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

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 differences 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 fibrosis, and pulmonary endothelium displayed significantly ultrastructural changes. In the isolated, perfused lung from db/db mice, filtration coefficient (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 NO2− were impaired, with no changes in immunostaining for eNOS expression. In turn, 6-keto-PGF1α 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 NO2− generation, and production of 6-keto-PGF1α 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.


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][2][3]. The detrimental effects of diabetes in the lungs are less clinically apparent. However, epidemiological and experimental data suggested that insulin resistance and diabetes affect the 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][6][7] and increased susceptibility to allergic response/inflammation induced with LPS or airway bacterial infection [8][9][10]. Structural changes in blood-alveolar barrier and diffusion impairment in vivo have also been reported [11,12]. Despite these reports, the possible detrimental effects 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 a better understanding of similarities and differences 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 conflicting. Both the presence and lack of impairment of NO-dependent pulmonary endothelial function have been reported [13,16,17]. In turn, an increase in pulmonary microvascular permeability without changes in eNOS or with increased iNOS expression has also been demonstrated [13,[16][17][18].
Similarly, reports on diabetes-induced changes in PGI 2 production in the pulmonary circulation are also not consistent. In streptozotocin-treated rats, basal PGI 2 production and stimulated PGI 2 production in pulmonary circulation were reported to increase or to remain unchanged [14,19,20]. Interestingly, PGI 2 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 activity of PGI 2 may result in the alteration in the activity of thrombomodulin, despite the fact that previous studies reported no changes [24][25][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 PGI 2 -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 PGI 2 activities were analyzed in the isolated, perfused diabetic lung, or in the aortic rings, respectively.

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 specific 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 Use of Laboratory Animals published by 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).

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, 5 min, 4°C). Complete blood count was analyzed within 15 minutes after collection (by automatic blood counter ABC Vet, HORIBA). HbA1c and total hemoglobin concentrations were measured using a biochemical analyser (ABX Pentra 400, HORIBA), and the ratio was given as a percentage of HbA1c. Glucose, aspartate aminotransferase, alanine aminotransferase, creatinine, albumin, and total protein were measured using colorimetric methods (ABX Pentra 400, HORIBA).

Histological
Analysis of the Lungs. The lungs were removed under anaesthesia and fixed in 4% buffered formalin (24 h) and were then dehydrated, embedded in paraffin, 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 with a digital camera.

Assessment of Changes in Lung
Ultrastructure. The chest of anaesthetized rats was opened, and samples of lung tissue were cut and fixed immediately using a mixture of 2.5% glutaraldehyde and 2% freshly prepared paraformaldehyde in 0.1 mol/L cacodylate buffer at pH 7.4. The lung tissue was fixed for 12 h at 4°C. Then, the lungs were postfixed in buffered 2% osmium tetroxide, dehydrated in a graded ethanol series and propylene oxide, and embedded in Epon 812. The ultrathin sections were stained according to routine protocol with uranyl acetate and lead citrate and were examined and documented by transmission electron microscopy (Jem 1011, JEOL, Japan).

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% CO 2 to the inspiratory air, using a peristaltic pump (ISM 834, HSE) at a constant flow (CF) of about 1.50 ml/min. The venous pressure was set between 2 and 5 cmH 2 O. The end-expiratory pressure in the chamber was set to be −3 cmH 2 O, and inspiratory pressure was adjusted between −6 and −10 cmH 2 O to 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 was 1 : 1 in each breath. Every 5 min throughout the experiments, a deep breath of end-inspiratory pressure of −21 cmH 2 O was automatically initiated by VCM module (HSE) to avoid atelectasis. Airflow velocity was measured with a pneumotachometer tube connected to a differential 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 artificial thorax. The PAP was set to be around 3 cmH 2 O. The venous pressure was set between 2 and 5 cmH 2 O.
Both arterial and venous pulmonary pressures (PAP, PVP) were continuously monitored by ISOTEC pressure transducers (HSE) connected to a perfusion line on arterial 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 first 15 min of perfusion with fresh buffer 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% N 2 and 5% CO 2 . 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 (ΔPAP) elicited by HPV was taken. TV, PVP, and weight did not change significantly during HPV.
2.6.2. Vasoreactivity. After equilibration, U46619 (1 μM) 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 (ΔPAP) 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 cmH 2 O 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 filtration coefficient (K f,c ) was calculated based on recordings for 5 minutes after PVP increase [28].
2.6.4. Biochemical Measurements. 6-keto-PGF 1α and NO − 2 / NO − 3 concentrations were measured in samples of effluents collected after 15 minutes of equilibration of isolated lungs perfused with constant flow, and then 5 and 45 minutes after recirculation of the perfusate was started. To assess the enzymatic source of 6-keto-PGF 1α , 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).

Assessment of Endothelial Function in the Isolated Aortic
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, CaCl 2 2.52, MgSO 4 1.16, NaHCO 3 24.88, K 2 PO 4 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 filled with 5 ml of KH solution chambers (37°C, pH 7.4, gassed with carbogen: 95% O 2 , 5% CO 2 ) of wire myograph (620 M, Danish Myo 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 10 mN, 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 30 mM, 60 mM), 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 0.001-1 μM) was assessed. The relaxation response was expressed as a percentage of the precontraction induced by phenylephrine.
2.8. Assessment of Prostacyclin (PGI 2 ) Production in the Isolated Aortic Rings. The concentration of PGI 2 , produced by aortic rings, was quantified on the basis of the formation of 6-keto-PGF 1α , a stable metabolite of PGI 2 . The aorta rings were preincubated for 15 minutes on the thermoblock (Liebisch Labortechnik) at a temperature of 37°C, in 250 μl KH buffer, 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 (1 μl/ml).
Aortic rings were then incubated for 60 minutes, and samples of effluents were collected after 3 and 60 minutes. After the experiment, aortic rings were dried (1 h, 50°C) and weighed. 6-keto-PGF 1α concentration in the effluents was measured using an EIA kit (Enzo, Life Technologies). Results were expressed as the change in 6-keto-PGF 1α concentration between 60 and 3 minutes of ring incubation and normalized to dry weight of aortic rings (pg/ml/mg).

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 μl KH buffer at 37°C, using a specially designed closed chamber (BIO-V(Noxygen)) that was equilibrated with carbogen gas mixture (95% O 2 , 5% CO 2 ). 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 NaNO 2 standard solution was prepared for every experiment before measurements of series of samples after each refilling of glass reaction chamber, according to the manufacturer's instructions (Sievers * Nitric Oxide Analyzer NOA 280i). The limit of detection was around 10 nM of nitrite. Multiple blank samples (without aortic rings) were used to monitor nitrite contamination in the buffer 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.

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 significance, 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.

Histology and Ultrastructure of Lungs in db/db Mice.
Lungs from db/db mice displayed inflammation, as evidenced by multicellular (including granulocytes, macrophages) infiltrations in the interstitial space (HE staining, Figure 1(b)) as compared to the control group (Figure 1(a)); mild fibrosis (increased amount of collagen), as evidenced by Trichrome staining (Figure 1(d)) as compared to the control group (Figure 1(c)); and endothelial inflammation, as evidenced by increased VCAM-1 expression in pulmonary endothelium (480,937 ± 70,112 versus 247,193 ± 64,821 AU, db/db and control, resp.; P = 0 07). Interestingly, ultrastructural investigations confirmed the presence of numerous macrophages (often lying next to each other) in the lungs from db/db mice, 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/db pulmonary microcirculation was the hyperplasia of basal lamina in db/db (Figures 1(f) and 1(h)) that was not evident in control samples (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 fibrils and probably contained also elastin fibrils 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)).

Alterations in Pulmonary Vascular Function and
Inflammation in the Isolated, Perfused Lung from db/db Mice

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 significant 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 (ΔPAP: 0.87 ± 0.32 versus 2.73 ± 0.44 cmH 2 O, before and after L-NAME, resp.; P < 0 05). However, in the isolated, perfused lung from db/db mice, the effect of L-NAME on HPV was substantially lost (ΔPAP: 0.60 ± 0.04 versus 1.17 ± 0.12 cmH 2 O, before and after L-NAME, resp.; P < 0 05) suggesting impaired NO-dependent function. L-NAME did not modify basal PAP (Δ basal PAP after L-NAME: 0.17 ± 0.03 versus 0.19 ± 0.06 cmH 2 O, in control and db/db mice, resp.).

Markers of Vascular Inflammation.
In diabetic lungs, immunohistochemical staining intensity of vascular adhesion molecule-1 (VCAM-1) was increased by a trend as 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 inflammatory state in the pulmonary system of diabetic mice.

Impairment of Endothelial Function and Inflammatory
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- (SNP-) induced response was preserved as compared to the control group (Figures 5(a) and 5(b)). Impairment of functional 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 buffer 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 effect 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 PGI 2 (measured as 6-keto-PGF 1α concentration in effluent) 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 PGI 2 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 endothelial dysfunction (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)).

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 PGIS immunostaining in diabetic lungs was also found (Figure 7(c)).

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 PGI 2 production with increased thrombomodulin expression. In contrast, endothelial dysfunction in the aorta was featured by impaired NOand PGI 2 -dependent function and diminished thrombomodulin (CD141) expression. These results suggest a differential response of pulmonary vasculature to diabetic insult in terms of PGI 2 -dependent function that might be associated with a lesser nonenzymatic protein nitration in the lung, as compared with peripheral endothelium and preserved PGI 2 synthase activity.
Our results are in line with the previous studies as regards phenotype 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 significantly 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 inflammatory cell infiltration 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 (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-PGF 1α product (control n = 6, diabetes n = 6). Data are presented as the means ± SEM. * P < 0 05.
reported here were also featured by activated endothelial cells 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 significant increase in endothelial permeability of diabetic pulmonary circulation, as evidenced by increase K f,c measurements [28] that seem also compatible with increased permeability of the human lungs from diabetic patients [42].
The important finding 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 reflect 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 effects of NOS inhibition on HPV response in the isolated, perfused lungs [43,44] supported also by lowered cumulative concentrations of NO − 2 in effluents from diabetic lungs. On the other hand, eNOS expression in the lungs from the control and db/db mice was not different, 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 PGI 2 is often released in a coupled manner with NO [45,46]. NO deficiency is sometimes linked with a decrease in PGI 2 production, but in many vascular pathologies, PGI 2 production may increase in response to nitric oxide deficiency [44,47]. Here, PGI 2 production in the aorta was reduced, but in the isolated lungs from the db/db mice, PGI 2 production was augmented. The major enzymatic source of PGI 2 in the aorta and lung was COX-2, as evidenced by the pronounced effect of COX-2 inhibition, and this is in line with the notion of COX-2 as the major source of systemic PGI 2 [48] and important contributor to pulmonary endothelial dysfunction [49,50]. PGI 2 amplifies CD141 expression [21][22][23]. As shown here, CD141 immunointensity in the lungs was increased, while it was diminished in the aorta, which supports the link between PGI 2 production and CD141. PGI 2 via CD141 activates protein C, thus enhancing the anticoagulant mechanism of the vascular wall. Reciprocally, activated protein C boosts PGI 2 production in endothelial cells [51].
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][67][68][69]. In systemic circulation in diabetes patients, protein nitration affects a number of enzymes, including PGIS [64,70]. Here, we present significantly increased general nitration of protein in the aorta, and a milder effect (nonstatistically significant) was noticed in the lungs. Nonenzymatic nitration may have less significance in the diabetic lungs as compared to 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.

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 inflammation and fibrosis. Impaired NO-dependent pulmonary vascular function was associated with upregulated PGI 2 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, PGI 2 , and CD141. Endothelial nitric oxide synthase H&E:

Data Availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.