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(Circulation Research. 1997;80:532-541.)
© 1997 American Heart Association, Inc.

Articles

Collagen VIII Is Expressed by Vascular Smooth Muscle Cells in Response to Vascular Injury

Nicholas E. S. Sibinga, Lauren C. Foster, Chung-Ming Hsieh, Mark A. Perrella, Wen-Sen Lee, Wilson O. Endege, E. Helene Sage, Mu-En Lee, , Edgar Haber

From the Cardiovascular Biology Laboratory, Harvard School of Public Health (N.E.S.S., L.C.F., C.-M.H., M.A.P., W.-S.L., W.O.E., M.-E.L., E.H.), the Department of Medicine, Harvard Medical School (N.E.S.S., M.A.P., M.-E.L., E.H.), and the Cardiovascular (N.E.S.S., M.-E.L.) and Pulmonary (M.A.P.) Divisions, Brigham and Women's Hospital, Boston, Mass, and the Department of Biological Structure, University of Washington School of Medicine (E.H.S.), Seattle, Wash.

Correspondence to Edgar Haber, MD, Cardiovascular Biology Laboratory, Harvard School of Public Health, 677 Huntington Ave, Boston, MA 02115. E-mail haber{at}cvlab.harvard.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract To identify genes involved in vascular remodeling, we applied differential mRNA display analysis to the rat carotid artery balloon injury model. One polymerase chain reaction product showing increased expression at days 2 to 14 after vascular injury was nearly identical to the mouse ₁ chain of type VIII collagen, a heterotrimeric short-chain collagen of uncertain function expressed by a limited number of cell types. By Northern analysis, expression of both chains of the type VIII collagen heterotrimer increased: collagen ₁ (VIII) mRNA expression was almost 4-fold higher than control by 7 days after vascular injury, and collagen ₂ (VIII) mRNA expression reached a maximum of almost 6-fold above baseline by 3 days after injury. By immunohistochemical analysis, type VIII collagen expression increased in the media and neointima in a localized pattern consistent with the distribution of activated dedifferentiated vascular smooth muscle cells (VSMCs). Cultured VSMCs expressed higher levels of type VIII collagen in response to serum and growth factors, notably platelet-derived growth factor (PDGF)-BB. VSMCs adhered significantly less to type VIII collagen than to type I collagen substrata and showed greater PDGF-BB–stimulated migration (by 2.2-fold) on type VIII collagen than on type I collagen. We hypothesize that increased expression of type VIII collagen by VSMCs after arterial injury may contribute to vascular remodeling through the promotion of VSMC migration.

Key Words: cardiovascular disease • artery • gene expression • extracellular matrix • cell movement

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The VSMC plays a central role in the pathogenesis of the most prevalent and costly diseases in developed societies, which include hypertension, peripheral and cerebral vascular disease, and coronary artery disease. Pathological VSMC activity, manifested as increased proliferation, migration, and synthesis of ECM proteins, is even more apparent in the syndromes of accelerated arteriosclerosis seen after coronary artery bypass surgery, cardiac transplantation, and coronary angioplasty. Despite intensive investigation, the factors controlling these fundamental VSMC activities remain incompletely understood. To identify new molecular species that might affect VSMC behavior during vascular remodeling, we applied differential mRNA display¹ to an in vivo model (RCBI) in which these VSMC activities have been described well.²

The response of VSMCs to injury is remarkable for a striking change in phenotype: quiescent contractile cells relatively stationary in the vessel wall start to proliferate, migrate from the media through the internal elastic lamina to the neointimal space, and synthesize more protein.² Over time, the neointima increases in volume and, by encroaching on the vessel lumen, may significantly compromise its function as a conduit. Synthetic activity by VSMCs after injury is particularly notable for changes in the expression of ECM proteins.³

ECM molecules can affect the growth and migration of a number of cell types, including VSMCs.^{4 5} Thrombospondin, a glycoprotein found in ECM from many tissues, promotes the progression of VSMCs through the cell cycle⁶ and leads to serum- and anchorage-independent growth when expressed at high levels in cultured fibroblasts,⁷ yet thrombospondin is not tumorigenic, possibly because it also negatively regulates angiogenesis.⁸ Type IV collagen, another ECM protein, may regulate smooth muscle differentiation and morphogenesis, as diffuse leiomyomatosis has been described in patients with chromosomal deletions that disrupt the genes encoding both the ₅ (IV) and ₆ (IV) collagen chains.⁹ A third ECM molecule, the glycoprotein osteopontin, contains an arginine-glycine-aspartate motif, is expressed at higher levels after vascular injury,¹⁰ and promotes directional VSMC migration in vitro.¹¹

Cell surface receptors that bind ECM proteins also have effects on cellular function. We recently reported that the interaction between hyaluronate and CD44, a cell surface protein expressed at higher levels after arterial injury, leads to increased VSMC proliferation.¹² Overexpression of RHAMM, another hyaluronate receptor, leads to transformation and development of a metastatic phenotype in fibroblasts,¹³ and it may also be important for VSMC migration.¹⁴ VSMC migration on collagen substrates has been shown to depend on expression by VSMCs of both the ₁ß₁¹⁵ and the ₂ß₁¹⁶ integrin receptors, and the above-mentioned chemotactic effect of osteopontin may depend on expression by VSMCs of the _vß₃ integrin.¹¹

Molecules that change the structural properties of the ECM, either by modifying the function of ECM proteins or by degrading them, may also affect cellular activity in normal development and in the pathogenesis of disease. For example, a truncated form of tropoelastin lacking the carboxy terminus is more soluble than full-length tropoelastin; production of truncated tropoelastin by VSMCs probably reduces the assembly of elastic laminae and thereby facilitates the migration of VSMCs that is necessary for the normal intimal thickening and closure that occurs in the ductus arteriosus.¹⁷ On the other hand, metalloproteinases produced by VSMCs, such as stromelysin, gelatinase, and collagenase (see reviews in References 18 through 20^{18 19 20} ), may impair ECM structural integrity, permit migration of VSMCs from the media across the basement membrane after vascular injury,^{21 22} and contribute to plaque rupture in arteriosclerotic lesions.^{23 24}

Type VIII collagen is a short-chain collagen thought to play a role in angiogenesis and cardiac morphogenesis. In the present report, we describe a marked increase in its expression by VSMCs in the injured arterial wall.* Moreover, the pattern of increased expression of type VIII collagen after injury that we found correlates well with the known distribution of VSMC activation in the RCBI model. In cultured VSMCs, expression of type VIII collagen mRNA increased in response to growth factors known to promote formation of neointima (especially in response to PDGF-BB, a potent stimulant of VSMC migration). Further cell culture analyses showed that VSMC adhered less to purified type VIII collagen than to type I collagen and migrated to a greater extent on type VIII collagen than on type I collagen. Taken together, these data suggest that the increased expression of type VIII collagen by VSMCs after injury may promote formation of neointima by facilitating VSMC migration.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Rat Carotid Artery Balloon Injury
Male Sprague-Dawley rats (Charles River Laboratories, Wilmington, Mass) weighing 350 to 400 g were anesthetized with ketamine (40 mg/kg) and xylazine (5 mg/kg). The left common carotid artery was denuded of endothelium and stretched by three passages of a 2F embolectomy catheter according to standard protocols.³ For some studies, balloon-injured Sprague-Dawley rats were also obtained from the Zivic-Miller Co (Zelienople, Pa). At specified time points after injury, animals were reanesthetized and killed, and both right (uninjured control) and left (injured) carotid arteries were harvested and snap-frozen in liquid nitrogen.

RNA Extraction and Differential mRNA Display
Total RNA was obtained from tissues by homogenization in RNazol B (BIOTECX) or from cultured cells by extraction with guanidinium isothiocyanate, followed by centrifugation through cesium chloride.²⁶ Differential mRNA display¹ was performed according to the protocol provided with the GenHunter kit (GenHunter Corp) by using 0.2 µg total RNA in a 20 µL reaction for reverse transcription with anchored oligo-dT primers. Random decamer oligonucleotides used for cDNA amplification were obtained from Operon. The PCR thermal cycling steps were 94°C for 15 seconds, 40°C for 30 seconds, and 72°C for 30 seconds for a total of 40 cycles. The resultant heterogeneous cDNA products were separated on a 6% DNA sequencing gel. Bands of the gel that appeared to correspond to regulated genes were excised, rehydrated, and amplified in a single-step 80 µL reaction for 40 cycles using conditions and primers identical to those used in the first PCR reaction. PCR products were analyzed by electrophoresis through 1.5% agarose gels stained with ethidium bromide and were subcloned into the pCRII vector with the TA cloning system (Invitrogen). Taq polymerase and [-³⁵S]dATP used in the differential display analysis were supplied respectively by Boehringer-Mannheim and Dupont/New England Nuclear.

DNA Sequencing and Homology Searching
PCR products were ligated into the pCRII cloning vector and were sequenced in both directions by the dideoxynucleotide chain termination technique.²⁶ Sequence homology searching was performed through the GenBank.

RNA Blot Hybridization
Total RNA (5 µg from tissues, 10 µg from cultured cells) was fractionated on 1.2% formaldehyde-agarose gels and then transferred to nitrocellulose filters. The filters were hybridized at 68°C for 2 hours with random-primed ³²P-labeled cDNA probes in QuikHyb solution (Stratagene). The 448-bp rat collagen ₁ (VIII) cDNA fragment was derived by differential mRNA display cloning, whereas the 324-bp rat collagen ₂ (VIII) fragment was generated by PCR with primers derived from regions showing (1) high homology to published human and mouse ₂ chain sequences and (2) low homology to published ₁ chain sequences (sense, 5'-TTCCCTGCCTCAGGCATGCC-3'; antisense, 5'-AGTGGATGTACTCCGTGGAGT-3'). The rat collagen ₁ (I) cDNA fragment was cloned by using primers designed according to the published mouse collagen ₁ (I) sequence²⁷ (sense, 5'-GGACCCCGAGGAAACAAT-3'; antisense, 5'-ACCACGGTCGCCATTCTT-3'); homology was confirmed by nucleotide sequencing. Hybridized filters were washed in 30 mmol/L sodium chloride, 3 mmol/L sodium citrate, and 0.1% SDS at 55°C and autoradiographed with Kodak XAR film for 24 to 72 hours or stored on phosphor screens for 12 to 16 hours. The filters were stripped in a 50% formamide solution at 80°C and rehybridized. An oligonucleotide (5'-ACGGTATCTGATCGTCTTCGAACC-3') complementary to 18S rRNA was end-labeled with ³²P and was hybridized to the filters to correct for differences in RNA loading. Phosphor screens were scanned, and radioactive signal intensity was measured on a PhosphorImager running the ImageQuant software (Molecular Dynamics).

Cell Culture and Cytokine Stimulation Studies
Arterial smooth muscle cells were harvested from the aortas of male Sprague-Dawley rats (200 to 250 g, Charles River Laboratories) by enzymatic dissociation.²⁸ The cells were cultured in DMEM (JRH Biosciences) and supplemented with 10% FCS (HyClone), 100 U/mL penicillin, 100 µg/mL streptomycin, and 10 mmol/L HEPES (pH 7.4) (Sigma Chemical Co). Rat VSMCs were passaged every 3 to 5 days, and experiments were performed on cells four to six passages from primary culture. Basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor I, and PDGF-BB were obtained from Collaborative Biomedical; tumor necrosis factor-, from Genzyme; and angiotensin II, ß-estradiol, dexamethasone, and prostaglandin E1, from Sigma. Recombinant rat interferon gamma was supplied by GIBCO-BRL. Transforming growth factor-ß1 was a gift from Bristol-Myers Squibb (Princeton, NJ). All cytokines and growth factors were dissolved according to the supplier's recommendations. For cytokine stimulation experiments, the FCS concentration was reduced to 2%, and cytokines or growth factors were added to the medium 16 hours after dilution. Control cultures received an equivalent amount of vehicle. Cells were harvested for RNA extraction 24 hours after the addition of cytokine or growth factor or at specified time points for time-course studies.

Immunohistochemistry
Adult male Sprague-Dawley rats were killed 4, 7, and 16 days after balloon injury. Carotid arteries were removed, fixed with 4% paraformaldehyde, and processed for paraffin embedding in an automated system. Polyclonal antiserum raised against native bovine type VIII collagen²⁹ was diluted 1:400 and incubated with 5-µm sections of the artery. Secondary antibody (biotinylated goat anti–guinea pig IgG, Vector Laboratories) was applied at a 1:1000 dilution. Staining was developed by the avidin-biotin–horseradish peroxidase method with 3,3'-diaminobenzidine used as chromogen. Positive staining was evinced by a brown color. Specificity of staining was ensured by incubation with the secondary antibody only.

Cell Adhesion Assays
Lyophilized rat-tail type I collagen (Collaborative Biomedical) was dissolved in dilute acetic acid according to the supplier's recommendations. Purified type VIII collagen protein (50-kD fragment) from bovine corneal subendothelium¹⁰ was dissolved in dilute acetic acid. Collagen concentrations were adjusted to 4 µg/mL, and 1 µg of protein was added to each well of a 24-well culture plate (Falcon). Plates were incubated overnight at 4°C and subsequently at room temperature for 1 hour before they were washed three times with PBS. For adhesion assays, VSMCs growing in 10% FCS–supplemented DMEM were harvested with a trypsin solution diluted 1:5 in PBS, washed with PBS, counted on a Coulter apparatus (Coulter Co), and resuspended in DMEM/2.5% bovine serum albumin. After the cells had been allowed to recover from harvesting for 60 minutes at 37°C, 50 000 cells in 1 mL were added to each well. Cells were allowed to adhere for 2 hours at 37°C. The medium was then aspirated, and each well was washed three times with 1 mL of PBS; the number of nonadherent cells was assessed by pooling the medium and washes for each well and counting the recovered cells on a Coulter apparatus. Analysis for each collagen chain was performed in triplicate; statistical analysis was by the unpaired t test (StatView analysis software, Abacus Concepts).

Cell Migration Assays
Migration of VSMCs on ECM proteins was assessed on Transwell polyethylene terephthalate cell culture inserts with 8-µm pores (Becton Dickinson). Substrate proteins were dissolved as described above, but the final concentrations were adjusted to 20 µg/mL. Each insert was coated with 5 µg of protein, incubated overnight at 4°C, and incubated at room temperature for 1 hour before being washed three times with PBS. For migration assays, VSMCs were cultured in 0.4% calf serum for 48 hours, harvested, washed, counted as described above, and resuspended in DMEM/2.5% bovine serum albumin at a concentration of 1x10⁶ cells/mL. Cells were allowed to recover from harvesting for 60 minutes at 37°C; 250 000 cells were subsequently added to each Transwell insert. Directed migration was stimulated by the addition of 400 µL of DMEM with 20 ng/mL PDGF-BB; cells were allowed to migrate for 4 hours at 37°C. Insert filters were fixed and stained by using the Hema 3 kit (Curtin Matheson); cells that did not migrate were removed from the top of the filter. To measure migration, we counted the number of nuclei on the underside of the filter under a Nikon Labophot-2 microscope. Ten randomly selected x20 fields were counted and averaged for each filter. Statistical analysis was by the unpaired t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Differential mRNA Display and Cloning
To identify genes involved in vascular remodeling, we applied differential mRNA display analysis to the RCBI model. A total of 35 bands that increased or decreased in density between days 2 and 14 after RCBI were excised and used as probes for Northern analysis of RNAs from injured carotid arteries and cultured quiescent or 10% FCS–stimulated VSMCs; of these, 24 identified RNAs showed no significant change in expression or gave no signal. The remaining 11 bands identified RNAs with changing levels of expression in Northern analysis, and their cDNAs were cloned and sequenced: four sequences showed no significant match on GenBank BLASTN and BLASTX searches, and one sequence identified an expressed sequence tag. Searches with five of the six remaining cDNAs identified homologies to the following sequences: rat senescence marker protein, rat 45S rRNA fragment (notable for polyadenylate sequences that presumably allowed its reverse transcription with oligo-dT primers), rat leukocyte common antigen, tropoelastin, and aldehyde dehydrogenase.

The sixth PCR product showed increased expression at days 2 to 14 after vascular injury (Fig 1). It was cloned and sequenced and found to have high homology to the ₁ chain of mouse type VIII collagen, an unusual collagen expressed by a limited number of tissues and thought to play a role in angiogenesis and cardiac morphogenesis.^{30 31 32} Two type VIII collagen chains, designated ₁ and ₂, have been identified.^{33 34} Because native type VIII collagen is thought to have a heterotrimeric form with a chain stoichiometry of (₁)₂:(₂)₁, we also used PCR to clone a fragment of the rat ₂ chain. This allowed us to study the expression of both chains after vascular injury. The nucleotide sequences of the rat ₁ and ₂ cDNA fragments, aligned to their respective mouse homologues, are shown in Fig 2. The level of nucleotide identity between species is 92.0% (358/389) for the ₁ chain fragment and 94.8% (308/325) for the ₂ chain fragment.

View larger version (49K): [in this window] [in a new window]   Figure 1. Differential mRNA display. Balloon-injured rat carotid arteries were harvested for RNA extraction from 6 hours to 14 days after injury. Control RNA was obtained from uninjured carotid arteries. Differential mRNA display analysis was performed as described in "Materials and Methods," and 35S-labeled PCR amplification products were resolved on a 6% denaturing polyacrylamide gel. The autoradiograph was used to localize band G2a (indicated with a line), which was then excised, eluted, reamplified, and ligated into a plasmid cloning vector for sequencing.

View larger version (49K): [in this window] [in a new window]   Figure 2. Nucleotide sequence analysis. DNA fragments cloned into the pCRII plasmid vector were sequenced in both directions using the dideoxynucleotide chain termination technique. Shown are sequence alignments with mouse type VIII collagen homologues identified by GenBank searching. A, The sequence of fragment G2a (upper line), identified from rat carotid artery RNA in differential display, aligned with mouse collagen 1 (VIII) (lower line). The box indicates the 6-nt sequence shared by the type VIII collagen fragment with the 5' primer used in differential display. The line above the sequence indicates the putative polyadenylation signal. B, The 325-nt fragment of rat collagen 2 (VIII) (upper line), amplified by reverse-transcription PCR from RNA from cultured rat VSMCs, aligned with the mouse homologue (lower line). The lines above the sequence indicate oligonucleotide primer sites.

Northern Analysis of Rat Carotid Artery Injury
Northern analysis of rat carotid artery specimens confirmed the increase in type VIII collagen mRNA expression after injury (Fig 3). In uninjured vessels (day 0), expression of both type VIII collagen chains was barely detectable. Both mRNAs were induced substantially after injury: collagen ₁ (VIII) mRNA levels increased 4-fold above baseline from days 7 to 14 after vascular injury, whereas collagen ₂ (VIII) mRNA levels were 2.5- to 6-fold greater than baseline between days 3 and 14. For reference, we also performed Northern analysis with a probe for the collagen ₁ (I) mRNA, which encodes a major structural component of the vascular wall.³⁵ In contrast with the expression pattern for type VIII collagen mRNA, collagen ₁ (I) mRNA was relatively abundant in the uninjured artery and after injury showed at most a 1.3-fold increase above baseline.

View larger version (56K): [in this window] [in a new window]   Figure 3. RNA blot analysis of collagen (Col) VIII expression in balloon-injured rat carotid arteries. Carotid arteries were harvested for RNA extraction at 0, 2, 3, 7, and 14 days after injury. Total RNA (5 µg) was fractionated for RNA blot analysis as described in "Materials and Methods." Nitrocellulose filters were hybridized to radiolabeled probes, washed in 30 mmol/L sodium chloride, 3 mmol/L sodium citrate, and 0.1% SDS at 55°C, and autoradiographed for 1 to 3 days at –80°C. Filters were reexposed to phosphor screens for 12 to 16 hours to quantify band density, as presented in the bar graph. The estimated sizes of bands identified are as follows: Col 1 (VIII), 5.5 and 4.2 kb (a minor band at 2.7 kb is not shown); Col 2 (VIII), 4.5 kb; and Col 1 (I), 6.0 and 4.7 kb.

Immunohistochemistry
To localize expression of type VIII collagen in the injured rat carotid artery, we performed immunohistochemical analysis with a polyclonal antiserum raised against native bovine type VIII collagen.³² This antiserum has been used to identify type VIII collagen expression in a number of species, including mouse, chicken, and human.^{32 36} Consistent with our observations by Northern analysis, type VIII collagen expression in uninjured rat carotid arteries was minimal (Fig 4A). In contrast, specific staining for type VIII collagen in sections from injured arteries showed robust but localized expression of type VIII collagen. Type VIII collagen–specific staining was present in the media 4 days after injury (Fig 4B), a point associated with the medial VSMC proliferation² that immediately precedes the large-scale migration of VSMCs from the media to the neointima.³⁷ Type VIII collagen expression was visible in the two medial layers closest to the lumen (Fig 4B) and was particularly high in cells adjacent to one of the elastic laminae (arrow). At 7 days after injury (Fig 4C), staining for type VIII collagen was visible throughout the neointima, whereas by 16 days after injury (Fig 4D), this staining was limited to that part of the neointima closest to the lumen.

View larger version (129K): [in this window] [in a new window]   Figure 4. Immunohistochemical analysis of type VIII collagen expression after injury of the rat carotid artery. Specimens were prepared as described in "Materials and Methods." Sections (5 µm) were incubated at room temperature for 1 hour and then overnight with a 1:400 dilution of polyclonal antiserum against purified bovine type VIII collagen, washed extensively, and incubated with a 1:1000 dilution of secondary antibody. Slides were developed by the avidin–biotin–horseradish peroxidase method. Red-brown color marks type VIII collagen immunoreactivity. A, Control (uninjured) carotid artery. B, Artery 4 days after injury. C, Artery 7 days after injury. D, Artery 16 days after injury. Specificity of staining was confirmed by incubation of the sections with secondary antibody alone (not shown). Arrows indicate the internal elastic lamina. Original magnification x400.

Serum, Cytokine, and Growth Factor Stimulation
The immunohistochemical studies implicated VSMCs as the likely source of substantial type VIII collagen expression during vascular remodeling. Type VIII collagen immunoreactivity has been identified in vascular subendothelium^{31 32} and in the tunica media of developing large vessels during embryogenesis.³² To further explore the possibility that VSMCs produce type VIII collagen, we assessed its expression in serum and in growth factor– and cytokine-stimulated rat VSMCs from primary culture. For the initial studies, these cells were made quiescent by incubation for 72 hours with medium containing 0.4% calf serum and were subsequently stimulated to grow by addition of medium containing 10% FCS. This treatment resulted in a 3.5-fold increase in the level of collagen ₁ (VIII) mRNA by Northern analysis (Fig 5A); the increase was maximal by 18 hours after stimulation. In contrast to our findings in vivo (Fig 3), expression of collagen ₂ (VIII) mRNA in cultured VSMCs was relatively difficult to detect by Northern analysis; therefore, we concentrated our efforts in vitro on evaluation of the expression of the ₁ (VIII) mRNA. We tested several biological molecules of potential importance during the vascular response to injury to identify specific factors that affect expression of type VIII collagen (Fig 5B). In cultured VSMCs, expression of collagen ₁ (VIII) mRNA increased after exposure to angiotensin II (1.52xcontrol), ß-estradiol (1.38xcontrol), basic fibroblast growth factor (1.28xcontrol), PDGF-BB (2.35xcontrol), and prostaglandin E₁ (1.46xcontrol) but decreased after treatment with dexamethasone (0.56xcontrol) and interferon gamma (0.6xcontrol). Lesser effects on expression were seen after treatment with transforming growth factor-ß1 (1.18xcontrol), epidermal growth factor (0.93xcontrol), insulin-like growth factor I (0.86xcontrol), and tumor necrosis factor- (0.84xcontrol). Time-course and dose-response studies of collagen ₁ (VIII) mRNA induction by PDGF-BB demonstrated a maximal response at 24 hours after treatment with 20 ng/mL (Fig 5C); in three separate experiments, the mean induction 24 hours after addition of this dose of PDGF-BB was 2.21±0.13xcontrol (P=.0008). A similar degree of induction was seen at doses ranging from 10 to 40 ng/mL (Fig 5C).

View larger version (55K): [in this window] [in a new window]   Figure 5. Expression of collagen (Col) VIII in cultured VSMCs. RNA blot hybridization (see "Materials and Methods") was performed with 10 µg of RNA per lane. After hybridization to radiolabeled type VIII collagen probes, both filters were hybridized to an 18S oligonucleotide probe to correct for variations in RNA loading. All signals were analyzed quantitatively by exposure to phosphor screens; normalized band densities are displayed in the bar graphs as a percentage of control. A, Growing rat VSMCs were made quiescent by incubation in medium containing 0.4% calf serum for 72 hours and then stimulated with medium containing 10% FCS until RNA extraction at the specified time points. A film-processing artifact appears as a horizontal line just under the upper Col 1 (VIII) band in the 32-hour lane. B, Growing rat VSMCs were incubated in medium containing 2% FCS for 16 hours, and then growth factors or cytokines were added to final concentrations as follows: angiotensin II (AT), 1 µmol/L; ß-estradiol (EST), 0.1 µmol/L; basic fibroblast growth factor (bFGF), 20 ng/mL; dexamethasone (DEX), 1 µmol/L; epidermal growth factor (EGF), 1 ng/mL; interferon gamma (IFN), 300 U/mL; insulin-like growth factor-I (IGF), 20 ng/mL; PDGF-BB, 20 ng/mL; prostaglandin E1 (PGE), 100 ng/mL; transforming growth factor-ß1 (TGFß), 10 ng/mL; and tumor necrosis factor- (TNF), 50 ng/mL. RNA was extracted 24 hours after addition of growth factors or cytokines for use in Northern analysis. C, Growing rat VSMCs were incubated in medium containing 2% FCS for 16 hours, PDGF-BB was added to the final concentrations indicated, and cells were harvested after 24 hours (upper panel), or PDGF-BB was added to a final concentration of 20 ng/mL, and cells were harvested at the time points indicated (lower panel).

Cell Adhesion and Migration
Of the growth factors and cytokines we evaluated, PDGF-BB was the most potent inducer of type VIII collagen expression by cultured VSMCs (Fig 5B). In addition to its mitogenic effects, PDGF-BB has potent promigratory effects on VSMCs both in vitro and in vivo.³⁸ Thus, we hypothesized that the increased expression of type VIII collagen after vascular injury, probably caused by locally active growth factors, might promote VSMC migration and therefore be important in vascular remodeling. Because the strength of adhesive interactions between cell and substrate has a critical effect on migration,³⁹ we first compared adhesion of cultured VSMCs to type I collagen– and type VIII collagen–coated wells. For both adhesion and migration studies, we used a highly purified fragment of type VIII collagen that includes its collagenous domain; in this preparation, the noncollagenous domains at either end have been removed by pepsin treatment during the purification protocol.⁴⁰ Cells were allowed to adhere to substrate-coated wells for 2 hours; subsequently, wells were washed with three changes of PBS to collect nonadherent cells. The number of nonadherent cells recovered from type VIII collagen–coated wells was nearly 5-fold greater than that recovered from type I collagen–coated wells (Fig 6A). We then determined whether this difference in VSMC adhesion between type VIII collagen and type I collagen was also reflected by a difference in cell migration on the two substrates.

View larger version (19K): [in this window] [in a new window]   Figure 6. Adhesion and migration of rat VSMCs on collagen (Col)-coated substrates. A, Proteins were dissolved in dilute acetic acid and used to coat wells of a 24-well culture plate. Cells (50 000 per well) were allowed to adhere for 2 hours at 37°C. Medium was aspirated, and each well was washed three times; nonadherent cells were counted by pooling the medium and washes from each well and counting the recovered cells in a Coulter apparatus. Analysis for each collagen was performed in triplicate. The data shown are representative of two separate experiments. *P<.0001. B, Migration of VSMCs on ECM proteins was assessed in a Transwell cell culture system. As described in "Materials and Methods," inserts were coated with 5 µg of protein. VSMCs (250 000) were added to each upper chamber, and migration was stimulated by adding PDGF-BB (20 ng/mL) to the bottom chamber. After 4 hours, filter inserts were fixed and stained. Nuclei on the underside of the filter (ie, cells that had migrated) in 10 randomly selected x20 fields were counted, and the values were averaged for each filter. The data shown here were combined from two separate experiments (n=5 per condition). Statistical analysis was by unpaired t test. *P<.002.

We used a Transwell system⁴¹ to evaluate cell migration on purified collagen type I and VIII substrates. Compared with the standard 48-well Boyden chamber unit, the Transwell system permits use of substantially smaller amounts of substrate protein to coat membranes, a practical advantage when a limited amount of protein is available. Nuclei of cells appearing on the underside of the porous filter separating the upper and lower chamber of this apparatus were counted, and the values were averaged and analyzed as described in "Materials and Methods." VSMCs in wells coated with type VIII collagen migrated toward a PDGF-BB gradient to a substantially greater degree (2.2-fold mean difference, P<.002) compared with those in wells coated with type I collagen (Fig 6B).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have identified an increase in the expression of type VIII collagen after vascular injury. By differential mRNA display analysis, an autoradiographic band corresponding to collagen ₁ (VIII) mRNA did not appear in samples taken from uninjured arteries but did appear in samples taken as early as 3 days after injury (Fig 1). By Northern analysis, strong expression of collagen ₁ (VIII) mRNA was first seen 7 days after carotid artery injury, and at 14 days, the amount of mRNA was still substantially greater than at baseline (Fig 3). We also used conventional PCR to generate a cDNA probe for the ₂ chain of rat type VIII collagen (Fig 2B) and evaluated its expression by Northern analysis. The level of rat collagen ₂ (VIII) mRNA was highest at 3 days after injury and declined somewhat at days 7 and 14 (Fig 3).

Thus, expression of the two type VIII collagen mRNAs increased after arterial injury, albeit with slightly different kinetics: the ₂ (VIII) mRNA showed an earlier and greater induction relative to the ₁ (VIII) mRNA. The stoichiometry of the native type VIII collagen chains is thought to be (₁)₂:(₂)₁.⁴² Although the similarity between the ₁ and ₂ chains in size and domain structure suggests that trimers with ratios other than 2:1 might be stable, there is no direct evidence of alternative forms. Further investigation with chain-specific antibodies would be necessary to determine whether the difference in mRNA expression patterns is reflected by a difference in protein expression patterns and should give insight into the biological significance of this differential mRNA induction.

To compare the induction of type VIII collagen with the regulation of other collagens after arterial injury, we also evaluated expression of a major fibrillar collagen, type I collagen. Relative to the two type VIII collagen mRNAs, collagen ₁ (I) mRNA was expressed at a high level in the uninjured artery and showed only a modest increase after injury (Fig 3). Type I collagen plays an essential role in maintaining the structural integrity of normal arteries³⁵ ; the pattern of expression of type VIII collagen that we found suggests that it may have a limited function in the normal arterial wall and a more significant role in the response of blood vessels to injury.

To localize expression of type VIII collagen within the vessel wall, we performed immunohistochemical analysis. Consistent with our Northern analysis, uninjured carotid arteries showed very limited expression of type VIII collagen (Fig 4A). By 4 days after injury, however, type VIII collagen was clearly present. Positive staining was seen in cells in expanded layers of the arterial media; in the example shown (Fig 4B), staining was particularly strong in cells at the base of a layer adjacent to the vessel lumen, with fainter staining in layers farther from the lumen. By 7 days after injury, type VIII collagen staining was present throughout the neointima, with relatively less staining in the medial layers. By day 16, staining was limited to cells on the luminal aspect of the neointima (Fig 4C and 4D). In the RCBI model, the thymidine labeling index (representing cellular replication) is maximal in the media at day 2 after injury and in the neointima at day 4.² Proliferative activity is still substantial in the media and neointima at 7 days after injury, but by 14 days, the overall level of activity decreases greatly, and residual activity is confined to the inner layers of the neointima.² We have found a similar spatial and temporal distribution of cells staining positive for another marker of proliferation, proliferating-cell nuclear antigen (data not shown). Thus, the distribution of type VIII collagen expression we found by immunohistochemical analysis in the RCBI model correlates with that of dedifferentiated proliferative VSMCs previously defined in this model.

To identify factors potentially responsible for the induction of type VIII collagen expression after vascular injury, we assessed the effect on type VIII collagen expression of serum and specific growth factors and cytokines. Collagen ₁ (VIII) mRNA was readily apparent in cultured quiescent VSMCs, a notable difference from its low level of expression in the uninjured carotid artery. Nevertheless, its expression increased markedly (up to 3.5-fold above baseline) through the first 12 to 18 hours after treatment with 10% FCS (Fig 5A). In an attempt to better approximate the state of VSMC quiescence in the uninjured artery, we also cultured VSMCs on Matrigel⁴³ ; to our surprise, the VSMCs expressed collagen ₁ (VIII) mRNA at a slightly higher level than did VSMCs grown on standard plastic culture dishes (data not shown), possibly because of the presence of PDGF-BB and transforming growth factor-ß1 in the Matrigel.⁴⁴ Although collagen ₂ (VIII) mRNA was readily identifiable by Northern analysis after arterial injury (Fig 3), its expression in cultured VSMCs was relatively low; therefore, we focused our subsequent efforts in vitro on evaluating the expression of the ₁ (VIII) mRNA.

The expression of collagen ₁ (VIII) mRNA in VSMCs growing in 2% FCS was robust (Fig 5B), and it was increased modestly by several growth factors, including angiotensin II, ß-estradiol, basic fibroblast growth factor, PDGF-BB, and prostaglandin E₁. PDGF-BB, which increased collagen ₁ (VIII) mRNA by >2.2-fold in multiple analyses, had the largest effect. This effect was both dose and time dependent (Fig 5C). Collagen ₁ (VIII) mRNA expression decreased in VSMCs treated with dexamethasone and interferon gamma (Fig 5B), which have been reported to inhibit the synthesis of other types of collagen.^{45 46} Of the factors that increased expression of collagen ₁ (VIII) mRNA, angiotensin II,^{19 20 21} basic fibroblast growth factor,^{22 23} and PDGF-BB⁴⁷ have been identified as important stimuli of VSMC growth after arterial injury. PDGF-BB, in addition to its mitogenic effects on VSMCs, has also been implicated as a major stimulus of VSMC migration after arterial injury.³⁸

The increased expression of type VIII collagen in response to these growth factors suggests that it may have effects on cellular proliferation or migration. Although the stimulatory effects of growth factors, including PDGF-BB, can result largely from direct effects on cellular growth–related⁴⁸ or migration-related^{49 50} signaling pathways, these factors can also mediate indirect effects by altering expression of cell surface receptors that bind to ECM proteins or by altering expression of secreted proteins that make the ECM environment more favorable for cellular growth or movement. For example, increased expression of tenascin, an ECM protein with antiadhesive properties,⁵¹ after VSMCs have been exposed to angiotensin II⁵² and PDGF-BB⁵¹ can contribute to enhanced VSMC migration. Osteopontin, a secreted glycoprotein that acts as a ligand for the integrins¹¹ and CD44,⁵³ promotes directional VSMC migration in vitro; increased expression of osteopontin after vascular injury may contribute to VSMC migration in vascular remodeling. Last, we have reported recently that the interaction between hyaluronate and CD44, a cell surface protein expressed at higher levels after arterial injury, leads to increased VSMC proliferation.¹² Oligosaccharides of hyaluronate have been reported to increase type VIII collagen expression in endothelial cells⁵⁴ ; although we have not tested it directly, it is conceivable that metabolites of hyaluronate may also increase type VIII collagen expression by endothelial cells or VSMCs in the remodeling artery.

To better understand the biological significance of increased type VIII collagen expression after vascular injury, we tested the effect of type VIII collagen on VSMC proliferation, adhesion, and migration. For these assays, we used a highly purified, 50-kD, pepsin-resistant collagen type VIII fragment.³⁰ Pepsin treatment is required to solubilize and purify the protein, which tends to copurify with type V collagen. As a result of treatment with pepsin, this form of type VIII collagen lacks the native protein's amino- and carboxy-terminal noncollagenous domains. Thus, it is possible that increased type VIII collagen expression in the injured arterial wall may affect VSMCs somewhat differently from what we have found in this cell culture system. On the other hand, given the susceptibility of type VIII collagen to protease degradation⁵⁵ and the reported increase in expression of degradative enzymes after vascular injury,²² it is also possible that a form of type VIII collagen similar to the 50-kD purified fragment may be present in the vessel wall during remodeling.

We first tried to demonstrate an effect of purified type VIII collagen (50 kD) on VSMC proliferation in culture. Repeated [³H]thymidine uptake assays at high- and low-serum concentrations showed no significant differences from control after supplementation of the culture medium or coating of the plastic wells with type VIII collagen (data not shown). However, type VIII collagen was expressed at relatively high levels by unstimulated cultured VSMCs, both by Northern analysis (Fig 5A and 5B) and by immunohistochemical analysis (authors' unpublished data, 1996). Because baseline type VIII collagen concentrations are high in culture, they could mask a proliferative effect due to additional type VIII collagen. It is also possible that a proliferative effect may be mediated through the noncollagenous domains, NC1 and NC2, that are missing in the 50-kD preparation of type VIII collagen. Thus, our experiments in culture cannot exclude an effect of type VIII collagen on VSMC growth.

We next assessed the effect of type VIII collagen on VSMC adhesion. Plastic wells were coated with equal quantities of purified type I collagen or type VIII collagen (50 kD). Fewer cells adhered to wells coated with type VIII collagen than with type I collagen (Fig 6A). We then hypothesized that the less adhesive quality of type VIII collagen could affect VSMC migration by changing the adhesive properties of the ECM. We first introduced type I or type VIII collagen (50 kD) into Matrigel, added VSMCs, and observed cell movement. VSMCs cultured in Matrigel plus type VIII collagen showed greater migration than did VSMCs cultured in Matrigel plus type I collagen, as assessed in serial photographs of the same microscopic fields over 24 to 48 hours (data not shown). To permit a quantifiable assessment of migration and to minimize the potentially confounding effects of ongoing collagen synthesis or the degradation of added collagens, we then used a Transwell system that allowed counting of cells that had migrated 4 hours after plating. We found that compared with type I collagen coating of migration chamber inserts, type VIII collagen coating supported a higher level of migration toward a PDGF-BB source (Fig 6B).

Because the strength of cellular attachment to various ECM proteins affects VSMC migration in vitro,³⁹ changes in the adhesiveness of the ECM environment are likely to affect VSMC migration in the injured arterial wall. Optimal VSMC migration reportedly occurs at intermediate attachment strengths.³⁹ We found that VSMCs adhered less to type VIII collagen than to type I collagen and that they migrated more readily on type VIII collagen than on type I collagen. Taken together, these findings suggest that the observed increase in expression of type VIII collagen by VSMCs after vascular injury may diminish adhesive interactions in the local neointimal environment and, as a consequence, may facilitate migration of VSMCs.

VSMC migration is an important component of the arterial wall's response to injury. We have found distinct patterns of expression of type I and type VIII collagens after arterial injury and have characterized the different effects of these collagens on the fundamental VSMC activities of adhesion and migration. The present study provides a new example of how VSMCs change gene expression after vascular injury and suggests how this altered expression may contribute to the observed increase in VSMC migration in this setting.

	Selected Abbreviations and Acronyms

 

ECM = extracellular matrix PCR = polymerase chain reaction PDGF = platelet-derived growth factor RCBI = rat carotid artery balloon injury VSMC = vascular smooth muscle cell

	Acknowledgments

 
This study was supported in part by National Institutes of Health grants K08 HL-03274 (Dr Sibinga), K08 HL-03194 (Dr Perrella), and RO1 GM-53249 (Dr Lee) and by a grant from the Bristol-Myers Squibb Pharmaceutical Research Institute. We wish to thank Dorothy Zhang for histological studies, Bonna Ith for technical assistance, and Thomas McVarish for editorial assistance.

	Footnotes

 
This manuscript was sent to Leslie A. Leinwand, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

¹ While this manuscript was in review, Bendeck et al²⁵ reported a similar identification, by differential mRNA display, of increased type VIII collagen expression in injured rat carotid arteries stimulated with PDGF-BB.

Received July 29, 1996; accepted December 30, 1996.

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