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(Circulation Research. 1997;80:159-169.)
© 1997 American Heart Association, Inc.

Articles

The Integrin Very Late Antigen-4 Is Expressed in Human Smooth Muscle Cell

Involvement of ₄ and Vascular Cell Adhesion Molecule-1 During Smooth Muscle Cell Differentiation

Cecile Duplaa, Thierry Couffinhal, Pascale Dufourcq, Brigitte Llanas, Catherine Moreau, Jacques Bonnet

the Institut National de la Sante et de la Recherche Medicale, Unite 441, Pessac, France.

Correspondence to Cecile Duplaa, INSERM U.441, Avenue du Haut-Leveque, 33600 Pessac, France. E-mail cecile.duplaa@bordeaux.inserm.fr

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Vascular cell adhesion molecule-1 (VCAM-1) and its counterreceptor, the integrin very late antigen-4 (VLA-4), have recently been identified in smooth muscle cells during intimal thickening in humans and in newly forming vessels during ontogeny in mice, respectively. We examined the coexpression of VCAM-1 and the ₄ integrin subunit in human smooth muscle cells. The expression of VCAM-1 and ₄ subunit were studied during development of the aorta. In the 10-week-old human fetal aorta, VCAM-1 and ₄ were strongly expressed in smooth muscle cells. Their expression was dramatically reduced within the 24th week of gestation and disappeared in the adult aortic media. However, smooth muscle cells from intimal atherosclerotic thickening of adult aorta reexpressed both VCAM-1 and ₄. In a culture model mimicking smooth muscle differentiation, VCAM-1 mRNA and protein and ₄ integrin protein were coexpressed with smooth muscle–specific variants of cytoskeletal and contractile proteins, smooth muscle myosin heavy chain, caldesmon heavy chain, and desmin. Treatment with antibodies against VCAM-1 or ₄ integrin subunit interfered with the mRNA induction of smooth muscle–specific markers of differentiation. These results in vitro, associated with the transitory expression of VCAM-1 and VLA-4 during vascular ontogeny and the atherosclerosis process, point to a possible role of VCAM-1 and VLA-4 in the induction of smooth muscle differentiation.

Key Words: atherosclerosis • human aortic development • cell-cell interaction • phenotype modulation • smooth muscle marker

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
A member of the integrin subfamily, VLA-4 (₄ß₁) was initially described on lymphoid and myeloid cells and, more recently, on adherent cell types. Its functional diversity is reflected by its intervention in cell-matrix interactions by binding to fibronectin¹ and in cell-cell interactions via an adhesion protein, VCAM-1.² VCAM-1 is a member of the immunoglobulin gene superfamily.³ It is expressed in endothelium and synovial fibroblasts, bone marrow stromal cells, follicular dendritic cells in lymph nodes, and macrophages. Interestingly, O'Brien et al⁴ have reported that VCAM-1 is expressed in a subset of intimal SMCs in human atherosclerotic plaque. In previous studies, we and others demonstrated that VCAM-1 expression in cultured SMCs was induced by cytokines⁵ and that besides its induction by tumor necrosis factor-, its expression was inhibited by growth-related factors, such as platelet-derived growth factor-BB or transforming growth factor-ß, at both mRNA and protein levels.⁶ The interaction between these two adhesion molecules is thought to be crucial to the recruitment, activation, and retention of ₄ß₁-expressing cells at inflammatory sites.^{7 8 9} In addition to VCAM-1/VLA-4's role in cell recruitment and migration, other reports have indicated that VCAM-1 and VLA-4 also mediated cell differentiation or maturation and tissue formation, as demonstrated by Rosen et al¹⁰ for skeletal muscle differentiation. Recent studies have focused on the role of VCAM-1 and VLA-4 during mouse embryonic development. Sheppard et al¹¹ described ₄ and VCAM-1 expression in developing tissues of the mouse, especially in the mesenchyme, giving rise to vascular endothelium and SMCs. VCAM-1 or ₄ knockout mice showed a failure of the fusion of the allantois with the chorion during placentation and a late effect, failure in the development of the epicardium and coronary vessels.^{12 13 14} Both processes clearly involved ₄ integrin and VCAM-1 interactions.

SMCs arise from the mesoderm during embryogenesis, subsequently differentiate into numerous different derivatives, increase in size during normal development, and acquire a differentiated phenotype characteristic of adulthood. SMC differentiation is characterized by the expression of smooth muscle–specific variants of cytoskeletal and contractile proteins and a coordinated organization of the extracellular matrix and membrane integrins.^{15 16 17 18} In the course of various vascular diseases (arterial hypertension, atherosclerosis, and restenosis after angioplasty), SMCs involved in neointimal development temporarily display a phenotype less differentiated, closer to an embryonic phenotype^{19 20} ; SMCs of the neointimal thickening have been shown to recover progressively a differentiated phenotype.^{17 21 22 23} The molecular basis for phenotypic modulation in vascular SMCs is poorly understood. Extracellular matrix and cell-matrix interactions are assumed to play a critical role in the regulation of gene expression and cytodifferentiation,^{24 25} although it is still not clear whether cell-cell interactions participate in the regulation of the SMC differentiation program.

In the present study, we show that ₄ and VCAM-1 are coexpressed in SMCs during human physiological embryonic development and pathological atherosclerotic neointimal development. Using a cell culture model mimicking SMC differentiation, we presented evidence that VCAM-1 and ₄ are coexpressed with smooth muscle–specific markers of differentiation and that blockade of their interaction inhibits the induction of the differentiation program.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
mAbs, Protein, and Peptide
mAbs against human ICAM-1 (84H10), VCAM-1 (1G11), ₄ integrin subunit (HP2/1), desmin (77), and -smooth muscle actin (IA4) were purchased from Immunotech. The cell type–specific mAbs against human T cells (anti–Leu-4) and human macrophages (HAM-56) were obtained from Becton Dickinson and Enzo Diagnostics, respectively. Anti–-smooth muscle actin alkaline phosphatase conjugate was provided by Sigma Chemical Co. The mAbs against human sm-MHC and h-caldesmon (high molecular weight of caldesmon) were kindly provided by Dr M. Glukhova (Frid et al²⁶ ). An irrelevant isotype-matched immunoglobulin (Sigma) was used as a negative control.

Tissue Samples
Normal human aortas were obtained from two 10-week-old embryos and five 24- to 27-week-old fetuses, aborted on medical grounds. Fifteen adult thoracic aorta samples and 11 plaques from the internal carotid artery of patients operated on because of transitory ischemic attacks were obtained at surgery. The tissue was frozen in isopentane, prechilled in liquid nitrogen, and stored at -70°C. Five-micron-thick sections were cut with a cryostat, air-dried, and used for immunostaining. At least five to eight sections of each specimen were examined.

Cell Culture and Differentiation Protocol
SMCs were isolated from media of human adult aortas by enzyme digestion, as previously described,^{27 28} and cultured in Ham's F10 medium (GIBCO BRL) supplemented with 5% FCS, 5% heat-inactivated human serum, 5 mmol/L HEPES, 50 U/mL penicillin, and 50 mg/mL streptomycin, at 37°C in a 5% CO₂/95% air atmosphere. The medium was changed every 3 days, and cells were passaged by 0.05% trypsin/0.02% EDTA solution. Studies were conducted on SMCs at passage 2, which were seeded at a high seeding density (5x10⁴ cells/cm²). At confluence, the cells were either maintained in medium with serum plus antibiotics or stimulated to differentiate in a defined serum-free medium containing a 1:1 mixture of DMEM (GIBCO BRL) and Ham's F10 plus insulin (10^-6 mol/L, Sigma), transferrin (200 µg/mL, Sigma), ascorbate (0.2 mmol/L, Sigma), and sodium selenite (6.25 ng/mL, Sigma) plus antibiotics for 1, 3, or 6 days. This growth condition maintains SMCs in a quiescent differentiated state and promotes the expression of smooth muscle–specific markers of differentiation.^{29 30} For immunofluorescence flow cytometry or mRNA analysis, SMCs were cultured in six-well plates (Falcon, Becton Dickinson); for the immunofluorescence microscopy study, SMCs were cultured on two-chamber slides (Lab-Tek, Nunc Inc).

In antibody blocking studies, antibodies against human VCAM-1 (1G11) and ₄ integrin subunit (HP 2/1) were added at a concentration of 50 µg/mL when the confluent cells were switched to defined serum-free medium. Irrelevant isotype-matched immunoglobulin (50 µg/mL) and antibody against human ICAM-1 (84H10, 50 µg/mL) were used as negative controls. Thirty-six hours later, the well plates were rinsed with PBS, and RNA was extracted and submitted to reverse-transcriptase PCR amplifications for each studied gene.

Immunohistochemistry
Immunoperoxidase staining was performed as previously described in detail.³¹ Briefly, 5-µm sections were fixed with 4% paraformaldehyde for 10 minutes at room temperature and washed in PBS. Endogenous peroxidase activity was blocked by incubating the sections in 0.3% hydrogen peroxide. To prevent nonspecific antibody binding, the sections were preincubated for 30 minutes in a solution containing 5% BSA in PBS. For antibody staining, sections were first incubated with a primary antibody at appropriate dilution for 1 hour at 37°C, rinsed with PBS, incubated with biotinylated rat anti-mouse IgG (Amersham) for 1 hour at room temperature, rinsed with PBS, and covered with streptavidin-HRP complex. After 30 minutes at room temperature, the sections were rinsed with PBS and revealed by incubation with 0.05% (wt/vol) 3,3'-diaminobenzidine tetrahydrochloride dihydrate. A counterstain of 10% Harris hematoxylin was applied before coverslipping. Negative control slides were prepared by substituting preimmune mouse serum for the primary mAb.

The immunoenzyme double-staining method was used to study SMC expression of VCAM-1 or ₄ integrin subunit in human adult arteries.^{32 33} Briefly, the first primary mAb (anti–VCAM-1 or anti-₄) was applied to the sections, followed by biotinylated IgG and then the streptavidin–biotinylated HRP complex. After incubation in 3,3'-diaminobenzidine to give a yellow-brown staining, sections were washed in PBS and incubated with monoclonal anti–-smooth muscle actin–alkaline phosphatase conjugate. After washes in straight PBS, the antibody conjugate was detected by incubating the sections in 100 mmol/L PBS, pH 9.5, with 1 mmol/l levamisole and chromogenic substrate BCIP/nitro blue tetrazolium to give a deep blue reaction (Vectastain ABC-AP kit, Vector Laboratories). Finally, the sections were dehydrated and coverslipped. All sections of the series were stained together in a single experiment for each antibody tested.

Immunofluorescence Microscopy
Monolayer cultures on glass sides, maintained in the different conditions of the differentiation protocol, were assayed for expression of VCAM-1 and ₄ integrin subunit. The cells were fixed in 2% paraformaldehyde in PBS for 10 minutes at 4°C, washed in PBS, and then blocked in 5% BSA for 20 minutes. After they were washed, the cells were sequentially incubated in primary mAb, biotinylated anti-mouse IgG (Amersham), and finally streptavidin–Texas red. All incubations were carried out in 1% horse serum/BSA for 1 hour at 4°C. After two 5-minute washes in PBS, stained cells were mounted in Fluoprep medium (BioMerieux) and examined with a Nikon Microphot-FXA microscope (Nikon).

Flow Cytometry
Expression of VCAM-1, ₄ integrin subunit, sm-MHC, h-caldesmon, and desmin was determined by flow cytometry at different times during the differentiation protocol. Monodispersed suspensions of SMCs were prepared by brief incubations in 5 mmol/L EDTA at 4°C after two washes with PBS, pH 7.2, containing 2% BSA with 0.2% sodium azide (NaN₃).²⁹ This buffer was used in all subsequent steps. sm-MHC, h-caldesmon, and desmin were assayed in cells permeabilized in 0.1% Triton X-100 in PBS for 5 minutes at 4°C. The cells were incubated with 100 µL of mAb in medium for 1 hour at 4°C, washed three times with cold medium to remove unbound antibody, resuspended in 100 µL of fluorescein isothiocyanate–conjugated goat anti-mouse IgG (Amersham), and incubated for a further 30 minutes on ice. After three additional washes, the samples were immediately analyzed with a FACS analyzer (ODAM-ATC 3000, Odam-Brucker). Before each experiment, the FACS analyzer was calibrated with FITC-calibrated beads.

RNA Preparation Procedure
Total cellular RNA was prepared from confluent or postconfluent cell monolayers using a single-step acid guanidinium isothiocyanate/phenol/chloroform extraction method.³⁴

PCR
After reverse transcription of 1 µg of total RNA by oligo(dT) priming, the resulting single stranded cDNA was amplified by PCR with incorporation of Bio-11-dUTP (Sigma) in 50 µL of PCR buffer containing 2.5 U of Thermus aquaticus (Taq) polymerase (Bioprobe systems) as previously described.^{35 36} For each set of primers, the number of cycles was chosen in the linear phase of amplification and subjected to defined rounds of temperature cycling (denaturation, primer annealing, and extension): 18 cycles (95°C/30 s, 56°C/5 s, and 72°C/1 min) for GAPDH, 20 cycles (95°C/30 s, 56°C/5 s, and 72°C/1 min) for VCAM-1 and h-caldesmon, 28 cycles (95°C/30 s, 60°C/5 s, and 72°C/1 min) for sm-MHC, 30 (95°C/30 s, 64°C/30 s, and 72°C/2 min) for the ₄ integrin subunit, and 30 cycles (95°C/30 s, 60°C/30 s, and 72°C/2 min) for desmin. The oligonucleotide primers for the PCR are shown in Table 1. The specificity of generated PCR products was attested by the correct size, based on the known cDNA sequences, the placement of the internal primers, and the predicted size of fragments produced by enzymatic restriction (data not shown).

View this table: [in this window] [in a new window]   Table 1. Oligonucleotides of 5' Primers and 3' Primers of Target Genes

Analysis and Quantification of the cDNA PCR-Amplified Products
The PCR products were subjected to a semiquantitative analysis as described elsewhere.³⁶ Briefly, PCR products (10 µL) were electrophoresed in a 1.5% agarose gel and then blotted onto positively charged nylon membranes (Hybond N+, Amersham). Blots were revealed using the HRP complex (Amersham)–diaminobenzidine (Sigma) procedure. The digitized signals of the PCR products were analyzed using GAPDH as an internal standard. The data were expressed as relative values (target gene/GAPDH).

Screening of a Human SMC cDNA Library
In order to determine the presence of ₄ mRNA in cultured SMCs, a PCR-amplified fragment of human ₄ integrin subunit was subcloned into pDirect vector (Clontech Laboratories), generating an ₄ probe (Alp5-Alp6). The specificity of the PCR amplification was checked by sequencing. A cDNA library from human cultured SMCs was constructed in the Uni-Zap XR vector (Stratagene Inc) using a cloning kit (Zap cDNA synthesis kit, Stratagene) and screened with -³²P–labeled Alp5-Alp6 probe. Several cDNA clones encompassing the carboxy terminus of human ₄ integrin were recovered, and their nucleotide sequences were determined (data not shown).

Ribonuclease Protection Assay
Total RNAs from freshly dispersed adult aortic SMCs and freshly dissociated fetal SMCs obtained from human umbilical cord veins were prepared as described above. The RNase protection assay was carried out according to the manufacturer's instructions (Ambion). A radiolabeled cRNA probe was produced using the Maxiscript kit (Ambion) by incubating the EcoRI-linearized Alp5-Alp6 plasmid with T7 RNA polymerase in the presence of 100 µCi uridine 5'-[-³²P]triphosphate (specific activity, 2x10⁸ cpm/µg) at 37°C for 1 hour. The ₄ integrin cRNA probe (2x10⁵ cpm) and 30 µg of total RNA were mixed and hybridized at 45°C overnight in hybridization buffer (80% formamide, 100 mmol/L sodium citrate, pH 6, 300 mmol/L sodium acetate, and 1 mmol/L EDTA). Unhybridized RNAs were digested with 5 µg/mL RNAse A and 100 U/mL RNase T1 at 37°C for 30 minutes. RNase-resistant hybrids were precipitated, run on a 5% polyacrylamide/8 mol/L urea gel, and exposed to Kodak XAR-5 film at -70°C with an intensifying screen. The size of the fragments was estimated from the positions of a DNA molecular marker, 5'–end-labeled with [-³²P]ATP, and run on the same gel.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Expression of VCAM-1 and ₄ in SMCs of Human Aorta
At 10 weeks of gestation, the aortic wall was stained with the mAbs to VCAM-1 and ₄ integrin subunit (Fig 1a and 1b). As confirmed by -actin staining on adjacent sections, VCAM-1 and ₄ were expressed in SMCs of the aortic embryonic media (Fig 1c). ₄ expression appeared more concentrated in smooth muscle layers surrounding the endothelium but was also present in numerous SMCs of the deeper media. VCAM-1 expression was distributed throughout the media. Medial SMCs of small caliber arteries displayed a more intense VCAM-1 and ₄ expression than did aortic medial SMCs (data not shown). In contrast, in the aorta of 24- and 27-week-old fetuses, scarce VCAM-1 and ₄ staining was observed in SMCs, and it was confined to the external portion of the media (as confirmed by -actin staining) (Fig 1e and 1f). Their expression disappeared in the medial SMCs of the normal adult aorta (see Fig 2). In the human adult atherosclerotic aorta, we attempted to locate VCAM-1 and ₄ expression and to define which kind of cell expressed these antigens. Serial sections were stained for VCAM-1 and ₄ integrin subunit, as well as cell-specific markers for SMCs (-actin and sm-MHC), macrophages, and T lymphocytes, in order to determine plaque cell populations. SMCs occupied two anatomic layers, the tunica media and tunica intima. As shown on serial sections in Fig 2, ₄ and VCAM-1 were not expressed in medial SMCs, whereas intimal expression was colocalized with -smooth muscle actin– and sm-MHC–positive staining (Fig 2a and 2d). The scattered T cells and macrophages that express ₄ and VCAM-1 could not account for the large distribution of VCAM-1 and ₄ labeling in the intima (Fig 2b and 2c). To confirm that intimal SMCs were able to express ₄ and VCAM-1, sections of human atherosclerotic arteries were double-stained with antibodies against VCAM-1 or ₄ and -smooth muscle actin. Some intimal cells clearly displayed a double-labeling VCAM-1/-actin and ₄/-actin; furthermore, ₄-positive and VCAM-1–positive SMCs were found in the same area in serial sections, as shown in Fig 2g through 2j. Double-labeled cells displayed the same typical spindle-shaped pattern as -actin–positive cells in the media (Fig 2h and 2j). These observations indicate that VCAM-1 and ₄ are temporarily expressed in the media during aortic development, disappear in the adult aortic media, and are reexpressed in SMCs involved in the intimal thickening of atherosclerotic lesions.

View larger version (73K): [in this window] [in a new window]   Figure 1. Expression of 4 integrin subunit and VCAM-1 in human vascular smooth muscle during ontogenic development. Serial sections of aorta from a 10-week-old embryo (E 10 W, a through d) and from a 27-week-old fetus (F 27 W, e through h) are stained with antibodies against 4 integrin subunit (a and e), VCAM-1 (b and f), -smooth muscle actin (c and g) (yellow-brown reaction product, hematoxylin counterstain), or nonimmune IgG (d and h). The bar in panel c represents 20 µm for panels a through d, and the bar in panel g represents 10 µm for panels e through h. At the early fetal stage of development, 4 and VCAM-1 are expressed on SMCs; in the 27-week-old embryonic aorta, their expression is only detected in the external part of the media.

View larger version (115K): [in this window] [in a new window]   Figure 2. 4 integrin subunit and VCAM-1 expressions in atherosclerotic plaque. Panels a through f show serial sections of a fibrofatty plaque stained by immunoperoxidase technique (yellow-brown reaction product, hematoxylin counterstain) with mAb against -smooth muscle actin (-actin) (a), human macrophages (M) (b), T cells (c), sm-MHC (d), 4 integrin subunit (e), and VCAM-1 (f). The bar in panel f represents 20 µm for panels a through f. Arrowheads denote the internal elastic lamina. M indicates media; I, intima; and L, lumen. Panels g through j show serial sections of another aortic atherosclerotic plaque, double-stained with antibodies against 4 integrin (g and h) or VCAM-1 (i and j) and -smooth muscle actin. Anti–4 integrin and anti–VCAM-1 were developed with a peroxidase technique to produce a yellow-brown stain; anti–-smooth muscle actin, with an alkaline phosphatase technique to yield a deep blue stain. Open arrows in panels g and i indicate the location of the higher magnification presented in panels h and i. The bar in panel i represents 10 µm for panels g and i; the bar in panel j represents 5 µm for panels h and j. Panels a through f demonstrate 4 integrin and VCAM-1 expression in intimal thickening in an area rich in SMCs (-actin– and sm-MHC–positive cells). 4 integrin and VCAM-1 expression could not be explained by the small number of macrophages or T cells. Panels g through j show intimal SMCs double-labeled with 4/-actin and VCAM-1/-actin in the same area.

Expression of ₄ Integrin Subunit in Cultured SMCs
We have previously reported that SMCs express VCAM-1 in vitro,⁶ whereas the ₄ integrin subunit has not been described in these cells. To identify the membrane expression of ₄, cultured medial SMCs (at confluence after the sixth day) were maintained either in medium with serum or in a defined serum-free medium. They were stained for ₄ and examined by immunofluorescence microscopy. No staining was observed in postconfluent cells cultured in medium supplemented with serum. In contrast, SMCs maintained 6 days in serum-free medium exhibited a different staining pattern with either a random distribution of the protein on the upper cell surface membrane or a patchy distribution on the cell edge (see below). Therefore, we compared ₄ mRNA expression of freshly dispersed adult aortic cells and freshly dissociated fetal SMCs using an RNase protection assay. This assay allowed us to detect ₄ mRNA in human SMCs and to compare the mRNA expression. 4 mRNA was barely detectable in adult SMCs, although its was strongly expressed in fetal cells, with a ratio between adult and fetal cells of 1:50 (Fig 3). In order to investigate the ₄ integrin gene sequence and expression, a human SMC cDNA library was screened with a human ₄ probe. Five clones that were >1.5 kb in length were isolated, and the longest ₄ clone, selected from this library, was shown by DNA sequencing to be identical to the previous published ₄ sequence. These findings indicated that SMCs express ₄ mRNA and protein in vivo and in vitro.

View larger version (35K): [in this window] [in a new window]   Figure 3. 4 integrin expression in fetal and adult SMCs. Total RNA (30 µg) from freshly dispersed adult aortic SMCs and freshly dissociated fetal SMCs was analyzed for the expression of 4 integrin mRNA by RNase protection. The size of the protected fragment was of 484 nucleotides. The high level of 4 mRNA in fetal cells is contrasted with the low level in adult aortic SMCs. This RNase protection experiment is representative of two separate assays with similar results. yRNA indicates yeast tRNA.

Modulation of ₄ Integrin Subunit and VCAM-1 mRNA and Protein Levels During Differentiation of Cultured SMCs
To investigate the regulation of VCAM-1 and VLA-4 expression in SMCs, we used a model of phenotypic modulation in culture.^{29 30} To characterize this model in human SMCs, mRNA and the protein contents of three markers of SMC differentiation (sm-MHC, h-caldesmon, and desmin) were analyzed by semiquantitative PCR and flow cytometry in confluent and 6-day-postconfluent cells maintained in medium supplemented with serum, and at 1, 3, or 6 days after confluence, cells were maintained in defined serum-free medium (Fig 4).

View larger version (9K): [in this window] [in a new window]   Figure 4. Schematic representation of SMC differentiation protocol. Second-passaged SMCs, seeded at high density, were maintained after confluence either in medium supplemented in serum or stimulated to differentiate in a defined serum-free medium. Cells were harvested at confluence (D 0) and at 6 days after confluence, when they were maintained in serum-free medium. In defined serum-free medium, SMCs were harvested at 1, 3, and 6 days after confluence (D 1, D 3, and D 6, respectively).

Analysis of mRNA content by semiquantitative PCR showed that confluent and 6-day postconfluent cells, cultured in medium supplemented with serum, exhibited a low constitutive expression of sm-MHC, h-caldesmon, and desmin mRNA. When confluent cells were cultured in defined serum-free medium, we observed a time-dependent increase in the mRNA content of the three smooth muscle–specific markers of differentiation (Fig 5A). sm-MHC mRNA was induced within 24 hours, continued to increase up to 3 days, and remained elevated at 6 days. After switching the medium, an increase in h-caldesmon and desmin mRNA was detected by PCR analysis by day 3, and the level continued to increase up to day 6 (Fig 5B). Flow cytometric analysis of the three markers on confluent and 6-day postconfluent SMCs maintained in medium with serum showed that these cells constitutively expressed low levels of proteins (Table 2). Cultivation of confluent cells in defined serum-free medium induced a time-dependent increase in protein content of the three smooth muscle markers of differentiation. An increase in sm-MHC expression was detectable at 3 days (2.5-fold) and continued to increase (4.6-fold at 6 days). Increases in h-caldesmon and desmin protein were also detectable 3 days after the switch of medium (4.5- and 2.6-fold, respectively) and continued to increase (12- and 3.5-fold, respectively, at 6 days) (Table 2). These results showed that confluent human SMCs, maintained in defined serum-free medium, promoted the expression of smooth muscle–specific markers of differentiation and progressed through their differentiation program.

View larger version (63K): [in this window] [in a new window]   Figure 5. Modulation of mRNA of smooth muscle–specific markers, VCAM-1, and 4 integrin subunit during differentiation of cultured SMCs. Confluent or postconfluent human SMCs were harvested at different times during the differentiation protocol as described in Fig 4. Total RNA (1 µg) was reverse-transcribed with poly(dT), and the resulting cDNA was subjected to separate PCR amplification using biotinylated dUTP nucleotide incorporation for sm-MHC, h-caldesmon, desmin, VCAM-1, 4 integrin, and GAPDH. Reaction mixture (10 µL was electrophoresed, transferred, and stained with streptavidin-HRP complex followed by diaminobenzidine. A, Southern transfer of biotinylated PCR products. The target gene amplified is indicated. Confluent (D 0 serum) and 6-day postconfluent (D 6 serum) cells were maintained in medium supplemented with serum. Postconfluent cells were harvested 1 (D 1), 3 (D 3), and 6 (D 6) days after the switch to defined serum-free medium. Total RNA from the medial layer of a human aorta was reverse-transcribed and used as positive control (media). A time-dependent incease in smooth muscle markers of differentiation and of VCAM-1 and 4 integrin was observed in cells cultured in defined serum-free medium. B, Graphs showing the target gene–to–GAPDH ratio for each culture condition as described in panel A. Results are representative of three independent PCR amplifications on two separate studies.

View this table: [in this window] [in a new window]   Table 2. Flow Cytometric Analysis of Smooth Muscle Markers of Differentiation, VCAM-1, and 4 Integrin on SMCs Submitted to Differentiation Protocol

In order to define the modulation of ₄ integrin subunit and VCAM-1 expression during SMC differentiation, ₄ and VCAM-1 mRNA and protein levels were analyzed by semiquantitative PCR and flow cytometry, respectively, on cells cultured under conditions similar to those described above. As noted previously, the basal expression of VCAM-1 mRNA in confluent or postconfluent cells maintained in medium supplemented with serum was rather low (Fig 5A). After confluence, when cells were cultured in growth conditions maintaining SMCs in a quiescent differentiated state, a strong induction of VCAM-1 mRNA was observed within 24 hours, continued to increase up to 3 days, and remained elevated at 6 days (Fig 5A and 5B). ₄ mRNA content in confluent and postconfluent cells, maintained in medium supplemented with serum, was rather low. We noted a lack of increased expression of ₄ mRNA in defined serum-free medium (Fig 5B). Flow cytometry with anti–VCAM-1 antibody showed that confluent or postconfluent SMCs maintained in medium with serum expressed low levels of VCAM-1 antigens (mean fluorescence, 1 and 8 arbitrary units, respectively) (Table 2). An increase in VCAM-1 membrane expression was detectable within 24 hours of culture in defined serum-free medium (5.9-fold), reached a plateau at 3 days (28-fold), and remained elevated between 3 and 6 days (Table 2). For ₄ membrane expression, no significant increase was detectable by flow cytometry in defined serum-free medium. The time-dependent modulation in both VCAM-1 and ₄ membrane expression was also analyzed in the culture model by immunofluorescence microscopy on cell monolayers. Confluent (Fig 6a and 6e) and postconfluent cells (data not shown) treated with serum did not express either ₄ or VCAM-1. On postconfluent cells treated with defined serum-free medium, a time-dependent increase in ₄ integrin membrane expression was observed. The expression began as a weak and patchy immunostaining on day 1 (Fig 6b), changing toward a clustering pattern at 3 and 6 days (Fig 6c and 6d). The expression level remained weak during the time course. We did not observe a dramatic change in the protein level but in the reorganization of ₄ expression along SMC membranes, which could explain the undetectable increase of ₄ integrin expression by FACS analysis. By the sixth day of culture in serum-free medium, a strong membrane expression of VCAM-1 was observed (Fig 6f). These results showed that under differentiated growth conditions, the expression of VCAM-1 mRNA and protein increased in a time-dependent manner in SMCs; ₄ expression was regulated through protein clustering and focal contact formation. These modifications preceded the expression of smooth muscle–specific markers of differentiation.

View larger version (72K): [in this window] [in a new window]   Figure 6. Immunofluorescence analysis of 4 integrin subunit and VCAM-1 in human cultured aortic SMCs at different times of the differentiation protocol. Cells were labeled by anti–4 integrin (a through d) and anti–VCAM-1 (e and f), followed by an anti-mouse phycoerythrine label. Confluent cells maintained in medium supplemented with serum show no expression of 4 integrin subunit (a) or VCAM-1 (e). A patchy and weak staining by mAb anti–4 integrin is observed on postconfluent cells maintained in defined serum-free medium 1 day after the switch of medium (b), with cell staining becoming more clustered at 3 (c) and 6 days (d). Under the same conditions, a strong staining is observed at 6 days with mAb anti–VCAM-1 (f).The bar in panel a represents 10 µm for panels a through f.

Antibodies to VCAM-1 and VLA-4 Inhibit the Expression of Smooth Muscle–Specific Markers of Differentiation
In order to determine more directly if VCAM-1–₄ interactions were involved in the induction of the expression of the smooth muscle differentiation markers, we examined the effects of 1G11 and HP2/1 mAb, which block VCAM-1 and ₄ interactions, respectively. Confluent SMCs were switched from medium with serum to defined serum-free medium to induce differentiation. At the same time, antibodies were added to the cultures, and cells were then maintained for an additional 36 hours before being harvested for extraction of total cellular RNA. The levels of mRNA expression of sm-MHC, h-caldesmon, desmin, VCAM-1, and ₄ integrin subunit were determined by semiquantitative PCR analysis. As shown in Fig 7, the significant increase in mRNA levels of sm-MHC and desmin from confluent serum-deprived versus confluent serum-growing cells (lanes 2 versus 1) was suppressed by anti–VCAM-1 (lane 4), anti-₄ (lane 5), or the combination of the two mAbs (lane 6). The blocking effect of the antibodies was less dramatic in h-caldesmon expression compared with the effect on sm-MHC and desmin expression. No significant effect was observed with the isotype-matched control (lane 3) or with mAb against ICAM-1. The strong mRNA induction of VCAM-1 observed during SMC redifferentiation was also suppressed by mAbs blocking VCAM-1–₄ interactions. ₄ mRNA expression remained almost the same.

View larger version (56K): [in this window] [in a new window]   Figure 7. Effects of antibodies to VCAM-1 and 4 integrin on SMC differentiation. Human SMCs were harvested 36 hours after confluence either in medium supplemented in serum or in defined serum-free medium. Total RNA (1 µg) was reverse-transcribed with poly(dT), and the resulting cDNA was subjected to separate amplification for sm-MHC, h-caldesmon, desmin, VCAM-1, 4 integrin, and GAPDH. Reaction mixture (10 µL) was electrophoresed, transferred, and stained. A, Southern transfer of biotinylated PCR products. The amplified target gene and the stimulation are indicated. After confluence in medium supplemented with serum, cultured human SMCs were maintained for 36 hours in the same medium (control with serum, lane 1) or in defined serum-free medium (lanes 2 through 7), without mAb (control, lane 2), with irrelevant mAb (50 µg/mL) (IgG1, lane 3), with anti–VCAM-1 mAb (50 µg/mL) (anti–VCAM-1, lane 4), with anti–4 integrin mAb (50 µg/mL) (anti–VLA-4, lane 5), with VCAM-1 (50 µg/mL) and 4 integrin (50 µg/mL) mAbs (anti–VCAM-1+VLA-4, lane 6), and with anti–ICAM-1 mAb (50 µg/mL) (anti–ICAM-1, lane 7). The mRNA induction of SMC markers (sm-MHC and desmin) and VCAM-1 after switching to defined serum-free medium is strongly inhibited by mAbs against VCAM-1 and 4 integrin. B, Graphs showing target gene–to–GAPDH ratio for each culture condition as described in panel A. Results are representative of three independent PCR amplifications on two separate studies.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we followed the expression of ₄ and VCAM-1 in SMCs during human vascular ontogeny and in atherosclerotic vessels. Our results indicate that ₄ integrin subunit and VCAM-1 are expressed in SMCs at the early embryonic stage of human aortic development (10 weeks of gestation). Their expression decreased dramatically between 10 and 24 weeks of gestation, moving to the external part of the media, and became undetectable in normal adult media. The pattern of ₄ integrin and VCAM-1 expression in human embryonic aortic tissue appears to be different from that described in the mouse.¹¹ During mouse development, ₄ is expressed in the aorta as early as embryonic day 10, predominantly in the smooth muscle layer surrounding the endothelium, and persists into adulthood. In contrast, VCAM-1 was not detected in vascular smooth muscle during development but was evident in the lung mesenchymal cell precursors of SMCs and endothelial cells. In human adult arteries, we showed that ₄ integrin was expressed in SMCs of thickened intima in the region of VCAM-1–positive SMCs. These results support the notion that atherosclerosis is accompanied by a reexpression of VCAM-1 and the ₄ integrin subunit in intimal SMCs. Expression of VCAM-1 on SMCs in atherosclerotic plaque has been reported previously,^{4 5} and it may reflect an "activated state" of SMCs, induced by cytokines derived from activated T lymphocytes or macrophages.³⁷ In the development of intimal thickening, the SMCs switch their phenotype toward a neonatal or embryonic genetic program, accompanied by the reexpression of earlier developmental events,²¹ and then SMCs evolve toward a redifferentiated phenotype.^{22 23} The presence of VCAM-1 and ₄ on intimal SMCs could be explained by their role in SMC phenotypic modulation.

During development and intimal hyperplasia, SMCs are interconnected by gap junctions and adherent junctions and, occasionally, by some tight junctions.^{21 38} Members of the cadherin family and immunoglobulin superfamily are thought to be involved in the organization of adherent junctions.³⁹ Although we lack a direct demonstration of the participation of VCAM-1 and VLA-4 in the organization of intercellular clefts between adjacent SMCs, the simultaneous presence of these two adhesion proteins during aortic development and in atherosclerotic plaque could suggest an interaction between VCAM-1 and VLA-4.

We demonstrated by cDNA cloning, RNase protection, and immunohistochemical assays that the ₄ integrin subunit can be expressed by human SMCs. Its transitory expression during vascular ontogenesis and the high mRNA level in fetal cells compared with adult cells suggest that VLA-4 expression is dependent on the state of SMC differentiation. Recent studies have shown that cell-cell adhesion and communication via VCAM-1–VLA-4 interaction appear to be important in cellular migration and tissue organization. For example, VCAM-1–VLA-4 interaction was implicated during myogenesis in the alignment of secondary myoblasts along primary myotubes and in myoblast-myotube fusion.¹⁰ VCAM-1 or ₄ integrin gene knockout mice demonstrated a failure in chorioallantoic fusion during placental development and abnormalities in the developing heart by nonadhesion of the epicardial layer to the myocardium.^{12 13 14} In the human atherosclerotic aorta, we hypothesized that the simultaneous expression of VCAM-1 and ₄ in intimal SMCs participates in the induction of SMC differentiation by their interaction.

In order to analyze the relationship between VCAM-1 and VLA-4 expression and SMC differentiation, we developed a cell culture model that mimics SMC differentiation and dedifferentiation. Cultured SMCs modulated their expression of smooth muscle contractile and cytoskeletal proteins in relation to their growth state. SMC differentiation can be tracked by the expression of a set of smooth muscle–specific markers.^{15 26 40 41} sm-MHC is expressed only in SMCs, starting in the fetus, with a dramatic increase observed in the postnatal period.^{42 43 44 45} h-Caldesmon, typically found in differentiated smooth muscle, is a thin filament–binding protein involved in contractile processes as an actomyosin regulatory protein.^{46 47 48} Desmin, a muscle-specific intermediate filament protein, is expressed in striated muscle and only in half of the vascular human SMCs of large arteries.¹⁵ The expression of sm-MHC, h-caldesmon, and desmin is downregulated (or even lost) in proliferating SMCs during atherosclerosis development or in culture.^{17 40 41 47 49 50 51} However, confluent SMCs in serum-free medium undergo redifferentiation with an increase in smooth muscle contractile proteins.^{29 30}

Under our experimental conditions, the switch of medium induces a significant and time-dependent increase in both mRNA and protein content of sm-MHC, h-caldesmon, and desmin in postconfluent SMCs. However, the rise in mRNA expression of sm-MHC was faster (increasing within day 1) than that of h-caldesmon and desmin (increasing at day 3). These findings are in line with other observations on the reexpression of differentiation markers in culture at subconfluence.^{30 45 52} In this model of SMC differentiation by defined serum-free medium, the functions of VCAM-1 seem to be regulated by changes at mRNA and protein levels, although the functions of ₄ seem to be regulated via protein clustering and focal contact formation. Immunofluorescence staining could point out a reorganization in ₄ integrin membrane expression with a patchy distribution within the first day after the switch of medium, evoking a clustering of the integrin. This expression pattern is completed by day 3. This demonstrates that VCAM-1 expression and ₄ integrin redistribution occurs early in the SMC differentiation process.

Induction of differentiation markers in vascular SMCs has been shown to be induced by cell-cell contact and dissociated from cell cycle withdrawal.⁵³ Blocking VCAM-1–₄ interaction by specific antibodies inhibited the defined serum-free medium–induced precocious expression of sm-MHC and desmin mRNA, together with the expression of VCAM-1 mRNA. We used a mAb against ICAM-1 as a control antibody. ICAM-1–like VCAM-1 is an adhesion molecule that belongs to the immunoglobulin superfamily and has been described on SMCs in vitro and in vivo in atherosclerotic lesions.^{5 32} Neither the mAb against ICAM-1 nor the isotype-matched antibody affected sm-MHC and desmin expression. These results suggest that VCAM-1–₄ interactions are specifically involved in SMC differentiation.

In conclusion, we have shown the following: (1) ₄ and VCAM-1 are transiently expressed in human SMCs during vascular development; (2) VCAM-1 and ₄ are reexpressed in human SMCs in intimal atherosclerotic thickening; (3) in a cell culture model mimicking SMC redifferentiation, VCAM-1 expression and ₄ redistribution precede smooth muscle–specific variant expression of contractile and cytoskeletal proteins; and (4) blockade of VCAM-1–₄ interactions at the early phase of SMC redifferentiation affects the reexpression of smooth muscle markers. These results all point to a role for VCAM-1–VLA-4 interactions in the control of SMC differentiation. As strongly suggested by the in vitro experiment, the interaction between VCAM-1 and VLA-4 may be a prerequisite in pathological processes for redifferentiation of vascular SMCs and perhaps a mediator of SMC growth arrest in intimal hyperplasia.

	Selected Abbreviations and Acronyms

 

FACS = fluorescence-activated cell sorter GAPDH = human glyceraldehyde-3-phosphate dehydrogenase h-caldesmon = heavy-caldesmon HRP = horseradish peroxidase ICAM-1 = intercellular adhesion molecule-1 mAb = monoclonal antibody PCR = polymerase chain reaction sm-MHC = smooth muscle myosin heavy chain SMC = smooth muscle cell VCAM-1 = vascular cell adhesion molecule-1 VLA = very late antigen

Received May 29, 1996; accepted November 15, 1996.

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