Columbia University NY), respectively.

Mouse Genotyping

Two-millimeter-long tail tissue was collected from isoflurane anesthetized mice and genomic DNA was extracted. Polymerase chain reaction (PCR) was performed to amplify the following primers: Mutant reverse 5’ CAC GAG ACT AGT GAG ACG TG 3’, wild type reverse 5’ CCG GGA ATA GAC AAA GAC CA 3’, wild type forward 5’ ACC AAG AAT AAG GCC CGA GT 3’. Genotyping of β2 null mice was performed using the protocol no. 29209 provided by the Jackson Laboratory (https://www.jax.org/Protocol?stockNumber=031496&protocolID=29209) using the primers listed previously. All three primers were used in a single reaction and the expected bands for WT mice and β2 null mice were 225 bp and 410 bp, respectively. PCR products were separated by gel electrophoresis on a 3% agarose gel.

Exposure to Hypobaric Hypoxia

Mice were treated with hypobaric hypoxia (HH; 0.4 atmospheres) for 18h. We chose 18h because this duration of HH elevates a) plasma NE and EPI levels in mice (7) and b) Epo levels in humans and mice return to control levels after 24 hours of hypoxia (4, 5). Mice in standard housing cages were placed in a hypobaric hypoxia chamber (between 17:00 PM and 11:00 AM next day). The hypobaric chamber contained an inlet port for entry of room air and an outlet port for administration of vacuum for induction of 0.4 ATM environments. Mice were unrestrained and fed ad libitum.

Measurements of renal Epo mRNA

Kidneys were harvested from anesthetized mice, and RNA was extracted using TRIZOL reagent and reverse transcribed using iScript reverse transcriptase super mix (Bio-Rad). Epo mRNA was determined by real-time polymerase chain reaction (RT-PCR) assay using a MiniOpticon system (Bio-Rad Labo-ratories) with SYBR Green ER two-step qRT-PCR kit (No.11764–100, Invitrogen) as described (8). The mRNA abundance was calculated with the comparative threshold (CT) method using the formula “2-ΔCT” where ΔCT is the difference between the threshold cycle of the Epo cDNA and the internal standard 18S gene. The purity and specificity of all products were confirmed by omitting the template and performing a standard melting curve analysis.

The primer used were 18S: Forward Primer, 5’- GTA ACC CGT TGA ACC CCA TT −3’ and 18S Reverse Primer, 5’- CCA TCC AAT CGG TAG TAG CG −3’; Epo Forward Primer, 5’- CAC CCT GCT GCT TTT ACT CT −3’ and Epo Reverse Primer, 5’- AAC CCA TCG TGA CAT TTT CT −3’

Measurements of Plasma EPO Protein

Blood samples were collected from anesthetized mice via cardiac puncture in heparinized tubes and plasma was separated and stored at −80°C until further analysis. Plasma erythropoietin (EPO) levels were measured using the Quantikine ELISA mouse erythropoietin immune assay kit (MEP00B, R&D Systems, Inc. Minneapolis, MN). The ELISA assay was based on the double-antibody sandwich method. Briefly, 70μl of each sample was diluted (twofold) with Calibrator Diluent (MEP00B). 50μl sample mixed with 50μl Assay Diluent (MEP00B) was loaded into a microplate well precoated with mouse EPO-specific monoclonal antibody. After washing unbound proteins, 100μl enzyme-linked EPO-specific monoclonal anti-body was introduced to each well (Mouse Epo conjugate, MEP00B) and allowed to incubate for 2h at room temperature on a shaker bath. Following removal of unbound reagent, 100μl substrate solution (MEP00B) was added to each well and the plate was incubated for 30min at room temperature while being protected from light. The enzymatic reaction between enzyme and substrate induced a blue-colored product that changed to yellow color after addition of 100μl stop solution (MEP00B). The intensity of the yellow color was measured by a microplate reader at 450nm and compared with a mouse EPO Standard (MEP00B). The intensity of the yellow color was proportional to the amount of Epo within each well. The detection limit of the ELISA Epo assay was 18pg/mL of plasma.

Catecholamine infusion

Alzet micro-osmotic pumps (model 2001D; Durect Corporation, Cupertino, Calif) were implanted subcutaneously in the intrascapular region of mice anesthetized with isoflurane. Each pump was loaded with 200μl of sterile saline with NE or EPI for 18hours (Sigma A7256; Sigma E4642). Infusion rate was 8 μl/h. NE and Epi were infused for 18h, the same duration as HH exposure. In experiments involving propranolol, 32mg/Kg of dl-propranolol was injected intraperitoneally (volume of 200μl) one hour prior to either HH or catecholamine infusion. Saline-treated mice served as controls.

Measurements of Plasma Catecholamines

Plasma Norepinephrine (NE) and epinephrine (EPI) levels were analyzed by high-performance liquid chromatography (HPLC) coupled with electrochemical detection (ECD). Blood samples (∼300μl) were collected from anaesthetized (urethane, 1.2 g/kg, i.p.) mice by cardiac puncture and placed in heparinized micro-centrifuge tubes. Plasma was separated by centrifugation and stored at −80°C until further analysis. Plasma NE and EPI were determined by HPLC-ECD using dihydroxybenzylamine as an internal standard. The NE and EPI levels were corrected for recovery loss and expressed as nanograms of NE and EPI per ml of plasma.

Data Analysis

Data are presented as individual data points along with mean or median and 95% confidence intervals(95%CI) wherever appropriate depending on the normality of the dataset. Charts were plotted using GraphPad Prism (version 8). Statistical analysis was performed testing assumptions of normal distribution (Shapiro-WilK test) and equal variances (Levene’s Median test). One-Way ANOVA followed by a post hoc test was performed if the assumptions were satisfied. Otherwise, One-Way ANOVA on Ranks followed by a post hoc test was applied. To determine whether the means/medians of two or more groups are affected by two different factors (i.e., genotype and treatment), two-Way ANOVA followed by Holm-Sidak test was performed. A transformation was applied to the datasets that did not meet the normal distribution and equal variances such that the transformed datasets satisfied the assumptions, and then two-Way ANOVA followed by Holm-Sidak test was performed. Liner regression was performed to assess the relationship between renal EPO mRNA or plasma EPO levels with doses of Epi tested. All statistical analysis used Sigma Plot (version 11) and P values <0.05 were considered significant.

RESULTS

Adrenal medullectomy (Adr) attenuates Epo responses to hypobaric hypoxia (HH)

Adrenal medulla synthesizes and stores primarily Epinephrine (Epi) and to a lesser extent Norepinephrine (NE). The role of Epi in the Epo responses was determined in mice with selective ablation of adrenal medulla. Both control (with intact adrenal medullae) and adrenal medullectomized (Adr) mice were treated with 18h of HH. Room air treated mice served as controls. We first measured plasma Epi and NE responses to HH (0.4 atm; 18h) in control and Adr mice. HH treated control mice with intact adrenal medullae showed elevated plasma NE and EPI. Conversely, in HH-treated Adr mice, plasma EPI levels were nearly undetectable, and the elevated NE levels observed in the control mice were absent (Fig. 1, A and B). These results confirm the ablation of adrenal medullae in Adr mice.

Fig. 1.

Fig. 1.

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Plasma catecholamine and erythropoietin (Epo) responses to hypobaric hypoxia (HH). Plasma Norepinephrine (NE) (A) and Epinephrine (EPI) (B), renal Epo mRNA (C) and plasma Epo levels (D) in control and adrenamedullectomized (Adr) mice. Data are presented as individual data points along with median and 95% confidence intervals (95%CI). Data in A and B were analyzed one-way ANOVA on Ranks followed by Student-Newman-Keuls test and in C and D with one-way ANOVA on ranks followed by Dunn’s test. *P<0.05; n.s., not significant; P>0.5. n=5–6 mice/group.

In adult mammals, Epo is primarily produced in the kidney. Renal Epo mRNA and plasma Epo levels were determined in control and Adr mice treated with HH. HH treated control mice with intact adrenal medullae showed increased renal Epo mRNA and plasma Epo levels (p < 0.05; n =5) and these responses were attenuated in HH treated Adr mice (p > 0.05; n = 5–6; Fig. 1, C and D).

EPI but NE mimic the effect of HH on renal Epo mRNA and plasma Epo

Epo responses to exogenous application of EPI and NE were examined. Epi and NE were infused using an Alzet pump for 18 hours (matching the duration of 18h HH) in mice breathing room air. The choice of Epi doses (0.3, 1.5, 3mg/kg/day) was based on a previous study on rats by Racotta et al. (9).

Infusion of Epi (1.5mg/kg/day) increased both the Epo mRNA and plasma Epo levels compared to vehicle treated controls (Fig. 2, A and B). In contrast, equivalent dose NE infusion had no effect either on the Epo mRNA or plasma Epo levels (Fig. 2 A and B). The Epo mRNA and plasma Epo correlated with the dose of Epi infusion (Fig. 2 C and D). Moreover, plasma Epi and NE levels with infusion of Epi or NE were comparable to that evoked by HH (Fig.2 E and F).

Fig. 2.

Fig. 2.

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Infusion of Epinephrine (EPI) but not Norepinephrine (NE) increases Epo mRNA and plasma Epo abundance. Renal Epo mRNA (A) and plasma Epo levels (B) in response to infusion of Epi and NE (1. 5mg/Kg/day). Dose-response of Epi infusion on Epo mRNA (C) and plasma Epo levels (D). Comparison of plasma Epi (E) and NE (D) responses to hypobaric hypoxia (HH) and with infusion of 1.5 mg/Kg/day of Epi and NE. Data are presented as individual data points along with mean and 95% confidence intervals (95%CI). *** P<0.001; **P<0.01; *P<0.05; n.s., not significant; P>0.05; Data were analyzed with one-way ANOVA followed by Student-Newman-Keul’s test. n=4–6 mice/group.

β2 adrenergic receptors (ARs) mediate Epo responses to Epi and HH

Thus far the results suggested that Epi activates Epo during HH. We sought to determine the role of adrenergic receptors mediating the response to Epi. Biological actions of Epi are mediated by β-adrenergic receptors (β ARs). The role of β ARs were assessed using two approaches: one with pharmacological blockade of β ARs with dl-propranolol and the second in mice with genetic knockout of β2 ARs.

Mice treated with dl-propranolol (32mg/Kg; i.p.) showed reduced Epo mRNA and plasma Epo responses to HH compared to vehicle-treated controls (Fig. 3, A and B).

Fig. 3.

Fig. 3.

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Dl-Propranolol attenuates Epo responses to hypobaric hypoxia (HH). DL- Propranolol (Prop; 32mg/kg) was given intraperitoneal (IP) one hour before subjecting mice to HH. Renal Epo mRNA (A) and plasma Epo levels (B). Data are presented as individual data points along with mean and 95% confidence intervals (95%CI). *** P<0.001; **P<0.01; *P<0.05; n.s., not significant; P>0.05; one-way ANOVA followed by Student-Newman-Keuls test. n=5 mice/group.

Kidney express β2-AR subunits in proximal tubules, glomeruli, and podocytes (10). Dl-propranolol is a non-selective β AR antagonist that inhibits both β1 and β2 ARs and may also have non-selective local anesthetic effects (11). To assess the specific role of β2 ARs without the interference of non-selective actions of dl-propranolol, Epo responses to HH were examined in global β2 AR knockout mice.

An example illustrating genotyping analysis of wild-type (WT) and β2 null mice is shown in Fig. 4A. DNA from WT mouse tissue displayed a band corresponding to 225 bp and β2 null mice showed a band corresponding to 410 bp (Fig. 4A), confirming the absence of the β2 AR gene in the mutant mice. β2 AR knockout mice showed significantly diminished Epo mRNA and plasma Epo responses to HH compared to wild-type controls (p<0.001; Fig. 4 B-C).

Fig. 4.

Fig. 4.

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β2 adrenergic receptor knock-out (β2 KO) mice show impaired Epo responses to hypobaric hypoxia (HH). A) An example illustrating genotyping analysis of DNA from wild-type (WT) and β2 null mice by polymerase chain reaction (PCR) using the primer pairs targeting WT (225 bp) and mutant β2 alleles (410 bp). B) Renal Epo mRNAs and C) plasma Epo protein levels in wild-type (WT) and β2 adrenergic receptor knock-out (β2 KO) mice treated with 18h of HH or room air (Nor). Data are presented as individual data points along with median and 95% confidence intervals (95%CI). *** P<0.001; n.s., not significant; P>0.05; Data were analyzed with two-Way ANOVA followed by Holm-Sidak test. n=4–6 mice/group.

Epi interacts with hypoxia-inducible factor 2α (Hif-2α)

Hypoxia-inducible factors (Hif-1 and Hif-2) play a crucial role in the Epo gene activation by hypoxia. We hypothesized that Epi interacts with the O2 sensitive regulatory α subunit of the Hif-1or Hif-2 complex. This possibility was examined by measuring Epo responses to HH as well as with Epi infusion in Hif-1α or Hif-2α heterozygous mice (Hif-1α +/− and Hif-2α +/−).

Wild type mice (WT) treated with HH responded with increased renal Epo mRNA and plasma EPO levels, and these responses were attenuated in Hif-2α +/− mice (Fig.5A–B). In contrast, Hif-1α +/− mice did not show such attenuation (Fig. 5, C and D).

Fig. 5.

Fig. 5.

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Hif-2a +/− mice exhibit impaired Epo response to hypobaric hypoxia (HH). Age matched wild type and Hif-2a+/− and Hif-1a+/− mice were treated with either HH or room air (Nor). Renal Epo mRNA and plasma Epo responses to HH in Hif-2a +/− in A and B and Hif-1a +/− in C and D. Data are presented as individual data points along with median and 95% confidence intervals (95%CI). *** P<0.001; n.s., not significant; P>0.05. Two-Way ANOVA followed by Holm-Sidak test. n=4–5 mice/group.

We then assessed the effects of Epi infusion (1.5mg/kg/day) on Epo mRNA and plasma Epo levels in Hif-2α +/− mice. Epi infusion, similar to hypoxia, increased renal Epo mRNA and plasma Epo levels in WT mice and these responses were significantly reduced in Hif-2α +/− mice (p<0.01; p <.0.001; Fig. 6, A and B).

Fig. 6.

Fig. 6.

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Impaired Epo responses to Epi infusion in Hif-2a+/− mice. A) Renal Epo mRNA and B) plasma Epo levels in wild type (Hif-2α+/+) and HIF-2α +/− mice with infusion of Epi (1.5mg/kg/day) for 18h. Data are presented as individual data points along with mean and 95% confidence intervals (95%CI). *** P<0.001; **P<0.01; n.s., not significant; P>0.05. Data were analyzed with two-Way ANOVA followed by Holm-Sidak test. n=5 mice/group.

DISCUSSION

This study tested the hypothesis that epinephrine (Epi) release from the adrenal medullae contributes to HH-induced Epo through interaction of adrenergic receptors with Hifs. Our results demonstrated a) Epo activation by HH was reduced in adrenalmedullectomized (Adr) mice b) Epi but not NE infusion mimic the effects of HH in mice breathing room air, c) Epo responses to HH were nearly absent in β2 AR knockout mice, and d) Epo responses to HH and Epi infusion were markedly attenuated in Hif-2α but not in Hif-1α heterozygous mice.

Consistent with an earlier report in humans (12), we found that HH (0.4 atm) elevated plasma NE levels (Fig. 1, A and B), suggesting HH activates the SNS. A previous study by Fisher and colleagues reported infusion of NE increase plasma Epo levels in anesthetized dogs, and this effect was attributed to reduced renal blood flow from vasoconstriction (1). However, in our study, NE infusion (1.5mg/kg/day) which resulted in elevation of plasma NE comparable to HH, neither activated renal Epo mRNA nor increased plasma Epo levels in unanesthetized mice (Fig. 2, C and D). This discrepancy might be due to unchanged renal blood flow in our study or species differences (mice versus dogs).

The following evidence suggest Epi released from adrenal medullae contributes to Epo activation by HH: 1) selective ablation of adrenal medulla (Adr mice) prevented Epi increase by HH and reduced the Epo gene activation and plasma Epo response to HH, and 2) Epi infusion at a dose comparable to that evoked by HH mimicked the Epo response to HH in mice breathing room air (Fig.2). A role for adrenal Epi is further supported by earlier studies showing elevated plasma Epo levels and erythropoiesis in patients with pheochromocytoma, a tumor of adrenal medulla (13). Moreover, Spontaneous hypertensive (SH) rats exhibit increased erythropoiesis and elevated serum erythropoietin (Epo) levels (14, 15) and adrenalectomy reduce both hypertension and red blood cell count in SH rats (15). The magnitude of the Epo response to Epi infusion was nearly half of that observed with HH (Fig. 2 A–B). Furthermore, Epo responses to HH were reduced but not absent in Adr mice (Fig. 1 C–D). These findings suggest that adrenal Epi might function as a modulator of Epo responses to HH. How might Epi modulate the Epo gene activation by HH?

Biological actions of Epi are mediated by β adrenergic receptors (βARs). We found dl propranolol, a pan β AR blocker reduced the Epo gene activation and plasma Epo responses to HH (Fig. 3, A and B), a finding consistent with an earlier report showing reduced plasma Epo response to HH in propranolol treated rabbits (16). The attenuated Epo gene activation is unlikely due to non-selective action of dl propranolol because HH treated β2-AR knockout mice showed reduced Epo gene activation by HH (Fig. 4, B and C). These findings demonstrate that Epi acting through β2-adrenoceptors contributes to the Epo gene activation by HH.

It is now established transcriptional activators, Hif-1 and Hif-2 mediate Epo gene activation hypoxia (17, 18). While homozygous knockdown of the O2 sensitive α subunit of either Hif-1 or Hif-2 is lethal, mice with partial knockdown of the α subunits survive. HH-induced activation of the Epo gene and plasma Epo responses were reduced in Hif-2α+/− but not in Hif-1α+/− heterozygous mice, a finding consistent with studies showing that Hif-2 is a major transcriptional activator of the Epo gene by hypoxia (18–22). These findings indicate interaction of β2ARs with Hif-2α.

How might β2AR interact with Hif-2? The signaling pathway of β2ARs involves G-proteins (Gs), which activate adenylyl cyclase (AC), leading to cAMP production and activation of protein kinase A (PKA). Bullen et al. reported that PKA interacts with Hif-1 signaling in HeLa cells and rat cardiomyocytes (23), and showed that PKA increases Hif-1 transcriptional activity by phosphorylating Hif-1α at Thr 63 and Ser 692, which stabilizes the Hif-1α protein and also facilitates the binding of the co-activator p300 to Hif-1α (23). Although we have not studied, similar signaling pathway likely accounts for interaction of β2AR with Hif-2α thereby contributing to the modulatory effect of Epi on HH-induced Epo gene activation. Such a possibility is partially supported by an earlier study reporting cAMP activates renal Epo production (17). Nevertheless, further studies are necessary to establish β2/PKA interaction with Hif-2α in the Epo gene activation by hypoxia.

Acknowledgement:

The authors thank Drs. Gregg L.Semenza, Joseph A. Garcia, and Gokhan Mutlu for providing Hif-a +/−; Hif-2a +/− and β2 adrenergic receptor knockout mice, respectively.

Funding:

This work was supported by the National Institutes of Health grant P01-HL144454.

Footnotes

Competing interests: Authors declare no competing interest.

Data and materials availability:

All data, code, and materials used in the analysis will be available upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

All data, code, and materials used in the analysis will be available upon reasonable request.

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