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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1737.)
© 2000 American Heart Association, Inc.

Vascular Biology

Expression of Allograft Inflammatory Factor-1 Is a Marker of Activated Human Vascular Smooth Muscle Cells and Arterial Injury

Michael V. Autieri; Christopher Carbone; Anbin Mu

From the Departments of Physiology and Cardiology, Heart Failure Research Group, Temple University School of Medicine, Philadelphia, Pa.

Correspondence to Michael Autieri, PhD, Departments of Cardiology and Physiology, Temple University School of Medicine, Room 809, MRB, 3420 N Broad St, Philadelphia PA 19140. E-mail mautieri{at}unix.temple.edu

	Abstract

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Abstract—The cytokine-induced activation and proliferation of medial vascular smooth muscle cells (VSMCs) leading to intimal hyperplasia is one of the most critical cellular events in the formation of transplant arteriopathy and balloon angioplasty-induced restenosis. Allograft inflammatory factor-1 (AIF-1) is a calcium-binding protein that we have previously shown to be expressed in balloon angioplasty–injured rat carotid arteries. We hypothesized that AIF-1 expression may be associated with the VSMC response to injury. In this study, we examined AIF-1 expression in immunologic and mechanical models of arterial injury. Reverse transcription–polymerase chain reaction and Western analysis demonstrated that AIF-1 is acutely and transiently expressed in aortic medial smooth muscle cells of rat cardiac allografts, with mRNA and protein peaking at 3 to 7 days after transplant and declining by 10 days after transplant. Immunohistochemical analysis identified abundant AIF-1 in the medial VSMCs of these vessels. Immunohistochemical analysis of balloon angioplasty–injured swine coronary arteries also demonstrates an acute AIF-1 expression detectable by 24 hours and continuing up to 14 days after the procedure. AIF-1 in these vessels also localizes to the medial VSMCs and cells of the developing neointima. AIF-1 protein is not expressed in quiescent cultured human VSMCs but is induced in cells challenged with various inflammatory cytokines, primarily by interferon-, interleukin-1ß, and T-cell–conditioned media. Transfection and overexpression of AIF-1 in human VSMCs result in enhanced growth of these cells. Taken together, these data indicate that AIF-1 expression is associated with vascular trauma and suggest that this protein may play a role in VSMC activation subsequent to arterial injury.

Key Words: allograft inflammatory factor-1 • vascular smooth muscle cells • cytokines • transplant arteriopathy • balloon angioplasty

	Introduction

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Vascular narrowing produced by graft vascular disease is a major complication that limits long-term survival of cardiac transplantation.^{1 2} A primary cause of cardiac allograft vasculopathy (CAV) subsequent to cardiac transplantation is the intimal proliferation of vascular smooth muscle cells (VSMCs) in large- and medium-sized arteries and veins. The initiation of CAV is believed to involve a chronic immune response of the recipient to the donor vasculature in which activated immune cells damage the endothelium, resulting in the production of cytokines.³ These cytokines in turn elicit the activation of medial VSMCs, which migrate into the intimal layer and proliferate. Similarly, the long-term efficacy of percutaneous transluminal coronary angioplasty as a treatment for advanced multivessel coronary artery disease is significantly limited by the high incidence of vascular restenosis observed in as many as 40% of the patients undergoing this procedure.⁴ Analogous to CAV, restenosis subsequent to balloon angioplasty injury is also a process mediated by activated VSMCs.⁵ It has been suggested that cytokine-induced activation and proliferation of medial VSMCs, leading to intimal hyperplasia, is the most critical cellular event in the formation of CAV and balloon angioplasty–induced restenosis.^{6 7 8 9} Accordingly, the activation of several selected genes may reflect the status of smooth muscle cell (SMC) activation and, thus, the development of arteriopathy indicative of the progression of neointimal hyperplasia. Identification and functional characterization of these gene products is a promising approach for the development of antirestenotic therapeutics.^{10 11 12}

Allograft inflammatory factor-1 (AIF-1) is a 143–amino acid calcium-binding protein.^{13 14} Initial investigations indicated that AIF-1 expression was limited to interferon- (IFN-)–stimulated macrophages and neutrophils, which infiltrated rat cardiac allografts.¹³ Our initial observations came from balloon angioplasty–damaged rat carotid arteries, in which we observed a rapid transient expression pattern in AIF-1 mRNA.¹⁴ The intimal proliferative response of balloon angioplasty–induced vascular injury in rats is similar to that observed in the rat cardiac transplant model with respect to endothelial cell loss, activation of medial VSMCs, and elaboration of neointimal hyperplasia.^{1 15} Considering the similarities between mechanical and immunologic injury, we hypothesized that AIF-1 would also be expressed in the VSMCs of allografted tissue and that its expression in CAV might be associated with the progression of vascular hypertrophy characteristic of balloon angioplasty–induced injury. It was our intention to focus our examination of AIF-1 expression to the SMC response to vascular injury, because VSMCs are the major cell type in the media and in the developing neointima in this tissue. In the present study, we examine AIF-1 expression in 2 in vivo models of arterial injury: immunologic (in rat aortic allografts) and mechanical (in porcine coronary artery overstretch). We also explore a more direct role for AIF-1 in human VSMC activation by examining its ability to influence VSMC growth. The goals of the present study are (1) to associate AIF-1 expression with arterial trauma in different models of arterial injury, (2) to compare this expression with progression of the restenotic lesion over time, (3) to localize AIF-1 expression to activated medial and intimal VSMCs, and (4) to characterize a role for AIF-1 in VSMC activation.

	Methods

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Heterotropic Rat Heart Transplantation
Lewis rats providing donor hearts were anesthetized with buprenorphine administered subcutaneously and 1% to 2% isoflurane gas inhalation. The hearts were removed and gently perfused with sodium heparin dissolved in saline and placed in a cold saline solution containing sodium heparin until transplantation. Recipient Wistar-Furth rats were similarly anesthetized, and the donor heart was transplanted into the recipient right abdominal cavity with the donor aorta connected to the recipient abdominal aorta and the donor pulmonary artery connected to the recipient inferior vena cava. Once completed, the wound was closed with sterile nonabsorbable sutures and swabbed with Betadine surgical scrub. At various times after transplant, the donor hearts were removed, and the aortas were isolated and freed of any adhesions and external material. Some samples were processed for RNA and protein isolation; others were fixed and processed for immunohistochemistry. All procedures were performed in accordance with guidelines approved by the Institutional Animal Care and Use Committee (IACUC).

Swine Coronary Artery Balloon Angioplasty
Domestic swine weighing 20 to 30 kg were sedated, and general anesthesia was induced. After an 8F introducer sheath with a hockey stick curve was placed in the right femoral artery of the anesthetized animals, they received a bolus of heparin (200 U/kg) and bretylium (2.5 mg/kg). A guiding catheter was advanced to the aortic root, intracoronary nitroglycerin (200 µg) was injected, and baseline angiograms of the left and right coronaries were obtained. Coronary injury was achieved by deliberate stretch of the vessel wall with an oversized angioplasty balloon inflated to a pressure of 8 to 10 atm for 30 seconds, with a 1-minute rest period, for a total of 3 times. After withdrawal of the catheter, the femoral sheath was removed, and the cutdown site was repaired. At various times after angioplasty, the animals were euthanized according to the standard protocol of massive pentobarbital overdose (100 mg/kg IV). The left main coronary artery was cannulated, and injured segments were located by examination, dissected in block, and processed for immunohistochemistry. All procedures were performed in accordance with IACUC-approved institutional guidelines.

Tissue Processing and Immunohistochemistry
Hearts excised from pigs were perfusion-fixed with 10% buffered formalin at physiological pressures (100 mm Hg), and injured segments were fixed in 10% formalin for 4 hours and then in 70% ethanol for 18 hours and paraffin-embedded. Rat aortas were dissected from the heart, cleared of adherent tissue, fixed, and processed as described for porcine tissue. Tissue sections were deparaffinized, rehydrated in PBS and ethanol, quenched with H₂O₂, and blocked with goat serum. For primary AIF-1 antibody, rabbits were immunized with a peptide corresponding to amino acids 17 to 33 (KAQQEERLDEINKQFLH) present in the AIF-1 protein. Antisera were affinity-purified with this peptide, and Western analysis of recombinant AIF-1 protein indicates that this antiserum recognizes a protein of the predicted 17 kDa. SMC -actin antibody (Sigma Chemical Co) was used at a 1:2500 dilution to identify VSMCs. AIF antibody was applied at a 1:1000 dilution in PBS/1% BSA, washed, and incubated with biotinylated goat anti-rabbit secondary antibody (Transduction Laboratories) at 1:2000 dilution. This was washed and incubated with an avidin-biotin enzyme complex and chromogenic substrate as described by the manufacturer (Vectastain Elite ABC peroxidase), which develops a reddish-brown stain. Sections were counterstained with hematoxylin. Sections treated with secondary antibodies only or nonimmune IgG did not show any staining. Coronary vessels from at least 2 swine and 3 sections per vessel were evaluated.

Cells and Culture
Human coronary VSMCs were obtained as cryopreserved secondary culture from Clonetics Corp and subcultured in growth medium as described previously.¹⁴ Cells from passages 3 to 6 were used in the described studies. The growth medium was changed every other day until cells approached confluence. Preconfluent VSMCs were serum-starved (0.25% FCS) for 48 hours and then exposed to 10% FCS, 10 ng/mL basic fibroblast growth factor (bFGF), 100 U/mL IFN-, 20 ng/mL interleukin (IL)-1ß, 20 ng/mL platelet-derived growth factor (PDGF)-AB, 2 ng/mL transforming growth factor (TGF)-ß, or T-cell–conditioned medium for 48 hours, at which times samples were processed for protein isolation. Some samples remained untreated and were used as controls. PDGF, bFGF, IFN-, and TGF-ß were purchased from GIBCO-BRL; IL-1ß was purchased from Boehringer-Mannheim; and T-cell–conditioned medium was purchased from Fisher Biotech.

Transfection and Proliferation Assay
The protein coding region of the AIF-1 cDNA was cloned by polymerase chain reaction (PCR) with the use of AIF-1 gene-specific primers. The 5' PCR primer also contained a Kozak consensus sequence (GCCGCCGCCATGG) to enhance translation. This was inserted into the expression vector pBK-CMV (Stratagene), and this purified DNA construct was termed pBK-CMV-AIF-1. Transfection of human VSMCs has been described.¹⁵ Briefly, human coronary artery SMCs grown in T-75 flasks were transfected with either no plasmid (mock control), pBK-CMV plasmid alone, or pBK-CMV-AIF-1 along with 3 µL/mL LipofectAMINE reagent (Life Technologies) and mixed with 1 µg/mL of either plasmid. Two days after transfection, the cells were trypsinized and split 1:2, with one half left to grow in the presence of growth medium+G418 (Geneticin) for 15 days. The other half was saved for RNA isolation. After selection for 15 days, the cells were then trypsinized and counted by use of a standard hemocytometer.

Western Blotting
To prepare cell extracts, human VSMCs grown in T-75 flasks were cultured and treated as described above, washed with PBS, and treated with 0.3 mL of ice-cold lysis buffer (50 mmol/L HEPES-KOH, pH 7.5, 150 mmol/L NaCl, and 0.1% Triton X-100) containing protease inhibitors, as described.¹⁶ Lysates were incubated on ice 20 minutes, withdrawn through a 21-gauge needle 3 times, then centrifuged at 3000 rpm for 15 minutes at 4°C, and stored at -20°C. Proteins were extracted from rat aorta by Tri-Reagent (Molecular Research Center). Equal protein concentrations of cell extracts were electrophoresed through an 18% polyacrylamide gel under reducing conditions and transferred to a nitrocellulose membrane. Membranes were blocked with 5% nonfat powdered milk in TBST buffer (0.1 mol/L Tris-HCl, pH 8.0, containing 1.5 mol/L NaCl and 0.5% Triton X-100). Membranes were incubated with a 1:2500 dilution of primary antibody and a 1:2000 dilution of goat anti-rabbit secondary antibody. Proliferating cell nuclear antigen and cyclooxygenase-2 antibody were from Santa Cruz, Inc. The membrane was washed with TBST, and reactive proteins were visualized by using the enhanced chemiluminescence method (Amersham) according to the manufacturer’s instructions.

RNA Isolation and Semiquantitative Reverse Transcription–PCR
For each time point studied, aorta from allografted rat hearts were isolated and cleared of adherent tissue. Total RNA was extracted by Tri-Reagent, which allowed simultaneous extraction of RNA and protein, and 4 µg of total RNA was reverse-transcribed by using random hexamers as described previously.¹⁴ One fifth of the cDNA was PCR-amplified by using the following primers: 5' TAT CAT GTC CTT GAA ACG AAT GCT GG AGA A 3' and 5' TTT GTC TTC TGT TTT AGC ATT CGG TCT CAG 3', which define a 330-bp region of the human AIF cDNA, for 32 cycles. This is in the linear assay range with respect to cycle number, template concentration, and dilution of cDNA. The glyceraldehyde-3-phosphate dehydrogenase (G3PDH) amplimers were purchased from Clonetech and define an amplicon of 450 bp. One fifth of the reaction was run on a 2.5% agarose gel, ethidium bromide–stained, and photographed. PCR products were Southern-transferred to the hybridization membrane and hybridized with an end-labeled 45-mer oligonucleotide probe complementary for a sequence internal to the PCR amplicons. For verification of transfection efficiency, total RNA was extracted from transfected cells, and 4 µg was reverse-transcribed. One fifth of the cDNA was PCR-amplified by using neomycin-specific amplimers, which define a 290-bp region of the neomycin cDNA, for 32 cycles.

	Results

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AIF-1 mRNA and Protein Are Expressed in Medial VSMCs in Allografted Rat Aorta
We initially examined the expression of AIF-1 mRNA in VSMCs subject to immunologic insult by using aortas isolated from rat cardiac allografts. Donor hearts from Lewis rats were heterotopically transplanted into the abdomen of Wistar-Furth recipient rats, and semiquantitative reverse transcription–PCR (RT-PCR) was performed on RNA isolated from aortas dissected from Lewis donor hearts at various times after transplant (Figure 1). Figure 1 indicates an acute expression pattern of AIF-1 mRNA expression, with mRNA levels increasing within 24 hours of transplantation, peaking at 3 to 7 days, and declining by 10 days after surgery. Seven-day syngeneic (Lewis to Lewis) control aortas also demonstrate a low, but detectable, increase in AIF-1 mRNA expression. AIF-1 is constitutively expressed in spleen tissue and was used as a positive control. The identity of this amplicon as human AIF-1 was confirmed by Southern analysis of the same gel hybridized with a probe specific for a sequence internal to the AIF-1 amplimers (Figure 1). These results indicate that AIF-1 mRNA is acutely and transiently induced in aortas from allografted rat hearts.

View larger version (35K): [in this window] [in a new window]   Figure 1. Expression of AIF-1 cDNA in aortas from allografted rat heart. Semiquantitative RT-PCR was performed on 4 µg total RNA isolated from aortas dissected from Lewis rat hearts allografted into Wistar-Furth hosts with use of human AIF-1 and G3PDH amplimers defining amplicons of 330 and 450 bp, respectively. One fifth of the reaction was run on a 2.5% agarose gel, ethidium bromide–stained, and photographed. Lanes are as follows: lane 1, no reverse transcriptase (no RT) template (negative control); lane 2, naive Lewis heart; lane 3, 1 day after transplant; lane 4, 3 days after transplant; lane 5; 7 days after transplant; lane 6, 10 days after transplant; lane 7, 7-day syngeneic (Lewis into Lewis); lane 8, spleen (positive tissue control); and lane 9, AIF-1 cDNA and G3PDH template (positive PCR control).

It was important to determine whether AIF-1 protein was expressed in a fashion similar to that for AIF-1 mRNA. Protein was extracted from the same aorta as used for RNA and subjected to Western analysis with AIF-1–specific antibody. Figure 2 indicates that AIF-1 protein expression is also inducible by cardiac allograft transplantation and follows a pattern similar to that for mRNA, with protein levels peaking at 3 to 7 days and declining by 10 days after surgery. No AIF-1 was observed 1 day after transplant, which may be due to the time necessary for mRNA to be translated into detectable amounts of protein. Again, a small increase in AIF-1 protein was detectable in 7-day Lewis to Lewis control rats. These results indicate that AIF-1 protein is also acutely and transiently expressed by immunologic insult and reflects that of mRNA expression.

View larger version (21K): [in this window] [in a new window]   Figure 2. Expression of AIF-1 protein in aortas from allografted rat hearts. Western analysis of protein from aortas dissected from Lewis rat hearts allografted into Wistar-Furth hosts by use of antibody directed to AIF-1 peptide sequence is shown. Lanes are as follows: lane 1, naive Lewis heart; lane 2, 1 day after transplant; lane 3, 3 days after transplant; lane 4, 7 days after transplant; lane 5, 10 days after transplant; lane 6, 7-day syngeneic (Lewis into Lewis); and lane 7, spleen (positive tissue control). Analysis of AIF-1 protein expression was detected by enhanced chemiluminescence.

We used immunohistochemistry to localize progression of AIF-1 protein accumulation in allografted aortas. Serial sections from Lewis to Wistar-Furth cardiac allografts were immunohistologically examined with AIF-1 antisera, and Figure 3 shows rat aortas before and at various times after transplant. Similar to protein levels determined by Western analysis, little to no AIF-1 protein was present in the uninjured aorta or in the aorta 24 hours after transplantation (Figure 3A and 3B). By 3 days after surgery, however, a marked induction of AIF-1 protein was observed in the medial VSMCs, continuing through day 7 (Figures 3C and 3D). By day 10, AIF-1 protein levels appeared to decline, and a modest amount of AIF-1 protein was detectable in a 7-day Lewis to Lewis control aorta (Figures 3E and 3F, respectively).

View larger version (57K): [in this window] [in a new window]   Figure 3. Immunohistochemical analysis of expression of AIF-1 protein in aortic VSMCs from Lewis rat hearts allografted into Wistar-Furth hosts. Cross sections of rat aorta (A, naive; B, 1 day after allograft; C, 3 days after allograft; D, 7 days after allograft; E, 10 days after allograft; and F, 7-day syngeneic [Lewis to Lewis]) were incubated with AIF-1 antisera. Panel G is secondary antibody only, and panel H is SMC -actin. Red-brown staining indicates AIF-1 expression, and sections were counterstained with hematoxylin. Original magnification x100.

It is important to note that positive AIF-1 staining localizes to SMCs, which are identified by their morphological features and positive staining for SMC -actin. (Figure 3H). These findings are also consistent with the cellular pathology of this model, in which monocytes and macrophages are frequently found only in the allograft adventitia in early lesions and are not detected in the media and intima until 20 days to 3 months onward.¹⁷ Overall, these results closely mirror those observed for AIF-1 mRNA and protein expression as detected by RT-PCR and Western analysis and indicate that AIF-1 is expressed acutely and transiently in aortic VSMCs by allograft transplantation.

AIF-1 Protein Is Induced in Swine Coronary Arteries by Balloon Angioplasty
The swine response to vascular injury presents many similarities to that of humans, particularly in the generation of neointimal formation. We performed immunohistochemistry on swine coronary arteries subject to oversized-balloon angioplasty and examined them for AIF-1 expression at several time points after surgery. Figure 4A demonstrates that AIF-1 protein was not detectable in uninjured arteries but was rapidly expressed in medial VSMCs by 1 day after injury (Figure 4B). This staining is distributed circumferentially around the vessel, and as with the aortic allografts, expression is almost exclusively limited to the medial VSMCs, because these cells also stain positively for SMC -actin (Figures 4G and 4L). Intense AIF-1 expression was apparent in the medial VSMCs and neointimal cells 3 days after injury (Figures 4C, 4H, and 4M). The most striking observation at this time point is that AIF-1 was present in cells of the developing neointima, whereas these same cells were negative for SMC -actin. Neointimal VSMCs that have switched from the contractile to the synthetic phenotype characteristically do not synthesize -actin.⁴ AIF-1 expression appeared to decline by 14 days after balloon injury (Figures 4D and 4I). At this time point, a large neointima was evident; however, the most intense AIF-1 immunostaining localized to the medial VSMCs compared with neointimal cells. The neointimal cells that did stain appeared to localize to the luminal side of the injury (Figure 4I). This neointima is a mix of SMC -actin–positive and –negative cells; however, not all SMC -actin–positive cells stained positively for AIF-1 (Figure 4N). This indicates that AIF-1 expression is not limited to rejecting allografts but is also induced by mechanical injury of the coronary arteries as well. These results are also similar to our findings in balloon-injured rat carotid arteries with respect to the acute and transient kinetics of AIF-1 expression.

View larger version (73K): [in this window] [in a new window]   Figure 4. Immunohistochemical analysis of expression of AIF-1 protein in VSMCs from balloon angioplasty–injured swine coronary arteries. Cross sections of balloon angioplasty–injured swine coronary arteries stained with AIF-1 antibody are shown: A, naive; B, 1 day after injury; C, 3 days after injury; and D, 14 days after injury. Panel E is secondary antibody only. Panels F to J are the same sections at higher magnification. Panels K to O are the same sections stained with SMC -actin to localize medial VSMCs (m). n indicates neointima. Red-brown staining indicates positive staining, and sections were counterstained with hematoxylin. Original magnification x40 (A to E) and x100 (F to O).

AIF-1 Protein Expression Is Induced by Cytokines in Human VSMCs
The lack of AIF-1 in uninjured vessels and its inducible expression in medial VSMCs after transplantation and mechanical injury suggested that expression of this protein in vivo is regulated by soluble factors. Because AIF-1 immunostaining localized to VSMCs, we examined induction of AIF-1 protein expression in primary cultured human coronary artery VSMCs stimulated with a variety of cytokines. In these experiments, cells were starved into quiescence by serum deprivation for 48 hours and exposed to 10% FCS, bFGF, IFN-, IL-1ß, PDGF, TGF-ß, and T-cell–conditioned media for 48 hours. Extracts were separated by SDS-PAGE, and AIF-1 was detected by Western analysis with specific antisera. The results of Figure 5 indicate that human AIF-1 is differentially induced by various soluble factors in cultured human VSMCs. Ten percent FCS, IFN-, and IL-1ß are capable of inducing this protein, whereas AIF-1 is not expressed in unstimulated cells. bFGF, PDGF, and TGF-ß can induce AIF-1 protein expression to a small degree. T-cell–conditioned media, which contains several soluble factors, elicited the strongest induction of AIF-1.

View larger version (29K): [in this window] [in a new window]   Figure 5. Expression of AIF-1 protein in serum- and cytokine-stimulated human VSMCs. Equal concentrations of protein extracted from unstimulated VSMCs and VSMCs treated for 48 hours with 10% FCS, bFGF, IFN-, IL-1ß, PDGF, TGF-ß, and T-lymphocyte–conditioned media (cond. media) were separated by SDS-PAGE, transferred to nitrocellulose, and incubated with antisera to human AIF-1. Analysis of AIF-1 protein expression was detected by enhanced chemiluminescence.

Because activated lymphocytes are among the first immune cells present in the arterial lesion and because T-lymphocyte–conditioned media elicited the strongest induction of AIF-1 protein in cultured VSMCs, we treated cultured human VSMCs with T-lymphocyte–conditioned media and examined extracts of these cells for AIF-1 protein by Western analysis at different time points. Figure 6 demonstrates that AIF-1 is not expressed in unstimulated cells but is induced by T-cell–conditioned media 24 hours after stimulation and peaks by 72 hours. These results indicate that in human VSMCs, AIF-1 is a cytokine-responsive protein and is particularly responsive to immune cell factors, suggesting that its induction in arteriopathic lesions is primarily dependent on inflammatory processes.

View larger version (46K): [in this window] [in a new window]   Figure 6. Expression of AIF-1 protein in human VSMCs stimulated with T-lymphocyte–conditioned media. Equal concentrations of protein were extracted from unstimulated VSMCs and VSMCs stimulated for 24, 48, and 72 hours, separated by SDS-PAGE, transferred to nitrocellulose, and incubated with antibody to human AIF-1. Analysis of AIF-1, proliferating cell nuclear antigen (PCNA), and cyclooxygenase (COX)-2 protein expression was by enhanced chemiluminescence.

Overexpression of AIF-1 in Human VSMCs Increases Their Proliferative Capacity
The expression of AIF-1 in activated VSMCs led us to investigate whether expression of this protein was linked to cell growth. As an initial approach toward understanding the function of AIF-1 in VSMC activation, we determined the effects of overexpression of this protein on VSMC proliferation. Primary human VSMCs were transfected with either no plasmid (mock control), with pBK-CMV plasmid alone, or with pBK-CMV containing AIF-1 protein coding region cDNA (pBK-CMV-AIF-1). Two days after transfection, cells were trypsinized and split 1:2, with one half left to grow in the presence of growth medium+G418 (Geneticin) for 15 days. The other half was saved for RNA isolation. After selection, the cells were trypsinized and counted. The results of 3 independent experiments are presented in the Table and demonstrate an average 2.6 fold-increase in pBK-CMV-AIF-1–containing cells compared with pBK-CMV–containing cells. Because the transfection efficiency of primary human cells is low, it was necessary to use RT-PCR of RNA isolated from newly transfected cells to indicate that equal amounts of plasmid are present in control and in pBKCMV-AIF-1–transfected cells. Figure 7 indicates that these results are not due to differences in transfection efficiency, suggesting that human VSMCs that overexpress AIF-1 proliferate at a more rapid rate than do cells that do not.

View this table: [in this window] [in a new window]   Table 1. Overexpression of AIF-1 Protein in Human VSMCs Enhances Their Proliferation

View larger version (26K): [in this window] [in a new window]   Figure 7. Increase in proliferation of VSMCs that overexpress AIF-1 is not due to differences in transfection efficiency. Total RNA was extracted from transfected cells 2 days after transfection, and 4 µg was reverse-transcribed and PCR-amplified by using neomycin (Neo)-specific amplimers, which define a 290-bp region of the Neo cDNA, and G3PDH amplimers, which define an amplicon of 450 bp. One fifth of the reaction was run on a 2.0% agarose gel, ethidium bromide–stained, and photographed. Lanes are as follows: lane 1, mock-transfected; lane 2, pBK-CMV; lane 3, pBK-CMV-AIF-1; lane 4, G3PDH PCR control; lane 5, Neo PCR control; and lane 6, no RT control.

	Discussion

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Two widely used and clinically relevant models of arterial injury are rat cardiac allograft rejection and swine balloon angioplasty.^{15 18} We initially focused on Lewis to Wistar-Furth allografts, which consistently develop an acute graft rejection within 12 days, and aortic tissue, which is almost exclusively composed of VSMCs. It has been shown that nonimmunosuppressed rat aortic allografts are not acutely rejected but develop arteriosclerotic alterations in the vascular wall that closely and histologically resemble the pathological changes observed in clinical transplants that undergo chronic rejection.^{17 19} These alterations include infiltration of mononuclear cells into the adventitia, endothelial cell hypertrophy, and SMC proliferation, resulting in thickening of the intima over time. Aortic allografts also resemble chronically rejecting human transplants biochemically with respect to plasma levels of thromboxane and prostacyclin.¹⁷ At the mRNA and protein level, AIF-1 is acutely and transiently expressed in aortic allografts (Figures 1 and 2). An induction of AIF-1 mRNA is detectable 24 hours after surgery, and AIF-1 protein is detectable shortly thereafter, at 3 days. mRNA and protein decline by 10 days after transplant. AIF-1 mRNA and protein are detectable at low levels in the Lewis to Lewis control animals, likely as a result of small numbers of macrophages and the moderate expression of growth factors that are found in isografted arteries.¹⁸

Immunohistochemical analysis of donor aortas with AIF-1 antibody closely reflects the acute and transient expression pattern obtained by Western analysis and RT-PCR. It has been demonstrated that in aortic allograft rejection, IL-2 receptors (T lymphocytes) and OX42-positive cells (monocytes/macrophages), although present in the adventitia, are generally absent in the media and intima until 20 days after transplant.¹⁷ Notwithstanding, it was possible that AIF-1 expression was due at least in part to infiltrating immune cells. Consequently, it was important to verify that the primary source of AIF-1 expression that we observed was derived from SMCs, and Figure 3 shows that AIF-1 is expressed in SMC -actin–positive medial VSMCs. It is also interesting to note that in allografted rat aortas, medial thickening and cellular proliferation in medial and intimal VSMCs does not begin until 14 days after surgery.¹⁷ The early presence of mononuclear cells in the adventitia of the grafted aorta may be the source of cytokines, which activate the medial VSMCs, which in turn induce AIF-1 expression in these cells. Because AIF-1 expression is readily detectable in VSMCs by 3 days after transplantation, expression of this protein may be an early marker of graft rejection originating from the vessel itself.

At the cellular and molecular level, the vascular responses to mechanical and immune injury are similar,^{1 15 16 18} and we hypothesized that balloon angioplasty–initiated injury would also evoke AIF-1 expression. In particular, the response produced by mechanical injury of porcine coronary arteries presents many similarities to that of humans, including coronary circulation and spontaneous development of atherosclerosis.^{5 20} Neointimal formation subsequent to balloon angioplasty is a dynamic process involving several different cell types and occurring in several phases. Early events are primarily inflammatory in nature and are initiated by T lymphocytes and macrophages, which secrete various cytokines and growth factors seminal to the local inflammatory response. Later events may last several weeks after injury and involve activated VSMCs, which at this stage are primarily fibroproliferative in nature and synthesize soluble factors and extracellular matrix proteins. Because several studies have suggested that the response of the injured vessel is related to the degree of injury,^{21 22} we examined swine coronary arteries at several time points after balloon angioplasty to correlate AIF-1 expression with the progression of restenosis. The rapid and robust expression of AIF-1 in 1-day balloon angioplasty–injured swine coronary arteries (Figures 4A to 4J) is associated with the initial arterial response to injury. Of particular note is the rapid and circumferential expression of AIF-1 in 1-day vessels. This expression is localized to the VSMC, as confirmed by morphology and SMC -actin staining. Studies have shown that very low numbers of proliferating cells are found in the media of angioplasty-damaged swine vessels.²³ This acute expression pattern is quite similar to that observed by us in balloon-injured rat carotid arteries,¹⁴ indicating that AIF-1 expression is independent from, or at least precedes, proliferation. In the more advanced 14-day lesions, AIF-1 expression is stronger in medial VSMCs and the luminal side of the neointima rather than in the fibroproliferative core of the lesion, suggesting that expression of this protein may responsive to soluble factors originating from inflammatory cells present in the adventitia and circulation. This is in contrast to that observed for SMC -actin–positive cells, which are more uniformly present throughout the neointima. Taken together, this infers that AIF-1 expression may be more of a marker of the initial VSMC response to inflammation rather than of the progression of intimal hyperplasia.

Immune cells secrete several soluble factors that stimulate VSMC migration, growth factor secretion, and cellular proliferation, and numerous studies have shown that VSMCs in allografted vessels and balloon angioplasty–injured arteries express several classes of cytokines and inflammatory factors.^{4 24} Inflammatory cytokines, such as IFN- and IL-1ß, along with T-cell–conditioned media and FCS, are proliferative as well as inflammatory and display the most potent induction of AIF-1 compared with other factors.^{4 25} Although cytokines such as bFGF-1, PDGF, and TGF-ß have been implicated as being responsible for the promotion of intimal hyperplasia in response to injury,^{1 4} these factors were capable of inducing AIF-1 protein expression only to a minor degree. Other studies have observed that in macrophages, AIF-1 mRNA expression is induced by T-cell factors, such as IFN-, and that this expression could be modulated by compounds that influence the inflammatory response.²⁶ These studies have suggested that AIF-1 is not expressed in VSMCs; however, the VSMCs in those experiments were not cytokine-stimulated.¹³ Our results indicating that AIF-1 is not expressed in unstimulated VSMCs corroborate that earlier work. T-lymphocyte–conditioned medium contains several factors, including granulocyte-macrophage colony–stimulating factor, IFN-, IL-1, IL-6, and TNF-ß, which are known to influence the VSMC phenotype, and it has been demonstrated that infiltration of the artery wall by T lymphocytes induces a change in VSMC phenotype, which is mediated by inflammatory, mitogenic, and antiproliferative factors.^{27 28} In agreement with what we observed with injured vessels in vivo, induction of AIF-1 occurs rapidly and is detectable at 24 hours of exposure to T-cell–conditioned medium (Figure 6).

Stimulation of cells with cytokines and mitogens results in a rapid transient increase in calcium levels,²⁹ and mitogenic stimulation of arterial SMCs involves a flux of calcium ions through the plasma membrane.^{30 31} Calcium and its primary receptor protein, calmodulin, are required for cellular growth and survival, because both of these are essential for the entry of quiescent cells into the cell cycle in response to mitogenic signals.^{32 33} Not surprisingly, calcium channel blockers and calcium antagonists inhibit restenosis subsequent to mechanical (balloon angioplasty) and immunologic (transplant vasculopathy) insult.^{34 35 36 37} Overexpression of calmodulin in cultured cells enhances cell proliferation, primarily through a reduction in the length of the G₁ phase of the cell cycle,^{8 9} and stable overexpression of several other calcium-binding proteins has also been shown to influence the cell cycle.^{12 13} With these observations as a starting point, we hypothesized a role for AIF-1 expression in VSMC growth.

Similar to other proteins that interact with calcium, AIF-1 is involved in cellular proliferation, because overexpression of this protein enhances the growth of human VSMCs. Stable transfection of AIF-1 cDNA into primary human VSMCs results in an average 2.6-fold increase in cell number (Table). Given the role of calcium regulation in growth control, one possible explanation for the growth-enhancing properties of AIF-1 is through its ability to interact with and possibly regulate the concentration of calcium ions in the cell.

The expression of AIF-1 in a localized cytokine-rich injury implies that AIF-1 may take part in inflammation-initiated activation events. The expression of AIF-1 in medial and intimal VSMCs before proliferation in vivo infers that this protein may function in the mediation of VSMC growth initiated by inflammatory stimuli. Additionally, although the vascular response to injury and development of restenosis has been shown to differ from species to species,³⁸ the present study also indicates that AIF-1 expression is conserved, suggesting a fundamental function for this protein in vascular injury. In immune and mechanical vascular injury, immune cells play an important role in the initiation and progression of restenosis. However, the vascular pathobiology resulting in intimal hyperplasia is due to VSMC activation, making AIF-1 not only a surrogate marker of arterial injury but also a potential target of antirestenotic therapies.

	Acknowledgments

 
The authors are grateful to Dr Howard Eisen for critical reading of the manuscript and Sheri Kelemen for technical advice.

Received August 17, 1999; accepted January 31, 2000.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Ip JH, Fuster V, Badimon L, Badimon J, Taubman M, Chesebro J. Syndromes of accelerated atherosclerosis: role of vascular injury and smooth muscle cell proliferation. J Am Coll Cardiol. 1990;15:1667–1687.[Abstract]
2. Miller LW. Allograft vascular disease, a disease not limited to hearts. J Heart Lung Transplant. 1992;11:932–936.
3. Brodsky FM, Guagliardi L. The cell biology of antigen processing and presentation. Annu Rev Immunol. 1991;9:707–744.[Medline] [Order article via Infotrieve]
4. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801–809.[Medline] [Order article via Infotrieve]
5. Schwartz RS, Murphy JG, Edwards WD, Camrud AR, Vlietstra RE, Holmes DR. Restenosis after balloon angioplasty: a practical proliferative model in porcine coronary arteries. Circulation. 1990;82:2190–2200.[Abstract/Free Full Text]
6. Liu MW, Roubin GS, King SB. Restenosis after coronary angioplasty: potential biologic determinants and role of intimal hyperplasia. Circulation. 1989;79:1374–1380.[Abstract/Free Full Text]
7. Austin GE, Ratliff NB, Hollman J, Tabei S, Phillips DF. Intimal proliferation of smooth muscle cells as an explanation for recurrent coronary artery stenosis after percutaneous transluminal coronary angioplasty. J Am Coll Cardiol. 1985;6:369–375.[Abstract]
8. Ventura HO, Mehra MR, Smart FW. Cardiac allograft vasculopathy: current concepts. Am Heart J. 1995;129:791–798.[Medline] [Order article via Infotrieve]
9. Libby P, Salomon R, Payne D, Schoen F, Pober J. Functions of vascular wall cells related to development of transplantation-associated coronary arteriosclerosis. Transplant Proc. 1989;21:3677–3684.[Medline] [Order article via Infotrieve]
10. Gregory C, Huie P, Billingham M, Morris R. Rapamycin inhibits arterial intimal thickening caused by both alloimmune and mechanical injury. Transplantation. 1993;55:1409–1418.[Medline] [Order article via Infotrieve]
11. Epstein SE, Speir E, Unger EF, Guzman RJ, Finkel T. The basis of molecular strategies for treating coronary restenosis after angioplasty. J Am Coll Cardiol. 1994;23:1278–1288.[Abstract]
12. Zhu NL, Wu L, Liu PX, Gordon EM, Anderson WF, Starnes VA, Hall FL. Downregulation of cyclin G1 expression by retrovirus-mediated antisense gene transfer inhibits vascular smooth muscle cell proliferation and neointima formation. Circulation. 1997;96:628–635.[Abstract/Free Full Text]
13. Utans U, Arceci R, Yamashita Y, Russell M. Cloning and characterization of allograft Inflammatory factor-1: a novel macrophage factor identified in rat cardiac allografts with chronic rejection. J Clin Invest. 1995;95:2954–2960.
14. Autieri MV. cDNA cloning of human allograft inflammatory factor-1: tissue distribution, cytokine induction, and mRNA expression in injured rat carotid arteries. Biochem Biophys Res Commun. 1996;228:29–37.[Medline] [Order article via Infotrieve]
15. Metha D, Angelini GD, Bryan AJ. Experimental models of accelerated atherosclerosis syndromes. Int J Cardiol. 1996;56:235–257.[Medline] [Order article via Infotrieve]
16. Autieri MV, Carbone C. 14–3-3 interacts with and is phosphorylated by multiple PKC isoforms in PDGF-stimulated human vascular smooth muscle cells. DNA Cell Biol. 1999;18:555–564.[Medline] [Order article via Infotrieve]
17. Mennander A, Tiisala S, Halttunen J, Yilmaz S, Paavonen T, Hayry P. Chronic rejection in rat aortic allografts: an experimental model for transplant arteriosclerosis. Arterioscler Thromb. 1991;11:671–680.[Abstract/Free Full Text]
18. Gregory CR, Huie P, Billingham ME, Morris RE. Rapamycin inhibits arterial intimal thickening caused by both alloimmune and mechanical injury. Transplantation. 1993;55:1409–1418.
19. Isik FF, McDonald TO, Ferguson M, Yamanaka E, Gordon D. Transplant arteriosclerosis in a rat aortic model. Am J Pathol. 1992;141:1139–1149.[Abstract]
20. Schwartz RS, Holmes DR, Topol EJ. The restenosis paradigm revisited: an alternative proposal for cellular mechanisms. J Am Coll Cardiol. 1992;20:1284–1293.[Abstract]
21. Schwartz R, Huber L, Murphy J. Restenosis and the proportional neointimal response to coronary artery injury: results in a porcine model. J Am Coll Cardiol. 1992;19:267–274.[Abstract]
22. Indolfi C, Esposito G, DiLorenzo E, Rapacciuolo A, Feliciello A, Porcellini A, Avedimento VE, Condorelli M, Chiariello M. Smooth muscle cell proliferation is proportional to the degrees of balloon injury in a rat model of angioplasty. Circulation. 1995;92:1230–1235.[Abstract/Free Full Text]
23. Scott N, Cipolla G, Ross C, Dunn B, Martin F, Simonet L, Wilcox J. Identification of a potential role for the adventitia in vascular lesion formation after balloon overstretch injury of porcine coronary arteries. Circulation. 1996;93:2178–2187.[Abstract/Free Full Text]
24. Libby P, Schwartz D, Brogi E, Tanaka H, Clinton SK. A cascade model for restenosis: a special case of atherosclerosis progression. Circulation. 1992;86(suppl III):III-47–III-52.
25. Demaeyer E, Demayer-Guignard J. Interferons and Other Regulatory Cytokines. New York, NY: John Wiley & Sons Inc; 1988.
26. Russell ME. Macrophages and transplant arteriosclerosis: known and novel molecules J Heart Lung Transplant.. 1995;14:S111–S115.[Medline] [Order article via Infotrieve]
27. Wang W, Chen HJ, Giedd KN, Schwartz A, Cannon PJ, Rabbani LE. T-cell lymphokines, interleukin-4, and gamma interferon, modulate the induction of vascular smooth muscle tissue plasminogen activator and migration by serum and platelet-derived growth factor. Circ Res. 1995;77:1095–1106.[Abstract/Free Full Text]
28. Rolfe BE, Campbell JH, Smith NJ, Cheong MW, Campbell GR. T lymphocytes affect smooth muscle cell phenotype and proliferation. Arterioscler Thromb Vasc Biol. 1995;15:1204–1210.[Abstract/Free Full Text]
29. Means A. Calcium, calmodulin, and cell cycle regulation. FEBS Lett.. 1994;347:1–4.[Medline] [Order article via Infotrieve]
30. Pauly R, Bilato C, Sollott S, Monticone R, Kelly P, Lakatta E, Crow M. Role of calcium/calmodulin-dependent protein kinase II in the regulation of vascular smooth muscle cell migration. Circulation. 1995;91:1107–1115.[Abstract/Free Full Text]
31. Block LH, Emmons LR, Vogt E, Sachinidis A, Vetter W, Hoppe J. Ca²⁺ channel blockers inhibit the action of recombinant platelet-derived growth factor in vascular smooth muscle cells. Proc Natl Acad Sci U S A. 1989;86:2366–2392.
32. Rasmussen C, Means AR. Calmodulin is required for cell-cycle progression during G1 and mitosis. EMBO J. 1989;8:73–82.[Medline] [Order article via Infotrieve]
33. Enslen H, Tokimitsu H, Stork P, Davis R, Soderling T. Regulation of mitogen-activated protein kinases by a calcium/calmodulin-independent protein kinase cascade. Proc Natl Acad Sci U S A. 1996 93;10803–10808.
34. Whitworth HB, Roubin GS. Effect of nifedipine on recurrent stenosis after percutaneous transluminal coronary angioplasty. J Am Coll Cardiol. 1986;8:1271–1276.[Abstract]
35. Hoberg E. The effects of calcium antagonists after PTCA. Eur Heart J. 1995;16(Suppl 11):9–12.
36. Jackson CL, Bush RC, Bowyer DE. Inhibitory effect of calcium antagonists on balloon catheter-induced arterial smooth muscle cell proliferation and lesion size. Atherosclerosis. 1988;69:115–122.[Medline] [Order article via Infotrieve]
37. Schroeder JS, Gao SZ, Alderman EL, Hunt SA, Johnstone I, Boothroyd DB, Wiederhold V, Stinson EB. A preliminary study of diltiazem in the prevention of coronary artery disease in heart transplant recipients. N Engl J Med. 1993;328:164–170.[Abstract/Free Full Text]
38. Schwartz RS. Neointima and arterial injury: dogs, rats, pigs, and more. Lab Invest. 1994;71:789–791.[Medline] [Order article via Infotrieve]

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