Mitochondrial hyperfusion induces metabolic remodeling in lung endothelial cells by modifying the activities of electron transport chain complexes I and III.

OBJECTIVE: Pulmonary hypertension (PH) is a progressive disease with vascular remodeling as a critical structural alteration. We have previously shown that metabolic reprogramming is an early initiating mechanism in animal models of PH. This metabolic dysregulation has been linked to remodeling the mitochondrial network to favor fission. However, whether the mitochondrial fission/fusion balance underlies the metabolic reprogramming found early in PH development is unknown. METHODS: Utilizing a rat early model of PH, in conjunction with cultured pulmonary endothelial cells (PECs), we utilized metabolic flux assays, Seahorse Bioassays, measurements of electron transport chain (ETC) complex activity, fluorescent microscopy, and molecular approaches to investigate the link between the disruption of mitochondrial dynamics and the early metabolic changes that occur in PH. RESULTS: We observed increased fusion mediators, including Mfn1, Mfn2, and Opa1, and unchanged fission mediators, including Drp1 and Fis1, in a two-week monocrotaline-induced PH animal model (early-stage PH). We were able to establish a connection between increases in fusion mediator Mfn1 and metabolic reprogramming. Using an adenoviral expression system to enhance Mfn1 levels in pulmonary endothelial cells and utilizing 13C-glucose labeled substrate, we found increased production of 13C lactate and decreased TCA cycle metabolites, revealing a Warburg phenotype. The use of a 13C5-glutamine substrate showed evidence that hyperfusion also induces oxidative carboxylation. The increase in glycolysis was linked to increased hypoxia-inducible factor 1α (HIF-1α) protein levels secondary to the disruption of cellular bioenergetics and higher levels of mitochondrial reactive oxygen species (mt-ROS). The elevation in mt-ROS correlated with attenuated ETC complexes I and III activities. Utilizing a mitochondrial-targeted antioxidant to suppress mt-ROS, limited HIF-1α protein levels, which reduced cellular glycolysis and reestablished mitochondrial membrane potential. CONCLUSIONS: Our data connects mitochondrial fusion-mediated mt-ROS to the Warburg phenotype in early-stage PH development.

Novel Relationship between Mitofusin 2-Mediated Mitochondrial Hyperfusion, Metabolic Remodeling, and Glycolysis in Pulmonary Arterial Endothelial Cells.

The disruption of mitochondrial dynamics has been identified in cardiovascular diseases, including pulmonary hypertension (PH), ischemia-reperfusion injury, heart failure, and cardiomyopathy. Mitofusin 2 (Mfn2) is abundantly expressed in heart and pulmonary vasculature cells at the outer mitochondrial membrane to modulate fusion. Previously, we have reported reduced levels of Mfn2 and fragmented mitochondria in pulmonary arterial endothelial cells (PAECs) isolated from a sheep model of PH induced by pulmonary over-circulation and restoring Mfn2 normalized mitochondrial function. In this study, we assessed the effect of increased expression of Mfn2 on mitochondrial metabolism, bioenergetics, reactive oxygen species production, and mitochondrial membrane potential in control PAECs. Using an adenoviral expression system to overexpress Mfn2 in PAECs and utilizing 13C labeled substrates, we assessed the levels of TCA cycle metabolites. We identified increased pyruvate and lactate production in cells, revealing a glycolytic phenotype (Warburg phenotype). Mfn2 overexpression decreased the mitochondrial ATP production rate, increased the rate of glycolytic ATP production, and disrupted mitochondrial bioenergetics. The increase in glycolysis was linked to increased hypoxia-inducible factor 1α (HIF-1α) protein levels, elevated mitochondrial reactive oxygen species (mt-ROS), and decreased mitochondrial membrane potential. Our data suggest that disrupting the mitochondrial fusion/fission balance to favor hyperfusion leads to a metabolic shift that promotes aerobic glycolysis. Thus, therapies designed to increase mitochondrial fusion should be approached with caution.

SOX17 Deficiency Mediates Pulmonary Hypertension: At the Crossroads of Sex, Metabolism, and Genetics.

Rationale: Genetic studies suggest that SOX17 (SRY-related HMG-box 17) deficiency increases pulmonary arterial hypertension (PAH) risk. Objectives: On the basis of pathological roles of estrogen and HIF2α (hypoxia-inducible factor 2α) signaling in pulmonary artery endothelial cells (PAECs), we hypothesized that SOX17 is a target of estrogen signaling that promotes mitochondrial function and attenuates PAH development via HIF2α inhibition. Methods: We used metabolic (Seahorse) and promoter luciferase assays in PAECs together with the chronic hypoxia murine model to test the hypothesis. Measurements and Main Results: Sox17 expression was reduced in PAH tissues (rodent models and from patients). Chronic hypoxic pulmonary hypertension was exacerbated by mice with conditional Tie2-Sox17 (Sox17EC-/-) deletion and attenuated by transgenic Tie2-Sox17 overexpression (Sox17Tg). On the basis of untargeted proteomics, metabolism was the top pathway altered by SOX17 deficiency in PAECs. Mechanistically, we found that HIF2α concentrations were increased in the lungs of Sox17EC-/- and reduced in those from Sox17Tg mice. Increased SOX17 promoted oxidative phosphorylation and mitochondrial function in PAECs, which were partly attenuated by HIF2α overexpression. Rat lungs in males displayed higher Sox17 expression versus females, suggesting repression by estrogen signaling. Supporting 16α-hydroxyestrone (16αOHE; a pathologic estrogen metabolite)-mediated repression of SOX17 promoter activity, Sox17Tg mice attenuated 16αOHE-mediated exacerbations of chronic hypoxic pulmonary hypertension. Finally, in adjusted analyses in patients with PAH, we report novel associations between a SOX17 risk variant, rs10103692, and reduced plasma citrate concentrations (n = 1,326). Conclusions: Cumulatively, SOX17 promotes mitochondrial bioenergetics and attenuates PAH, in part, via inhibition of HIF2α. 16αOHE mediates PAH development via downregulation of SOX17, linking sexual dimorphism and SOX17 genetics in PAH.

LPS-induced Acute Lung Injury Involves NF-κB-mediated Downregulation of SOX18.

One of the early events in the progression of LPS-mediated acute lung injury in mice is the disruption of the pulmonary endothelial barrier resulting in lung edema. However, the molecular mechanisms by which the endothelial barrier becomes compromised remain unresolved. The SRY (sex-determining region on the Y chromosome)-related high-mobility group box (Sox) group F family member, SOX18, is a barrier-protective protein through its ability to increase the expression of the tight junction protein CLDN5. Thus, the purpose of this study was to determine if downregulation of the SOX18-CLDN5 axis plays a role in the pulmonary endothelial barrier disruption associated with LPS exposure. Our data indicate that both SOX18 and CLDN5 expression is decreased in two models of in vivo LPS exposure (intraperitoneal, intratracheal). A similar downregulation was observed in cultured human lung microvascular endothelial cells (HLMVECs) exposed to LPS. SOX18 overexpression in HLMVECs or in the mouse lung attenuated the LPS-mediated vascular barrier disruption. Conversely, reduced CLDN5 expression (siRNA) reduced the HLMVEC barrier-protective effects of SOX18 overexpression. The mechanism by which LPS decreases SOX18 expression was identified as transcriptional repression through binding of NF-κB (p65) to a SOX18 promoter sequence located between -1,082 and -1,073 bp with peroxynitrite contributing to LPS-mediated NF-κB activation. We conclude that NF-κB-dependent decreases in the SOX18-CLDN5 axis are essentially involved in the disruption of human endothelial cell barrier integrity associated with LPS-mediated acute lung injury.

Hyper-activation of pp60Src limits nitric oxide signaling by increasing asymmetric dimethylarginine levels during acute lung injury.

The molecular mechanisms by which the endothelial barrier becomes compromised during lipopolysaccharide (LPS) mediated acute lung injury (ALI) are still unresolved. We have previously reported that the disruption of the endothelial barrier is due, at least in part, to the uncoupling of endothelial nitric oxide synthase (eNOS) and increased peroxynitrite-mediated nitration of RhoA. The purpose of this study was to elucidate the molecular mechanisms by which LPS induces eNOS uncoupling during ALI. Exposure of pulmonary endothelial cells (PAEC) to LPS increased pp60Src activity and this correlated with an increase in nitric oxide (NO) production, but also an increase in NOS derived superoxide, peroxynitrite formation and 3-nitrotyrosine (3-NT) levels. These effects could be simulated by the over-expression of a constitutively active pp60Src (Y527FSrc) mutant and attenuated by over-expression of dominant negative pp60Src mutant or reducing pp60Src expression. LPS induces both RhoA nitration and endothelial barrier disruption and these events were attenuated when pp60Src expression was reduced. Endothelial NOS uncoupling correlated with an increase in the levels of asymmetric dimethylarginine (ADMA) in both LPS exposed and Y527FSrc over-expressing PAEC. The effects in PAEC were also recapitulated when we transiently over-expressed Y527FSrc in the mouse lung. Finally, we found that the pp60-Src-mediated decrease in DDAH activity was mediated by the phosphorylation of DDAH II at Y207 and that a Y207F mutant DDAH II was resistant to pp60Src-mediated inhibition. We conclude that pp60Src can directly inhibit DDAH II and this is involved in the increased ADMA levels that enhance eNOS uncoupling during the development of ALI.

Asymmetric Dimethylarginine Stimulates Akt1 Phosphorylation via Heat Shock Protein 70-Facilitated Carboxyl-Terminal Modulator Protein Degradation in Pulmonary Arterial Endothelial Cells.

Asymmetric dimethylarginine (ADMA) induces the mitochondrial translocation of endothelial nitric oxide synthase (eNOS) through the nitration-mediated activation of Akt1. However, it is recognized that the activation of Akt1 requires phosphorylation events at threonine (T) 308 and serine (S) 473. Thus, the current study was performed to elucidate the potential effect of ADMA on Akt1 phosphorylation and the mechanisms that are involved. Exposure of pulmonary arterial endothelial cells to ADMA enhanced Akt1 phosphorylation at both threonine 308 and Ser473 without altering Akt1 protein levels, phosphatase and tensin homolog activity, or membrane Akt1 levels. Heat shock protein (Hsp) 90 plays a pivotal role in maintaining Akt1 activity, and our results demonstrate that ADMA decreased Hsp90-Akt1 interactions, but, surprisingly, overexpression of a dominant-negative Hsp90 mutant increased Akt1 phosphorylation. ADMA exposure or overexpression of dominant-negative Hsp90 increased Hsp70 levels, and depletion of Hsp70 abolished ADMA-induced Akt1 phosphorylation. ADMA decreased the interaction of Akt1 with its endogenous inhibitor, carboxyl-terminal modulator protein (CTMP). This was mediated by the proteasomal-dependent degradation of CTMP. The overexpression of CTMP attenuated ADMA-induced Akt1 phosphorylation at Ser473, eNOS phosphorylation at Ser617, and eNOS mitochondrial translocation. Finally, we found that the mitochondrial translocation of eNOS in our lamb model of pulmonary hypertension is associated with increased Akt1 and eNOS phosphorylation and reduced Akt1-CTMP protein interactions. In conclusion, our data suggest that CTMP is directly involved in ADMA-induced Akt1 phosphorylation in vitro and in vivo, and that increasing CTMP levels may be an avenue to treat pulmonary hypertension.

Nitric oxide induces hypoxia ischemic injury in the neonatal brain via the disruption of neuronal iron metabolism.

We have recently shown that increased hydrogen peroxide (H2O2) generation is involved in hypoxia-ischemia (HI)-mediated neonatal brain injury. H2O2 can react with free iron to form the hydroxyl radical, through Fenton Chemistry. Thus, the objective of this study was to determine if there was a role for the hydroxyl radical in neonatal HI brain injury and to elucidate the underlying mechanisms. Our data demonstrate that HI increases the deposition of free iron and hydroxyl radical formation, in both P7 hippocampal slice cultures exposed to oxygen-glucose deprivation (OGD), and the neonatal rat exposed to HI. Both these processes were found to be nitric oxide (NO) dependent. Further analysis demonstrated that the NO-dependent increase in iron deposition was mediated through increased transferrin receptor expression and a decrease in ferritin expression. This was correlated with a reduction in aconitase activity. Both NO inhibition and iron scavenging, using deferoxamine administration, reduced hydroxyl radical levels and neuronal cell death. In conclusion, our results suggest that increased NO generation leads to neuronal cell death during neonatal HI, at least in part, by altering iron homeostasis and hydroxyl radical generation.

Endothelin-1 induces a glycolytic switch in pulmonary arterial endothelial cells via the mitochondrial translocation of endothelial nitric oxide synthase.

Recent studies have indicated that, during the development of pulmonary hypertension (PH), there is a switch from oxidative phosphorylation to glycolysis in the pulmonary endothelium. However, the mechanisms underlying this phenomenon have not been elucidated. Endothelin (ET)-1, an endothelial-derived vasoconstrictor peptide, is increased in PH, and has been shown to play an important role in the oxidative stress associated with PH. Thus, in this study, we investigated whether there was a potential link between increases in ET-1 and mitochondrial remodeling. Our data indicate that ET-1 induces the redistribution of endothelial nitric oxide synthase (eNOS) from the plasma membrane to the mitochondria in pulmonary arterial endothelial cells, and that this was dependent on eNOS uncoupling. We also found that ET-1 disturbed carnitine metabolism, resulting in the attenuation of mitochondrial bioenergetics. However, ATP levels were unchanged due to a compensatory increase in glycolysis. Further mechanistic investigations demonstrated that ET-1 mediated the redistribution of eNOS via the phosphorylation of eNOS at Thr495 by protein kinase C δ. In addition, the glycolytic switch appeared to be dependent on mitochondrial-derived reactive oxygen species that led to the activation of hypoxia-inducible factor signaling. Finally, the cell culture data were confirmed in vivo using the monocrotaline rat model of PH. Thus, we conclude that ET-1 induces a glycolytic switch in pulmonary arterial endothelial cells via the redistribution of uncoupled eNOS to the mitochondria, and that preventing this event may be an approach for the treatment of PH.

Asymmetric dimethylarginine induces endothelial nitric-oxide synthase mitochondrial redistribution through the nitration-mediated activation of Akt1.

We have recently demonstrated that asymmetric dimethylarginine (ADMA) induces the translocation of endothelial nitric-oxide synthase (eNOS) to the mitochondrion via a mechanism that requires protein nitration. Thus, the goal of this study was elucidate how eNOS redistributes to mitochondria and to identify the nitrated protein responsible for this event. Our data indicate that exposure of pulmonary arterial endothelial cells to ADMA enhanced eNOS phosphorylation at the Akt1-dependent phosphorylation sites Ser(617) and Ser(1179). Mutation of these serine residues to alanine (S617A and S1179A) inhibited nitration-mediated eNOS translocation to the mitochondria, whereas the phosphormimic mutations (S617D and S1179D) exhibited increased mitochondrial redistribution in the absence of ADMA. The overexpression of a dominant-negative Akt1 also attenuated ADMA-mediated eNOS mitochondrial translocation. Furthermore, ADMA enhanced Akt1 nitration and increased its activity. Mass spectrometry identified a single nitration site in Akt1 located at the tyrosine residue (Tyr(350)) located within the client-binding domain. Replacement of Tyr(350) with phenylalanine abolished peroxynitrite-mediated eNOS translocation to mitochondria. We also found that in the absence of ADMA, eNOS translocation decreased mitochondrial oxygen consumption and superoxide production without altering cellular ATP level. This suggests that under physiologic conditions, eNOS translocation enhances mitochondria coupling. In conclusion, we have identified a new mechanism by which eNOS translocation to mitochondria is regulated by the phosphorylation of eNOS at Ser(617) and Ser(1179) by Akt1 and that this is enhanced when Akt1 becomes nitrated at Tyr(350).

Disruption of endothelial cell mitochondrial bioenergetics in lambs with increased pulmonary blood flow.

AIMS: The mitochondrial dysfunction in our lamb model of congenital heart disease with increased pulmonary blood flow (PBF) (Shunt) is associated with disrupted carnitine metabolism. Our recent studies have also shown that asymmetric dimethylarginine (ADMA) levels are increased in Shunt lambs and ADMA increases the nitration of mitochondrial proteins in lamb pulmonary arterial endothelial cells (PAEC) in a nitric oxide synthase (NOS)-dependent manner. Thus, we determined whether there was a mechanistic link between endothelial nitric oxide synthase (eNOS), ADMA, and the disruption of carnitine homeostasis in PAEC. RESULTS: Exposure of PAEC to ADMA induced the redistribution of eNOS to the mitochondria, resulting in an increase in carnitine acetyl transferase (CrAT) nitration and decreased CrAT activity. The resulting increase in acyl-carnitine levels resulted in mitochondrial dysfunction and the disruption of mitochondrial bioenergetics. Since the addition of L-arginine prevented these pathologic changes, we examined the effect of L-arginine supplementation on carnitine homeostasis, mitochondrial function, and nitric oxide (NO) signaling in Shunt lambs. We found that the treatment of Shunt lambs with L-arginine prevented the ADMA-mediated mitochondrial redistribution of eNOS, the nitration-mediated inhibition of CrAT, and maintained carnitine homeostasis. In turn, adenosine-5'-triphosphate levels and eNOS/heat shock protein 90 interactions were preserved, and this decreased NOS uncoupling and enhanced NO generation. INNOVATION: Our data link alterations in cellular L-arginine metabolism with the disruption of mitochondrial bioenergetics and implicate altered carnitine homeostasis as a key player in this process. CONCLUSION: L-arginine supplementation may be a useful therapy to prevent the mitochondrial dysfunction involved in the pulmonary vascular alterations secondary to increased PBF.

GTP cyclohydrolase I expression is regulated by nitric oxide: role of cyclic AMP.

Our previous studies have demonstrated that nitric oxide (NO) leads to nitric oxide synthase (NOS) uncoupling and an increase in NOS-derived superoxide. However, the cause of this uncoupling has not been adequately resolved. The pteridine cofactor tetrahydrobiopterin (BH(4)) is a critical determinant of endothelial NOS (eNOS) activity and coupling, and GTP cyclohydrolase I (GCH1) is the rate-limiting enzyme in its generation. Thus the initial purpose of this study was to determine whether decreases in BH(4) could underlie, at least in part, the NO-mediated uncoupling of eNOS we have observed both in vitro and in vivo. Initially we evaluated the effect of inhaled NO levels on GCH1 expression and BH(4) levels in the intact lamb. Contrary to our hypothesis, we found that there was a significant increase in both plasma BH4 levels and peripheral lung GCH1 protein levels. Furthermore, in vitro, we found that exposure to the NO donor spermine NONOate (SPNONO) led to an increase in GCH1 protein and BH(4) levels in both COS-7 and pulmonary arterial endothelial cells. However, SPNONO treatment also caused a significant increase in phospho-cAMP response element binding protein (CREB) levels, as detected by Western blot analysis, and significantly increased cAMP levels, as detected by enzyme immunoassay. Furthermore, utilizing GCH1 promoter fragments fused to a luciferase reporter gene, we found that GCH1 promoter activity was enhanced by SPNONO in a CREB-dependent manner, and electromobility shift assays revealed an NO-dependent increase in the nuclear binding of CREB. These data suggest that NO increases BH(4) levels through a cAMP/CREB-mediated increase in GCH1 transcription and that the eNOS uncoupling associated with exogenous NO does not involved reduced BH(4) levels.

Estradiol increases guanosine 5'-triphosphate cyclohydrolase expression via the nitric oxide-mediated activation of cyclic adenosine 5'-monophosphate response element binding protein.

A number of studies have demonstrated that estradiol can stimulate endothelial nitric oxide synthase expression and activity, resulting in enhanced nitric oxide (NO) generation. However, its effect on the NO synthase cofactor, tetrahydrobiopterin are less clear. Cellular tetrahydrobiopterin levels are regulated, at least in part, by GTP cyclohydrolase 1 (GCH1). Thus, the purpose of this study was to determine the effect of estradiol on GCH1 expression and the regulatory mechanisms in pulmonary arterial endothelial cells. Our data indicate that 17beta-estradiol (E2) increases GCH1 transcription in a dose- and time-dependent manner, whereas estrogen receptor antagonism or NO synthase inhibition attenuated E2-stimulated GCH1 expression. Analysis of the GCH1 promoter fragment responsive to E2 revealed the presence of a cAMP response element, and we found that E2 triggers a rapid but transient elevation of phospho-cAMP response element-binding protein (CREB; <1 h) followed by a second sustained rise after 6 h. EMSA analysis revealed an increase in the binding of CREB during E2 treatment and mutation of the cAMP response element in the GCH1 promoter attenuated the E2-mediated increase in transcription. Furthermore, inhibition of the cAMP-dependent kinase, protein kinase A (PKA) completely abolished the E2-stimulated GCH1 promoter activity, whereas the stimulation of cAMP levels with forskolin increased GCH1 promoter activity, indicating the key role of cAMP in regulating GCH1 promoter activity. In conclusion, our results demonstrate that estradiol can modulate GCH1 expression via NO-mediated activation of CREB in pulmonary arterial endothelial cells. These findings provide new insight into the vascular protective effect of estradiol.

Hydrogen peroxide decreases endothelial nitric oxide synthase promoter activity through the inhibition of AP-1 activity.

Previously, we have reported that endothelial nitric oxide synthase (eNOS) promoter activity is decreased in pulmonary arterial endothelial cells (PAECs) in response to hydrogen peroxide (H(2)O(2)). Thus the objective of this study was to identify the cis-element(s) and transcription factor(s) responsible for oxidant-mediated downregulation of the eNOS gene. Initial promoter experiments in PAECs treated with H(2)O(2) revealed a significant decrease in activity of a promoter fragment containing 840 bp of upstream sequence of the human eNOS gene fused to a luciferase reporter. However, a promoter construct containing only 640 bp of upstream sequence had a significantly attenuated response to H(2)O(2) challenge. As the 840-bp promoter construct had a putative binding site for the transcription factor activator protein-1 (AP-1) that was lacking in the 640-bp construct, we evaluated the effect of H(2)O(2) on promoter activity after mutation of the AP-1 binding sequence (TGAGTCA at -661 to TGAGTtg in the 840-bp construct). Similar to the results seen with the 640 bp, the AP-1 mutant promoter had a significantly attenuated response to H(2)O(2). EMSA revealed decreased binding of AP-1 during H(2)O(2) treatment. Supershift analysis indicated that the AP-1 complex consisted of a c-Jun and FosB heterodimer. Furthermore, in vitro EMSA analysis indicated the c-Jun binding was significantly decreased after H(2)O(2) exposure. Using chromatin immunoprecipitation analysis, we demonstrated decreased binding of AP-1 to the eNOS promoter in vivo in response to H(2)O(2). These data suggest a role of decreased AP-1 binding likely through c-Jun in the H(2)O(2)-mediated decrease in eNOS promoter activity.