Biochem. J. (2014) 462, 103–112 (Printed in Great Britain)
103
doi:10.1042/BJ20140486
miR-21/DDAH1 pathway regulates pulmonary vascular responses to hypoxia
The NOS (nitric oxide synthase) inhibitor ADMA (asymmetric dimethylarginine) contributes to the pathogenesis of pulmonary hypertension. Reduced levels of the enzymes metabolizing ADMA, dimethylarginine dimethylaminohydrolases (DDAH1 and DDAH2) and increased levels of miR-21 are linked to disease pathology, but the mechanisms are not understood. In the present study we assessed the potential role of miR-21 in the regulation of hypoxia-induced changes in ADMA metabolism in vitro and in vivo. Hypoxia inhibited DDAH1 and DDAH2 expression and increased ADMA levels in cultured human pulmonary endothelial cells. In contrast, in human pulmonary smooth muscle cells, only DDAH2 was reduced whereas ADMA levels remained unchanged. Endothelium-specific down-regulation of DDAH1 by miR-21 in hypoxia induced endothelial dysfunction and was prevented by overexpression of DDAH1 and miR-
21 blockade. DDAH1, but not DDAH2, mRNA levels were reduced, whereas miR-21 levels were elevated in lung tissues from patients with pulmonary arterial hypertension and mice with pulmonary hypertension exposed to 2 weeks of hypoxia. Hypoxic mice treated with miR-21 inhibitors and DDAH1 transgenic mice showed elevated lung DDAH1, increased cGMP levels and attenuated pulmonary hypertension. Regulation of DDAH1 by miR-21 plays a role in the development of hypoxia-induced pulmonary hypertension and may be of broader significance in pulmonary hypertension.
INTRODUCTION
disruption of vascular homoeostasis. DDAH1 (dimethylarginine dimethylaminohydrolase 1) and DDAH2 are enzymes that metabolize ADMA and play an important role under pathological conditions [15–19]. DDAH1 heterozygous knockout mice show abnormalities associated with pulmonary hypertension, including increase in right ventricular pressures and structural changes in the pulmonary vasculature [15]. Evidence from mice with the endothelium-specific deletion of DDAH1 showed that in the lung DDAH1 predominantly localizes in the vascular endothelium and plays a key role in the regulation of ADMA metabolism [19]. On the other hand, DDAH2 gene silencing in mice was shown to reduce vascular NO generation and endothelium-derived relaxation factor responses [20]. DDAH expression levels are reduced in animal and human pulmonary hypertensive lung [12,14,15], but mechanisms leading to DDAH suppression remain to be elucidated. miRNAs play vital roles in regulating gene expression in the cardiovascular system and dysregulation of miRNA expression contributes to the pathogenesis of cardiovascular diseases, including PAH (pulmonary arterial hypertension) [21– 26]. Two miRNA candidates potentially targeting the DDAH1 3 -UTR, miR-21 and miR-128, have previously been implicated in the pathogenesis of pulmonary hypertension [25–27]. miR-21 expression is increased in the hypoxic pulmonary hypertensive mouse lung, but its actions are complicated and not well
Chronic hypoxia induces pulmonary hypertension in humans and animals [1,2]. Pulmonary hypertension is characterized by increased right ventricular pressure resulting from pulmonary vascular resistance due to pulmonary vasoconstriction and pulmonary vascular remodelling [1,2]. Endothelial dysfunction and PASMC (pulmonary arterial smooth muscle cell) hypertrophy and proliferation in pulmonary hypertension are attributed, at least in part, to inhibition of NO (nitric oxide) signalling [3]. NO produced by eNOS (endothelial NO synthase) acts as a vasorelaxant and an inhibitor of vascular smooth muscle cell proliferation in vitro and in vivo [4,5]. In addition, NO exerts barrier-protective, antioxidant, anti-thrombotic and antiinflammatory effects on the pulmonary vascular endothelium [6,7]. Activation of PKG (protein kinase G) and a subsequent increase in the intracellular cGMP levels constitute a key part of NO signalling [8]. Methylarginines, e.g. ADMA (asymmetric dimethylarginine) and L-NMMA (N G -monomethyl-L-arginine), are competitive inhibitors of all NOSs (NO synthases), whereas SDMA (symmetric dimethylarginine) has no effect [9,10]. Increased plasma and lung tissue levels of ADMA in animals and patients with hypoxia-induced pulmonary hypertension and IPAH (idiopathic pulmonary arterial hypertension) [11– 14], are associated with reduced NO bioavailability and
Key words: asymmetric dimethylarginine (ADMA), dimethylarginine dimethylaminohydrolase (DDAH), endothelium, hypoxia, pulmonary hypertension, smooth muscle.
Abbreviations: ADMA, asymmetric dimethylarginine; BrdU, bromodeoxyuridine; D7-ADMA, [2,3,3,4,4,5,5-2 H]asymmetric dimethylarginine; DDAH, dimethylarginine dimethylaminohydrolase; DDAH1tg, DDAH1 transgenic; HPAEC, human pulmonary arterial endothelial cell; HPASMC, human pulmonary artery smooth muscle cell; IPAH, idiopathic pulmonary arterial hypertension; LNA, locked nucleic acid anti-miRNA; MOI, multiplicity of infection; L-NMMA, N G -monomethyl-L-arginine; NOS, nitric oxide synthase; PAH, pulmonary arterial hypertension; PASMC, pulmonary arterial smooth muscle cell; PCNA, proliferating cell nuclear antigen; PKG, protein kinase G; RVH, right ventricular hypertrophy; RVSP, right ventricular systolic pressure; SDMA, symmetric dimethylarginine; t 1/ , half-life; VEGF, vascular endothelial growth factor. 2 1 To whom correspondence should be addressed (email
[email protected]). c The Authors Journal compilation c 2014 Biochemical Society
Biochemical Journal
*Centre for Pharmacology and Therapeutics, Experimental Medicine, Imperial College London, Du Cane Road, London W12 0NN, U.K. †MRC Clinical Sciences Centre, Imperial College London, Du Cane Road, London W12 0NN, U.K.
www.biochemj.org
Lucio IANNONE*, Lan ZHAO*, Olivier DUBOIS*, Lucie DULUC*, Christopher J. RHODES*, John WHARTON*, Martin R. WILKINS*, James LEIPER† and Beata WOJCIAK-STOTHARD*1
104
L. Iannone and others
understood as both protective [24] and detrimental [25,26] effects were postulated. Interestingly, a recent study revealed that miR-21 regulates DDAH1 expression in cultured human umbilical vein endothelial cells [23]. miR-128 levels were up-regulated in the plasma of patients with PAH [27]. In the present study we assessed the potential role of miR-21 in regulating hypoxia-induced changes in DDAH activity (thus ADMA metabolism) in the pulmonary vasculature, employing cultured pulmonary vascular cells and a mouse model of hypoxiainduced pulmonary hypertension. We show that a hypoxiainduced increase in miR-21 levels contributes to degradation of DDAH1 mRNA and endothelial dysfunction associated with a reduction in NO/cGMP signalling. Importantly, exposure of mice to 2 weeks of hypoxia induces pulmonary hypertension accompanied by increased miR-21 and ADMA levels and decreased DDAH1 and cGMP levels in lung tissues. miR21 blockade or DDAH1 overexpression restore DDAH1/NO signalling and attenuate the development of hypoxia-induced pulmonary hypertension in mice. Lung tissues from IPAH patients show significantly elevated levels of miR-21 and reduced levels of DDAH1 mRNA, suggesting a potential role for DDAH1 in the human condition. MATERIALS AND METHODS
The extended Materials and methods section is available in the Supplementary Online Data (at http://www.biochemj.org/bj/ 462/bj462103add.htm).
preventing the miRNA from binding to the 3 -UTR of its target mRNAs [28]. The inhibitor of miR-21 (LNA21) and the inhibitor of miR-128 (LNA128) (Exiqon) were transfected into HPAECs at 10 nmol/106 cells. Following transfection, cells were left in normoxia or in hypoxia for 24 h. Luciferase reporter assays
The 3 -UTRs of the human DDAH1 gene were cloned by GenScript into the 3 -UTR of the Firefly luciferase gene in a luciferase pGL3-CMV plasmid (Promega). Manipulation of DDAH1 expression and activity in cultured cells
Adenoviral vectors for DDAH1, DDAH2 and the inactive mutants of DDAH1 (DDAH1–GFP) and DDAH2 (DDAH2–GFP) were constructed as described previously [7]. The cells were infected at a MOI (multiplicity of infection) of 1:100 and allowed to express recombinant proteins for 24 h prior to hypoxic exposure. ADMA and cGMP measurement
ADMA levels in cell culture medium and in lung tissues were measured using HPLC. cGMP levels were measured with the cGMP enzyme immunoassay Biotrak (EIA) System (GE Healthcare) according to the manufacturer’s instructions. DDAH activity measurement
Cell culture
HPAECs (human pulmonary artery endothelial cells) and HPASMCs (human pulmonary artery smooth muscle cells) were cultured under normoxic (20 % O2 , 5 % CO2 ) or hypoxic (2 % O2 , 5 % CO2 , 92 % N2 ) conditions for 1–48 h.
Cell lysates were incubated with 500 μM of the DDAH substrate D7-ADMA ([2,3,3,4,4,5,5-2 H]asymmetric dimethylarginine) and DDAH activity was assessed by quantification of D7-citrulline, a product of DDAH metabolism [29]. Data are expressed as the peak area ratio of D7-ADMA to D7-citrulline normalized to the protein concentration in each sample.
RT (reverse transcription)–PCR
RNA was isolated using the PureLink RNA Mini Kit (Invitrogen) and cDNA was synthesized using oligo(dT) and Superscript II Reverse Transcriptase (Invitrogen). Quantitative real-time PCR was carried out using a HT-7500 thermocycler (Applied Biosystems). TaqMan primers/probes set for DDAH1 (Hs00201707_m1), DDAH2 (Hs00967863_g1), VEGF (vascular endothelial growth factor) (Hs00900055_m1) and miR21 (Hs04231424_s1) were from Applied Biosystems. To assess mRNA stability, RNA synthesis was terminated by the addition of 5 mg/l actinomycin D. Western blot analysis and immunostaining
DDAH1, DDAH2, RhoA, RhoA(pSer188 ), VE-cadherin (vascular endothelial cadherin), PCNA (proliferating cell nuclear antigen), α-tubulin and β-actin protein expression was studied by Western blotting and immunofluorescence of lung lysates and cultured cells. Plasmids, lock nucleic acids and cell transfection
HPAECs and HPASMCs were transfected with pCMV-miRNA21 (p-miRNA21), pCMV-miRNA128 (p-miRNA-128) and, as a negative control, pCMV-EGFP (p-EGFP), expression vectors (Origene). LNA (locked nucleic acid anti-miRNA) probes were used to inhibit miRNA activity. These bind to and form heteroduplexes with target miRNA, thereby sequestering and c The Authors Journal compilation c 2014 Biochemical Society
Endothelial permeability and morphology
Endothelial cell permeability and morphology were studied as in [7]. In some experiments, HPAECs grown in Transwell chambers (3 μm; Corning Life Sciences) were treated with the PKG inhibitor Rp-8-Br-PET-cGMPS (0.5 μM; R&D Systems) for 24 h. Cell proliferation
Cell proliferation was evaluated in HPAECs and HPASMCs using a BrdU (bromodeoxyuridine) assay (Millipore). In some experiments, HPASMCs were grown in the presence of the untreated or adenovirus-treated HPAECs in Transwell dishes. In some experiments, HPASMCs were incubated with 0.5 μM Rp8-Br-PET-cGMPS for 24 h [30]. Chronic hypoxia studies in vivo
Studies were conducted in accordance with the U.K. Home Office Animals (Scientific Procedures) Act 1986 and institutional guidelines. Male C57BL mice, 12–15 weeks old (Charles River) and DDAH1tg (DDAH1 transgenic) mice (Jackson Laboratory) were housed in normal air or placed in a normobaric hypoxic chamber [FiO2 (fraction of inspired oxygen) 10 %] for 2 weeks (n = 6–8/group). Development of pulmonary hypertension was confirmed as described previously [31]. Intraperitoneal injection of LNA21 (10 mg/kg of body weight) was given once every other
miR-21 and DDAH1 in pulmonary vascular responses to hypoxia
Figure 1
105
The effect of hypoxia (1–48 h) on DDAH1 gene and protein expression in pulmonary vascular cells
DDAH1 gene (a and b) and protein (c and d) expression in HPAECs and HPASMCs are shown, as indicated. Representative examples of Western blots are underneath the histograms. DDAH1 mRNA levels in (a) and (b) were normalized to the expression levels of the housekeeping gene TATA-binding protein (TBP), whereas in (c) and (d) DDAH1 protein expression was normalized to α-tubulin (band absorbance, arbitrary units). *P < 0.05, **P < 0.001 and ***P < 0.0001 in comparison with normoxic controls; n = 6–8.
day for a total of eight times. Following the first administration of LNA21, mice were exposed to hypoxia for 2 weeks (n = 8/group). Human samples
Lung tissues were acquired at lung transplantation from treatment-naive patients with IPAH (n = 12) and from uninvolved regions of lobectomy specimens from bronchial carcinoma patients (n = 12) [32] with the informed consent of the patients and the approval of the Brompton and Harefield National Heart and Lung Institutes and Hammersmith Hospitals Research Ethics committees (Supplementary Table S1 at http://www.biochemj.org/bj/462/bj4620103add.htm). Further information regarding human samples is provided in the Supplementary Materials and methods section. Statistical analysis
All of the experiments were performed in triplicate and data are presented as the mean + − S.E.M. Comparisons were carried out using two-tailed Student’s t tests or one-way ANOVA as appropriate. Statistical significance was accepted for P < 0.05 and tests were performed using GraphPad Prism version 6.0. RESULTS Hypoxia decreases DDAH mRNA and protein levels in cultured pulmonary vascular cells
Hypoxia (1–48 h) led to a 3–4-fold decrease in DDAH1 mRNA levels in HPAECs, whereas DDAH1 mRNA levels in HPASMCs
were unaffected (Figures 1a and 1b). Consistent with changes in mRNA levels, DDAH1 protein levels were significantly reduced in hypoxic HPAECs, whereas in hypoxic HPASMCs DDAH1 protein levels remained unchanged (Figures 1c and 1d). Hypoxia reduced DDAH2 mRNA and protein levels in both HPAECs and HPASMCs (Supplementary Figure S1 at http://www.biochemj.org/bj/462/bj4620103add.htm).
Hypoxia decreases ADMA metabolism in pulmonary endothelial cells, but not in pulmonary smooth muscle cells
ADMA levels in HPAEC culture medium were significantly increased following 24 and 48 h of hypoxia (2- and 3-fold increase respectively), whereas ADMA levels in HPASMCs were unchanged (Figures 2a and 2b). L-NMMA and SDMA levels in either cell type were not affected (Supplementary Figures S2a–S2d at http://www.biochemj.org/bj/462/bj4620103add.htm). In HPAECs, DDAH activity was reduced by 5-fold in hypoxia, whereas DDAH activity in HPASMCs did not change (Figures 2c and 2d). To verify further the role of DDAH expression in hypoxiainduced ADMA metabolism, DDAH1, DDAH2 and inactive DDAH (DDAH) mutants were overexpressed in HPAECs using adenoviral gene transfer [7]. The MOI and expression time were established experimentally to induce a physiological (∼3-fold) increase in DDAH expression (Supplementary Figure S2e). DDAH1 significantly reduced ADMA levels by 1.3-fold in HPAECs exposed to 24 h of hypoxia, whereas DDAH2 or inactive DDAH mutants had no effect (Supplementary Figure S2f), confirming the key role of DDAH1 in ADMA metabolism. c The Authors Journal compilation c 2014 Biochemical Society
106
Figure 2
L. Iannone and others
The effect of hypoxia on ADMA levels and DDAH activity in HPAECs and HPASMCs
Hypoxia induces changes in ADMA levels (a and b) and DDAH activity (c and d) in normoxic and hypoxic HPAECs and HPASMCs, as indicated. DDAH activity was measured directly by using labelled D7-ADMA as a substrate for DDAH and measuring the levels of the ADMA metabolite D7-L-citrulline. *P < 0.05 and ***P < 0.0001 in comparison with normoxic controls; n = 6
miR-21 regulates ADMA metabolism in pulmonary endothelial cells
To verify whether hypoxia-induced changes in DDAH mRNA levels result from changes in mRNA stability, and not transcriptional regulation, we measured the half-life (t 12 ) of DDAH mRNA in the presence of the transcription inhibitor actinomycin D. In HPAECs, t1/2 of DDAH1 mRNA was reduced from 1.5 h in normoxia to 1 h in hypoxia, whereas in HPASMCs t 12 of DDAH1 mRNA remained unchanged (Figures 3a and 3b). Consistent with the changes in gene expression, t 12 of DDAH2 mRNA was reduced both in HPAECs and in HPASMCs (Figures 3c and 3d). In contrast, t 12 of VEGF mRNA, acting as a positive control, increased from 0.4 h to 2 h (Supplementary Figure S3 at http://www.biochemj.org/bj/462/bj4620103add.htm). Exposure of HPAECs to hypoxia for 0.5–2h induced an increase in miR-21 that preceded a decrease in DDAH1 mRNA (Supplementary Figures S4a and S4b at http://www.biochemj.org/bj/462/bj4620103add.htm). In order to study the role of miR-21 in the regulation of DDAH1 3 -UTR, HPAECs were transfected with expression plasmids (pCMV) containing pre-miR-21 or pre-miR-128. Both miRNAs have predicted binding sites in the DDAH1 3 -UTR and have been implicated in the pathogenesis of PAH [24,33]. Imperfect base pairing of miRNAs to target RNAs often results in inaccurate predictions and therefore the role of miRNAs has to be verified experimentally. Overexpression of miR-21 mimicked the effects of hypoxia on DDAH1 gene and protein expression, but miR128 had no effect (Figures 4a and 4b). DDAH2 expression was unaffected (Supplementary Figures S4c and S4d). c The Authors Journal compilation c 2014 Biochemical Society
Conversely, miR-21-knockdown probes were used to examine the effects of miR-21 inhibition on hypoxia-induced DDAH1 degradation. As shown in Figure 4(c), treatment of HPAECs transfected with EcoRI/FseI 3 -UTR luciferase with LNA21 completely prevented hypoxia-induced decrease in DDAH1 3 -UTR-dependent luciferase expression. Consistent with the postulated endothelium-specific effect, 24 h of hypoxia significantly increased miR-21 levels in HPAECs (4-fold increase, P < 0.0001), whereas the change seen in HPASMCs was not significant (Figure 4d).
Increase in DDAH1 expression attenuates hypoxia-induced endothelial dysfunction in vitro
We hypothesized that hypoxia-induced vascular dysfunction results, at least in part, from inhibition of DDAH expression and activity. Hypoxia increased HPASMC proliferation in vitro, whereas HPAEC proliferation was unaffected (Figures 5a and 5b). Overexpression of DDAH1- or DDAH1-inactive mutants did not affect cell proliferation or apoptosis in either normoxic or hypoxic conditions (Supplementary Figure S5 at http://www.biochemj.org/bj/462/bj4620103add.htm and results not shown). Endothelium-derived NO inhibits PASMC proliferation in vitro and in vivo [4,5] and we hypothesized that this NOdependent effect may be attenuated in hypoxia as a result of DDAH down-regulation. Hypoxia (24 h) stimulated the growth of HPASMCs co-cultured with HPAECs, but the effect was inhibited in the presence of HPAECs overexpressing
miR-21 and DDAH1 in pulmonary vascular responses to hypoxia
Figure 3
107
Hypoxia regulates DDAH mRNA stability
The t 1 of DDAH1 mRNA in hypoxia (triangles) or normoxia (circles) in HPAECs and HPASMCs is shown in (a) and (b) respectively. DDAH2 t 1 in hypoxia and in normoxia in HPAECs and HPASMCs 2 2 is shown in (c) and (d). The broken line indicates the values of t 1 of DDAH mRNA in normoxia and in hypoxia. The cells were pre-incubated with actinomycin D (5.0 mg/l) and exposed to hypoxia 2 (2 % O2 ) for different time intervals (0–2.5 h). The fold change in DDAH mRNA expression was measured by real-time PCR and is represented as the percentage decrease compared with the untreated sample; n = 5.
Figure 4
The effect of miR-21 on DDAH1 3 -UTR
Overexpression of miR-21 mimics the effect of hypoxia on (a) gene and (b) protein expression of DDAH1, whereas miR-128 has no effect. miR-21 and miR-128 were overexpressed in normoxic HPAECs using shuttle plasmids (p-miRNA21 and p-miRNA128). (c) LNA21 represses the effects of hypoxia on the DDAH1 3 -UTR, whereas LNA128 has no effect. The luciferase reporter vector EcoRI/FseI DDAH1 3 -UTR was co-transfected into HPAECs with 10 nmol LNA21 and LNA128. (d) miR-21 levels are increased by hypoxia (24 h) in HPAECs, but not in HPASMCs. n = 5; *P < 0.05; **P < 0.001 and ***P < 0.001 in comparison with the transfection controls or as indicated.
c The Authors Journal compilation c 2014 Biochemical Society
108
Figure 5
L. Iannone and others
The effects of hypoxia and DDAH on cell proliferation and endothelial barrier function
Proliferation (BrDU incorporation) in HPAECs (a) and HPASMCs (b) in normoxia or in hypoxia is indicated. HPASMC (S) proliferation in co-culture with HPAECs (E) in normoxia and in hypoxia is shown in (c). Overexpression of DDAH1 attenuates hypoxia-induced increase in endothelial permeability (d). In (a–d) HPAECs were left untreated or overexpressed with GFP (adenoviral control), AdDDAH1, AdDDAH2 or the inactive DDAH mutants DDAH1 or DDAH2. *P < 0.05 and **P < 0.001 in comparison with normoxic controls; # P < 0.05 in comparison with hypoxic untreated controls. n = 5.
DDAH1, whereas DDAH2 or DDAH mutants had no effect (Figure 5c). Hypoxia also induced a 1.5-fold increase in HPAEC permeability accompanied by a dispersion of intercellular adherens junctions (Figure 5d and Supplementary Figure S6 at http://www.biochemj.org/bj/462/bj4620103add.htm). DDAH1 overexpression significantly attenuated the effect of hypoxia, whereas DDAH2 or inactive mutants of DDAH had no significant effect (Figure 5d and Supplementary Figure S6). The protective effects of endothelial DDAH1 overexpression were attenuated in the presence of the PKG inhibitor and the phosphodiesteraseresistant cGMP antagonist, Rp-8-cGMP (Supplementary Figure S7 at http://www.biochemj.org/bj/462/bj4620103add.htm). In summary, the results demonstrate that hypoxia-induced changes in miR-21/DDAH1 expression have a critical impact on ADMA levels, endothelial barrier function and the endotheliumdependent modulation of PASMC proliferation.
miR-21 inhibitors increase DDAH1 levels in the lung and attenuate development of hypoxia-induced pulmonary hypertension
Mice exposed to 2 weeks of hypoxia developed pulmonary hypertension, confirmed by the measurement of RVSP (right ventricular systolic pressure), RVH (right ventricular hypertrophy) and pulmonary vascular remodelling [31] (Figures 6a–6d). Hypoxia c The Authors Journal compilation c 2014 Biochemical Society
increased miR-21 levels and decreased DDAH1 protein levels by 3-fold in lung tissues, whereas miR-21/DDAH1 levels in the kidney and liver remained unaffected (Supplementary Figure S8 at http://www.biochemj.org/bj/462/bj4620103add.htm). The in vivo inhibition of miR-21 with specific LNA probes reduced the hypoxia-induced increase in RVH, RVSP and muscularization of small intrapulmonary arteries (Figure 6). LNA21 treatment restored DDAH1 expression in the lung and reduced protein expression levels of PCNA, a nuclear protein that participates in active cell proliferation [34] (Supplementary Figures S9a and S9b at http://www.biochemj.org/ bj/462/bj4620103add.htm). DDAH2 levels were not affected by LNA21 treatment (results not shown). LNA21-treated hypoxic mice also showed significantly elevated lung RhoA(pSer188 ) and cGMP levels (Supplementary Figures S9c–S9e), indicative of improved NO signalling [35,36]. The potential therapeutic effect of increased DDAH1 expression was investigated further in hypoxic DDAH1tg mice. No significant differences in RVSP were observed between DDAH1tg mice and their wild-type littermates kept in normal air (Figure 7a), consistent with previous reports [37]. Similar to the effects seen in LNA21-treated mice, the hypoxia-induced increases in RVH, RVSP, vessel muscularization and PCNA levels were attenuated in DDAHtg mice compared with hypoxic wildtype controls (Figure 7 and Supplementary Figures S10a and
miR-21 and DDAH1 in pulmonary vascular responses to hypoxia
Figure 6
109
LNA21 treatment attenuates the development of hypoxia-induced pulmonary hypertension in mice
Treatment with LNA21 reduces RVSP (a), RVH (b) and muscularization of intrapulmonary arteries in hypoxic pulmonary hypertensive wild-type mice (c). *P < 0.05 and ***P < 0.001 in comparison with untreated normoxic controls; # P < 0.05, ## P < 0.01 and ### P