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Soluble receptor for advanced glycation end products (sRAGE) attenuates haemodynamic changes to chronic hypoxia in the mouse
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Soluble receptor for advanced glycation end products (sRAGE) attenuates haemodynamic changes to chronic hypoxia in the mouse Q5
David G.S. Farmer, Marie-Ann Ewart, Kirsty M. Mair, Simon Kennedy* Institute of Cardiovascular and Medical Sciences, College of Medical, Veterinary & Life Sciences, University of Glasgow, G12 8QQ Glasgow, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 June 2013 Received in revised form 20 December 2013 Accepted 1 January 2014
The calgranulin-like protein MTS1/S100A4 and the receptor for advanced glycation end-products (RAGE) have recently been implicated in mediating pulmonary arterial smooth muscle cell proliferation and vascular remodelling in experimental pulmonary arterial hypertension (PH). Here, the effects of RAGE antagonism upon 2 weeks of hypobaric hypoxia (10% O2)-induced PH in mice were assessed. Treatment with sRAGE was protective against hypobaric hypoxia-induced increases in right ventricular pressure but distal pulmonary vascular remodelling was unaffected. Intralobar pulmonary arteries from hypobaric hypoxic mice treated with sRAGE showed protection against a hypoxia-induced reduction in compliance. However, a combination of sRAGE and hypoxia also dramatically increased the force of contractions to KCl and 5-HT observed in these vessels. The acute addition of sRAGE to the organ bath produced a small, sustained contraction in intralobar pulmonary vessels and produced a synergistic enhancement of the maximal force of contraction in subsequent concentrationeresponse curves to 5-HT. sRAGE had no effect on 5-HT-induced proliferation of Chinese hamster lung fibroblasts (CCL39), used since they have a similar pharmacological profile to mouse pulmonary fibroblasts but, surprisingly, produced a marked increase in hypoxia-induced proliferation. These data implicate RAGE as a modulator of both vasoreactivity and of proliferative processes in the response of the pulmonary circulation to chronic-hypoxia. Ó 2014 Published by Elsevier Ltd.
Keywords: Pulmonary hypertension RAGE MTS1/S100A4 Hypoxia Fibroblasts
1. Introduction Q1
Pulmonary hypertension (PH) is a rare, progressive disease of the small pulmonary arteries characterised by pulmonary vascular remodelling, vasoconstriction and thrombosis [34]. Pulmonary vascular remodelling is associated with smooth muscle proliferation, muscularisation of peripheral pulmonary arteries and medial thickening in larger pulmonary arteries. Muscularisation is associated with fibroelastosis, ablated responses to vasodilators and formation of obliterative plexiform lesions. Sustained elevation of pulmonary vascular pressure increases afterload in the right ventricle, ultimately leading to right heart failure [14,15,34].
Abbreviations: 5-HT, 5-hydroxytryptamine; PH, pulmonary hypertension; sRAGE, soluble RAGE; RAGE, receptor for advanced glycation end-products; PASMC, pulmonary arterial smooth muscle cells; HMGB-1, high mobility group box-1; RVH, right ventricular hypertrophy; CCL39, Chinese hamster lung fibroblasts; LME, linear mixed effects model; RVP, right ventricular pressure; RV, right ventricle; LV, left ventricle; BrdU, bromodeoxyuridine; SNP, sodium nitroprusside; MTS1, metastasis associated 1. * Corresponding author. Tel.: þ44 141 330 4763. E-mail addresses: david.farmer@florey.edu.au (D.G.S. Farmer), marie-ann. [email protected] (M.-A. Ewart), [email protected] (K.M. Mair), simon. [email protected] (S. Kennedy).
Recent studies have suggested a role for the receptor for advanced glycation end-products (RAGE) in experimental models of PH [11,18,24,37]. In systemic vascular disease, RAGE activation is associated with the generation of reactive oxygen species, activation of inflammatory transcription factors (e.g. NF-kB), increased vascular permeability, adhesion molecule expression and increased expression of RAGE itself. RAGE is therefore implicated in both the induction and amplification of inflammation [1,31,36]. RAGE is basally expressed in small arteries and arteriolar capillaries of the lung [2,26]. The calcium-binding, calgranulin-like protein MTS1/S100A4, a RAGE ligand, is expressed in remodelled pulmonary vessels and advanced occlusive vascular lesions in PH patients [11]. Thus RAGE ligands by their interaction with RAGE may modulate vascular remodelling in PH. In vitro, MTS1/S100A4 produces proliferation in human pulmonary arterial smooth muscle cells (PASMC) via an action at RAGE. Additionally, expression and secretion of MTS1/S100A4 re part of a downstream pathway producing proliferation in response to 5-HT; an established mitogen which is implicated in PH [7,18,21]. In vivo, overexpression of MTS1/S100A4 in transgenic mice is associated with spontaneous formation of obliterative, plexiform-like lesions in a subset (w5%) of animals [11]. All MTS1/S100A4 mice show impaired recovery from a chronic hypoxic challenge, reduced pulmonary vascular lumen diameter and increased elastin deposition occurring downstream of RAGE activation [24].
1094-5539/$ e see front matter Ó 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.pupt.2014.01.002
Please cite this article in press as: Farmer DGS, et al., Soluble receptor for advanced glycation end products (sRAGE) attenuates haemodynamic changes to chronic hypoxia in the mouse, Pulmonary Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pupt.2014.01.002
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Much work to date has focussed on the role of RAGE in PASMC proliferation. In the hypoxic lung in vivo however, adventitial fibroblasts demonstrate a more immediate and dramatic proliferation before migrating to the media and secreting substances which are mitogenic to PASMCs [33,38]. The role of RAGE in pulmonary fibroblast proliferation is unknown but HMGB-1, a RAGE ligand, was shown to induce proliferation and migration of mouse 3T3 fibroblasts which were prevented by a RAGE antibody [32]. In contrast, RAGE blockade increased proliferation in cultured human pulmonary fibroblasts [30]. RAGE activation may be prevented experimentally using soluble RAGE (sRAGE). sRAGE possesses the RAGE ligand binding domains but lacks the cytoplasmic and transmembrane domains. sRAGE will therefore compete for ligands which bind to cell-bound RAGE [35]. Additionally, sRAGE can prevent RAGE signal transduction directly by preventing the homodimerisation of RAGE on the cell surface [46]. In this study we tested the hypothesis that treatment with sRAGE would attenuate the effect of chronic hypoxia in mice. Additionally, we attempted to characterise the proliferative response of pulmonary fibroblasts to mitogenic stimuli in the presence of sRAGE in vitro. 2. Methods 2.1. In vivo experiments All animal care and experimental procedures were in accordance with the UK Animals (Scientific Procedures) Act 1986 and conformed to institutional regulations at the University of Glasgow. All mice used in the study were bred in the University of Glasgow, kept on a 12 h light/dark cycle and fed ad libitum. 2.2. Exposure to hypoxia In humans, idiopathic and familial PH occurs with a greater frequency in females than males. However, in experimental animals, males are more prone than females to hypoxia-induced PH [44]. For that reason, we chose to use male mice in this study. C57Bl/6 mice, aged 2e3 months (n ¼ 8e10 mice per group) were maintained in normoxic conditions or hypobaric/hypoxic conditions for 14 days as previously described [22,25]. The hypobaric chamber was depressurised over the course of 2 days to 550 mbar (equivalent to 10% O2). Temperature was maintained at 21 Ce 22 C, and the chamber was ventilated with air at 45 L min1. 2.3. Plasma RAGE determination In order to establish the effect of chronic hypoxia on plasma RAGE concentration an ELISA assay was run (R&D Quantikine ELISA for mouse RAGE; R&D, Abingdon, UK). Five plasma samples each were assayed from normoxic and chronic hypoxic mice in duplicate and compared against a calibration curve (31.3e2000 pg ml1 RAGE). All samples fell within the calibration range and are reported as plasma RAGE in ng ml1. 2.4. Administration of sRAGE Normoxic and hypoxic mice were randomly assigned to daily intraperitoneal injection with either vehicle (phosphate-buffered saline; PBS) or 20 mg/day sRAGE. This dose was chosen based on the work of [12] where it was demonstrated that daily dosing with sRAGE significantly impaired the worsening of disease in a mouse model of atherosclerosis at doses of 20 mg/day and 40 mg/day with no further benefit derived from a dose of 100 mg/day. Mice in the hypoxic groups were returned to normoxia once a day to facilitate
dosing with either vehicle or sRAGE over the 14 day course of the intervention. sRAGE was kindly supplied by Dr Anne-Marie Schmidt (Columbia University Medical Centre, New York, USA). 2.5. Characterisation of PH 2.5.1. Pressure measurements After 14 days hypobaric hypoxia, systolic right ventricular pressure (sRVP) was measured under isoflurane (1.5% in O2) anaesthesia via a 25-gauge needle advanced into the right ventricle trans-diaphragmatically [16,22]. RVP and heart rate (derived from the RVP) were recorded on a data acquisition system (MP 100, Biopac Systems). Systemic arterial pressure was recorded via a cannula placed in the carotid artery. It was not possible to obtain a full set of readings in all animals so the final n number for each group was: normoxic vehicle group ¼ 7, hypoxic vehicle group ¼ 9, normoxic sRAGE group ¼ 7, hypoxic sRAGE group ¼ 6. 2.5.2. Measurement of RVH and pulmonary vascular remodelling The ratio of right ventricular weight to left ventricular weight plus septum [RV/(LV þ S)] was used as an index of right ventricular hypertrophy (RVH) [22,25]. Briefly, the atria were removed and the right ventricle separated from the left ventricle and septum. The wet weights of the right ventricle and the left ventricle were measured on an analytical balance. The right lung was dropped into chilled 10% neutral buffer formalin (Composition: 90% dH2O, 10% formalin, 4 g per litre NaH2PO4, 6.5 g per litre Na2HPO4) without inflation and agitated for a period of 3 days. Sagittal sections were obtained, stained with Elastic Van Gieson stain and microscopically assessed for muscularisation of pulmonary vessels (<80 mm external diameter) as described previously [22,25]. Vessels were considered muscularised if they possessed a distinct double-elastic lamina visible for at least half the circumference of the vessel in cross-section. The percentage of vessels containing double-elastic lamina (and hence deemed remodelled) was calculated as the number of muscularised vessels/total number of vessels counted per section 100. Two to three sections from each left lung were assessed for every mouse. Lung sections from four to six mice from each group were studied. 2.5.3. Pulmonary artery myography Pulmonary arteries from normoxic and hypoxic controls and sRAGE-treated mice were studied. The animals were killed by cervical dislocation on completion of in vivo experiments and the lungs removed. Small pulmonary arteries (third order, first intralobar) of w250 mm internal diameter were dissected from the left lung, cut into 2 mm long segments, and mounted in an isometric myograph using 40 mm stainless steel wires (Danish Myo Technology, Aarhus, Denmark) as described previously [16,25]. The vessels were maintained at 37 C in Krebs’ buffer solution (pH 7.4) of the following composition (in mM): NaCl 118.4, NaHCO3 25, KCl 4.7, KH2PO4 1.2, MgSO4 0.6, CaCl2 2.5, glucose 11.0 and EDTA 0.023. Pulmonary arteries were aerated with 16% O2/5% CO2 balance N2. Aerating with this composition of gases results in an oxygen tension in the bath equivalent to that of the pulmonary circulation in vivo and has been used in previous studies from our laboratory [25]. Vessels were placed under ascending gradations of known tension equivalent to the mean in vivo RVP (12e15 mmHg). The diameter of the vessel corresponding to each of these tensions was recorded and forceediameter graphs plotted as a measure of compliance. Vessel responses were recorded onto a computer using a data acquisition and recording software system (Chart, ADInstruments, Oxford, UK). After a 45 min equilibration period, the response to 50 mM KCl, the concentration that produced maximal contraction in these vessels, was measured. For studies involving
Please cite this article in press as: Farmer DGS, et al., Soluble receptor for advanced glycation end products (sRAGE) attenuates haemodynamic changes to chronic hypoxia in the mouse, Pulmonary Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pupt.2014.01.002
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contractile agents, ascending concentrations of drug were added to artery rings under resting tension (5-HT: 300 pMe30 mM) cumulatively and the responses recorded. To measure the effect of hypoxia and sRAGE treatment on endothelium-independent relaxation, ascending concentrations of sodium nitroprusside (SNP; 300 pMe300 mM) were added to preconstricted rings and the responses calculated as a percentage loss of the initial constrictorinduced tone. In some experiments, endothelium-dependant relaxation to acetylcholine (1 mM) was measured. Typically this resulted in less than 10% relaxation so we conclude that the intralobar pulmonary arteries used in this study are denuded. 2.6. Fibroblast proliferation For studies on the effects of sRAGE on fibroblast proliferation, Chinese hamster lung fibroblasts (CCL39 cells) were used as these cells exhibit a very similar pharmacological profile to mouse pulmonary arterial fibroblasts [23] and can be grown in sufficient quantity for such experiments. At confluence, CCL39 cells were plated into 24-well plates at 20,000 cells/well and grown in full medium (Dulbecco’s modified Eagle’s medium supplemented with 10% v/v foetal bovine serum, penicillin, streptomycin, 1 mg ml1 fungizone and 50 mg ml1 gentamycin) at 37 C for 24 h before serum starvation for 24 h. For studies on the effects of sRAGE on proliferative stimuli, cells were preincubated with sRAGE for a period of 30 min before addition of the proliferative stimulus. Cells were then washed with sterile PBS, harvested by trypsinisation and collected by centrifugation. The cell pellet was resuspended in medium and 1 mL was added to a haemocytometer. Cell number was quantified by manual counting of the number of cells in four 1 mm2 sectors. Cell counts were made in duplicate for each well and proliferation was expressed as an elevation above the mean number of cells in control wells. To study the effects of hypoxia sRAGE on CCL39 proliferation, cells were grown under normobaric conditions in a humidified, temperature controlled and nitrogen-supplemented, Galaxy R incubator (Wolf Laboratories, York, UK). This allowed control of internal O2 levels at 5%, and CO2 levels were maintained at 5%. This achieves, for the hypoxic conditions, a tissue culture supernatant PO2 of 35 mmHg. In experiments involving sRAGE, this was added 30 min prior to transferring the fibroblasts to the hypoxic environment. The fibroblasts were exposed to hypoxia for 24 h before proliferation was measured. In some experiments, CCL39 proliferation was measured using a BrdU assay (Calbiochem, Nottingham, UK). Briefly, CCL39 cells were plated around 40% confluence and grown in DMEM medium as above. Cells were quiesced when around 70% confluent in DMEM þ0.2% serum for 24 h then treated with either sRAGE (2.5 or 10 mg ml1) or vehicle for 30 min. In some wells, the RAGE agonist S100B, a protein whose effects in alveolar cell lines are antagonised by sRAGE [29] was then incubated at 1 mg ml1 [19] for 30 min. Proliferation was then stimulated in all wells by addition of either 0.2% or 2.5% serum plus BrdU for 24 h at 37 C in the incubator. The plates were then fixed and the BrdU assay was run. Proliferation index was assayed spectrophotometrically and results reported as optical densities. 2.7. Statistical analysis All data are presented as mean SEM. Analysis of all haemodynamic parameters, the extent of RVH and of remodelling, and comparisons between EC50 values and maximal responses to pharmacological agents was by one-way ANOVA followed by posthoc analysis with Fisher’s least significant difference (LSD) test for multiple comparisons. The effect of sRAGE treatment and hypoxia combined was analysed by two-way ANOVA. A significant
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interaction between barometric treatment group and drug treatment group was taken as evidence for an effect of sRAGE. Plasma RAGE was analysed using Student’s unpaired t-test. Differences between concentration response curves were assessed by a linear mixed-effects (LME) model. For the purpose of making statistical comparisons of the effects of various stimuli on cell proliferation, normalised cell counts were assessed by means of a linear model to identify treatments which induced a statistically significant increase above baseline. The effects of treatments were compared using Student’s t-test or Fisher’s LSD test where appropriate. BrdU data for proliferation was analysed by one-way ANOVA with Dunnett’s correction for multiple comparisons. A p-value of less than 0.05 was considered significant and where appropriate, p-values were adjusted for multiple comparisons using the Bonferroni method. 3. Results 3.1. Effects of hypoxia 2 weeks chronic hypoxia markedly elevated the concentration of RAGE detected by ELISA in the plasma (3.21 0.77 ng ml1 in control mice vs. 11.97 1.67 ng ml1 in hypoxic mice; n ¼ 5 p < 0.05 vs. control). Systemic arterial pressure was not significantly altered by chronic hypoxia or by daily sRAGE injection in either normoxic or hypoxic animals (data not shown). Two weeks of hypobaric hypoxia produced significant increases in sRVP (Normoxia: 22.65 1.01 mmHg; Hypoxia: 31.02 1.51 mmHg; p < 0.01), proportion of remodelled vessels (Normoxic: 12.67 2.13%; Hypoxic: 20.83 1.94%; p < 0.05) and RVH (Normoxic: 0.27 0.01; Hypoxic: 0.34 0.01; p < 0.01) in vehicle-treated animals. sRAGE protected animals against the increase in sRVP in response to hypoxia as compared to the response in vehicle-treated animals (p < 0.05; two-way ANOVA; Fig. 1A). Furthermore, the sRVP of hypoxic sRAGE-treated mice did not differ significantly from normoxic mice receiving the same treatment (normoxic: 24.41 0.73 mmHg; hypoxic: 26.92 1.53 mmHg; p ¼ ns) but did not protect the animals against hypoxia-induced remodelling (Fig. 1B) or increased RVH (Fig. 1C). Under normoxic conditions, sRAGE produced no significant changes in sRVP (24.41 0.73 mmHg; Fig. 1A), proportion of remodelled vessels (13.92 2.16%; Fig. 1B), or RVH (0.28 0.011; Fig. 1C) when compared to vehicle-treated controls. Heart rate was not significantly altered by sRAGE treatment in normoxic animals (Vehicle: 337.37 16.48 bpm; sRAGE: 293.44 14.87; p ¼ ns, Student’s ttest). Chronic hypoxia significantly lowered heart rate in both vehicle and sRAGE-treated animals to a similar extent (vehicle: 276.3 12.28; sRAGE: 269.25 10.21; p < 0.01 vs. control for both groups, LME model). Remodelled vessels possessing a double elastic lamina were evident in sections from both hypoxic groups (Fig. 2) but the extent of vascular remodelling in sRAGE-treated hypoxic mice was not significantly different from vehicle-treated hypoxic mice. Compliance of intrapulmonary vessels, measured as the increase in diameter caused by the application of increasing force was unchanged by sRAGE treatment under normoxic conditions (Fig. 3). Exposure to 2 weeks of hypoxia significantly reduced the compliance of these vessels in vehicle-treated mice (Fig. 3A) while sRAGE prevented this reduction in compliance (Fig. 3B). 3.2. Pulmonary artery reactivity In these vessels, contraction to 5-HT was not altered by hypoxia alone (Fig. 4A). However in animals treated with sRAGE during
Please cite this article in press as: Farmer DGS, et al., Soluble receptor for advanced glycation end products (sRAGE) attenuates haemodynamic changes to chronic hypoxia in the mouse, Pulmonary Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pupt.2014.01.002
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Fig. 2. Representative small pulmonary vessels from mice treated with vehicle or sRAGE under normoxic conditions or after 2 weeks hypoxia. Small intrapulmonary vessels possessing a double elastic lamina were evident from both groups of animals after hypoxic challenge. sRAGE had no effect on the appearance of remodelled vessels. Photographs taken under 40 objective magnification. Scale bar ¼ 50 mm.
normoxic control) but significantly increased the contraction in hypoxic animals (3.11 0.39 mN; p < 0.05; two-way ANOVA; p < 0.05 vs. normoxic sRAGE-treated mice; Fisher’s LSD test; Fig. 5). When the concentrationeresponse to 5-HT from each experiment was normalised to its corresponding KCl response, no significant enhancement of the response to 5-HT was observed in sRAGE-treated, hypoxic animals when compared to normoxic,
Fig. 1. A e Effect of two weeks of chronic hypobaric hypoxia with daily sRAGE or vehicle (PBS) treatment on systemic right ventricular pressure, B e % remodelled vessels or C e right ventricular hypertrophy. Daily treatment with i.p. sRAGE (20 mg kg1) attenuated the hypoxia induced rise in sRVP but had no effect on vessel remodelling or ventricular hypertrophy. Data are expressed as mean SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs. normoxic, PBS treated mice; yyp < 0.01 vs. normoxic, sRAGE-treated mice, xp < 0.05 two-way ANOVA vs. vehicle-treated mice (vehicle normoxic: n ¼ 7; vehicle hypoxic: n ¼ 9; sRAGE normoxic: n ¼ 7; sRAGE hypoxic: n ¼ 6).
exposure to hypoxia, there was a significant exaggeration in the contractile response to 5-HT (Fig. 4B) compared to sRAGE treatment under normoxic conditions. Emax values in vessels from hypoxic sRAGE-treated animals were significantly elevated above their normoxic counterparts (Emax ¼ 3.94 0.57 mN; p < 0.05). Responses in vessels from hypoxic sRAGE-treated mice were also significantly different from those in normoxic, vehicle-treated mice (p < 0.01) and from those in hypoxic, vehicle-treated mice (p < 0.05) (Fig. 4). No difference in the log EC50 values in these responses was detected (log EC50 for vehicle: 7.56 0.21 vs. sRAGE: 7.65 0.11; p ¼ ns). To ascertain if this increase in contractile force was specific to 5-HT, we examined the magnitude of responses to KCl obtained at the start of each experiment. Two weeks of hypoxia had no significant effect on the maximal contraction to 50 mM KCl (normoxic: 1.72 0.22 mN; hypoxic: 1.61 0.10 mN, p ¼ ns). sRAGE treatment under normoxic conditions did not alter the contraction to KCl (2.00 0.29 mN; p ¼ ns vs.
Fig. 3. A e Compliance of intrapulmonary arteries is significantly reduced in vehicletreated mice exposed to 2 weeks hypoxia (*p < 0.05 vs. normoxia; n ¼ 7e11). B e Compliance of arteries was unchanged following hypoxia in animals treated with daily sRAGE for two weeks. In normoxic animals, sRAGE had no effect on vessel compliance (p ¼ ns). Statistical analysis was by LME model. (Vehicle-treated and sRAGE-treated mice, n ¼ 7; n ¼ 11 at force ¼ 1.2, 1.5 and 1.8 mN; n ¼ 7; n ¼ 11 at force ¼ 2.1 mN; n ¼ 6; n ¼ 9 at force ¼ 2.4 mN).
Please cite this article in press as: Farmer DGS, et al., Soluble receptor for advanced glycation end products (sRAGE) attenuates haemodynamic changes to chronic hypoxia in the mouse, Pulmonary Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pupt.2014.01.002
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Fig. 4. The maximal force of contraction is elevated in hypoxic mice treated with sRAGE but not PBS. (A) Hypoxia produced no alteration in the concentration-response curve to 5-HT in vessels from vehicle-treated animals (p ¼ ns). (B) Responses in vessels treated with sRAGE were significantly altered by hypoxia (*p < 0.05). Responses in vessels from hypoxic sRAGE-treated were also significantly different from those in normoxic, vehicle-treated mice (p < 0.01) and from those in hypoxic, vehicle-treated mice (p < 0.05). Emax in hypoxic, sRAGE treated vessels was significantly increased in comparison to hypoxic, vehicle treated mice (p < 0.05) but not normoxic, vehicletreated or sRAGE-treated vessels. Data are expressed as mean SEM. Analysis was by LME model. (Vehicle normoxic: n ¼ 4; Vehicle hypoxic: n ¼ 4; sRAGE normoxic: n ¼ 5 sRAGE hypoxic: n ¼ 4).
vehicle-treated or sRAGE-treated animals. Relaxation to the nitrovasodilator SNP was not significantly different between vehicletreated and sRAGE-treated animals under normoxic conditions (data not shown). Hypoxia had no significant effect on the relaxation to SNP and sRAGE treatment did not affect the response in hypoxic animals (Emax ¼ 80.24 14.26% in vehicle-treated vs. 69.14 13.75% in sRAGE treated; n ¼ 3e4, p ¼ ns). In some experiments the effects of acute addition of sRAGE (10 mg ml1) on vessel reactivity were studied. In intralobar vessels isolated from normoxic mice, addition of sRAGE produced a small, sustained contraction which reached a maximum at approximately 30 min (Fig. 6A). Similarly, preincubating arteries with sRAGE strongly
Fig. 5. Daily treatment with sRAGE (20 mg kg1) results in an increased force of contraction in response to 50 mM KCl in intrapulmonary arteries from animals exposed to 2 weeks of chronic hypoxic. Data are presented as mean SEM. p < 0.05, Fisher’s LSD test vs. normoxic sRAGE-treated mice; xp < 0.05, two-way ANOVA vs. vehicle-treated mice (Vehicle normoxic: n ¼ 6; Vehicle hypoxic: n ¼ 6; sRAGE normoxic: n ¼ 7; sRAGE hypoxic: n ¼ 6).
Fig. 6. A e Addition of sRAGE to intrapulmonary arteries at resting tension induced a small, sustained contraction. ***p < 0.001 vs. baseline; yp < 0.05 vs. control; control: n ¼ 5; sRAGE: n ¼ 4. B e In vitro addition of sRAGE also significantly augmented the contraction to 5-HT when analysed by LME model. ***p < 0.001 Data are presented as mean SEM; (Control: n ¼ 5; sRAGE: n ¼ 4).
enhanced the maximal contraction to 5-HT although the log EC50 value was unchanged (Fig. 6B). 3.3. CCL39 proliferation Cells in 0% FCS allowed to proliferate for 24 h in normoxia showed a 2.95 1.21 fold increase over the number of plated cells across both experimental groups. 5-HT induced an increase in cell number which was maximal at a concentration of 10 mM. Addition of 2.5 mg ml1 sRAGE had no effect on 5-HT-induced proliferation (Fig. 7A). Interestingly, 2.5 mg ml1 sRAGE alone produced a modest increase in cell number to 31.90 8.12% above baseline (p < 0.01; linear model). Twenty-four hours of hypoxia significantly increased cell number (Fig. 7B) while a combination of hypoxia and 2.5 mg ml1 sRAGE produced a strong proliferative response which was of greater magnitude than hypoxia or sRAGE alone (Fig. 7B). The effect of the RAGE agonist S100B and sRAGE was also assayed using BrdU incorporation as an index of proliferation. When cells were stimulated with low serum (0.2%), S100B alone tended to increase proliferation though this was not significant. sRAGE alone had no effect and the combination of S100B and sRAGE did not have any effect on proliferation (Fig. 7C). When cells were stimulated with 2.5% serum, S100B, sRAGE or a combination of the two had no effect on proliferation compared to control wells (data not shown).
Please cite this article in press as: Farmer DGS, et al., Soluble receptor for advanced glycation end products (sRAGE) attenuates haemodynamic changes to chronic hypoxia in the mouse, Pulmonary Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pupt.2014.01.002
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Fig. 7. A e In CCL39 Chinese hamster lung fibroblasts, 5-HT and 2.5 mg ml1 sRAGE alone induced a significant proliferation compared to serum-free conditions. sRAGE did not modify the proliferative response to 5-HT and there was no evidence of an additive effect of the two agents. B e Incubation of CCL39 cells in a hypoxic environment for 24 h produced a significant proliferation which was significantly augmented by addition of 2.5 mg ml1 sRAGE. C e When 0.2% serum was used as the proliferative stimulus, s100, sRAGE up to 10 mg ml1 or a combination of the two had no significant effect on proliferation measured by BrdU incorporation (data is mean of 8 replicates). Data are presented as mean SEM; *p < 0.05; **p < 0.01, ***p < 0.001 vs. serum-free control (baseline; n ¼ 4); yp < 0.05 vs. hypoxia. (n ¼ 3).
4. Discussion The aim of the current study was to characterise the effects of sRAGE in two novel settings: as an intervention to hypoxia-induced pulmonary hypertension in vivo, and as an influence on proliferation in pulmonary fibroblasts. Since the RAGE agonist MTS1/ S100A4 appears to mediate proliferation in PASMCs through RAGE and can alter structural and functional parameters which may affect pulmonary vascular resistance (i.e. remodelling, vasoreactivity) and influence the pulmonary blood pressure [18], we hypothesised that daily treatment with sRAGE would reduce pulmonary vascular remodelling and decrease the effect of hypoxia on sRVP. We found that sRAGE treatment reduced the magnitude of
the hypoxia-induced elevation in sRVP. However, this change was not accompanied by inhibition of distal vascular remodelling. It is important to note that sRVP was assessed after animals had been returned to normoxia. Therefore, elevation of the sRVP is not due to acute hypoxic pulmonary vasoconstriction but to persistent structural and/or functional alterations of the pulmonary vasculature. In the heart, right ventricular hypertrophy also persists when animals are returned to normoxia. In the current study, reduced sRVP associated with sRAGE treatment was not accompanied by a reduction in hypoxia-induced right ventricular hypertrophy. Since RVH is presumably caused, at least in part, by increased afterload associated with increased pulmonary vascular resistance during the hypoxic challenge, this result suggests that sRAGE treatment did not reduce acute pulmonary vascular pressures during this time. Similarly, the lowered sRVP in sRAGE-treated mice is unlikely to be due to changes in heart rate or cardiac output since both groups showed a similar drop in heart rate on exposure to chronic hypoxia. Although we did not measure cardiac output, a recent study [3] used echocardiography and found that there was no change in RV fractional shortening or cardiac output despite raised sRVP in mice exposed to 6 weeks chronic hypoxia. Alterations in vascular compliance provide a possible explanation for the reduction in sRVP by sRAGE after hypoxia. In vehicletreated animals, chronic hypobaric hypoxia produced a fall in compliance of the intralobar pulmonary arteries (Fig. 3) that was completely prevented by sRAGE. Larger distal conducting vessels contribute to elevated pulmonary pressures through smooth muscle cell hyperplasia and hypertrophy resulting in a loss of elasticity or compliance. The lost ability of these vessels to buffer the pulmonary pressure at right ventricular systole through stretch increases pressure in the pulmonary artery at systole and increases shear stress on more distal vessels [13,47]. Thus, it may be that the number of vessels possessing a double-elastic lamina was similar in sRAGE and vehicle-treated animals but that vessels from sRAGEtreated animals retained basal levels of compliance throughout and after the hypoxic insult. In MTS1/S100A4 overexpressing mice, [24] reported elevated pulmonary pressures in the absence of enhanced remodelling which they attributed to increased elastin deposition in pulmonary vessels. In the current study, we would speculate that an sRAGE-driven inhibition of elastin deposition may have allowed pulmonary arteries from hypoxic animals to retain their compliance. An important point to note is that we did not inflate lungs in any experimental group prior to fixation. This can complicate matters since it can be difficult to determine vessel size and very small vessels may remain collapsed. Thus, although we report data on number of remodelled vessels in this study, we cannot comment on the effect of sRAGE treatment on vessel size. Pulmonary fibroblasts appear to be key mediators of pulmonary vascular remodelling in response to hypoxia. In the hypoxic lung in vivo, adventitial fibroblasts demonstrate an immediate and dramatic proliferation and migration to the tunica media. Here they secrete mitogens for PASMC and contribute to the remodelling process [33,38]. In vitro, it has been reported that PASMCs in culture do not proliferate in response to hypoxia when cultured alone but will do so when co-cultured with fibroblasts [33]. Here, we report that 24 h of hypoxia induced a doubling of the number of fibroblasts compared to baseline; in line with previous in vitro studies on pulmonary fibroblasts [39,41e43]. Surprisingly, preincubation with sRAGE produced a further increase in the number of cells after hypoxia and sRAGE alone produced a small but significant increase in the number of cells over baseline. This suggests that either nominal release of a RAGE ligand or constitutive RAGE activity is an antiproliferative stimulus in pulmonary fibroblasts and that this process may also be an important part of the proliferative response of these cells to hypoxia. The reduction of RAGE ligand
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bioavailability by sRAGE in solution would reduce the former while inhibition of RAGE signalling through heterodimerization of RAGE and sRAGE on the cell surface would inhibit either process. Interestingly, the proliferative effect of sRAGE on fibroblasts was not observed when serum was used as the stimulus. This may be the effect of serum stimulation overcoming the modest proliferative effect of sRAGE under conditions where proliferation was allowed to proceed in the absence of serum. Alternatively, since cell counting was used in the initial experiments with sRAGE and despite adequately controlling the experiments with cells counts made in the absence of sRAGE, caution must be exercised in ascribing the effect wholly to proliferation. BrdU incorporation with serum stimulation is likely to be a more sensitive and physiologically plausible index of proliferation and under these conditions we found no effect of sRAGE alone. In addition, under these conditions the RAGE ligand S100B also had no effect on proliferation and so perhaps the multitude of growth factors contained within serum is too potent a stimulus to be affected by the S100/RAGE axis. Similarly, the basal activation of RAGE by an unknown mechanism appears to be important in the acute regulation of tone in pulmonary arteries since sRAGE produced a small amount of contraction alone and greatly enhanced the force of contraction in response to 5HT when added to the bathing solution in the myograph. The magnitude of the effect of sRAGE upon the response to 5-HT appeared to be greater than a summation of the effects of each compound alone which suggests increased sensitivity of the contractile apparatus of PASMCs to Ca2þ. Another possibility is an action on the vascular endothelium producing either an increase in the basal release of contractile mediators (e.g. endothelin-1, thromboxane A2) or decreased release of relaxatory mediators (e.g. NO, prostacyclin). However, the vessels used in this study were of a small diameter making it difficult to preserve the integrity of the endothelium and an effect here is unlikely. A direct effect of sRAGE upon the smooth muscle therefore seems more likely. We also observed some evidence of an action of sRAGE on SMCs in that vascular contractility was greatly enhanced by a combination of chronic-hypoxia and sRAGE treatment in vivo. A recent study demonstrated that chronic hypoxia enhances both receptormediated calcium entry and store-operated calcium entry in pulmonary arteries [27]. It is therefore possible that sRAGE is able to modulate intracellular calcium to enhance sensitivity to contractile stimuli. The interpretation of the current in vivo data remains difficult. It is not clear what, if fibroblast proliferation is also enhanced in vivo, the overall impact of sRAGE treatment on pulmonary vascular structure and function will be at the level of the vascular cells as they respond to the hypoxic stimulus. sRAGE is reported to reduce PASMC proliferation, yet an increase in fibroblast proliferation might be expected to enhance PASMC proliferation and hence vascular remodelling.
5. Conclusions Although sRAGE-treatment caused a significant, beneficial effect on sRVP after chronic hypoxia, the mechanism does not appear to be via effects on small vessel remodelling or on vascular reactivity in the lung as these were both enhanced. We speculate that sRAGE may, by acting directly upon PAMSCs or indirectly through enhancing proliferation of fibroblasts in hypoxia, produce enhanced sensitivity of the vasculature to contractile stimuli whilst simultaneously protecting against hypoxiainduced reductions in vascular compliance. What remains to be determined in future studies is the effect of sRAGE treatment on RAGE signalling at a cellular levels under control and particularly hypoxic conditions.
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66 67 68 None of the authors has any conflict of interest to declare. 69 70 Uncited references Q4 71 72 [4e6,8e10,17,20,28,40,45]. 73 74 Acknowledgements 75 76 This study was supported by the Integrative Mammalian Biology 77 Initiative e jointly funded by the BBSRC and MRC. 78 79 References 80 81 [1] Basta G. Receptor for advanced glycation endproducts and atherosclerosis: 82 from basic mechanisms to clinical implications. Atherosclerosis 2008;196:9e 21. 83 [2] Brett J, Schmidt AM, Yan SD, Zou YS, Weidman E, Pinsky D, et al. Survey of the 84 distribution of a newly characterized receptor for advanced glycation end 85 products in tissues. Am J Pathol 1993;143:1699e712. [3] Brown RD, Ambler SK, Li M, Sullivan TM, Henry LN, Crossno Jr JT, et al. MAP 86 kinase kinase kinase-2 (MEKK2) regulates hypertrophic remodeling of the 87 right ventricle in hypoxia-induced pulmonary hypertension. Am J Physiol 88 Heart Circ Physiol 2013;304:H269e81. [4] Cadogan E, Hopkins N, Giles S, Bannigan JG, Moynihan J, McLoughlin P. 89 Enhanced expression of inducible nitric oxide synthase without vasodilator 90 effect in chronically infected lungs. Am J Physiol 1999;277:L616e27. Q2 91 [5] Cooper AL, Beasley D. Hypoxia stimulates proliferation and interleukin-1a production in human vascular smooth muscle cells. Am J Physiol 1999;277: 92 H1326e37. 93 [6] Dempsey EC, McMurtry IF, O’Brien RF. Protein kinase C activation allows 94 pulmonary artery smooth muscle cells to proliferate to hypoxia. Am J Physiol 1991;260:L136e45. 95 [7] Dempsie Y, MacLean MR. Pulmonary hypertension: therapeutic targets within 96 the serotonin system. Br J Pharmacol 2008;155:455e62. 97 [8] Gibbons GH, Dzau VJ. The emerging concept of vascular remodeling. N Engl J 98 Med 1994;330:1431e8. [9] Glagov S, Weisenberg E, Zarins CK, Stankunavicius R, Kolettis GJ. Compensa99 tory enlargement of human atherosclerotic coronary arteries. N Engl J Med 100 1987;316:1371e5. 101 [10] Graham LM, Vasil A, Vasil ML, Voelkel NF, Stenmark KR. Decreased pulmonary vasoreactivity in an animal model of chronic pseudomonas pneumonia. Am J 102 Respir Crit Care Med 1990;142:221e9. 103 [11] Greenway S, van Suylen RJ, Du Marchie Sarvaas G, Kwan E, Ambartsumian N, 104 Lukanidin E, et al. S100A4/Mts1 produces murine pulmonary artery changes resembling plexogenic arteriopathy and is increased in human plexogenic 105 arteriopathy. Am J Pathol 2004;164:253e62. 106 [12] Harja E, Bu D-X, Hudson BI, Jong SC, Shen X, Hallam K, et al. Vascular and 107 inflammatory stresses mediate atherosclerosis via RAGE and its ligands in apoE-/- mice. J Clin Invest 2008;118:183e94. 108 [13] Hopkins N, McLoughlin P. The structural basis of pulmonary hypertension in 109 chronic lung disease: remodelling, rarefaction or angiogenesis? J Anat 110 2002;201:335e48. [14] Humbert M, Morrell NW, Archer SL, Stenmark KR, MacLean MR, Lang IM, et al. 111 Cellular and molecular pathobiology of pulmonary arterial hypertension. J Am 112 Coll Cardiol 2004;43:13Se24S. 113 [15] Jeffery TK, Morrell NW. Molecular and cellular basis of pulmonary vascular 114 remodeling in pulmonary hypertension. Prog Cardiovasc Dis 2002;45:173e 202. 115 [16] Keegan A, Morecroft I, Smillie D, Hicks MN, MacLean MR. Contribution of the 116 5-HT(1B) receptor to hypoxia-induced pulmonary hypertension: converging 117 evidence using 5-HT(1B)-receptor knockout mice and the 5-HT(1B/1D)-receptor antagonist GR127935. Circ Res 2001;89:1231e9. 118 [17] Lannér MC, Raper M, Pratt WM, Rhoades RA. Heterotrimeric g proteins and 119 the platelet-derived growth factor receptor-b contribute to hypoxic prolifer120 ation of smooth muscle cells. Am J Respir Cell Mol Biol 2005;33:412e9. [18] Lawrie A, Spiekerkoetter E, Martinez EC, Ambartsumian N, Sheward WJ, 121 MacLean MR, et al. Interdependent serotonin transporter and receptor path122 ways regulate S100A4/Mts1, a gene associated with pulmonary vascular 123 disease. Circ Res 2005;97:227e35. [19] Leclerc E, Fritz G, Weibel M, Heizmann CW, Galichet A. S100B and S100A6 124 differentially modulate cell survival by interacting with distinct RAGE (re125 ceptor for advanced glycation end products) immunoglobulin domains. J Biol 126 Chem 2007;282:31317e31. [20] Lu S-Y, Wang D-S, Zhu M-Z, Zhang Q-H, Hu Y-Z, Pei J-M. Inhibition of hypoxia127 induced proliferation and collagen synthesis by vasonatrin peptide in cultured 128 rat pulmonary artery smooth muscle cells. Life Sci 2005;77:28e38. 129 [21] Maclean MR, Dempsie Y. The serotonin hypothesis of pulmonary hyperten130 sion revisited. Adv Exp Med Biol 2010;661:309e22. Conflict of interest
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Soluble receptor for advanced glycation end products (sRAGE) attenuates haemodynamic changes to chronic hypoxia in the mouse Q5
David G.S. Farmer, Marie-Ann Ewart, Kirsty M. Mair, Simon Kennedy* Institute of Cardiovascular and Medical Sciences, College of Medical, Veterinary & Life Sciences, University of Glasgow, G12 8QQ Glasgow, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 June 2013 Received in revised form 20 December 2013 Accepted 1 January 2014
The calgranulin-like protein MTS1/S100A4 and the receptor for advanced glycation end-products (RAGE) have recently been implicated in mediating pulmonary arterial smooth muscle cell proliferation and vascular remodelling in experimental pulmonary arterial hypertension (PH). Here, the effects of RAGE antagonism upon 2 weeks of hypobaric hypoxia (10% O2)-induced PH in mice were assessed. Treatment with sRAGE was protective against hypobaric hypoxia-induced increases in right ventricular pressure but distal pulmonary vascular remodelling was unaffected. Intralobar pulmonary arteries from hypobaric hypoxic mice treated with sRAGE showed protection against a hypoxia-induced reduction in compliance. However, a combination of sRAGE and hypoxia also dramatically increased the force of contractions to KCl and 5-HT observed in these vessels. The acute addition of sRAGE to the organ bath produced a small, sustained contraction in intralobar pulmonary vessels and produced a synergistic enhancement of the maximal force of contraction in subsequent concentrationeresponse curves to 5-HT. sRAGE had no effect on 5-HT-induced proliferation of Chinese hamster lung fibroblasts (CCL39), used since they have a similar pharmacological profile to mouse pulmonary fibroblasts but, surprisingly, produced a marked increase in hypoxia-induced proliferation. These data implicate RAGE as a modulator of both vasoreactivity and of proliferative processes in the response of the pulmonary circulation to chronic-hypoxia. Ó 2014 Published by Elsevier Ltd.
Keywords: Pulmonary hypertension RAGE MTS1/S100A4 Hypoxia Fibroblasts
1. Introduction Q1
Pulmonary hypertension (PH) is a rare, progressive disease of the small pulmonary arteries characterised by pulmonary vascular remodelling, vasoconstriction and thrombosis [34]. Pulmonary vascular remodelling is associated with smooth muscle proliferation, muscularisation of peripheral pulmonary arteries and medial thickening in larger pulmonary arteries. Muscularisation is associated with fibroelastosis, ablated responses to vasodilators and formation of obliterative plexiform lesions. Sustained elevation of pulmonary vascular pressure increases afterload in the right ventricle, ultimately leading to right heart failure [14,15,34].
Abbreviations: 5-HT, 5-hydroxytryptamine; PH, pulmonary hypertension; sRAGE, soluble RAGE; RAGE, receptor for advanced glycation end-products; PASMC, pulmonary arterial smooth muscle cells; HMGB-1, high mobility group box-1; RVH, right ventricular hypertrophy; CCL39, Chinese hamster lung fibroblasts; LME, linear mixed effects model; RVP, right ventricular pressure; RV, right ventricle; LV, left ventricle; BrdU, bromodeoxyuridine; SNP, sodium nitroprusside; MTS1, metastasis associated 1. * Corresponding author. Tel.: þ44 141 330 4763. E-mail addresses: david.farmer@florey.edu.au (D.G.S. Farmer), marie-ann. [email protected] (M.-A. Ewart), [email protected] (K.M. Mair), simon. [email protected] (S. Kennedy).
Recent studies have suggested a role for the receptor for advanced glycation end-products (RAGE) in experimental models of PH [11,18,24,37]. In systemic vascular disease, RAGE activation is associated with the generation of reactive oxygen species, activation of inflammatory transcription factors (e.g. NF-kB), increased vascular permeability, adhesion molecule expression and increased expression of RAGE itself. RAGE is therefore implicated in both the induction and amplification of inflammation [1,31,36]. RAGE is basally expressed in small arteries and arteriolar capillaries of the lung [2,26]. The calcium-binding, calgranulin-like protein MTS1/S100A4, a RAGE ligand, is expressed in remodelled pulmonary vessels and advanced occlusive vascular lesions in PH patients [11]. Thus RAGE ligands by their interaction with RAGE may modulate vascular remodelling in PH. In vitro, MTS1/S100A4 produces proliferation in human pulmonary arterial smooth muscle cells (PASMC) via an action at RAGE. Additionally, expression and secretion of MTS1/S100A4 re part of a downstream pathway producing proliferation in response to 5-HT; an established mitogen which is implicated in PH [7,18,21]. In vivo, overexpression of MTS1/S100A4 in transgenic mice is associated with spontaneous formation of obliterative, plexiform-like lesions in a subset (w5%) of animals [11]. All MTS1/S100A4 mice show impaired recovery from a chronic hypoxic challenge, reduced pulmonary vascular lumen diameter and increased elastin deposition occurring downstream of RAGE activation [24].
1094-5539/$ e see front matter Ó 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.pupt.2014.01.002
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Much work to date has focussed on the role of RAGE in PASMC proliferation. In the hypoxic lung in vivo however, adventitial fibroblasts demonstrate a more immediate and dramatic proliferation before migrating to the media and secreting substances which are mitogenic to PASMCs [33,38]. The role of RAGE in pulmonary fibroblast proliferation is unknown but HMGB-1, a RAGE ligand, was shown to induce proliferation and migration of mouse 3T3 fibroblasts which were prevented by a RAGE antibody [32]. In contrast, RAGE blockade increased proliferation in cultured human pulmonary fibroblasts [30]. RAGE activation may be prevented experimentally using soluble RAGE (sRAGE). sRAGE possesses the RAGE ligand binding domains but lacks the cytoplasmic and transmembrane domains. sRAGE will therefore compete for ligands which bind to cell-bound RAGE [35]. Additionally, sRAGE can prevent RAGE signal transduction directly by preventing the homodimerisation of RAGE on the cell surface [46]. In this study we tested the hypothesis that treatment with sRAGE would attenuate the effect of chronic hypoxia in mice. Additionally, we attempted to characterise the proliferative response of pulmonary fibroblasts to mitogenic stimuli in the presence of sRAGE in vitro. 2. Methods 2.1. In vivo experiments All animal care and experimental procedures were in accordance with the UK Animals (Scientific Procedures) Act 1986 and conformed to institutional regulations at the University of Glasgow. All mice used in the study were bred in the University of Glasgow, kept on a 12 h light/dark cycle and fed ad libitum. 2.2. Exposure to hypoxia In humans, idiopathic and familial PH occurs with a greater frequency in females than males. However, in experimental animals, males are more prone than females to hypoxia-induced PH [44]. For that reason, we chose to use male mice in this study. C57Bl/6 mice, aged 2e3 months (n ¼ 8e10 mice per group) were maintained in normoxic conditions or hypobaric/hypoxic conditions for 14 days as previously described [22,25]. The hypobaric chamber was depressurised over the course of 2 days to 550 mbar (equivalent to 10% O2). Temperature was maintained at 21 Ce 22 C, and the chamber was ventilated with air at 45 L min1. 2.3. Plasma RAGE determination In order to establish the effect of chronic hypoxia on plasma RAGE concentration an ELISA assay was run (R&D Quantikine ELISA for mouse RAGE; R&D, Abingdon, UK). Five plasma samples each were assayed from normoxic and chronic hypoxic mice in duplicate and compared against a calibration curve (31.3e2000 pg ml1 RAGE). All samples fell within the calibration range and are reported as plasma RAGE in ng ml1. 2.4. Administration of sRAGE Normoxic and hypoxic mice were randomly assigned to daily intraperitoneal injection with either vehicle (phosphate-buffered saline; PBS) or 20 mg/day sRAGE. This dose was chosen based on the work of [12] where it was demonstrated that daily dosing with sRAGE significantly impaired the worsening of disease in a mouse model of atherosclerosis at doses of 20 mg/day and 40 mg/day with no further benefit derived from a dose of 100 mg/day. Mice in the hypoxic groups were returned to normoxia once a day to facilitate
dosing with either vehicle or sRAGE over the 14 day course of the intervention. sRAGE was kindly supplied by Dr Anne-Marie Schmidt (Columbia University Medical Centre, New York, USA). 2.5. Characterisation of PH 2.5.1. Pressure measurements After 14 days hypobaric hypoxia, systolic right ventricular pressure (sRVP) was measured under isoflurane (1.5% in O2) anaesthesia via a 25-gauge needle advanced into the right ventricle trans-diaphragmatically [16,22]. RVP and heart rate (derived from the RVP) were recorded on a data acquisition system (MP 100, Biopac Systems). Systemic arterial pressure was recorded via a cannula placed in the carotid artery. It was not possible to obtain a full set of readings in all animals so the final n number for each group was: normoxic vehicle group ¼ 7, hypoxic vehicle group ¼ 9, normoxic sRAGE group ¼ 7, hypoxic sRAGE group ¼ 6. 2.5.2. Measurement of RVH and pulmonary vascular remodelling The ratio of right ventricular weight to left ventricular weight plus septum [RV/(LV þ S)] was used as an index of right ventricular hypertrophy (RVH) [22,25]. Briefly, the atria were removed and the right ventricle separated from the left ventricle and septum. The wet weights of the right ventricle and the left ventricle were measured on an analytical balance. The right lung was dropped into chilled 10% neutral buffer formalin (Composition: 90% dH2O, 10% formalin, 4 g per litre NaH2PO4, 6.5 g per litre Na2HPO4) without inflation and agitated for a period of 3 days. Sagittal sections were obtained, stained with Elastic Van Gieson stain and microscopically assessed for muscularisation of pulmonary vessels (<80 mm external diameter) as described previously [22,25]. Vessels were considered muscularised if they possessed a distinct double-elastic lamina visible for at least half the circumference of the vessel in cross-section. The percentage of vessels containing double-elastic lamina (and hence deemed remodelled) was calculated as the number of muscularised vessels/total number of vessels counted per section 100. Two to three sections from each left lung were assessed for every mouse. Lung sections from four to six mice from each group were studied. 2.5.3. Pulmonary artery myography Pulmonary arteries from normoxic and hypoxic controls and sRAGE-treated mice were studied. The animals were killed by cervical dislocation on completion of in vivo experiments and the lungs removed. Small pulmonary arteries (third order, first intralobar) of w250 mm internal diameter were dissected from the left lung, cut into 2 mm long segments, and mounted in an isometric myograph using 40 mm stainless steel wires (Danish Myo Technology, Aarhus, Denmark) as described previously [16,25]. The vessels were maintained at 37 C in Krebs’ buffer solution (pH 7.4) of the following composition (in mM): NaCl 118.4, NaHCO3 25, KCl 4.7, KH2PO4 1.2, MgSO4 0.6, CaCl2 2.5, glucose 11.0 and EDTA 0.023. Pulmonary arteries were aerated with 16% O2/5% CO2 balance N2. Aerating with this composition of gases results in an oxygen tension in the bath equivalent to that of the pulmonary circulation in vivo and has been used in previous studies from our laboratory [25]. Vessels were placed under ascending gradations of known tension equivalent to the mean in vivo RVP (12e15 mmHg). The diameter of the vessel corresponding to each of these tensions was recorded and forceediameter graphs plotted as a measure of compliance. Vessel responses were recorded onto a computer using a data acquisition and recording software system (Chart, ADInstruments, Oxford, UK). After a 45 min equilibration period, the response to 50 mM KCl, the concentration that produced maximal contraction in these vessels, was measured. For studies involving
Please cite this article in press as: Farmer DGS, et al., Soluble receptor for advanced glycation end products (sRAGE) attenuates haemodynamic changes to chronic hypoxia in the mouse, Pulmonary Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pupt.2014.01.002
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contractile agents, ascending concentrations of drug were added to artery rings under resting tension (5-HT: 300 pMe30 mM) cumulatively and the responses recorded. To measure the effect of hypoxia and sRAGE treatment on endothelium-independent relaxation, ascending concentrations of sodium nitroprusside (SNP; 300 pMe300 mM) were added to preconstricted rings and the responses calculated as a percentage loss of the initial constrictorinduced tone. In some experiments, endothelium-dependant relaxation to acetylcholine (1 mM) was measured. Typically this resulted in less than 10% relaxation so we conclude that the intralobar pulmonary arteries used in this study are denuded. 2.6. Fibroblast proliferation For studies on the effects of sRAGE on fibroblast proliferation, Chinese hamster lung fibroblasts (CCL39 cells) were used as these cells exhibit a very similar pharmacological profile to mouse pulmonary arterial fibroblasts [23] and can be grown in sufficient quantity for such experiments. At confluence, CCL39 cells were plated into 24-well plates at 20,000 cells/well and grown in full medium (Dulbecco’s modified Eagle’s medium supplemented with 10% v/v foetal bovine serum, penicillin, streptomycin, 1 mg ml1 fungizone and 50 mg ml1 gentamycin) at 37 C for 24 h before serum starvation for 24 h. For studies on the effects of sRAGE on proliferative stimuli, cells were preincubated with sRAGE for a period of 30 min before addition of the proliferative stimulus. Cells were then washed with sterile PBS, harvested by trypsinisation and collected by centrifugation. The cell pellet was resuspended in medium and 1 mL was added to a haemocytometer. Cell number was quantified by manual counting of the number of cells in four 1 mm2 sectors. Cell counts were made in duplicate for each well and proliferation was expressed as an elevation above the mean number of cells in control wells. To study the effects of hypoxia sRAGE on CCL39 proliferation, cells were grown under normobaric conditions in a humidified, temperature controlled and nitrogen-supplemented, Galaxy R incubator (Wolf Laboratories, York, UK). This allowed control of internal O2 levels at 5%, and CO2 levels were maintained at 5%. This achieves, for the hypoxic conditions, a tissue culture supernatant PO2 of 35 mmHg. In experiments involving sRAGE, this was added 30 min prior to transferring the fibroblasts to the hypoxic environment. The fibroblasts were exposed to hypoxia for 24 h before proliferation was measured. In some experiments, CCL39 proliferation was measured using a BrdU assay (Calbiochem, Nottingham, UK). Briefly, CCL39 cells were plated around 40% confluence and grown in DMEM medium as above. Cells were quiesced when around 70% confluent in DMEM þ0.2% serum for 24 h then treated with either sRAGE (2.5 or 10 mg ml1) or vehicle for 30 min. In some wells, the RAGE agonist S100B, a protein whose effects in alveolar cell lines are antagonised by sRAGE [29] was then incubated at 1 mg ml1 [19] for 30 min. Proliferation was then stimulated in all wells by addition of either 0.2% or 2.5% serum plus BrdU for 24 h at 37 C in the incubator. The plates were then fixed and the BrdU assay was run. Proliferation index was assayed spectrophotometrically and results reported as optical densities. 2.7. Statistical analysis All data are presented as mean SEM. Analysis of all haemodynamic parameters, the extent of RVH and of remodelling, and comparisons between EC50 values and maximal responses to pharmacological agents was by one-way ANOVA followed by posthoc analysis with Fisher’s least significant difference (LSD) test for multiple comparisons. The effect of sRAGE treatment and hypoxia combined was analysed by two-way ANOVA. A significant
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interaction between barometric treatment group and drug treatment group was taken as evidence for an effect of sRAGE. Plasma RAGE was analysed using Student’s unpaired t-test. Differences between concentration response curves were assessed by a linear mixed-effects (LME) model. For the purpose of making statistical comparisons of the effects of various stimuli on cell proliferation, normalised cell counts were assessed by means of a linear model to identify treatments which induced a statistically significant increase above baseline. The effects of treatments were compared using Student’s t-test or Fisher’s LSD test where appropriate. BrdU data for proliferation was analysed by one-way ANOVA with Dunnett’s correction for multiple comparisons. A p-value of less than 0.05 was considered significant and where appropriate, p-values were adjusted for multiple comparisons using the Bonferroni method. 3. Results 3.1. Effects of hypoxia 2 weeks chronic hypoxia markedly elevated the concentration of RAGE detected by ELISA in the plasma (3.21 0.77 ng ml1 in control mice vs. 11.97 1.67 ng ml1 in hypoxic mice; n ¼ 5 p < 0.05 vs. control). Systemic arterial pressure was not significantly altered by chronic hypoxia or by daily sRAGE injection in either normoxic or hypoxic animals (data not shown). Two weeks of hypobaric hypoxia produced significant increases in sRVP (Normoxia: 22.65 1.01 mmHg; Hypoxia: 31.02 1.51 mmHg; p < 0.01), proportion of remodelled vessels (Normoxic: 12.67 2.13%; Hypoxic: 20.83 1.94%; p < 0.05) and RVH (Normoxic: 0.27 0.01; Hypoxic: 0.34 0.01; p < 0.01) in vehicle-treated animals. sRAGE protected animals against the increase in sRVP in response to hypoxia as compared to the response in vehicle-treated animals (p < 0.05; two-way ANOVA; Fig. 1A). Furthermore, the sRVP of hypoxic sRAGE-treated mice did not differ significantly from normoxic mice receiving the same treatment (normoxic: 24.41 0.73 mmHg; hypoxic: 26.92 1.53 mmHg; p ¼ ns) but did not protect the animals against hypoxia-induced remodelling (Fig. 1B) or increased RVH (Fig. 1C). Under normoxic conditions, sRAGE produced no significant changes in sRVP (24.41 0.73 mmHg; Fig. 1A), proportion of remodelled vessels (13.92 2.16%; Fig. 1B), or RVH (0.28 0.011; Fig. 1C) when compared to vehicle-treated controls. Heart rate was not significantly altered by sRAGE treatment in normoxic animals (Vehicle: 337.37 16.48 bpm; sRAGE: 293.44 14.87; p ¼ ns, Student’s ttest). Chronic hypoxia significantly lowered heart rate in both vehicle and sRAGE-treated animals to a similar extent (vehicle: 276.3 12.28; sRAGE: 269.25 10.21; p < 0.01 vs. control for both groups, LME model). Remodelled vessels possessing a double elastic lamina were evident in sections from both hypoxic groups (Fig. 2) but the extent of vascular remodelling in sRAGE-treated hypoxic mice was not significantly different from vehicle-treated hypoxic mice. Compliance of intrapulmonary vessels, measured as the increase in diameter caused by the application of increasing force was unchanged by sRAGE treatment under normoxic conditions (Fig. 3). Exposure to 2 weeks of hypoxia significantly reduced the compliance of these vessels in vehicle-treated mice (Fig. 3A) while sRAGE prevented this reduction in compliance (Fig. 3B). 3.2. Pulmonary artery reactivity In these vessels, contraction to 5-HT was not altered by hypoxia alone (Fig. 4A). However in animals treated with sRAGE during
Please cite this article in press as: Farmer DGS, et al., Soluble receptor for advanced glycation end products (sRAGE) attenuates haemodynamic changes to chronic hypoxia in the mouse, Pulmonary Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pupt.2014.01.002
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Fig. 2. Representative small pulmonary vessels from mice treated with vehicle or sRAGE under normoxic conditions or after 2 weeks hypoxia. Small intrapulmonary vessels possessing a double elastic lamina were evident from both groups of animals after hypoxic challenge. sRAGE had no effect on the appearance of remodelled vessels. Photographs taken under 40 objective magnification. Scale bar ¼ 50 mm.
normoxic control) but significantly increased the contraction in hypoxic animals (3.11 0.39 mN; p < 0.05; two-way ANOVA; p < 0.05 vs. normoxic sRAGE-treated mice; Fisher’s LSD test; Fig. 5). When the concentrationeresponse to 5-HT from each experiment was normalised to its corresponding KCl response, no significant enhancement of the response to 5-HT was observed in sRAGE-treated, hypoxic animals when compared to normoxic,
Fig. 1. A e Effect of two weeks of chronic hypobaric hypoxia with daily sRAGE or vehicle (PBS) treatment on systemic right ventricular pressure, B e % remodelled vessels or C e right ventricular hypertrophy. Daily treatment with i.p. sRAGE (20 mg kg1) attenuated the hypoxia induced rise in sRVP but had no effect on vessel remodelling or ventricular hypertrophy. Data are expressed as mean SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs. normoxic, PBS treated mice; yyp < 0.01 vs. normoxic, sRAGE-treated mice, xp < 0.05 two-way ANOVA vs. vehicle-treated mice (vehicle normoxic: n ¼ 7; vehicle hypoxic: n ¼ 9; sRAGE normoxic: n ¼ 7; sRAGE hypoxic: n ¼ 6).
exposure to hypoxia, there was a significant exaggeration in the contractile response to 5-HT (Fig. 4B) compared to sRAGE treatment under normoxic conditions. Emax values in vessels from hypoxic sRAGE-treated animals were significantly elevated above their normoxic counterparts (Emax ¼ 3.94 0.57 mN; p < 0.05). Responses in vessels from hypoxic sRAGE-treated mice were also significantly different from those in normoxic, vehicle-treated mice (p < 0.01) and from those in hypoxic, vehicle-treated mice (p < 0.05) (Fig. 4). No difference in the log EC50 values in these responses was detected (log EC50 for vehicle: 7.56 0.21 vs. sRAGE: 7.65 0.11; p ¼ ns). To ascertain if this increase in contractile force was specific to 5-HT, we examined the magnitude of responses to KCl obtained at the start of each experiment. Two weeks of hypoxia had no significant effect on the maximal contraction to 50 mM KCl (normoxic: 1.72 0.22 mN; hypoxic: 1.61 0.10 mN, p ¼ ns). sRAGE treatment under normoxic conditions did not alter the contraction to KCl (2.00 0.29 mN; p ¼ ns vs.
Fig. 3. A e Compliance of intrapulmonary arteries is significantly reduced in vehicletreated mice exposed to 2 weeks hypoxia (*p < 0.05 vs. normoxia; n ¼ 7e11). B e Compliance of arteries was unchanged following hypoxia in animals treated with daily sRAGE for two weeks. In normoxic animals, sRAGE had no effect on vessel compliance (p ¼ ns). Statistical analysis was by LME model. (Vehicle-treated and sRAGE-treated mice, n ¼ 7; n ¼ 11 at force ¼ 1.2, 1.5 and 1.8 mN; n ¼ 7; n ¼ 11 at force ¼ 2.1 mN; n ¼ 6; n ¼ 9 at force ¼ 2.4 mN).
Please cite this article in press as: Farmer DGS, et al., Soluble receptor for advanced glycation end products (sRAGE) attenuates haemodynamic changes to chronic hypoxia in the mouse, Pulmonary Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pupt.2014.01.002
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Fig. 4. The maximal force of contraction is elevated in hypoxic mice treated with sRAGE but not PBS. (A) Hypoxia produced no alteration in the concentration-response curve to 5-HT in vessels from vehicle-treated animals (p ¼ ns). (B) Responses in vessels treated with sRAGE were significantly altered by hypoxia (*p < 0.05). Responses in vessels from hypoxic sRAGE-treated were also significantly different from those in normoxic, vehicle-treated mice (p < 0.01) and from those in hypoxic, vehicle-treated mice (p < 0.05). Emax in hypoxic, sRAGE treated vessels was significantly increased in comparison to hypoxic, vehicle treated mice (p < 0.05) but not normoxic, vehicletreated or sRAGE-treated vessels. Data are expressed as mean SEM. Analysis was by LME model. (Vehicle normoxic: n ¼ 4; Vehicle hypoxic: n ¼ 4; sRAGE normoxic: n ¼ 5 sRAGE hypoxic: n ¼ 4).
vehicle-treated or sRAGE-treated animals. Relaxation to the nitrovasodilator SNP was not significantly different between vehicletreated and sRAGE-treated animals under normoxic conditions (data not shown). Hypoxia had no significant effect on the relaxation to SNP and sRAGE treatment did not affect the response in hypoxic animals (Emax ¼ 80.24 14.26% in vehicle-treated vs. 69.14 13.75% in sRAGE treated; n ¼ 3e4, p ¼ ns). In some experiments the effects of acute addition of sRAGE (10 mg ml1) on vessel reactivity were studied. In intralobar vessels isolated from normoxic mice, addition of sRAGE produced a small, sustained contraction which reached a maximum at approximately 30 min (Fig. 6A). Similarly, preincubating arteries with sRAGE strongly
Fig. 5. Daily treatment with sRAGE (20 mg kg1) results in an increased force of contraction in response to 50 mM KCl in intrapulmonary arteries from animals exposed to 2 weeks of chronic hypoxic. Data are presented as mean SEM. p < 0.05, Fisher’s LSD test vs. normoxic sRAGE-treated mice; xp < 0.05, two-way ANOVA vs. vehicle-treated mice (Vehicle normoxic: n ¼ 6; Vehicle hypoxic: n ¼ 6; sRAGE normoxic: n ¼ 7; sRAGE hypoxic: n ¼ 6).
Fig. 6. A e Addition of sRAGE to intrapulmonary arteries at resting tension induced a small, sustained contraction. ***p < 0.001 vs. baseline; yp < 0.05 vs. control; control: n ¼ 5; sRAGE: n ¼ 4. B e In vitro addition of sRAGE also significantly augmented the contraction to 5-HT when analysed by LME model. ***p < 0.001 Data are presented as mean SEM; (Control: n ¼ 5; sRAGE: n ¼ 4).
enhanced the maximal contraction to 5-HT although the log EC50 value was unchanged (Fig. 6B). 3.3. CCL39 proliferation Cells in 0% FCS allowed to proliferate for 24 h in normoxia showed a 2.95 1.21 fold increase over the number of plated cells across both experimental groups. 5-HT induced an increase in cell number which was maximal at a concentration of 10 mM. Addition of 2.5 mg ml1 sRAGE had no effect on 5-HT-induced proliferation (Fig. 7A). Interestingly, 2.5 mg ml1 sRAGE alone produced a modest increase in cell number to 31.90 8.12% above baseline (p < 0.01; linear model). Twenty-four hours of hypoxia significantly increased cell number (Fig. 7B) while a combination of hypoxia and 2.5 mg ml1 sRAGE produced a strong proliferative response which was of greater magnitude than hypoxia or sRAGE alone (Fig. 7B). The effect of the RAGE agonist S100B and sRAGE was also assayed using BrdU incorporation as an index of proliferation. When cells were stimulated with low serum (0.2%), S100B alone tended to increase proliferation though this was not significant. sRAGE alone had no effect and the combination of S100B and sRAGE did not have any effect on proliferation (Fig. 7C). When cells were stimulated with 2.5% serum, S100B, sRAGE or a combination of the two had no effect on proliferation compared to control wells (data not shown).
Please cite this article in press as: Farmer DGS, et al., Soluble receptor for advanced glycation end products (sRAGE) attenuates haemodynamic changes to chronic hypoxia in the mouse, Pulmonary Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pupt.2014.01.002
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Fig. 7. A e In CCL39 Chinese hamster lung fibroblasts, 5-HT and 2.5 mg ml1 sRAGE alone induced a significant proliferation compared to serum-free conditions. sRAGE did not modify the proliferative response to 5-HT and there was no evidence of an additive effect of the two agents. B e Incubation of CCL39 cells in a hypoxic environment for 24 h produced a significant proliferation which was significantly augmented by addition of 2.5 mg ml1 sRAGE. C e When 0.2% serum was used as the proliferative stimulus, s100, sRAGE up to 10 mg ml1 or a combination of the two had no significant effect on proliferation measured by BrdU incorporation (data is mean of 8 replicates). Data are presented as mean SEM; *p < 0.05; **p < 0.01, ***p < 0.001 vs. serum-free control (baseline; n ¼ 4); yp < 0.05 vs. hypoxia. (n ¼ 3).
4. Discussion The aim of the current study was to characterise the effects of sRAGE in two novel settings: as an intervention to hypoxia-induced pulmonary hypertension in vivo, and as an influence on proliferation in pulmonary fibroblasts. Since the RAGE agonist MTS1/ S100A4 appears to mediate proliferation in PASMCs through RAGE and can alter structural and functional parameters which may affect pulmonary vascular resistance (i.e. remodelling, vasoreactivity) and influence the pulmonary blood pressure [18], we hypothesised that daily treatment with sRAGE would reduce pulmonary vascular remodelling and decrease the effect of hypoxia on sRVP. We found that sRAGE treatment reduced the magnitude of
the hypoxia-induced elevation in sRVP. However, this change was not accompanied by inhibition of distal vascular remodelling. It is important to note that sRVP was assessed after animals had been returned to normoxia. Therefore, elevation of the sRVP is not due to acute hypoxic pulmonary vasoconstriction but to persistent structural and/or functional alterations of the pulmonary vasculature. In the heart, right ventricular hypertrophy also persists when animals are returned to normoxia. In the current study, reduced sRVP associated with sRAGE treatment was not accompanied by a reduction in hypoxia-induced right ventricular hypertrophy. Since RVH is presumably caused, at least in part, by increased afterload associated with increased pulmonary vascular resistance during the hypoxic challenge, this result suggests that sRAGE treatment did not reduce acute pulmonary vascular pressures during this time. Similarly, the lowered sRVP in sRAGE-treated mice is unlikely to be due to changes in heart rate or cardiac output since both groups showed a similar drop in heart rate on exposure to chronic hypoxia. Although we did not measure cardiac output, a recent study [3] used echocardiography and found that there was no change in RV fractional shortening or cardiac output despite raised sRVP in mice exposed to 6 weeks chronic hypoxia. Alterations in vascular compliance provide a possible explanation for the reduction in sRVP by sRAGE after hypoxia. In vehicletreated animals, chronic hypobaric hypoxia produced a fall in compliance of the intralobar pulmonary arteries (Fig. 3) that was completely prevented by sRAGE. Larger distal conducting vessels contribute to elevated pulmonary pressures through smooth muscle cell hyperplasia and hypertrophy resulting in a loss of elasticity or compliance. The lost ability of these vessels to buffer the pulmonary pressure at right ventricular systole through stretch increases pressure in the pulmonary artery at systole and increases shear stress on more distal vessels [13,47]. Thus, it may be that the number of vessels possessing a double-elastic lamina was similar in sRAGE and vehicle-treated animals but that vessels from sRAGEtreated animals retained basal levels of compliance throughout and after the hypoxic insult. In MTS1/S100A4 overexpressing mice, [24] reported elevated pulmonary pressures in the absence of enhanced remodelling which they attributed to increased elastin deposition in pulmonary vessels. In the current study, we would speculate that an sRAGE-driven inhibition of elastin deposition may have allowed pulmonary arteries from hypoxic animals to retain their compliance. An important point to note is that we did not inflate lungs in any experimental group prior to fixation. This can complicate matters since it can be difficult to determine vessel size and very small vessels may remain collapsed. Thus, although we report data on number of remodelled vessels in this study, we cannot comment on the effect of sRAGE treatment on vessel size. Pulmonary fibroblasts appear to be key mediators of pulmonary vascular remodelling in response to hypoxia. In the hypoxic lung in vivo, adventitial fibroblasts demonstrate an immediate and dramatic proliferation and migration to the tunica media. Here they secrete mitogens for PASMC and contribute to the remodelling process [33,38]. In vitro, it has been reported that PASMCs in culture do not proliferate in response to hypoxia when cultured alone but will do so when co-cultured with fibroblasts [33]. Here, we report that 24 h of hypoxia induced a doubling of the number of fibroblasts compared to baseline; in line with previous in vitro studies on pulmonary fibroblasts [39,41e43]. Surprisingly, preincubation with sRAGE produced a further increase in the number of cells after hypoxia and sRAGE alone produced a small but significant increase in the number of cells over baseline. This suggests that either nominal release of a RAGE ligand or constitutive RAGE activity is an antiproliferative stimulus in pulmonary fibroblasts and that this process may also be an important part of the proliferative response of these cells to hypoxia. The reduction of RAGE ligand
Please cite this article in press as: Farmer DGS, et al., Soluble receptor for advanced glycation end products (sRAGE) attenuates haemodynamic changes to chronic hypoxia in the mouse, Pulmonary Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pupt.2014.01.002
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bioavailability by sRAGE in solution would reduce the former while inhibition of RAGE signalling through heterodimerization of RAGE and sRAGE on the cell surface would inhibit either process. Interestingly, the proliferative effect of sRAGE on fibroblasts was not observed when serum was used as the stimulus. This may be the effect of serum stimulation overcoming the modest proliferative effect of sRAGE under conditions where proliferation was allowed to proceed in the absence of serum. Alternatively, since cell counting was used in the initial experiments with sRAGE and despite adequately controlling the experiments with cells counts made in the absence of sRAGE, caution must be exercised in ascribing the effect wholly to proliferation. BrdU incorporation with serum stimulation is likely to be a more sensitive and physiologically plausible index of proliferation and under these conditions we found no effect of sRAGE alone. In addition, under these conditions the RAGE ligand S100B also had no effect on proliferation and so perhaps the multitude of growth factors contained within serum is too potent a stimulus to be affected by the S100/RAGE axis. Similarly, the basal activation of RAGE by an unknown mechanism appears to be important in the acute regulation of tone in pulmonary arteries since sRAGE produced a small amount of contraction alone and greatly enhanced the force of contraction in response to 5HT when added to the bathing solution in the myograph. The magnitude of the effect of sRAGE upon the response to 5-HT appeared to be greater than a summation of the effects of each compound alone which suggests increased sensitivity of the contractile apparatus of PASMCs to Ca2þ. Another possibility is an action on the vascular endothelium producing either an increase in the basal release of contractile mediators (e.g. endothelin-1, thromboxane A2) or decreased release of relaxatory mediators (e.g. NO, prostacyclin). However, the vessels used in this study were of a small diameter making it difficult to preserve the integrity of the endothelium and an effect here is unlikely. A direct effect of sRAGE upon the smooth muscle therefore seems more likely. We also observed some evidence of an action of sRAGE on SMCs in that vascular contractility was greatly enhanced by a combination of chronic-hypoxia and sRAGE treatment in vivo. A recent study demonstrated that chronic hypoxia enhances both receptormediated calcium entry and store-operated calcium entry in pulmonary arteries [27]. It is therefore possible that sRAGE is able to modulate intracellular calcium to enhance sensitivity to contractile stimuli. The interpretation of the current in vivo data remains difficult. It is not clear what, if fibroblast proliferation is also enhanced in vivo, the overall impact of sRAGE treatment on pulmonary vascular structure and function will be at the level of the vascular cells as they respond to the hypoxic stimulus. sRAGE is reported to reduce PASMC proliferation, yet an increase in fibroblast proliferation might be expected to enhance PASMC proliferation and hence vascular remodelling.
5. Conclusions Although sRAGE-treatment caused a significant, beneficial effect on sRVP after chronic hypoxia, the mechanism does not appear to be via effects on small vessel remodelling or on vascular reactivity in the lung as these were both enhanced. We speculate that sRAGE may, by acting directly upon PAMSCs or indirectly through enhancing proliferation of fibroblasts in hypoxia, produce enhanced sensitivity of the vasculature to contractile stimuli whilst simultaneously protecting against hypoxiainduced reductions in vascular compliance. What remains to be determined in future studies is the effect of sRAGE treatment on RAGE signalling at a cellular levels under control and particularly hypoxic conditions.
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Please cite this article in press as: Farmer DGS, et al., Soluble receptor for advanced glycation end products (sRAGE) attenuates haemodynamic changes to chronic hypoxia in the mouse, Pulmonary Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pupt.2014.01.002
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