PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Circ Res. Author manuscript; available in PMC 2013 May 25.
Published in final edited form as:
PMCID: PMC3539779
NIHMSID: NIHMS385405

Dynamin-Related Protein 1 (DRP1)-Mediated Mitochondrial Mitotic Fission Permits Hyperproliferation of Vascular Smooth Muscle Cells and Offers a Novel Therapeutic Target in Pulmonary Hypertension

Abstract

Rationale

Pulmonary arterial hypertension (PAH) is a lethal syndrome characterized by pulmonary vascular obstruction due in part to pulmonary artery smooth muscle cell (PASMC) hyperproliferation. Mitochondrial fragmentation and normoxic activation of hypoxia-inducible factor-1α (HIF-1α) have been observed in PAH PASMCs, however their relationship and relevance to the development of PAH is unknown. Dynamin-related protein-1 (DRP1) is a GTPase that, when activated by kinases that phosphorylate Serine-616, causes mitochondrial fission. It is however unknown whether mitochondrial fission is a prerequisite for proliferation.

Objective

We hypothesize that DRP1 activation is responsible for increased mitochondrial fission in PAH PASMCs and that DRP1 inhibition may slow proliferation and have therapeutic potential.

Methods and Results

Experiments were conducted using human control and PAH lungs (n=5) and PASMCs in culture. Parallel experiments were performed in rat lung sections and PASMCs and in rodent PAH models induced by the HIF-1α activator, cobalt, chronic hypoxia, and monocrotaline. HIF-1α activation in human PAH leads to mitochondrial fission by cyclin B1/CDK1-dependent phosphorylation of DRP1 at Serine-616. In normal PASMC, HIF-1α activation by CoCl2 or desferrioxamine causes DRP1-mediated fission. HIF-1α inhibition reduces DRP1 activation, prevents fission and reduces PASMC proliferation. Both the DRP1 inhibitor Mdivi-1 and siDRP1 prevent mitotic fission and arrest PAH PASMCs at the G2/M interphase. Mdivi-1 is antiproliferative in human PAH PASMC and in rodent models. Mdivi-1 improves exercise capacity, right ventricular function and hemodynamics in experimental PAH.

Conclusion

DRP-1-mediated mitotic fission is a cell cycle checkpoint that can be therapeutically targeted in hyperproliferative disorders such as PAH.

Keywords: Mitochondrial fission, Hypoxia-inducible factor 1, Mitotic check point, CDK1-cyclin B1, Mitochondrial division inhibitor-1 (Mdivi-1)

Introduction

Pulmonary arterial hypertension (PAH) is a syndrome in which obstructed, constricted and inflamed small pulmonary arteries (PA) increase pulmonary vascular resistance (PVR), leading to right ventricular hypertrophy and ultimately failure. The cause of PAH remains unclear, despite important advances in understanding the genetic and epigenetic basis of this syndrome. The discovery of mutations in bone morphogenetic protein receptor-2 (BMPR-2) in familial PAH1, 2, the recognition of somatic chromosomal abnormalities in sporadic PAH3 and the discovery of epigenetic silencing of mitochondrial superoxide dismutase 2 (SOD2) and the resulting activation of hypoxiainducible factor-1α (HIF-1α4 have yet to alter treatment. Moreover, despite effective vasodilator therapies, mortality rates remain high (15% at 1-year)5 suggesting a new treatment paradigm is needed. Excessive pulmonary artery smooth muscle cell (PASMC) proliferation plays an important role in PAH (reviewed in 6), suggesting that antiproliferative therapies are needed. We recently discovered that the hyperproliferative phenotype in PAH PASMCs (human and rodent) is associated with normoxic activation of HIF-1α and fragmentation of the mitochondrial network7. Normoxic activation of HIF-1α in human PAH occurs in PASMC4, 7 and endothelial cells8. HIF-1α activation can result from the redox changes initiated by epigenetic silencing of SOD2 in PAH4, consistent with prior descriptions of redox-regulation of HIF-1α9, 10. Activated HIF-1α suppresses mitochondrial oxidative metabolism (by increasing the expression of pyruvate dehydrogenase kinases, PDKs, thereby blocking pyruvate uptake into the Krebs cycle)11 while simultaneously upregulating enzymes and transporters that favor glycolysis (i.e. hexokinase-2 and the glucose transporter-1, GLUT1) Targeting these mitochondrial-metabolic abnormalities in PAH, using the PDK inhibitor dichloroacetate, reduces glycolysis, restores oxidative metabolism and regresses. PAH in several experimental models12-14. This demonstrates the susceptibility of PAH to mitochondrial-metabolic therapies. HIF-1α is activated early in experimental models of PAH and leads to a pulmonary glycolytic pattern which can be detected with a 18F-fluoro-deoxy-glucose positron emission tomography (FDG-PET) scan. This glycolytic switch is reversible with therapies including PDK inhibitors and Gleevec15. However, the link between HIF-1α, mitochondrial fragmentation, and cell proliferation is unknown.

Mitochondria exist in a dynamic network comprised of individual organelles that continuously join (fusion) and fragment (fission). The GTPases Mitofusin-1 (MFN1), Mitofusin-2 (MFN2), and Optic atrophy-1 (OPA1) regulate fusion. Conversely, Fission-1 (FIS1) and Dynamin-related protein-1 (DRP1) mediate mitochondrial fission16. DRP1’s activity results from phosphorylation by regulatory kinases, including the mitosis regulator, cyclin B1/Cyclin-dependent kinase 1 (CDK1), which causes mitotic phosphorylation of Serine 616 and activation of DRP1. Upon activation DRP1 moves from the cytosol to the mitochondria where it assembles in multimers that constrict and divide the mitochondria. Conversely, phosphorylation of DRP1 at Serine 637, by protein kinase A, inhibits fission17, 18.

Here we determine the molecular basis for mitochondrial fragmentation in PAH, its relationship to HIF-1α and assess whether mitochondrial fission offers a novel antiproliferative therapeutic target in PAH. Studies are conducted in human PASMCs and lung sections from PAH and control patients and in 3 rodent models of PAH, including a model induced by chemical activation of HIF-1α. We demonstrate that HIF-1α activation is a proliferative stimulus that causes DRP1-mediated mitochondrial fission. In vivo HIF-1α activation (by cobalt) is sufficient to produce PAH in rats and Mdivi-1, a small-molecule DRP1 inhibitor19, restores mitochondrial fusion and reduces the hemodynamic and histological severity of PAH in this model. Increased fission in PAH PASMC reflects DRP1 activation by cyclin B1/CDK1 activity and accompanies cell cycle progression from G2 to mitosis. We show that fission is required for normal cell division and that Mdivi-1 slows proliferation by locking mitochondria in fusion and inhibiting cell-cycle progression, causing G2/M arrest, as we recently also observed in lung cancer cells20. Mdivi-1 also has antiproliferative therapeutic benefit in monocrotaline- and hypoxia-induced pulmonary hypertension. We conclude that normoxic activation of HIF-1α creates a fissogenic, proliferative milieu in PAH. Cyclin B1/CDK1, which is upregulated in PAH, coordinates mitosis and mitochondrial fission, permitting rapid proliferation; conversely inhibition of mitotic fission causes cell cycle arrest, and regresses PAH. Thus, mitotic fission is a checkpoint that can be therapeutically targeted by inhibiting DRP1 in hyperproliferative syndromes.

Methods

Human lung samples

Paraffin-embedded tissue sections and fresh tissue for PASMCs isolation were obtained from autopsied patients with idiopathic PAH or control patients (without PAH) under an IACUC-approved protocol (Online Table I).

Animal studies

The University of Chicago Animal Care Committee approved all protocols. Male Sprague-Dawley rats were purchased from Charles Rivers Laboratories (Wilmington, MA). To induce pulmonary hypertension in rats, we injected 2 mg CoCl2 i.p./day for 4-weeks (Sigma Aldrich, St. Louis, MO). This dose was well tolerated and did not induce mortality. Higher doses caused death or severe lethargy. Control rats received saline injections. Other animal models for PAH were chronic hypoxia (10% oxygen) and monocrotaline (60mg/kg i.p.). The DRP1 inhibitor Mdivi-1 (Enzo Life Sciences, Plymouth Meeting, PA) was dissolved in DMSO and injected at a dose based on the literature (50mg/kg)21 for prevention studies (weekly injections for CoCl2 and bi-weekly injections for chronic hypoxia), while 5 daily injections were given 3 weeks after monocrotaline injection for regression studies. Control animals received intraperitoneal DMSO injections.

Immunoblotting and immunofluorescence

Immunoblotting was performed on 25μg of protein from PASMCs. The primary antibodies used were anti-cyclin B1 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-cyclin E (Cell Signaling Technology, Boston, MA), anti-DRP1 (Novus Biologicals, Littleton, CO), anti-phospho Ser616 DRP1 (Cell Signaling Technology, Boston, MA), anti-phospho Ser637 DRP1 (Cell Signaling Technology, Boston, MA), anti-HIF-1α (Novus Biologicals, Littleton, CO), anti-smooth muscle cell actin (MP Biomedicals, Solon, OH), and anti-vWF (Abcam, Cambridge, MA). To confirm equal protein loading, blots were stripped in Restore Western Blot stripping buffer (Thermo Scientific, Rockford, IL) for 15’ and then reprobed with anti-Actin (Millipore, Billerica, MA). Antigen retrieval, secondary antibodies, signal amplification and imaging parameters are described in detail in the Online Data Supplement.

Small interfering RNA (siRNA) treatment of PASMCs

For siRNA treatment, PASMCs were grown to ~80% confluence and then transfected using oligofectamine (Invitrogen, Carlsbad, CA) and 25nM validated Silencer Select siRNAs (Applied Biosystems, Carlsbad, CA). After 4 hours, normal culture medium was added and gene knockdown was assessed after 48 hours using qRT-PCR and immunocytochemistry. For each experiment there was a scrambled siRNA control (Applied Biosystems, Carlsbad, CA).

Live cell imaging to assess mitochondrial networks

Mitochondria of live PASMCs were imaged with a Zeiss 510META confocal laser scanning microscope equipped with an environmental chamber to maintain humidity, 37°C, and 5% CO2. We used co-expression of mitochondrial matrix-targeted photoactivatable GFP (mito-PA-GFP, a kind gift of Dr. Richard Youle, NIH, Bethesda, MD)22 and a mitochondrial matrix-targeted DsRed Protein (mito-DsRed, a kind gift of Dr. Michael Frohman, Stony Brook University, Stony Brook, NY)23 to assess the degree of mitochondrial connectivity in live cells. PASMCs were transfected using Fugene HD transfection reagent (Roche, Indianapolis, IN). After acquiring 3 control images mito-PA-GFP was photoactivated with 10 passes of the 2-photon laser (set to 700nm) in a confined 200×60 pixel area. Photoactivated GFP was measured at 488nm excitation, 500-550nm emission. For assessing mitochondrial morphology in the whole cell, we used a mitochondrial matrix-targeted GFP protein (mito-GFP, a kind gift of Dr. Michael Frohman, Stony Brook University, Stony Brook, NY)23.

Image analysis of mitochondrial morphology

Two recently validated techniques were used for assessing mitochondrial morphology in the whole cell: measurement of the mitochondrial fragmentation count (MFC) and the mitochondrial networking factor (MNF)20. Briefly, images of mito-GFP transfected PASMCs were obtained, thresholded and binarized to identify mitochondrial segments using ImageJ (NIH, Bethesda, MD). The mitochondrial fragmentation count (MFC) was calculated by normalizing the number of individual mitochondria to the entire mitochondrial pixel count20. To quantify functional networking of the mitochondria, PASMCs were transfected with mitochondrial-targeted, photoactivatable green fluorescent protein (mito-PA-GFP) and mitochondrial-targeted DsRed protein. The extent of mitochondrial networking is reflected in the diffusion of the photoactivated mitochondrial GFP away from the site of photoactivation, as measured after 17.6 seconds using ImageJ (NIH, Bethesda, MD). A highly interconnected network allows the photoactivated GFP to distribute in a larger area, resulting in a higher MNF20.

Replication deficient adenoviruses

The HIF-1α Dominant Negative adenovirus was constructed using a plasmid from Dr. Jian Chen (Hokkaido University, Japan24). Viral methodology is described in the Online Data Supplement.

Proliferation assay

Proliferation was quantified using the Click-It EdU kit according the manufacturer’s instructions (Invitrogen, Carlsbad, CA).

Transmission electron microscopy

Freshly isolated pulmonary arteries (3rd-4th order) were fixed and processed for electron microscopy (see Online Data Supplement). Images were collected using a scanning transmission electron microscope at 300KV (Tecnai F30; FEI) with a high-performance Gatan CCD camera.

Measurement of cyclin B1/CDK1 activity by Förster resonance energy transfer (FRET)

A biosensor for cyclin B1/CDK1 activity was recently developed. It consists of a plasmid encoding 2 fluorescent proteins (mCerulean and YPet) linked by a cyclin B1/CDK1 phosphorylation site and a phosphobinding domain25. Phosphorylation induces a conformational change which facilitates energy transfer from mCerulean to YPet. PASMCs were transfected with this FRET biosensor plasmid and mCerulean was exited with a 458 nm Argon laser. Emission was recorded at 465-510nm for mCerulean and 520-555nm for YPet. Increased YPet/mCerulean signal indicates cyclin B1/CDK1 activation25.

Statistics

Values are stated as mean±SEM. Inter-group differences were assessed by an unpaired Student’s t-test, simple or repeated-measures ANOVA with post hoc analysis using Tukey’s test, as appropriate. When applicable, normality was confirmed with a Kolmogorov-Smirnov test. For comparing the number of cyclin E positive cells after Mdivi-1 treatment, a Fisher’s exact test was used. A p<0.05 was considered statistically significant.

Supplemental methods

Human and rat PASMC culture, qRT-PCR, O2-consumption and metabolic measurements, electrophysiology, cytosolic calcium measurements, hemodynamic measurements and histology were performed as previously described4, 26, and details are in the Online Data Supplement.

Results

DRP1-Mediated Mitochondrial Fragmentation in Human PAH PASMCs

Mitochondria are more fragmented in PASMCs from idiopathic PAH patients versus control PASMCs (Figure 1A). Quantitative RT-PCR for the genes that regulate mitochondrial fission and fusion16 shows a profile that favors fission (increased DRP1 and FIS1 and decreased MFN2) (Figure 1B). OPA1 and MFN1 expression are unchanged (Online Figure IA). We focused on the role of DRP1 because preliminary data indicated that Mdivi-1, a small molecule inhibitor of DRP1, substantially rescues the mitochondrial network and slows proliferation.

Figure 1
DRP1 activation causes excessive fission in human PAH PASMCs

Expression of total and activated DRP1 (phosphorylated at Ser616) are upregulated in PAH PASMC (Figure 1C-E). Direct inhibition of CDK1, using 10μM of the specific inhibitor RO-330627, significantly reduces DRP1 Ser616 phosphorylation, implicating CDK1 as the key kinase driving DRP1 activation in PAH. In contrast, inhibition of other putative mediators of DRP1 phosphorylation, such as calmodulin, with 5μM W7, or calcium/calmodulin-dependent protein kinase II, with 20μM KN93, has no effect on DRP1 Ser616 phosphorylation (Figure 1E). Serum withdrawal (like CDK1 inhibition) reduces both the percentage of PASMCs with DRP1 Ser616 phosphorylation and the expression of the G2/mitotic-specific cyclin B1 in PAH PASMCs (Figure 1E-F), indicating that external growth factors/mitogens promote DRP1 activation. Using a FRET biosensor for cyclin B1/CDK1, we confirmed that increased levels of cyclin B1 in PAH PASMCs translate into higher cyclin B1/CDK1 activity (Figure 1G).

Mitochondrial networking, measured using the MNF, is decreased in human PAH PASMC and this is reversed by 25μM Mdivi-1 (Figure 1H). Online Movies I-III show representative 8-minute time-lapse movies of control, PAH, and PAH+Mdivi-1-treated PASMCs immediately prior to and following photoactivation, documenting the more rapid spread of the signal in cells treated with Mdivi-1.

Molecular or Pharmacologic DRP1 Inhibition Blocks Cell Cycle Progression

Compared to control human PASMCs, PAH PASMCs have increased proliferation rates at baseline, confirming the persistence of their hyperproliferative phenotype in vitro. DRP1 inhibition by Mdivi-1 causes a dose-dependent reduction in proliferation rates (Figure 2A). Mdivi-1’s antiproliferative effects are due to induction of cell-cycle arrest in the G2/M phase (Figure 2B and Online Figure IIA), suggesting that cell-cycle progression is dependent on fission of mitochondria. To exclude non-specific effects of Mdivi-1, we used siRNA directed against DRP1 and found a similar reduction in proliferation (Figure 2C) due to accumulation of PASMCs at the G2/M phase of the cell cycle (Figure 2D). To more exactly establish at which point the cell cycle is interrupted, we used immunoblotting to assess whether CDK1 activation is altered by Mdivi-1. CDK1 activation during mitosis depends on dephosphorylation of Thr14 and Tyr1528. This dephosphorylation is not impaired by Mdivi-1 (Figure 2E), suggesting that cell cycle arrest occurs specifically in prometaphase (between late G2 and metaphase) since this is the last phase in which cells have dephosphorylated CDK1. Subsequently, CDK1 becomes phosphorylated/inactivated due to proteosomal degradation of cyclin B129 and this was not observed with Mdivi-1. Mdivi-1-induced G2/M arrest is reminiscent of the state observed with cell synchronization by the microtubule assembly inhibitor, nocodazole. Therefore, we synchronized PASMCs in prometaphase with 50ng/ml nocodazole for 16-hours. Synchronization was confirmed by an increase in cyclin B1-positive PASMCs (Figure 2F). After removal of nocodazole, PAH cells rapidly move through mitosis, evident in parallel decreases in cyclin B1 levels and DNA content; however, Mdivi-1 prevents decreases in cyclin B1 levels (Figure 2F), confirming that Mdivi-1 specifically blocks cell cycle progression beyond prometaphase in PAH PASMCs.

Figure 2
DRP1 inhibition reduces proliferation by causing G2/M arrest

Mdivi-1 binds to an allosteric site on DRP1 and thereby blocks a conformational change required for self-assembly19. Since self-assembly is downstream of DRP1 Ser616 phosphorylation, Mdivi-1 should not decrease DRP1 Ser616 phosphorylation. Indeed, Mdivi-1 actually increases DRP1 Ser616 phosphorylation (Online Figure IB-C), consistent with the observed increased CDK1 activation in response to Mdivi-1 (Figure 2E). Thus Mdivi-1 is not interfering with CDK1-mediated phosphorylation of DRP1 Ser616. Recently, Mitra et al. described increased levels of the S-phase promoting cyclin E in response to Mdivi-1 (which we confirmed, Online Figure IIB)30. They also noted increased entry into S-phase, which supports our finding that Mdivi-1 inhibits proliferation mainly by inhibiting a G2/M checkpoint in the cell cycle.

DRP1 Activation in Human PAH Pulmonary Arteries

We obtained human lung tissue from 5 autopsied idiopathic PAH patients and 5 age-matched controls (patient characteristics in Online Table I) and confirmed the presence of plexiform lesions and increased pulmonary artery muscularization (Figure 3A). Plexiform lesions and muscularized pulmonary arteries from idiopathic PAH patients have 10±7% and 12±3% increases in DRP1 expression, respectively compared to arteries from normal subjects (Online Figure IIIA). DRP1 Ser616 is clearly enriched in pulmonary arteries (versus lung parenchyma) in PAH and is greater in PAH versus control lungs (Figure 3B), consistent with the PASMC data (Figure 1D-E). Moreover, the G2/mitotic-specific cyclin B1 is not only upregulated in PAH PASMCs, but also activated, evident from its nuclear translocation (Figure 3C), which is in line with the increased cyclin B1/CDK1 activity measured in PAH PASMCs using a FRET biosensor (Figure 1G). Increased cyclin B1/CDK1 directly leads to DRP1 Ser616 phosphorylation31. While endothelial cells stain strongly for the inhibitory Ser637 form of DRP1, we did not detect this form of DRP1 in the media of control or PAH lungs (Online Figure IIIB) and therefore focused on DRP1 Ser616 in subsequent experiments.

Figure 3
Activated DRP1 is present in remodeled pulmonary arteries of idiopathic PAH patients

HIF-1α Activation Increases Proliferation and Glycolysis in Human PAH PASMCs

We confirmed that HIF-1α activation occurs in PASMCs in lungs from PAH but not control patients (Figure 4A). Even when PAH PASMCs are cultured in vitro at oxygen concentrations of ~140mmHg, most have HIF-1α activation, evident from its nuclear translocation, greatly exceeding the rate in control PASMCs (Figure 4B). Metabolically, HIF-1α activation increased glycolysis, as measured with a Seahorse Bioscience XF24 analyzer (Online Data Supplement). Compared to control PASMCs, we observed a more than 3-fold higher lactate production rate and a 2-fold lower ratio of oxygen consumption/lactate production in PAH versus control human PASMCs (Online Figure IVA). The upregulated glycolysis is also evident in increased gene expression of GLUT1, PDK2 and PDK4 (Online Figure IVB).

Figure 4
HIF-1α is activated in lungs and PASMCs from PAH patients which is necessary for proliferation and leads to mitochondrial fragmentation

We next examined the consequences of HIF-1α activation on proliferation and metabolic function in human PASMCs. The importance of HIF-1α activation for proliferation was studied using chetomin, a small molecule that inhibits the interaction of HIF-1α with its p300 coactivator, preventing hypoxia-inducible gene transcription32. Chetomin caused minimal cell death (Figure 4C) consistent with the low doses (5-50nM) we used, relative to those reported in the literature33, 34. Chetomin dose-dependently inhibited proliferation (Figure 4D) and DRP1 Ser616 phosphorylation (Figure 4E).

HIF-1α Activation in Normal PASMCs Recapitulates the PAH Phenotype and Induces Mitochondrial Fragmentation

We used two chemically discrete HIF-1α activators (CoCl and desferrioxamine)35 2 to establish a direct relationship between normoxic HIF-1α activation and mitochondrial fragmentation in normal rat PASMCs. This approach avoids confounders, such as genetic abnormalities and upregulation of growth factor pathways that might exist in human PAH cells. CoCl2 activated HIF-1α in most PASMCs (evident from nuclear accumulation of HIF-1α, Online Figure VA). Whole-cell patch clamp traces show a reduction in voltage-gated K+ currents (Kv) with CoCl2, which is rescued with a HIF-1α dominant-negative virus (HIF-1α DN, Online Figure VIA-B). The reduced Kv currents lead to depolarization of the PASMC (lower EM, Online Figure VIC) and increased cytosolic calcium levels (Online Figure VID). Both abnormalities can be prevented by overexpression of a HIF-1α DN construct, confirming the role of HIF-1α in the current inhibition caused by ex vivo CoCl2 treatment. These HIF-1α-induced electrophysiologic changes recapitulate those seen in PAH PASMCs7, 36-39.

HIF-1α-Dependence of Cobalt-Induced Mitochondrial Fission

CoCl2 (500μM) rapidly induces mitochondrial fission, again mimicking PAH (Figure 4F). To exclude nonspecific effects of the chemical HIF-1α activators, we first showed that 50μg/ml cycloheximide, an inhibitor of protein translation, prevents cobalt or desferrioxamine-induced mitochondrial fragmentation. This indicates that protein synthesis is required for HIF-1α-induced fission (Figure 5A-B). Next we showed that HIF-1α siRNA largely prevents cobalt-induced fission, proving the fragmentation is indeed due to HIF-1α activation (Figure 5C). Efficacy of HIF-1α siRNA is demonstrated in Online Figure VB-C. Finally, we showed that, cobalt increases the phosphorylation of DRP1 Ser616 (Figure 5D). Thus cobalt-induced HIF-1α activation mimics the PAH phenotype described in Figure 1D.

Figure 5
Fragmentation of mitochondria by cobalt is dependent on HIF-1α and DRP1 activation

Mdivi-1 Reverses HIF-1α-Induced Mitochondrial Fragmentation

We used Mdivi-1 to confirm that the mitochondrial fragmentation observed after CoCl2-induced HIF-1α activation is dependent on DRP1. Mdivi-1 treatment (25μM) not only normalizes mitochondrial morphology after CoCl2 treatment (Figure 5E-F) but also enhances networking, as demonstrated by the more rapid and broader spread of photoactivated, mitochondrial matrix-targeted GFP (Figure 5E-G). This is concordant with the effects of Mdivi-1 in human PAH PASMCs (Figure 1H).

Chronic Treatment with CoCl2 Causes Experimental PAH

Since cobalt recapitulates many of the features of PAH PASMCs in culture, we determined whether activation of HIF-1α using cobalt would cause PAH in vivo. As expected, chronic cobalt treatment increased hematocrit, confirming that the selected dose of CoCl2 was biologically active (Figure 6A). Moreover, cobalt decreased maximal walking distance (Figure 6B). Echocardiography confirmed that the pulmonary artery acceleration time (PAAT), a measure of vascular compliance that is inversely related to pulmonary artery pressures26, decreases significantly after cobalt treatment (Figure 6C, Online Figure VIIA), while right and left heart catheterization confirmed that cobalt increased pulmonary vascular resistance (Figure 6D-F, Online Figure VIIB-C). Cobalt-induced PAH is not caused by left ventricular dysfunction as the left ventricular end diastolic pressures did not change between groups (Figure 6F). Post mortem analysis revealed increasedright ventricular hypertrophy and percentage medial thickness of small pulmonary arteries (Figure 6G-I).

Figure 6
Inhibition of DRP1 using Mdivi-1 prevents cobalt-induced PAH in vivo

DRP1 Inhibition Attenuates Pulmonary Hypertension in vivo

To determine whether inhibition of mitochondrial fission in vivo would attenuate cobalt-induced PAH, we co-injected 50mg/kg Mdivi-1 (or the DMSO vehicle) on a weekly basis in rats receiving chronic CoCl2 injections. Exercise capacity, which is reduced in cobalt-induced PAH, is normalized by Mdivi-1 treatment (Figure 6B). The PAAT increases significantly after Mdivi-1 treatment (Figure 6C, Online Figure VIIA). Similarly, Mdivi-1 significantly decreases PVR (Figure 6D). Mdivi-1 treatment decreases right ventricular hypertrophy (Figure 6G) and the percentage medial thickness of small pulmonary arteries, consistent with an antiproliferative effect (Figure 6H-I).

Mitochondrial Fragmentation in vivo in Response to CoCl2

To prove that Mdivi-1’s antiproliferative effects in vivo reflected inhibition of fission, we performed transmission electron microscopy studies on pulmonary arteries isolated from control rats and rats receiving CoCl2 or CoCl2+Mdivi-1. These images demonstrate that mitochondrial fragmentation is present in vivo in the cobalt PAH model and that these morphologic changes are reversed with Mdivi-1 (Figure 6J).

Mdivi-1 Attenuates the Development of Chronic Hypoxia Induced Pulmonary Hypertension

We also studied whether similar benefits of Mdivi-1 therapy could be observed in additional experimental models of PAH. As shown in Figure 7A, Mdivi-1 prevents the decrease in exercise capacity caused by chronic hypoxia-induced pulmonary hypertension. Echocardiography confirms that Mdivi-1 improved PAAT (consistent with a reduction in pulmonary artery pressure) and TAPSE (indicating improved RV function), while reducing right ventricular hypertrophy (Figure 7A). Mdivi-1 also has anti-proliferative effects in the chronic hypoxia model. In line with our previous findings, we observed decreased muscularization and fewer proliferating PASMCs in rats treated with Mdivi-1 (Figure 7B-D).

Figure 7
Therapeutic benefit of Mdivi-1 in the chronic hypoxia model

Mdivi-1 Reduces Proliferation in the Monocrotaline Model of PAH

Having shown that Mdivi-1 can attenuate the development of CoCl2 and chronic hypoxia-induced PAH, we tested Mdivi-1 as regression therapy in the monocrotaline model. More specifically, we wanted to test whether inhibiting fission reduces PASMC proliferation. Daily Mdivi-1 treatment was started 3 weeks after monocrotaline injection, when PAH is already established (confirmed by echocardiography), and continued for 5 days. This relative short treatment significantly improved TAPSE, and there were trends towards improved PAAT and reduced right ventricular hypertrophy, although neither achieved statistical significance (Online Figure VIIIA). In addition, muscularization of small pulmonary arteries was significantly decreased by Mdivi-1 and there was a pronounced decrease in PASMC proliferation (Online Figure VIIIB). HIF-1α activation was also present in the MCT model (Online Figure VIIIC).

Discussion

We demonstrate that the hyperproliferative diathesis of human PAH PASMCs reflects increased DRP1 expression and activation (Figures 1--2).2). The molecular mechanism of DRP1 activation in human PAH patients is increased DRP1 Ser616 phosphorylation (Figures 1D-E and and3B),3B), reflecting increased activation of DRP1’s regulatory kinase (cyclin B1/CDK1). DRP1 Ser616 phosphorylation and fission can be induced in normal PASMCs by chemically activating HIF-1α. The relevance of HIF-1α-induced DRP-1 activation to PAH is evident in the discovery that cobalt-induced HIF-1α activation elicits PAH in normal rats and that PAH is attenuated by Mdivi-1. Notably, the hemodynamic benefits were associated with restoration of mitochondrial fusion in vivo (Figure 6J). The antiproliferative effects of blocking mitotic fission are also evident in two well-established models of pulmonary hypertension. By locking the mitochondria in fusion, Mdivi-1 and siDRP1 prevent mitotic fission and cause cell-cycle arrest in the G2/M phase (Figure 2A-D), which is consistent with the recent demonstration that inhibition of DRP1 regresses lung cancer cell xenografts20. Thus excessive fission, which occurs in human PAH PASMCs in vitro and in vivo, contributes to the disease by driving proliferation and can be therapeutically targeted.

Mitochondrial morphology reflects the balance of fission and fusion. Fusion merges the contents of adjoined mitochondria, creating a homogenous network of elongated mitochondria allowing the redistribution of mitochondrial DNA and proteins40. Conversely, fission is necessary during cell division to evenly distribute mitochondria between daughter cells31. Fission occurs when DRP1 is recruited from the cytoplasm to the mitochondrial surface where it forms oligomeric complexes that hydrolyse GTP and mechanically constrict the mitochondria causing fragmentation41

Taguchi et al. showed that the interconnected mitochondrial. network structures in interphase HeLa cells become fragmented in the early mitotic phase via cyclin B1/CDK1-mediated DRP1 phosphorylation31. In the current study we show that in human PAH there is both an upregulation of total and Ser616 phosphorylated DRP1. The pro-fission phenotype is seen in lungs from patients with PAH and persists in human PAH PASMCs in culture (Figure 1D-E, Figure 3B). The elevation of cyclin B1 and CDK1, which coordinate mitosis and mitotic fission, is a major stimulus for Ser616 phosphorylation in PAH (Figure 1F-G and Figure 3C). Cell proliferation is driven by many factors in PAH, including elevated levels of mitogens and growth factors (reviewed in 6) and mutations of the bone morphogenetic protein receptor (in familial PAH)42.

In carefully controlled experiments, using molecular tools including siHIF-1α and a HIF-1α dominant-negative virus, we showed that HIF-1α activation is sufficient to confer a PAH phenotype on previously normal PASMCs (Online Figure VI) and induce mitochondrial fission (Figure 5). The increased HIF-1α in human PAH lungs (Figure 4A) persists in culture, despite PASMCs being cultured at ambient oxygen concentrations (~140mmHg). This is consistent with the recently described epigenetic suppression of mitochondrial superoxide dismutase, which is an upstream, heritable cause of the HIF-1α activation and proliferation in human and fawn-hooded rat PAH that persist in cells in culture4. In addition, normoxic HIF-1α activation can be induced in aortic SMC by growth factors such as serotonin and PDGF-BB43.

HIF-1α activation causes a glycolytic shift that confers a proliferative advantage in PASMCs. Similar HIF-1α-dependent glycolytic metabolic changes also occur in the endothelial cells of PAH patients8, 44. Proof that HIF-1α underlies the proliferation advantage comes from experiments in which HIF-1α inhibition by chetomin32 reduces PASMC proliferation (Figure 4D). Activation of HIF-1α results in glycolytic metabolic changes which can be detected using lung FDG-PET scans. These glycolytic changes occur simultaneously with the development of pulmonary hypertension after monocrotaline administration and appear to be related to increased proliferation15. Reversing the glycolytic phenotype with dichloroacetate also regresses experimental PAH15. Our findings are consistent with the observations that HIF-1α haploinsufficient mice are relatively protected from chronic hypoxic pulmonary hypertension45 whilst HIF-1α activation increases proliferation of human PASMC in response to platelet-derived growth factor46. These data provide compelling evidence that HIF-1α activation contributes to disease progression in human PAH.

While CoCl2 clearly induces HIF-1α in PASMCs (Online Figure VA), it is possible that non-specific effects of cobalt contribute to our observations. Indeed gene expression arrays in fibroblasts and carcinoma cells have shown that CoCl2 and HIF-1α induce a common set of genes, but some genes are specifically induced by cobalt47, 48. Yet, desferrioxamine, another HIF-1α activator35, similarly leads to mitochondrial fragmentation (Figure 5A) and conversely, siRNA-mediated knockdown of HIF-1α prevents CoCl2-induced mitochondrial fragmentation (Figure 5C), indicating that mitochondrial fragmentation depends on HIF-1α activation. Moreover, changes in K+ currents, membrane potential, and cytoplasmic calcium concentrations induced by CoCl2 are almost completely negated by adenoviral overexpression of a HIF-1α dominant negative construct (Online Figure VI), again suggesting that in PASMCs most effects of CoCl2 are HIF-1α mediated. In addition, in vivo, CoCl2 will induce HIF-1α in other tissues, as exemplified by increased hematocrit which is dependent on renal erythropoietin production. Despite possible non-specific effects, the net result of chronic CoCl2 administration is pulmonary vascular remodeling (Figure 6).

We acknowledge that while Mdivi-1 clearly improves mitochondrial networking both in human (Figure 1H) and rat PASMCs (Figure 5E) and in vivo (Figure 6J), it could potentially have off-target effects. However, knockdown of DRP1 expression by using a specific siRNA causes a similar arrest of PAH PASMC in the G2/M phase and has the same antiproliferative effects as Mdivi-1 on PAH PASMC (Figure 2C-D). Since Mdivi-1 is currently the only commercially available small molecule inhibitor of mitochondrial fragmentation, the development of other inhibitors will be necessary to confirm the specificity of our findings. Even though DRP1 knockout mice have been generated, they die around embryonic day 12.5, precluding their use to study the role of DRP1 in the development of PAH49.

In patient-derived PAH PASMCs as well as cobalt treated PASMCs, Mdivi-1 prevents mitochondrial fragmentation and enhances fusion (Figure 1H and 5E-G). Mdivi-1 or knockdown of DRP1 prevent proliferation in human PAH PASMCs (Figure 2A,C). The notion that mitochondrial fragmentation is essential for cell cycle progression past the G2/M checkpoint is an important and possibly controversial discovery. In support of our findings, gene therapy with Mitofusin-2, which causes mitochondrial fusion, also slows SMC proliferation in systemic arterial injury50. Moreover, DRP1 activation has recently been shown to be crucial to the hyperproliferative phenotype in lung cancer and Mdivi-1 is therapeutic in regressing tumors in a xenotransplantation model20. More study is required to determine whether mitotic division of organelles (not just mitochondria) is relevant to proliferative disorders. Supporting this notion, blocking Golgi fragmentation prevents progression through mitosis51. Interestingly, CDK1 is involved in controlling Golgi fragmentation as well52.

Electron microscopy confirms that CoCl2 induces mitochondrial fragmentation in vivo (Figure 6J), and chronic cobalt administration leads to pulmonary hypertension in line with previous publications that describe its ability to increase hematocrit, pulmonary hypertension and right ventricular hypertrophy in rats53 and right ventricular hypertrophy in broiler chickens54. Importantly, Mdivi-1 has already been shown to protect against ischemia-induced kidney damage and cardiac ischemia-reperfusion injury21, 55. For the first time, we used Mdivi-1 for long-term treatment (during the 4 weeks of CoCl2 administration) and observed that it is well tolerated and significantly improves exercise capacity and reduces PVR and right ventricular hypertrophy (Figure 6B-G). Moreover, muscularization of the pulmonary small blood vessels is attenuated by Mdivi-1 treatment, consistent with its antiproliferative effects on PAH PASMCs (Figure 6H-I). We only looked at the effect of Mdivi-1 on PASMC proliferation and it is possible that Mdivi-1 could influence other cell types as well. However, we did observe increased hematocrit in CoCl2 rats treated with Mdivi-1, indicating that hematopoietic stem cell proliferation is not or only minimally impaired. Moreover, impaired intestinal stem cell proliferation would result in impaired nutrient absorption and weight loss, which we did not observe. After intraperitoneal injection, Mdivi-1 is taken up in the mesenteric vein and transported to the right ventricle and subsequently to the pulmonary circulation. Therefore, Mdivi-1 concentrations are expected to be higher in the pulmonary vascular bed compared to other vascular beds. A similar mechanism is responsible for the relative pulmonary specificity of monocrotaline-induced pulmonary hypertension. Future pharmacological studies are necessary to evaluate tissue distribution and retention times of Mdivi-1. Mdivi-1 reduces PASMC proliferation in both monocrotaline and chronic hypoxic pulmonary hypertension (Figure 7 and Online Figure VIII). Therefore, it is reasonable to anticipate that Mdivi-1 is more effective in preventing than in reversing PAH. Thus inhibition of mitochondrial fission may have broad therapeutic benefit in preventing disease progression in various forms of PAH. Future research is necessary to elucidate the role of FIS1 and MFN-2 in the hyperproliferative phenotype of PAH.

In conclusion, HIF-1α stimulated, DRP1-mediated mitochondrial fission leads to increased proliferation in human PAH PASMCs. This is the first direct evidence that an acquired abnormality of mitochondrial fission contributes to the maintenance of human PAH and demonstrates the feasibility of targeting mitochondrial dynamics as a therapeutic option for PAH. Taken together, our data indicate that mitotic fission is required for rapid proliferation and conversely, that locking the mitochondria in a networked state, prevents mitotic fission causing cell cycle arrest. A schematic representation of our conclusions Is provided in Figure 8. Our findings have great therapeutic potential in PAH, evidenced by the effective and well-tolerated response to Mdivi-1 in the human PAH PASMC and several experimental models of pulmonary hypertension (Figures 6--77 and Online Figure VIII). This work also demonstrates an important interaction between the mitochondrial cycle and the cell cycle, which can perhaps be exploited in other proliferative disorders, such as cancer.

Figure 8
Schematic representation of how the cell and mitochondrial cycles interact

Novelty and Significance

What is known?

  • Excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) contributes to the development of pulmonary arterial hypertension (PAH).
  • Mitochondria form a dynamic network which is controlled by a balance between fusion and fission, and mitochondria in PASMCs from PAH patients are excessively fragmented.
  • Normoxic activation of hypoxia-induced signaling pathways and the transcription factor hypoxia-inducible factor 1α (HIF-1α occur in PAH PASMCs.

What new information does this article contribute?

  • Mitochondrial fragmentation in PAH PASMCs reflects increased fission resulting largely from activation of the GTPase dynamin-related protein 1 (DRP1) which upon activation translocates to the mitochondria, multimerizes, and causes fission.
  • A fragmented mitochondrial network can be induced by activation of HIF-1α.
  • Mitochondrial fragmentation is crucial for PAH PASMC proliferation because inhibiting mitochondrial fragmentation arrests PAH PASMCs in the G2/M phase of the cell cycle and thus slows down proliferation.

Supplementary Material

1

Online Movie I: Control PASMCs were cotransfected with mitochondrial matrix-targeted DsRed and mitochondrial matrix-targeted photoactivatable GFP. Upon photoactivation, GFP spreads throughout fused mitochondria. Images were obtained during 8 minutes following photoactivation.

2

Online Movie II: Photoactivation experiment in PAH PASMC.

3

Online Movie III: Photoactivation experiment in PAH PASMC treated with 25μM Mdivi-1.

4

Online Figure I. Expression levels of additional mitochondrial fusion mediators and Mdivi-1 does not inhibit phosphorylation of DRP1 Ser 616.

A) Mitofusin-1 (MFN1) and Optic atrophy-1 (OPA1) are proteins involved in mitochondrial fusion and are present in the outer and inner mitochondrial membrane, respectively. Their expression levels were not changed between control and PAH human PASMCs.

B-C) Mdivi-1 does not prevent cyclin B1/CDK1 mediated phosphorylation of DRP1 at Ser616. Immunocytochemistry (B) and immunoblotting (C) demonstrated increased Ser616 phosphorylation, in agreement with the increased CDK1 activation in response to Mdivi-1.

Online Figure II. Cell cycle analysis after Mdivi-1 treatment of PAH PASMCs and increased cyclin E expression after Mdivi-1 treatment.

A) Human PASMCs were incubated for 1 hour with the thymidine analog 5-ethynyl-2’-deoxyuridine (EdU) to assess proliferation and 7-Amino-Actinomycin D (7-AAD) was used to determine DNA content. The percentage of cells in the G2/M phase of the cell cycle (no EdU incorporation, more than diploid DNA content) increased in response to 25μM Mdivi-1.

B) Increased percentage of PASMCs are positive for nuclear cyclin E staining after Mdivi-1 treatment, asterisks point at PASMCs with nuclear cyclin E staining. The nuclear intensity in approximately 60 nuclei was analyzed in each group. Based on the no antibody control, nuclei with a mean relative fluorescence intensity >1200 were considered positive. Scale bar = 100μm.

Online Figure III. Immunostaining for DRP1 and DRP1 phosphorylated at Ser637 of human lungs.

A) Immunofluorescent staining for DRP1 in human lung sections. Double staining for DRP1 (green) and smooth muscle cell actin (red) allows identification of the smooth muscle cell layer and permitted quantification of DRP1 staining in the PASMCs. PASMCs in PAH lungs have a 12±3% increased DRP1 staining compared to PASMCs in control lungs. Approximately 50 blood vessels were analyzed per group. Scale bar = 100μm.

B) Immunohistochemistry for DRP1 phosphorylation at Ser637. When phosphorylated at this Serine amino acid, DRP1 is inactivated. While there is strong staining in endothelial cells and inflammatory cells, we could not detect Ser637 phosphorylation in small precapillary resistance PASMCs of control or PAH lungs. Scale bar = 50μm.

Online Figure IV. Glycolytic shift in PAH PASMCs.

A) PAH PASMCs have increased lactate production and a decreased oxygen consumption/lactate production ratio as measured using the Seahorse analyzer. This suggests that PAH PASMCs generate a larger proportion of their ATP from glycolysis compared to control PASMCs.

B) This glycolytic switch is confirmed by the upregulation of glucose transporter 1 (GLUT1), and pyruvate dehydrogenase kinases (PDK) 2 and 4.

Online Figure V. Confirmation of HIF-1α induction by CoCl2 and of the effectiveness of siRNA mediated HIF-1α knockdown.

A) CoCl2 leads to a strong induction and nuclear accumulation of HIF-1α in the majority of PASMCs.

B) siRNA mediated HIF-1α downregulation prevents HIF-1α accumulation in response to CoCl2. Arrowheads point to examples of nuclei without HIF-1α, while asterisks point at nuclei with HIF-1α accumulation. There is a clear reduction in HIF-1α signal intensity when cells are pretreated with siHIF-1α. Scale bar = 100μm.

C) The glucose transporter 1 (GLUT1) is upregulated in response to HIF-1α and siRNA against HIF-1α prevent this increase, confirming the efficacy of this HIF-1α inhibition strategy.

Online Figure VI. HIF-1α recapitulates the features described for PAH PASMCs.

A-B) Representative patch clamp traces show a reduction in voltage-gated potassium (Kv) currents after chronic (24 hours) cobalt treatment of rat PASMCs. This is rescued with a HIF-1α dominant-negative virus (HIF-1α DN, n=4). The 4-aminopyridine (4-AP)-sensitive current is diminished in cobalt-treated cells, demonstrating decreased Kv currents. * P < 0.05 versus control.

C-D) Chronic (24 hours) cobalt treatment depolarizes cells (lower membrane potential, n=4) and increases cytosolic calcium concentrations ([Ca2+]cyt). Both effects are prevented by the HIF-1α DN virus (n=11).

Online Figure VII. Echocardiography and catheterization measurements in CoCl2/Mdivi-1 treated animals.

A) Representative pulse wave Doppler tracings of the pulmonary artery. These traces were used to measure the pulmonary artery acceleration time (PAAT).

B-C) Representative catheterization traces of the pulmonary artery (B) and the right ventricle (C). The mean pulmonary artery pressure was used to calculate pulmonary vascular resistance. These traces were obtained using a 22 gauge fluid-filled catheter in anesthetized, open-chest rats.

Online Figure VIII. Therapeutic benefit of Mdivi-1 in the chronic hypoxia model.

A) Rats were injected with monocrotaline and 3 weeks later, when pulmonary hypertension is present, we started daily treatment for 5 days with Mdivi-1. We found that Mdivi-1 improves functional capacity measured on a treadmill. There is a trend for improved pulmonary artery acceleration time (PAAT) and decreased right ventricular fractional weight, while there is a significant improvement in tricuspid annular plane systolic excursion (TAPSE).

B) Rats injected with monocrotaline develop pulmonary hypertension characterized by excessive PASMC proliferation. Mdivi-1 both reduces the degree of muscularization of the pulmonary arteries and the number proliferating cell nuclear antigen (PCNA) positive PASMCs. Scale bar = 50μm

C) We observed nuclear accumulation of HIF-1α in PASMCs of monocrotaline-treated rats. Scale bar = 50μm.

Online Table I. Characteristics of patients of which lung tissue was studied by immunohistochemistry

Acknowledgments

Funding Sources

Dr. Archer is supported by NIH-RO1-HL071115, 1RC1HL099462-01, and the American Heart Association. Dr. Rehman is supported by NIH-R01-GM094220.

Non-standard Abbreviations and Acronyms

BMPR-2
Bone morphogenetic protein receptor-2
CDK1
Cyclin-dependent kinase 1
DRP1
Dynamin-related protein-1
FDG-PET
18F-fluoro-deoxy-glucose positron emission tomography
FIS1
Fission-1
FRET
Förster resonance energy transfer
GLUT1
Glucose transporter-1
HIF-1α
Hypoxia-inducible factor-1α
HIF-1α DN
HIF-1α dominant negative adenovirus
Kv
Voltage-gated K+ channel
Mdivi-1
Mitochondrial division inhibitor-1
MFC
Mitochondrial fragmentation count
MFN1
Mitofusin-1
MFN2
Mitofusin-2
Mito-DsRed
Mitochondrial matrix-targeted DsRed
Mito-GFP
Mitochondrial matrix-targeted GFP
Mito-PA-GFP
Mitochondrial matrix-targeted photoactivatable GFP
MNF
Mitochondrial networking factor
OPA1
Optic atrophy-1
PA
Pulmonary artery
PAH
Pulmonary arterial hypertension
PASMC
Pulmonary artery smooth muscle cell
PDK
Pyruvate dehydrogenase kinase
PVR
Pulmonary vascular resistance
SOD2
Superoxide dismutase 2

Footnotes

Disclosures

None

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

1. Lane KB, Machado RD, Pauciulo MW, Thomson JR, Phillips JA, 3rd, Loyd JE, Nichols WC, Trembath RC. Heterozygous germline mutations in bmpr2, encoding a tgf-beta receptor, cause familial primary pulmonary hypertension. Nat Genet. 2000;26:81–84. [PubMed]
2. Thomson JR, Machado RD, Pauciulo MW, Morgan NV, Humbert M, Elliott GC, Ward K, Yacoub M, Mikhail G, Rogers P, Newman J, Wheeler L, Higenbottam T, Gibbs JS, Egan J, Crozier A, Peacock A, Allcock R, Corris P, Loyd JE, Trembath RC, Nichols WC. Sporadic primary pulmonary hypertension is associated with germline mutations of the gene encoding bmpr-ii, a receptor member of the tgf-beta family. J Med Genet. 2000;37:741–745. [PMC free article] [PubMed]
3. Aldred MA, Comhair SA, Varella-Garcia M, Asosingh K, Xu W, Noon GP, Thistlethwaite PA, Tuder RM, Erzurum SC, Geraci MW, Coldren CD. Somatic chromosome abnormalities in the lungs of patients with pulmonary arterial hypertension. Am J Respir Crit Care Med. 2010;182:1153–1160. [PMC free article] [PubMed]
4. Archer SL, Marsboom G, Kim GH, Zhang HJ, Toth PT, Svensson EC, Dyck JR, Gomberg-Maitland M, Thebaud B, Husain AN, Cipriani N, Rehman J. Epigenetic attenuation of mitochondrial superoxide dismutase 2 in pulmonary arterial hypertension: A basis for excessive cell proliferation and a new therapeutic target. Circulation. 2010;121:2661–2671. [PMC free article] [PubMed]
5. Thenappan T, Shah SJ, Rich S, Tian L, Archer SL, Gomberg-Maitland M. Survival in pulmonary arterial hypertension: A reappraisal of the nih risk stratification equation. Eur Respir J. 2010;35:1079–1087. [PubMed]
6. Archer SL, Weir EK, Wilkins MR. Basic science of pulmonary arterial hypertension for clinicians: New concepts and experimental therapies. Circulation. 2010;121:2045–2066. [PMC free article] [PubMed]
7. Bonnet S, Michelakis ED, Porter CJ, Andrade-Navarro MA, Thebaud B, Haromy A, Harry G, Moudgil R, McMurtry MS, Weir EK, Archer SL. An abnormal mitochondrial-hypoxia inducible factor-1alpha-kv channel pathway disrupts oxygen sensing and triggers pulmonary arterial hypertension in fawn hooded rats: Similarities to human pulmonary arterial hypertension. Circulation. 2006;113:2630–2641. [PubMed]
8. Fijalkowska I, Xu W, Comhair SA, Janocha AJ, Mavrakis LA, Krishnamachary B, Zhen L, Mao T, Richter A, Erzurum SC, Tuder RM. Hypoxia inducible-factor1alpha regulates the metabolic shift of pulmonary hypertensive endothelial cells. Am J Pathol. 2010;176:1130–1138. [PubMed]
9. Wang GL, Jiang BH, Semenza GL. Effect of altered redox states on expression and DNA-binding activity of hypoxia-inducible factor 1. Biochem Biophys Res Commun. 1995;212:550–556. [PubMed]
10. Huang LE, Arany Z, Livingston DM, Bunn HF. Activation of hypoxia-inducible transcription factor depends primarily upon redox-sensitive stabilization of its alpha subunit. J Biol Chem. 1996;271:32253–32259. [PubMed]
11. Kim JW, Tchernyshyov I, Semenza GL, Dang CV. Hif-1-mediated expression of pyruvate dehydrogenase kinase: A metabolic switch required for cellular adaptation to hypoxia. Cell Metab. 2006;3:177–185. [PubMed]
12. Michelakis ED, Dyck JR, McMurtry MS, Wang S, Wu XC, Moudgil R, Hashimoto K, Puttagunta L, Archer SL, McMurtry MS, Bonnet S, Wu X, Dyck JR, Haromy A, Hashimoto K, Michelakis ED. Gene transfer and metabolic modulators as new therapies for pulmonary hypertension. Increasing expression and activity of potassium channels in rat and human models. Dichloroacetate prevents and reverses pulmonary hypertension by inducing pulmonary artery smooth muscle cell apoptosis. Adv Exp Med Biol. Circ Res. 2001;2004;50295:401–418.
14. Piao L, Fang YH, Cadete VJ, Wietholt C, Urboniene D, Toth PT, Marsboom G, Zhang HJ, Haber I, Rehman J, Lopaschuk GD, Archer SL. The inhibition of pyruvate dehydrogenase kinase improves impaired cardiac function and electrical remodeling in two models of right ventricular hypertrophy: Resuscitating the hibernating right ventricle. J Mol Med. 2010;88:47–60. [PMC free article] [PubMed]
15. Marsboom G, Wietholt C, Haney CR, Toth PT, Ryan JJ, Morrow E, Thenappan T, Bache-Wiig P, Piao L, Paul J, Chen CT, Archer SL. Lung 18f-fluorodeoxyglucose positron emission tomography for diagnosis and monitoring of pulmonary arterial hypertension. American journal of respiratory and critical care medicine. 2012;185:670–679. [PMC free article] [PubMed]
16. Westermann B. Mitochondrial fusion and fission in cell life and death. Nat Rev Mol Cell Biol. 2010;11:872–884. [PubMed]
17. Chang CR, Blackstone C. Cyclic amp-dependent protein kinase phosphorylation of drp1 regulates its gtpase activity and mitochondrial morphology. J Biol Chem. 2007;282:21583–21587. [PubMed]
18. Cribbs JT, Strack S. Reversible phosphorylation of drp1 by cyclic amp-dependent protein kinase and calcineurin regulates mitochondrial fission and cell death. EMBO Rep. 2007;8:939–944. [PubMed]
19. Cassidy-Stone A, Chipuk JE, Ingerman E, Song C, Yoo C, Kuwana T, Kurth MJ, Shaw JT, Hinshaw JE, Green DR, Nunnari J. Chemical inhibition of the mitochondrial division dynamin reveals its role in bax/bak-dependent mitochondrial outer membrane permeabilization. Dev Cell. 2008;14:193–204. [PMC free article] [PubMed]
20. Rehman J, Zhang HJ, Toth PT, Zhang Y, Marsboom G, Hong Z, Salgia R, Husain AN, Wietholt C, Archer SL. Inhibition of mitochondrial fission prevents cell cycle progression in lung cancer. The FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 2012 [PubMed]
21. Brooks C, Wei Q, Cho SG, Dong Z. Regulation of mitochondrial dynamics in acute kidney injury in cell culture and rodent models. J Clin Invest. 2009;119:1275–1285. [PMC free article] [PubMed]
22. Karbowski M, Lee YJ, Gaume B, Jeong SY, Frank S, Nechushtan A, Santel A, Fuller M, Smith CL, Youle RJ. Spatial and temporal association of bax with mitochondrial fission sites, drp1, and mfn2 during apoptosis. J Cell Biol. 2002;159:931–938. [PMC free article] [PubMed]
23. Choi SY, Huang P, Jenkins GM, Chan DC, Schiller J, Frohman MA. A common lipid links mfn-mediated mitochondrial fusion and snare-regulated exocytosis. Nat Cell Biol. 2006;8:1255–1262. [PubMed]
24. Chen J, Zhao S, Nakada K, Kuge Y, Tamaki N, Okada F, Wang J, Shindo M, Higashino F, Takeda K, Asaka M, Katoh H, Sugiyama T, Hosokawa M, Kobayashi M. Dominant-negative hypoxia-inducible factor-1 alpha reduces tumorigenicity of pancreatic cancer cells through the suppression of glucose metabolism. Am J Pathol. 2003;162:1283–1291. [PubMed]
25. Gavet O, Pines J. Progressive activation of cyclinb1-cdk1 coordinates entry to mitosis. Developmental cell. 2010;18:533–543. [PMC free article] [PubMed]
26. Urboniene D, Haber I, Fang YH, Thenappan T, Archer SL. Validation of high-resolution echocardiography and magnetic resonance imaging vs. High-fidelity catheterization in experimental pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol. 2010;299:L401–412. [PubMed]
27. Vassilev LT, Tovar C, Chen S, Knezevic D, Zhao X, Sun H, Heimbrook DC, Chen L. Selective small-molecule inhibitor reveals critical mitotic functions of human cdk1. Proceedings of the National Academy of Sciences of the United States of America. 2006;103:10660–10665. [PubMed]
28. Lindqvist A, Rodriguez-Bravo V, Medema RH. The decision to enter mitosis: Feedback and redundancy in the mitotic entry network. The Journal of cell biology. 2009;185:193–202. [PMC free article] [PubMed]
29. Peters JM. The anaphase promoting complex/cyclosome: A machine designed to destroy. Nature reviews. Molecular cell biology. 2006;7:644–656. [PubMed]
30. Mitra K, Wunder C, Roysam B, Lin G, Lippincott-Schwartz J. A hyperfused mitochondrial state achieved at g1-s regulates cyclin e buildup and entry into s phase. Proceedings of the National Academy of Sciences of the United States of America. 2009;106:11960–11965. [PubMed]
31. Taguchi N, Ishihara N, Jofuku A, Oka T, Mihara K. Mitotic phosphorylation of dynamin-related gtpase drp1 participates in mitochondrial fission. J Biol Chem. 2007;282:11521–11529. [PubMed]
32. Kung AL, Zabludoff SD, France DS, Freedman SJ, Tanner EA, Vieira A, Cornell-Kennon S, Lee J, Wang B, Wang J, Memmert K, Naegeli HU, Petersen F, Eck MJ, Bair KW, Wood AW, Livingston DM. Small molecule blockade of transcriptional coactivation of the hypoxia-inducible factor pathway. Cancer Cell. 2004;6:33–43. [PubMed]
33. Staab A, Loeffler J, Said HM, Diehlmann D, Katzer A, Beyer M, Fleischer M, Schwab F, Baier K, Einsele H, Flentje M, Vordermark D. Effects of hif-1 inhibition by chetomin on hypoxia-related transcription and radiosensitivity in ht 1080 human fibrosarcoma cells. BMC Cancer. 2007;7:213. [PMC free article] [PubMed]
34. Spirig R, Djafarzadeh S, Regueira T, Shaw SG, von Garnier C, Takala J, Jakob SM, Rieben R, Lepper PM. Effects of tlr agonists on the hypoxia-regulated transcription factor hif-1alpha and dendritic cell maturation under normoxic conditions. PLoS One. 2010;5:e0010983. [PMC free article] [PubMed]
35. Wang GL, Semenza GL. Desferrioxamine induces erythropoietin gene expression and hypoxia-inducible factor 1 DNA-binding activity: Implications for models of hypoxia signal transduction. Blood. 1993;82:3610–3615. [PubMed]
36. Pozeg ZI, Michelakis ED, McMurtry MS, Thebaud B, Wu XC, Dyck JR, Hashimoto K, Wang S, Moudgil R, Harry G, Sultanian R, Koshal A, Archer SL. In vivo gene transfer of the o2-sensitive potassium channel kv1.5 reduces pulmonary hypertension and restores hypoxic pulmonary vasoconstriction in chronically hypoxic rats. Circulation. 2003;107:2037–2044. [PubMed]
37. Yuan XJ, Wang J, Juhaszova M, Gaine SP, Rubin LJ. Attenuated k+ channel gene transcription in primary pulmonary hypertension. Lancet. 1998;351:726–727. [PubMed]
38. Archer SL, Souil E, Dinh-Xuan AT, Schremmer B, Mercier JC, El Yaagoubi, A Nguyen, Huu L, Reeve HL, Hampl V. Molecular identification of the role of voltage-gated k+ channels, kv1.5 and kv2.1, in hypoxic pulmonary vasoconstriction and control of resting membrane potential in rat pulmonary artery myocytes. J Clin Invest. 1998;101:2319–2330. [PMC free article] [PubMed]
39. Yuan XJ. Voltage-gated k+ currents regulate resting membrane potential and [ca2+]i in pulmonary arterial myocytes. Circ Res. 1995;77:370–378. [PubMed]
40. Chen H, Chomyn A, Chan DC. Disruption of fusion results in mitochondrial heterogeneity and dysfunction. J Biol Chem. 2005;280:26185–26192. [PubMed]
41. Chan DC. Mitochondrial dynamics in disease. N Engl J Med. 2007;356:1707–1709. [PubMed]
42. Yang J, Davies RJ, Southwood M, Long L, Yang X, Sobolewski A, Upton PD, Trembath RC, Morrell NW. Mutations in bone morphogenetic protein type ii receptor cause dysregulation of id gene expression in pulmonary artery smooth muscle cells: Implications for familial pulmonary arterial hypertension. Circulation research. 2008;102:1212–1221. [PubMed]
43. Richard DE, Berra E, Pouyssegur J. Nonhypoxic pathway mediates the induction of hypoxia-inducible factor 1alpha in vascular smooth muscle cells. The Journal of biological chemistry. 2000;275:26765–26771. [PubMed]
44. Xu W, Koeck T, Lara AR, Neumann D, DiFilippo FP, Koo M, Janocha AJ, Masri FA, Arroliga AC, Jennings C, Dweik RA, Tuder RM, Stuehr DJ, Erzurum SC. Alterations of cellular bioenergetics in pulmonary artery endothelial cells. Proc Natl Acad Sci U S A. 2007;104:1342–1347. [PubMed]
45. Yu AY, Shimoda LA, Iyer NV, Huso DL, Sun X, McWilliams R, Beaty T, Sham JS, Wiener CM, Sylvester JT, Semenza GL. Impaired physiological responses to chronic hypoxia in mice partially deficient for hypoxia-inducible factor 1alpha. J Clin Invest. 1999;103:691–696. [PMC free article] [PubMed]
46. ten Freyhaus H, Dagnell M, Leuchs M, Vantler M, Berghausen EM, Caglayan E, Weissmann N, Dahal BK, Schermuly RT, Ostman A, Kappert K, Rosenkranz S. Hypoxia enhances platelet-derived growth factor signaling in the pulmonary vasculature by down-regulation of protein tyrosine phosphatases. American journal of respiratory and critical care medicine. 2011;183:1092–1102. [PubMed]
47. Vengellur A, Woods BG, Ryan HE, Johnson RS, LaPres JJ. Gene expression profiling of the hypoxia signaling pathway in hypoxia-inducible factor 1alpha null mouse embryonic fibroblasts. Gene Expr. 2003;11:181–197. [PubMed]
48. Vengellur A, Phillips JM, Hogenesch JB, LaPres JJ. Gene expression profiling of hypoxia signaling in human hepatocellular carcinoma cells. Physiol Genomics. 2005;22:308–318. [PubMed]
49. Ishihara N, Nomura M, Jofuku A, Kato H, Suzuki SO, Masuda K, Otera H, Nakanishi Y, Nonaka I, Goto Y, Taguchi N, Morinaga H, Maeda M, Takayanagi R, Yokota S, Mihara K. Mitochondrial fission factor drp1 is essential for embryonic development and synapse formation in mice. Nature cell biology. 2009;11:958–966. [PubMed]
50. Guo YH, Chen K, Gao W, Li Q, Chen L, Wang GS, Tang J. Overexpression of mitofusin 2 inhibited oxidized low-density lipoprotein induced vascular smooth muscle cell proliferation and reduced atherosclerotic lesion formation in rabbit. Biochemical and biophysical research communications. 2007;363:411–417. [PubMed]
51. Sutterlin C, Hsu P, Mallabiabarrena A, Malhotra V. Fragmentation and dispersal of the pericentriolar golgi complex is required for entry into mitosis in mammalian cells. Cell. 2002;109:359–369. [PubMed]
52. Rabouille C, Kondylis V. Golgi ribbon unlinking: An organelle-based g2/m checkpoint. Cell Cycle. 2007;6:2723–2729. [PubMed]
53. Ono S, Westcott JY, Voelkel NF. Paf antagonists inhibit pulmonary vascular remodeling induced by hypobaric hypoxia in rats. J Appl Physiol. 1992;73:1084–1092. [PubMed]
54. Diaz GJ, Julian RJ, Squires EJ. Cobalt-induced polycythaemia causing right ventricular hypertrophy and ascites in meat-type chickens. Avian Pathol. 1994;23:91–104. [PubMed]
55. Ong SB, Subrayan S, Lim SY, Yellon DM, Davidson SM, Hausenloy DJ. Inhibiting mitochondrial fission protects the heart against ischemia/reperfusion injury. Circulation. 2010;121:2012–2022. [PubMed]