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. Author manuscript; available in PMC: 2026 May 5.
Published in final edited form as: Am J Physiol Lung Cell Mol Physiol. 2026 Feb 25;330(4):L404–L415. doi: 10.1152/ajplung.00229.2025

Decreased AMP-Kinase Function in Pulmonary Artery Smooth Muscle Cells Contributes to Pulmonary Artery Remodeling in Persistent Pulmonary Hypertension of the Newborn

Emily A Mooers 1, Ujala Rana 1, Chintamani Joshi 1, Adeleye J Afolayan 1, Ru-Jeng Teng 1, Girija G Konduri 1
PMCID: PMC13138608  NIHMSID: NIHMS2154416  PMID: 41740187

Abstract

Pulmonary vascular remodeling contributes to Persistent Pulmonary Hypertension of the Newborn (PPHN); the mechanisms remain unknown. 5’AMP-Activated Protein Kinase (AMPK) is a critical regulator of energy balance and metabolism. We investigated the hypothesis that decreased AMPK function in pulmonary artery smooth muscle cells (PASMCs) leads to impaired mitochondrial capacity to perform oxidative phosphorylation and altered notch ligand expression, which together promote vascular remodeling in PPHN. Studies were performed in fetal lambs with PPHN induced by prenatal ductus arteriosus constriction and gestation-matched controls. For in vitro studies, PPHN PASMCs were treated with AMPK agonists, A769662 or metformin, and compared to untreated control and PPHN PASMCs. Expression of phosphorylated-AMPK (p-AMPK) and its downstream mediators, PGC-1α, mitochondrial electron transport chain (ETC) complexes, differentiation markers, and notch ligands, were assessed using immunoblotting in control and PPHN PASMCs. For in vivo studies, PPHN lambs were treated with metformin, and were compared to untreated control and PPHN lambs. Lung sections from in vivo experiments were evaluated via immunofluorescence. Compared to controls, p-AMPK, PGC-1α and ETC complexes were decreased in PPHN PASMCs and lung sections. PASMC differentiation marker, MYH11, was reduced in PPHN lung sections while dedifferentiation marker, vimentin, was increased. Expression of Jag1 and Hey1 in Notch pathway were reduced in PPHN PASMCs and lung sections. A769662 and metformin increased the expression of PGC-1α, ETC complexes I and IV, Jag1 and Hey1 in PPHN PASMCs. Decreased AMPK function contributes to reduced mitochondrial oxidative phosphorylation capacity, less differentiated PASMCs, and imbalanced notch signaling, promoting remodeling in PPHN.

Keywords: smooth muscle cells, mitochondrial bioenergetics, AMPK, vascular remodeling, proliferation

NEW & NOTEWORTHY

Our manuscript reveals a novel mechanism for pulmonary vascular remodeling in Persistent Pulmonary Hypertension of the Newborn (PPHN). We identify a decrease in the function of a key energy sensor, AMPK, as contributing to pulmonary vascular remodeling through decreased mitochondrial oxidative phosphorylation capacity, altered differentiation marker expression, and notch ligand imbalance. Our studies may provide translational significance as restoring AMPK function offers a new therapeutic target in PPHN to improve postnatal transition in PPHN.

Graphical Abstract

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INTRODUCTION

Persistent Pulmonary Hypertension of the Newborn (PPHN) occurs when pulmonary vascular resistance (PVR) fails to decrease at birth, resulting in extra-pulmonary right-to-left shunting and severe hypoxemia (1, 2). Reduced vasoreactivity, impaired angiogenesis, and remodeled vascular structure all contribute to PPHN pathogenesis (3, 4). Vascular remodeling is a consequence of pulmonary artery smooth muscle cell (PASMC) hyperproliferation, increased migration, and apoptosis resistance (5), which lead to a thickened media in the pulmonary arteries (PAs). We utilize a fetal lamb model of PPHN in which prenatal ductus arteriosus (PDA) constriction 8 days before birth results in hemodynamically significant elevation in PVR, consistent with elevated right ventricle (RV) pressure and Fulton Index (6). This PPHN model, shows medial hypertrophy and an extension of smooth muscle into the normally non-muscular distal arteries (7), resulting in reduced vessel density and PA remodeling (6). The remodeling decreases the ability of PA to dilate in response to oxygen (O2) and vasodilators at birth, leading to severe PPHN (8). Elucidating the signaling pathways involved in the development of vascular remodeling in PPHN is, therefore, of particular significance.

Pulmonary vascular remodeling develops due to PASMC dedifferentiation (9), resulting in a shift towards a higher proliferation state (10). PASMC dedifferentiation can be triggered by reactive oxygen species (ROS) (11, 12) and metabolic dysregulation (13, 14). In adult pulmonary hypertension (PH), oxidant stress damages mitochondria, resulting in a change from oxidative phosphorylation to glycolysis for energy production (13). As a result, PASMCs can resist apoptosis while they proliferate and migrate excessively, creating the maladaptive phenotype of vascular remodeling in PH characterized by dedifferentiated and proliferative PASMCs (13, 15). The mechanism by which energy depletion and oxidant damage contribute to the pathogenesis of PPHN is unknown but is key to understanding the PASMC reprogramming in pulmonary vascular remodeling.

5’ AMP-Activated Protein Kinase (AMPK) is a critical metabolic and redox sensor through its maintenance of mitochondrial function and integrity and was reported to play a role in adult PH (16, 17). Failure of its induction in the presence of decreased adenosine triphosphate (ATP) levels and elevated ROS in PPHN PASMCs may lead to excess proliferation, which has been previously reported in adult PH (16). We previously reported that decreased AMPK levels in pulmonary artery endothelial cells (PAECs) contribute to impaired metabolic homeostasis, with resultant reduction in mitochondrial function and decrease in angiogenesis in PPHN (18). Despite smooth muscle cells having higher mitochondrial content compared to endothelial cells, the role of AMPK in PASMCs affected by PPHN has not been well studied.

The potential downregulation of AMPK as a mechanistic link to pulmonary vascular remodeling in PPHN is a plausible hypothesis. AMPK function is usually upregulated with increased metabolic demands and oxidative stress, which leads to increased oxidative phosphorylation and upregulation of antioxidants (19). Failure to increase AMPK function in the setting of energy deprivation and oxidant damage results in a failure to upregulate oxidative phosphorylation for efficient energy production; and as a result, PASMCs may dedifferentiate and undergo aberrant proliferation through alternate energy sources. Since hyperproliferation is a central feature of pulmonary vascular remodeling in PPHN, defining this mechanism is critical to improve our understanding of PPHN. We hypothesize that downregulated AMPK function, with subsequent alteration of Peroxisome Proliferator-Activated Receptor Gamma Co-Activator-1α (PGC-1α) expression, leads to reduced mitochondrial oxidative phosphorylation capacity and excess PASMC proliferation in pulmonary vascular remodeling in PPHN. We further posit that enhancing AMPK activity will inhibit pulmonary vascular remodeling via restoration of mitochondrial integrity and PASMC differentiation.

MATERIALS AND METHODS

Experiments were performed in control and PPHN PASMCs from lambs with or without fetal PH induced by prenatal ductus arteriosus (PDA) constriction. All fetal lamb studies were performed in compliance with NIH guidelines and were approved by the Medical College of Wisconsin IACUC.

Fetal lamb model:

Constriction of fetal DA was performed at 128 ± 2 days of gestation (term = 144 days), as previously described (20, 21). Sham-operated, gestation-matched twin fetal lambs were used as controls. After 8 days of ductal constriction, fetal lungs were harvested for PASMC isolation. Previous studies have shown that these lambs consistently develop severe PPHN with high PVR and pulmonary vascular remodeling with this intervention (6, 18, 20, 22). For AMPK activation, metformin was infused via Alzet mini-osmotic infusion pumps in PPHN lambs at 128 ± 2 days of gestation to provide 5 mg/d and 40 mg over 8 days.

Histology for Lung and Pulmonary Artery (PA):

For each lamb, right lower lobe was inflated with 10% neutral buffer formalin for 30 minutes using gravity and was later incubated in the formalin fixative for 48 hours. The formalin-fixed lungs were subjected to paraffin embedding and sectioning to obtain formalin-fixed paraffin-embedded (FFPE) tissues, which were cut at 4-μm thickness on a frosted glass slide. For assessment of PA thickness, the FFPE sections were stained with hematoxylin and eosin (H&E). PAs were identified by position adjacent to the airway (AW). The medial thickness/external diameter ratio for medium-sized PAs (50–200 μm external diameter) was measured using ImageJ, as previously described (23). For IF staining of lungs, the FFPE sections were deparaffinized and antigen retrieved before staining with DAPI and IF for alpha-actin-2 (also known as α-smooth muscle actin, α-SMA), phospho-Threonine (Thr) 172-AMPK (p-AMPK), Liver Kinase B1 (LKB1), Peroxisome Proliferator-Activated Receptor Gamma Co-activator-1α (PGC-1α), Sirtuin-1 (SIRT-1), Superoxide Dismutase 2 (SOD2), Myosin Heavy Chain 11 (MYH11), Vimentin, phos-(Ser10)-Histone H3 (pH3), Antigen Kiel 67 (Ki-67), Jagged1 (Jag1), and Hairy/enhancer-of-split related with YRPW motif 1 (Hey1), followed by confocal imaging. Images were obtained under a fluorescence microscope (Olympus IX51, Center Valley, PA) with the appropriate filter. Complete list of antibodies is provided in Supplemental data 1. To evaluate the expression of the proteins of interest in only the PASMC layer, and not including the fluorescence from the PAEC layer, the IF calculation was performed as follows: IF signal intensity for protein of interest relative to DAPI in SMC layer = IF protein signal of interest for SMC - IF protein signal of interest for EC layer ÷ IF DAPI signal intensity for SMC layer- IF DAPI signal intensity for EC layer. Additional explanation and schematic provided in supplemental Fig 1 (https://doi.org/10.6084/m9.figshare.31145077. IF signal intensities were calculated relative to DAPI, a nuclear stain, for consistency across control, PPHN, and PPHN + Metformin images, as was performed in our previous publications, and ImageJ was used to determine IF signal intensities (18, 24).

Smooth Muscle Cell (SMC) Isolation and Culture:

PASMCs were isolated from fetal lungs by 20-minute digestion with 0.1% collagenase. The cells were flushed with Dulbecco’s modified eagle medium (DMEM) and were selected for endothelial cells using magnetic beads coated with CD31. The negative selection was cultured as PASMCs and then identity was verified using a specific SMC marker, α-SMA.

Treatment of PASMCs:

PASMCs in 60-mm dishes were treated when sub-confluent. PPHN PASMCs were treated with either: 1) 25 μM A769662 (Tocris, Minneapolis, MN), the allosteric AMPK activator which interacts with the AMP binding domain of the γ subunit (25, 26); or 2) 10−4 M metformin. Both treatment groups were harvested after 48 hours.

Immunoblotting (IB):

PASMCs treated for their respective analyses were lysed in modified radio immune precipitation assay (RIPA) buffer. Using the modified Bradford method, protein concentrations were determined in the supernatants via the Pierce™ BCA Protein Assay Kit (23225). Proteins were separated by SDS PAGE and transferred to a nitrocellulose membrane. Proteins were blotted with specific antibodies for LKB1, p-AMPK, AMPK, PGC-1α, mitochondrial electron transport chain (ETC) complexes I-V, SIRT-1, SOD2, Jag1, Hey1, and β-Actin (Cell Signaling). Membranes were blotted with horseradish peroxidase (HRP)-conjugated anti-mouse or -rabbit IgG antibody and exposed to CL-XPosure films (Pierce) after treatment with SuperSignal West Pico (Pierce). Signals were analyzed with ImageJ and normalized to β-Actin. Complete list of antibodies provided in Supplemental data 1.

Mitochondrial Bioenergetics:

PASMCs were seeded at 5 × 103 cells/well in normal growth media in a Seahorse XF96 Cell Culture Microplate. The plate was incubated at 37 °C for 3–4 hours prior to the PPHN PASMCs being treated with metformin or A769662, as described above; the four groups were: control, PPHN, PPHN+ Metformin, and PPHN+A769662. Following 48 hours, the growth media was exchanged with Seahorse Phenol Red-free DMEM and an XF Cell Mito stress test (Agilent) was performed according the manufacturer’s instructions. The measurements of basal respiration, spare respiratory capacity, maximum respiration, and ATP production were determined using the software, and the results were graphed in the Wave application with normalization for protein concentration in each well.

Assessment of Mitochondrial Superoxide (O2−) Levels:

We assessed mitochondrial O2− levels using MitoSOX Red Mitochondrial Superoxide Indicator (Invitrogen, M36007) as previously described (27). After 48 hours of each respective treatment in a 6-well plate, cells were incubated with MitoSOX for 30 minutes to detect mitochondrial O2− levels. Fluorescence signals were quantified using fluorescence detection microscopy and analyzed with ImageJ. DAPI (4’,6-diamidino-2-phneylinodole) was used as a contrasting fluorescent signal to highlight the nucleus blue, and results were expressed as ratio of MitoSOX/DAPI intensity signals.

Statistical Analyses:

Data are shown as mean ± SD. Comparison of data among the experiments groups was done by one-way ANOVA with Tukey’s post hoc test for significant differences (P < 0.05). Data were analyzed using GraphPad Prism version 10.

RESULTS

PPHN induced via Patent Ductus Arteriosus (PDA) Constriction in Fetal Lambs:

Lambs with in vitro DA constriction developed medial hypertrophy of the pulmonary arteries (PAs) compared to control lambs, with a significant increase in medial width/external diameter ratio (Fig 1A). In utero infusion of metformin mitigated the increase in medial width in the PPHN lambs (Fig 1A), as we previously reported in this model (18). Supplemental Figure 2 shows these same findings of pulmonary vascular remodeling at a lower magnification (20X) for improved delineation of the structural differences in the lung vasculature between control and PPHN lambs. Additionally, IF for α-SMA, a SMC differentiation marker that is associated with a PASMC profile of lower rates of proliferation and migration (28), is significantly decreased in PPHN PASMCs relative to controls (Fig 1B). In contrast, in utero infusion of metformin restored α-SMA levels in PPHN PASMCs (Fig 1B). Since in utero metformin treatment restores α-SMA expression in PPHN PASMCs and improves the medial width of PAs, our data support the concept that AMPK dysfunction contributes to impairment of vascular development in utero in PPHN.

Figure 1.

Figure 1.

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(A) Hematoxylin and eosin (H&E) staining of lung sections showing pulmonary artery (PA) and adjacent airway (AW) in control, persistent pulmonary hypertension of the newborn (PPHN), and metformin treated PPHN (PPHN + Met) lambs. Arrows point to PA lumen. Scale bars: 100 μm. PPHN lambs have increased medial width/external diameter compared to controls. Metformin attenuates the increase in medial width in PPHN lambs. Summarized data are for n = 6 lambs in each group, with results representing three vessels analyzed per animal. The vessels analyzed have an external diameter of 50–200 μm, with an average external vessel diameter of 100 μM. (B) Immunofluorescence staining for α-smooth muscle actin (α-SMA) in representative lung sections from control, PPHN, and PPHN + Met lambs. Lung sections were also stained with DAPI to localize nuclei. Scale bars: 200 μm. α-SMA expression is reduced in PPHN lungs compared to controls. Metformin restores α-SMA expression in PPHN lungs. Summarized data are for n = 6 lambs in each group, showing integrated fluorescence density. For (A and B), *indicates p <0.05 from control lambs and #from PPHN lambs by ANOVA and Tukey’s post hoc test.

Effects of PDA constriction and Metformin/A769662 on AMPK expression and function in PPHN PASMCs:

Protein levels of LKB1, the main upstream kinase of AMPK (29), are decreased in PPHN PASMCs compared to controls (Fig 2A). Treatment with either metformin or A769662 increased LKB1 protein levels in PPHN PASMCs relative to untreated PPHN PASMCs (Fig 2A). Supplemental Figure 3 shows representative images with triple staining of LKB1 with SMA and CD31 and demonstrates that LKB1 is reduced specifically in the smooth muscle layer of PAs. In PPHN PASMCs, phosphorylated (Thr172)-AMPK (p-AMPK), the functionally active form of AMPK, was decreased compared to controls (Figure 2B). Native AMPK protein levels were unchanged in PPHN PASMCs (Fig 2C). In PPHN PASMCs treated with metformin, the protein level of p-AMPK was significantly increased (Fig 2B), whereas native AMPK remained unchanged (Fig 2C). Similarly, PPHN PASMCs treated with A769662, an allosteric AMPK activator, show a significant increase in p-AMPK (Fig 2B) without altering native AMPK levels (Fig 2C).

Figure 2.

Figure 2.

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(A-C) Protein levels of LKB1 (A), phos(Thr172)-AMPK (p-AMPK, B), and AMPK (C) in control and PPHN PASMCs and the effect of allosteric AMPK agonist, A769662, and metformin on the LKB1, p-AMPK, and AMPK protein levels in PPHN PASMCs. LKB1 and p-AMPK protein levels are decreased in PPHN PASMCs compared to controls, while AMPK levels are unaltered. Both A769662 and metformin increase the protein levels of LKB1 and p-AMPK in PPHN PASMCs, but not AMPK. For (A, B, and C), all results are normalized to β-Actin. The ß-actin loading control for p-AMPK was also the loading control for Sirt1 protein in Fig 3 as these 2 proteins and corresponding ß-actin were run on the same gel. *Indicates p <0.05 from control PASMCs and #from PPHN PASMCs by ANOVA and Tukey’s post hoc test for n = 3 in each group representing distinct cell lines from 3 animals per group. (D and E) Immunofluorescence staining for LKB1 (D) and p-AMPK (E) in representative lung sections from control, PPHN, and metformin-treated PPHN (PPHN + Met) lambs. Lung sections were also stained with smooth muscle cell maker, α-smooth muscle actin (α-SMA), to assess colocalization of these proteins within capillaries, and DAPI to outline nuclei. Scale bars: 200 μm. Both LKB1 and p-AMPK that colocalize to the blood vessels in the lung are decreased in the smooth muscle layer in PPHN. Metformin infusion in utero increases fluorescence of LKB1 and p-AMPK in the PPHN lamb lungs. For (D and E), summarized data are for n = 6–10 in each group, showing integrated fluorescence density. *Indicates p <0.05 from control lambs and #from PPHN lambs by ANOVA and Tukey’s post hoc test.

LKB1 and p-AMPK levels are both decreased in PPHN lamb lung sections in the PASMC layer, identified by α-SMA IF staining (Fig 2D and 2E). Prenatal treatment of PPHN lambs with metformin increased both the LKB1 and p-AMPK signals in the PASMC layer, verifying the changes observed in PPHN PASMCs via IB (Fig 2D and 2E). These data suggest that AMPK dysfunction may be due to decreased activation by LKB1 in PPHN PASMC.

Effects of PDA constriction and Metformin/A769662 on Mitochondrial Bioenergetics:

Downstream of AMPK, Peroxisome Proliferator-Activated Receptor Gamma Coactivator-1α (PGC-1α) functions as a mitochondrial transcription co-factor responsible for maintaining oxidative phosphorylation (27, 30). In PPHN PASMCs, PGC-1α protein expression was decreased compared to controls (Fig 3C). Additionally, the mitochondrial ETC complexes I-V are decreased in PPHN PASMCs compared to controls (Fig 3A), suggesting impaired oxidative phosphorylation capacity. When PPHN PASMCs were treated with metformin or A769662, both PGC-1α and the mitochondrial ETC complexes I and IV are significantly increased, indicating a restoration of mitochondrial oxidative phosphorylation capacity (Fig 3C and 3A, respectively). Sirtuin-1 (SIRT-1), an NAD+-dependent deacetylase, acts in conjunction with AMPK to activate PGC-1α in times of energy or nutrient deprivation (31, 32). SIRT-1 protein level was not decreased in PPHN PASMCs compared to controls (Fig 3B). SIRT-1 protein expression is unaffected in PPHN PASMCs treated with either metformin or A769662 (Fig 3B).

Figure 3.

Figure 3.

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(A) Protein levels of the mitochondrial ETC complex proteins I-V in control and PPHN PASMCs and the effect of metformin and allosteric AMPK agonist, A769662, on these proteins in PPHN PASMCs. Mitochondrial ETC complex proteins I-V are decreased in PPHN PASMCs relative to controls. Both A769662 and metformin increase complexes I and IV in PPHN PASMCs relative to untreated PPHN PASMCs. (B-C) Protein levels of SIRT1 (B) and PGC-1α (C) in control and PPHN PASMCs and the effect of A769662 and metformin on these protein levels in PPHN PASMCs. SIRT1 protein level is unchanged in PPHN PASMCs relative to controls while PGC-1α protein level is decreased in PPHN PASMCs. Neither A769662 nor metformin altered SIRT1 protein levels in PPHN PASMCs relative to untreated PPHN PASMCs. Both A769662 and metformin increased PGC-1α protein levels in PPHN PASMCs relative to untreated PPHN PASMCs. For (A-C), all results were normalized to β-Actin. The ß-actin loading control for Sirt1 was also used for p-AMPK as the loading control in Fig 2 as these 2 proteins and corresponding ß-actin were run on the same gel. *Indicates p <0.05 from control PASMCs and #from PPHN PASMCs by ANOVA and Tukey’s post hoc test for n = 3 in each group, representing cells from 3 different lambs in each group. (D and E) Immunofluorescence staining for PGC-1α (D) and SIRT1 (E) in representative lung sections from control, PPHN, and metformin-treated PPHN (PPHN + Met) lambs. Lung sections were also stained with smooth muscle cell marker, α-smooth muscle actin (α-SMA), to assess colocalization of these proteins within capillaries, and DAPI to outline nuclei. Scale bars: 200 μm. Both PGC-1α and SIRT1 that colocalize to the blood vessels in the lung are decreased in the smooth muscle layer in PPHN. Metformin infusion in utero increases fluorescence of PGC-1α and SIRT1 in the PPHN lamb lungs. For (D and E), summarized data are for n = 8 in each group, showing integrated fluorescence density. (F) Oxygen consumption measurements in an extracellular Flux analyzer (Seahorse Fe96 Analyzer) reveal that basal respiration, spare respiratory capacity, maximum respiration, and ATP production were all significantly reduced in PPHN PASMCs compared to controls. Metformin and A769662 did not alter them significantly compared to untreated PPHN PASMCs. For (F), summarized data are for n = 3 representing cells from 3 different lambs in each group. *Indicates p <0.05 from control lambs and #from PPHN lambs by ANOVA and Tukey’s post hoc test.

IF signals for PGC-1α and SIRT-1 were decreased in PPHN lamb lung sections in the PASMC layer (Fig 3D and 3E). Prenatal treatment of PPHN lambs with metformin increased both the PGC-1α and SIRT-1 signals in the PASMC layer of the lung sections (Fig 3D and 3E). While the IF results are consistent with the findings found with IB done on PASMC treated in vitro with metformin and A769662 for PGC-1α, SIRT-1 levels increased only in lung sections of metformin treated PPHN lambs. These data suggest that regulation of SIRT-1 expression in vivo may be different than in vitro under culture conditions.

Lastly, we assessed mitochondrial bioenergetics in the four groups, and found that basal respiration, spare respiratory capacity, maximum respiration, and ATP linked O2 consumption were all significantly reduced in PPHN PASMCs compared to controls (Fig 3F). While Metformin and A769662 increased the spare respiratory capacity in PPHN PASMCs, changes were not significantly different from untreated PPHN PASMCs (Fig 3F). These data suggest that mitochondrial bioenergetic function is compromised in PPHN PASMCs, but neither AMPK targeted treatment improved the capacity of the mitochondria to perform oxidative phosphorylation, in our in vitro studies.

Superoxide Dismutase 2 (SOD2) Expression and Superoxide (O2−) Levels:

Since AMPK is a well-recognized regulator of metabolic homeostasis (33), we investigated whether AMPK affected mitochondrial ROS via its regulation of expression of SOD2, a mitochondrial antioxidant. In PPHN PASMCs, we found SOD2 protein expression to be significantly decreased in PPHN PASMCs compared to controls (Fig 4A), which was associated with elevated O2− levels in PPHN PASMCs compared to controls (Fig 4C). SOD2 protein levels increased in response to treatment with both metformin and A769662 in PPHN PASMCs (Fig 4A), which correlated with reduction in O2− levels (Fig 4C), suggesting that SOD2 expression is regulated by AMPK.

Figure 4.

Figure 4.

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(A) Protein level of SOD2 in control and PPHN PASMCs and the effect of metformin and allosteric AMPK agonist, A769662, on SOD2 protein levels in PPHN PASMCs. SOD2 protein levels are decreased in PPHN PASMCs relative to controls. Both metformin and A769662 increased SOD2 protein levels in PPHN PASMCs relative to untreated PPHN PASMCs. All results are normalized to β-Actin. *Indicates p <0.05 from control PASMCs and #from PPHN PASMCs by ANOVA and Tukey’s post hoc test for n = 3 representing cells from 3 different lambs in each group. (B) Immunofluorescence staining for SOD2 in pulmonary arteries in representative lung sections from control, PPHN, and metformin treated PPHN (PPHN + Met) lambs. Lung sections were also stained with smooth muscle cell marker, α-smooth muscle actin (α-SMA), to assess colocalization of these proteins within pulmonary arteries, and DAPI to outline nuclei. Scale bars: 200 μm. SOD2 colocalized to pulmonary arteries in the lung is decreased in the smooth muscle layer in PPHN. Metformin infusion in utero increases fluorescence of SOD2 in the PPHN lamb lungs. For (B and C), summarized data are for n = 6 in each group, showing integrated fluorescence density. (C) Mitochondrial Superoxide (O2−) Levels assessed by MitoSOX – There is significantly higher detection of O2− levels (red) in PPHN lamb PASMCs relative to controls. The levels of O2− were lower in PPHN lamb PASMCs treated with metformin or A769662 compared to those without treatment. Images also stained with DAPI to highlight the nucleus. Images analyzed and shown at 40x, and scale bar is 20 μm. For (C), summarized data are for n = 7 in each group. *Indicates p <0.05 from control lambs and #from PPHN lambs by ANOVA and Tukey’s post hoc test.

IF signal for SOD2 in PPHN lamb lung sections in the smooth muscle layer, identified by α-SMA, is decreased compared to controls (Fig 4B). In utero treatment of PPHN lambs with metformin increased the SOD2 signal in the PASMC layer of PPHN lung sections, confirming the changes we observed in PPHN PASMCs via IB (Fig 4B). These data suggest that downregulation of AMPK may cause reduced SOD2 expression resulting in oxidative stress, which is well known to impair mitochondrial integrity and induce hyperproliferation (34).

Expression of PASMC Differentiation and Proliferation Markers:

In PPHN, PASMCs are predominantly in the dedifferentiated, proliferative state, which contributes to pulmonary vascular remodeling (15, 35). Using IF, we found that Myosin Heavy Chain 11 (MYH11), a marker for the quiescent, differentiated state, is significantly decreased in PPHN PASMCs while vimentin, an intermediate filament protein of mesenchymal cells which is a marker for the proliferative, dedifferentiated state (36–38), is significantly increased in PPHN PASMCs (Fig 5A and 5B, respectively). In utero treatment of PPHN lambs with metformin reversed this ratio, increasing MYH11 and decreasing vimentin (Fig 5A and 5B, respectively). Moreover, since dedifferentiated PASMCs tend to have higher proliferation activity (9), we evaluated proliferation markers, phos(Ser10)-Histone H3 (pH3) and antigen Kiel 67 (Ki-67), which we found to be increased in PPHN PASMCs compared to controls, and prenatal treatment with metformin reduced their expression in PPHN PASMCs (Figure 5C and 5D). Our findings suggest that AMPK may regulate the expression of differentiation markers in PASMCs in neonatal PH.

Figure 5.

Figure 5.

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(A-D) Immunofluorescence staining for MYH11 (A), vimentin (B), phos(Ser10)-histone H3 (pH3, C), and antigen Kiel 67 (Ki-67, D) in the pulmonary arteries in representative lung sections from control, PPHN, and metformin-treated PPHN (PPHN + Met) lambs. Lung sections were also stained with smooth muscle cell marker, α-smooth muscle actin (α-SMA), to assess colocalization of these proteins within pulmonary arteries, and DAPI to outline nuclei. Scale bars: 200 μm. MYH11 colocalized to the pulmonary arteries in the lung is decreased in the smooth muscle layer in PPHN while vimentin, pH3, and Ki-67 are increased relative to controls. Metformin increased MYH11 in PPHN lamb lungs and decreased vimentin, pH3, and Ki-67, compared to untreated PPHN lungs. Summarized data are for n = 6–16 in different studies, showing integrated fluorescence. *Indicates p <0.05 from control lambs and #from PPHN lambs by ANOVA and Tukey’s post hoc test.

Notch/Jag1 Signal Expression:

The expression of Notch ligand, Jagged1 (Jag1), which inhibits PASMC proliferation and restores its differentiated state (39), is significantly decreased in PPHN PASMCs compared with control PASMCs in both IB of PASMC and IF of PA in lung sections (Fig 6A and 6C). Both metformin and A769662 increased Jag1 expression in PPHN PASMCs, while in utero treatment of PPHN lambs with metformin increased Jag1 expression in the smooth muscle layer of PPHN lung sections compared to untreated PPHN lambs (Fig 6A and 6C). Another Notch signaling protein, Hey1/Herp2, was also decreased in PPHN PASMCs compared to controls in both IB and IF (Fig 6B and 6D). Both metformin and A769662 increased Hey1 expression in PPHN PASMCs compared to untreated PPHN PASMCs while metformin treatment in utero increased Hey1 in the smooth muscle layer of PPHN lung sections compared to untreated PPHN lungs (Fig 6B and 6D). These data suggest that AMPK regulates Jag1 and Hey1 expression in PASMCs. Similar to previous studies (39), these data suggest that AMPK preserves the quiescent, differentiated state of PASMCs.

Figure 6.

Figure 6.

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(A and B) Protein levels of Jagged1 (Jag1, A) and Hey1 (B) in control and PPHN PASMCs and the effect of metformin and allosteric AMPK agonist, A769662, on Jag1 and Hey1 protein levels in PPHN PASMCs. Both Jag1 and Hey1 protein levels are decreased in PPHN PASMCs relative to controls. Both metformin and A769662 increased Jag1 and Hey1 in PPHN PASMCs relative to untreated PPHN PASMCs. For (A and B), all results are normalized to β-Actin. *Indicates p <0.05 from control PASMCs and #from PPHN PASMCs by ANOVA and Tukey’s post hoc test for n = 3 representing cells from 3 different lambs in each group. (C and D) Immunofluorescence staining for Jag1 (C) and Hey1 (D) in representative lung sections from control, PPHN, and metformin treated PPHN (PPHN + Met) lambs. Lung sections were also stained with smooth muscle cell marker, α-smooth muscle actin (α-SMA), to assess colocalization of these proteins within capillaries, and DAPI to outline nuclei. Scale bars: 200 μm. The IF for both Jag1 and Hey1 colocalized to the pulmonary arteries in the lung was decreased in the smooth muscle layer in PPHN. Metformin infusion in utero increased both Jag1 and Hey1 IF in the pulmonary arteries in PPHN lamb lungs. Summarized data are for n = 6, showing integrated fluorescence intensity. *Indicates p <0.05 from control lambs and #from PPHN lambs by ANOVA and Tukey’s post hoc test.

DISCUSSION

Our studies provide the evidence that PPHN is associated with a decrease in the function of AMPK, a critical sensor and regulator of mitochondrial integrity and redox balance in PASMCs (40). Our results are consistent with findings previously reported in pulmonary arterial hypertension (PAH) in adults (41, 42). We observed that decreased p-AMPK protein level in PPHN is associated with reduced expression of LKB1, a required upstream AMPK kinase (29). We also found that reduced AMPK function in PPHN PASMCs correlated with decreased expression of PGC-1α and the mitochondrial ETC complexes I-V. These changes were associated with a reduction in mitochondrial bioenergetic parameters, including basal respiration, spare respiratory capacity, maximum respiration, and ATP production. These results suggest that oxidative phosphorylation capacity of PASMCs is compromised in PPHN. AMPK function appears to be linked to the PASMC differentiation marker profile based on the decreased ratio of MYH11 to vimentin and an increase in proliferative markers, pH3 and Ki-67, in PPHN. Lastly, we found that decreased AMPK function in PASMCs is associated with reduced expression of Notch ligand, Jag1 and its downstream mediator, Hey1, which impairs angiogenesis function (18) and also alters the proliferation potential and differentiation state (39). Our in vitro and in vivo studies together reveal that AMPK downregulation is potentially reversible with metformin treatment. Our studies suggest a coordinated decrease in LKB1- AMPK signaling and compromised mitochondrial bioenergetics in PPHN PASMCs, which may provide the mechanism by which skewing of the differentiation markers occurs, resulting in pulmonary vascular remodeling.

In a recent publication, we also reported downregulation of AMPK-PGC-1α signaling in PPHN PAECs, resulting in impaired mitochondrial oxidative phosphorylation capacity and decreased angiogenesis function, which was reversible with metformin and A769662 treatment (18). Similarly, it appears that in PASMCs, the AMPK-PGC-1α pathway is also reduced, which we found to be associated with impaired mitochondrial bioenergetics and reduced protein expression of ETC complexes I-V, suggesting impaired oxidative phosphorylation capacity in PPHN PASMCs. While metformin and A769662 increased the expression of ETC complexes I and IV, mitochondrial bioenergetic function did not significantly improve with either treatment in our in vitro studies. However, our in vitro experiments examined these effects of AMPK agonists in cultured PPHN PASMCs after only 48 hours of treatment. This brief in vitro treatment may be insufficient to have functional effects on mitochondrial respiration. Future in vivo studies are needed to better assess the effects of these treatments on mitochondrial bioenergetics. Additionally, we found that proliferation markers, pH3 and Ki-67, were increased in PPHN PASMCs, which declined significantly with in vivo treatment of metformin. This finding suggests that PPHN PASMCs exhibit upregulation of proliferation markers. However, since we did not perform specific proliferation assays, it remains unclear whether PPHN PASMCs are more proliferative. It is interesting that while downregulation of AMPK-PGC-1α signaling in PAECs causes reduced proliferation and tube formation (18), a reduction of this same signaling pathway in PASMCs results in increased proliferation marker expression with ensuing pulmonary vascular remodeling. In adult PAH, it was found that reduced capacity to perform oxidative phosphorylation can result in both endothelial dysfunction and smooth muscle hyperproliferation, which lead to pulmonary vascular remodeling (43). Of note, a study involving a smooth muscle-specific AMPK knockout (KO) mouse showed that the mitochondrial dysfunction occurring in this model was not due to PGC-1α downregulation (44). However, this study only assessed redox stress resulting in mitochondrial dysfunction (44). Our study supports the hypothesis that PGC-1α signaling regulates oxidative phosphorylation capacity of mitochondria while SOD2 regulates excess ROS.

Our studies also support our concept that AMPK upregulation can decrease the PASMC shift toward increasing the expression of proliferative markers, which may induce a phenotypic change, which has also been shown in adult animal models of PH (45). However, our current data are novel in demonstrating that AMPK expression may preserve PASMC differentiation to prevent excess proliferation in PPHN. We show that when AMPK expression is reduced in PPHN, PASMCs shift towards a dedifferentiated state, as shown by the reduced ratio of MYH11 to vimentin. Moreover, we found AMPK downregulation to be associated with increased pH3 and Ki-67 expression, which indicates upregulation of proliferation marker expression. With metformin infusion in utero, the dedifferentiation and proliferation markers were reversed. The inverse relationship between AMPK and vimentin has been well-described in cancer studies characterizing endothelial-to-mesenchymal transition, a feature of vascular remodeling (46). Interestingly, in an adult PAH model using hypoxia and Sugen (anti-VEGF) treatment in rats to create the PAH phenotype, AMPK has been found to promote, rather than rescue from, phenotypic reprogramming of PASMCs (47). Our findings may be different due to 1) species differences, as these previous studies used mouse and human cells; and 2) difference in disease model, i.e., fetal and neonatal PPHN vs. adult PAH.

SIRT-1, a longevity protein that serves as an NAD+-dependent deacetylase, works with AMPK and PGC-1α to enhance mitochondrial health (31, 32). In our model of PPHN, we found SIRT-1 levels to be downregulated along with AMPK and PGC-1α, but only to a significant level in the in vivo IF studies in lung sections. SIRT-1 has been shown in adult PAH studies to prevent excess PASMC proliferation via AMPK signaling (48). SIRT-1 expression did not respond to either treatment in the IB of PASMC in vitro samples but responded to metformin in vivo as shown in the IF staining of lung sections. The interaction between SIRT-1 and AMPK is more complex and may be related to SIRT-1 posttranslational modifications by AMPK after activation of AMPK by agonists. Additionally, our studies only assessed a change in the protein expression of SIRT-1, not a functional alteration.

We have also recently reported that altered metabolic and redox balance function in PAECs is associated with reduced expression of notch ligand Jag1 (18) and Hey1/Herp2 (49), which normally regulate angiogenesis in PAECs (50). We now report that the altered notch ligand function in PAECs is replicated in PASMCs and may be contributing to PASMC dedifferentiation and excess proliferation in PPHN. It is well known that Notch ligands play an important role in endothelial cell biology, but their role in PPHN PASMCs is not well understood. We found that the Notch ligand, Jag1, an inhibitor of PASMC proliferation and marker of the differentiated PASMC state (39), is downregulated in PPHN PASMCs. Similar to our findings, two studies utilizing a smooth muscle-specific Jag1 knockout (KO) mouse found that these mice have dedifferentiated smooth muscle cells, as supported by reduced SMA expression (51), and increased proliferation (51, 52). Interestingly, another study with an endothelial-specific Jag1 KO mouse found that SMCs had reduced Jag1 which was associated with a dedifferentiated state (53). Moreover, Hey1, a downstream mediator of Notch function that is also referred to as Herp2, is suppressed in PPHN PASMCs compared to controls, consistent with decreased Jag1 levels. Interestingly, in adult PAH, Hey1 is elevated to promote PASMC proliferation and migration (39, 54).

The factors regulating the process of pulmonary vascular remodeling in PPHN remain unclear. Our study demonstrates that reduced differentiation and increased proliferation marker expression is associated with downregulation of AMPK and a decrease in Jag1 signaling. Whether this altered balance is mechanistically caused by decreased AMPK is still unknown, but it appears that both in vitro and in utero treatments with metformin can restore the proper balance of proliferative and differentiation markers, which was associated with improved mitochondrial electron transport chain complex and decreased O2− levels. Determining a mechanistic relationship between decreased AMPK function and these changes will require additional gain and loss of function studies.

Our study has some limitations. While our animal model represents one condition that leads to the PPHN phenotype, it does not encompass all causes of PPHN. Our studies in cultured PASMCs reveal changes in AMPK signaling, and these findings were replicated in the lung sections from our PPHN model; however, it remains unclear whether these alterations apply to infants with PPHN. Changes in these signaling pathways in the setting of adjacent endothelial and smooth muscle cells with intact cell-cell signaling are unknown. Lastly, since metformin exerts its effects via both AMPK-dependent and -independent mechanisms, it is possible that the improvement in mitochondrial bioenergetics and mitigation of excess cell proliferation marker expression may be due to AMPK-independent effects.

In conclusion, our experiments provide evidence that PPHN in our fetal lamb model is associated with decreased AMPK function in PASMCs with an altered balance of proliferation and differentiation markers along with a decrease in mitochondrial bioenergetic function. Restoring AMPK function, via metformin administration, restores the balance of proliferation and differentiation markers along with mitochondrial proteins. Whether metformin will serve as an effective treatment for infants suffering from PPHN requires further studies.

Supplementary Material

Supplemental table of antibodies used in the study, supplemental figures and legends for figures are available at the link for DOI: https://doi.org/10.6084/m9.figshare.31145077.

GRANTS

National Heart, Lung, & Blood Institute (NHLBI), 1R01HL 136597–01 and 1R01 HL174635 (Konduri), Advancing a Healthier Wisconsin Foundation Endowment (Konduri), Children’s Research Institute of Children’s Wisconsin and New Faculty Pilot Grant, Research Affairs Committee (Mooers)

Footnotes

DISCLOSURES

No documented financial relationships to disclose or conflicts of interest (COIs) to resolve for all authors.

DISCLAIMERS

This manuscript does not contain any required disclaimers.

DATA AVAILABILITY

Data sharing is not applicable to this article as no unique datasets were generated or analyzed during the current study.

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Data Availability Statement

Data sharing is not applicable to this article as no unique datasets were generated or analyzed during the current study.

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