PDGF-BB regulates the pulmonary vascular tone: impact of prostaglandins, calcium, MAPK- and PI3K/AKT/mTOR signalling and actin polymerisation in pulmonary veins of guinea pigs

Female Dunkin Hartley GPs (350 ± 50 g) were delivered from Charles River (Sulzfeld, Germany). All animal experiments were approved by the Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen (ID: 84–02.04.2013A146, 8.87–51.05.20.10.245 and 50066A4) and strictly performed according to the rules of the Directive 2010/63/EU of the European Parliament.

GPs’ lungs were prepared as described [7, 13, 14]. In brief, intraperitoneal anaesthesia was performed (pentobarbital: 95 mg kg^− 1) and verified by missing reflexes. The animal was exsanguinated, the trachea cannulated and the lung ventilated with positive pressure (70 breaths/min). The apex of the left ventricle was cut and cannulas were placed in the PA (perfusion inflow) and in the left atrium (perfusion outflow). The lung was perfused at constant flow (20 mL/min) with Krebs-Henseleit buffer, containing 2% bovine serum albumin, 0.1% glucose, 0.3% HEPES and 50 nM salbutamol to prevent bronchoconstriction [31]. The temperature of the perfusate was maintained at 37 °C with a water bath and the pH was adjusted between 7.35 and 7.45 by gassing with CO₂. Heart and lungs were withdrawn and transferred into a negative-pressure chamber, the so-called artificial thorax chamber. Next, ventilation was switched from positive pressure to negative pressure. To prevent the formation of lung oedema during constant flow perfusion and negative pressure ventilation, a pressure balancing chamber was established in the perfusion outflow which was connected by tubing to the artificial thorax chamber. To prevent atelectasis of the lung, every 5 min a deep breath was applied. Tidal volume (TV), dynamic compliance (Cdyn), resistance (Res), pulmonal arterial pressure (P_PA), left atrial pressure (P_LA) and the flow were continuously monitored. Further, the capillary pressure (P_cap) was measured every 10 min by the double occlusion method [14] and the precapillary (R_pre) and postcapillary resistance (R_post) were calculated by the following equations: R_pre = \( \frac{PPA- Pcap}{flow} \) and R_post = \( \frac{Pcap- PLA}{flow} \).

As soon as respiratory and haemodynamic parameters remained stable over 10 min (baseline), imatinib (10 μM) was perfused at time point 10 min. At a buffer volume of 200 mL, a concentration of 10 μM imatinib corresponds to a total dose of 1.18 mg imatinib or to 3.5 mg/kg body weight imatinib, respectively. Control lungs remained untreated. Next, PDGF-BB (10 nM) was added to the recirculating perfusion buffer (total volume 200 mL) at time point 30 min and perfused in untreated lungs and imatinib-pre-treated lungs. Beyond that, imatinib mesylate was nebulised in some lungs prior to the perfusion of PDGF-BB. Therefore, 29.38 mg imatinib mesylate were solved in 3 ml aqua to obtain a solution of 16.6 mM and nebulised 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 nebulised amount of imatinib [32], namely 1.18 mg, corresponding to 3.5 mg/kg body weight imatinib, respectively. To measure PGI₂ and TXA₂, IPL-perfusate samples were obtained at time point 0, 30 (before the application of PDGF-BB) and 120 min. The different groups and the timeline of the experiments are illustrated in Fig. 1.

In GPs, intraperitoneal anaesthesia 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. Thereafter, PCLS were prepared as described before [13, 16, 30]. Whole lungs were filled via the trachea with 1.5% low-melting agarose and cooled on ice to harden the lungs. Afterwards, tissue cores (diameter 11 mm) were prepared and cut into 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.

Pulmonary vessels from GPs were identified by their anatomical landmarks; e.g. PAs accompany the airways and PVs lie aside [13, 16].

To evaluate the contractile effect of PDGF-BB in PAs/PVs from GPs, PCLS were exposed for 60 min to 100 nM PDGF-BB (Figs. 3, 4 and 5, Figs 7, 8 and 9). If a signalling pathway was evaluated (Figs. 3,4 and 5, Figs 7, 8 and 9), PCLS were additionally pre-treated for 1 h with one of the following inhibitors at concentrations about 10–100 fold above the IC₅₀ value of the target: PDGFR-α: 100 nM ponatinib (IC₅₀: 1.1 nM) [33‐35]; PDGFR-β: 5 μM SU6668 (IC₅₀: 0.008–0.1 μM) [36‐38]; PDGFR-α/β: 100 μM imatinib (IC₅₀: 0.6–1.8 μM) [39]; L-Type Ca²⁺-channels: 100 nM amlodipine (IC₅₀: 1.9 nM) [40]; Rho-Kinase: 10 μM fasudile (IC₅₀: 1.4 μM) [41]; protein kinase C (PKC): 5 μM calphostin C (IC₅₀: 50 nM) [42]; cyclooxygenase 1/2: 3 μM indomethacin (IC₅₀: 13–26 nM) [43, 44]; EP₁: 1 μM SC51322 (IC₅₀: 13.8 nM) [45]; EP₂: 1 μM PF04418948 (IC₅₀: 2.7 nM) [46, 47]; EP₃: 1 μM L798106 (IC₅₀: 10 nM) [48, 49]; EP₄: 1 μM L161982 (IC₅₀: 3.2 nM) [48]; TP: 10 μM SQ29548 (IC₅₀ 10 nM) [48]; IP: 1 μM RO-1138452 (IC₅₀: 5–10 nM) [50]; MAP2K: 50 μM PD98059 (IC₅₀: 2–7 μM) [51]; MAP2K: 5 μM U0126 (IC₅₀: 58–72 nM) [52]; p38-MAPK: 5 μM SB203580 (IC₅₀: 0.5 μM, for AKT/PKB 3–5 μM) [53, 54]; PI3K-α: 100 nM GSK 1059625 (IC₅₀: 2 nM); PI3K-γ: 100 nM AS252424 (IC₅₀: 33 nM) [55]; AKT/PKB: 10 μM 10-DEBC (IC₅₀: 2 μM) [56]; actin polymerisation: 10 μM cytochalasin D (IC₅₀: 100 nM) [57] or 1 μM latrunculin A [58]; adenyl cyclase (AC): 100 μM SQ22536 (IC₅₀: 1.4–200 μM) [59] and NO-synthase (NOS): 100 μM L-NAME (IC₅₀: 25 μM).

In PCLS, all changes of the initial vessel area (IVA) were quantified in % and indicated as “Change [% of IVA]”. Thus, an IVA < 100% indicates contraction and an IVA > 100% indicates relaxation. To compare the contractile effect of PDGF-BB in pre-treated vessels, the intraluminal area was defined after pre-treatment again as 100%. In the graphs, all pre-treatments were indicated. The intraluminal area of PAs and PVs was monitored with a digital video camera (Leica Viscam 1280, Leica DFC 280). The images were analysed with Optimas 6.5 (Media Cybernetics, Bothell, WA).

To analyse cAMP, PVs were isolated out of tissue cores guided by their anatomical landmarks, e.g. the PAs accompany the airways and PV lies aside. PVs were incubated in medium, flushed with PDGF-BB (100 nM) and after 30 min frozen by liquid nitrogen. Cyclic AMP was quantified with ELISA-kits following the manufacturer’s protocol. Samples/standards were acetylated for stabilisation. To measure cAMP, all samples were diluted 1:2 with 0.1 M HCL. The ELISA was analysed at 405 nM (GENIOS, Tecan, Switzerland).

To analyse prostacyclin (synonym: prostaglandine I₂ (PGI₂)) and thromboxane A₂ (TXA₂), IPL perfusate samples were obtained at 0, 30 (before PDGF-BB was applied) or 120 min and stored at − 80 °C. PGI₂ and TXA₂ are quickly metabolised, hence the metabolites 6-keto prostaglandin F_1α (6-keto PGF_1α) and 11-dehydro TXB₂ and 2,3-dinor (TXB₂) were measured to estimate the generation of PGI₂ and TXA₂, respectively. Prostaglandin metabolites were quantified with ELISA-kits following the manufacturer’s protocol and measured at 412 nM (GENIOS, Tecan, Switzerland).

PDGF-BB was provided by Peprotech (Hamburg, Germany). Imatinib mesylate, amlodipine, fasudile, calphostin C, indomethacin, SC51322, PF04418948, L798106, L161982, GSK 1059615, AS 252424, 10-DEBC and SQ22536 were purchased from Tocris Bioscience (Ellisville, Missouri, USA). Ponatinib was acquired from SelleckChem (Munich, Germany). SQ29548, RO-1138452, SU6668, SB203580, PD98059 and U0126 were acquired from Cayman Europe (via Biomol, Hamburg, Germany). The cAMP ELISA-kit was acquired from Enzo (Lörrach, Germany), whereas all ELISA-kits applied to quantify prostaglandin generation were acquired from Cayman Europe (via Biomol, Hamburg, Germany). L-Name, cytochalasin D, latrunculin A or standard laboratory chemicals were provided by Sigma (Steinheim, Germany).

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 Fig. 6a/c were analysed by the Wilcoxon signed rank test (matched pairs), whereas the data in Fig. 6b/dor Fig. 8c were analysed by the Mann-Whitney U test (no matched paired). All other data were analysed using a linear mixed model analysis (LMM) with the covariance structure AR(1). 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.

Perfusion of PDGF-BB (final concentration in the buffer: 10 nM) increased P_PA up to 116% (p < 0.05), whereas P_PA remained stable over 140 min in untreated control lungs (Fig. 2a). Pre-treatment with perfused imatinib (final concentration in the buffer: 10 μM) completely prevented the PDGF-BB-related increase of P_PA (p < 0.05) (Fig. 2a) and even decreased P_PA compared to baseline values (p < 0.001). Pre-treatment with nebulised imatinib also prevented the PDGF-BB-induced increase of P_PA (p < 0.05) (Fig. 2a). In addition, the soley perfusion of imatinib significantly decreased P_PA compared to baseline values (p < 0.001) (Fig. 2a).

Perfusion of PDGF-BB (final concentration in the buffer: 10 nM) increased P_cap up to 193% (p < 0.001) compared to control lungs and to baseline values (Fig. 2b). According to the effects on P_PA, the PDGF-BB-induced increase of P_cap was completely prevented (p < 0.001), if the lungs were pre-treated with perfused or nebulised imatinib (Fig. 2b). Further, perfusion of imatinib lowered P_cap, at time points 60, 100 or 120 min (for all: 0.04) (Fig. 2b). Neither perfusion of PDGF-BB, nor perfusion of imatinib affected anyhow R_pre (Fig. 2c).

Perfusion of PDGF-BB significantly increased R_post (p < 0.001). This effect was completely prevented, if lungs were pre-treated with imatinib (p < 0.001), either perfused or nebulised. Further, perfusion of imatinib alone decreased R_post (p < 0.05) compared to control lungs and to baseline values (Fig. 2d). The addition of PDGF-BB did not alter P_LA (data not shown).

In IPLs, PDGF-BB contracted the pulmonary vascular bed and this was preventable by the TKI imatinib (Fig. 2). Next, we tried to find out in PCLS if PDGF-BB contracts PAs and if this contraction predominantly depends on PDGFR-β, as it was shown for PVs (Fig. 3a) [7]. PDGF-BB contracted PAs up to 87% of IVA (p < 0.05) and this contraction was prevented, if PCLS were pre-treated with the PDGFR-β inhibitor SU6668 (p < 0.01), whereas pre-treatment with the PDGFR-α inhibitor ponatinib had no effect (Fig. 3b). In PVs, PDGF-BB-induced contraction was stronger than in PAs (p < 0.01, Fig. 3c).

To get insights if there is a link between PDGF-BB-induced contraction and the pathogenesis of PH, we focused the mechanisms beyond PDGF-BB-induced contraction. Due to the weak contractile effect of PDGF-BB in PAs; we studied PDGF-BB downstream-signalling in PVs.

PVs were pre-treated for 60 min with 100 nM amlodipine (L-Type Ca²⁺-channels), 10 μM fasudile (Rho kinase inhibitor) or 5 μM calphostin C (PKC) prior to the application of 100 nM PDGF-BB. Amlodipine significantly reduced the contractile effect of PDGF-BB (p < 0.05) (Fig. 4a), whereas fasudile (Fig. 4b) or calphostin C (Fig. 4c) were without significant effect (p > 0.05 for both).

Next, we studied, whether the contractile effect of PDGF-BB is mediated via contractile prostaglandins. PCLS were pre-treated with the non-selective cyclooxygenase-inhibitor indomethacin (3 μM), with the EP₁-receptor antagonist SC51322 (1 μM), with the EP₂-receptor antagonist PF04418948 (1 μM), with the EP₃-receptor antagonist L798106 (1 μM), with the EP₄-receptor antagonist L161982 (1 μM), with the TP-receptor antagonist SQ29548 (10 μM) and with the IP-receptor antagonist RO-1138454 (1 μM). Inhibition of prostaglandin synthesis (indomethacin) did not significantly alter PDGF-BB-induced contraction (Fig. 5a), although the sustained effect of PDGF-BB appeared to be reduced. PDGF-BB-induced contraction was significantly reduced, if EP₁-receptors (p < 0.01; Fig. 5b), EP₃-receptors (p < 0.001; Fig. 5b) or TP-receptors (p < 0.01; Fig. 5e) were blocked. In contrast, inhibition of EP₄-receptors (Fig. 5d) only attenuated PDGF-BB-induced contraction from time point 45 min, whereas inhibition of EP₂-receptors (Fig. 5c) or IP-receptors (Fig. 5f) did not affect the maximal contractile effect of PDGF-BB. However, pre-treatment with the IP-receptor antagonist RO-1138454 strongly contracted PVs to 75.5% of IVA (p < 0.001; data not shown). Finally, inhibition of EP₃-receptors (Fig. 5b) was most potent and nearly completely prevented PDGF-BB-induced contraction.

In IPL-perfusate samples, we studied the effect of PDGF-BB on the generation of prostaglandins, e.g. TXB₂ for TXA₂ and 6-keto PGF_1α for PGI₂ (Fig. 6). After 120 min of perfusion, PDGF-BB enhanced TXB₂ compared to basic values (p < 0.05; Fig. 6a). Further at 120 min, TXB₂ was significantly increased compared to 1) the control, 2) the imatinib/PDGF-BB and 3) the imatinib group (p < 0.05; Fig. 6b), whereas at 0 or 30 min, the four treatment groups did not differ (Fig. 6b). In contrast, the PGI₂-metabolite 6-keto PGF_1α was in all groups significantly increased (p < 0.05) in dependence to the perfusion time (Fig. 6c). At 120 min, 6-keto PGF_1α reached a level of 341 pg/ml in the PDGF-BB group compared to 193 pg/ml in the control group (p > 0.05) and to 124 pg/ml in the imatinib/PDGF-BB group (p < 0.05; Fig. 6d). Hence, 6-keto PGF_1α was significantly lower, if IPLs were pre-treated with imatinib compared to PDGF-BB alone, although PDGF-BB did not significantly increase 6-keto PGF_1α compared to the control group. With regard to 6-keto PGF_1α, no differences were found at 0 or 30 min (Fig. 6d).

Next, we studied if the PDGF-BB downstream-signalling involved in proliferation [3] also contributes to PDGF-BB-induced contraction. Therefore, we inhibited cellular pathways such as MAP2K (5 μM U0126 / 50 μM PD98059), p-38 MAPK (5 μM SB203580), PI3K-α (100 nM GSK 1059625), PI3K-γ (100 nM AS252424) and AKT/PKB (10 μM DEBC). Inhibition of MAP2K (p < 0.05, p < 0.001; Fig. 7a), p38-MAPK (p < 0.05; Fig. 7b), AKT/PKB (p < 0.001; Fig. 7e), PI3K-α (p < 0.5; Fig. 7c) and PI3K-γ (p < 0.05) reduced the contractile effect of PDGF-BB (Fig. 7d).

The data from Fig. 7a/e reveal weak relaxation due to PDGF-BB leading to an IVA > 100%. Together with the observations that some PVs contract to less than 80% of IVA (Fig. 7a; p = 0.003), the idea was obvious that PDGF-BB downstream-signalling might involve relaxant mediators different from PGI₂ (Fig. 6c/d), e.g. cAMP or NO. To study this issue, PVs were pre-treated with the AC-inhibitor SQ22536 or with the endothelial NOS-inhibitor L-NAME prior to the application of PDGF-BB. Inhibition of cAMP-generation (SQ22536) increased the contractile effect of PDGF-BB until time point 35 min (p < 0.001) (Fig. 8a), whereas inhibition of endothelial NOS (eNOS) only slightly enhanced it (Fig. 8b), as this increase only reached statistical significance between the time points 30–45 min (p < 0.05; Fig. 8b). Further, 100 nM PDGF-BB increased cAMP (p < 0.001) in PVs (Fig. 8c) suggesting that PDGF-BB-induced generation of cAMP influences the pulmonary venous tone, whereas PDGF-BB-related NO-synthesis appears to play a minor role in PVs.

PDGF-BB-induced contraction appears to depend on complex intracellular pathways. Next, we analysed the role of actin polymerisation by 10 μM cytochalasin D or 1 μM latrunculin A. Inhibition of actin polymerisation significantly lowered the contractile effect of PDGF-BB, as indicated for cytochalasin D (p < 0.001; Fig. 9a) and latrunculin A (p < 0.05; Fig. 9b).

In the IPL, recirculating perfusion of 10 nM PDGF-BB significantly enhanced P_PA up to 116% (Fig. 2a). These results confirmed those from PCLS; where 100 nM PDGF-BB contracted PAs up to 87% of IVA (Fig. 3b). According to the Hagen-Poiseuille law, the resistance increases 16 fold, if the radius is splitted in half. Hence, P_PA would have increased even above 116%, if lungs were perfused with 100 nM PDGF-BB. Further in the IPL, PDGF-BB-induced alteration of the vascular tone is detected even at lower concentrations compared to PCLS. This is supported by the fact that 10 nM PDGF-BB did not contract GPs’ PAs in PCLS (data not shown), whereas 10 nM PDGF-BB enhanced P_PA to 116% in the IPL (Fig. 2a). In contrast to these results, PDGF-BB did not alter R_pre (Fig. 2c) indicating a minor effect on the cavine precapillary pulmonary vascular bed. Possible reasons for this observation might be 1) a lower receptor density; 2) a varying receptor equipment with reduced sensitivity. Most probably, our results regarding the effect of PDGF-BB on R_pre are not transferable to the human situation, as small human PAs are equipped with PDGFR-β [1]. In general, GPs’ PCLS allow to study more central pulmonary vessel, but do not represent the precapillary part of the pulmonary circulation [13]. In contrast, the IPL allows addressing the entire pulmonary vascular bed (except central PVs); particularly, it enables to determine the segmental vascular resistance (R_pre / R_post) by the double occlusion method [7, 13, 14]. Beyond PDGF-BB-induced pulmonary arterial contraction, PDGF-BB increased P_cap (Fig. 2b) and R_post (Fig. 2d) up to 200 and 140% of baseline values, respectively. Further, PDGF-BB contracted central PVs from GPs up to 70% (Fig. 3a).

Hence, our GP’ data from both models suggest that PDGF-BB exerts significant contraction along the pulmonary vascular bed and give strong evidence that PDGF-BB-induced contraction is accentuated in the pulmonary venous system PVs (Fig. 2; Fig. 3). This result is of high clinical relevance, as the pulmonary venous bed contributes about 40% to pulmonary vascular resistance (PVR) [60] and plays a major role in PH due to left heart disease [61], the most common cause of PH [62, 63].

PDGF-BB-induced contraction of the pulmonary vascular bed was completely prevented if IPLs (Fig. 2) were pre-treated with the PDGFR-α/β inhibitor imatinib, either perfused or nebulised; indicating that PDGF-BB-induced contraction specifically relies on the activation of PDGFR-α/β. With regard to GPs’ PVs, we recently showed that PDGF-BB-induced contraction mainly depends on the activation of PDGFR-β (Fig. 3a), whereas activation of PDGFR-α only plays a minor role [7]. In this work, we validated this also for GPs’ PAs as we found that inhibition of PDGFR-β (SU6668) completely prevented PDGF-BB-induced contraction of PAs (Fig. 3b) and inhibition of PDGFR-α (ponatinib) had no effect (Fig. 3b). In the IPL, perfusion of imatinib did not only prevent the PDGF-BB-induced increase of P_PA, P_cap and R_post, but also decreased P_PA, R_post and in part P_cap (time points 90 and 110 min after addition) compared to untreated control lungs. These data suggest the existence of endogenously produced PDGF-BB and the permanent activation of PDGFR.

After identification of PDGF in the seventies, PDGF-BB-induced contraction [8‐10, 64] and relaxation [11, 12] was proven in systemic arteries. Afterwards, the vascular effects of PDGF disappeared in the background and research focused on the proliferative effects of PDGF [1, 2] leading to the introduction of TKIs in the therapy of PH [2, 4, 65]. With this regard, TKI-induced relaxation has been uncovered [5‐7] and the contractile effects of PDGF-BB have been proven in PVs [7]. Thus, it becomes apparent that PDGF-BB promotes aside proliferation also contraction of PAs/PVs, both promoting the progress of PH. Therefore we studied the mechanisms beyond the contractile effect of PDGF-BB in GPs’ PVs.

In PVs, PDGF-BB-induced contraction depended on the activation of L-Type Ca²⁺-channels (Fig. 4a), whereas Ca²⁺-sensitisation did not play a role, as inhibition of Rho-Kinase or PKC did not alter PDGF-BB-induced contraction (Fig. 4b/c). In line with our results, PDGF-AB or PDGF-BB contract extra pulmonary vessels in a calcium dependent manner [8‐10, 64, 66] and vice versa, TKIs modulate the activity of L-Type Ca²⁺-channels in portal veins [67, 68]. Anyhow, differences exist within the PDGF dimers AA, AB or BB; e.g. Sachinidis et al. [10] reported in rats’ aortic rings that PDGF-BB contracts stronger and rises intracellular calcium more potently than PDGF-AB, whereas PDGF-AA acts only poorly contractile and does not rise intracellular calcium [10]. In rat aortic smooth muscle cells (SMCs), the same group [10, 69] found that PDGF-AA potently stimulates PKC, whereas PDGF-BB activates PKC only in a minor degree. These findings suggest that PDGF-AA rather acts via Ca²⁺-sensitisation, whereas PDGF-BB mainly exerts contraction via the increase of calcium. Moreover, with regard to tone or endothelial barrier, pulmonary and systemic vessels are diversely regulated [70]. This circumstance might also explain contrasting results.

So far, it is unknown, if prostaglandins mediate the contractile effect of PDGF-BB in pulmonary vessel. Although, PDGFR downstream-signalling is linked to the generation of prostaglandins [71‐73] and prostaglandin receptors, e.g. TP- or EP_1/3-receptors are involved within the regulation of the tone of human PAs [74] and PVs [75, 76].

Now, our data reveal that PDGF-BB-induced contraction goes ahead with the activation of TP-receptors, as 1) inhibition of TP-receptors strongly reduced the contractile effect of PDGF-BB and as 2) TXB_2, the inactive metabolite of TXA₂ was significantly enhanced in PDGF-BB perfused lungs. TXA₂ acts as a potent vasoconstrictor and highly contributes to increased vascular tone in PH [77‐79]. TP-receptors represent G-Protein-coupled receptors (GPCR) which are mainly coupled to G_αq/11 and G_α12/13, but also to G_αs/i, G_h and G_βγ; finally their stimulation leads to the regulation of phospholipase C (PLC)/inositol trisphosphate (IP₃)/calcium, Rho and AC [80]. Beyond that, MAPK- and PI3K-signalling is also involved [80]. In line with our data, Sachinidis et al. [10] showed in systemic vessels that PDGF-BB leads to the generation of TXA₂ which exerts as well as the TP-agonist U46619 a strong and long-lasting contraction along the pulmonary vascular bed [81‐84]. Our data show no involvement of Rho/PKC in PDGF-BB downstream-signalling. This is opposing to Murtha et al. [84] who showed in rabbits’ pulmonary arterial rings that the contractile effect of TXA₂ depends on PKC. Further, TP-receptors are coupled to G_α12/13, hence their stimulation should activate Rho [85], unless TP-receptors are primarily coupled to G_αq/11 [80]. Notably, vasoconstrictors such as endothelin-1 or platelet-activating factor also mediate their contractile effect via the release of TXA₂ [86, 87].

Our results indicate that PDGF-BB-induced contraction goes ahead with the activation of EP_1/3/4-receptors, whereas stimulation of EP₂-receptors does not play a role (Fig. 5c). Hence, PGE₂ as the most widely produced prostaglandin of the body binding to EP_1–4-receptors (GPCRs) [25] appears to be highly involved in PDGF-BB-induced contraction. EP₁-receptors are coupled to G_αq/11 [25, 88] and their activation triggers the intracellular increase of PLC, IP₃ and calcium [88]. In contrast, EP₃-receptors are mainly coupled to G_αi and their activation inhibits the AC leading to decreased cAMP-levels [25, 88]. Here, inhibition of EP₃-receptors nearly completely prevented PDGF-BB-induced contraction, although inhibition of EP₁- or TP-receptors was also very effective in preventing the contractile effect of PDGF-BB. So, the question comes up if the EP₃-receptor antagonist L798106 acts unspecific and also binds to EP₁- or TP-receptors. According to IC₅₀ values, this is not the case [48]. However, the prominent effect of EP₃-inhibition on PDGF-BB-induced contraction might be explainable by the consideration that EP₃-inhibition may provoke an overwhelming cAMP-generation counteracting other contractile mediators activated by PDGF-BB. In general, EP₃-agonists strongly contract human PAs [74]. Beyond that, they influence the progress of PH, as EP₃-receptor deficiency attenuates the expression of PH [89]. Aside EP_1/3-receptors, PGE₂ also activates EP₂- and EP₄-receptors which are both coupled to G_αs leading to the activation of AC, to the increase of cAMP and to reduced vessel tone [25, 88]. Moreover, EP₄-receptors also couple to G_αi representing the counter player of G_αs [25, 88]. Here, inhibition of EP₂-receptors did not enhance the contractile effect of PDGF-BB, inhibition of EP₄-receptors reduced PDGF-BB-induced contraction and PVs treated with PDGF-BB showed increased cAMP-levels. These data suggest 1) a possible minor expression of EP₂-receptors in GPs’ PVs or 2) a possible dominant coupling of EP₄-receptors to G_αi [90] which has been already reported in the hypoxic pulmonary arterial bed of the rat. In addition, although EP₄-receptors appear to couple dominantly to G_αi, G_αs-coupling seems to be of relevance, as PDGF-BB significantly increased cAMP-levels in PVs. This idea is supported by the circumstance that PGI₂, as a main source for cAMP [91] did not significantly increase due to PDGF-BB. Thus, the production of cAMP should derive from PDGF-BB-induced activation of prostaglandin receptors others than IP, but also coupled to G_αs.

In general, the various prostaglandins mediate contraction or relaxation. In this work, EP_1/3/4- and TP-receptors reduced the contractile effect of PDGF-BB, whereas none of the inhibitors, including the IP-receptor antagonist RO-1138452, did increase PDGF-BB-induced contraction. At first glance, PDGF-BB-downstream-signalling appears to be dominated by the generation of contractile prostaglandins. At second view, this assumption is opposed, as inhibition of prostaglandin synthesis (indomethacin) did not significantly alter PDGF-BB-induced contraction (Fig. 5a); a fact which suggests that PDGF-BB-dependent prostaglandin generation is well-balanced between contractile and relaxant ones. Further, inhibition of MAP2K- and AKT/PKB-signalling (Fig. 7a/e) unmasked a slight relaxant effect of PDGF-BB supporting the hypothesis that PDGFR-downstream-signalling is anyhow related to relaxant pathways. Our results indicate that PGI₂ is of less relevance within the regulation of the pulmonary vascular tone by PDGF-BB, as 1) inhibition of IP-receptors did not enhance the contractile effect of PDGF-BB (Fig. 5f). 2) PDGF-BB did not increase PGI₂ (Fig. 6c/d), though a trend appears to be evident which is enforced by the fact that pre-treatment with imatinib significantly lowered PGI₂-levels compared to PDGF-BB perfusion alone. The observation that PGI₂ increased time-dependently in the perfusate of all IPL-groups (Fig. 6c), including control lungs is explainable by the endothelial release of PGI₂ counteracting the increased shear stress in perfused lungs [92, 93]. In general, the release of PGI₂ strongly depends on shear stress [94]. In contrast, in PCLS shear stress is hardly effective [95], hence time-dependent PGI₂-release is not expected. However, we could show that basal PGI₂-release should occur, as inhibition of IP-receptors increased the tone of PVs. Finally, our data indicate that PDGF-BB-induced PGI₂-release is less relevant, although we cannot exclude that PDGF-BB potentiates anyhow the release of PGI₂ due to shear stress. Our results are different from those of Yamawaki et al. [12] who proved that PDGF-BB relaxes rat mesenteric arteries in dependence to the release of PGI₂. Finally, the role of PDGF-BB-induced PGI₂-release might depend on the vessel localisation, e.g. pulmonary vessels versus systemic vessels and on the species.

In spite of the fact that PDGF-BB contracts GPs’ PVs, the generation of relaxant mediators such as cAMP and cGMP plays a relevant role in PDGFR-downstream-signalling. 1) Inhibition of AC (Fig. 8a) enforced the contractile effect of PDGF-BB, 2) PVs treated with PDGF-BB had higher cAMP-levels than control PVs (Fig. 8c) and 3) inhibition of eNOS slightly enforced the contractile effect of PDGF-BB (Fig. 8b). Our results are supported by those of Graves et al. [96] who found in human arterial SMCs that PDGF-BB downstream-signalling goes ahead with the generation of cAMP/PKA, just as the cAMP generation depends on the release of arachidonic acid, probably activating EP_2/4- or IP-receptors. These results were also proven in rat myometrial cells [71] and in GPs’ airway SMCs [97]. Usually, stimuli which activate prostaglandin receptors coupling to G_αs; e.g. EP_2/4, IP or DP should increase intracellular cAMP [98, 99].

Aside from cAMP, NO seems to be of impact in PDGF-BB downstream-signalling. Though, NO-inhibition only slightly enhanced PDGF-BB-induced contraction (Fig. 8b). Our results are in line with those from Takase et al. [11] who perfused rat mesenteric arteries; there PDGF-BB stimulated NO-release even relaxed rat mesenteric arteries. The different characteristic of PDGF-BB-induced NO-release might be due to two facts; 1) Takase et al. [11] exposed rat mesenteric arteries to shear stress, generally going ahead with endothelial NO-release [100], 2) systemic and pulmonary vessel behave different to similar stimuli [70].

With regard to cellular regulation (migration, differentiation, proliferation, growth or survival of cells), PDGFR downstream-signalling mainly activates two pathways: 1) the MAPK-pathway and 2) the PI3K/AKT/mTOR pathway [101]. Our data show that both inhibition of MAP2K by PD98059 or U-0126, as well as inhibition of p38-MAPK by SB 203580 almost prevented the contraction by PDGF-BB (Fig. 7a/b). Notably, GPs’ PVs even relaxed slightly (Fig. 7a). In line with our data, Schaafsma et al. [102] showed in GPs’ tracheal strips that PDGF-BB-induced contraction highly depends on the activation of MAP2K. Next, Boulven et al. [71] proved in rat myometrial cells that PDGF-BB-dependent synthesis of prostaglandins is up to MAP2K. Conversely, the stimulating effect of PDGF-BB on phospholipase A₂ (PLA₂) and subsequent prostaglandin synthesis also depends on MAP2K [71, 103‐105]. Finally, MAPK-signalling is highly involved in PDGF-BB-induced prostaglandin synthesis.

The PI3K/AKT/mTOR pathway highly contributes to mediate the proliferative aspects of PDGFR [3, 101]. Here both, PI3K-α which is expressed ubiquitously [106] and PI3K-γ which is expressed in the cardiovascular system [106] contribute to the contractile effect of PDGF-BB. Hence, PI3K-γ does not only regulate the systemic vascular tone [106], but is also of impact for the regulation of the pulmonary vascular tone. Together with the fact that inhibition of AKT/PKB (Fig. 7e) prevented the contractile effects in PVs, our results suggest a role of PI3K/AKT/mTOR-signalling within the contractile effect of PDGF-BB. Our data are supported by Hua et al. [107] who showed that AKT prevents the degradation of cytosolic PLA₂ (cPLA₂), finally promoting prostaglandin synthesis. Beyond that, activation of AKT is linked to the activation of eNOS [108], an issue which seems to be negligible within PDGF-BB-induced regulation of the pulmonary venous tone, as 1) inhibition of AKT did not contract PVs and 2) as inhibition of eNOS enhanced the contractile effect of PDGF-BB only slightly (Fig. 8b). Further, our results are contrasting to those of Macrez et al. [109] who showed in vascular SMCs that the PDGF-BB-induced intracellular increase of calcium depends on PI3K-β, but not on PI3K-α which are both coupled to receptor tyrosine kinases (RTKs) [110‐112]. Regarding PDGF-BB-signalling, non-direct activation of MAPK and PI3K is conceivable, as TP-receptors are also linked to G_βγ, finally leading to the activation of MAPK- or PI3K/Akt/mTOR signalling [80].

In conclusion, prostaglandin generation appears to be a major mechanism beyond PDGF-BB-induced regulation of the pulmonary venous tone. Within this context, there are several possibilities to activate cPLA₂, 1) by the increase of intracellular calcium [71, 104, 113], 2) by PDGF-BB-induced MAPK-signalling [71, 103‐105] and 3) by the inhibitory properties of AKT on cPLA₂-degradation [107]. In addition, 4) the transactivation of PDGFR by the G_αq-coupled AngII is described [101], leading to the activation of PLC and IP₃ and to the subsequent increase of intracellular calcium. For an overview, please see Fig. 10.

Aside MLC-phosphorylation, actin polymerisation plays an important role within the contractile process of SMCs [26, 27]. Within this context, it is of interest that PDGF-BB – via activation of SRC - stimulates the abelson tyrosine kinase (ABL) [114‐116] which itself promotes actin polymerisation [27]. In contrast, the TKI imatinib is known to inhibit ABL [27].

Our data indicate that PDGF-BB-induced actin polymerisation contributes to the contractile effect of PDGF-BB, as inhibition of actin polymerisation by cytochalasin D (Fig. 9a) or latrunculin A (Fig. 9b) strongly reduced PDGF-BB-induced contraction in PVs. With regard to the stimulating effect of PDGF-BB on ABL, our results are comprehensible. They are even less unexpected, as MAPK-signalling which represents a cornerstone of PDGF-BB downstream signalling influences actin polymerisation [117, 118]. So far, the impact of actin polymerisation for the regulation of the pulmonary venous tone has not been shown. Although, its relevance for SMC-contraction was shown in SMCs of various vessel and species; e.g. canine carotids [119], ferret aorta [120], rat mesenteric arteries [121, 122], rat thoracic aorta [57] or rat extrapulmonary PAs [123], as well as in SMCs from airways [124].

PDGFR-downstream-signalling is associated with the increase of intracellular calcium [9, 10, 64]. Here we show that the contractile effect of PDGF-BB also depends on it. Increased calcium-levels represent a major trigger for vasoconstriction, proliferation and migration of vascular SMCs [18, 80, 125‐127]. Thus, stimuli which increase calcium-levels, e.g. TXA₂ or ET-1 [80, 128], but also hypoxia [125, 127] or PDGF-BB [2, 64] do not constrain to increase the pulmonary vascular tone, but also promote pulmonary vascular remodelling. Hence, enhanced tone and remodelling are closely linked with each other. In PH, this circumstance could be beneficial in view of new therapeutics, e.g. therapeutics addressing the mechanisms beyond PDGF-BB-induced contraction may attenuate vasoconstriction and remodelling. Ultimately, calcium-levels could be adressed directly by amlodipine or nifedipine, but also indirectly via inhibition of TP/EP_1/3- receptors. In respect thereof it is worth mentioning that EP₃-receptor deficiency attenuates PH [89] and that prednisolone inhibits PDGF-BB-induced proliferation of PAs’ SMCs [129]. Further, inhibition of MAP2K-/AKT-signalling and cPLA₂ could be of interest. Notably, the mentioned pathways could be addressed in a systemic way, but also topically, e.g. via inhalation to reduce systemic side effects.