Cardiovascular Research (2015) 107, 20–31 doi:10.1093/cvr/cvv143

Perlecan heparan sulfate deficiency impairs pulmonary vascular development and attenuates hypoxic pulmonary hypertension Ya-Ting Chang 1*, Chi-Nan Tseng 1, Philip Tannenberg 1, Linne´a Eriksson 1, Ke Yuan 2,3, Vinicio A. de Jesus Perez2,3, Johan Lundberg 4,5, Mariette Lengquist1, Ileana Ruxandra Botusan 1, Sergiu-Bogdan Catrina 1, Phan-Kiet Tran 6, Ulf Hedin 1, and Karin Tran-Lundmark 1,7 1

Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden; 2Division of Pulmonary and Critical Care Medicine, Stanford University, Stanford, CA, USA; Stanford Cardiovascular Institute, Stanford University, Stanford, CA, USA; 4Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden; 5Department of Neuroradiology, Karolinska University Hospital, Stockholm, Sweden; 6Department of Cardiothoracic Surgery, Uppsala University, Uppsala, Sweden; and 7Department of Experimental Medical Science, Lund University, Lund, Sweden 3

Received 17 September 2014; revised 19 April 2015; accepted 1 May 2015; online publish-ahead-of-print 7 May 2015 Time for primary review: 40 days

Excessive vascular cell proliferation is an important component of pulmonary hypertension (PH). Perlecan is the major heparan sulfate (HS) proteoglycan in the vascular extracellular matrix. It binds growth factors, including FGF2, and either restricts or promotes cell proliferation. In this study, we have explored the effects of perlecan HS deficiency on pulmonary vascular development and in hypoxia-induced PH. ..................................................................................................................................................................................... Methods In normoxia, Hspg2 D3/D3 mice, deficient in perlecan HS, had reduced pericytes and muscularization of intra-acinar vessels. Pulmonary angiography revealed a peripheral perfusion defect. Despite these abnormalities, right ventricular and results systolic pressure (RVSP) and myocardial mass remained normal. After 4 weeks of hypoxia, increases in the proportion of muscularized vessels, RVSP, and right ventricular hypertrophy were significantly less in Hspg2 D3/D3 compared with wild type. The early phase of hypoxia induced a significantly lower increase in fibroblast growth factor receptor-1 (FGFR1) protein level and receptor phosphorylation, and reduced pulmonary artery smooth muscle cell (PASMC) proliferation in Hspg2 D3/D3. At 4 weeks, FGF2 mRNA and protein were also significantly reduced in Hspg2 D3/D3 lungs. Ligand and carbohydrate engagement assay showed that perlecan HS is required for HS –FGF2 –FGFR1 ternary complex formation. In vitro, proliferation assays showed that PASMC proliferation is reduced by selective FGFR1 inhibition. PASMC adhesion to fibronectin was higher in Hspg2 D3/D3 compared with wild type. ..................................................................................................................................................................................... Conclusions Perlecan HS chains are important for normal vascular arborization and recruitment of pericytes to pulmonary vessels. Perlecan HS deficiency also attenuates hypoxia-induced PH, where the underlying mechanisms involve impaired FGF2/ FGFR1 interaction, inhibition of PASMC growth, and altered cell –matrix interactions.

----------------------------------------------------------------------------------------------------------------------------------------------------------Keywords

Pulmonary hypertension † Heparan sulfate † Perlecan † Hypoxia † FGF2

1. Introduction Pulmonary hypertension (PH) is characterized by a progressive increase in pulmonary vascular resistance, which leads to right ventricular failure and death. Despite the increase in treatment options in recent years, the long-term outcome of patients with PH remains poor. 1 The aetiologies of PH are diverse, and PH can be divided into five

groups according to the WHO classification.2 Hypoxia is the major contributing factor in Group 3.2 While acute hypoxia causes vasoconstriction,3 chronic hypoxia results in vascular remodelling, including muscularization of distal non-muscular pulmonary arterioles, as well as medial and adventitial thickening of muscular vessels.4,5 Current therapy consists mainly of pulmonary vasodilators. However, recent studies have shown that anti-proliferative agents can

* Corresponding author. Vascular Biology Laboratory, CMM L8:03, Karolinska University Hospital, SE-17176, Stockholm, Sweden. Tel: +46 8 517 73561; fax: +46 8 339 309. Email: [email protected] Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2015. For permissions please email: [email protected].

Downloaded from by guest on November 14, 2015

Aims

21

Perlecan heparan sulfate in pulmonary vasculature

2. Methods For some of the methods, a detailed description is available in Supplementary material online.

2.1 Animals Hspg2 D3/D3 mice were generated as previously described by Rossi, et al. 22 Wild-type and homozygous mutant (Hspg2 D3/D3) offspring from N12/F2 mice (second generation of homozygous breeding between littermates) were used. The animal experiments were conducted under the approval of the Stockholm Animal Research Committee (N64/09, N24/11, and N471/12, Karolinska Institutet, A5002-01) according to the Swedish Law on Protection of Animals and the Directive 2010/63/EU of the European Parliament.

2.2 Exposure to chronic hypoxia Female mice at 8 – 11 weeks of age were divided into four experimental groups, wild-type normoxia, mutant normoxia, wild-type hypoxia, and mutant hypoxia. Hypoxic groups were kept in a ventilated hypoxic cabinet (10% O2, Coy Laboratory Products) for 3.5 days or 4 weeks. Normoxic groups were kept in room air.

2.3 Right ventricular systolic pressure Under anaesthesia with 1.5% isoflurane, a micro catheter (Millar) was inserted into the right ventricle through the right external jugular vein. Right ventricular systolic pressure (RVSP) was recorded using a PowerLabw data acquisition system (ADInstruments).

2.4 In vivo mouse lung angiography Under anaesthesia with 1.5% isoflurane, lung angiography was performed by an injection of contrast (Visipaque 270, GE Healthcare) into the right atrium. Live angiographic series (6 fps) in frontal projections were captured at least six times for each animal. Regions of interest were chosen in the right lower lobe, and contrast attenuation was measured and normalized to total pulmonary area. Details are provided in Supplementary material online.

2.5 Tissue preparation After RVSP measurements and collection of heart and lung tissues, the right lung lobes were snap-frozen in liquid nitrogen for RNA and protein extraction. Left lungs were perfusion-fixed by infusion of 4% zinc– formaldehyde via the right ventricle and the trachea (perfusion pressure: 18 cmH2O), and further processed for paraffin sectioning. For the hearts, the right ventricle was separated from the left ventricle plus septum, and the samples were dried and weighed to obtain the right ventricle to left ventricle plus septum ratio [RV/(LV + S)].

2.6 Immunohistochemistry and IF Enzyme-based probe-polymer systems were used for staining of paraffinembedded lung sections. Fluorochrome-conjugated secondary antibodies were used for IF of frozen sections. Details are provided in Supplementary material online.

2.7 Morphometric analysis and detection of proliferation Double staining for von Willebrand factor and smooth muscle a-actin (a-SMA) was used for assessment of muscularization. For cell proliferation, Ki-67 staining was quantified by manual counting of positive and negative nuclei within the pulmonary vessels. Two proliferation indices were calculated: (i) total vascular cell proliferation index [(total Ki-67-positive nuclei in the entire vascular wall)/(total nuclei)] and (ii) pulmonary artery SMC proliferation index [(total Ki67-positive PASMC nuclei)/(PASMC nuclei)].

Downloaded from by guest on November 14, 2015

reduce muscularization and thickening of vessels, improve haemodynamics, and reduce right ventricular hypertrophy. Modulators of growth factor signalling could therefore have a role as add-on therapy for PH.6 The importance of growth factor pathways is well founded in the literature. PDGF-B regulates proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) and plays a critical role in the progression of PH.7 Increased expression and activation of PDGF receptor b (PDGFRb) has been shown in patients with PH and in animal models of hypoxia-induced PH.8 Imatinib mesylate is a tyrosine kinase inhibitor that inhibits PDGFRb signalling and cell proliferation.8 Recently, a phase III clinical trial reported improved right ventricular function in patients with advanced disease after 24 weeks of imatinib treatment as add-on therapy.9 Imatinib is, however, not specific for the PDGF pathway, and it has not been approved for PH treatment because of serious side effects and frequent drug discontinuations.10 Fibroblast growth factor-2 (FGF2) is another potent mitogen for smooth muscle cells (SMCs) that have been shown to accumulate in human pulmonary vascular lesions. The use of the pharmacological agent SU5402, a pan-FGF receptor inhibitor, successfully attenuates monocrotaline-induced PH in rats.11 FGF2 has also recently been shown to mediate pericyte coverage of distal pulmonary arterioles in PH.12 Growth factor signalling may be modulated by interactions with proteoglycans. Perlecan is a complex proteoglycan composed of a core protein (470 kDa) containing five globular domains and three GAG chains in the N-terminal and one in the C-terminal.13 The GAG chains are mainly heparan sulfate (HS), but may also be chondroitin sulfate depending on cell type-specific post-translational modifications.14,15 Perlecan-null mutation is lethal with severe cardiac and neuromuscular disorders.16,17 In blood vessels, perlecan is deposited in the basement membranes underneath endothelial cells and around SMCs.18 In the vascular wall, there are also cell membrane-bound HS proteoglycans (syndecans and glypicans), but perlecan is the major HS proteoglycan in the extracellular matrix. Perlecan HS has been shown to bind various growth factors, including FGF2 and PDGF-B,19,20 and thus serves as an important modulator of growth factor signalling. The HS chains may either sequester the growth factor and inhibit downstream signalling, or present the ligand to facilitate receptor activation.21 To investigate the role of perlecan HS in pulmonary vascular development, and in the regulation of growth factor signalling in hypoxiainduced PH, we used a mouse strain that expresses perlecan deficient in HS (Hspg2 D3/D3).22 The HS deficiency was generated through targeted deletion of the HS attachment sites in exon 3 (domain I). This results in loss of all three N-terminal HS chains.22 The mice have been shown to develop spontaneous cataract,22 impaired tumour angiogenesis, and wound healing.23 Our group has previously shown that perlecan HS inhibits SMC proliferation in muscular arteries in the systemic circulation. In the carotid ligation model, the Hspg2 D3/D3 mice developed significantly more intimal hyperplasia compared with controls, possibly related to defective sequestration of FGF2 in the vascular extracellular matrix.24 Previously, no apparent vascular pathology as an immediate consequence of the HS deficiency has been described in the Hspg2 D3/D3 mice, and the role of perlecan HS in the pulmonary circulation had not been explored. In this study, we hypothesized that perlecan HS can modulate growth factor – receptor interactions in vascular remodelling in PH. We show impaired pulmonary vascular development in mice expressing HS-deficient perlecan. When challenged with hypoxia, the PH is less severe in Hspg2 D3/D3 mice.

22

Y.-T. Chang et al.

2.8 Proximity ligation assay

2.15 Statistics analysis

Duolink proximity ligation assay (PLA) was performed on paraffin-embedded lung sections following the manufacturer’s instructions (Sigma-Aldrich). After blocking and primary antibody incubation with mouse anti-FGF2 antibody (5 mg/mL, clone bFM-2303, Millipore), and rabbit anti-perlecan antibody (1 mg/mL, sc-25848, Santa Cruz) overnight at 48C, PLA probes were added. Ligation and amplification were performed using the Duolink bright field detection kit. After conjugation with horseradish peroxidase, NovaRed substrate was used for detection.

Data are presented as mean + SEM. IBM SPSS statistics version 21 software was used for statistical analyses. Sample size was determined by power analysis (G*Power, version 3.1.3; Heinrich-Heine-Universita¨t Du¨sseldorf, Germany) with a ¼ 0.05 and b ¼ 0.2 in two tails. All data were also analysed with the normality test. Unpaired Student’s t-test was used for comparisons between two groups. For multiple comparisons when data passed the normality test, two-way ANOVA with Bonferroni’s multiple comparison test was used. The Kruskal – Wallis test with Dunn’s multiple comparison post hoc test was applied for non-normally distributed data. A P-value of ,0.05 was considered significant.

2.9 FGF ligand and carbohydrate engagement assay25

2.10 RNA extraction and quantitative PCR About 20 mg of lung tissue from each mouse was homogenized for RNA extraction. Quantitative real-time PCR was performed with Applied Biosystems’ 7900HT system. Details are provided in Supplementary material online.

2.11 Protein extraction and western blot Snap-frozen lungs were homogenized for protein extraction and western blot. The anti-FGFR1 antibody is specific to the C-terminus of FGFR1 protein and detected several bands as previously described. The antibody against phospho-FGFR1 has high specificity for phospho-Y654, the phosphorylation site essential for signalling. Details are provided in Supplementary material online.

2.12 Primary cell isolation and cell culture Age-matched littermates of wild-type and Hspg2 D3/D3 mice were anaesthetized with isoflurane and euthanized by cervical dislocation. For each cell isolation, pulmonary arteries from six mice were pooled and enzyme digestion was used to obtain PASMCs as previously described.24 Three different batches of cell isolates were used for all in vitro experiments. Details are provided in Supplementary material online.

2.13 Proliferation assay Two methods were used for quantification of cell proliferation, BrdU incorporation (colorimetric ELISA assay, Roche), and manual cell counting. For hypoxia incubation, cells were cultured in an Invivo2 Hypoxia Workstation with 1% O2 and 5% CO2 (Ruskinn).

2.14 Adhesion assay PASMCs from wild-type and mutant mice were allowed to adhere in 96-well plates coated with collagen IV, EHS laminin, or fibronectin. Details are provided in Supplementary material online.

3. Results 3.1 Muscularization defect in intra-acinar vessels of Hspg2 D3/D3 mice Gross histology of lungs and hearts from wild-type and Hspg2 D3/D3 mice (n ¼ 6 per group) was analysed by Masson’s trichrome and haematoxylin–eosin staining, and showed no signs of abnormalities in structure or collagen content in the Hspg2 D3/D3 mice (Figure 1A). Total vessel number per alveoli was equivalent to the wild-type (data not shown). In contrast, double staining for endothelial cells and SMCs with von Willebrand factor and a-SMA revealed a lower percentage of a-SMA-positive pulmonary vessels in Hspg2 D3/D3 mice (Figure 1B). Western blot for three markers for SMC differentiation, a-SMA, myosin heavy chain 11, and SM22a, using aortic samples, showed no significant difference between wild-type and Hspg2 D3/D3 mice (Figure 1C), indicating that the lack of perlecan HS does not cause a general defect in SMC differentiation. The muscularization defect is likely to be caused by the cell number loss, and not by the change in cell protein content. In addition, confocal imaging confirmed loss of pericyte coverage in the distal pulmonary arterioles in Hspg2 D3/D3 mice (Figure 1D).

3.2 Peripheral perfusion defect demonstrated by in vivo pulmonary angiography Defective muscularization of pulmonary vessels in the Hspg2 D3/D3 mice suggests a possible vascular developmental defect. Pulmonary angiograms showed perfusion defects in the peripheral parts of the lungs (Figure 2A), and a significant reduction in contrast attenuation (Figure 2B), which indicates a possible arborization abnormality. The lumen diameter of pulmonary arteries was measured and normalized to the size of the thoracic aorta. In proximal pulmonary arteries up to third-degree branches, there were no detectable differences between Hspg2 D3/D3 and wild type mice (data not shown). This suggests that the possible arborization defect in Hspg2 D3/D3 lungs resides in more distal parts of the arterial tree.

3.3 Hypoxia induces perlecan expression Perlecan mRNA isolated from whole lung was significantly increased after 3.5 days in hypoxia, and returned to baseline level after 4 weeks. These changes in perlecan mRNA expression in response to hypoxia were nearly identical between Hspg2 D3/D3 and wild-type mice (Figure 3A). Immunohistochemical analysis of lung sections showed a small amount of perlecan in the basement membranes of pulmonary vessels in normoxia (Figure 3B), which then increased significantly following 4 weeks of hypoxia (Figure 3C). No differences were observed between Hspg2 D3/D3 and wild type (not shown).

Downloaded from by guest on November 14, 2015

For ligand and carbohydrate engagement (LACE) assay on tissues, 5 mm paraffin-embedded lung sections were deparaffinized and rehydrated, followed by incubation in 0.1 M glycine in PBS for 30 min. To ensure that the observed binding was mediated by HS, control sections were treated with heparinase III (2 U/mL; Sigma-Aldrich) in heparinase buffer (50 mM HEPES, 50 mM NaOAc, 150 mM NaCl, 5 mM CaCl2, and 0.1% BSA, pH 6.8) at 378C for 4 h. All other sections were treated with heparinase buffer only. After blocking with 5% BSA in PBS, the sections were incubated with 2 mM recombinant human FGF2 and 2 mM human FGF receptor-1 (FGFR1) IIIc-Fc chimera (both from R&D systems) in blocking buffer overnight at 48C, and then washed with PBS, followed by incubation with the Cy3conjugated anti-human Fc IgG secondary antibody (1 : 30, Sigma-Aldrich) at room temperature for 2 h. After washing with PBS and drying, the slides were mounted with fluorescence mounting medium (DAKO) and examined using a Nikon Optiphot-2 fluorescence microscope. For LACE on cell cultures, HRP-conjugated anti-human Fc IgG secondary antibody (1 : 5000, Sigma-Aldrich) was used instead. Details are provided in Supplementary material online.

Perlecan heparan sulfate in pulmonary vasculature

23

Downloaded from by guest on November 14, 2015

Figure 1 Vascular phenotype of Hspg2 D3/D3 mice. (A) Masson’s Trichrome staining of intra-acinar pulmonary artery from wild-type control (WT) and Hspg2 D3/D3 mice (Mut). (B) Significantly reduced proportion of intra-acinar pulmonary vessels covered by a-SMA-positive cells in Hspg2 D3/D3 mice (Mut) compared with wild type. Ninety to 160 vessels per mouse were analysed (n ¼ 12; *P , 0.01 by unpaired Student’s t-test). (C ) Representative western blots from aortic lysates and quantification data for SMC differentiation markers: myosin heavy chain-11 (MYH-11), smooth muscle a-actin (a-SMA), and transgelin (SM-22). Tubulin was used as a loading control. No significant differences between genotypes by unpaired Student’s t-test (n ¼ 4 – 5). (D) Representative confocal images of IF for pericyte markers, PDGFRb, desmin, and a-SMA showed loss of pericytes in Hspg2 D3/D3 mice (Mut). CD31 was used to locate vascular endothelial cells.

24

Y.-T. Chang et al.

Figure 2 Peripheral perfusion defect in Hspg2 D3/D3 mice demon-

3.4 Perlecan HS deficiency attenuates hypoxia-induced PH In normoxia, there were no differences in RVSP between Hspg2 D3/D3 mice and wild type. After 4 weeks of hypoxia, both developed PH with significant elevations of RVSP, but RVSP was significantly lower in Hspg2 D3/D3 animals compared with wild type (32.5 + 1.4 vs. 37.8 + 0.9 mmHg; Figure 4A). This result was corroborated by a concomitant development of significant right ventricular hypertrophy as determined by the RV/(LV + S) ratio. Also here, Hspg2 D3/D3 had less right ventricular hypertrophy. The ratios increased from 0.25 + 0.01 to 0.35 + 0.01 and from 0.26 + 0.004 to 0.40 + 0.01 in Hspg2 D3/D3 and wild type, respectively (Figure 4B).

3.5 Reduction in hypoxia-induced vascular muscularization and SMC proliferation Muscularization of intra-acinar vessels was quantified following double stainings for endothelial cells and SMCs as described by Schermuly et al. 8 Under normoxia, the proportion of non-muscularized vessels was significantly higher in the Hspg2 D3/D3 (76%) compared with the wild type (58%), and the proportion of partially and fully muscularized vessels was significantly lower in Hspg2 D3/D3 (Figure 4C). After 4 weeks of hypoxia, a significant increase in fully muscularized vessels was observed in both genotypes, and this increase was significantly less pronounced in Hspg2 D3/D3 (Figure 4C). To evaluate cell proliferation, mice were exposed to 3.5 days of hypoxia. Previously published data have indicated that a peak in

Figure 3 Increased perlecan expression in hypoxia-induced PH. (A) Relative quantification of perlecan mRNA in whole lung homogenates. A significant increase was seen in both genotypes following 3.5 days of hypoxia. The data were normalized to age-matched, wild-type normoxic controls. b2-microglobulin was used as a reference gene (n ¼ 5; *P , 0.05 by two-way ANOVA with Bonferroni’s multiple comparisons test). (B and C) Immunohistochemistry staining for perlecan core protein (red) in intra-acinar pulmonary vessels of wild-type mice under normoxia (B) and after 4 weeks of hypoxia (C ) showed perlecan accumulation in basement membranes of hypoxic vessels. White arrowheads indicate the location of perlecan staining.

proliferation occurs between days 3 and 4.26 Lung sections were double-stained for Ki-67 and a-SMA (Figure 4D). Hypoxia induced significant increases in proliferation of pulmonary vascular cells. However, the increased proliferation was significantly less in Hspg2 D3/D3 compared with wild type (Figure 4E and F ).

3.6 Perlecan co-localization with FGF2 in pulmonary arteries Double staining showed that perlecan and FGF2 are co-localized along the endothelium and in the extracellular matrix of pulmonary arteries (Figure 5A). In addition, PLA demonstrated positive amplification signals in pulmonary arteries, which confirmed that endogenous FGF2 and perlecan are within range for interaction with each other (Figure 5B).

3.7 Reduced HS –FGF2 –FGFR1 ternary complex formation in hypoxic Hspg2 D3/D3 mice LACE assay, with the addition of both chimeric FGFR1 and FGF2 on wild-type hypoxic lung sections, detected widespread binding of

Downloaded from by guest on November 14, 2015

strated by in vivo pulmonary angiography. (A) Representative in vivo angiographic images of wild-type control (WT) and Hspg2 D3/D3 mice (Mut). Circles indicate the region of interest for quantitative image analysis. (B) Image quantification of contrast attenuation in the region of interest showed a significant reduction in the Hspg2 D3/D3 mice (n ¼ 3; *P , 0.01 compared with wild type by unpaired Student’s t-test).

Perlecan heparan sulfate in pulmonary vasculature

FGFR1. Corresponding signals were significantly reduced on Hspg2 D3/D3 hypoxic lung sections, which suggested that perlecan HS contributes to FGFR1 binding. This was confirmed as heparinase III treatment of wild-type lung sections nearly abolished the binding of FGFR1 (Figure 5C). Further experiments with added chimeric FGFR1 and no FGF2 to lung sections yielded no signal. This indicates

25 that both FGF2 and perlecan HS are required for ternary complex formation with FGFR1 (data not shown). The above findings could be replicated on hypoxia-treated PASMC cultures. Addition of FGF2, but with a lack of HS as in the Hspg2 D3/D3 or by heparinase III treatment of the wild type, resulted in poor binding of FGFR1 (Figure 5D).

Downloaded from by guest on November 14, 2015

26

Y.-T. Chang et al.

3.8 Attenuated hypoxia-induced increase in FGFR1 protein and FGFR1 phosphorylation

3.11 Reduced proliferation and increased adhesion in vitro

Following 3.5 days of hypoxia, a significant and equivalent increase in FGFR1 mRNA expression was observed in both Hspg2 D3/D3 and wildtype lungs (Figure 6A), whereas western blot demonstrated a significant increase in FGFR1 protein only in the wild type (Figure 6B and C). In addition, there was an increased level of phosphorylated FGFR1 in both Hspg2 D3/D3 and wild-type lungs, although significantly less in Hspg2 D3/D3 (Figure 6B and D). These findings indicate that perlecan HS is required for increased levels of FGFR1 protein and for FGFR1 phosphorylation. Analysis of PDGFRb protein levels under the same conditions as above revealed no differences between Hspg2 D3/D3 and wild type (see Supplementary material online, Figure S1).

PASMC proliferation was studied in vitro. In normoxia (20% O2 and 5% CO2) and hypoxia (1% O2 and 5% CO2), both Hspg2 D3/D3 and wild type showed a significant and equivalent increase in DNA synthesis and cell numbers after stimulation with PDGF-B and FGF2 (Figure 7C, data not shown for normoxic conditions). Stimulation with 20% fetal bovine serum under hypoxic conditions also resulted in a significant increase in DNA synthesis for both wild-type and Hspg2 D3/D3 PASMCs, but the increase was significantly smaller in Hspg2 D3/D3 (Figure 7C). Altered cell adhesion was analysed as this may affect cell proliferation. PASMCs of Hspg2 D3/D3 displayed a significantly increased adhesion to fibronectin as well as at baseline, without any coating protein. Adhesion to collagen IV or laminin was similar between Hspg2 D3/D3 and wild type (Figure 7D).

3.9 Abrogated hypoxia-induced increase in FGF2 mRNA and decreased FGF2 protein

3.10 Selective FGFR1 inhibition reduces PASMC proliferation To determine the role of FGFR1 for PASMC growth, a selective FGFR1 inhibitor27 was used in vitro. A dose of PD166866 (1 mM) significantly inhibited FGF-2-induced wild-type PASMC proliferation both in normoxia (data not shown) and in hypoxia (Figure 7A). To confirm the effect of the inhibitor on FGFR1 phosphorylation, cells were pretreated with PD166866 2 h prior to FGF-2 stimulation and cell lysates were collected 15 min after FGF-2 stimulation for western blot analysis (Figure 7A). At higher concentrations, up to 10 mM, PD166866 inhibited 20% FBS-stimulated PASMC proliferation (Figure 7B). A pan-FGFR inhibitor, SU5402, was also tested. A dose of SU5402 (10 mM) significantly reduced FGF2-induced PASMC proliferation, but had no effect on 20% FBS-stimulated PASMCs (data not shown).

Perlecan is a highly conserved basement membrane HS proteoglycan with diverse biological roles.21 The negatively charged HS chains can sequester growth factors and thereby restrict their range of action,24 but they may also serve as co-receptors to stabilize the growth factor –receptor complex for signalling.28 The intact perlecan molecule is pro-angiogenic,29 whereas a cleavage product from the C-terminal of the core protein (endorepellin) is anti-angiogenic.30 In this study, we show that perlecan HS deficiency not only leads to a pulmonary arborization defect and loss of pericytes, but also attenuates hypoxia-induced PH accompanied by reduced vascular remodelling and less right ventricular hypertrophy. FGF2, a prototype HS/heparin-binding growth factor, has been reported to be elevated in patients with pulmonary arterial hypertension,31 in monocrotaline-treated rats,11 and in lambs with shuntinduced PH. 32 FGF2-siRNA and pharmacological FGFR inhibition have also been shown to reverse monocrotaline-induced PH in rats.11 Additionally, our data here show that the FGF2/FGFR1 signalling pathway is involved in PH in mice. Hypoxia induces a significant increase in FGFR1 mRNA, protein level, and receptor phosphorylation in wild-type lungs. We have also observed a significant increase in FGF2 mRNA after 4 weeks of hypoxia in wild-type lungs. In contrast, in a previous study, no increase in FGF2 mRNA was observed in lasermicrodissected intrapulmonary arteries from Balb/cAnNCrlBR mice exposed to chronic hypoxia. The discrepancy between our findings may be explained by differences in mouse strain and methodology.33

Figure 4 Perlecan HS deficiency attenuates hypoxia-induced PH. (A) RVSP in mice under isoflurane anaesthesia after 4 weeks of hypoxia and agematched normoxia controls (n ¼ 5 – 6; *P , 0.05 by two-way ANOVA with Bonferroni’s multiple comparisons test). (B) Dry weight ratio of right ventricle (RV) to left ventricle plus septum (LV + S) after 4 weeks of hypoxia and age-matched normoxia controls, as an index of RV hypertrophy (n ¼ 5 – 6; *P , 0.05 by two-way ANOVA with Bonferroni’s multiple comparisons test). (C) Morphometric analysis of double staining immunohistochemistry for muscularization in intra-acinar pulmonary vessels. Non: intra-acinar pulmonary vessels without a-SMA-positive cells; partial: intra-acinar pulmonary vessels that were incompletely covered with a-SMA-positive cells; full: intra-acinar pulmonary vessels that were completely covered with a-SMA-positive cells. Ninety to 160 vessels per mouse were analysed (n ¼ 5 –6; *P , 0.05 compared with WT hypoxia, §P , 0.05 compared with normoxic control for the same genotype, and #P , 0.05 compared with WT normoxia by the Kruskal – Wallis test with Dunn’s multiple comparison). (D) Representative images of double staining immunohistochemistry for cell proliferation in paraffin-embedded lung sections from mice after 3.5 days of hypoxia and normoxia. Red: Ki-67; green: a-SMA. (E and F) Quantification of proliferating cells in intra-acinar pulmonary vessels from double staining immunohistochemistry. Twenty vessels per mouse were analysed (n ¼ 4; *P , 0.05 compared with WT hypoxia, §P , 0.05 compared with normoxic control for the same genotype by the Kruskal –Wallis test with Dunn’s multiple comparison). (E) Proportion of Ki-67-positive cells among all cells in the vascular wall, as a proliferation index for vascular cells. (F ) Proportion of Ki-67-positive cells among a-SMA-positive cells, as a proliferation index for SMCs.

Downloaded from by guest on November 14, 2015

Following 4 weeks of hypoxia, FGF2 mRNA expression in wild-type lungs was significantly increased compared with normoxic controls. This increase was absent in Hspg2 D3/D3 lungs, where instead a trend of gradual decrease in FGF2 mRNA was observed (Figure 6E). Further analysis with western blot showed that the level of high-molecular-weight FGF2 protein was significantly decreased in Hspg2 D3/D3, but remained unchanged in the wild type (Figure 6F and G). There were no significant changes in low-molecular-weight FGF2 protein (Figure 6F and G).

4. Discussion

Perlecan heparan sulfate in pulmonary vasculature

27

Downloaded from by guest on November 14, 2015

Figure 5 Perlecan HS–FGF2–FGFR1 interaction. (A) Double staining of wild-type hypoxic lung sections. Perlecan (bluish-green) and FGF2 (red) co-localize in the extracellular space of the pulmonary artery. (B) PLA for perlecan–FGF2 interaction (positive signal in brown). (C) FGF LACE assay on hypoxic lung tissue sections. Cy3-fluorescence indicates tissue binding of FGF2–FGFR1 complex. Heparinase III treatment was used to confirm HS-dependent binding. (D) Quantification of LACE assay on PASMC cultures incubated with 1% O2 (n ¼ 3, *P , 0.05 compared with wild type by unpaired Student’s t-test).

On the protein level, our immunohistochemistry results showed that FGF2 is located predominantly in vessels in hypoxic lungs. We could, however, not demonstrate any significant increase in FGF2 protein in whole lung homogenates following hypoxic treatment.

In this study, we have found up-regulation of whole lung perlecan mRNA following 3.5 days of hypoxia and perlecan accumulation in the vascular basement membrane in response to chronic hypoxia. To our knowledge, this is the first report to show increased perlecan in

28

Y.-T. Chang et al.

Downloaded from by guest on November 14, 2015

Figure 6 Perlecan HS deficiency alters FGF2/FGFR1 in Hspg2 D3/D3 mice. (A) qPCR of FGFR1 mRNA from whole lung homogenates. The data were normalized to age-matched, wild-type normoxic controls (n ¼ 5). (B) Representative western blots for phospho-FGFR1 (Y654) and total FGFR1 from whole lung lysates at day 3.5 of hypoxia and age-matched normoxia control. (C and D) Quantitative results from western blots for total FGFR1 (n ¼ 6) and phospho-FGFR1 (n ¼ 4). (E) qPCR of FGF2 mRNA from whole lung homogenates. The data were normalized to age-matched, wild-type normoxic controls (n ¼ 5). (F ) Representative western blot for FGF2 in whole lung lysates at 4 weeks of hypoxia and age-matched normoxia controls. (G) Quantification of western blots for FGF2 (n ¼ 4). *P , 0.05 by two-way ANOVA with Bonferroni’s multiple comparisons test.

hypoxia-induced PH. We also demonstrate that perlecan co-localizes with FGF2 in pulmonary arteries, and significantly reduced HS – FGF2 – FGFR1 ternary complex formation in Hspg2 D3/D3 lungs. This suggests that perlecan HS mediates FGF2 – FGFR1 interaction. This finding is in agreement with a previous report in which perlecan HS facilitates FGF2 signalling in the neointima of rat kidney allografts.34 A study on human coronary artery SMCs also showed that perlecan HS promotes FGF2 signalling through ternary complex formation

between FGF2 and FGFR1.15 We speculate that perlecan HS serves as a co-receptor for FGFR1 or prevents FGFR1 proteolysis by ternary complex formation. The inability of Hspg2 D3/D3 mice to increase the FGFR1 protein level and phosphorylation in response to hypoxia may, in part, explain the observed reduction in PASMC proliferation and attenuated vascular remodelling. In addition, our results show significantly reduced HMW FGF2 following hypoxia in Hspg2 D3/D3 lungs. Apart from being regulated by perlecan HS, HMW FGF2 is also known

Perlecan heparan sulfate in pulmonary vasculature

29

to be induced by PDGF-B in SMCs,35 and has been shown to become pro-proliferative after thrombin cleavage.36 HMW FGF2 can be found extracellularly and has been linked to myocardial hypertrophy.37 Little is known regarding the role of HMW FGF2 in hypoxic PH. More studies will be needed to clarify its function. Here, we have demonstrated that perlecan HS deficiency leads to an arborization defect seen by angiography and to a reduction of a-SMA-positive cells in pulmonary vessels with a diameter of 20 – 40 mm. In the embryo, pulmonary vascular development involves sprouting angiogenesis as well as vasculogenesis.38 These processes are regulated by growth factors like VEGF, and also FGF2.39 A regulatory role for perlecan in FGF2 signalling and angiogenesis is supported by a study by Gustafsson et al., where addition of FGF2 but not VEGF was shown to rescue defective angiogenesis in perlecan-null embryoid bodies.40 Our LACE assay data emphasize the critical role of perlecan HS for the HS – FGF2 –FGFR1 ternary complex formation and is supported by our previous study, where FGF2 binding to the extracellular matrix from Hspg2 D3/D3 SMCs was significantly reduced.24 The loss of

pericyte coverage and defective arborization in Hspg2 D3/D3 mice may be a result of defective FGF2 storage and positioning in the matrix. The reduced recruitment of pericytes seen with perlecan HS deficiency may also contribute to reduced development of hypoxic PH. Ricard et al. recently published a study, showing that recruitment of pericytes is of importance for the development of PH. The number of pericytes substantially increases in distal pulmonary arteries of patients with idiopathic pulmonary arterial hypertension. The data also suggested that this increase is mediated by FGF2.12 Other growth factors may, however, also be of importance. PDGF-B, dependent on its retention motif that mediates binding to HS, has been shown to recruit pericytes to the vascular wall. Defective pericyte attachment in microvessels has been demonstrated in retention motif knockout mice (Pdgfb ret/ret),41 and a similar phenotype has been reported in mice with defective N-sulfation of HS.42 It is therefore quite possible that perlecan HS deficiency also affects PDGF storage in the extracellular matrix. Our findings of equivalent levels of PDGFRb in Hspg2 D3/D3 and wild type do not exclude this possibility.

Downloaded from by guest on November 14, 2015

Figure 7 In vitro proliferation and adhesion assay. (A) Upper panel: the effect of PD166866, a selective FGFR1 inhibitor, on FGF2-induced proliferation by BrdU assay (n ¼ 3). The drug exposure time was 49 h. Lower panel: western blot from cell lysates pretreated with PD166866 2 h prior to FGF-2 stimulation. (B) The effect of PD166866 on 20% FBS-induced proliferation by BrdU assay (n ¼ 3). (C) PASMCs of wild-type (WT) and Hspg2 D3/D3 mice (Mut) were treated with 10 ng/mL of PDGF-B, 10 ng/mL of FGF2, or 20% fetal bovine serum for 24 h. Cell proliferation under 1% hypoxia was measured using BrdU. Results are presented as the mean value of quadruplicates normalized by the control group with 0.5% fetal bovine serum (n ¼ 3). (D) PASMC adhesion to 20 mg/mL of collagen IV, 20 mg/mL of EHS laminin, 10 mg/mL of fibronectin, and 20 mg/mL of fibronectin (n ¼ 3). *P , 0.05 by unpaired Student’s t-test.

30

Supplementary material Supplementary material is available at Cardiovascular Research online.

Acknowledgements We thank Malin Kronqvist for excellent technical assistance. Conflict of interest: none declared.

Funding This work was supported by the Swedish Heart-Lung Foundation (to U.H. and K.T.-L.); Uppsala and Ska˚ne County Councils (to P.K.-T. and K.T.-L.); Odd Fellows Sweden (to Y.-T.C.); the Swedish Brain Foundation (to J.L.); Thelma Zoega’s Foundation (to K.T.-L.); Stiftelsen Konsul Thure Carlssons Minne (to K.T.-L); Fanny Ekdahl’s Foundation (to K.T.-L); and Greta and Johan Kock Foundation (to K.T.-L.).

References 1. Benza RL, Miller DP, Barst RJ, Badesch DB, Frost AE, McGoon MD. An evaluation of long-term survival from time of diagnosis in pulmonary arterial hypertension from the REVEAL Registry. Chest 2012;142:448 –456. 2. Simonneau G, Gatzoulis MA, Adatia I, Celermajer D, Denton C, Ghofrani A, Gomez Sanchez MA, Krishna Kumar R, Landzberg M, Machado RF, Olschewski H, Robbins IM, Souza R. Updated clinical classification of pulmonary hypertension. J Am Coll Cardiol 2013;62:D34 – D41. 3. Sylvester JT, Shimoda LA, Aaronson PI, Ward JP. Hypoxic pulmonary vasoconstriction. Physiol Rev 2012;92:367 – 520. 4. Stenmark KR, Fagan KA, Frid MG. Hypoxia-induced pulmonary vascular remodeling: cellular and molecular mechanisms. Circ Res 2006;99:675 –691. 5. Sheikh AQ, Lighthouse JK, Greif DM. Recapitulation of developing artery muscularization in pulmonary hypertension. Cell Rep 2014;6:809 – 817. 6. Archer SL, Weir EK, Wilkins MR. Basic science of pulmonary arterial hypertension for clinicians: new concepts and experimental therapies. Circulation 2010;121:2045 –2066. 7. Perros F, Montani D, Dorfmuller P, Durand-Gasselin I, Tcherakian C, Le Pavec J, Mazmanian M, Fadel E, Mussot S, Mercier O, Herve P, Emilie D, Eddahibi S, Simonneau G, Souza R, Humbert M. Platelet-derived growth factor expression and function in idiopathic pulmonary arterial hypertension. Am J Respir Crit Care Med 2008;178:81 –88. 8. Schermuly RT, Dony E, Ghofrani HA, Pullamsetti S, Savai R, Roth M, Sydykov A, Lai YJ, Weissmann N, Seeger W, Grimminger F. Reversal of experimental pulmonary hypertension by PDGF inhibition. J Clin Invest 2005;115:2811 –2821. 9. Shah AM, Campbell P, Rocha GQ, Peacock A, Barst RJ, Quinn D, Solomon SD, IMPRES Investigators. Effect of imatinib as add-on therapy on echocardiographic measures of right ventricular function in patients with significant pulmonary arterial hypertension. Eur Heart J 2015;36:623–632. 10. Hoeper MM, Barst RJ, Bourge RC, Feldman J, Frost AE, Galie N, Gomez-Sanchez MA, Grimminger F, Grunig E, Hassoun PM, Morrell NW, Peacock AJ, Satoh T, Simonneau G, Tapson VF, Torres F, Lawrence D, Quinn DA, Ghofrani HA. Imatinib mesylate as add-on therapy for pulmonary arterial hypertension: results of the randomized IMPRES study. Circulation 2013;127:1128 –1138. 11. Izikki M, Guignabert C, Fadel E, Humbert M, Tu L, Zadigue P, Dartevelle P, Simonneau G, Adnot S, Maitre B, Raffestin B, Eddahibi S. Endothelial-derived FGF2 contributes to the progression of pulmonary hypertension in humans and rodents. J Clin Invest 2009;119:512 – 523. 12. Ricard N, Tu L, Le Hiress M, Huertas A, Phan C, Thuillet R, Sattler C, Fadel E, Seferian A, Montani D, Dorfmuller P, Humbert M, Guignabert C. Increased pericyte coverage mediated by endothelial-derived fibroblast growth factor-2 and interleukin-6 is a source of smooth muscle-like cells in pulmonary hypertension. Circulation 2014;129: 1586– 1597. 13. Noonan DM, Fulle A, Valente P, Cai S, Horigan E, Sasaki M, Yamada Y, Hassell JR. The complete sequence of perlecan, a basement membrane heparan sulfate proteoglycan, reveals extensive similarity with laminin A chain, low density lipoprotein-receptor, and the neural cell adhesion molecule. J Biol Chem 1991;266:22939 –22947. 14. Knox S, Fosang AJ, Last K, Melrose J, Whitelock J. Perlecan from human epithelial cells is a hybrid heparan/chondroitin/keratan sulfate proteoglycan. FEBS Lett 2005;579: 5019– 5023. 15. Lord MS, Chuang CY, Melrose J, Davies MJ, Iozzo RV, Whitelock JM. The role of vascular-derived perlecan in modulating cell adhesion, proliferation and growth factor signaling. Matrix Biol 2014;35:112 –122. 16. Costell M, Carmona R, Gustafsson E, Gonzalez-Iriarte M, Fassler R, Munoz-Chapuli R. Hyperplastic conotruncal endocardial cushions and transposition of great arteries in perlecan-null mice. Circ Res 2002;91:158 –164. 17. Arikawa-Hirasawa E, Rossi SG, Rotundo RL, Yamada Y. Absence of acetylcholinesterase at the neuromuscular junctions of perlecan-null mice. Nat Neurosci 2002;5: 119 –123. 18. Murdoch AD, Liu B, Schwarting R, Tuan RS, Iozzo RV. Widespread expression of perlecan proteoglycan in basement membranes and extracellular matrices of human tissues as detected by a novel monoclonal antibody against domain III and by in situ hybridization. J Histochem Cytochem 1994;42:239 –249. 19. Aviezer D, Hecht D, Safran M, Eisinger M, David G, Yayon A. Perlecan, basal lamina proteoglycan, promotes basic fibroblast growth factor-receptor binding, mitogenesis, and angiogenesis. Cell 1994;79:1005 –1013. 20. Gohring W, Sasaki T, Heldin CH, Timpl R. Mapping of the binding of platelet-derived growth factor to distinct domains of the basement membrane proteins BM-40 and perlecan and distinction from the BM-40 collagen-binding epitope. Eur J Biochem 1998;255: 60 –66. 21. Whitelock JM, Melrose J, Iozzo RV. Diverse cell signaling events modulated by perlecan. Biochemistry 2008;47:11174 –11183. 22. Rossi M, Morita H, Sormunen R, Airenne S, Kreivi M, Wang L, Fukai N, Olsen BR, Tryggvason K, Soininen R. Heparan sulfate chains of perlecan are indispensable in the lens capsule but not in the kidney. EMBO J 2003;22:236–245. 23. Zhou Z, Wang J, Cao R, Morita H, Soininen R, Chan KM, Liu B, Cao Y, Tryggvason K. Impaired angiogenesis, delayed wound healing and retarded tumor growth in perlecan heparan sulfate-deficient mice. Cancer Res 2004;64:4699 –4702.

Downloaded from by guest on November 14, 2015

Here, we have shown that perlecan HS deficiency inhibits PASMC proliferation. However, the findings from systemic arteries would predict perlecan to be anti-proliferative rather than pro-proliferative.43,44 We previously reported that Hspg2 D3/D3 mice develop increased intimal hyperplasia in carotid arteries after flow cessation, and the role of perlecan HS in the carotid is likely to be to bind and sequester FGF2 and thereby limit signalling.24 Because there is variation of glycanation of perlecan HS in different cell types and different sulfation patterns can modulate FGF2 signalling in different ways,15,34,45 it is highly possible that HS chains in pulmonary arteries have a different glycan structure compared with those in systemic arteries. The relative amount of endothelial-derived vs. SMC-derived perlecan is also most likely higher in the intra-acinar pulmonary vessels compared with the carotid artery. These two sources of HS have recently been shown to have different signalling properties.14 Moreover, our data show significantly reduced Hspg2 D3/D3 PASMC proliferation in vitro in hypoxia, but not in normoxia. This finding suggests that the sulfation pattern of HS may be subjected to further modification after hypoxia exposure. Other types of cell –matrix interactions may also be of importance. Hspg2 D3/D3 PASMCs adhere more strongly to fibronectin, indicating that perlecan HS may be involved in the regulation of the adhesive strength of cells to the extracellular matrix. Adhesive strength has been shown to affect cell proliferation and may in part explain the different response in proliferation between wild-type and Hspg2 D3/D3 cells in this study. Our study has its limitations. The data presented only demonstrates that basement membrane perlecan HS regulates FGF2 –FGFR1 interaction at the extracellular level. The effect of perlecan HS deficiency on pathways downstream of FGF2–FGFR1 awaits further investigation. Interactions between other FGFs and FGF receptors are also likely to be altered in Hspg2 D3/D3 mice, and the impacts of perlecan HS deficiency on other heparin/HS-binding factors such as PDGF, VEGF, Wnt, and BMP need to be addressed in future studies. FGF2/FGFR1 is an example of the importance of growth factor – proteoglycan interactions in hypoxia-induced PH. In conclusion, perlecan HS in the basement membrane is important for normal vascular arborization and recruitment of pericytes to pulmonary vessels. Perlecan HS deficiency attenuates hypoxia-induced PH, where the underlying mechanisms involve impaired FGF2/FGFR1 ternary complex formation and receptor phosphorylation, inhibition of PASMC growth, and altered cell –matrix interactions.

Y.-T. Chang et al.

Perlecan heparan sulfate in pulmonary vasculature

35. Pintucci G, Yu PJ, Saponara F, Kadian-Dodov DL, Galloway AC, Mignatti P. PDGF-BB induces vascular smooth muscle cell expression of high molecular weight FGF-2, which accumulates in the nucleus. J Cell Biochem 2005;95:1292 –1300. 36. Yu PJ, Ferrari G, Pirelli L, Galloway AC, Mignatti P, Pintucci G. Thrombin cleaves the high molecular weight forms of basic fibroblast growth factor (FGF-2): a novel mechanism for the control of FGF-2 and thrombin activity. Oncogene 2008;27:2594 –2601. 37. Santiago JJ, Ma X, McNaughton LJ, Nickel BE, Bestvater BP, Yu L, Fandrich RR, Netticadan T, Kardami E. Preferential accumulation and export of high molecular weight FGF-2 by rat cardiac non-myocytes. Cardiovasc Res 2011;89:139 –147. 38. Jain RK. Molecular regulation of vessel maturation. Nat Med 2003;9:685 –693. 39. Shing Y, Folkman J, Sullivan R, Butterfield C, Murray J, Klagsbrun M. Heparin affinity: purification of a tumor-derived capillary endothelial cell growth factor. Science 1984; 223:1296 –1299. 40. Gustafsson E, Almonte-Becerril M, Bloch W, Costell M. Perlecan maintains microvessel integrity in vivo and modulates their formation in vitro. PLoS ONE 2013;8:e53715. 41. Lindblom P, Gerhardt H, Liebner S, Abramsson A, Enge M, Hellstrom M, Backstrom G, Fredriksson S, Landegren U, Nystrom HC, Bergstrom G, Dejana E, Ostman A, Lindahl P, Betsholtz C. Endothelial PDGF-B retention is required for proper investment of pericytes in the microvessel wall. Genes Dev 2003;17:1835 –1840. 42. Abramsson A, Kurup S, Busse M, Yamada S, Lindblom P, Schallmeiner E, Stenzel D, Sauvaget D, Ledin J, Ringvall M, Landegren U, Kjellen L, Bondjers G, Li JP, Lindahl U, Spillmann D, Betsholtz C, Gerhardt H. Defective N-sulfation of heparan sulfate proteoglycans limits PDGF-BB binding and pericyte recruitment in vascular development. Genes Dev 2007;21:316 –331. 43. Kinsella MG, Tran PK, Weiser-Evans MC, Reidy M, Majack RA, Wight TN. Changes in perlecan expression during vascular injury: role in the inhibition of smooth muscle cell proliferation in the late lesion. Arterioscler Thromb Vasc Biol 2003;23:608–614. 44. Walker HA, Whitelock JM, Garl PJ, Nemenoff RA, Stenmark KR, Weiser-Evans MC. Perlecan up-regulation of FRNK suppresses smooth muscle cell proliferation via inhibition of FAK signaling. Mol Biol Cell 2003;14:1941 – 1952. 45. Lai J, Chien J, Staub J, Avula R, Greene EL, Matthews TA, Smith DI, Kaufmann SH, Roberts LR, Shridhar V. Loss of HSulf-1 up-regulates heparin-binding growth factor signaling in cancer. J Biol Chem 2003;278:23107 –23117.

Downloaded from by guest on November 14, 2015

24. Tran PK, Tran-Lundmark K, Soininen R, Tryggvason K, Thyberg J, Hedin U. Increased intimal hyperplasia and smooth muscle cell proliferation in transgenic mice with heparan sulfate-deficient perlecan. Circ Res 2004;94:550 –558. 25. Friedl A, Filla M, Rapraeger AC. Tissue-specific binding by FGF and FGF receptors to endogenous heparan sulfates. Methods Mol Biol 2001;171:535–546. 26. White TA, Witt TA, Pan S, Mueske CS, Kleppe LS, Holroyd EW, Champion HC, Simari RD. Tissue factor pathway inhibitor overexpression inhibits hypoxia-induced pulmonary hypertension. Am J Respir Cell Mol Biol 2010;43:35 –45. 27. Panek RL, Lu GH, Dahring TK, Batley BL, Connolly C, Hamby JM, Brown KJ. In vitro biological characterization and antiangiogenic effects of PD 166866, a selective inhibitor of the FGF-1 receptor tyrosine kinase. J Pharmacol Exp Ther 1998;286:569 –577. 28. Aviezer D, Iozzo RV, Noonan DM, Yayon A. Suppression of autocrine and paracrine functions of basic fibroblast growth factor by stable expression of perlecan antisense cDNA. Mol Cell Biol 1997;17:1938 –1946. 29. Zoeller JJ, Whitelock JM, Iozzo RV. Perlecan regulates developmental angiogenesis by modulating the VEGF-VEGFR2 axis. Matrix Biol 2009;28:284–291. 30. Willis CD, Poluzzi C, Mongiat M, Iozzo RV. Endorepellin laminin-like globular 1/2 domains bind Ig3-5 of vascular endothelial growth factor (VEGF) receptor 2 and block pro-angiogenic signaling by VEGFA in endothelial cells. FEBS J 2013;280:2271 –2284. 31. Benisty JI, McLaughlin VV, Landzberg MJ, Rich JD, Newburger JW, Rich S, Folkman J. Elevated basic fibroblast growth factor levels in patients with pulmonary arterial hypertension. Chest 2004;126:1255 –1261. 32. Wedgwood S, Devol JM, Grobe A, Benavidez E, Azakie A, Fineman JR, Black SM. Fibroblast growth factor-2 expression is altered in lambs with increased pulmonary blood flow and pulmonary hypertension. Pediatr Res 2007;61:32– 36. 33. Kwapiszewska G, Wilhelm J, Wolff S, Laumanns I, Koenig IR, Ziegler A, Seeger W, Bohle RM, Weissmann N, Fink L. Expression profiling of laser-microdissected intrapulmonary arteries in hypoxia-induced pulmonary hypertension. Respir Res 2005;6:109. 34. Katta K, Boersema M, Adepu S, Rienstra H, Celie JW, Mencke R, Molema G, van Goor H, Berden JH, Navis G, Hillebrands JL, van den Born J. Renal heparan sulfate proteoglycans modulate fibroblast growth factor 2 signaling in experimental chronic transplant dysfunction. Am J Pathol 2013;183:1571 –1584.

31

Perlecan heparan sulfate deficiency impairs pulmonary vascular development and attenuates hypoxic pulmonary hypertension.

Excessive vascular cell proliferation is an important component of pulmonary hypertension (PH). Perlecan is the major heparan sulfate (HS) proteoglyca...
999KB Sizes 0 Downloads 5 Views