Imatinib relaxes the pulmonary venous bed of guinea pigs

Background Recently, the IMPRES study revealed that systemic imatinib improves exercise capacity in patients with advanced pulmonary arterial hypertension. Imatinib blocks the tyrosine kinase activity of the platelet-derived growth factor (PDGF)-receptor (PDGFR), acts antiproliferative and relaxes pulmonary arteries. However so far, the relaxant effects of imatinib on pulmonary veins (PVs) and on the postcapillary resistance are unknown, although pulmonary hypertension (PH) due to left heart disease (LHD) is most common and primarily affects PVs. Next, it is unknown whether activation of PDGFR alters the pulmonary venous tone. Due to the reported adverse effects of systemic imatinib, we evaluated the effects of nebulized imatinib on the postcapillary resistance. Methods Precision-cut lung slices (PCLS) were prepared from guinea pigs. PVs were pre-constricted with Endothelin-1 (ET-1) and the imatinib-induced relaxation was studied by videomicroscopy; PDGF-BB-related vascular properties were evaluated as well. The effects of perfused/nebulized imatinib on the postcapillary resistance were studied in cavine isolated perfused lungs (IPL). Intracellular cAMP/cGMP was measured by ELISA in PVs. Results In PCLS, imatinib (100 μM) relaxed pre-constricted PVs (126%). In PVs, imatinib increased cAMP, but not cGMP and inhibition of adenyl cyclase or protein kinase A reduced the imatinib-induced relaxation. Further, inhibition of KATP-channels, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {\mathrm{BK}}_{\mathrm{Ca}}^{2+} $$\end{document}BKCa2+-channels or Kv-channels diminished the imatinib-induced relaxation, whereas inhibition of NO-signaling was without effect. In the IPL, perfusion or nebulization of imatinib reduced the ET-1-induced increase of the postcapillary resistance. In PCLS, PDGF-BB contracted PVs, which was blocked by imatinib and by the PDGFR-β kinase inhibitor SU6668, whereas inhibition of PDGFR-α (ponatinib) had no significant effect. Conversely, PDGFR-β kinase inhibitors (SU6668/DMPQ) relaxed PVs pre-constricted with ET-1 comparable to imatinib, whereas the PDGFR-α kinase inhibitor ponatinib did not. Conclusions Imatinib-induced relaxation depends on cAMP and on the activation of K+-channels. Perfused or nebulized imatinib significantly reduces the postcapillary resistance in the pre-constricted (ET-1) pulmonary venous bed. Hence, nebulization of imatinib is feasible and might reduce systemic side effects. Conversely, PDGF-BB contracts PVs by activation of PDGFR-β suggesting that imatinib-induced relaxation depends on PDGFR-β-antagonism. Imatinib combines short-term relaxant and long-term antiproliferative effects. Thus, imatinib might be a promising therapy for PH due to LHD.


Background
Pulmonary hypertension (PH) due to left heart disease (LHD) is the most common cause of PH [1][2][3][4][5]. In response to elevated left ventricular (LV) or atrial filling pressure, PH due to LHD is associated with increased pulmonary capillary wedge pressure (P pcw ) >15 mmHg leading by backwards transmission to elevated pulmonary arterial pressure (P PA ) ≥25 mmHg, even though the transpulmonary gradient (TPG) often remains ≤12 mmHg [6]. Though, some patients develop severe PH characterized by a TPG ≥12, by proliferative pulmonary vascular disease and elevated pulmonary vascular resistance (PVR) [6]. PH due to LHD primarily affects the pulmonary veins (PVs) [7], thus it is also called postcapillary PH or pulmonary venous hypertension (PVH) [1,4,8]. This feature is pivotal for the therapy of PH due to LHD, as vasodilators acting primarily in the pulmonary arterial bed may increase pulmonary perfusion leading to elevated hydrostatic pressure, pulmonary edema and elevated PVR [8]. Until now, specific agents to treat PH due to LHD are missing, although some agents, e.g. the PDE-III inhibitor milrinone exert relaxant effects in human PVs [9]. Thus, current guidelines recommend the management of the underlying condition, e.g. the therapy of LHD, the repair of valvular heart disease or LV-assist device implantation [3,6]. However, cardiac surgery is at high risk. Therefore, new therapeutic approaches are warranted.
The tyrosine kinase inhibitor (TKI) imatinib represents such a novel approach which demonstrated already promise in experimental models [10] and clinical trials [11][12][13]. Moreover, two studies in rats show that imatinib may act also as a vasodilator in rat pulmonary arteries (PAs) and aortas [14,15]. Recently, the IMPRES study showed that oral imatinib improves exercise capacity and hemodynamics in patients with advanced pulmonary arterial hypertension (PAH) [16]. So far, although relevant, the effects of imatinib have been never studied in the pulmonary venous bed or in PH due to LHD. Of note, the IMPRES study uncovered also severe imatinib-related adverse effects such as subdural hematoma or QTc prolongation [16,17]. Thus, a local application, e.g. by nebulization could be advantageous to improve the safety of imatinib [18].
Imatinib resembles an intriguing approach in PH, as it targets the vascular remodeling, a major pathophysiologic condition of PH. It inhibits the receptor tyrosine kinase (RTK) platelet derived growth factor (PDGF)-receptor (PDGFR) and counteracts thereby PDGF [19,20]. Both PDGF and its receptor are overexpressed in pulmonary arterial smooth muscle cells (SMCs) and to lesser extent in endothelial cells of patients with idiopathic PAH [21]. Further, PDGFR-β immunoreactivity is increased in small arterioles and venules, as well as in capillaries of patients with pulmonary venous occlusive disease and systemic sclerosis associated PAH [22]. PDGFR consists of two subunits (αα, αβ, ββ) among which PDGFR-β mediates proliferation [23]. At present it is not known whether PDGF alters the pulmonary vascular tone. In systemic vessels, PDGF acts as a vasodilator in mesenteric arteries [24,25] and as a vasoconstrictor in aortas [26]. However, these discrepant findings do not allow to predict the action of PDGF in the lungs, in particular, because the pulmonary and the systemic vasculature are remarkably dissimilar [27].
With regard to PH due to LHD, there are several unsolved issues concerning the effects of imatinib and PDGF on the pulmonary venous tone. 1) Does imatinib relax PVs? 2) Which are the possible underlying mechanisms? 3) Does imatinib affect the postcapillary resistance (R post )? 4) Does imatinib exert pulmonary relaxant effects when applied by nebulization? 5) Does PDGF act as a pulmonary vasoconstrictor? -A finding that would in part explain the relaxant effects of imatinib. In the present study, we address these questions to evaluate the value of imatinib within the context of PH due LHD.

PCLS
PCLS were prepared as described [28,29]. Briefly, intraperitoneal anesthesia was performed with 95 mg kg −1 pentobarbital (Narcoren; Garbsen, Germany) and verified by missing reflexes. The GP was bled, the trachea cannulated and the diaphragm opened. The lungs were filled via the trachea with 1.5% low melting agarose. To harden the lungs, they were cooled with ice. Then, tissue cores (diameter 11 mm) were prepared and cut into about 300 μM thick slices with a Krumdieck tissue slicer (Alabama Research & Development, Munford, AL, USA). PCLS were incubated at 37°C and repeated medium changes were performed to wash out the agarose.

Cyclic AMP and cGMP enzyme immunoassay
To analyze cAMP/cGMP, PVs were isolated out of tissue cores guided by their anatomical landmarks, e.g. the PAs accompany the airways, whereas PV lies aside. PVs were incubated in medium, flushed with imatinib (100 μM) and after 30 min frozen by liquid nitrogen. Cyclic AMP/ cGMP was quantified with ELISA-kits following the manufacturer's protocol. For stabilization, all samples or standards were acetylated. To measure cAMP, all samples were diluted 1:2 with 0.1 M HCL. The ELISA was evaluated at 405 nM (GENIOS, Tecan, Switzerland).

Isolated perfused lungs of the GP
Cavine lungs were prepared as described [9,38]. Briefly, intraperitoneal anesthesia was performed (pentobarbital: 95 mg kg −1 ) and verified by missing reflexes. The GP was bled, the trachea cannulated and the lung ventilated with positive pressure (70 breaths/min). The left ventricle's apex was cut and cannulas were placed in the pulmonary artery (perfusion inflow) and in the left atrium (perfusion outflow). The lung was perfused at constant flow (12,5 mL/min) with Krebs-Henseleit buffer, containing 2% bovine serum albumin, 0.1% glucose, 0.3% HEPES and 50 nM salbutamol to prevent bronchoconstriction [39]. The temperature of the perfusate was maintained at 37°C with a water bath and the pH was maintained between 7.35 and 7.45 by gassing with carbon dioxide. Heart and lungs were removed and transferred into a negative-pressure chamber. Tidal volume, compliance, resistance, pulmonal arterial pressure (P PA ), left atrial pressure (P LA ), and the flow were continuously monitored. As soon as the respiratory and hemodynamic variables remained stable over 10 min, ET-1 (20 nM) was added to the recirculating perfusion buffer (total volume 200 mL) to enhance R post [40]. Ten minutes after the application of ET-1, imatinib (10 μM) was perfused. At a buffer volume of 200 mL, this corresponds to total amount of 1.18 mg imatinib or to 3.5 mg/kg body weight imatinib, respectively. Thereafter, changes of the capillary pressure (P cap ) were measured every 10 min by the double occlusion method [38], R post and the precapillary resistance (R pre ) were calculated by the following equations: Flow . In order to examine the effects of nebulized imatinib on R post , isolated perfused GP lungs were perfused as described before. After the increase of R post by ET-1 (20 nM), 3 mL of imatinib (10 mM), corresponding to a total amount of 17,691 mg imatinib were nebulized over a period of 130 min. Assuming a lung flow of 0,21 L/ min (70 breaths à 3 mL) and a pressure of 1.5 bar, the total amount of inhaled imatinib corresponds to less than 4% of the nebulized amount of imatinib [41], namely 0,71 mg, corresponding to 2 mg/kg body weight imatinib, respectively. The contractile effect of ET-1 agonists strongly varies. Therefore, the maximal ET-1-induced increase of R post was normalized to 100% and all submaximal increases of R post were indicated in % of the maximal increase of R post .

Statistical analysis
Statistics were conducted using SAS software 9.3 (SAS Institute, Cary, North Carolina, USA) and GraphPad Prism 5.01 (GraphPad, La Jolla, USA). The data in Figs. 4, 5 and 6a,b,e,f were analyzed using a linear mixed model analysis (LMM) with the covariance structures VC or AR(1); EC 50 values (Figs. 2b, c, e, f, 3b and 6c, d) were calculated by the standard 4-parameter logistic non-linear regression model (GraphPad, La Jolla, USA). The AIC-criterion was used to select the most parsimonious model, i.e. a common top, bottom, slope and EC 50 value in the regression model or the covariance matrix with the least number of parameters. Non-parametric analysis (Figs. 2a, d and 3a, c) was performed by the Mann-Whitney U test. All p-values were adjusted for multiple comparisons by the false discovery rate and are presented as mean ± SEM; n indicates the numbers of animals. P <0.05 was considered as significant.

Results
We studied the relaxant effects of imatinib in naïve (not pre-constricted) and in pre-constricted PVs.

Involvement of the cAMP/PKA-pathway to the vasorelaxant effect of imatinib
In PVs, imatinib increased intracellular cAMP (Fig. 2a). The functional role of the cAMP/PKA-pathway was addressed by pre-treatments with the adenyl cyclaseinhibitor SQ22536 (100 μM) and the PKA-inhibitor KT5720 (1 μM). Both inhibitors alone do not alter the contractile effect of ET-1 [29], but here they decreased the imatinib-induced relaxation (Fig. 2b, c). To study the contribution of NO or NO-downstream products (cGMP/PKG) to the imatinib-induced relaxation, PVs were pre-treated with the NO-synthase-inhibitor L-NAME (100 μM) or the PKG-inhibitor KT5823 (2 μM). Both inhibitors do not affect the ET-1-induced contraction [29]. Here, L-NAME and KT5823 did also not alter the relaxant effect of imatinib (Fig. 2e, f). Accordingly, imatinib did not increase intracellular cGMP-levels in PVs (Fig. 2d).

Involvement of K + -channels to the vasorelaxant effect of imatinib
We further evaluated the impact of K + -channel-activation in the imatinib-induced relaxation of cavine PVs. K ATP -channel-inhibition by glibenclamide (10 μM), BK Ca 2þ -channel-inhibition by iberiotoxin (100 nM) and K v -channel-inhibition by 5 mM 4-aminopyridine (4-AP) do not affect ET-1-induced contraction [29]. However, inhibition of K ATP -channels, BK Ca 2þ -channels or K v -channels significantly reduced the relaxant effect of imatinib (Fig. 3a-c); inhibition of K ATP -or K v -channels was most effective.

Imatinib lowers the R post in the IPL
To elucidate whether and how imatinib affects the pulmonary venous bed in an intact organ, we perfused and nebulized imatinib in the IPL after raising R post by ET-1. In the IPL, perfusion of 20 nM ET-1 increased P PA about 600% of baseline from 1.5 cmH 2 O to 9.5 cmH 2 O (p <0.0001), whereas control lungs had no increase of P PA and remained stable (Fig. 4a). ET-1 did not affect P LA (Fig. 4b). Further, the experiments with the double occlusion method revealed that ET-1 significantly enhanced R pre up to 312% and R post up to 169% of baseline (Fig. 4c, d). Next we studied if perfused or nebulized imatinib significantly affects R post . Perfusion of imatinib significantly lowered the ET-1-induced increase of R post (Fig. 5a), even though R post was still increased (p <0.05) compared to control lungs (Fig. 5a). Nebulization of imatinib (calculated dosage: 2 mg/kg body weight) reduced the ET-1-induced increase of R post from the time points 110 -150 min, but R post was still significantly increased (p <0.001) compared to control lungs (Fig. 5b).

The role of PDGFR-β and interaction of ET-1 with PDGFR
The antiproliferative properties of imatinib are likely explained by PDGFR-inhibition. Thus, we hypothesized that the imatinib-induced relaxation may depend on inhibition of PDGFR kinase activity and further that PDGF-BB may possibly exert contractile properties in pulmonary vessels. To test this hypothesis and to identify, which PDGFR-subunit might be responsible for a possible contractile effect, we pre-treated PVs with 100 μM imatinib (PDGFR), 5 μM SU6668 (PDGFR-β) or 100 nM ponatinib (PDGFR-α) prior to the treatment with 10 or 100 nM PDGF-BB. Control PVs treated with 10 or 100 nM PDGF-BB contracted up to 70% of IVA. In contrast, pre-treatment with imatinib and SU6668 abolished the contractile effect of PDGF-BB, whereas pre-treatment with ponatinib only insignificantly reduced the contractile effect of PDGF-BB (Fig. 6a, b). Next, we analyzed the effects of the PDGFR-α inhibitor ponatinib and the PDGFR-β inhibitors SU6668 and DMPQ on PVs pre-constricted with ET-1. Treatment with SU6668 and DMPQ relaxed PVs pre-constricted with ET-1 up to 140% of IVA (Fig. 6c), whereas treatment with the PDGFR-α inhibitor ponatinib had no significant relaxant effect (Fig. 6c). Further, concomitant pre-treatment with 5 μM SU6668 and 1 nM ET-1 or 5 μM DMPQ and 1 nM ET-1 (Fig. 6d) abolished imatinib-induced relaxation, exceptional if it was applied at 100 μM. Next, we studied whether stimulation of ET-1 receptors interacts anyhow with PDGFR or conversely, if activation of PDGFR contributes to the contractile effect of ET-1. In order to inhibit PDGFR, we pre-treated PVs 60 min with imatinib (10 or 100 μM), 5 μM SU6668 or 100 nM ponatinib prior to the treatment of 1 nM ET-1 (Fig. 6e, f ). We found, irrespective of inhibition of PDGFR-αβ, ET-1 contracted PVs without significant difference (Fig. 6e, f ).

Discussion
The tyrosine kinase inhibitor imatinib represents an intriguing approach to treat PH. Initially, research focused on the antiproliferative effects of imatinib, in line with the reversal of vascular remodeling in experimental studies [42]. Interestingly, in clinical trials and case reports, imatinib also ameliorated hemodynamics [11,12,16] and restored heart function [13] suggesting that it may also improve vascular tone. Meanwhile, corroborating studies in PAs from healthy or hypertensive rats demonstrated the relaxant effects of imatinib [14,15]. Clinically, however, effects on PVs may be more relevant since the most common cause of PH is LHD, a condition that primarily affects PVs [8,43]. Therefore, in the present study we focused on the relaxant effects of imatinib in the pulmonary venous bed. We selected GPs, as the access to human lung tissue is limited and the pulmonary smooth muscle pharmacology of GPs resembles human tissue much better than rats or mice [28,44]. Most studies were performed using PCLS, a method [28,29] that allows to study PVs within their natural tissue anatomy [45]. To study the effects of imatinib   on the R post , we used the double occlusion method [38] in isolated perfused lungs (IPL) of GPs. To mimic a pathophysiological aspect of PH, namely the overexpression of ET-1 and its receptor [46,47], we pre-constricted PVs with ET-1.
In the present study, imatinib relaxed pre-constricted PVs with an EC 50 value of 32 μM in line with a previous study in PAs [14]. Imatinib-induced relaxation was dependent on the generation of cAMP and on the activation of K + -channels. In the IPL, perfused or nebulized  (Fig. 6a, b, e, f). Asterics indicate different EC 50 values (Fig. 6c, d). P <0.05 are considered as significant: *** < 0.001 imatinib reduced the ET-1-induced increase of R post . Notably, nebulized imatinib might prevent perfusion/ ventilation mismatches and imatinib-related systemic side effects such as subdural hematoma or hypotension. Further, PDGF-BB contracts PVs, a finding which might explain in part why imatinib exerts relaxation. Our results suggest the use of imatinib in the therapy of PH due to LHD.

Mechanisms contributing to imatinib-induced relaxation
The tone of vascular SMCs is regulated by various signaling cascades, which ultimately modulate intracellular Ca 2+ -levels or/and Ca 2+ -sensitivity of the myofilaments. In this context, the nucleotides cAMP and cGMP play a leading part. Cyclic AMPvia activation of PKAcauses relaxation by K + -channel-stimulation. Further, cAMP acts in a Ca 2+ -desensitizing manner: 1) cAMP blocks myosin light chain kinase (MLCK); 2) cAMP activates myosin light chain phosphatase (MLCP) [48]. It has been reported that Ca 2+ -desensitization plays a role in imatinib-induced relaxation [14]. Here we show that imatinib leads to increased intracellular cAMP levels in cavine PVs. The functional relevance of this cAMP-increase was shown by inhibiting adenyl cyclase (SQ22536) or PKA (KT5720); both inhibitors attenuated the relaxant effect of imatinib. The generation of cAMP has not yet been reported within the context of imatinib-or TKI-induced vasorelaxation, even though they are supported by own unpublished results from human pulmonary vessels and by Kim et al. [49] who proved in murine interstitial cell of Cajals that the imatinib-induced inhibition of pacemaker potentials depends on the generation of cAMP [49]. These results raise the question, how to explain the imatinib-induced increase of cAMP or more precisely how PDGFR-inhibition is linked to the activation of G αs, the inhibition of G αi or the inhibition of PDE-III: 1) There is evidence that RTKs and GPCR interact together, e.g. via transactivation [50], as it was already shown for EGF and G αi [51]. 2) Recently it was reported that PDGF inhibits regulators of G protein signaling (RGS) [52]. In particular, it was shown that PDGF suppresses the expression of RGS-5 in vascular SMC; since downregulation of RGS-5 activates G αi , adenyl cyclase is blocked and the generation of intracellular cAMP reduced.
Cyclic AMP/PKA-signaling appears critical for the imatinib-induced relaxation, although inhibition of adenyl cyclase or PKA did not completely prevent it. This suggests the involvement of other signaling cascades such as NO/cGMP/PKG. PKG promotes Ca 2+ -desensitization via MLCP-activation and stimulates K + -channels [48]. The NO/cGMP/PKG-signaling highly depends on an intact endothelium releasing aside NO [53] also prostacyclin [54,55]. In PCLS, PVs dispose of an intact endothelium [56], even though NO/cGMP/PKG-signaling did not contribute to imatinib-induced relaxation, as 1) imatinib failed to increase cGMP; 2) inhibition of NOsynthesis (L-NAME) or PKG (KT5823) did not attenuate imatinib-induced relaxation. Despite these results, we cannot conclude if imatinib-induced relaxation is endothelium independent or dependent, as it is highly based on cAMP/PKA-signaling and within this context, the endothelial release of prostacyclin might be of impact. Our results corroborate in part those of Abe et al. [14], who showed that imatinib relaxes PAs from hypertensive rats, despite NO-inhibition. Aside that, they [14] proved in endothelial denuded PAs (rats) that the relaxant effect of imatinib does not depend on the endothelium or the endothelial prostacyclin release, as well. In contrast, in SMCs from human corpus cavernosum [57] and in prostatic SMCs [58], imatinib-induced relaxation appears to depend on NO-signaling. Thus, the role of NO/PKG/ cGMP in the imatinib-induced relaxation appears to be species and organ-specific.
To study further mechanisms, we evaluated the impact of K ATP -, BK Ca 2þ -and K v -channels, all of which are expressed in pulmonary vessels [59,60]. Activation of K + -channels leads to cell membrane hyperpolarization resulting in reduced cytosolic Ca 2+ -influx and in relaxation of vascular SMCs. In GP PVs, inhibition of all three K + -channels attenuated imatinib-induced relaxation, with K ATP -and K v -channel inhibitors being more effective. The minor role of BK Ca 2þ -channels might be explained by the fact that BK Ca 2þ -channels dominate in conduit PAs [59], although in rat PAs K + -channel-activation could not explain the imatinibinduced relaxation [15]. In line with our results, a dominant role of K ATP -versus BK Ca 2þ -channels was demonstrated for the imatinib-induced relaxation in human prostatic SMCs [58]. Conversely, PDGF has been shown to decrease K v -channel currents [61]. Moreover, the imatinib-induced activation of K + -channels does not only mediate relaxation, but also contributes to its antiproliferative effects, as hyperpolarization of the cell membrane lowers the intracellular Ca 2+ -influx/content and counteracts the vascular remodeling [59]. A similar relationship has also been shown for the Ca 2+ -sensitizer levosimendan that develops pulmonary vasorelaxant effects [29] and antiproliferative properties via K + -channel-activation [62]. Finally, imatinib-induced relaxation is based on cAMP/ PKA dependent mechanisms and on K + -channel activation. Both signaling pathways may interact to some extent in an additive manner [48,63].

Pulmonary vasorelaxant effects of perfused and nebulized Imatinib in the IPL
The resistance of the pulmonary venous bed, precisely the postcapillary resistance (R post ) is increased in PH due to LHD and mainly responsible for the generation of hydrostatic edema [6]. Vice versa, reduction of R post is one of the major therapeutic goals in this disease. To address the vasorelaxant effects of imatinib on R post in situ, we perfused imatinib in isolated lungs. This set-up enables: 1) to address the pulmonary arterial and venous segment independent from each other; 2) to monitor P PA , P LA and the capillary pressure (P cap ) and 3) to calculate R post from P LA and P cap . In order to access the resistance of the pulmonary venous bed, the calculation of R post is highly relevant, because it includes the small PVs. Further, it is superior to the P pcw , which rather reflects the pressure in large PVs [64] or P LA [65].
Perfusion of 20 nM ET-1 significantly increased P PA , R pre and R post in the IPL. In contrast, P LA did not change, as it is expected due to the reservoir function of the left atrium in this model. In the present study, perfusion of 10 μM imatinib significantly lowered R post . This finding is in line with the results of the IMPRES study that showed improved hemodynamics after the application of imatinib, expressed as a decrease in PVR and an increase in the 6 min walk distance [16]; of note, the IMPRES study only included patients with PAH [16]. Our results are further supported by Shah et al. [13], who found that imatinib improves right but also left heart function. However, the IMPRES study [16], as well as the long-term extension of the IMPRES study [17] further revealed undesirable imatinib-related side effects such as vomiting, nausea or peripheral edema that decrease patient compliance; and even more critical were the occurrence of subdural hematoma, QTc prolongation, cardiac failure and syncope, the latter two most probably being caused by systemic hypotension [16,17]. Unfortunately, however in PH, systemic hypotension is quite problematic, as it decreases the coronary perfusion pressure and consequently worsens left and right ventricular function [66]. To find a way to prevent systemic side effects, we evaluated the possibility and efficacy to apply imatinib locally via the airways [18], as it is already usual in PH for NO-donors, prostanoids or PDE-inhibitors [67,68]. In the IPL, we could show that nebulized imatinib reduces the ET-1-induced increase of R post . The results from the IPL (perfusion/nebulization) correspond to our data from PCLS, even though nebulization of imatinib was less effective in reducing R post than perfusion. As mentioned above (methods), these differences are probably explained by the maximal imatinib up-take: the imatinib up-take via nebulization is 0.71 mg corresponding to 2 mg/kg body weight imatinib and via perfusion 1.18 mg corresponding to 3.5 mg/kg body weight imatinib (the ratio is 0.6). Other effects may also play a role such as an insufficient imatinib-uptake through the alveo-capillary membrane or the degradation of imatinib by intraalveolar macrophages. Here, we performed an IPL model; thus the extent to which nebulized imatinib provokes systemic adverse effects remains to be shown. In summary, we focused on central PVs in PCLS and we addressed R post (IPL) , resembling the peripheral part of the pulmonary venous bed [69,70]. In both models, we demonstrated that imatinib relaxes the pulmonary venous bed of GPs.

Conclusions
Imatinib relaxes PVs by activation of cAMP/PKA and potassium channels (K ATP -, BK Ca 2þ -, K v -channels), mechanisms that are related in an unknown way to the inhibition of PDGFR-β. The clinical importance of imatinib-induced relaxation is supported by reports that imatinib improved the clinical condition of a patient with pulmonary venous occlusive disease within 24 h [12] and ameliorated PH due to congenital hernia [11]. The prompt improvement in both cases favors relaxant effects of imatinib in contrast to the reversal of vascular remodeling which lasts longer [10]. However, until now studies with human pulmonary vessels are still missing and quite difficult to obtain. Our data suggest a beneficial role of imatinib in PH due to LHD. Since imatinib combines short-term relaxant, even if inhaled and long-term antiproliferative effects, it may represent a promising approach to treat PH due to LHD.