Versican Accumulates in Vascular Lesions in Pulmonary Arterial Hypertension

Abstract

Pulmonary arterial hypertension (PAH) is a lethal condition for which there is no effective curative pharmacotherapy. PAH is characterized by vasoconstriction, wall thickening of pulmonary arteries, and increased vascular resistance. Versican is a chondroitin sulfate proteoglycan in the vascular extracellular matrix that accumulates following vascular injury and promotes smooth-muscle cell proliferation in systemic arteries. Here, we investigated whether versican may play a similar role in PAH. Paraffin-embedded lung sections from patients who underwent lung transplantation to treat PAH were used for immunohistochemistry. The etiologies of PAH in the subjects involved in this study were idiopathic PAH, scleroderma, and congenital heart disease (atrial septal defect) with left-to-right shunt. Independent of the underlying etiology, increased versican immunostaining was observed in areas of medial thickening, in neointima, and in plexiform lesions. Western blot of lung tissue lysates confirmed accumulation of versican in patients with PAH. Double staining for versican and CD45 showed only occasional colocalization in neointima of high-grade lesions and plexiform lesions. In vitro, metabolic labeling with [³⁵S]sulfate showed that human pulmonary artery smooth-muscle cells (hPASMCs) produce mainly chondroitin sulfate glycosaminoglycans. In addition, hypoxia, but not cyclic stretch, was demonstrated to increase both versican messenger RNA expression and protein synthesis by hPASMCs. Versican accumulates in vascular lesions of PAH, and the amount of versican correlates more with lesion severity than with underlying etiology or inflammation. Hypoxia is a possible regulator of versican accumulation, which may promote proliferation of pulmonary smooth-muscle cells and vascular remodeling in PAH.

Keywords

pulmonary arterial hypertension versican vascular smooth-muscle cell hypoxia mechanical strain

Pulmonary hypertension is defined as a mean pulmonary arterial pressure ≥25 mmHg at rest. In the subgroup of patients with pulmonary arterial hypertension (PAH), the pathological changes behind the increased pressure are precapillary.¹ The vasculopathy in PAH is characterized by endothelial dysfunction, inflammation, and vascular remodeling, which leads to an increase in vascular resistance and eventually to right heart failure. Histopathological findings in PAH include extension of muscularization distally into previously nonmuscularized arteries, medial hypertrophy (vascular wall thickening), intimal hyperplasia (neointima), fibroproliferative changes in the adventitia, and plexiform lesions. In plexiform lesions, the hallmark of PAH, abnormal proliferation of endothelial cells and α-actin–positive cells and recruitment of many other cell types, including progenitor cells (e.g., CD31⁺/c-kit⁺ endothelial progenitors and circulating fibrocytes), contribute to obliteration of small pulmonary arteries and formation of complex capillary-like channels.^2–5 Pulmonary artery smooth-muscle cell (PASMC) hypertrophy, proliferation, migration, and antiapoptosis properties are thought to play key roles in the pathology of PAH.⁶

PASMCs synthesize and secrete bioactive extracellular matrix (ECM) proteins (e.g., elastin and collagens), which have been shown to play important roles in control of cell migration and proliferation during development of PAH.^7,8 Versican is a large and complex chondroitin sulfate proteoglycan produced by smooth-muscle cells (SMCs).⁹ The versican core protein contains an N-terminal G1 domain containing a hyaluronan binding region, a C-terminal G3 domain, and glycosaminoglycan (GAG)–attachment regions between the two, the αGAG and βGAG domains.¹⁰ Alternative splicing generates at least 4 isoforms, which vary in the GAG attachment regions. While both αGAG and βGAG are present in V0, a single GAG-attachment domain is found in V1 (βGAG) and V2 (αGAG), and V3 lacks both αGAG and βGAG. All 4 isoforms can be present in the vascular ECM.^11–13 However, whether versican has a role in the vascular remodeling process of PAH is unclear.

Versican has a pivotal role in modulating cell behavior, including cell adhesion, migration, proliferation, and assembly of ECM.¹⁴ Versican expression is low in most tissues but increases dramatically in a number of different pathologies, such as diseases of the lung and the cardiovascular system, cancer, and autoimmune conditions.^15–18 In vascular pathology in the systemic circulation, versican is upregulated in restenosis after vascular intervention as well as in atherosclerotic plaques, where versican mediates cell proliferation and lipid retention.^16,19,20 Experimentally, inhibition of versican synthesis in SMCs leads to reduced proliferation.²¹ On the other hand, overexpression of V3, the versican variant without GAG-attachment domains, inhibits SMC growth and migration.²² In pulmonary pathology, versican has been identified as a macrophage activator in lung cancer, and increased versican production by bronchial fibroblasts has been linked to airway hyperresponsiveness in patients with asthma.^23,24 Fibroblasts derived from distal airway from patients with chronic obstructive pulmonary disease (COPD) have also been shown to produce increased levels of versican compared with cells from control subjects.²⁵ Furthermore, increased immunostaining for versican in patients with moderate COPD has been shown to correlate with a decrease in elastin-binding protein.²⁶ This indicates that versican may be involved in elastin regulation in pulmonary pathology.

Recent studies have shown that inflammation is likely to be one of the principal factors driving vascular remodeling in PAH.²⁷ Aberrant CD4⁺ T cell function and accumulation of CD68⁺ macrophages and immature dendritic cells lead to perivascular inflammation in PAH. In addition, overproduction of cytokines and chemokines (e.g., interleukin [IL] type 1, IL-6, and macrophage inhibitory factor) has been shown to influence the proliferative response of PASMCs.^28–30 Versican may affect inflammatory responses by interactions with receptors on inflammatory cells (e.g., CD44, P-selectin glycoprotein ligand 1, and P- or L-selectins) or by interactions with other ECM components of importance for inflammation (e.g., hyaluronan and fibronectin).³¹ Versican, together with hyaluronan, tumor necrosis factor–inducible gene 6 protein, and inter-α-trypsin inhibitor, can form organized filaments permissive for leukocyte adhesion.³² A viscous hyaluronan- and versican-rich matrix has also been shown to impede T cell migration, which could play a role in T cell trafficking and function in inflamed tissues.³³ High levels of hyaluronan, which can bind to versican G1 domain, have been found in plasma and explanted human pulmonary artery smooth-muscle cells (hPASMCs) from patients with PAH.³⁴ In addition, Lauer et al.³⁵ recently showed that the hyaluronan that accumulates in patients' pulmonary vessels is modified by covalently bound heavy chains from plasma-derived inter-α-trypsin inhibitor. This heavy chain–hyaluronan complex was also shown to partially colocalize with leukocyte infiltration areas in plexiform lesions of PAH, indicating that hyaluronan may influence the inflammatory response in PAH.³⁵ However, the role of versican in the immuno-pathology of PAH has not been explored.

Since the pulmonary circulation is a low-resistance and low-oxygen system, the knowledge gained from studies of systemic arteries about the roles of vascular proteoglycans may not be fully applicable. In this study, we aimed to investigate the role of versican in pulmonary vascular remodeling in PAH. We examined whether versican is increased in different types of vascular lesions in PAH of different etiology in humans and explored the association between versican and inflammation. We also tested whether increased mechanical strain or hypoxia could influence versican production in hPASMCs, given the pathophysiological environment in the pulmonary vasculature in PAH.

METHODS

Lung tissues from patients with pulmonary hypertension and failed donor lungs

Paraffin-embedded lung sections and frozen lung tissue from patients with PAH who had undergone lung transplantation and control samples from unused donor lungs were provided by Marlene Rabinovitch under the Pulmonary Hypertension Breakthrough Initiative and obtained from the pathology biobank at Skåne University Hospital, Lund, Sweden (approved by the regional ethical review board, 534/2005 and 248/2010). Samples used for this project had been completely deidentified. In total, tissue from 15 patients with idiopathic PAH (IPAH), 3 patients with scleroderma, 4 patients with PAH associated with congenital heart disease, and 15 control subjects were used for this study.

Immunohistochemistry

After deparaffinization and rehydration, endogenous peroxidase activity was quenched using 0.7% hydrogen peroxide in 100% methanol for 20 minutes, and then the sections were pretreated with chondroitinase ABC (0.2 units/mL) in 18 mM Tris-acetate buffer pH 8.0 containing 0.1% bovine serum albumin (BSA) for 1 hour at 37°C, followed by 1 hour of blocking with 3% nonfat dry milk and 2% BSA in phosphate-buffered saline (PBS) buffer. Primary antibody (mouse monoclonal antiversican, clone 2B1, Seikagaku) was applied for overnight incubation at 4°C. PBS buffer was used for all washing steps. The sections were then incubated with a biotinylated goat anti-mouse secondary antibody (1 : 300, Zymed) for 1 hour at room temperature. After detection with Vectastain Elite ABC kit and DAB HRP substrates (Vector Labs), the sections were counterstained with hematoxylin.

For double staining of versican and leukocytes, a mouse monoclonal antihuman CD45 antibody was used (1 : 100, clones 2B11 + PD7/26, Dako) and heat-induced antigen retrieval with citrate buffer for 20 minutes was added to the protocol above. The double staining was performed using a Leica Bond-Max automated immunostainer. The incubation with primary antibodies was done for 1 hour at room temperature, and DAB HRP was used as substrate for versican and Vector Red for CD45.

Double immunofluorescence

After deparaffinization and heat-induced antigen retrieval with citrate buffer, the lung sections were treated with chondroitinase ABC (0.2 units/mL) in 18 mM Tris-acetate buffer pH 8.0 containing 0.1% BSA for 1 hour at 37°C, followed by 20 minutes of blocking with BlockAid blocking solution (Thermo Fisher Scientific). The sections were then incubated with rabbit monoclonal antiversican G3 domain antibody (1 : 100, ab177480, Abcam) overnight at 4°C. After washing with PBS buffer, cyanine Cy3 goat antirabbit immunoglobulin G (IgG; 1 : 200, Jackson ImmunoResearch) and fluorescein isothiocyanate–conjugated mouse monoclonal anti–smooth-muscle α-actin antibody (1 : 500, ab8211, Abcam) were applied simultaneously for 1 hour at room temperature. Following washing with PBS buffer, the slides were mounted with ProLong Diamond Antifade Mountant with 4′,6-diamidino-2-phenylindole (Thermo Fisher Scientific).

Proteoglycan extraction and Western blot

Proteoglycan extraction from cells . Cells were solubilized in 8 M urea buffer (8 M urea, 50 mM Tris-base, 0.5% Triton X-100, 0.25 M NaCl, 2 mM EDTA, pH 7.4) with protease inhibitors (5 mM benzamidine, 100 mM 6-aminohexanoic acid, and 1 mM phenyl-methylsulfonyl fluoride). After measuring the protein concentration by Coomassie (Bradford) protein assay (Pierce), equal protein aliquots from each sample were used for purification of proteoglycans by ion-exchange chromatography on diethylaminoethyl (DEAE) Sephacel in 8 M urea buffer. The columns were washed with 8 M urea buffer and eluted with 8 M urea buffer containing 2 M NaCl. Isolated proteoglycans were purified by ethanol precipitation as described elsewhere.³⁶ After chondroitinase ABC digestion (1.67 units/mL) for 3 hours at 37°C, the samples were reduced and electrophoresed on a 4%–12% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel. Proteins were then transferred to nitrocellulose membranes using a Bio-Rad Transblot SD Semi-Dry Transfer Cell. After incubation with the blocking buffer (10% Aqua Block in Tris-buffered saline [TBS] containing 0.1% Tween-20) for 2 hours at room temperature, membranes were incubated with a goat polyclonal antibody against the human versican βGAG domain (1 : 2000, AF 3054, R&D systems) overnight at 4°C in blocking buffer. TBS containing 0.1% Tween-20 was used for all washing steps. After incubation with fluorescence-conjugated secondary antibody for 1 hour and washing, membranes were scanned by Odyssey CLx infrared imaging system (LI-COR). Protein band signal intensities were measured by Image Studio Lite software (LI-COR).

Proteoglycan extraction from tissues. Frozen lung tissue from each subject was homogenized with 4 M guanidine buffer (4 M guanidine HCl, 100 mM sodium sulfate, 100 mM Tris-base, 2.5 mM Na₂ EDTA, 0.5% Triton X-100, pH 7.0) and then dialyzed extensively against 7 M urea buffer (7 M urea, 50 mM Tris-base, 2 mM EDTA, 0.5% Triton X-100, pH 7.5). Protein concentration was determined by Coomassie (Bradford) protein assay. Equal amounts of total proteins of each sample were subjected to ion exchange chromatography as described above. Equal volumes of isolated proteoglycans were ethanol precipitated, digested with chondroitin ABC lyase, and electrophoresed on 4%–12% SDS-PAGE gels with 3.5% stacking. The samples were then subjected to Western blot analysis as described above. Two different primary antibodies were used to detect versican, a goat polyclonal antibody against βGAG domain (1 : 3000, AF 3054, R&D systems) and a mouse monoclonal antibody against C-terminal G3 domain (2B1, 1 : 3000, Seikagaku).

Cell culture

The hPASMCs and aortic SMCs were obtained from Life Technologies. Medium 231 and smooth-muscle growth supplement were used as growth medium for cell culture propagation. Final concentration of fetal bovine serum in the growth medium was 5%. Cells with passage 5–9 were used for in vitro experiments.

Cyclic stretch

Silicon chambers (self-made; inner dimensions: 4 cm × 4 cm × 4 cm) with newly attached bottoms were sterilized and coated with bovine fibronectin (10 μg/cm²) as described elsewhere.³⁷ The hPASMCs were plated on the coated chambers with the cell density of 8,000 cells/cm². After 72 hours of culture with growth medium, the medium was changed to DMEM containing 0.4% fetal bovine serum, and 10% of biaxial cyclic stretch was applied to the silicone chambers with the frequency of 0.5 cycles per second. After 6 hours of cyclic stretch, the medium was collected, and the cell layer was lysed for additional studies. For metabolic labeling experiments, 50 μCi/mL of [³⁵S]sulfate was added during 6 hours of cyclic stretch. To ensure that our biaxial mechanical stretch device had the same effect on versican production in aortic SMCs as previously described with a different device and lower degree of mechanical stretch, human aortic SMCs from the same donor as hPASMCs were used as positive controls.³⁸

One percent hypoxia

The hPASMCs were plated on 100-mm Petri dishes (8,000 cells/cm²) and cultured in growth medium for 72 hours. At this time point, the cultures were subconfluent. The medium was then changed to DMEM with 0.4% fetal bovine serum. Cells in the hypoxic group were incubated in an InvivO₂ 300 hypoxia workstation (Ruskinn) with 1% O₂ and 5% CO₂ for 24 hours at 37°C. Normoxic control cells were incubated in a regular cell incubator (5% CO₂). After 24 hours, the medium was collected, and the cell layer was lysed for RNA purification or proteoglycan extraction.

Metabolic labeling and gel filtration chromatography

A total of 1 × 10⁶ SMCs were metabolically labeled with 50 μCi/mL of [³⁵S]sulfate (PerkinElmer) in DMEM with 0.4% fetal bovine serum for 6 hours. After labeling, the medium was collected. The cells were washed with PBS, incubated in solubilization buffer (50 mM Tris-HCl pH 7.5, 1% Triton X-100, 0.15 M NaCl) for 30 minutes at 4°C, and then centrifuged at 800 g for 10 minutes. The supernatants from the cell lysates and the medium samples were applied on 0.2-mL columns of DEAE-Sephacel (GE Healthcare), equilibrated with 50 mM Tris-HCl pH 8, 0.1% Triton X-100, 0.15 M NaCl. Following washing of the DEAE-Sephacel column with equilibration buffer and a second washing with sodium acetate buffer (50 mM NaAc pH 4, 0.1% Triton X-100, 0.15 M NaCl), the [³⁵S] sulfate-labeled proteoglycans were eluted with 50 mM NaAc pH 4, 0.1% Triton X-100, 2 M NaCl. Protease inhibitor cocktail (Roche) was used during solubilization and the whole process of ion exchange chromatography.

To release the [³⁵S]sulfate-labeled GAG chains from their core proteins, a portion of the elute was treated with 0.5 M NaOH overnight at 4°C.³⁹ After neutralization to pH 7.4, desalting in ultra-pure water on an NAP-10 column, followed by lyophilization, half volume of the samples were treated with chondroitinase ABC digestion (0.01 units/μL) overnight at 37°C. The treated and untreated samples were subsequently analyzed by gel filtration on Sephadex G50 eluted with 0.2 M NaCl. For analysis of [³⁵S]sulfate-labeled proteoglycans, the DEAE-Sephacel purified samples were applied to a Sepharose CL-2B column (10 mm × 90 cm) in the buffer containing 50 mM Tris-HCl pH 7.5, 0.1% Triton X-100, 1 M NaCl; 0.5 mL-fractions were collected and analyzed for [³⁵S]sulfate-radioactivity.

RNA isolation and quantitative polymerase chain reaction (qPCR)

RNA extraction was performed using RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. Deoxyribonuclease was applied to remove genomic DNA. Total RNA integrity, RNA purity, and concentration were measured by a 2100 Bioanalyzer (Agilent Technologies) and a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). To make complementary DNA (cDNA), 1 μg of each RNA sample with RNA integrity number 10 was used for reverse transcription by a high-capacity RNA-to-cDNA kit (Applied Biosystems). For each 20-μL amplification reaction, 50 ng of cDNA was used. Real-time PCR was done using Applied Biosystems 7900HT. The following TaqMan gene expression assays were applied: total versican, Hs00171642_m1; versican V0, Hs01007944_m1; versican V1, Hs01007937_m1; versican V2, Hs01007943_m1; and versican V3, Hs01007941_m1. β2 microglobulin (Hs99999907_m1) was used as reference gene. Fold changes in gene expression were calculated using the relative quantification (ΔΔC_t) method. Individual sample values were normalized to β2 microglobulin levels. Gene expression data from 3 independent batches of cells in quadruplicates were combined before statistical analyses.

Statistical analysis

IBM SPSS statistics version 21 software was used for statistical analyses. Student t test was used for comparisons between two groups. Data were presented as mean ± SEM. A P value <0.05 was considered significant.

RESULTS

Versican accumulates in vascular lesions of patients with PAH

The presence of versican in vascular lesions of PAH was demonstrated by immunohistochemistry (Fig. 1). In PAH of patients with IPAH, in PAH secondary to scleroderma, and in PAH secondary to congenital heart disease, strong staining for versican was seen in neointima, in hypertrophic media, and in plexiform lesions (Fig. 1A, 1B). In contrast, little versican was found in the vessels of control subjects (Fig. 1B). The increased deposition of versican in PAH was confirmed by Western blotting of lung tissue homogenates using 2 different antibodies, one for the G3 domain (same as for immunohistochemistry) and one for the βGAG domain (Fig. 2). The Western blots revealed multiple bands in lung homogenates from patients with IPAH, which may represent breakdown products of versican. Similar results have been reported in differentiating embryoid bodies accompanied with increased expression levels of versican degradation enzymes.³⁶ Notably, versican levels were consistently lower in one of the patients with IPAH, possibly due to fewer vascular lesions in the specimen.

Figure 1.

Versican accumulates in the vascular lesions of individuals with pulmonary arterial hypertension (PAH). A, Immunohistochemistry for versican (in brown) in different vascular lesions. B, Representative images of versican staining in lungs from patients with PAH of different etiologies. A monoclonal antiversican antibody (clone 2B1) against the C-terminal G3 domain was used for immunostaining. Images were captured with a x20 objective. One section per subject was analyzed (n = 7 for idiopathic PAH [IPAH], n = 7 for failed donor lung as control, n = 3 for atrial septal defect with PAH [ASD], and n = 3 for scleroderma with PAH [Scl]). Donor: healthy donor lungs unused for transplantation as controls; Neg: mouse immunoglobulin G used as negative control.

Figure 2.

Western blots confirm increased levels of versican in the lungs of patients with pulmonary arterial hypertension (PAH). Proteoglycan extracts from lung tissue homogenates treated with the chondroitinase ABC were used. For each sample, 1.5 mg of protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions, and immunoblotting for versican was performed using two different antibodies, mAb 2B1 for the C-terminal G3 domain (A) and pAb 3054 for the βGAG domain (B). Samples from 4 patients with idiopathic PAH (IPAH) were compared with age-matched unused donor lungs (control). Increased versican fragments (lanes 5, 6, and 8) were observed in subjects with IPAH compared with age-matched control subjects. C, Schematic drawing of versican splice variants and recognition sites for mAb 2B1 and pAb 3054. Versican has two globular domains, the G1 domain at the amino terminus and the G3 domain at the carboxy terminus. The splice variants vary in the GAG-attachment domains (αGAG and βGAG). C: complement regulatory region; E: epidermal growth factor–like domain; HABR: hyaluronan-binding region; Ig: immunoglobulin-like domain; L: lectin-binding domain.

Only sporadic colocalization of versican immunostaining with leukocyte infiltration in PAH

To examine the relationship between inflammation and versican in PAH, double staining for CD45 (leukocyte common antigen) and versican was performed. However, there was no clear correlation between versican staining in the vasculature and the inflammatory status of the lungs. In both patients and controls, vessels with clear accumulation of CD45⁺ cells in the adventitia often showed little versican staining (Fig. 3A–3C), and most of the versican deposition did not colocalize with CD45 in plexiform lesions (Fig. 3D–3F). In neointima, the majority of areas positive for versican were not positive for CD45 (Fig. 3G, 3H), although a few regions with positive staining for both antibodies were observed (Fig. 3I–3K).

Figure 3.

Versican and inflammation in pulmonary arterial hypertension (PAH). Representative images of double immunostaining for versican (in brown) and CD45 (leukocyte common antigen, in red). A and B, Pronounced perivascular inflammation with little versican staining. C, Infiltration of inflammatory cells in the adventitia without colocalization with versican. D–F, Versican and CD45 are rarely colocalized in plexiform lesions. G and H, Versican accumulates in the neointima, but there is almost no colocalization with inflammatory cells. I–K, Some degree of colocalization in neointima, but it is not consistent. Arrows indicate the colocalization of versican and CD45. The letter L indicates the lumen of pulmonary vessels. Mouse immunoglobulin G (IgG) was used as a negative control. The images were captured with a x4 objective for K and a x20 objective for A–J. There were 4–6 sections per slide, and 1 slide per subject was analyzed (n = 13 for PAH group; n = 7 for control group).

Versican accumulates in ECM surrounding smooth-muscle α-actin–positive cells

The abundance of versican in the neointima and medial layer of pulmonary vessels implies that hPASMCs may play a key role in versican production. Immunofluorescent double staining for versican and smooth-muscle α-actin revealed that the majority of versican was in the extracellular space in close proximity to smooth-muscle α-actin–positive cells, both in neointimal areas (Fig. 4A–4C) and in plexiform lesions (Fig. 4D–4F). In the healthy control subjects (Fig. 4G, 4H), little versican was observed in areas with smooth-muscle α-actin–positive cells.

Figure 4.

Versican and smooth-muscle α-actin–positive cells. Representative images of double immunofluorescence staining for smooth-muscle α-actin (in green) and versican (in red). Nuclear staining was shown in blue. A, Smooth-muscle α-actin staining of neointimal lesion (idiopathic pulmonary arterial hypertension [IPAH]). B, Strong versican immunostaining in the same neointima. C, Merged image (A and B), which shows versican accumulation in areas with smooth-muscle α-actin–positive cells within the neointima. D, Smooth-muscle α-actin staining of plexiform lesion (IPAH). E, Prominent versican staining in the same plexiform lesion. F, Merged image (D and E). G and H, Double immunofluorescence staining of vessels from healthy control subjects, with very low levels of versican in areas with smooth-muscle α-actin–positive cells. I, Rabbit immunoglobulin G–negative control. The images were captured with a ×20 objective. One section per subject was analyzed (n = 4 for IPAH; n = 3 for healthy control subjects).

Majority of GAGs secreted by hPASMCs are chondroitin sulfate and dermatan sulfate

Since little is known regarding the composition of proteoglycans secreted by hPASMC, proteoglycan profiles were examined by metabolic labeling with [³⁵S]sulfate. Sephadex G50 chromatography after digestion of isolated GAG chains with chondroitinase ABC showed that almost all GAGs secreted by hPASMC were susceptible to the treatment, indicating that the proteoglycans carried chondroitin sulfate and dermatan sulfate and not heparan sulfate side chains (Fig. 5A). In the cell-layer samples, but not in medium samples, there was a small peak resistant to digestion, representing heparan sulfate. Sepharose CL-2B chromatography showed that, compared with aortic SMCs from the same healthy donor, proteoglycans produced by hPASMCs were of smaller hydrodynamic size, suggesting that the composition of proteoglycans may differ between the two cell types (Fig. 5B).

Figure 5.

Characterization of proteoglycans produced by human pulmonary artery smooth-muscle cells (hPASMCs). A, Gel filtration chromatography on Sephadex G50 of [³⁵S]sulfate-labeled glycosaminoglycan chains. [³⁵S]sulfate-labeled glycosaminoglycans were released from their core proteins by alkaline treatment. The graph represents profiles before and after chondroitinase ABC digestion. B, [³⁵S]sulfate-labeled intact proteoglycans synthesized by hPASMCs and aortic smooth-muscle cells (AoSMCs) from the same donor were characterized by gel filtration chromatography on Sepharose CL-2B. Samples were applied on the basis of equal amount of radioactivity (measured in counts per minute [CPM]). Similar results were seen in 3 independent experiments.

Mechanical strain has no immediate effect on versican production in hPASMCs in vitro

Since we observed that versican was increased in patients with PAH independent of whether the PAH was idiopathic or associated with scleroderma or congenital heart disease, we hypothesized that an initial increase in pressure, possibly mainly caused by vasoconstriction, may induce versican deposition and further vascular remodeling. To test our hypothesis, hPASMCs were subjected to 10% biaxial cyclic stretch for 6 hours. Under these conditions, no obvious cell death was found as examined by Trypan blue staining. As a positive control, aortic SMCs from the same donor were used.³⁸

During the 6 hours of 10% cyclic stretch, SMCs were labeled with [³⁵S]sulfate, and [³⁵S]sulfate-labeled proteoglycans were analyzed by Sepharose CL-2B chromatography. The results showed that mechanical strain did not affect the hydrodynamic size of proteoglycans. However, the peak representing small proteoglycans clearly disappeared after stretch (Fig. 6A). The results for hPASMCs were similar to control aortic SMCs from the same donor (Fig. 6B).

Figure 6.

The hydrodynamic size of proteoglycans after mechanical strain. Sepharose CL-2B chromatography of [³⁵S]sulfate-labeled proteoglycans. A, Results for stretched human aortic smooth-muscle cells (hAoSMCs) compared with nonstretched controls. B, Stretched human pulmonary artery smooth-muscle cells (hPASMCs) compared with nonstretched controls. CPM: counts per minute.

For the aortic control SMCs, qPCR and Western blot revealed that stretch significantly increased both versican mRNA and protein levels after 6 hours of cyclic stretch, compared with static conditions (Fig. 7A, 7B). However, for hPASMCs, the same stretch conditions did not affect versican mRNA levels immediately after 6 hours of cyclic stretch (not shown). Following prolonged mechanical strain up to 12 hours, a trend toward increase in versican protein and mRNA was seen in hPASMCs compared with static conditions, but the differences did not reach statistical significance with the exception of an increase in V0 mRNA (Fig. 7C, 7D).

Figure 7.

Upregulation of versican messenger RNA and protein by mechanical strain in human aortic smooth-muscle cells (SMCs) but not in human pulmonary artery smooth-muscle cells (hPASMCs). Results of quantitative polymerase chain reaction (qPCR) for versican isoforms (V0, V1, V2, and V3; A) and a representative Western blot (B) for versican from cell cultures of stretched aortic SMCs and nonstretched controls. Samples were collected after 6 hours of cyclic stretch. Results of qPCR (C) for versican isoforms and a representative Western blot (D) for versican from cell cultures of stretched hPASMCs and nonstretched controls. Samples were collected for another 6 hours after 6 hours of cyclic stretch. For qPCR (A and C), fold changes were calculated using ΔΔC_t method compared with nonstretched controls. β₂-microglobulin was used as reference gene (n = 3; asterisk indicates P < 0.05 by Student t test). For Western blots (B and D), equal amount of total proteins from conditioned medium and cell lysates, respectively, were applied to ion-exchange chromatography for proteoglycan purification. Chondroitinase ABC-treated samples were then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing condition and probed with goat antihuman versican β glycosaminoglycan domain antibody for versican isoforms V0 and V1. N: nonstretched control group; S: stretched group; (+): positive control extracted from human aortic SMC–conditioned medium.

Hypoxia influences versican expression in hPASMCs in vitro

Hypoxia is a known contributing factor for vascular remodeling in PAH.⁴⁰ To determine whether versican expression can be regulated by hypoxia in hPASMCs, cells were cultured in an incubator with 1% O₂ and 5% CO₂. After 24 hours of 1% O₂, versican isoforms V0 and V1 were both significantly increased at the mRNA level, whereas V2 transcripts were significantly decreased compared with the normoxic group of PASMCs (Fig. 8A). Western blot analysis confirmed the increase of versican protein following hypoxia exposure (Fig. 8B). The results suggest that hypoxia influences versican production in hPASMCs.

Figure 8.

Versican is regulated by hypoxia. A, quantitative polymerase chain reaction (qPCR) for versican isoforms (V0, V1, V2, and V3) from cell lysates of hypoxic human pulmonary artery smooth-muscle cells (hPASMCs). Data were analyzed using ΔΔC_t method compared with normoxic controls. β₂-microglobulin was used as reference gene (n = 3; asterisk indicates P < 0.05 by Student t test). B, Representative Western blot for versican from cell cultures of hypoxic hPASMCs and normoxic controls. Purified, chondroitinase-treated samples were probed with goat antihuman versican β glycosaminoglycan domain antibody for versican isoforms V0 and V1. H: hypoxia; N: normoxia; (+): positive control extracted from human aortic smooth-muscle cell–conditioned medium.

DISCUSSION

In this study, we show versican accumulation in neointima, in medial hypertrophy, and in plexiform lesions of patients with IPAH, as well as in PAH secondary to connective tissue disease or congenital heart disease. In addition, our data show that hypoxia, but not mechanical strain, induces versican expression in hPASMCs in vitro. Hypoxia may be an important factor for versican deposition during vascular remodeling of PAH.

SMCs have previously been demonstrated to be the main source of versican in blood vessels.⁴¹ Here, versican was mainly found in areas with positive staining for smooth-muscle α-actin. Therefore, hPASMCs are likely to be a major source of versican during vascular remodeling in PAH. In plexiform lesions, there were often some strongly stained areas and some almost negative areas, possibly indicating different cell populations, because the origin of cells in plexiform lesions has been shown to be very diverse.³

Earlier studies of vascular development and remodeling support the idea that increased vessel wall thickness is an adaptation to higher blood pressure, which may also apply to vascular remodeling in PAH.^42,43 Our hypothesis was that versican, secreted by vascular cells exposed to increased strain, contributes to vascular wall thickening. Mechanical strain may have a direct impact on versican production, which may facilitate vascular remodeling. Lee and coworkers have previously shown that versican protein is increased in aortic SMCs after 36 hours of 4% cyclic stretch.³⁸ In our study, we increased the mechanical strain to 10% stretch, and an upregulation of versican protein was observed in aortic SMCs already after 6 hours of stretch exposure. However, no significant increase in versican protein was found at this time point in hPASMCs. We also observed that hPASMCs (but not aortic SMCs) began to detach from the fibronectin-coated silicone chambers after 12 hours of 10% cyclic stretch. We were therefore unable to examine long-term effects of mechanical strain on hPASMCs using our system. It should be noted that the cells used for this study are from a healthy donor, and there is a possibility that hPASMCs from patients with pulmonary hypertension would respond differently to mechanical strain.

Hypoxia may partly explain versican accumulation in pulmonary vessels. Hypoxia-inducible factors (HIFs) are transcriptional factors that respond to cellular oxygen deficiency to maintain cell survival. HIF-1α has been shown to be an important factor in PAH.⁴⁴ Asplund et al.⁴⁵ reported that the versican gene, VCAN, has a hypoxia response element, DNA sequence 5′-[AG]CGTG-3′, in the promoter region and that HIF-1α and HIF-2α can mediate versican transcription. Here, we show that hypoxia induces increases in versican V0 and V1 mRNA levels but a decrease in V2 mRNA, indicating that selective splicing between exons encoding αGAG and βGAG domains exists under hypoxia. Whether HIFs have a direct impact on alternative RNA splicing awaits additional investigations.

In addition to mechanical strain and hypoxia, several growth factors and transcription factors are likely to be involved in the regulation of versican expression. Transforming growth factor β and platelet-derived growth factor subunit B have, for example, previously been demonstrated to increase versican secretion.^46,47 In Wnt signaling, the β-catenin/T cell factor complex has been identified as a transcriptional activator of versican in SMCs.⁴⁸ These factors are also known to contribute to the proliferative vasculopathy of PAH.^49–51 So far, we can only speculate about the function of versican in vascular remodeling in PAH. Increased versican accumulation makes the vascular ECM enriched in chondroitin sulfate. The negatively charged GAG chains may attract proteoglycan-binding mitogens and act as indirect regulators of growth factor signaling. The GAG chains also absorb water and provide a water-rich pericellular matrix, which can facilitate cell proliferation and migration by creating space for cell movement.⁵²

In this study, we saw little colocalization of versican with CD45 staining for inflammatory cells. However, this does not exclude a role for versican in inflammation in PAH, because vascular remodeling is a dynamic process, and our methodology did not allow us to analyze more than a single time point for each patient (i.e., the time of transplantation). An association with inflammation may be more evident earlier in the remodeling process. A close association between versican and hyaluronan has been shown in inflammation, as mentioned above, and it has also been reported in several other pathological processes, such as intimal hyperplasia in the systemic arteries.⁵³ However, in the case of PAH, versican staining is mainly localized in the tunica media and in neointima and is less pronounced in the adventitia, where hyaluronan deposits have been reported.³⁴ Hyaluronan-versican complexes are therefore not likely to be present in PAH, except possibly in plexiform lesions, where staining for both molecules is seen. However, as with inflammation, it is also possible that the two molecules may be more closely associated during certain stages of the remodeling, such as during rapid neointima expansion. This would be interesting to explore in animal models where lesions from different time points can be studied.

It is not easy to answer the question of whether versican plays an important functional role in PAH or whether it is just a marker for the disease and part of the normal response of the vessels when there is a pathological insult. However, in a recent study by de Jesus Perez et al.,⁵⁴ point mutations in the VCAN gene were described in patients with IPAH. Versican was one of the top 3 mutated genes detected in samples from patients with IPAH by whole-exome sequencing, indicating that versican could be a major player in PAH. It is possible that point mutations in the VCAN gene can explain why some people are more susceptible to developing pulmonary hypertension when exposed to other factors, such as high altitude and certain drugs. It is also known that a majority of family members who carry BMPR2 mutations, the most common type of mutations in hereditary PAH, will never develop PAH, so other factors are clearly necessary for penetrance and disease progression.⁵⁵

In summary, we show that versican accumulates in vascular lesions of PAH and that it can be regulated by hypoxia. Additional studies on the expression and localization of different versican splice variants, as well as studies of cell-type-specific or inducible-versican mutant mice, will be needed to clarify the diverse roles of this large chondroitin sulfate proteoglycan in the pathobiology of PAH. More detailed studies of the molecular mechanisms behind the regulation by hypoxia may also be helpful to elucidate the control of versican metabolism, a potential antiremodeling target for treatment of PAH.

Footnotes

ACKNOWLEDGMENTS

Tissue samples were provided by Marlene Rabinovitch under the Pulmonary Hypertension Breakthrough Initiative and were obtained from the pathology biobank at Skåne University Hospital in Lund. We thank the patients and everyone involved in the tissue collection. We also especially thank Nancy Ferreira Tojais, Michal Roof, Lingli Wang, Hans Brunnström, and Lena Thiman for excellent technical assistance and Marlene Rabinovitch and Leif Eriksson for facilitating the study.

Source of Support: Funding for the Pulmonary Hypertension Breakthrough Initiative is provided by the Cardiovascular Medical Research and Education Fund. This project was also funded by the National Institutes of Health (P01 HL098067 to TNW), the Swedish Heart-Lung Foundation, Thelma Zoéga's Foundation for Medical Research, Greta and Johan Kock's Foundation, Thure Carlsson Memorial Foundation, Fanny Ekdahl Foundation for Pediatric Research, and Karolinska Institutet.

Conflict of Interest: None declared.

References

Hoeper

, Bogaard

, Condliffe

, Frantz

, Khanna

, Kurzyna

, Langleben

. Definitions and diagnosis of pulmonary hypertension. J Am Coll Cardiol 2013; 62(25 suppl.): D42–D50.

Jonigk

, Golpon

, Bockmeyer

, Maegel

, Hoeper

, Gottlieb

, Nickel

. Plexiform lesions in pulmonary arterial hypertension composition, architecture, and microenvironment. Am J Pathol 2011; 179(1): 167–179.

Yeager

, Frid

, Stenmark

. Progenitor cells in pulmonary vascular remodeling. Pulm Circ 2011; 1(1): 3–16.

Toshner

, Voswinckel

, Southwood

, Al-Lamki

, Howard

, Marchesan

, Yang

. Evidence of dysfunction of endothelial progenitors in pulmonary arterial hypertension. Am J Respir Crit Care Med 2009; 180(8): 780–787.

Nikam

, Schermuly

, Dumitrascu

, Weissmann

, Kwapiszewska

, Morrell

, Klepetko

. Treprostinil inhibits the recruitment of bone marrow-derived circulating fibrocytes in chronic hypoxic pulmonary hypertension. Eur Respir J 2010; 36(6): 1302–1314.

Tajsic

, Morrell

. Smooth muscle cell hypertrophy, proliferation, migration and apoptosis in pulmonary hypertension. Compr Physiol 2011; 1(1): 295–317.

Aiello

, Gutierrez

, Chaves

, Lopes

, Higuchi

, Ramires

. Morphology of the internal elastic lamina in arteries from pulmonary hypertensive patients: A confocal laser microscopy study. Mod Pathol 2003; 16(5): 411–416.

Botney

, Liptay

, Kaiser

, Cooper

, Parks

, Mecham

. Active collagen synthesis by pulmonary arteries in human primary pulmonary hypertension. Am J Pathol 1993; 143(1): 121–129.

Chang

, Yanagishita

, Hascall

, Wight

. Proteoglycans synthesized by smooth muscle cells derived from monkey (Macaca nemestrina) aorta. J Biol Chem 1983; 258(9): 5679–5688.

10.

Zimmermann

, Ruoslahti

. Multiple domains of the large fibroblast proteoglycan, versican. EMBO J. 1989; 8(10): 2975–2981.

11.

Ito

, Shinomura

, Zako

, Ujita

, Kimata

. Multiple forms of mouse PG-M, a large chondroitin sulfate proteoglycan generated by alternative splicing. J Biol Chem 1995; 270(2): 958–965.

12.

Wight

. Versican: A versatile extracellular matrix proteoglycan in cell biology. Curr Opin Cell Biol 2002; 14(5): 617–623.

13.

Zimmermann

. Versican. In: Iozzo

, ed. Proteoglycans: structure, biology and molecular interactions. New York: Dekker, 2000: 327–341.

14.

Wight

, Kinsella

, Evanko

, Potter-Perigo

, Merrilees

. Versican and the regulation of cell phenotype in disease. Biochim Biophys Acta 2014; 1840(8): 2441–2451.

15.

Andersson-Sjoland

, Hallgren

, Rolandsson

, Weitoft

, Tykesson

, Larsson-Callerfelt

, Rydell-Tormanen

. Versican in inflammation and tissue remodeling: The impact on lung disorders. Glycobiology 2015; 25(3): 243–251.

16.

Wight

, Merrilees

. Proteoglycans in atherosclerosis and restenosis: Key roles for versican. Circ Res 2004; 94(9): 1158–1167.

17.

Ricciardelli

, Sakko

, Ween

, Russell

, Horsfall

. The biological role and regulation of versican levels in cancer. Cancer Metastasis Rev 2009;28(1–2): 233–245.

18.

Bogdani

, Johnson

, Potter-Perigo

, Nagy

, Day

, Bollyky

, Wight

. Hyaluronan and hyaluronan-binding proteins accumulate in both human type 1 diabetic islets and lymphoid tissues and associate with inflammatory cells in insulitis. Diabetes 2014; 63(8): 2727–2743.

19.

Wight

, Lara

, Riessen

, Le Baron

, Isner

. Selective deposits of versican in the extracellular matrix of restenotic lesions from human peripheral arteries. Am J Pathol 1997; 151(4): 963–973.

20.

Halpert

, Sires

, Roby

, Potter-Perigo

, Wight

, Shapiro

, Welgus

, Wickline

, Parks

. Matrilysin is expressed by lipidladen macrophages at sites of potential rupture in atherosclerotic lesions and localizes to areas of versican deposition, a proteoglycan substrate for the enzyme. Proc Natl Acad Sci U S A 1996; 93(18): 9748–9753.

21.

Huang

, Merrilees

, Braun

, Beaumont

, Lemire

, Clowes

, Hinek

, Wight

. Inhibition of versican synthesis by antisense alters smooth muscle cell phenotype and induces elastic fiber formation in vitro and in neointima after vessel injury. Circ Res 2006; 98(3): 370–377.

22.

Lemire

, Merrilees

, Braun

, Wight

. Overexpression of the V3 variant of versican alters arterial smooth muscle cell adhesion, migration, and proliferation in vitro. J Cell Physiol 2002; 190(1): 38–45.

23.

Kim

, Takahashi

, Lin

, Descargues

, Grivennikov

, Kim

, Luo

, Karin

. Carcinoma-produced factors activate myeloid cells through TLR2 to stimulate metastasis. Nature 2009; 457(7225): 102–106.

24.

Westergren-Thorsson

, Chakir

, Lafreniere-Allard

, Boulet

, Tremblay

. Correlation between airway responsiveness and proteoglycan production by bronchial fibroblasts from normal and asthmatic subjects. Int J Biochem Cell Biol 2002; 34(10): 1256–1267.

25.

Hallgren

, Nihlberg

, Dahlback

, Bjermer

, Eriksson

, Erjefalt

, Lofdahl

, Westergren-Thorsson

. Altered fibroblast proteoglycan production in COPD. Respir Res 2010;11: 55.

26.

Merrilees

, Ching

, Beaumont

, Hinek

, Wight

, Black

. Changes in elastin, elastin binding protein and versican in alveoli in chronic obstructive pulmonary disease. Respir Res 2008;9: 41.

27.

Rabinovitch

, Guignabert

, Humbert

, Nicolls

. Inflammation and immunity in the pathogenesis of pulmonary arterial hypertension. Circ Res 2014; 115(1): 165–175.

28.

Huertas

, Perros

, Tu

, Cohen-Kaminsky

, Montani

, Dorfmuller

, Guignabert

, Humbert

. Immune dysregulation and endothelial dysfunction in pulmonary arterial hypertension: A complex interplay. Circulation 2014; 129(12): 1332–1340.

29.

Ricard

, Tu

, Le Hiress

, Huertas

, Phan

, Thuillet

, Sattler

. 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(15): 1586–1597.

30.

Zhang

, Shen

, Xu

, Liu

, Luo

, Xu

, Wang

. Role of macrophage migration inhibitory factor in the proliferation of smooth muscle cell in pulmonary hypertension. Mediators Inflamm 2012;2012: 840737.

31.

Wight

, Kang

, Merrilees

. Versican and the control of inflammation. Matrix Biol 2014; 35: 152–161.

32.

Evanko

, Potter-Perigo

, Johnson

, Wight

. Organization of hyaluronan and versican in the extracellular matrix of human fibroblasts treated with the viral mimetic poly I:C. J Histochem Cytochem 2009; 57(11): 1041–1060.

33.

Evanko

, Potter-Perigo

, Bollyky

, Nepom

, Wight

. Hyaluronan and versican in the control of human T-lymphocyte adhesion and migration. Matrix Biol 2012; 31(2): 90–100.

34.

Aytekin

, Comhair

, de la Motte

, Bandyopadhyay

, Farver

, Hascall

, Erzurum

, Dweik

. High levels of hyaluronan in idiopathic pulmonary arterial hypertension. Am J Physiol Lung Cell Mol Physiol 2008; 295(5): L789–L799.

35.

Lauer

, Aytekin

, Comhair

, Loftis

, Tian

, Farver

, Hascall

, Dweik

. Modification of hyaluronan by heavy chains of inter-alpha-inhibitor in idiopathic pulmonary arterial hypertension. J Biol Chem 2014; 289(10): 6791–6798.

36.

Shukla

, Nair

, Rolle

, Braun

, Chan

, Johnson

, Wight

, McDevitt

. Synthesis and organization of hyaluronan and versican by embryonic stem cells undergoing embryoid body differentiation. J Histochem Cytochem 2010; 58(4): 345–358.

37.

Zeller

, Riaz

, Sarve

, Li

, Tengholm

, Johansson

. The role of mechanical force and ROS in integrin-dependent signals. PLoS One 2013; 8(5):e64897.

38.

Lee

, Yamamoto

, Feng

, Potter-Perigo

, Briggs

, Landschulz

, Turi

. Mechanical strain induces specific changes in the synthesis and organization of proteoglycans by vascular smooth muscle cells. J Biol Chem 2001; 276(17): 13847–13851.

39.

Hascall

, Calabro

, Midura

, Yanagishita

. Isolation and characterization of proteoglycans. Methods Enzymol 1994; 230: 390–417.

40.

Bonnet

, Michelakis

, Porter

, Andrade-Navarro

, Thebaud

, Bonnet

, Haromy

. An abnormal mitochondrial-hypoxia inducible factor-1alpha-Kv channel pathway disrupts oxygen sensing and triggers pulmonary arterial hypertension in fawn hooded rats: Similarities to human pulmonary arterial hypertension. Circulation 2006; 113(22): 2630–2641.

41.

Yao

, Moody

, Schonherr

, Wight

, Sandell

. Identification of the proteoglycan versican in aorta and smooth muscle cells by DNA sequence analysis, in situ hybridization and immunohistochemistry. Matrix Biol 1994; 14(3): 213–225.

42.

Wolinsky

. Long-term effects of hypertension on the rat aortic wall and their relation to concurrent aging changes: Morphological and chemical studies. Circ Res 1972; 30(3): 301–309.

43.

Hoefer

, den Adel

, Daemen

. Biomechanical factors as triggers of vascular growth. Cardiovasc Res 2013; 99(2): 276–283.

44.

Farha

, Asosingh

, Xu

, Sharp

, George

, Comhair

, Park

. Hypoxia-inducible factors in human pulmonary arterial hypertension: A link to the intrinsic myeloid abnormalities. Blood 2011; 117(13): 3485–3493.

45.

Asplund

, Stillemark-Billton

, Larsson

, Rydberg

, Moses

, Hulten

, Fagerberg

, Camejo

, Bondjers

. Hypoxic regulation of secreted proteoglycans in macrophages. Glycobiology 2010; 20(1): 33–40.

46.

Oktem

, Sercan

, Guven

, Uslu

, Uysal

, Goksel

, Ayla

, Bilir

. Cancer stem cell differentiation: TGFbeta1 and versican may trigger molecules for the organization of tumor spheroids. Oncol Rep 2014; 32(2): 641–649.

47.

Cardoso

, Little

, Ballinger

, Chan

, Braun

, Potter-Perigo

, Bornfeldt

, Kinsella

, Wight

. Platelet-derived growth factor differentially regulates the expression and post-translational modification of versican by arterial smooth muscle cells through distinct protein kinase C and extracellular signal-regulated kinase pathways. J Biol Chem 2010; 285(10): 6987–6995.

48.

Rahmani

, Read

, Carthy

, McDonald

, Wong

, Esfandiarei

, Si

. Regulation of the versican promoter by the beta-catenin-T-cell factor complex in vascular smooth muscle cells. J Biol Chem 2005; 280(13): 13019–13028.

49.

Machado

, Aldred

, James

, Harrison

, Patel

, Schwalbe

, Gruenig

. Mutations of the TGF-beta type II receptor BMPR2 in pulmonary arterial hypertension. Hum Mutat 2006; 27(2): 121–132.

50.

Barst

. PDGF signaling in pulmonary arterial hypertension. J Clin Invest 2005; 115(10): 2691–2694.

51.

de Jesus Perez

, Alastalo

, Wu

, Axelrod

, Cooke

, Amieva

, Rabinovitch

. Bone morphogenetic protein 2 induces pulmonary angiogenesis via Wnt-beta-catenin and Wnt-RhoA-Rac1 pathways. J Cell Biol 2009; 184(1): 83–99.

52.

Evanko

, Angello

, Wight

. Formation of hyaluronan and versican-rich pericellular matrix is required for proliferation and migration of vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 1999; 19(4): 1004–1013.

53.

Wight

. Arterial remodeling in vascular disease: A key role for hyaluronan and versican. Front Biosci 2008; 13: 4933–4937.

54.

de Jesus Perez

, Yuan

, Lyuksyutova

, Dewey

, Orcholski

, Shuffle

, Mathur

. Whole-exome sequencing reveals TopBP1 as a novel gene in idiopathic pulmonary arterial hypertension. Am J Respir Crit Care Med 2014; 189(10): 1260–1272.

55.

Cogan

, Austin

, Hedges

, Womack

, West

, Loyd

, Hamid

. Role of BMPR2 alternative splicing in heritable pulmonary arterial hypertension penetrance. Circulation 2012; 126(15): 1907–1916.