Hindawi Oxidative Medicine and Cellular Longevity Volume 2018,Article ID 4036709, 15 pages https://doi.org/10.1155/2018/4036709 


ResearchArticle 

Comparison of Pulmonary and Systemic NO-and PGI2-Dependent Endothelial Function in Diabetic Mice 
Andrzej Fedorowicz 

, 1,2 Elżbieta Buczek 
, 1 Łukasz Mateuszuk,1 Elzbieta Czarnowska,3 
Barbara Sitek,1 Agnieszka Jasztal,1 Antonina Chmura-Skirlińska,1 Mobin Dib,4 1,2
Sebastian Steven,4 Andreas Daiber 

, 4 and Stefan Chlopicki 
1Jagiellonian Centre for Experimental Pharmacology (JCET), Jagiellonian University, Bobrzyńskiego 14, 30-348 Krak, Poland 2Chair of Pharmacology, Jagiellonian University Medical College, Grzegzecka 16, 31-531 Krak, Poland 3Department of Pathology, The Children’s Memorial Health Institute, Al. Dzieci Polskich 20, 04-730 Warsaw, Poland 4Center for Cardiology 1, Laboratory of Molecular Cardiology, University Medical Center of the Johannes Gutenberg University, 
Mainz 55131, Germany 

Correspondence should be addressed to Stefan Chlopicki; stefan.chlopicki@jcet.eu 
Received 17 January 2018; Revised 3 April 2018; Accepted 16 April 2018; Published 4 June 2018 
Academic Editor: Valeria Conti 

Copyright©2018 Andrzej Fedorowicz et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 
Diabetes increases the risk of pulmonary hypertension and is associated with alterations in pulmonary vascular function. Still, it is not clear whether alterations in the phenotype of pulmonary endothelium induced by diabetes are distinct, as compared to peripheral endothelium. In the present work, we characterized di.erences between diabetic complications in the lung and aorta in db/db mice with advanced diabetes. Male, 20-week-old db/db mice displayed increased HbA1c and glucose concentration compatible with advanced diabetes. Diabetic lungs had signs of mild .brosis, and pulmonary endothelium displayed signi.cantly ultrastructural changes. In the isolated, perfused lung from db/db mice, .ltration coe.cient (Kf,c)and contractile response to TXA2 analogue were enhanced, while endothelial NO-dependent modulation of pulmonary response to hypoxic ventilation and cumulative production of NO- 2 were impaired, with no changes in immunostaining for eNOS expression. In turn, 6-keto-PGF1. release from the isolated lung from db/db mice was increased, as well as immunostaining of thrombomodulin (CD141). In contrast to the lung, NO-dependent, acetylcholine-induced vasodilation, ionophore-stimulated NO- 2 generation, and production of 6-keto-PGF1. were all impaired in aortic rings from db/db mice. Although eNOS immunostaining was not changed, that of CD141 was clearly lowered. Interestingly, diabetes-induced nitration of proteins in aorta was higher than that in the lungs. In summary, diabetes induced marked ultrastructural changes in pulmonary endothelium that were associated with the increased permeability of pulmonary microcirculation, impaired NO-dependent vascular function, with compensatory increase in PGI2 production, and increased CD141 expression. In contrast, endothelial dysfunction in the aorta was featured by impaired NO-, PGI2-dependent function and diminished CD141 expression. 
1. Introduction 
Diabetes induces profound alterations in systemic circulation and is the leading cause of macro-and microangiopathies such as diabetic retinopathy, nephropathy, and myocardial infarct, as well as peripheral artery disease [1–3]. The detri­mental e.ects of diabetes in the lungs are less clinically apparent. However, epidemiological and experimental data suggested that insulin resistance and diabetes a.ectthe lung. 
In diabetic subjects, the risk of pulmonary hypertension and pulmonary embolism was increased [4]. Interestingly, several studies have suggested that diabetes results in the impairment of respiratory function [5–7] and increased susceptibility to allergic response/in.ammation induced with LPS or airway bacterial infection [8–10]. Structural changes in blood-alveolar barrier and di.usion impairment in vivo have also been reported [11, 12]. Despite these reports, the possible detrimental e.ects of insulin resistance and diabetes on the pulmonary circulation have received little attention, and therefore research on this aspect of the pathophysiology of diabetes has largely been neglected. To the best of our knowledge, there are only few reports directly comparing systemic and pulmonary circulation response to diabetes in the same experimental model. Such an approach could give abetter understanding of similarities and di.erences between diabetes-induced changes in pulmonary and peripheral circulation [13, 14]. 
Furthermore, although peripheral endothelial dysfunc­tion represents a well-recognized hallmark of peripheral dia­betic macro-and microangiopathies [2, 7, 15], the evidence on the development of pulmonary endothelial dysfunction in diabetes is rather con.icting. Both the presence and lack of impairment of NO-dependent pulmonary endothelial functionhavebeen reported[13,16,17].Inturn,an increase in pulmonary microvascular permeability without changes in eNOS or with increased iNOS expression has also been dem­onstrated [13, 16–18]. 
Similarly, reports on diabetes-induced changes in PGI2 production in the pulmonary circulation are also not consistent.In streptozotocin-treated rats, basal PGI2 produc­tion and stimulated PGI2 production in pulmonary circulation were reported to increase or to remain unchanged [14, 19, 20]. Interestingly, PGI2 is a major regulator of the expression of thrombomodulin (CD141) [21, 22], which complexes with thrombin (IIa) and activates protein C to act as an anticoagulant and endothelial protective mediator [23]. Thus, the changes in the activityof PGI2 may result in the alteration in the activity of thrombomodulin, despite the fact that previous studies reported no changes [24–26]. 
Given the nonconsistent literature, the aim of the present work was to characterize changes in pulmonary endothelial function in comparison with changes in peripheral endothe­lial function in the aorta, with special focus on NO-and PGI2-dependent pathways. For this purpose, male db/db mice at the age of 20 weeks with features of advanced diabetes were used, and pulmonary and peripheral endothelial functions and NO and PGI2 activities were analyzed in the isolated, perfused diabetic lung, or in the aortic rings, respectively. 


2. Material and Methods 
2.1. Animals. 20-week-old db/db (BKS.Cg-Dock7m +/+LeprdbJ) and C57BL/6J mice, purchased from Charles River Laboratories, were housed in speci.c pathogen-free conditions (SPF) and fed with a standard laboratory diet and water ad libitum. 
All experimental procedures used in the present study were conducted according to the Guidelines for Animal Care and Treatment of the European Communities and the Guide for the Care and Useof Laboratory Animals publishedby the US National Institutes of Health (NIH Publication number 85-23, revised 1996). All procedures were approved by the local Jagiellonian University Ethical Committee on Animal Experiments (number 53/2009). 

2.2. Blood Count, HbA1c, and Basal Biochemistry in Plasma. Blood was collected from anaesthetized animals 
(pentobarbital, 140 mg/kg, i.p.) via the right ventricle to a syringe with nadroparine (end concentration: 10 U/ml) for analysis of blood count and HbA1c, and the rest of the sample were centrifuged to obtain plasma (1000g, 5min, 4 ° C). Complete blood count was analyzed within 15 minutes after collection (by automatic blood counter ABC Vet, HORIBA). HbA1c andtotalhemoglobin concentrations were measured using a biochemical analyser (ABX Pentra 400, HORIBA),andtheratio wasgiven asapercentageofHbA1c. Glucose, aspartate aminotransferase, alanine aminotrans­ferase, creatinine, albumin, and total protein were measured using colorimetric methods (ABX Pentra 400, HORIBA). 
2.3. Histological Analysis of the Lungs. The lungs were removedunder anaesthesia and .xed in 4% bu.ered forma­lin (24h) and were then dehydrated, embedded in para.n, cut into 5 µm sections on Accu-Cut® SRM™ 200 Rotary Microtome, and stained with either hematoxylin and eosin (H&E), Masson Trichrome, Orcein and Methyl Scarlet Blue (OMSB[27]), or Picro Sirius Red. Light microscopic exami­nation and photographic documentation were performed using an Olympus BX53F microscope equipped witha digital camera. 
2.4. Assessment of Changes in Lung Ultrastructure. The chest of anaesthetized rats was opened, and samples of lung tissue were cut and .xed immediately using a mixture of 2.5% glutaraldehyde and 2% freshly prepared paraformaldehyde in 0.1 mol/L cacodylate bu.er at pH7.4. The lung tissue was .xed for 12h at4 ° C. Then, the lungs were post.xed in bu.ered 2% osmium tetroxide, dehydrated in a graded ethanol series and propylene oxide, and embedded in Epon 
812. The ultrathin sections were stainedaccording to routine protocol with uranyl acetate and lead citrate and were exam­ined and documented by transmission electron microscopy (Jem 1011, JEOL, Japan). 
2.5. Immunohistochemistry of Lung Tissue. After excision, lung tissues were .xed with 4% formalin solution (10 min) and placed in 50% OCT for cryopreservation (24 h), then snap frozen at -80 ° C. Blocks were cut into 10 µm-thick cross-sectional slides. 5% normal goat serum (Jackson Immuno) or 2.5% horse serum (Vector Labs) and 2% .ltered dry milk were applied to minimalize nonspeci.c binding of antibodies. For indirect immunohistochemical detection of von Willebrand factor (vWF), thrombomodulin (CD141), endothelial nitric oxide synthase (eNOS), vascular cell adhesion molecule 1 (VCAM-1), and macrophage content (MAC3), sections were incubated with rabbit anti-vWF polyclonal Ig (Abcam), rat polyclonal anti-CD141 Ig (BD Bioscience), mouse monoclonal anti-eNOS Ig (BD Biosci­ence), rat anti-VCAM-1 monoclonal Ig (Millipore), or rat anti-MAC3 monoclonal Ig (Thermo), respectively (1h). Antibodies were applied at concentrations of 5µg/ml or 10µg/ml (dilution1:100–1:300 of stock solution). After rinsing in PBS, secondary biotinylated horse anti-rabbit (Vector Labs), goat anti-mouse, goat anti-rat, or goat anti­rabbit (Jackson Immuno) antibodies were applied for 30min. At a third step of staining, Cy3-conjugated streptavidin (Jackson Immuno) and Hoechst 33258 solution were used. 
2.6. Assessment of Endothelial Function in the Isolated Lung Preparation. Trachea in anesthetized mice were cannulated, and the lungs were ventilated with positive pressures at a rate of 90 breaths/min (VCM module from Hugo Sachs Electronic (HSE)). After laparotomy, the diaphragm was cut and nadroparine at a dose of 600 I.U. was injected into the right ventricle to prevent microthrombi formation during the surgical procedure. Then, the animals were exsangui­nated by incision of the left renal artery. The lungs were exposed via a median sternotomy. The pulmonary artery and left atrium were cannulated via the right and left atrium, respectively. 
Immediately after cannulation, the lung/heart block was dissected from the thorax. Using tracheal cannula, the iso­lated lung was mounted in a water-jacketed (38 ° C), air-tight glass chamber (HSE) and ventilated with negative pressures. The lungs were perfused with low-glucose DMEM with 4% albumin and 0.3% HEPES; the pH of perfusate was maintained at 7.35 throughout the whole experiment by continuous addition of 5% CO2 to the inspiratory air, using a peristaltic pump (ISM 834, HSE) at a constant .ow (CF) of about 1.50 ml/min. The venous pressure was set between 2and5cmH2O. The end-expiratory pressurein the chamber was set to be -3cmH2O, and inspiratory pressure was adjusted between -6and-10 cmH2Oto yield the initial tidal volume (TV) of about 0.2 ml. Breathing frequency was set to be 90 breaths/min, and a duration of inspiration versus expiration was1:1 in each breath. Every5min throughout the experiments, a deep breath of end-inspiratory pressure of -21 cmH2O was automatically initiated by VCM module (HSE) to avoid atelectasis. Air.ow velocity was measured with a pneumotachometer tube connected to a di.erential pressure transducer (HSE), from which the value of respira­tory tidal volume was determined. In experiments with constant pressure perfusion (CP), CP mode was turned on just after placing the lungs in the arti.cial thorax. The PAP wassettobe around3cmH2O.Thevenous pressurewasset between2and5cmH2O. 
Both arterial and venous pulmonary pressures (PAP, PVP) were continuously monitored by ISOTEC pressure transducers (HSE) connected to a perfusion line on arte-rial and venous sides, respectively. The weight of the lungs was monitored by a weight transducer (HSE). TC, PAP, PVP, and lung weight data were acquired by the PC trans­ducer card and subsequently analyzed by Pulmodynpulmo software (HSE). 
All lung preparations were allowed to equilibrate for the .rst 15 min of perfusion with fresh bu.er until baseline PAP, PVP, TV, and weight were stable. At this time point, weight of the lung (the value of which varied considerably between experiments) was set to zero. 
2.6.1. Hypoxic Pulmonary Vasoconstriction (HPV). HPV was evoked by 10-minute intervals of hypoxic ventilation with a mixture of 95%N2 and 5% CO2. HPV, measured as changes in PAP, was stabilized after 5 minutes. After cessation of acute hypoxia, PAP returned to a basal level. There was a 10-minute interval of normal ventilation between HPV procedures. HPV was repeated twice, then L-NAME (300 µM) was added to the perfusate and recirculated through the lung for 10 minutes, and HPV response was repeated twice again. Although TV, PAP, PVP, and weight were continuously monitored throughout the experiment, for data analysis, only maximum increase in PAP(.PAP) elicited by HPV was taken. TV, PVP, and weight did not change signi.cantly during HPV. 
2.6.2. Vasoreactivity. After equilibration, U46619 (1µM) was added to the perfusate, which resulted in an increase of PAP, but other parameters of isolated lungs did not change. For data analysis, only maximum increase of PAP(.PAP) was taken. 
2.6.3. Pulmonary Microcirculation Permeability. In equili­brated isolated lung, perfused with constant pressure, the pulmonary venous pressure was increased to obtain PVP 
1.5 cmH2O above PAP and was maintained at this level through 15 minutes. This resulted in an increase in weight of the lungs; other parameters were stable. After 15 minutes, PVP was set to basal value, and the process was repeated. The .ltration coe.cient(Kf,c)was calculated based on recordings for5minutes after PVP increase [28]. 
2.6.4. Biochemical Measurements. 6-keto-PGF1. and NO- 2/ NO- 3 concentrations were measured in samples of e.uents collected after 15 minutes of equilibration of isolated lungs perfused with constant .ow, and then 5 and 45 minutes after recirculation of the perfusate was started. To assess the enzymatic source of 6-keto-PGF1., a sample was taken before and after administration of COX-2 selective inhibitor (DuP-697, 1 µM) or nonselective COX-1/COX-2 inhibitor (indomethacin,1 µM). 
2.7. Assessment of Endothelial Function in the IsolatedAortic Rings. The thoracic aorta was quickly dissected out of the chest of anaesthetized mice, and the surrounding fat/connec­tive tissue was removed in Krebs-Henseleit (KH) solution (mM: NaCl 118.0, CaCl2 2.52, MgSO4 1.16, NaHCO3 24.88, K2PO4 1.18, KCl 4.7, glucose 10.0, pyruvic acid 2.0, and EDTA 0.5). Then, the aorta was cut into 2-3 mm rings, which were mounted between two pins .lled with 5ml of KH solution chambers (37 ° C, pH7.4, gassed with carbogen: 95%O2,5%CO2)of wire myograph(620M, DanishMyo Technology, Denmark). The unstretched aortic rings were allowed to equilibrate for 30 minutes. Then, the resting tension of the rings was increased stepwise to reach 10mN, and the rings were washed with fresh KH solution and incubated to equilibrate for the next 30 mins. 
After equilibration, the viability of the tissue was exam­ined by contractile responses to potassium chloride (KCl 30mM, 60mM), and then the aortic rings were contracted with phenylephrine (Phe 0.01–3.0 µM) to obtain maximal possible constriction of the rings. All tissue responses were recorded, using a data acquisition system and recording soft­ware (PowerLab, LabChart, and ADInstruments, Australia). The aortic rings were next contracted with phenylephrine to obtain 80–90% of maximal contraction, and the endothelial-dependent response was assessed using cumula­tive concentrations of acetylcholine (ACh 0.01–10 µM). After washout, the vessels were again contracted with phenyleph­rine, and endothelial-independent vasodilation to cumulative concentrations of sodium nitroprusside (SNP 0.001–1µM) was assessed. The relaxation response was expressedasa per­centage of the precontraction induced by phenylephrine. 
2.8. Assessment of Prostacyclin (PGI2) Production in the Isolated Aortic Rings. The concentration of PGI2, produced by aortic rings, was quanti.ed on the basis of the formation of 6-keto-PGF1., a stable metabolite of PGI2. The aorta rings were preincubated for 15 minutes on the thermoblock (Liebisch Labortechnik) at a temperature of 37 ° C, in 250µl KH bu.er, gassed with carbogen in the absence or in the presence of COX-2 selective inhibitor (DuP-697, 1 µM) or nonselective COX-1/COX-2 inhibitor(indomethacin,5 µM). All inhibitors were dissolved in DMSO, and then control rings were incubated with addition of the same amount of DMSO (1µl/ml). 
Aortic rings were then incubated for 60 minutes, and samples of e.uents were collected after 3 and 60 minutes. After the experiment, aortic rings were dried (1h, 50 ° C) and weighed. 6-keto-PGF1. concentration in the e.uents was measured using an EIA kit (Enzo, Life Technologies). Results were expressed as the change in 6-keto-PGF1. con­centration between 60 and 3 minutes of ring incubation and normalized to dry weight of aortic rings (pg/ml/mg). 
2.9. Assessment of Nitrite Production in the Isolated Aortic Rings. Basal NO production by the aorta was estimated by measurements of nitrite, a primary stable product of nitric oxide oxidation, and thus considered relevant for estimation of NO synthesis by the aortic endothelium. Segments from the aortic arch were longitudinally opened, placed in 96­well plates facing up with endothelium, and incubated for one hour in 120 µlKHbu.er at37 ° C, using a specially designed closed chamber (BIO-V(Noxygen)) that was equilibrated with carbogen gas mixture (95%O2,5%CO2). The nitrite concentration after back reduction to NO was measured using the gas-phase chemiluminescent reaction between NO and ozone using a Sievers* Nitric Oxide Ana­lyzer NOA 280i. The reduction of nitrites was performed in a closed glass chamber containing a reducing agent (1% wt/ vol of KI in acetic acid) to convert nitrite to NO. The inde­pendent calibration on fresh NaNO2 standard solution was prepared for every experiment before measurements of series of samples after each re.lling of glass reaction chamber, according to the manufacturer’s instructions (Sievers* Nitric Oxide Analyzer NOA 280i). The limit of detection was around 10nM of nitrite. Multiple blank samples (without aortic rings) were used to monitor nitrite contamination in the bu.er and/or by laboratory atmosphere in every set of experiments. The averaged blank signal from a blank sample in a given experiment was subtracted as a background signal. Samples were kept on ice and measured directly after experiments. Nitrite concentration was expressed as ng/ml/mg of dry weight of aortic rings. 
2.10. Immunohistochemistry in Aorta. Dissected thoracic aorta, cleared of the surrounding fat/connective tissue in Krebs-Henseleit (KH) solution, was .xed with 4% formalin solution (10 min), embeddedin 50% OCT for cryopreserva­tion (24h), snap frozen at -80 ° C, and cut in 10µm-thick cross-sectional slides for immunohistochemistry. 2.5% horse serum (Vector Labs) and 2% dry milk were applied to mini­malize nonspeci.cbinding of antibodies. For von Willebrand factor (vWF) staining, rabbit anti-mouse vWF polyclonal Ig (Abcam) was used, followed by biotinylated horse anti­rabbit Ig (Vector Labs) and Cy3-streptavidin (Jackson Immuno), as described above. Nuclei were counterstained with Hoechst 33258 (Sigma-Aldrich). Images were acquired using an Axio Observer D2 .uorescent microscope, and .uorescence parameters were analyzed automatically by Columbus software (PerkinElmer). 
2.11. Dot Blot Analysis in Aorta and Lungs. Protein expres­sion and modi.cation were assessed by standard dot blot analysis using established protocols [29]. 3-Nitrotyrosine­
(3NT-) positive proteins were assessed by dot blot analysis of protein homogenates in aorta and lungs, which were transferred to a Protran BA85 (0.45 µm) nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) by a MinifoldI vacuum dot-blot system (Schleicher&Schuell, Dassel, Germany) [30].A mouse monoclonal 3NT antibody (1:1000, Upstate Biotechnology, MA, USA) was used for dot blot analysis. Detection and quanti.cation of all blots were performed by ECL with peroxidase anti-mouse (1:10,000, Vector Lab., Burlingame, CA). Densitometric quanti.cation of antibody-speci.c bands was performed with a ChemiLux Imager (CsX-1400 M, Intas, Gtingen, Germany) and Gel-Pro Analyzer software (Media Cyber­netics, Bethesda, MD). 
3. 
Statistical Analysis 

Results are presented as the mean ±SEM. The normality of the results was analysed using the D’Agostino & Pearson omnibus normality test and the Shapiro-Wilk test. To calculate statistical signi.cance, a paired Student’s t-test, Mann-Whitney test, or unpaired Student t-test was used. Post hoc analysis was calculated using Dunn’s multiple comparisons test. 

4. 
Results 


4.1. Basal Characteristics of db/db Mice. 20-week-old db/ db mice were obese (body weight: 53.77 ±0.18 versus 
29.5 ±0.12g, db/db and control, resp.; P<0 
05) and had increased HbA1c (15.38 ±1.7 versus 4.12 ±1.36%, db/db and control, resp.; P<0 
05)and fasting glucose concentra­tion in plasma (40.64 ±5.41 versus 8.66 ±1.24 mmol/l, db/ db and control, resp.; P<0 
05)as compared to control mice. In addition to hyperglycaemic pro.le, db/db mice displayed signs of liver injury (increased plasma AST, ALT, e.g., for ALT: 141.90 ±19.68 versus 38.02 ±2.80, db/db and control, resp.; P<0 
001) and kidney injury (increased plasma 


(a) (b) 



(c) 


(d) 


(e) (f) 


(g) (h) 

Figure 1: Histology of the lungs and ultrastructure of pulmonary endothelial cells from the control (a, c, e, g) and diabetic (b, d, f, h) lungs (db/db mice). (a) Histological structure of the control lungs. (b) In.ammation in the lung tissue: increased amount of cells (including granulocytes and macrophages) and (d) collagen in parenchyma of the diabetic lung tissue as compared to control; visible decreased aerial space in diabetic lungs as compared to the control (c). (e) Microphotographs of ultrastructure of the control lungs—blood-air barrier (alveolar–capillary barrier) with normal endothelial layer. (f, g, h) Microphotographs of ultrastructure of the lungs from the db/db mice. 
(f) Capillary endothelial cells (arrows) with numerous plasmalemmal vesicles (caveolae) on thickened blood-air barrier (bab). In the center: collapsed pulmonary alveoulus, on the right: blood platelet (plt) inside the vessel. (g) A presence of convoluted apical region in endothelial cells with cytoplasmic extensions on hyperplastic basal laminae enriched with elastine (el); on the bottom: red blood cell (rbc). 

(h) Endothelial cells of various heights (arrows) separated by thickened blood-air barrier (bab) from pulmonary alveolus; neighbouring red blood cell (rbc). Representative images of at least3independent experiments. 


8.0 6.0 
6.0 
4.0 4.0

Aft er U46619 (cmH2O) 
db/db Basal PAP(cmH2O) Control




.PAP
2.0 
0.0 2.0 
0.0 
Controldb/db
Control
Kf,c 
(ml/min/cmH2O/100 g of body weight)
db/db 
15.0 

(a) (b) 
0.04 
. 1.5 
. 
Resistance(cmH2O/ml/s)Compliance(ml/cmH2O)
0.03 
0.00 

0.0 

10.0 
5.0 
0.0 1.0 

0.02 0.50.01 
Controldb/dbControl
db/db 

(c) (d) (e) 
Figure 2: Comparison of basal parameters of the isolated, perfused lungs amongst diabetic and control animals. (a) No change in basal pulmonary pressure, although (b) enhanced reactivity to thromboxane analogue in the lungs of the diabetic mice (U-44619,1 µM, control n=5, diabetes n=5). (c) Decreased compliance without changes in (d) resistance of the lungs (control n=5, diabetes n=6). (e) Increased .ltration coe.cient in the diabetic pulmonary circulation (control n=5, diabetes n=5). Data are presented as the means±SEM. *P<0
05. 
creatinine 62.90 ±63.61 versus 46.74 ±7.02 µmol/(L* cm2), db/db and control, resp.; P<0
05). 
4.2. Histology and Ultrastructure of Lungs in db/db Mice. 
Lungs from db/db mice displayed in.ammation, as evidenced by multicellular (includinggranulocytes, macrophages) in.l­trations in the interstitial space (HE staining, Figure 1(b)) as compared to the control group (Figure 1(a)); mild .brosis (increased amount of collagen), as evidenced by Trichrome staining (Figure 1(d)) as compared to the control group (Figure 1(c)); and endothelial in.ammation, as evidenced byincreased VCAM-1expression in pulmonary endothelium (480,937 ±70,112 versus 247,193 ±64,821 AU, db/db and control, resp.; P=0
07). Interestingly,ultrastructural investi­gations con.rmed the presence of numerous macrophages (oftenlyingnexttoeach other)inthelungsfromdb/dbmice, as compared to control mice, and they were also detected as adhering to endothelium (data not shown). Moreover, semi­thin sections of lung tissue revealed diminished area of alveoli 
(34.13 ±4.25 versus 50.75 ±3.83, db/db and control, resp.). Capillary endothelial cells displayed protruded apical regions into the capillary lumen, increased area of sarcoplasmic retic­ulum, plasmalemmal vesicles (caveolae), and sometimes presence of multivesicular bodies or lysosomes. 
One of the typical features of db/dbpulmonary microcir­culation was the hyperplasia of basal lamina in db/db (Figures 1(f)and 1(h)) that was not evident in control sam­
ples (Figures 1(e) and 1(g)). Thickness of capillaries’ basal lamina ranges from 0.1 µm to 0.35 µm, compared to 0.05 µm in controls. Additionally, septa separating lung alve­oli in db/db were thicker, with abundant collagen .brils and probably contained also elastin .brils marked by an uncon­trasted area in the thin sections routinely stained with uranyl acetate and lead citrate (Figures 1(f) and 1(h)), which was also not seen in control samples (Figures 1(e) and 1(g)). 
4.3. Alterations in Pulmonary Vascular Function and In.ammation in the Isolated, Perfused Lung from db/db Mice 
4.3.1. Changes in Basal Pulmonary Parameters and Vasoreactivity. The basal pulmonary artery pressures in the isolated, perfused lungs (bPAP) were comparable in db/db and control mice (PAP: 4.70 ±0.62 versus 5.20 ±0.30 cmH2O, db/db and control, resp., Figure 2(a)). Vasoreactivity to thromboxane A2 analogue U46619 was increased threefold in the isolated lungs from db/db mice (.PAP: 
4.83 ±0.32 versus 1.43 ±0.69 cmH2O, db/db and control, resp.; P=0
073)(Figure 2(b)). Compliance but not resistance was decreased in db/db mice (compliance: 0.016 ±0.002 versus 0.030 ±0.003 ml/cmH2O, db/db and control, resp.; P<0
05; resistance: 1.09 ±0.04 versus 0.99 ±0.04 cmH2O/ ml/s, db/db and control, resp.; Figures 2(c) and 2(d)). 
4.3.2. Changes in Permeability Coe.cient (Kf,c). In the isolated, perfused lung, an increase in pulmonary venous pressure resulted in slow, reversible weight gain of the lungs, bothin control and db/db mice. CalculationofKf,c revealeda higher .ltration coe.cient in the diabetic lungs as compared with the control lungs (8.70 ±1.33 versus 5.02 ±0.27 ml/ 
6.0
.. 0.6
4.0 
p = 0.357 . 





3.0 0.4 4.0 
1.0 

0.0 


0.0
0.0 

.NO3Basal NO2­During 25 min of recirculation ( 
. M) (
. M) 
. 6-keto-PGF1
. Basal NO3­In 40 min of recirculation (pg/ml)(
. M)

(cmH2O).PAP
2.0 0.2 2.0 
-+-+
L-NAME 
Control
db/db 


db/db
Control
db/dbControl

db/db
db/db 

(a) (b) (c) 
.NO2
During 25 min of recirculation ( 
. M)
500 .. 
0.3 . 0.0 
-0.5 
-1.0 400 



0.2 
0.1 
0.0 

0 


- + - + DuP-697 
300 
200 
100 
-0.1 
-0.2 -1.5 -100 
Control
db/dbControlControl

(d) (e) (f) 
Figure 3: NO-and PGI2-dependent function in the diabetic isolated lungs. (a) Impaired NO-dependent hypoxic pulmonary vasoconstriction response after L-NAME in the lungs of the diabetic mice (control n=5,diabetesn=5).(b,c)Lackofchangeinbasal productionof nitriteand nitrate and (d, e) impaired capacity to cumulative production of nitrite but not nitrate in e.uents from the isolated, perfused lungs of the diabetic mice (control n=6, diabetes n=7). (f) Increased COX-2-dependent prostacyclin production in e.uents from the isolated, perfused lungs and e.ects of a COX-2 inhibitor (DuP-697, 1 µM, control n=6, diabetes n=7). Data are presented as the means±SEM. *P<0 
05. 
min/cmH2O/100g of body weight, db/db and control, resp.; P<0 
05, Figure 2(e)). 
4.3.3. Impairment of NO-Dependent Regulation of Hypoxic Pulmonary Vasoconstriction (HPV). In the isolated, per­fused lung from control mice, episodes of hypoxic ventila­tion resulted in an increase of pulmonary arterial pressure (PAP) without signi.cant changes in other parameters of isolated lung preparation (Figure 3(a)). In control lungs, the nonselective inhibitor of nitric oxide synthases, L-NAME, augmented HPV response (.PAP: 0.87 ±0.32 versus 2.73 ±0.44 cmH2O, before and after L-NAME, resp.; P<0 
05). However, in the isolated, perfused lung from db/db mice, the e.ect of L-NAME on HPV was substan­tially lost (.PAP: 0.60 ±0.04 versus 1.17 ±0.12 cmH2O, before and after L-NAME, resp.; P<0 
05) suggesting impaired NO-dependent function. L-NAME did not modify basal PAP(. basal PAP after L-NAME: 0.17 ±0.03 versus 
0.19 ±0.06 cmH2O, in control and db/db mice, resp.). 
4.3.4. Nitrite/Nitrate (NO- 2/NO3-) and Prostacyclin (PGI2) Production. In e.uents from the isolated, perfused lungs, basal NO2-/NO- 3 concentrations were comparable in both groups (e.g., NO- 2 0.40 ±0.04 versus 0.56 ±0.19 µM, db/db and control, resp., Figures 3(b) and 3(c)). The cumulative concentrations of NO- 2 from the diabetic lungs (see Methods for details) were signi.cantly lower (.NO- 2: -0.04±0.04 versus 0.11 ±0.05 µM, db/db and control, resp.), but there were no changes in NO3 - concentrations (Figures 3(d) and 3(e)). The cumulative concentration of stable PGI2 metabolite, 6-keto-PGF1., in the e.uents from the iso­lated diabetic lungs was higher than that in the control (.6-keto-PGF1.: 223.5±57.91 versus 95.06 ±24.00 pg/ml, db/db and control, resp.) and was blunted after COX-2 inhibitor, DuP-697 (Figure 3(f)). 
4.3.5. Markers of Vascular In.ammation. In diabetic lungs, immunohistochemical staining intensity of vascular adhe­sion molecule-1 (VCAM-1) was increasedbya trendas com­pared to the control samples (Figure 4(a)). Von Willebrand factor (vWF) and thrombomodulin (CD141) (but not eNOS) were higher in the diabetic lungs than in the control lungs (Figures 4(b)–4(d)). All together these results support an increased in.ammatory state in the pulmonary system of diabetic mice. 
4.4. Impairment of Endothelial Function and In.ammatory Markers in the Aorta of db/db Mice. Vasoreactivity to phen­ylephrine (30 µM) in the aortic rings from db/db mice was increased (not shown). Acetylcholine-(ACh-) induced endothelium-dependent vasodilation was decreased for all concentrations in db/db mice, while sodium nitroprusside­
1.0 × 107 1.0 × 106 vWF
VCAM-1 
8.0 × 106 8.0 × 105 
6.0 × 105 
... 
p= 0.070 6.0 × 106




Cy3 fl uorescence (AU)
Cy3 fl uorescence (AU)
Cy3 fl uorescence (AU)


4.0 × 106 
2.0 × 106 4.0 × 105 

db/db  0  db/dbntrol 
(a)  Co(b)  

 
 

 


2.0 × 105 
0 
Control


4 × 106 
eNOS 
3 × 106 
2 × 106 

1 × 106 
0 
Control
db/db
Control
db/db 

(c) (d) 
Figure 4: Immunohistochemical pro.le of the diabetic lungs. (a) In.ammation in vascular wall related to increased immunostaining of vascular cell adhesion molecule 1 (VCAM-1) and (b) von Willebrand factor in the lungs of the diabetic mice (both for VCAM-1 and vWF: control n=4, diabetes n=4). (c) No change in eNOS immunostaining in the diabetic lungs as compared to the control (control n=4, diabetes n=4). (d) Increased thrombomodulin (CD141) immunostaining in the diabetic lungs as compared to the control (control n=4, diabetes n=4). Data are presented as the means±SEM. **P<0
01, *** P<0
001
. 
(SNP-) induced response was preserved as compared to the control group (Figures 5(a) and 5(b)). Impairment of func­
tional response was supported by a decline in ionophore­stimulated NO- 2 production in the aortic rings from the db/ db mice (33.88±10.01 versus 173.30 ±77.79 nM, db/db and control, resp.; P<0
05). Basal NO- 2 concentrations in bu.er from the incubated aortic rings were comparable in both groups (27.17 ±13.43 versus 32.75 ±11.68 nM, db/db and control, resp.), whereas the e.ect of the NOS inhibitor, L-NIO, was striking in the aorta of the control mice and absent in the diabetic group (Figure 5(c)). COX-2­dependent production of PGI2 (measured as 6-keto-PGF1. concentration in e.uent) was decreased in the aortic rings from the db/db mice as compared to the control 
(148.80 ±27.20 versus 329.30 ±68.18 pg/ml, db/db and control, resp.; P<0
05), and COX inhibitors decreased PGI2 production in the aorta of the control but not the diabetic mice (Figure 5(d)). Furthermore, endothelium in the aorta displayed increased VCAM-1 expression compati­ble with endothelialdysfunction (Figure 6(a)), although there were no changes in the eNOS immunostaining intensity (Figure 6(c)). In turn, in contrast to the pulmonary endothelium, thrombomodulin (CD141) immunostaining intensity was decreased (Figure 6(b)). 
4.5. Nonenzymatic Nitration in the Lungs and Aorta. Dot­blot-assessed general protein nitration was increased in the aorta but not in the lungs from the db/db mice, as compared with the control; in the lung, only a trend of increased nitration was observed (Figure 7(a) and 7(b)). Immunohistochemical staining showed only a slight increase in the signal of nitrated proteins in the lungs. A slight increase in PGISimmunostainingin diabetic lungs was also found (Figure 7(c)). 
5. Discussion 
In the present work, we characterized the phenotype of endo­thelial dysfunction in pulmonary endothelium, as compared with peripheral endothelium in the diabetic mice (db/db mice). We demonstrated that diabetes induced marked ultrastructural changes in pulmonary endothelium that were associated with the increased permeability of pulmonary microcirculation and impaired NO-dependent function, as well as compensatory increase in PGI2 production with increased thrombomodulin expression. In contrast, endothe­lial dysfunction in the aorta was featured by impaired NO-and PGI2-dependentfunction and diminished thrombomod­ulin (CD141) expression. These results suggest a di.erential response of pulmonary vasculature to diabetic insult in terms of PGI2-dependent function that might be associated with a lesser nonenzymatic protein nitration in the lung, as compared with peripheral endothelium and preserved PGI2 synthase activity. 
Endothelial dysfunction induced by diabetes in periph­eral circulation in db/db mice has been well documented [31–33] and involves (1) increased reactive oxygen species production, scavenging of endothelial NO, and increased 


0 
25 25 


Relaxation (% of
NO2- concentration (nM) 
phenylephrine induced contraction) 
phenylephrine induced contraction)

.6-keto-PGF1
. in 30 min of incubation (pg/ml)Relaxation (% of
50 
75 50 
75 p < 0.01 

100 100 

10-9 10-8 10-7 10-6 10-5 10-4 10-9 10-8 10-7 10-6 10-5 10-4 ACh (M) SNP (M) 

db/db 

db/db 

Control 

Control 

(a) (b) 
500300 


400 
300 
200 
100 
0 200 
100 
0 
BasalIonophoreL-NIOBasalIonophoreL-NIO 

Control  
Control  db/db  
db/db  
(c)  (d)  

Figure 5: Endothelial dysfunction in systemic conduit vessel (aorta). (a, b) Impaired endothelium-dependent response to acetylcholine (ACh) with preserved endothelium-independent vasodilation in response to sodium nitroprusside (SNP) (control n=6, diabetes n=7). 
(c) Preserved basal but impaired ionophore-stimulated production of nitrite in the aortic rings of diabetic mice (control n=6, diabetes n=7). (d) Impaired basal production of prostacyclin as assessed by concentrations of its stable 6-keto-PGF1. product (control n =6, diabetes n=6). Data are presented as the means±SEM. *P<005. 
nonenzymatic protein nitration[31, 32]; (2) decreased pro­
duction of PGI2 and CD141 expression and impaired endothelial-dependent functional responses [31, 34–36]. Ourresultsareinlinewiththeprevious studiesasregardsphe­notype of endothelial dysfunction in the aorta. Importantly, we evaluated the peripheral endothelial phenotype for com­parison with the analysis of the phenotype of endothelial dys­function in pulmonary circulation that has been signi.cantly less studied, including only few reports in the db/db mice [13, 17, 18] and studies in models of diabetes in rats [16, 37]. 
In the present work, we demonstrated that diabetic lungs from db/db mice displayed mild in.ammatory cell in.ltra­tion and ultrastructural alterations featured by profound thickening of the basal membrane, compatible with the pre­vious reports on diabetic lungs in humans [11, 38, 39]. Indeed, thickening of the basal membrane and an impair­ment of permeability of the alveolar basement membrane coexist in diabetes type II in the human lungs, and these changes are followed by a decrease in respiratory function [6, 40]. Ultrastructural changes of pulmonary endothelium 


Control db/db 
(a) 
8.0 × 105


3.0 × 104 eNOS 
CD141
Cy3 fl uorescence (AU)

Cy3 fl uorescence (AU) 
6.0 × 105 
4.0 × 105 
2.0 × 105 0 

.
2.0 × 104 

1.0 × 104 
0 Control db/db Control db/db 

(b) (c) 
Figure 6: Immunohistochemical pro.le of systemic conduit vessel (aorta). (a) Increased VCAM-1 immunostaining intensity suggesting aortic wall in.ammation in the diabetic mice (control n=3, diabetes n=4). (c) Comparable eNOS immunostaining intensity in the diabetic and control aortic rings (control n=3, diabetes n=4). (b) Decreased immunostaining intensity of thrombomodulin (CD141) in the aortic wall from the diabetic mice (control n=3, diabetes n=4). Data are presented as the means±SEM. *P<0
05, **P<0
01. 
reported here were also featuredbyactivated endothelialcells that were, however, less pronounced as high-convoluted apical plasmalemma and numerous plasmalemmal vesicles reported in transgenic mice model of diabetes type I [41], suggesting milder pulmonary endothelial activation in the db/db mice. Nevertheless, we found a signi.cant increase in endothelial permeability of diabetic pulmonary circula­tion, as evidenced by increase Kf,c measurements [28] that seem also compatible with increased permeability of the human lungs from diabetic patients [42]. 
The important .nding of this work was the demonstra­tion of the impairment of NO-dependent function in the lungs from the db/db mice. We took advantage of the domi­nant role of endogenous NO in blunting HPV [43] to study functional NO-dependent response in the whole isolated lung, instead of choosing isolated pulmonary arteries that may re.ect NO-dependent function only in the selected part of the pulmonary circulation. Our original approach to detect impaired NO-dependent function was based on diminished modulatory e.ects of NOS inhibition on HPV response in the isolated, perfused lungs [43, 44] supported also by low­
ered cumulative concentrations of NO- 2 in e.uents from diabetic lungs. On the other hand, eNOS expression in the lungs from the control and db/db mice was not di.erent, suggesting that alteration of the NO bioavailability was responsible for functionally impaired NO-dependent response in pulmonary circulation from the db/db mice. 
Endothelium-derived PGI2 is often released in a coupled manner with NO [45, 46]. NO de.ciencyis sometimes linked with a decrease in PGI2 production, but in many vascular pathologies, PGI2 production may increase in response to nitric oxide de.ciency [44, 47]. Here, PGI2 production in the aorta was reduced, but in the isolated lungs from the db/db mice, PGI2 production was augmented. The major enzymatic source of PGI2 in the aorta and lung was COX-2, as evidenced by the pronounced e.ect of COX-2 inhibition, and this is in line with the notion of COX-2 as the major source of systemic PGI2 [48] and important contributor to pulmonary endothelial dysfunction [49, 50]. PGI2 ampli.es CD141 expression[21–23].As shown here, CD141 immunointensity in the lungs was increased, while it was diminished in the aorta, which supports the link between PGI2 production and CD141. PGI2 via CD141 activates protein C, thus enhancing the anticoagulant mechanism of the vascular wall. Reciprocally, activated protein C boosts PGI2 production in endothelial cells [51]. 
Thus, pulmonary PGI2 a.ords potent antiplatelet and vasoprotective activity, activating also CD141-dependent 
1000 

Lungs 
Aorta 
800 
Pos. 
Total tyrosinenitration (%) 
600 

Lung control 
400 


Lungs db/db200 

1 2 3 4Ctr. 
1+2 3+4 
. 



1+2+4 3+5
12345 
0 Control 


db/db 

(a) (b) 
3-Nitrotyrosine staining PGI2 synthase 




(c) 
Figure 7: Protein nitration in the lungs and aorta. (a,b)Increased general protein nitration in the aorta but not in the lungs of diabetic mice as revealed by dot blot analysis (control n=5, diabetes n=5). Representative original blot images are also shown. (c) Immunohistochemical determination of 3-nitrotyrosine (3-NT) and PGIS with the slight increase in 3-NT immunostaining in the diabetic lungs—the comparison of similar regions of tissues. Data are presented as the means ±SEM. *P<0
05. Representative images of at least 2 
independent experiments. 
anticoagulant mechanisms which might constitute an impor­tant compensatory mechanism in diabetes o.setting in.am­matory and thrombotic processes in diabetes involving also detrimental COX-2-derived metabolites contributing to endothelial dysfunction in diabetes [49, 50, 52] . 
Interestingly, 1-MNA exerts antithrombotic [53] and anti-in.ammatory[54] properties mediatedbytheactivation of COX-2 and PGI2 pathways. It could well be that the therapeutic e.cacy of 1-MNA reported previously [53–62] is linked with the capacity of 1-MNA to stimulate compensa­tory mechanisms linked to pulmonary PGI2 [44]. Obviously, this hypothesis needs to be veri.ed in further studies. 
It is well known that diabetes is associated with increased local ROS production in intrapulmonary arteries, as well as in systemic circulation [16, 63, 64]. Superoxide anions and NO by forming peroxynitrite may lead to nonenzymatic nitration of proteins[63, 65] including PGIS; the nitration-mediated inactivation of which plays an important role in the develop­ment of endothelial dysfunction [66–69]. In systemic circula­
tionin diabetes patients,protein nitrationa.ectsanumberof enzymes, including PGIS [64, 70]. Here, we present signi.­cantly increased general nitration of protein in the aorta, and a milder e.ect (nonstatistically signi.cant) was noticed in the lungs. Nonenzymatic nitration may have less signi.­cance in the diabetic lungs as comparedto systemic circula­tion. Therefore, despite locally increased ROS generation in pulmonary vessels [16, 37, 71], in the whole lungs, ROS may not play such an important role in pulmonary circula­tion of the db/db mice as compared to systemic endothelium. 
6. Conclusions 
In conclusion, our results demonstrate that diabetes induced profound changes in the lung in the db/db mice involving endothelial ultrastructural changes, increased endothelial permeability, and increased vasoreactivity, as well as lung in.ammation and .brosis. Impaired NO-dependent pulmo­nary vascular function was associated with upregulated PGI2 and CD141 that might constitute an important com­pensatory mechanism in pulmonary circulation in diabetes that does not operate in endothelium in the aorta, whereby endothelial dysfunction is featured by impaired NO, PGI2, and CD141. 
Abbreviations 
6-keto-PGF1.: Stable prostacyclin metabolite Ang: Angiotensin AST/ALT: Aspartate transaminase/alanine 
transaminase CD141: Thrombomodulin CF: Constant .ow CP: Constant pressure COX: Cyclooxygenase DuP-697: Selective COX-2 inhibitor eNOS: Endothelial nitric oxide synthase H&E: Hematoxylin and eosin HPV: Hypoxic pulmonary vasoconstriction iNOS: Inducible nitric oxide synthase 1-MNA: 1-Methylnicotinamide NO: Nitric oxide NO- 2/NO- 3: Nitrite/nitrate OMSB: Orcein and methyl scarlet blue staining PAP: Pulmonary arterial pressure .PAP: The change in pulmonary arterialpressure PGI2: Prostacyclin PVP: Pulmonary venous pressure RVW/BW: Right ventricular to body weight ratio SNP: Sodium nitroprusside TV: Tidal volume U46619: ThromboxaneA2 analogue vWF: von Willebrandfactor. 
Data Availability 
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. 
Conflicts of Interest 
The authors declared that no con.ict of interest exists. 
Authors’ Contributions 
Andrzej Fedorowicz and Stefan Chlopicki conceived and designed the research; Andrzej Fedorowicz, Elżbieta Buczek, Barbara Sitek, Łukasz Mateuszuk, Agnieszka Jasztal, Anto­nina Chmura-Skirlińska, Mobin Dib, and Sebastian Steven carried out the experiments; Elzbieta Czarnowska, Agnieszka Jasztal, and Andreas Daiber contributed with the analytic tools; Andrzej Fedorowicz, Elzbieta Czarnowska, Agnieszka Jasztal, Antonina Chmura-Skirlińska, Mobin Dib, Andreas Daiber, and Sebastian Steven performed the data analysis; Andrzej Fedorowicz and Stefan Chlopicki drafted the manu­script; Elżbieta Buczek, Łukasz Mateuszuk, Elzbieta Czar­nowska, Mobin Dib, Sebastian Steven, and Andreas Daiber revised the manuscript; Andrzej Fedorowicz and Stefan Chlopicki wrote the .nal version of the manuscript. All authors readand approved the .nal manuscript. 
Acknowledgments 
The work was supported by a grantfrom the resources of the European Regional Development Fund under the Innovative Economy Programme (grant coordinated by JCET-UJ, no. POIG.01.01.02-00-069/09) and partiallyby National Science Centre Grant Symfonia no. DEC-2015/16/W/NZ4/00070. 
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