Elevated pulmonary arterial pressure in Zucker diabetic fatty rats

RESEARCH ARTICLE Elevated pulmonary arterial pressure in Zucker diabetic fatty rats Daniel Morales-CanoID 1,2,3 , Maria Callejo 1,2,3 , Bianca Barreira 1,2,3 , Gema MondejarParreño 1,2,3 , Sergio Esquivel-Ruiz 1,2,3 , Sonia Ramos 4 , Marı ´a A ´ngeles Martı ´n 4,5 , Angel Cogolludo 1,2,3 , Laura Moreno 1,2,3 , Francisco Perez-VizcainoID 1,2,3 * 1Departament of Pharmacology and Toxicology, School of Medicine, Universidad Complutense de Madrid, Madrid, Spain, 2Ciber Enfermedades Respiratorias (Ciberes), Madrid, Spain, 3Instituto de Investigacio ´n Sanitaria Gregorio Maraño ´n (IISGM), Madrid, Spain, 4Department of Metabolism and Nutrition, Institute of Food Science and Technology and Nutrition (ICTAN), Consejo Superior de Investigaciones Cientı ´ficas (CSIC), Madrid, Spain, 5Ciber de Diabetes y Enfermedades Metabo ´licas Asociadas (CIBERDEM), Madrid, Spain *[email protected] Abstract Diabetes is a very strong predictor of chronic systemic vascular diseases and acute cardiovascular events. Recently, associations between metabolic disorders and pulmonary hypertension have also been reported in both humans and animal models. In order to get some further insight into the relationship of pulmonary hypertension with obesity, insulin resistance and hyperglycemia, herein we have used the Zucker diabetic fatty rats (ZDF/clr-lepr fa) at 20 weeks fed a standard diet and compared to their lean Zucker littermates (ZL). ZDF rats were obese, had elevated plasma glucose levels and insulin resistance, i.e. a clinically relevant model of type 2 diabetes. They presented elevated systolic, diastolic and mean pulmonary arterial pressures and a parallel increase in the Fulton index. Systemic arterial pressures were also increased but the left ventricle plus septum weight was similar in both groups and the heart rate was reduced. Wall media thickening was observed in the small pulmonary arteries from the ZDF rats. Isolated pulmonary arteries mounted in a wire myograph showed similar vasoconstrictor responses to phenylephrine and 5-HT and similar responses to the endothelium-dependent vasodilator acetylcholine. However, the iNOS inhibitor 1400W enhanced the vasoconstrictor responses in ZDF but not in ZL rats. The protein expression of eNOS and iNOS was not significantly different in the lungs of the two groups. The lung expression of Bmpr2 mRNA was downregulated. However, the mRNA expression of Kcna5,Kcnk3,Kcnq1,Kcnq4 or Kcnq5, which encode for the potassium channels Kv1.5, TASK-1, Kv7.1, Kv7.4 and Kv7.5, respectively, was similar in ZL and ZDF rats. In conclusion, ZDF rats show increased pulmonary arterial pressure, right ventricular hypertrophy, pulmonary arterial medial thickening and downregulated lung Bmpr2 despite leptin resistance. These changes were mild but are consistent with the view that diabetes is a risk factor for pulmonary hypertension. PLOS ONE | https://doi.org/10.1371/journal.pone.0211281 January 28, 2019 1 / 16 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Morales-Cano D, Callejo M, Barreira B, Mondejar-Parreño G, Esquivel-Ruiz S, Ramos S, et al. (2019) Elevated pulmonary arterial pressure in Zucker diabetic fatty rats. PLoS ONE 14(1): e0211281. https://doi.org/10.1371/journal. pone.0211281 Editor: Michael Bader, Max Delbruck Centrum fur Molekulare Medizin Berlin Buch, GERMANY Received: September 11, 2018 Accepted: January 10, 2019 Published: January 28, 2019 Copyright: ©2019 Morales-Cano et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and/or Supporting Information files. Funding: This study was supported by grants from Ministerio de Economı ´a y Competitividad (Mineco, https://sede.micinn.gob.es SAF2016-77222-R to FPV and AC and AGL2015–67087-R to SR and MAM), Instituto de Salud Carlos III (www.isciii.es, PI15/01100 to LM), with funds from the European Union (Fondo Europeo de Desarrollo Regional FEDER). M.C., G.M-P. and S.E-R. are funded by Introduction Pulmonary hypertension (PH) is characterized by an increase in pulmonary arterial pressure (PAP) and pulmonary vascular resistance determined by right heart catheterization at rest [1]. It is classified into 5 groups according to its origin: 1) pulmonary arterial hypertension (PAH), 2) PH hypertension associated with left heart disease, 3) PH associated with lung diseases and hypoxia, 4) PH associated to chronic thromboembolism (CTEPH), and 5) PH of unknown origin or multifactorial [1]. With the exception of idiopathic PAH, in all groups and subgroups of PH there is a known factor, such as a mutation, infection, hypoxia, drugs, embolism or other diseases, that is associated with the development of the disease. However, none of these factors by itself is sufficient to trigger the disease [2]. The clinical risk factors that predict the development of PH in patients at risk, i.e. the so called “second hits”, have not yet been fully identified. In recent years there has been a worldwide increase in the prevalence of type 2 diabetes [3], which is a very well-known predictor of chronic systemic vascular diseases and acute cardiovascular events [4]. Recently, associations between metabolic disorders and pulmonary hypertension have also been reported [5]. Several studies have suggested that insulin resistance and type 2 diabetes are associated with pulmonary hypertension in humans [6–9]. However, the relation of PH with obesity, which is very frequently associated to insulin resistance and diabetes, is unclear. Systolic PAP has been positively correlated with body mass index in 3790 echocardiographically normal subjects [10]. Paradoxically, obesity has also been suggested as a protective prognostic factor in patients with PH [11]. Several studies in rodents have also shown pulmonary vascular dysfunction in diabetes. Type 1 diabetic animals show pulmonary endothelial dysfunction, BMPR2 downregulation and lung inflammation [12,13]. These factors alone are insufficient to increase pulmonary arterial pressure but potentiate the effect of hypoxia [14]. The insulin resistant ApoE knockout mice fed on a high fat diet, which have increased blood glucose levels but normal or moderately increased body weight, show PA remodeling and increased PAP [15]. In contrast, the obese non-diabetic Zucker model (OZR), characterized by a mutation in the leptin receptor yielding high circulating leptin levels, obesity and insulin resistance but normal fasting blood glucose, does not present any of the characteristic features of pulmonary vascular disease but rather a hyporresponsiveness to several pulmonary vasoconstrictors [16]. However, at Denver’s altitude, in OZR, overfeeding elicited PA remodeling, neomuscularization of distal arterioles, and elevated PA pressure, accompanied by right ventricular hypertrophy [17]. The Zucker diabetic fatty rats (ZDF/ crl-lepr fa) is an inbred strain derived from OZR carrying an additional yet unidentified mutation leading to hyperglycemia after 10–12 weeks of age [18– 20]. At 4 weeks of age, ZDF rats with moderate weight gain, normoglycemia and normoinsulinemia not only had normal PAP and right ventricular weight but also were partially protected from hypoxia-induced PH [21]. In order to get some further insight into the relationship of PH with obesity, insulin resistance, leptin and hyperglycemia, the present study was designed to analyze the key features of the pulmonary circulation in ZDF at 20 weeks of age, a rat model of obesity, hyperlipidemia, insulin resistance and hyperglycemia, i.e. a clinically relevant model for type 2 diabetes in humans. Materials and methods Animals and ethics statement All experimental procedures utilizing animals were carried out according to the Spanish Royal Decree 1201/2005 and 53/2013 on the Care and Use of Laboratory Animals and approved by Pulmonary vascular dysfunction in diabetes PLOS ONE | https://doi.org/10.1371/journal.pone.0211281 January 28, 2019 2 / 16 Universidad Complutense (www.ucm.es) and Ciberes (www.Ciberes.org) grant with funds from Fundacio ´n Contra la Hipertensio ´n Pulmonar (Enpathy, www.fchp.es) and a FPU grant from Ministerio de Educacio ´n (www.mecd.gob.es), respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. the institutional Ethical Committees of the Universidad Complutense de Madrid (Madrid, Spain) and the regional Committee for Laboratory Animals Welfare (Comunidad de Madrid, Ref. number PROEX-304-15). Male ZDF rats (ZDF/crl-lepr fa, n = 6) and their lean littermates (ZL, +/?, n = 6) were obtained from Charles River Laboratories (L’Arbresle, France). Animals were maintained on standard chow diet and weekly monitored for blood glucose and body weight. Hemodynamic measurements and right ventricular hypertrophy Rats were anesthetized (80 mg/kg ketamine and 8 mg/kg xylacine i.p.), tracheostomyzed and ventilated with room air (tidal volume 9 mL/kg, 60 breaths/min, and a positive end-expiratory pressure of 2 cm H 2 O, Nemi Scientific Inc, Medway, USA). Systolic, diastolic and mean systemic arterial pressure (sSAP, dSAP and mSAP) was measured by cannulation of the carotid artery in closed chest animals. Then, after sternotomy, a catheter was placed in the pulmonary artery (PA) through the right ventricle for systolic, diastolic and mean PA pressure (sPAP, dPAP and mPAP) recording [22]. It should be noted that open-chest measurements in anaesthetized animals underestimate real PAP. We estimated oxygen consumption by the rate pressure product as the product of heart rate and systolic pressure multiplied by 10 −3 at systemic and pulmonary levels [23,24], and pulse pressure was defined as the difference between systolic and diastolic arterial pressure. At the end of the experiment, the right ventricle (RV) and the left ventricle plus the septum (LV+S) were dissected and weighed. The Fulton Index [RV/ (LV+S)] was calculated to assess the right ventricular hypertrophy. Lung histology The right lung was inflated in situ with formol saline through the right bronchus and embedded in paraffin. Lung sections were stained with haematoxylin and eosin and examined by light microscopy, and elastin was visualized by its green auto-fluorescence. Small arteries (25– 100 mm outer diameter) were analyzed in a blinded fashion and categorized as muscular, partially muscular or non-muscular as previously described [22]. Media thickness was measured using image-J software. Four to eight photographs were taken and at least eight arteries were analyzed from each animal. Vascular reactivity Resistance PA rings (diameter ~0.3–0.5 mm and length ~2 mm) were mounted in Krebs solution at 37˚C gassed with a 21% O 2 -5% CO 2 mixture in a wire myograph. After stretching to give an appropriate resting tension (equivalent to 30 mm Hg) vessels were sequentially exposed to different vasoconstrictor agents to test the vascular response, KCl (80 mmol/L), phenylephrine (Phe, 1 nmol/L-10 μmol/L) and serotonin (5-HT, 30 nmol/L-30 μmol/L). The contractile responses were performed by cumulative drug addition and the tissues were washed 3 times with drug-free Krebs after each stimulus and allowed to recover for 30 min. The endothelial function was estimated by the analysis of the relaxant response to the cumulative addition of acetylcholine (1 nmol/L-10 μmol/L) after precontraction with a concentration of phenylephrine titrated to induce a contraction 75% of the response to KCl. Some experiments were carried out in the presence of the inducible nitric oxide synthase inhibitor iNOS inhibitor 1400W (10 μmol/L). RNA extraction and quantitative RT-PCR Total RNA was extracted from lung tissue using miRNeasy Mini Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s instructions. RNA concentration and quality Pulmonary vascular dysfunction in diabetes PLOS ONE | https://doi.org/10.1371/journal.pone.0211281 January 28, 2019 3 / 16 was checked using NanoDropTM 1000 Spectrophotometer (Thermo Scientific). One μg of RNA was reverse transcribed into cDNA using iScriptTM cDNA Synthesis Kit (Biorad, California, USA) following manufacturer’s instructions. Gene expression was determined in triplicates by quantitative real-time PCR (qRT-PCR) with a Taqman Gene Expression Master Mix (Ref: 4369016, Applied Biosystems), with specific primers from Applied Biosystems (Table 1) in the Genomic Unit of the Universidad Complutense de Madrid. A standard Taqman amplification protocol was used as follows: 10 min at 95˚C followed by 40 cycles of 15 s at 95˚C (denaturation) and 1 min at 60˚C (annealing). The efficiency was calculated in preliminary experiments by serial sample dilutions. The delta-delta Ct method was used to quantify relative changes. mRNA expression was normalized by the geometrical mean of the expression of βactin and β2-microglobulin. [25] Protein expression Whole lungs were homogenized with a lysis buffer (Trizma Pre-set cystals pH 7.5, DL-dithiothreitol (DTT, 1M), NP40 (1%) and supplemented with protease (Protease inhibitor cocktail tablets, Roche Diagnosis GmbH) and phosphatase (PhosSTOP, Roche Diagnostics GmbH) inhibitors in a Tissuelyser device (Qiagen, Hilden, Germany). After four short pulses (30 seconds, stopping 15 seconds between each pulse) of sonication, the lysates were centrifuged for 10 min at 10000 rpm. Protein concentration was determined by a colorimetric assay based on the Lowry method (Biorad, California, USA). Twenty μg for lung homogenates were run on a sodium dodecyl sulphate-polyacrilamide electrophoresis and proteins were transferred to polyvinylidene difluoride membranes (Biorad, California, USA). Membranes were blocked by incubation with 5% of BSA or milk for one hour, and were incubated overnight at 4˚C with primary antibodies (Table 2). Membranes were then incubated with the appropriate secondary antibodies conjugated with horseradish peroxidase at room temperature for one hour. Antibody binding was detected by an ECL system (SuperSignal West Fento Chemiluminescent Substrate, Thermo Scientific, USA). Blots were imaged using an Odissey Fc System (Li-COR Biosciences, USA) and were quantified by densitometry using Quantity One software. Results were normalized to the relative expression of smooth muscle β-actin. Table 1. Taqman primers used in this study (all from Applied Biosystems). Gen Reference Gene Bank Amplicon lenght R 2 Efficiency Actin, beta (Actb)Rn00667869_m1 NM_031144.3 91 0.99 87% Beta-2 microglobulin (B2m) Rn00560865_m1 NM_012512.2 58 0.99 103% Bone morphogenetic protein receptor type 2 (Bmpr2) Rn01437214_m1 NM_080407.1 104 0.99 108% Potassium two pore domain channel subfamily K member 3 (Kcnk3) Rn04223042_m1 NM_033376.1 72 0.99 99% Potassium voltage-gated channel subfamily A member 5 (Kcna5) Rn00564245_s1 NM_012972.1 63 0.99 102% Potassium voltage-gated channel subfamily Q member 1 (Kcnq1) Rn00583376_m1 NM_032073.1 85 0.99 97% Potassium voltage-gated channel subfamily Q member 4 (Kcnq4) Rn01518851_m1 XM_008764109.1 71 0.99 102% Potassium voltage-gated channel subfamily Q member 5 (Kcnq5) Rn01512013_m1 NM_001134643.2 89 0.99 102% https://doi.org/10.1371/journal.pone.0211281.t001 Table 2. Primary antibodies used in this study. Primary antibody Protein Specie Supplier Reference Dilution Anti-β-Actin β-actin Mouse Sigma-Aldrich A1978 1:5000 Anti-eNOS eNOS Mouse BD Transduction Laboratories 610296 1:1000 Anti-iNOS iNOS Rabbit Santa Cruz Biotechnology SC-650 1:200 https://doi.org/10.1371/journal.pone.0211281.t002 Pulmonary vascular dysfunction in diabetes PLOS ONE | https://doi.org/10.1371/journal.pone.0211281 January 28, 2019 4 / 16 Measurement of proinflammatory cytokines Tumor necrosis factor-α(TNF-α) and interleukin-6 (IL-6) levels were quantified in serum samples and lung homogenates by specific rat TNF-alpha Quantikine ELISA Kit (RTA00, R&D System, USA) and IL-6 DuoSet ELISA Kit (DY506, R&D System, USA) according to the manufacturer’s instruction. Drugs All drugs were from Sigma-Aldrich Quimica (Spain). Statistical analysis Data are expressed as means ±s.e.m. All data passed normality test. Statistical comparisons were performed, unless otherwise stated, using two-tailed unpaired t tests for continuous variables. The Chi square test was used for the analysis of arterial muscularization. P <0.05 was considered statistically significant. Results Body weight, glucose, insulin and cytokines At the age of 20 weeks, ZDF showed marked increases in body weight, plasma glucose and plasma insulin as compared to ZL (Table 3, P <0.01 for all comparisons). As expected, insulin resistance, measured by the HOMA-IR index, was significantly increased in ZDF rats compared to lean controls (P <0.01). The circulating levels of the cytokines IL-6 and TNF-alpha in serum or lung IL-6 were not significantly different between ZL and ZDF rats (Table 3). Hemodynamics and RV hypertrophy The systolic, diastolic and mean PAP were significantly elevated in ZDF when compared with ZL rats (Fig 1A and 1B). This was accompanied by increased systolic, diastolic and mean systemic arterial pressures (SAP, Fig 1C). Differences were more marked for systolic than for diastolic pressures and, therefore, pulse pressure was significantly elevated in the systemic but not in in the pulmonary circulation (Fig 1D). ZDF rats also showed decreased heart rate (Fig 1E). The rate pressure product as an indicator of the myocardial oxygen consumption did not show differences between ZL and ZDF rats at systemic and pulmonary levels (Fig 1F). Absolute RV but not LV+S weight was significantly increased in ZDF rats (Fig 2A). Notably, the RV weight referred to LV+S, i.e. the Fulton index, was also elevated in ZDF rats (Fig 2B). RV contractility estimated by (dP/dt max)/P calculated from the right ventricular pressure wave was not different in the two groups (37.6 ±1.5 vs 35.2 ±1.2 s -1 , respectively). Fig 2C shows that there is a good correlation between the mPAP and the Fulton index, which was consistent when data from both groups of rats were analyzed individually or when all values were pooled. Table 3. Body weight, plasma glucose, insulin, HOMA-IR index and cytokine levels. Body weight (g) Plasma glucose (mg/dL) Insulin (μg/L) Homa-IR index Serum IL-6 (pg/ml) Serum TNFα (pg/ml) Lung IL-6 (ng/mg protein) ZL 328 ±6 79 ±5 0.38 ±0.02 2.7 ±0.1 166 ±20 13 ±1 1.67 ±0.28 ZDF 455 ±11 �� 262 ±17 �� 4.50 ±0.50 �� 87 ±11 �� 197 ±87 13 ±1 1.40 ±0.33 Values are expressed as means ±s.e.m. n = 6 per group. �� indicates P <0.01, ZDF versus ZL (unpaired t test). https://doi.org/10.1371/journal.pone.0211281.t003 Pulmonary vascular dysfunction in diabetes PLOS ONE | https://doi.org/10.1371/journal.pone.0211281 January 28, 2019 5 / 16 Fig 1. ZDF rats show increased pulmonary and systemic arterial pressure. (A) Typical pulmonary arterial pressure (PAP) recordings. (B) Systolic, diastolic and mean PAP. (C) Systolic, diastolic, and mean systemic arterial pressure (SAP). Pulmonary vascular dysfunction in diabetes PLOS ONE | https://doi.org/10.1371/journal.pone.0211281 January 28, 2019 6 / 16 Lung histology Small PA from lung sections from ZL and ZDF rats (Fig 3A) were classified in a blinded fashion as muscular, partially muscular and non-muscular arteries. The percentage of muscularized arteries was not significantly different (Fig 3B) in ZL vs ZDF. However, the small muscularized arteries in ZDF rats of lumen less than 75 μm (Fig 3C) showed a modest but significant increase in pulmonary arterial wall thickness compared to ZL rats (Fig 3D, P<0.05). We did not observe other apparent histological changes. (D) Pulse pressure, (E) Heart rate, (F) Rate pressure product. Data are shown as scatterplots and means of 6 animals (except SAP could not be recorded in one ZDF rat). �� and ��� indicate P <0.01 and P<0.001, respectively, ZDF versus ZL (unpaired t test). https://doi.org/10.1371/journal.pone.0211281.g001 Fig 2. ZDF rats show right ventricular hypertrophy. (A) Right ventricular (RV) weight and left ventricular plus septum (LV +S) weight as absolute values and (B) Fulton index [RV/(LV+S) ratio]. (C) Correlation between mPAP and Fulton index. The dotted line represents the linear regression for pooled data from both groups (r 2 = 0.72, p<0.001. Results are expressed as scatter plots and means of 6 animals, �,�� and ��� indicate P <0.05, P <0.01 and P<0.001, respectively, ZDF versus ZL (unpaired t test). https://doi.org/10.1371/journal.pone.0211281.g002 Pulmonary vascular dysfunction in diabetes PLOS ONE | https://doi.org/10.1371/journal.pone.0211281 January 28, 2019 7 / 16 Vascular reactivity The responses to vasoconstrictor and vasodilators were analyzed in isolated PA mounted in a wire myograph (Figs 4and 5). The response to KCl, which is regarded as an index of the contractile capacity of the vessel, was borderline significantly increased in ZDF rats (Fig 4A). However, we found no differences in the concentration-response curves for the vasoconstrictor responses to the α-adrenergic receptor phenylephrine (Fig 4B) or to serotonin (5-HT, Fig 4C) in ZDF compared to ZL. The endothelium-dependent vasodilator responses to acetylcholine (Fig 5A) were also similar in the two groups. Consistently, the protein expression of eNOS in Fig 3. Histological changes in the pulmonary circulation. (A) Representative images of cross-sections of lungs stained with hematoxylin and eosin. On the left a non muscularized small PA from a ZL rat. On the right a muscularized small PA from a ZDF rat. The insets show the green autofluorescence of elastin. (B) Percentage of muscular, partially muscular and non-muscular PA. (C) Lumen and (D) Media wall thickness in PA <75μm. Results are expressed as scatter plots of media thickness in each individual artery from 6 ZL and 6 ZDF rats. �indicates P <0.05, ZDF versus ZL (unpaired t test). https://doi.org/10.1371/journal.pone.0211281.g003 Pulmonary vascular dysfunction in diabetes PLOS ONE | https://doi.org/10.1371/journal.pone.0211281 January 28, 2019 8 / 16 the lung was also similar (Fig 5B). We also analyzed the effect of the iNOS inhibitor 1400W on the contractile responses induced by phenylephrine in ZL (Fig 5C) and ZDF (Fig 5D) rats. This inhibitor modestly increased the response to the vasoconstrictor in ZDF which was only significant at the highest concentration analyzed (paired t test) but had no significant effect in the ZL rats. However, iNOS expression in whole lung homogenates was not significantly different between groups (Fig 5E). Gene expression We analyzed the expression of several genes involved in PH such as Bmpr2,Kcna5,Knck3, Kcnq1,Kcnq4 and Kcnq5 in the lungs of ZL and ZDF rats (Fig 6). Bmpr2 (Fig 6A) was significantly reduced in ZDF rats but the other genes were unchanged. There was a trend for reduced Kcna5, which encodes the voltage-gated potassium channel Kv1.5, but the expression was Fig 4. ZDF rats show normal PA pulmonary artery contraction. (A) Contractile responses to 80 mM KCl. (B and C) Cumulative concentration-response curves to (B) the alpha-adrenergic agonist phenylephrine and (C) serotonin (5-HT). Results are expressed as means ±SEM of 5 (ZDF) and 4 (ZL) rats in duplicate. 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