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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:1421.)
© 2001 American Heart Association, Inc.

Vascular Biology

Overexpression of Allograft Inflammatory Factor-1 Promotes Proliferation of Vascular Smooth Muscle Cells by Cell Cycle Deregulation

Michael V. Autieri; Christopher M. Carbone

From the Departments of Physiology (M.V.A.), and Cardiology (M.V.A., C.M.C.), Cardiovascular Research Group, Temple University School of Medicine, Philadelphia, Pa.

Correspondence to Michael Autieri, PhD, Department of Physiology, Room 810, MRB, Temple University School of Medicine, 3420 N. Broad St, Philadelphia, PA 19140. E-mail mautieri{at}unix.temple.edu

	Abstract

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Abstract—— Allograft inflammatory factor-1 (AIF-1) is not present in normal arteries, but its expression is induced in vascular smooth muscle cells (VSMCs) in several models of arterial injury. The proliferation of VSMCs is a major component of neointimal hyperplasia in many arteriopathies, and the purpose of this study was to determine the role of AIF-1 in the growth of VSMCs. Transfection and constitutive expression of AIF-1 in a primary and a rat VSMC line results in enhanced growth of those cells as measured by cell number and is proportional to the amount of AIF-1 expressed. Constitutive expression of AIF-1 results in a shorter cell cycle, as measured by flow cytometry, and aberrant expression of cell cycle proteins, as determined by Western blot. AIF-1 overexpression also permits growth of these cells in serum-reduced media. Collectively, these data suggest that AIF-1 may participate in the progression of vascular proliferative disease on the basis of its ability to regulate the growth of VSMCs.

Key Words: allograft inflammatory factor-1 • vascular smooth muscle cells • vascular injury • proliferation • cell cycle

	Introduction

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The neointimal formation associated with vasculopathy subsequent to immune and mechanical injury is a complex process involving several cell types that secrete various cytokines and growth factors seminal to the local inflammatory response.^1,2 The activation of vascular smooth muscle cells (VSMCs) is responsible for most of the arterial intimal thickening in solid organ allografts.^1,3 A popular hypothesis is that the cytokine-induced activation and proliferation of VSMCs in the media, leading to intimal hyperplasia, is one of the most critical cellular events in the development of transplant arteriopathy and balloon angioplasty-induced restenosis.^2,4

Several calcium-binding proteins have been shown to play a significant role in the regulation of cellular growth and proliferation.⁵ Overexpression of calmodulin (CaM) in cultured cells enhances cell proliferation by reducing the length of the G₁ phase of the cell cycle.^6,7 Concomitantly, transient reduction of CaM levels results in a transient inhibition of cell growth at multiple phase transitions in the cell cycle.⁷ Stable overexpression of other calcium-binding proteins has also been shown to influence the cell cycle.^8,9 Overall, identification and functional characterization of gene products involved in VSMC proliferation, particularly those that may modulate calcium metabolism, are regarded as a promising approach for the identification of targets for anti-restenotic therapeutics.

Human allograft inflammatory factor-1 (AIF-1) is an 143–amino acid, cytoplasmic, EF-hand–containing protein.¹⁰ The EF-hand calcium-binding motif is present in many calcium-binding proteins, in which it is assumed to play a role in the modulation of Ca²⁺-dependent signaling, cytosolic Ca²⁺ buffering, and structural stabilization of Ca²⁺-dependent enzymes.¹¹ AIF-1 is a recently discovered protein, and ever since its initial recognition, data from several groups in diverse systems advocate an important role for AIF-1 in inflammatory processes. AIF-1 expression has been associated with infiltrating macrophages in rat cardiac allografts,¹² with inflammatory lesions of the central nervous system and experimental autoimmune encephalomyelitis,^13,14 with the pancreas of prediabetic BB rats,¹⁵ and with the allograft response of phylogenetically diverse species, such as carp and marine sponges.¹⁶ AIF-1 transcript levels are significantly decreased in allografted animals that receive immunosuppressive and immunomodulatory regimens, suggesting a tight association with the inflammatory process.¹⁷ Interestingly, a 44–amino acid segment of AIF-1 contains an amidation signal flanked by a cluster of paired basic residues, which are characteristic cleavage motifs for peptide hormone precursors.¹⁵ AIF-1 is mapped to the major histocompatibility complex class III region of chromosome 6.¹⁸ The location and function of many genes mapping to this region are known to contribute to immune function and disease pathophysiology. We have previously reported the acute and transient expression of AIF-1 in medial and intimal VSMCs in mechanical and immune models of arterial injury in several species.^10,19 AIF-1 is not expressed in unstimulated cultured human VSMCs but is strongly induced in response to inflammatory cytokines and T-lymphocyte–conditioned media.¹⁹ A precise function for this protein in inflammation has yet to be elucidated. Although we have correlated AIF-1 expression with proliferative and inflammatory gene expression in activated VSMCs, questions remain as to its role in the VSMC response to injury.²⁰

The specific aim of the present study was to test the hypothesis that AIF-1 expression plays a direct role in the progression of vascular proliferative disease by characterizing its role in the growth regulation of VSMCs.

	Methods

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Cell Culture, Stable Transfection, and Proliferation Assay
Primary rat arterial VSMCs were isolated from rat aorta and subcultured in DMEM with 10% FBS and 100 IU/mL penicillin and 50 µg/mL streptomycin. VSMCs from passage 3 were used in these studies. The protein coding region of AIF-1 cDNA (429 nt) was inserted into the expression vector pBK-CMV (Stratagene) and termed pBK-CMV-AIF-1. Vector alone (pBK-CMV) was used as a negative control. VSMCs were transfected with pBK-CMV plasmid alone or with pBK-CMV-AIF-1 with the use of 3 µL/mL lipofectin reagent (Life Technologies) and mixed with 1 µg/mL of either plasmid. After antibiotic selection (300 µg/mL G418; Fisher Biotechnology) to eliminate untransfected cells, transfectants were pooled and expanded in 150 µg/mL G418, and AIF-1 content was determined by Western blot. For proliferation assays, equal numbers of stable transfectants were seeded into 12-well plates at a density of 7500 cells per milliliter. The medium was changed on the fourth day, and after 1, 4, and 7 days, cells were trypsinized and counted by using a standard hemocytometer. Rat aortic VSMCs (A10 cell line obtained from American Type Culture Collection) were cultured in DMEM supplemented with 10% FCS as described.²¹ Transfection and selection were identical to those described for primary VSMCs, except that after antibiotic selection, individual colonies were selected and expanded in 150 µg/mL G418, and AIF-1 content was determined by Western blot. Doubling times for all VSMCs by using day 7 cell counts were calculated by the following formula: , where T is time, F is final cell number, and I is initial cell number.

Antibodies and Western Blotting
Cell extracts, SDS-PAGE, and Western blotting were performed as described.¹⁹ Equal protein concentrations of cell extracts were determined by Bradford assay, and equal loading on gels was verified by ponceau S staining of the membrane. AIF-1 antibody has been described.¹⁹ Proliferating cell nuclear antigen (PCNA) and secondary antibody were purchased from Transduction Laboratories, and cyclins D1, E, and B were from Santa Cruz. Immunoreactive proteins were visualized by using enhanced chemiluminescence (Amersham) according to the manufacturer’s instructions.

Flow Cytometry
VSMCs were serum-starved for 48 hours, and after 30 hours of stimulation with growth media, cells were harvested by trypsin treatment, collected by centrifugation, washed in 1 mL of 1% BSA in PBS, and fixed with ice-cold 70% ethanol for 1 hour. The fixed cells were centrifuged and treated with 0.1 mg/mL RNase A and 50 µg/mL propidium iodide for 30 minutes at room temperature. The flow cytometric analysis was performed on a Becton-Dickinson flow cytometer. For each sample, at least 35 000 nuclei were analyzed. The percentage of the cells in each cell cycle was determined by the CellFIT cell-cycle analysis version 2.01 from Becton-Dickinson. Three separate experiments were performed with 3 different transfections with similar results.

	Results

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Primary VSMCs That Constitutively Overexpress AIF-1 Have an Enhanced Proliferative Capacity
We transfected the protein coding region of AIF-1 cDNA or pBK-CMV empty vector into cultured primary rat VSMCs, isolated the stable transfectants by antibiotic selection, and pooled populations of resistant cells to avoid the effects of clonal selection. Equal numbers of these cells were seeded into replicate 12-well plates. Growth medium containing 10% FCS was replaced on the fourth day, and after 1, 4, and 7 days, cells were counted. The results of 3 independent transfection experiments demonstrate that primary VSMCs that overexpress AIF-1 grow at a more rapid rate than do control cells (P<0.0001, Figure 1A). The average doubling times for these cells are 28.6±0.56 and 24.1±0.24 hours for pBK-CMV and AIF-1, respectively. Western analysis of protein extracts from pooled, independently derived, stable transfectants confirmed overexpression of AIF-1 protein versus empty vector control cells (Figure 1B). Unstimulated VSMCs do not express AIF-1 protein,²⁰ and empty vector–transfected VSMCs express detectable levels of AIF-1 protein only when stimulated with growth medium containing 10% FCS, which induces endogenous AIF-1 expression. AIF-1 is constitutively expressed in transfected cells, and this amount increases on serum stimulation.

View larger version (36K): [in this window] [in a new window]   Figure 1. Primary VSMCs that stably overexpress AIF-1 grow more rapidly than those that do not. A, Equal numbers of pooled VSMCs stably transfected with pBK-CMV (plasmid control) or pBK-CMV-AIF-1 were seeded into 12-well plates. After 1, 4, and 7 days, cells were trypsinized and counted in triplicate. Numbers on the y-axis indicate cells per well. This is a representative experiment from 3 independent transfections with similar results (P<0.0001 for all experiments). B, Overexpression of AIF-1 protein in stably transfected primary VSMCs is shown. Extracts from pooled VSMCs stably transfected with pBK-CMV and AIF-1 were separated by SDS-PAGE, and AIF-1 content was determined by Western analysis with AIF-1–specific antisera. Some cells were treated with growth medium to induce endogenous AIF-1 expression.

Growth-Enhancing Effects of AIF-1 Are Dose Dependent
The limited lifespan of primary VSMCs necessitated the use of a VSMC line to more closely characterize the effects of AIF-1 expression on VSMC growth. Rat A10 VSMCs were stably transfected and selected as described for primary VSMCs. Several selected individual colonies (clones) were established, and the steady-state levels of AIF-1 protein were determined by Western analysis. Serum-starved VSMCs were used so that endogenously induced AIF-1 would not be detected. Figure 2A shows that different clones exhibited a wide range of AIF-1 expression, as quantified by scanning densitometry (Figure 2B). Selected AIF-1–overexpressing VSMC clones were then examined for their ability to influence the proliferation of VSMCs. Several cells expressing AIF-1 and pBK-CMV vector only were examined to diminish any effects of clonal selection. These experiments were conducted as described for primary VSMCs, and Figure 3 demonstrates that every AIF-1–overexpressing clone grew more rapidly than did cells expressing pBK-CMV vector alone, which had roughly identical growth rates. Clones C8 and C3, which express the highest levels of AIF-1, had more cells after 7 days than did clones C1 and C10, which are the lowest AIF-1–expressing clones. Clones C9 and C12, which are the median AIF-1–expressing clones, demonstrated cell numbers between the highest and lowest AIF-1–expressing clones. Between-group differences that were analyzed by Poisson regression with AIF-1 protein expression used as a covariant indicate that the observed differences in growth effect of each of these clones is highly dependent on the amount of AIF-1 expressed (P<0.0001). The doubling times for these clones were calculated just before reaching density-dependent growth inhibition (7 days for clones 8 and 3, Table) and indicate that the highest AIF-1–expressing clones have a shorter doubling time than do the median AIF-1–expressing, lowest AIF-1–expressing, and control cells. We also quantified the amount of PCNA expressed in each of these clones by scanning densitometry of Western blots. The Table illustrates that the fastest growing clones (C8, C3, and C12) contained more PCNA than did the slowest growing clone (C10) as well as all 3 pBK-CMV clones. Taken together, this indicates that the growth rate of the AIF-1–expressing cells corresponds with the level of AIF-1 expressed, indicating a close relationship between AIF-1 expression and the proliferation rate of VSMCs.

View larger version (49K): [in this window] [in a new window]   Figure 2. Expression of AIF-1 protein in A10 VSMCs. A, Rat VSMCs were transfected with AIF-1 cDNA or plasmid alone, 12 individual clones were selected and expanded, and AIF-1 levels in serum-starved cells were determined by Western blot. Numbers above each lane indicate clone number. N indicates a representative empty vector (pBK-CMV)–transfected clone; P, recombinant AIF-1 protein. B, Quantification of AIF-1 protein in stably transfected VSMCs by scanning densitometry is shown.

View larger version (29K): [in this window] [in a new window]   Figure 3. Dose-dependent growth effects of AIF-1 overexpression in VSMCs. Selected clones detailed in Figure 2 were expanded and, along with empty vector–transfected cells, were grown in growth medium. After 1, 4, and 7 days, cells were trypsinized and counted in triplicate. Numbers on y-axis indicate cells per well. Standard deviations are from 4 experiments.

View this table: [in this window] [in a new window]   Table 1. Growth Characteristics of Stably Transfected Rat VSMCs

Constitutive AIF-1 Expression Shortens the VSMC Cell Cycle
AIF-1 clone 8 (expressing highest AIF-1) was chosen to characterize the growth-enhancing characteristics of AIF-1 in more detail. VSMCs were first synchronized by serum deprivation for 48 hours and then stimulated with 10% FCS, and cell cycle distribution was assessed at several time points after stimulation by flow cytometry. Figures 4A and 4B reveal several differences between VSMCs that constitutively express AIF-1 versus control cells. First, even when they are serum-starved (time 0), 19.3% of AIF-1–expressing cells are replicating (S+G₂/M), whereas only 7.2% of pBK-CMV clone 1 VSMCs are replicating. Second, AIF-1–expressing cells enter the S phase more rapidly than do control cells, peaking at 16 hours versus 24 hours for pBK-CMV. Third, for AIF-1–expressing cells, the G₂/M phase peak contains 32.1% of the total cell population, whereas for vector control cells, the G₂/M peak contains only 18.3% of the population. This shift in the cell cycle profile implies that AIF-1 overexpression drives VSMCs more rapidly through the cell cycle.

View larger version (28K): [in this window] [in a new window]   Figure 4. Effect of AIF-1 overexpression on cell cycle distribution of rat VSMCs. Stably transfected A10 cells were serum-starved for 48 hours and then stimulated for the indicated periods with 10% FCS. Cells were processed for flow cytometry by propidium iodide staining. Numbers on x-axis indicate hours after stimulation; numbers on y-axis, percentage of cells in each phase. A, Percentage of cells in S phase. B, Percentage of cells in G2+M phase. AIF-1 is denoted by connected solid squares; pBK-CMV, by connected shaded diamonds.

Constitutive AIF-1 Expression Alters Expression of Cell Cycle–Regulatory Proteins
The effects of AIF-1 expression on the kinetics and degree of expression of cell cycle proteins were examined in more detail by Western analysis. VSMCs were synchronized by serum reduction for 48 hours, and cellular proteins were extracted at several time points after stimulation with 10% FCS. Figure 5 shows that comparable to the flow cytometry experiments, AIF-1 overexpression upregulates the expression of cell cycle proteins. pBK-CMV–transfected cells display a more conventional cell cycle protein expression profile: expression of the G₁-phase marker cyclin D1 is expressed at 10 to 16 hours, the G₁/S-phase marker cyclin E peaks at 16 hours, the S-phase marker PCNA begins to rise at 16 hours and peaks 24 hours after stimulation, and the G₂/M-phase marker cyclin B peaks at 24 hours after stimulation. In contrast, the induction and degree of expression of PCNA and cyclins D1, E, and B are atypical in AIF-1–expressing cells. The first noticeable difference in these cells are the appreciable basal levels of PCNA and cyclins in the unstimulated samples. Furthermore, rapid increases in expression of these proteins are noticeable at 1 hour after stimulation and peak between 10 and 16 hours after simulation. PCNA and cyclin B levels remain elevated up to 32 hours after stimulation, and they decline by 48 hours after stimulation.

View larger version (57K): [in this window] [in a new window]   Figure 5. Effect of AIF-1 on expression of cell cycle–regulatory proteins in VSMCs. AIF-1 clone 8 (highest AIF-1 expression) and pBK-CMV clone 1 were serum-starved for 48 hours and then stimulated for the indicated periods of time with 10% FCS. Protein extracts were separated by SDS-PAGE, transferred to nitrocellulose, and Western-blotted with antibodies to the indicated cell cycle proteins.

AIF-1–Expressing Cells Can Proliferate in Serum-Reduced Media
Previous experiments showing the expression of cell cycle proteins and the presence of replicating cells in serum-reduced media have suggested that serum-deprivation alone might not be sufficient to achieve full quiescence in AIF-1–expressing cells. To more directly investigate a direct effect of AIF-1 on cell growth in the absence of growth-stimulatory factors, equal numbers of pBK-CMV-AIF-1 clones 8 and 3 (high AIF-1 expression), clone 1 (low AIF-1 expression), and pBK-CMV clones 1 and 3 were seeded into replicate 12-well plates. After 24 hours, the medium was replaced with medium containing 0.3% FCS, and cells were counted at 1, 3, and 6 days. Figure 6 shows that the highest AIF-1–expressing clones can proliferate in 0.3% FCS up to 6 days, whereas similar to wild-type A10 cells, the lower AIF-1–expressing clone and pBK-CMV–expressing clones cannot.^21,22 The differences between the highest AIF-1–expressing and the low AIF-1– and empty vector–expressing clones are significant at 6 days (P<0.0001). An important observation is that control cells increase only 7% from day 3 to day 6, whereas the highest AIF-1–expressing cells increase an average of 65% from 3 to 6 days. These data are in agreement with data obtained by flow cytometry and Western analysis of cell cycle proteins and, taken together, indicate that constitutive expression of high levels of AIF-1 allows VSMCs to overcome the growth-inhibitory effects of serum deprivation, implying that AIF-1 can influence entry into the cell cycle.

View larger version (24K): [in this window] [in a new window]   Figure 6. Growth of AIF-1–expressing clones in serum-free medium. AIF-1 clones 8 and 3 (highest AIF-1 expression), clone 1 (low AIF-1 expression), and pBK-CMV clones 1 and 2 were seeded into 12-well plates in the presence of 0.3% FCS. After 1, 3, and 6 days, they were trypsinized and counted in triplicate. Numbers on y-axis indicate cells per well. Standard deviations are from 3 experiments.

	Discussion

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We have previously shown that AIF-1 expression is induced in several models of vascular proliferative disease, including rat aortic allografts and balloon-injured swine coronary arteries.¹⁹ The present study extends those observations by indicating that constitutive overexpression of AIF-1 in primary rat VSMCs results in an enhanced ability of these cells to proliferate. It was important to determine whether the amount of AIF-1 overexpressed could be directly correlated with the degree of growth enhancement of the transfected cells. The use of a VSMC line facilitated the isolation of individual clones to permit differential selection of the amount of AIF-1 being expressed and allowed growth comparison between each clone. Clonal selection of stably transfected A10 VSMCs has been used to correlate the proliferative effects of c-myc and protein kinase C isoforms and the inhibition of proliferation by abrogation of cell cycle proteins.^22–24 The close relationship observed between the amount of AIF-1 overexpression in each clone and its proliferative capacity points to a central role for this protein in VSMC growth.

The use of stably transfected VSMC clones also allowed us to expand the cell population for more comprehensive studies. Cell cycle examination by flow cytometry can indicate what percentage of cells are present in a particular phase at a particular point in time. Two key points taken from this experiment suggest an AIF-1–mediated deregulation of the mechanisms that regulate growth factor–driven cell cycle progression. The first point indicates that AIF-1–expressing cells move more rapidly through the cell cycle than do control cells, inasmuch as they peak in S phase earlier. The second point suggests that unlike control and wild-type VSMCs, AIF-1–expressing cells cannot achieve complete quiescence by serum-reduced media. Both of these observations led us to more direct investigation of the impact of AIF-1 on VSMC growth. Because the cell cycle is controlled by the precisely coordinated expression and turnover of a particular set of proteins, examination of cell cycle protein expression can generate insight into the molecular mechanisms responsible for the changes we see with the flow data. In this regard, the observed deregulation of cyclin and the PCNA expression in AIF-1–transfected cells not only show a clear change in cell cycle kinetics but corroborate the flow cytometry data. Upregulation of cyclin D1 suggests that this effect is early in the cell cycle, but direct effects on other cell cycle phases cannot be ruled out at this time. Quantification of cell number and PCNA content of several AIF-1–containing versus empty vector–containing clones and distinctions in growth capacity in the absence of serum, together with observed growth enhancement in pooled primary VSMCs, suggest that the differences in growth are not a function of clonal selection but are dependent on the amount of AIF-1 being expressed.

To summarize the flow cytometry and Western data, 2 distinct, but complementary, effects of AIF-1 overexpression are evident. First, AIF-1 expression enhances growth factor–induced proliferation. Second, the fact that VSMCs, which constitutively express AIF-1, also express low levels of cell cycle proteins and continue to proliferate in low serum leads us to conclude that AIF-1 expression also maintains VSMCs in a state of activation, in which they are primed to readily proliferate on the addition of growth factors.

The exact mechanism by which AIF-1 imparts growth-promoting effects in VSMCs is still being elucidated. The AIF-1 protein contains 1 EF-hand calcium-binding sequence and binds calcium. Calcium, its primary receptor protein CaM, and calcium-dependent protein kinases are essential for the entry of quiescent cells into the cell cycle in response to mitogenic signals.^5,25–27 In some studies, calcium channel blockers and calcium antagonists reduce restenosis subsequent to mechanical and immunologic insult, most likely through the inhibition of growth factor–stimulated VSMC migration and proliferation.²⁸ Overexpression of CaM in cultured cells enhances cell proliferation, primarily through a reduction in the length of the G₁ phase of the cell cycle.^6,7 Overexpression of the calcium-binding protein sphingosine kinase (SPHK) in 3T3 fibroblasts enhances proliferation by promoting the G₁- to S-phase transition.⁹ Flow cytometric analysis indicated that stable overexpression of SPHK shortened the G₁ phase of the cell cycle by 31%, and these investigators concluded that SPHK is important for nontransformed cells to progress through the G₁-S boundary. Stable overexpression of several other calcium-binding proteins has also been shown to influence the cell cycle.^8,9 Analogous to CaM and SPHK, overexpression of AIF-1 results in a 28% reduction in doubling time for clone 8, which is comparable to the 10% to 20% reduction in doubling times reported for cell lines that overexpress CaM and the 21% reduction time reported for SPHK.^6,9 Because AIF-1 has only 1 EF-hand, its calcium binding is more likely to dictate its tertiary structure and thus to have an impact on its interactions with other proteins rather than to sequester calcium directly.

Overexpression of other proteins has been reported to support growth of VSMCs in serum-reduced media, including the oncoprotein c-myc and the hyaluronan-binding protein TSG-6 (tumor necrosis factor–stimulated gene-6).^22,29 Interestingly, similar to AIF-1, both of these proteins are rapidly induced in cytokine-stimulated VSMCs and arterial injury. Expression of c-myc in particular is analogous to AIF-1, in that it is not limited to the early growth factor response but continues throughout the first and following cell cycles, suggesting that it is important not only in the initiation but also in the maintenance of VSMC proliferation.³⁰ Thus, its expression may be part of the VSMC response to injury, resulting in proliferation and intimal hyperplasia.

On the basis of the expression pattern and genomic location of AIF-1, several investigators have proposed a fundamental role for AIF-1 in inflammatory reactions, although a function for this protein has yet to be elucidated.^20,31 Similar to the aforementioned proteins, overexpression of AIF-1 in VSMCs may not approximate its cytokine-inducible expression in response to injury in vivo. However, the present study is the first to attempt to define a function for this protein and presents several novel findings concerning the role of AIF-1 in vascular proliferative diseases. First, overexpression of AIF-1 protein in VSMCs results in the enhanced growth of those cells. Second, AIF-1 expression correlates with the degree of VSMC proliferation. Third, constitutive AIF-1 expression leads to a shortening of the cell cycle and aberrant expression of cell cycle proteins. Finally, AIF-1 overexpression facilitated VSMC growth in the absence of serum growth factors. The cytokine and injury-inducible expression of AIF-1 and its ability to modulate the growth of VSMCs support the hypothesis that the expression of AIF-1 in injured arteries may participate in the proliferative response of VSMCs to inflammatory stimuli.

	Acknowledgments

 
This work was supported in part by the W.W. Smith Charitable Trust (grant H9606 to M.V.A.).

Received March 19, 2001; accepted June 27, 2001.

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