This Article Abstract Full Text (PDF) All Versions of this Article: 94/4/550    most recent 01.RES.0000117772.86853.34v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tran, P.-K. Articles by Hedin, U. Search for Related Content PubMed PubMed Citation Articles by Tran, P.-K. Articles by Hedin, U. Related Collections Coagulation Restenosis Mechanism of atherosclerosis/growth factors Genetically altered mice Smooth muscle proliferation and differentiation

(Circulation Research. 2004;94:550.)
© 2004 American Heart Association, Inc.

Integrative Physiology

Increased Intimal Hyperplasia and Smooth Muscle Cell Proliferation in Transgenic Mice With Heparan Sulfate–Deficient Perlecan

Phan-Kiet Tran, Karin Tran-Lundmark, Raija Soininen, Karl Tryggvason, Johan Thyberg^*, Ulf Hedin^*

From the Department of Surgical Sciences (P.-K.T., K.T.-L., U.H.), Karolinska Hospital, and Departments of Medical Biochemistry and Biophysics (R.S., K.T.) and Cell and Molecular Biology (J.T.), Karolinska Institutet, Stockholm, Sweden.

Correspondence to Phan-Kiet Tran, Department of Surgical Sciences, Karolinska Hospital, SE-17176 Stockholm, Sweden. E-mail kiet.tran{at}kirurgi.ki.se

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Smooth muscle cell (SMC) proliferation is a critical process in vascular disease. Heparan sulfate (HS) proteoglycans inhibit SMC growth, but the role of endogenous counterparts in the vessel wall in control of SMC function is not known in detail. Perlecan is the major HS proteoglycans in SMC basement membranes and in vessel wall extracellular matrix (ECM). In this study, transgenic mice with HS-deficient perlecan were analyzed with respect to vascular phenotype and intimal lesion formation. Furthermore, SMC cultures were established and characterized with respect to morphology, immunocytochemical features, proteoglycan synthesis, proliferative capacity, and ECM binding of basic fibroblast growth factor (FGF-2). In vitro, mutant SMCs formed basement membranes with perlecan core protein, but with decreased levels of HS, they showed diminished secretion of HS-containing perlecan into the medium and a defective ECM-binding capacity of FGF-2. In vitro, mutant SMCs showed increased proliferation compared with wild-type cells, and in vivo, enhanced SMC proliferation and intimal hyperplasia were observed after flow cessation of the carotid artery in mutant mice. The results indicate that the endogenous HS side-chains of perlecan contribute to SMC growth control both in vitro and during intimal hyperplasia, possibly by sequestering heparin-binding mitogens such as FGF-2.

Key Words: smooth muscle cells • perlecan • heparan sulfate proteoglycans • intimal hyperplasia • cell proliferation

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Smooth muscle cell (SMC) proliferation contributes to development of atherosclerotic and restenotic lesions. In normal vessels, SMCs are surrounded by basement membranes composed of laminin (LN), type IV collagen, and heparan sulfate proteoglycans (HSPGs) such as perlecan.^1,2 Basement membrane components retain differentiated properties and prevent proliferation of SMCs in vitro.^3,4 SMC activation after arterial injury is coupled to disappearance of LN and basement membrane structures, whereas fibronectin (FN) accumulates around proliferating cells in the arterial media and intima.⁵ These observations indicate that basement membrane components take part in the regulation of the differentiated and quiescent state of SMCs.⁶

Perlecan is the predominant HSPG in basement membranes and vessels.^7,8 It has a core protein with five globular domains, and HS glycosaminoglycan (GAG) side-chains are attached to exon-3 in domain-I, resulting in a molecule of 800 kDa.^7,9 Heterogeneity with respect to GAG side-chains has been described, and additional attachment sites are found in domain V.¹⁰

Perlecan is important for the structural integrity of basement membranes but also influences cellular function and has been attributed a critical role in vascular disease.^11,12 It may both suppress and promote cell proliferation.^13,14 Heparin and HS inhibit SMC migration and proliferation in intimal hyperplasia after arterial injury by binding to and modulating the activity of growth factors, such as fibroblast growth factor (FGF)-2.^15–18 In vivo, purified arterial HSPGs inhibit intimal lesion formation after arterial injury, and antisense-mediated suppression of perlecan production in endothelial cells abolished inhibition of intimal hyperplasia.^18,19 It was recently shown that deposition of perlecan in the neointima coincides with reduced SMC proliferation late after balloon injury, whereas heparinase treatment of explanted neointima promoted SMC growth.²⁰ Perlecan may therefore be essential in control of SMC function, but the specific role for perlecan and its HS side-chains in vascular disease has not been determined.¹¹ In this study, transgenic mice with targeted deletion of the major HS attachment sites in the perlecan core protein (Hspg2^3/3)²¹ were used to study SMC proliferation in vitro and in vivo in intimal hyperplasia after flow cessation of the carotid artery.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Ham’s medium F-12, fetal calf serum (FCS), and trypsin were purchased from Life Technologies; collagenase type II from Worthington; cell culture and ELISA plastic ware from Corning Incorporated; gel-electrophoresis reagents equipment and mini-columns from Bio-Rad Laboratories; ³⁵S-sulfate, ³H-thymidine, ¹²⁵I-FGF-2, nitrocellulose membrane (Hybond), ECL kit, and X-Ray film (Hyperfilm) from Amersham Biosciences; autoradiography reagents from Kodak MSIG; enhancer from New England Biolabs; FGF-2 from Boehringer; and heparin-binding epidermal growth factor (HB-EGF) from R&D Systems. Rat monoclonal antibody against mouse perlecan core protein (HK-102) and against HS (Hep-ss1) were purchased from Seikagaku Corporation (Tokyo, Japan); rat monoclonal antibody against mouse Ki67, HRP-conjugated mouse anti-SMC -actin, and rhodamine-labeled donkey anti-rat IgG from DakoCytomation (Glostrup, Denmark); biotinylated rabbit anti-rat IgG (H+L), ABC Elite immunodetection kit, diaminobenzidine (DAB), Nova Red, and Vectashield from Vector Laboratories (Burlingame, Calif); and chondroitinase ABC lyase (EC No 4.2.2.4) from ICN Biomedicals. All other reagents, including heparinase I (CAS 9025-39-2) and II (CAS 149371-12-0) and diethylaminoethyl (DEAE) sephacel, were from Sigma.

Transgenic Mice
Mice lacking exon 3 of the perlecan (Hspg2) gene, thus lacking attachment sites for 3 HS side chains, were generated by gene targeting without altering the expression or the reading frame, previously described by Rossi et al.²¹ Hspg2^3/3 were back-bred nine generations to the C57BL/6 background. C57BL/6 mice were purchased from Scanbur-BK, Sweden. Wild-type and homozygous mutant offspring from N9/F2 (second-generation homozygous breeding between littermates) animals were studied. All experiments were approved by the local animal ethics committee.

Light and Electron Microscopy
Animals were euthanized by CO₂ asphyxiation. The heart, aorta, and its main branches were rinsed with PBS and perfusion-fixed with 4% formaldehyde in PBS or 3% glutaraldehyde in 0.1 mol/L sodium cacodylate-HCl buffer (pH 7.3) and 0.05 mol/L sucrose. For light microscopy, the specimens were dehydrated in ethanol (70% to 100%), cleared in xylene, and embedded in paraffin. Sections of 5 µm were cut on a standard microtome, stained with H&E or Masson’s trichrome, and studied in a Nikon 800 Eclipse microscope (Nikon). For electron microscopy, the specimens were postfixed in 1.5% cacodylate-buffered osmium tetroxide containing 0.7% potassium ferrocyanate, dehydrated in ethanol, stained with 2% uranyl acetate, and embedded in epoxy resin. Sections were cut on a Leica Ultracut, stained, and examined in a Philips CM120 EM.

SMC Culture
The thoracic aorta was separated in situ from its adventitia by microdissection and digested for 8 to 10 hours in 0.1% collagenase in F-12 with 50 µg/mL L-ascorbic acid, 50 µg/mL streptomycin, 50 IU/mL penicillin, and 0.1% BSA (F-12/0.1% BSA). The cell suspension was filtered, centrifuged, washed in F-12/0.1% BSA, seeded in F-12/20% FCS for primary culture, and fixed for electron microscopy or passaged by trypsinization. Passages 2 through 5 were used for experiments. To confirm stable euploidy, SMCs from passage 6 were analyzed for DNA content by flow cytometry.

Immunocytochemistry and Immunohistochemistry
SMCs were fixed for 60 minutes in PBS/2% formaldehyde, incubated for 15 minutes in 50 mmol/L ammonium chloride, and permeabilized for 3 minutes in PBS/0.2% Triton X-100. Nonspecific binding was blocked with PBS/0.2% BSA. Hidden epitopes were unmasked for 30 minutes in PBS/hyaluronidase (15 000 U/mL) at 37°C, followed by 3 hours of incubation at 20°C with anti-perlecan (1:100) in PBS/0.1% BSA, washing in PBS, and incubation for 1 hour with rhodamine-labeled donkey anti-rat IgG in PBS/0.2% BSA at 20°C; coverslips were mounted in Vectashield. Paraffin-embedded sections were dewaxed and rehydrated, peroxidase activity was quenched with 0.3% hydrogen peroxide in 70% methanol, and the specimens were boiled for 10 minutes in citrate buffer (pH 6.0) or treated with hyaluronidase to facilitate detection of perlecan core protein. After overnight incubation with anti-perlecan, anti-HS, anti--actin, or anti-Ki67 in TBS/0.1% BSA (1:100), slides were washed with TBS and incubated with secondary biotinylated rabbit anti-rat IgG (1:50 in TBS/2% rabbit serum), followed by ABC immunodetection using DAB or Nova Red (for HS staining) as substrate, and counterstained with hematoxylin.

Metabolic Labeling and Proteoglycan Extraction
SMCs were grown to subconfluence, synchronized for 24 hours in F-12/0.5% FCS, and labeled for 48 hours with 50 µCi/mL ³⁵S-sulfate in F-12/0.5% FCS. Medium was collected, cell layer–rinsed with PBS, solubilized in 8 mol/L urea, 50 mmol/L Tris-HCl (pH 7.5), 0.5% Triton X-100, 2 mmol/L EDTA, and 0.25 mol/L NaCl (8 mol/L urea/0.25 mol/L NaCl) for 20 minutes, and scraped off the dishes, and protease and phosphatase inhibitors were added (5 mmol/L benzamidine-HCl, 1 mmol/L phenylmethylsulfonylfluoride, and 100 mmol/L 6-aminocaproic acid). PGs in the extracts were concentrated by anion chromatography on DEAE-sephacel in 8 mol/L urea/0.25 mol/L NaCl and eluted with 8 mol/L urea/3 mol/L NaCl.²²

SDS-PAGE and Western Blotting
Equal amounts (75 000 cpm) of PG concentrates were precipitated in 70% ethanol and 1.0% potassium acetate for 2 hours at -72°C. The pellets were digested with 0.08 U chondroitinase ABC lyase (chondroitinase) in 30 µL of 330 mmol/L Tris-HCl (pH 8.0), 17 mmol/L sodium acetate, and 0.1% BSA or 0.8 U each of heparinase I and heparinase II (heparinases) with or without 0.5 U chondroitinase in 30 µL of 33 mmol/L Tris-HCl (pH 7.0), 3 mmol/L calcium acetate, and 6 mmol/L sodium acetate (heparinase buffer) for 2 hours at 37°C and 1 hour at 42°C. SDS-PAGE was performed on 50 000 cpm of digested materials on a 4–12% gradient gel with a 3.5% stacking gel. The whole gel was fixed, treated for 45 minutes with enhancer, rinsed, dried onto Whatman filter paper, and exposed for 7 days to Kodak film. For Western blotting, digested samples were applied, under nonreducing conditions, on a 5% SDS-PAGE gel (Bio-Rad Mini Protean-II) and transferred to nitrocellulose membranes. The membrane was blocked for 3 hours with 2% BSA in 50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, and 0.5% Tween (TTBS) and incubated overnight at 4°C with anti-perlecan (1:2500 in TTBS/1.0% goat serum/0.15% BSA). The membrane was washed in TTBS, incubated with HRP-conjugated goat anti-rat IgG (1:3000 in TTBS/2% BSA) for 2 hours at 20°C, and washed, and HRP activity was detected by ECL.²²

ELISA
SMCs were plated on 96-well ELISA plates at increasing concentrations (2500 to 25 000 cells/cm²) and grown for 4 days in F-12/20% FCS. The cultures were rinsed with PBS and fixed for 20 minutes in 4% formaldehyde. After blocking for 2 hours with 2% BSA in PBS and hyaluronidase treatment, wells were incubated with anti-perlecan or anti-HS, followed by biotinylated secondary antibody as described above. For colorimetric detection, each well was incubated for 20 minutes with 200 µL of 4 mg/mL O-phenylenediamine in 0.103 mol/L monosodium phosphate and 0.0485 mol/L citric acid (pH 5.0; McIlvaines buffer).²³ The reaction was arrested with 50 µL of 4 mol/L sulfuric acid. Total protein was quantified by Pierce’s BCA method (Pierce). Perlecan and HS were quantified at 490 nm, and total protein was quantified at 562 nm in SpectraMax 340 ELISA plate reader (GTF AB).

DNA Synthesis
SMCs grown in F-12/20% FCS were synchronized for 48 hours in F-12/1% FCS and then incubated for 24 hours with 10% FCS, platelet-derived growth factor (PDGF)-AA (20 ng/mL), PDGF-BB (20 ng/mL), FGF-2 (20 ng/mL), or HB-EGF (20 ng/mL) with 1 µCi/mL ³H-thymidine. The cells were fixed in 3% glutaraldehyde, dehydrated in ethanol, air dried, exposed for 2 days to Kodak NTB2 emulsion (Eastman Kodak Co) at 4°C, developed in Kodak D-19, fixed, and stained with 1% methylene blue. Labeling index was determined by counting 500 cells in each specimen.

Flow-Cessation Model by Ligation of Distal Carotid Artery
Mice were anesthetized with 3% isoflurane, and left common carotid artery was exposed by an anterior midline neck incision and ligated with 6-0 silk proximal to the carotid bifurcation.²⁴ One (n=5) and 6 (n=12) weeks after surgery, the animals were euthanized, the vessels were fixed and embedded in paraffin, and 5-µm serial sections were obtained from the ligature in a proximal direction. A total length of 1.2 and 2.0 mm from the knot was examined for the 1- and 6-week experiments, respectively.

Morphological Analysis
All samples were blinded by randomly assigning each animal a letter code and the sections a serial number indicating distance from the ligature. For each animal, 12 sections at equal distance proximal to the ligature were analyzed within a 400-µm region to avoid quantification of regions with massive lesions or no lesions at all.²⁴ This region lay between 800- to 1200-µm and 400- to 800-µm proximal to the ligature in the 1- and 6-week groups, respectively. The internal elastic lamina (IEL), external elastic lamina (EEL), and lumen were traced in digitized images. Luminal area (L; remaining open lumen), neointimal area (I; area inside IEL minus L), and medial area (M; area between IEL and EEL) were calculated and expressed in square micrometers. Variations between repeated tracings of the same section were <1% for length and area determinations.

Analysis of SMC Proliferation In Vivo
One-week-old lesions were analyzed for proliferative activity. Twelve randomly selected sections from four animals per group were stained for Ki67. All nuclei in the neointima were counted, and replication index was determined by dividing the number of Ki67-positive nuclei by the total number of nuclei.

ECM Preparation and ¹²⁵I-FGF-2 Binding Assay
Twenty-four-well plates were coated overnight with 0.1 mg/mL type I collagen in 0.45 mol/L acetic acid and rinsed with PBS, and SMCs were seeded in F-12/20% FCS (10 000 cells/cm²) and grown for 5 days. Wells without cells were used as background control. Cell-free ECM was prepared by treatment with 0.5% Triton X-100 in PBS and 25 mmol/L ammonium hydroxide in PBS, as previously described.²⁵ Total protein in the ECM was quantified as described above and was equivalent between Hspg2^3/3 and wild-type. The ECM was blocked for 2 hours in F-12/4% BSA and treated with heparinase buffer or digested with chondroitinase or heparinases, as described above, and then incubated for 1 hour with 50 nmol/L ¹²⁵I-FGF-2 in F-12/4% BSA. After repeated washes with PBS, ¹²⁵I-FGF-2 bound to the ECM was extracted with heparinases and counted in Wizard 1470 counter (Wallac Sverige AB).

Statistical Analysis
Differences between the experimental groups analyzed in vitro and in vivo were tested by unpaired Student’s t test and ANOVA, respectively. Data are expressed as mean with SEM or SD, as indicated. P<0.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Vascular Phenotype of Hspg2^3/3 Mice and SMCs
Mutant mice showed no birth defects, were bred normally, and developed without abnormalities except for congenital cataracts.²¹ To study vascular development, Hspg2^3/3 and wild-type mice were kept on chow diet, and the heart and aorta, including main branches, were analyzed by light and electron microscopy at different times up to 1 year. No differences were noted between wild-type and Hspg2^3/3 animals in vessel development and endothelial cells, and SMCs exhibited normal ultrastructural features with a basement membrane adjacent to the cells (not shown).

SMCs isolated from wild-type and Hspg2^3/3 mice aorta were seeded in F12/20% FCS and processed for light and electron microscopy after 3 and 5 days of primary culture. No differences with respect to adhesion efficiency and cell morphology were observed. Ultrastructurally, both cell types showed a typical transition from contractile into synthetic phenotype during the first week of culture.²⁶ Cells from the sixth passage were analyzed by FACS and showed no aneuploidy (not shown).

Reduced HSPG Production by Hspg2^3/3 SMCs
Wild-type and Hspg2^3/3 SMCs were labeled in vitro with ³⁵S-sulfate, and PG production was analyzed by concentrating medium and cell-layer material over DEAE columns followed by digestion with chondroitinase or heparinases and SDS-PAGE. This protocol yields PGs with attached GAG side-chains only, and no nonglycosylated material (HS-deficient perlecan) is recovered. After chondroitinase digestion of medium samples from wild-type SMCs, large HSPG, presumably perlecan, was identified as a smear in the stacking gel and an intense band at the top of the resolving gel (Figure 1A).²² The identity of perlecan was confirmed by immunoblotting (Figure 1B). Large HS-perlecan was not detectable in medium samples from Hspg2^3/3 SMCs (Figure 1A). The smear at the top of the resolving gel with diminishing intensity toward 213 kDa, possibly representing small cell-surface–bound HSPGs (syndecans, glypicans, or perlecan with HS chains attached to domain V), was similar in the medium from wild-type and Hspg2^3/3 (Figure 1A).^10,27 Treatment with both chondroitinase and heparinase yielded complete digestion of the samples, confirming that the analyzed material indeed represented the HS (Figure 1A). Analysis of material from the cell layer revealed nearly identical results (not shown). The results show that the Hspg2^3/3 mutation results in a depletion of large HS-perlecan and a significant reduction in the overall secretion of HS by SMCs.

View larger version (31K): [in this window] [in a new window]   Figure 1. Identification and analysis of large HS perlecan in the medium of wild-type (wt) and Hspg23/3 (3) SMCs in vitro. A, Gradient gel electrophoresis and autoradiography of 35S-sulfate–labeled and DEAE-purified PGs after digestion with chondroitinase and heparinases I and II. B, Immunoblotting with monoclonal antibody against perlecan core protein (HK-102) after digestion with heparinase I and II with and without chondroitinase.

Because nonglycosylated proteins are lost with DEAE purification, the production and deposition of perlecan core protein in wild-type and Hspg2^3/3 SMC cultures was analyzed by immunocytochemistry and a modified ELISA. A slight increase in perlecan staining was observed in Hspg2^3/3 SMC cultures compared with wild-type cells (Figures 2A and 2B). In support of this observation, 20% more perlecan was detected in the cell layer of Hspg2^3/3 SMCs by ELISA compared with wild-type cells (Figure 2C). In addition, ELISA showed a significant reduction of HS in Hspg2^3/3 cultures (Figure 2D), confirming the SDS-PAGE analysis of ³⁵S-labeled material.

View larger version (73K): [in this window] [in a new window]   Figure 2. Immunocytochemical demonstration (A and B) and quantification of perlecan core protein (C) and HS (D) by a modified ELISA in the cell layer of cultured SMCs. Wild-type () and Hspg23/3 (). Representative results from 3 independent experiments in duplicates are presented as mean±SD.

Reduced Production of Large HS-Perlecan Is Associated With Increased Proliferation of SMCs In Vitro
Because heparin and HS-containing molecules have been shown to inhibit SMC proliferation, the growth potential of Hspg2^3/3 was compared with wild-type SMCs. The lag phase to DNA synthesis after serum stimulation was found to be 24 hours in both cell types (Figure 3A). Hspg2^3/3 SMCs responded with increased DNA synthesis compared with wild-type cells after stimulation with 10% FCS, PDGF-AA, PDGF-BB, FGF-2, EGF, and HB-EGF (Figure 3B).

View larger version (21K): [in this window] [in a new window]   Figure 3. Determination of lag phase to DNA synthesis in wild-type () and Hspg23/3 () SMCs after stimulation with 10% FCS (A). DNA synthesis in wild-type (white bars) and Hspg23/3 (black bars) SMCs after stimulation with 10% FCS, PDGF-AA (20 ng/mL), PDGF-BB (20 ng/mL), FGF-2 (20 ng/mL), or HB-EGF (20 ng/mL). Cells were stimulated with mitogen in the presence of 3H-thymidine at increasing time intervals (A) or 24 hours (B). DNA synthesis was analyzed by autoradiography. Five hundred cells in each specimen (n=5) were counted, and results are expressed as mean±SD (**P<0.01).

Increased Intima Formation in Hspg2^3/3 Mice After Carotid Artery Flow Cessation
In vivo SMC growth was analyzed using a carotid artery ligation model earlier shown to generate reproducible intimal lesions attributable to SMC proliferation.²⁴ The carotid artery was ligated and prepared for morphological analysis after 1 and 6 weeks. Vessels in Hspg2^3/3 mice developed an increase in intimal area and ratio of intima to media area compared with wild-type controls already at 1 week after surgery. At 6 weeks, these differences were even more pronounced (Figures 4B and 4D). No differences were found in residual lumen or medial area (Figures 4A and 4C). Examination of sections collected 12 hours after flow cessation did not reveal any thrombus formation in either Hspg2^3/3 or wild-type mice (not shown).

View larger version (22K): [in this window] [in a new window]   Figure 4. Determination of residual lumen area (A), intimal area (B), medial area (C), and intima/media area ratio (D) after flow cessation for 1 (n=5) and 6 (n=12) weeks in the carotid artery of wild-type (white bars) and Hspg23/3 (black bars) mice. Results are expressed as mean±SEM (*P<0.05).

Intimal Lesions in Hspg2^3/3 Mice Contain Perlecan Core Protein and Proliferating SMCs
To determine the mechanisms behind the enhanced intimal hyperplasia in mutant mice, lesions were also analyzed by electron microscopy and immunocytochemistry. The ultrastructure of intimal lesions from wild-type and Hspg2^3/3 animals was equivalent. After 1 week, the lesions were a few cell layers thick and consisted of leukocytes (granulocytes, monocytes, and lymphocytes) and polygonal cells with abundant synthetic organelles (sometimes observed in mitosis), dispersed in a scanty and loosely arranged ECM. After 6 weeks, the lesions were larger and built up by synthetic cells with a large endoplasmic reticulum (ER) and Golgi complex and differentiated SMCs surrounded by a dense ECM rich in collagen and elastic fibers (Figures 5A through 5C).

View larger version (152K): [in this window] [in a new window]   Figure 5. Electron micrographs of intimal lesions 6 weeks after flow cessation in Hspg23/3 mice. A, Overview showing cells with a differentiated SMC appearance (black arrowhead) at the bottom, covered by a few layers of synthetic SMCs (white arrowheads) and endothelial-like cells (EC) at the top. B, Detail of a synthetic SMC with a large ER and Golgi complex (GC). C, Detail of a differentiated SMC with a cytoplasm filled by myofilaments (F). E indicates elastic fibers; L, vessel lumen; N, nuclei. Bars=1 µm.

Immunohistochemical analysis of 6-week-old lesions showed a similar pattern of deposition of perlecan core protein in Hspg2^3/3 as in wild-type mice, with more staining in the neointima than in the media (Figures 6A and 6B). With the exception of the endothelial cell layer, possibly representing cell surface–bound syndecans and glypicans, HS staining was absent in the vessel wall of Hspg2^3/3 animals, whereas a diffuse staining was seen in the wild-type (Figures 6C and 6D). SMC -actin was expressed by almost all cells in the media and in the intima both in Hspg2^3/3 and wild-type animals (Figures 6E and 6F).

View larger version (128K): [in this window] [in a new window]   Figure 6. Immunohistochemical demonstration of perlecan (A and B), HS (C and D), and SMC -actin (E and F) in the carotid artery of wild-type (A, C, and E) and Hspg23/3 (B, D, and F) mice 6 weeks after flow cessation. The IEL and border between the intima and media are indicated with arrowheads.

Proliferative activity in the lesions was quantified at 1 week after ligation by staining for Ki67. Only nuclei in the intima were counted, because hardly any nuclei in the media stained positively for Ki67 at this time point. The replication index was significantly higher in Hspg2^3/3 mice compared with wild-type controls (Figures 7A through 7C).

View larger version (66K): [in this window] [in a new window]   Figure 7. Immunohistochemical demonstration of Ki-67 staining (A and B) and quantification (C) of cell replication in the intima of the carotid artery in wild-type (n=4) and Hspg23/3 (n=4) mice 1 week after flow cessation (white arrowheads, IEL; black arrowheads, positive nuclei). C, Results are expressed as mean of percent positive nuclei with SEM (*P<0.05).

Reduced Binding of FGF-2 to the ECM of Hspg2^3/3 SMCs
FGF-2 has been established as a potent mitogen for SMC proliferation in intimal hyperplasia.¹⁶ FGF-2 may bind to HS chains in the ECM, which then serves as a reservoir for mitogens.²⁸ In this study, binding of ¹²⁵I-FGF-2 to cell-free ECM prepared from Hspg2^3/3 and wild-type SMC cultures was analyzed. Hspg2^3/3 ECM was found to bind 75% less ¹²⁵I-FGF-2 than wild-type. Pretreatment with chondroitinase did not change the binding capacity of ¹²⁵I-FGF-2 to either ECM, whereas pretreatment with heparinases reduced binding down to background level (Figure 8)

View larger version (23K): [in this window] [in a new window]   Figure 8. 125I-FGF-2 binding to cell-free ECM from wild-type (white bars) and Hspg23/3 (black bars) SMCs and type I collagen (control; gray bars). The ECM was pretreated with buffer, chondroitinase, and heparinase I and II (heparinase), as indicated, and bound 125I-FGF-2 was released with heparinase I and II and measured as described in Materials and Methods. Representative results from 3 independent experiments in quadruplicates are presented as mean±SD (**P<0.01).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
SMC proliferation can be inhibited by heparin or HSPGs and promoted after removal of HS with heparinases both in vitro and in animal models.^15,18 These observations suggest that endogenous perlecan may regulate SMC function in the vessel wall, possibly by modulating the accessibility or activity of heparin-binding mitogens such as FGF-2.^{16,18,20,28,29} In this study, the role of large HS-perlecan in the control of SMC proliferation was studied in Hspg2^3/3 mice lacking attachment sites for HS side-chains in domain-I of the perlecan core protein.²¹ In vitro, Hspg2^3/3 SMCs deposited an ECM depleted of HS with a defective FGF-2 binding ability but with normal amounts of perlecan core protein. Moreover, they showed higher proliferative capacity than wild-type cells. In vivo, Hspg2^3/3 mice displayed normal cardiovascular development but enlarged intimal lesions and increased SMC proliferation after flow cessation in the carotid artery.

Homozygous-null mutation in the perlecan gene results in an embryonically lethal phenotype attributable to cardiovascular malformations and ruptures of the heart and the large vessels.³⁰ Transgenic mice lacking other basement membrane components, such as the laminin ₁ chain or dystroglycan, develop abnormal basement membranes and are also embryonically lethal. The same is true for mice lacking N-deacetylase/N-sulfotransferase-1, an enzyme engaged in HS biosynthesis.^12,31–33 However, Hspg2^3/3 mice displayed normal vascular development, indicating that the HS chains of domain I in perlecan are not required in this process. Because the HS chains of perlecan have been shown to interact with laminin and support the structural integrity of basement membranes, it is possible that GAG chains attached to other domains of the molecule are sufficient for basement membrane assembly.⁸

Analysis of PG production in SMCs isolated from Hspg2^3/3 mice demonstrated depletion of large HS perlecan, and reduced levels of HS were found in the cell layer of Hspg2^3/3 SMCs by ELISA, indicating that perlecan is the predominant large HSPG produced by mouse SMCs. As expected, deletion of exon-3 did not interfere with secretion of perlecan core protein, and mutant cells deposited more core protein in the cell layer, as determined by ELISA and immunocytochemistry. Possibly the lack of HS chains in domain I promotes perlecan retention at the cell surface through integrin receptor binding.³⁴

Hspg2^3/3 and wild-type SMCs showed a similar ultrastructure and passed through the transition from a myofilament-rich, contractile phenotype into an ER/Golgi-rich, synthetic phenotype at identical rates. Cell adhesion, focal adhesion formation, and cell spreading were not affected in mutant cells, even though HS has been suggested to be necessary for focal adhesion formation. Most likely, this property is associated with cell-surface HSPGs such as syndecans, whereas the HS chains of perlecan rather prevent SMC adhesion.^23,35 Reduced secretion of large HS-perlecan by Hspg2^3/3 SMCs was associated with increased proliferation. Perlecan has previously been demonstrated to prevent SMC replication in vitro, and induction of perlecan synthesis by apolipoprotein E was coupled to SMC growth inhibition.^36–39 The antiproliferative properties of perlecan probably reside in the HS chains, because heparinase abolishes the ability of endothelial cell–conditioned medium to inhibit SMC proliferation and because heparin as well as purified HSPGs prevents SMC proliferation.^15,18,40 In contrast to cell-surface HSPGs, which potentiate the mitogenic activity of several growth factors, the HS chains of perlecan may sequester growth factors and prevent receptor binding.^{27,38,39,41,42} In support of this notion, ECM prepared from Hspg2^3/3 SMCs had a defective capacity to bind FGF-2, whereas a heparinase-sensitive binding to wild-type SMC ECM was observed. In the rat carotid balloon injury model, FGF-2 has been shown to be critical for early SMC proliferation.¹⁶ In addition, increased deposition of perlecan has recently been observed in the mature neointima, where SMC proliferation is insensitive to FGF-2 but enhanced after heparinase treatment.^20,29 These observations suggest that HS-perlecan may regulate FGF-2 activity and SMC proliferation in the intima by sequestering the mitogen. Because we observed increased proliferation of Hspg2^3/3 SMCs stimulated with non–heparin-binding mitogens, it cannot be excluded that these cells also have a defective capacity to sequester endogenously released mitogens such as HB-EGF in the ECM, which may favor a direct interaction with cell-surface receptors and promote cell proliferation.⁴³

Intimal lesions were more prominent in Hspg2^3/3 mice after flow cessation in the carotid artery. Previously, lesion formation in this model has been attributed to an increase in cell number.²⁴ Accordingly, electron microscopy revealed that the early lesions were made up of leukocytes and ER/Golgi-rich cells, whereas mature lesions primarily contained a mixture of synthetic and differentiated SMCs. The intima was found to contain perlecan core protein without HS staining together with SMC -actin–positive and Ki-67–positive cells. In addition, the proliferation index was increased in the intima of Hspg2^3/3 mice, suggesting that lesion formation indeed was dependent on SMCs proliferating in an environment lacking large HS-perlecan. In support of our findings, arterial HSPGs were previously found to reduce intimal hyperplasia in rabbits, and endothelial cells transfected with antisense vector against domain III of perlecan were observed to have a reduced capacity to limit intimal hyperplasia when implanted adjacent to an injured porcine artery.^18,19 Under these conditions, a reduced ability to prevent thrombus formation was also reported. In contrast, we did not observe any increased thrombosis in mutant mice. In support of our findings, hemostasis was recently shown to be preserved in transgenic mice lacking anticoagulant HS.⁴⁴

In summary, the results indicate that the HS side-chains of perlecan contribute to SMC growth control both in vitro and in vivo, possibly by sequestering heparin-binding mitogens such as FGF-2.

	Acknowledgments

 
Financial support was obtained from the Swedish Research Council (grants 06537 and 12233), the Swedish Heart-Lung Foundation, the King Gustaf V 80th Birthday Fund, the King Gustaf V and Queen Victoria’s Fund, and Karolinska Institutet, the Swedish Institute, and the Sweden-America Foundation.

	Footnotes

 
*Both authors contributed equally to this study.

Original received June 6, 2003; resubmission received December 8, 2003; accepted January 9, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Timpl R. Macromolecular organization of basement membranes. Curr Opin Cell Biol. 1996; 8: 618–624.[CrossRef][Medline] [Order article via Infotrieve]
2. Lusis AJ. Atherosclerosis. Nature. 2000; 407: 233–241.[CrossRef][Medline] [Order article via Infotrieve]
3. Li X, Tsai P, Wieder ED, Kribben A, Van Putten V, Schrier RW, Nemenoff RA. Vascular smooth muscle cells grown on Matrigel: a model of the contractile phenotype with decreased activation of mitogen-activated protein kinase. J Biol Chem. 1994; 269: 19653–19658.[Abstract/Free Full Text]
4. Hedin U, Bottger BA, Forsberg E, Johansson S, Thyberg J. Diverse effects of fibronectin and laminin on phenotypic properties of cultured arterial smooth muscle cells. J Cell Biol. 1988; 107: 307–319.[Abstract/Free Full Text]
5. Thyberg J, Blomgren K, Roy J, Tran PK, Hedin U. Phenotypic modulation of smooth muscle cells after arterial injury is associated with changes in the distribution of laminin and fibronectin. J Histochem Cytochem. 1997; 45: 837–846.[Abstract/Free Full Text]
6. Hedin U, Roy J, Tran PK, Lundmark K, Rahman A. Control of smooth muscle cell proliferation: the role of the basement membrane. Thromb Haemost. 1999; 82 (suppl 1): 23–26.[Medline] [Order article via Infotrieve]
7. Iozzo RV, Cohen IR, Grassel S, Murdoch AD. The biology of perlecan: the multifaceted heparan sulphate proteoglycan of basement membranes and pericellular matrices. Biochem J. 1994; 302(pt 3): 625–639.[Medline] [Order article via Infotrieve]
8. Timpl R, Brown JC. Supramolecular assembly of basement membranes. Bioessays. 1996; 18: 123–132.[CrossRef][Medline] [Order article via Infotrieve]
9. Dolan M, Horchar T, Rigatti B, Hassell JR. Identification of sites in domain I of perlecan that regulate heparan sulfate synthesis. J Biol Chem. 1997; 272: 4316–4322.[Abstract/Free Full Text]
10. Tapanadechopone P, Hassell JR, Rigatti B, Couchman JR. Localization of glycosaminoglycan substitution sites on domain V of mouse perlecan. Biochem Biophys Res Commun. 1999; 265: 680–690.[CrossRef][Medline] [Order article via Infotrieve]
11. Pillarisetti S. Lipoprotein modulation of subendothelial heparan sulfate proteoglycans (perlecan) and atherogenicity. Trends Cardiovasc Med. 2000; 10: 60–65.[CrossRef][Medline] [Order article via Infotrieve]
12. Forsberg E, Kjellen L. Heparan sulfate: lessons from knockout mice. J Clin Invest. 2001; 108: 175–180.[CrossRef][Medline] [Order article via Infotrieve]
13. Klein G, Conzelmann S, Beck S, Timpl R, Muller CA. Perlecan in human bone marrow: a growth-factor-presenting, but anti-adhesive, extracellular matrix component for hematopoietic cells. Matrix Biol. 1995; 14: 457–465.[CrossRef][Medline] [Order article via Infotrieve]
14. Mathiak M, Yenisey C, Grant DS, Sharma B, Iozzo RV. A role for perlecan in the suppression of growth and invasion in fibrosarcoma cells. Cancer Res. 1997; 57: 2130–2136.[Abstract/Free Full Text]
15. Clowes AW, Karnowsky MJ. Suppression by heparin of smooth muscle cell proliferation in injured arteries. Nature. 1977; 265: 625–626.[CrossRef][Medline] [Order article via Infotrieve]
16. Lindner V, Olson NE, Clowes AW, Reidy MA. Inhibition of smooth muscle cell proliferation in injured rat arteries: interaction of heparin with basic fibroblast growth factor. J Clin Invest. 1992; 90: 2044–2049.[Medline] [Order article via Infotrieve]
17. Koyama N, Kinsella MG, Wight TN, Hedin U, Clowes AW. Heparan sulfate proteoglycans mediate a potent inhibitory signal for migration of vascular smooth muscle cells. Circ Res. 1998; 83: 305–313.[Abstract/Free Full Text]
18. Bingley JA, Hayward IP, Campbell JH, Campbell GR. Arterial heparan sulfate proteoglycans inhibit vascular smooth muscle cell proliferation and phenotype change in vitro and neointimal formation in vivo. J Vasc Surg. 1998; 28: 308–318.[CrossRef][Medline] [Order article via Infotrieve]
19. Nugent MA, Nugent HM, Iozzo RV, Sanchack K, Edelman ER. Perlecan is required to inhibit thrombosis after deep vascular injury and contributes to endothelial cell-mediated inhibition of intimal hyperplasia. Proc Natl Acad Sci U S A. 2000; 97: 6722–6727.[Abstract/Free Full Text]
20. 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.[Abstract/Free Full Text]
21. 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.[CrossRef][Medline] [Order article via Infotrieve]
22. Castillo GM, Cummings JA, Ngo C, Yang W, Snow AD. Novel purification and detailed characterization of perlecan isolated from the Engelbreth-Holm-Swarm tumor for use in an animal model of fibrillar A ß amyloid persistence in brain. J Biochem (Tokyo). 1996; 120: 433–444.[Abstract/Free Full Text]
23. Lundmark K, Tran PK, Kinsella MG, Clowes AW, Wight TN, Hedin U. Perlecan inhibits smooth muscle cell adhesion to fibronectin: role of heparan sulfate. J Cell Physiol. 2001; 188: 67–74.[CrossRef][Medline] [Order article via Infotrieve]
24. Kumar A, Lindner V. Remodeling with neointima formation in the mouse carotid artery after cessation of blood flow. Arterioscler Thromb Vasc Biol. 1997; 17: 2238–2244.[Abstract/Free Full Text]
25. Bar-Shavit R, Eldor A, Vlodavsky I. Binding of thrombin to subendothelial extracellular matrix. Protection and expression of functional properties. J Clin Invest. 1989; 84: 1096–1104.[Medline] [Order article via Infotrieve]
26. Thyberg J. Differentiated properties and proliferation of arterial smooth muscle cells in culture. Int Rev Cytol. 1996; 169: 183–265.[Medline] [Order article via Infotrieve]
27. Rosenberg RD, Shworak NW, Liu J, Schwartz JJ, Zhang L. Heparan sulfate proteoglycans of the cardiovascular system: specific structures emerge but how is synthesis regulated? J Clin Invest. 1997; 100: S67–S75.[Medline] [Order article via Infotrieve]
28. Bashkin P, Doctrow S, Klagsbrun M, Svahn CM, Folkman J, Vlodavsky I. Basic fibroblast growth factor binds to subendothelial extracellular matrix and is released by heparitinase and heparin-like molecules. Biochemistry. 1989; 28: 1737–1743.[CrossRef][Medline] [Order article via Infotrieve]
29. Olson NE, Chao S, Lindner V, Reidy MA. Intimal smooth muscle cell proliferation after balloon catheter injury: the role of basic fibroblast growth factor. Am J Pathol. 1992; 140: 1017–1023.[Abstract]
30. 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.[Abstract/Free Full Text]
31. Smyth N, Vatansever HS, Murray P, Meyer M, Frie C, Paulsson M, Edgar D. Absence of basement membranes after targeting the LAMC1 gene results in embryonic lethality due to failure of endoderm differentiation. J Cell Biol. 1999; 144: 151–160.[Abstract/Free Full Text]
32. Williamson RA, Henry MD, Daniels KJ, Hrstka RF, Lee JC, Sunada Y, Ibraghimov-Beskrovnaya O, Campbell KP. Dystroglycan is essential for early embryonic development: disruption of Reichert’s membrane in Dag1-null mice. Hum Mol Genet. 1997; 6: 831–841.[Abstract/Free Full Text]
33. Ringvall M, Ledin J, Holmborn K, van Kuppevelt T, Ellin F, Eriksson I, Olofsson AM, Kjellen L, Forsberg E. Defective heparan sulfate biosynthesis and neonatal lethality in mice lacking N-deacetylase/N-sulfotransferase-1. J Biol Chem. 2000; 275: 25926–25930.[Abstract/Free Full Text]
34. Hayashi K, Madri JA, Yurchenco PD. Endothelial cells interact with the core protein of basement membrane perlecan through ß₁ and ß₃ integrins: an adhesion modulated by glycosaminoglycan. J Cell Biol. 1992; 119: 945–959.[Abstract/Free Full Text]
35. Woods A, McCarthy JB, Furcht LT, Couchman JR. A synthetic peptide from the COOH-terminal heparin-binding domain of fibronectin promotes focal adhesion formation. Mol Biol Cell. 1993; 4: 605–613.[Abstract]
36. Benitz WE, Kelley RT, Anderson CM, Lorant DE, Bernfield M. Endothelial heparan sulfate proteoglycan, I: inhibitory effects on smooth muscle cell proliferation. Am J Respir Cell Mol Biol. 1990; 2: 13–24.[Medline] [Order article via Infotrieve]
37. Kojima T, Leone CW, Marchildon GA, Marcum JA, Rosenberg RD. Isolation and characterization of heparan sulfate proteoglycans produced by cloned rat microvascular endothelial cells. J Biol Chem. 1992; 267: 4859–4869.[Abstract/Free Full Text]
38. Forsten KE, Courant NA, Nugent MA. Endothelial proteoglycans inhibit bFGF binding and mitogenesis. J Cell Physiol. 1997; 172: 209–220.[CrossRef][Medline] [Order article via Infotrieve]
39. Paka L, Goldberg IJ, Obunike JC, Choi SY, Saxena U, Goldberg ID, Pillarisetti S. Perlecan mediates the antiproliferative effect of apolipoprotein E on smooth muscle cells: an underlying mechanism for the modulation of smooth muscle cell growth? J Biol Chem. 1999; 274: 36403–36408.[Abstract/Free Full Text]
40. Ettenson DS, Koo EW, Januzzi JL, Edelman ER. Endothelial heparan sulfate is necessary but not sufficient for control of vascular smooth muscle cell growth. J Cell Physiol. 2000; 184: 93–100.[CrossRef][Medline] [Order article via Infotrieve]
41. Nugent MA, Karnovsky MJ, Edelman ER. Vascular cell-derived heparan sulfate shows coupled inhibition of basic fibroblast growth factor binding and mitogenesis in vascular smooth muscle cells. Circ Res. 1993; 73: 1051–1060.[Abstract/Free Full Text]
42. Salmivirta M, Jalkanen M. Syndecan family of cell surface proteoglycans: developmentally regulated receptors for extracellular effector molecules. Experientia. 1995; 51: 863–872.[CrossRef][Medline] [Order article via Infotrieve]
43. Kalmes A, Vesti BR, Daum G, Abraham JA, Clowes AW. Heparin blockade of thrombin-induced smooth muscle cell migration involves inhibition of epidermal growth factor (EGF) receptor transactivation by heparin-binding EGF-like growth factor. Circ Res. 2000; 87: 92–98.[Abstract/Free Full Text]
44. HajMohammadi S, Enjyoji K, Princivalle M, Christi P, Lech M, Beeler D, Rayburn H, Schwartz JJ, Barzegar S, de Agostini AI, Post MJ, Rosenberg RD, Shworak NW. Normal levels of anticoagulant heparan sulfate are not essential for normal hemostasis. J Clin Invest. 2003; 111: 989–999.[CrossRef][Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				K. Tran-Lundmark, P.-K. Tran, G. Paulsson-Berne, V. Friden, R. Soininen, K. Tryggvason, T. N. Wight, M. G. Kinsella, J. Boren, and U. Hedin Heparan Sulfate in Perlecan Promotes Mouse Atherosclerosis: Roles in Lipid Permeability, Lipid Retention, and Smooth Muscle Cell Proliferation Circ. Res., July 3, 2008; 103(1): 43 - 52. [Abstract] [Full Text] [PDF]

				Y. Nakashima, T. N. Wight, and K. Sueishi Early atherosclerosis in humans: role of diffuse intimal thickening and extracellular matrix proteoglycans Cardiovasc Res, July 1, 2008; 79(1): 14 - 23. [Abstract] [Full Text] [PDF]

				J. J. Zoeller, A. McQuillan, J. Whitelock, S.-Y. Ho, and R. V. Iozzo A central function for perlecan in skeletal muscle and cardiovascular development J. Cell Biol., April 21, 2008; 181(2): 381 - 394. [Abstract] [Full Text] [PDF]

				A. Kadenhe-Chiweshe, J. Papa, K. W. McCrudden, J. Frischer, J.-O Bae, J. Huang, J. Fisher, J. H. Lefkowitch, N. Feirt, J. Rudge, et al. Sustained VEGF Blockade Results in Microenvironmental Sequestration of VEGF by Tumors and Persistent VEGF Receptor-2 Activation Mol. Cancer Res., January 1, 2008; 6(1): 1 - 9. [Abstract] [Full Text] [PDF]

				H. Morita, A. Yoshimura, K. Inui, T. Ideura, H. Watanabe, L. Wang, R. Soininen, and K. Tryggvason Heparan Sulfate of Perlecan Is Involved in Glomerular Filtration J. Am. Soc. Nephrol., June 1, 2005; 16(6): 1703 - 1710. [Abstract] [Full Text] [PDF]

				A. Segev, N. Nili, and B. H Strauss The role of perlecan in arterial injury and angiogenesis Cardiovasc Res, September 1, 2004; 63(4): 603 - 610. [Abstract] [Full Text] [PDF]

				Z. Zhou, J. Wang, R. Cao, H. Morita, R. Soininen, K. M. Chan, B. Liu, Y. Cao, and K. Tryggvason Impaired Angiogenesis, Delayed Wound Healing and Retarded Tumor Growth in Perlecan Heparan Sulfate-Deficient Mice Cancer Res., July 15, 2004; 64(14): 4699 - 4702. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 94/4/550    most recent 01.RES.0000117772.86853.34v1