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(Circulation. 2003;107:98.)
© 2003 American Heart Association, Inc.

Basic Science Reports

Adenoviral Gene Transfer of Fortilin Attenuates Neointima Formation Through Suppression of Vascular Smooth Muscle Cell Proliferation and Migration

David A. Tulis, PhD; Zakar H. Mnjoyan, PhD; Rachel L. Schiesser, BS; Harnath S. Shelat, MS; Alida J. Evans, BS; Pierre Zoldhelyi, MD; Ken Fujise, MD

From the Department of Medicine, Baylor College of Medicine (D.A.T., R.L.S., A.J.E.); Veterans Affairs Medical Center (D.A.T., R.L.S.); The Institute of Molecular Medicine for the Prevention of Human Diseases (Z.H.M., K.F.); and Division of Cardiology, Department of Internal Medicine (H.S.S., P.Z., K.F.), The University of Texas Health Science Center at Houston, Houston, Tex.

Correspondence to David A. Tulis, PhD, Baylor College of Medicine, Department of Medicine, 2002 Holcombe Blvd, Bldg 109, Room 118, Houston, TX 77030. E-mail dtulis{at}bcm.tmc.edu

	Abstract

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Background— Fortilin, a recently characterized nuclear antiapoptotic factor structurally distinct from inhibitor of apoptosis proteins (IAPs) and Bcl-2 family member proteins, has been suggested to be involved in cell survival and regulation of apoptosis within the cardiovascular system. In this continued investigation, we characterized the influence of adenovirus-mediated fortilin (Ad-fortilin) gene delivery on vascular remodeling after experimental angioplasty.

Methods and Results— Vessel wall expression of Ad-fortilin or adenoviral luciferase (Ad-luc) was demonstrated 72 hours and 14 days after rat carotid artery (CA) balloon angioplasty. Morphometric analyses 14 days after injury revealed significantly diminished neointima development in the Ad-fortilin–treated CAs compared with Ad-luc or PBS controls, with no changes in medial wall morphometry observed between the 3 groups. The Ad-fortilin–treated CAs demonstrated a 50% reduction in medial wall proliferating cell nuclear antigen (PCNA) labeling after 72 hours, with significantly reduced neointimal and medial wall PCNA labeling and cell counts after 14 days. Terminal dUTP nick-end labeling results and morphological changes characteristic of programmed cell death suggest a trend toward reduced apoptosis in the fortilin-transfected balloon-injured vessels compared with Ad-luc injured controls. Temporal analysis of human aorta smooth muscle cell (SMC) proliferation demonstrated a marked time-dependent inhibition in Ad-fortilin treated SMCs without the influence of elevated apoptosis. Thymidine incorporation was significantly inhibited in the Ad-fortilin–treated cells compared with Ad-luc controls. Ad-fortilin transfected SMCs also demonstrated significantly decreased migration compared with Ad-luc controls.

Conclusions— These cumulative results suggest that the novel antiapoptotic protein fortilin may play important redundant pathophysiological roles in modulating the vascular response to experimental angioplasty through suppression of SMC proliferation and migration concomitant with reduction of vessel wall apoptosis.

Key Words: angioplasty • carotid arteries • proteins • gene therapy • remodeling

	Introduction

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The coordinated interplay of cellular proliferation and apoptosis is fundamental to normal tissue and organ system development and homeostasis, as well as to the pathophysiological adaptations often observed after onset of disease or injury. Within the cardiovascular system, eutrophic vasculature typically demonstrates quiescent cell cycling with a minimal degree of apoptosis; however, under the inimical conditions after interventional catheterization, vascular cells undergo an array of phenotypic modifications, including enhanced proliferation, migration, apoptosis, and synthesis of extracellular matrix components. These synchronized processes eventuate in the formation of an adaptive neointima, which, along with constrictive inward remodeling of the vessel, contribute to restenosis of the vessel. Although redundant experimental approaches have identified many features intrinsic to this process, discordant and confusing results necessitate further investigation.

Fortilin is a recently characterized hydrophilic nuclear protein that has been suggested to be involved in cell survival as a negative regulator of apoptosis.¹ Fortilin is theorized to be a new class of apoptosis modulator, structurally unique from inhibitor of apoptosis proteins (IAPs) and Bcl-2 family member proteins. Sequence analysis reveals that fortilin is a highly conserved protein, ubiquitously expressed in a wide range of normal tissue and especially abundant in tumorigenic tissue. Functional analyses reveal that fortilin can prevent mammalian cells from undergoing chemically induced apoptosis and cytotoxicity, and overexpression of fortilin was shown to inhibit pro-apoptotic effector caspases. In addition, antisense depletion of fortilin from a malignant human ductal carcinoma cell line stimulated spontaneous and massive cell death.

Our laboratory² and others³ have proposed that, after experimental vascular injury, acute stimulation of apoptosis reduces the viable smooth muscle cell (SMC) population resident within the medial wall, thereby attenuating ensuing neointima formation. Alternatively, it has been suggested that early onset of apoptosis stimulates subsequent proliferation and migration of surviving medial SMCs, thereby contributing to ensuing neointimal formation.⁴ This process of enhanced apoptosis, however, reduces the structural integrity of the vessel wall through medial wall degeneration, and may dispose the injured artery to progressive dilatation and aneurysm.⁵ Herein, we report that localized adenovirus-mediated gene delivery of antiapoptotic fortilin (Ad-fortilin) to balloon-injured rat carotid arteries (CAs) significantly attenuates neointima development through suppression of vascular SMC proliferation and reduced migration. We believe that fortilin confers this vasoprotection without compromising vessel wall integrity from deleterious pro-apoptotic actions. We suggest that fortilin may play a pivotal pathophysiological role in modulating the arterial response to experimental angioplasty through redundant antiproliferative and antiapoptotic properties, and it represents a robust multifunctional therapeutic agent aimed at minimizing the pathobiological adaptive response to vascular intervention.

	Methods

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Rat Carotid Artery Balloon Angioplasty
Our procedure for balloon angioplasty in the rat CA has been previously described.^2,6 Male Sprague Dawley rats (435±4.8 g; Harlan, Indianapolis, Ind) were anesthetized (ketamine, xylazine, and acepromazine; 0.5 to 0.7 mL/kg intramuscularly; VetMed Drugs), and the left CA vasculature isolated. A Fogarty 2F embolectomy catheter (Baxter Healthcare Corp) was introduced into the common CA through the external carotid branch, advanced, inflated, and withdrawn thrice. The catheter was removed, and the injured vessel was exposed to the various treatments. Animals were closed and given buprenorphine (0.5 mg/kg subcutaneously; Greenpark Pharmacy) for analgesia. On full recovery, animals were returned to the animal care facility and provided standard rat chow and water ad libitum. At specific times, rats were euthanized by exsanguination, and the tissues were harvested for specific protocols. All experimental protocols used complied with guidelines of the institutional animal care and use committee.

Adenovirus-Fortilin Gene Preparation and Delivery
Using standard polymerase chain reaction techniques, the full-length human fortilin cDNA with a FLAG sequence (DYKDDDDK) at the 5'-end was inserted into the shuttle plasmid pDC515 containing the mouse cytomegalovirus promoter (Microbix, Ontario, Canada). Recombinant adenoviruses were generated by co-transfection into 293 cells of pDC515-FLAG-fortilin and pBHGfrtE1, 3FLP (Microbix). A viral stock of Ad-fortilin was plaque-purified, further propagated in 911 cells (IntroGene), and purified by CsCl centrifugation.⁷ Purified virions were suspended in sucrose (2% wt/vol) and MgCl₂ (2 mmol/L) in PBS, desalted by Sepharose CL4B chromatography (Pharmacia), supplemented with 5% glycerol, and stored at -80°C. The concentration of infectious viral particles was determined as described previously.⁸ Recombinant adenoviruses containing the luciferase gene (Ad-luc) were prepared and titrated in a similar fashion.

The protocol used for treating balloon-injured rat CAs with adenovirus has been previously described.² Immediately after injury, a polyethylene catheter was introduced through the external carotid branch and the CA section was flushed. Fifty µL (2x10⁹ plaque-forming units/mL) of Ad-luc, Ad-fortilin, or PBS was infused and incubated for 30 minutes. The adenovirus (or PBS) was removed, the CA section flushed, and blood flow restored through the CA.

Morphometric Analysis
Standard histological staining procedures were performed.² Microscopic analyses were performed using Image Pro Plus 4.1.1.2 (Media Cybernetics, L.P.) and Adobe Photoshop 5.5 (Adobe Systems) linked through a realtime XCAP-Lite digital color camera (EPIX, Inc) to a Zeiss Axioskop 2 Plus light microscope (Carl Zeiss).

Ad-fortilin and Ad-luc Immunohistochemistry
Immunohistochemical analysis of injured CAs treated with Ad-fortilin or Ad-luc was performed.¹ Perfusion-fixed, paraffin-embedded CA sections were deparaffinized, rehydrated, and subjected to antigen retrieval (Citra Plus microwave method, BioGenex). Endogenous peroxidase activity was quenched, and tissues were incubated with anti-FLAG (fortilin) monoclonal (1:400) or anti-luciferase polyclonal antibodies (1:800) for 60 minutes. Negative control tissues were incubated with PBS and did not contain primary antibody. Bound antibodies were detected using EnVision system (DAKO) and diaminobenzidine (DAB; Sigma). Tissues were lightly counterstained with hematoxylin and analyzed under light microscopy.

Proliferating Cell Nuclear Antigen Immunohistochemistry
Immunostaining for proliferating cell nuclear antigen (PCNA) was performed as previously described.² An anti-PCNA monoclonal antibody (PC-10; 1:25; Sigma) complimented by a biotinylated anti-mouse secondary antibody (1:100) was applied to perfusion-fixed, paraffin-embedded tissues. Slides were treated with an avidin-biotin block and exposed to DAB black with nuclear fast red counterstain and analyzed under light microscopy. Data are represented as a PCNA labeling index (LI), defined as the percentage of total cells within a given area positive for PCNA staining.

Western Blot for Fortilin Protein
Western blot analyses of injured CAs treated with Ad-fortilin or Ad-luc were performed.¹ Snap-frozen specimens were pulverized, incubated in protein extraction buffer (20 mmol/L NaCl, 100 mmol/L Tri-HCl, 5% SDS, 5% NP-40, phenylmethylsulfonyl fluoride, aprotinin, benzamidine; pH 7.6), and centrifuged to remove insoluble fractions. Protein concentrations were measured using Micro BCA Protein Assay kit (Pierce). Thirty micrograms of protein for each specimen were subjected to SDS-PAGE and Western blotting with the use of anti-FLAG (fortilin) (1:1000) or anti-luc (1:250) antibodies. Protein loading controls were established by probing the same membrane with an -actin-specific antibody (1:10 000; Chemicon).

Vascular SMC Proliferation Assay
Human aortic SMCs (2x10⁵) were seeded in duplicate in 6-well plates. Cells were transduced with either Ad-fortilin or Ad-luc (100 pfu/cell) for 24 hours, serum-starved for 48 hours, and then serum-stimulated for 24 hours. After growth stimulation, cells were washed with PBS, harvested by trypsinization, and counted using a hemocytometer.

Thymidine Incorporation Assay
Serum-stimulated human aortic SMCs (2x10⁴/well) were seeded in triplicate in 24-well plates, treated with Ad-fortilin, Ad-luc, or PBS for 24 hours, serum-starved for 48 hours, and serum-stimulated for 24 hours in the presence of 1 µCi/mL [methyl-³H]-thymidine (Amersham Biosciences). Cells were harvested into ristocetin-induced platelet agglutination buffer (50 mmol/L Tris-Cl, pH 7.2, 1% NP-40, 150 mmol/L NaCl, 1% sodium deoxycholate, 0.1% SDS). Protein concentrations of lysates were measured using a Micro BCA Protein Assay kit (Pierce), and lysates were counted (LS6500 Multipurpose Scintillation Counter, Beckman) to determine tritium content. The thymidine index was calculated as [total counts (dpm)]/[total protein amount (µg)].

Terminal dUTP Nick-End Labeling Staining
Terminal dUTP nick-end labeling (TUNEL) was performed on cultured human aorta SMCs using a FragEL DNA Fragmentation Detection Kit (Oncogene Research Products). Cells (5x10⁴/well) were seeded in duplicate in 8-well Laboratory-Tech chamber slides (Nalge Nunc International), treated with Ad-fortilin, Ad-luc, etoposide, or PBS for 24 hours, and harvested. Cells were serially treated with paraformaldehyde, peroxidase, a biotin-conjugated deoxynucleotide mixture, and a streptavidin-horseradish peroxidase (HRP) conjugate with hematoxylin and eosin counterstain. Bound HRP was detected by DAB, and a minimum of 500 cells per chamber was counted. Data are represented as a TUNEL LI.

Balloon-injured CAs were stained for apoptotic nuclei using an In Situ Cell Death Detection kit.² Perfusion-fixed, paraffin-embedded tissues were treated with proteinase K, endogenous peroxidase activity was blocked, and tissues exposed to avidin-biotin block with antigen retrieval. Tissues were incubated with TUNEL reaction mixture and treated with converter-peroxidase solution. Tissues were treated with DAB, counterstained with hematoxylin, and analyzed under light microscopy. Data are represented as a TUNEL LI and are normalized for background (contralateral right CA). Conventional light microscopy was performed on TUNEL-positive cells to confirm morphological characteristics of apoptosis.

Migration Assay
Vascular SMC migration was assayed using a modified Boyden’s chamber method.⁹ Briefly, human aortic SMCs were transduced with Ad-luc or Ad-fortilin for 24 hours, trypsinized, and washed with medium 231 without serum. Exactly 1x10⁴ cells per group were aliquotted onto the upper surface of a 5-µm pore size ChemoTx plate (Neuro Probe, Inc). Medium 231 containing varying percentage of serum was aliquotted into the lower chamber of the plate. Cells were allowed to migrate for 6 hours at 37°C. Cells on the upper surface were removed, and migrated cells on the lower surface were fixed in 4% paraformaldehyde, permeabilized with 70% methanol, and stained with hematoxylin. Experiments were performed in triplicate, and numbers of migrated cells were counted by light microscopy (400x magnification). The punctate appearance of the surface (ie, dark blue dots) represents the 5-µm pores, whereas the lighter blue diffuse staining (lower right panel) signifies migrated vascular SMCs.

Endothelial Regrowth
Experiments examining the influence of adenoviral fortilin on endothelial regrowth after balloon injury were performed.⁶ Animals were balloon-injured with intravenous injection of Evans blue dye (0.5 mL in 5% PBS; Sigma) 15 minutes before sacrifice. At various time points, animals were perfuse-fixed and the injured left CA and its contralateral right CA removed. The area of the unstained re-endothelialized section was measured and normalized to the entire area of the vessel.

Statistical Analyses
Data were stored and analyzed on personal computers using Excel 2000 (Microsoft) and Sigma Plot 8.0 with Sigma Stat 2.03 (SPSS, Inc). In vivo data were grouped according to treatment and analyzed using an unpaired Student’s t test. In vitro data were compared using a one-way ANOVA with Tukey’s post-hoc multiple comparison test. All data are represented as mean±standard error of the mean (SEM). A probability value 0.05 is considered statistically significant for all comparisons.

	Results

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Successful adenoviral transfection is demonstrated for balloon-injured rat CAs treated with Ad-fortilin or Ad-luc 72 hours and 14 days after injury by Western blot and immunohistochemistry (Figure 1A and 1B, respectively). Western analysis indicates robust fortilin and luciferase protein expression in the injured, treated left CA segments compared with the uninjured, untreated contralateral CAs. Residual neointimal expression of fortilin and luciferase is observed 14 days after injury (Figure 1B). These results verify efficient and prolonged protein expression from localized luminal gene delivery after ipsilateral experimental angioplasty.

View larger version (53K): [in this window] [in a new window]   Figure 1. Robust protein expression for Ad-fortilin or Ad-luciferase is demonstrated in balloon-injured rat carotid arteries by Western blot analysis 72 hours after injury (A) and by immunostaining 14 days after injury (B).

Representative photomicrographs of Verhoeff Van Gieson-stained, balloon-injured rat CA cross-sections 14 days after injury are shown in Figure 2. Figure 2A illustrates an injured CA exposed to PBS immediately after injury. A significant concentric neointima is evident, with the media clearly defined by the internal and external elastic laminae. Figure 2B and 2C show injured CAs treated with Ad-luc or Ad-fortilin, respectively. Histomorphometric analyses for these data are graphically illustrated in Figure 2D through 2G. Although a slight trend for an increased neointima is evident in the Ad-luc vessels compared with the PBS controls, this elevation does not approach statistical significance.

View larger version (47K): [in this window] [in a new window]   Figure 2. Representative photomicrographs (magnification 100x) of balloon-injured, perfusion-fixed, Verhoeff-van Gieson-stained left carotid arteries 14 days after injury. Immediately after injury, arteries were treated with PBS (A), Ad-luciferase (B), or Ad-fortilin (C). No statistically significant differences were detected between the Ad-luciferase and PBS control arteries for any morphometric parameter; however, the Ad-fortilin–treated arteries exhibit significant reduction in neointima formation (D, E, and F) compared with either the PBS- or Ad-luciferase arteries. No observed changes in medial wall area (G) were detected between the 3 treatment groups. nequals;6 for PBS, 9 for Ad-luciferase, and 9 for Ad-fortilin groups. *P<0.05 versus PBS; #P<0.05 versus Ad-luciferase.

Parameters for balloon-injured CA neointimal area (Figure 2D), neointimal thickness (Figure 2E), and neointimal to medial wall area ratio (Figure 2F) were all significantly attenuated in the Ad-fortilin CAs compared with vessels treated with PBS or Ad-luc. No significant differences in medial wall area were observed between the PBS, Ad-luc, or Ad-fortilin CAs (Figure 2G). No differences were detected between the Ad-luc and Ad-fortilin CAs for circumferences of the left CA internal elastic laminae (2.69±0.04 versus 2.64±0.03 mm, respectively) or external elastic laminae (3.01±0.04 versus 2.97±0.03 mm, respectively).

Figure 3 represents medial wall and neointimal cellular changes 14 days after balloon injury for CAs treated with Ad-luc or Ad-fortilin. Figure 3A shows significantly decreased PCNA labeling in the Ad-fortilin vessels in both the medial wall and neointima. Similarly, Figure 3B illustrates significant reduction in total cell counts in both the media and neointima of Ad-fortilin CAs. Figure 3C shows a marked reduction in medial wall cellular density in the Ad-fortilin vessels only, whereas interestingly, no differences in neointimal cellular density were observed between the Ad-luc and Ad-fortilin CAs. Cellular analyses 72 hours after injury reveal approximately a 50% reduction in medial wall PCNA labeling in the Ad-fortilin vessels compared with the Ad-luc vessels (data not shown); however, no differences were observed at this time point in absolute or normalized cell counts between the treatment groups. TUNEL results on balloon-injured CAs 72 hours after injury demonstrate a trend (P=0.06) toward a significant reduction of apoptosis in fortilin-treated vessels (-1.87±2.8%) compared with Ad-luc controls (8.95±3.1%). Complimenting the TUNEL results, positively labeled cells consistently revealed the presence of intracellular cytoplasmic blebbing, membrane detachment from surrounding cells and cell shrinkage, and nuclear condensation (data not shown).

View larger version (40K): [in this window] [in a new window]   Figure 3. Medial wall and neointimal cellular changes 14 days after balloon injury in arteries treated with Ad-luciferase or Ad-fortilin. PCNA labeling index (A), absolute cell counts, and cell counts normalized to area (C). At 72 hours after injury, balloon-injured arteries treated with Ad-fortilin showed approximately a 50% reduction in medial wall PCNA LI compared with Ad-luciferase-treated arteries, with no differences in absolute or normalized cell counts (data not shown). n=3 per treatment group. *P<0.05, **P<0.01, ***P<0.001.

The temporal influence of Ad-luc or Ad-fortilin on human aorta SMC proliferation is shown in Figure 4A. Starting at 72 hours and progressing through 120 hours, SMCs exposed to Ad-fortilin demonstrate a significant time-dependent reduction in cell number compared with the Ad-luc controls. Supplementary analyses indicate that this reduction in cell number is not due to fortilin-induced apoptosis (Figure 4B) or to cellular necrosis and detachment (data not shown). Figure 5 illustrates results from a thymidine incorporation assay and shows that DNA replication is inhibited approximately 40% in the Ad-fortilin cells compared with the Ad-luc cells. No significant differences in thymidine uptake were observed in the non-stimulated, quiescent cell populations exposed to Ad-luc or Ad-fortilin. Evans blue endothelial cell exclusion staining showed no observable differences in the extent of endothelial cell regeneration between the Ad-luciferase-treated (40.8±0.8%) and the Ad-fortilin–treated (40.3± 0.3%) arteries.

View larger version (17K): [in this window] [in a new window]   Figure 4. A, Temporal analysis of human aorta SMC proliferation indicates that, starting 72 hours after treatment and progressively increasing through 120 hours, Ad-fortilin–treated cells are significantly inhibited compared with Ad-luciferase–treated controls. n=2 per treatment group. *P<0.05, **P<0.01, Tukey’s multiple comparison test after one-way ANOVA. B, TUNEL apoptosis assay for human aorta SMCs demonstrates that the inhibition of SMC proliferation experienced by Ad-fortilin-transfected cells was not due to increased apoptosis. A negative control and an etoposide-induced positive control are shown for comparison. n=2 per treatment group. ***P<0.001 versus negative control.

View larger version (24K): [in this window] [in a new window]   Figure 5. Thymidine incorporation assay in human aorta SMCs. Data indicate that vascular SMC DNA replication is inhibited approximately 40% in the Ad-fortilin cells compared with Ad-luciferase controls. n=4 per treatment group. *P<0.05 versus serum-stimulated controls; #P<0.05 versus serum-stimulated Ad-luciferase cells.

As shown in Figure 6A, human aorta SMCs did not migrate into the lower chamber without serum; however, at 5% serum, numerous SMCs exposed to Ad-luc migrated, whereas in those treated with Ad-fortilin, migration was significantly inhibited. Dose-dependency in terms of percentage serum-stimulation is illustrated in Figure 6B, showing a highly attenuated migration of fortilin-transfected SMCs compared with Ad-luc controls.

View larger version (39K): [in this window] [in a new window]   Figure 6. A, Migration assay using human aorta SMCs. Data demonstrate significantly reduced migration in Ad-fortilin–treated SMCs compared with those treated with Ad-luciferase after 5% serum stimulation. B, Significant inhibition of migration is observed in Ad-fortilin–transfected SMCs compared with Ad-luciferase controls. n=3 per group per % serum. *P<0.05, **P<0.01, ***P<0.001.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We describe here results from our continued investigation into the potential vascular regulatory functions of a recently described antiapoptotic protein, fortilin. Using a highly characterized experimental model of arterial injury, we report that targeted adenoviral fortilin significantly minimizes neointima development without altering medial wall dimensions. Fortilin exerted potent antiproliferative actions in the medial wall and in the neointima, which were corroborated by reduction of SMC proliferation and DNA replication under culture conditions. We also provide data suggesting an antimigratory function for fortilin under controlled conditions, and an antiapoptotic function for fortilin after arterial injury. We propose that fortilin may represent an important vasoprotective protein with therapeutically effective and redundant actions capable of attenuating the stenotic neointimal response to injury.

The novel antiapoptotic functions of fortilin were recently described in scientifically comprehensive studies by this laboratory.^1,10 Fortilin was originally described as a growth-related protein in murine cells that was subjected to translational repression processes¹¹ and was termed "translationally controlled tumor protein" (TCTP). Subsequent investigations have discovered that TCTP was originally cloned from non-tumorigenic cells,¹² and that TCTP is widely expressed in a variety of normal, non-cancerous tissues.¹³ The regulation of TCTP is also under transcriptional control.¹⁴ On the basis of accumulating evidence, we advocate use of the term "fortilin" (from Latin fortis, strong, robust) for this unique and important protein.^1,10

For these initial experiments addressing the potential physiological impact of fortilin on injured vasculature, we chose to use the well-characterized rat CA balloon angioplasty model that has been established in our laboratory.^2,6 Anatomic constraints of the rat CA include a lower percentage of medial wall elastin, a condensed subintimal layer, and lack of an existing vasa vasorum.¹⁵ Moreover, experimental angioplasty using the rat CA model results in formation of a primary neointimal lesion without preexisting atherogenic or vasoproliferative components, and the adaptive response entails incomplete endothelial regeneration. Despite these inherent limitations compared with the human system, however, this model is still widely applied for the study of molecular mechanisms and presents a useful "proof-of-concept" tool. Ipsilateral adenovirus-mediated luminal delivery of fortilin or luciferase was performed immediately after rat CA balloon angioplasty, and efficiency of transfection was verified through Western blotting and immunostaining. Results confirm successful transfection of the arterial wall occurred after injury, with significant gene expression after 72 hours that persisted through 14 days.

Vessel wall remodeling and neointimal development resultant from pathological insult or onset of disease are intricate processes largely dependent on the balance between cell growth and cell loss. Reduction of the proliferative capacity of medial wall SMCs subject to arterial intervention has been suggested to provide protection against intimal thickening under experimental conditions.^2,16 In this report, we demonstrate that fortilin exerts robust antiproliferative actions on arterial SMCs under both injurious and controlled cell culture conditions. Ad-fortilin arteries from balloon-injured animals exhibited marked inhibition of medial wall DNA replication 72 hours after injury. At 14 days after injury, the Ad-fortilin vessels demonstrated significantly reduced DNA replication, as well as cell proliferation in both the medial wall and in the neointima. These data provide strong evidence that fortilin is functionally active in injured arterial SMCs and suggest that fortilin confers protection against aberrant cell proliferation under pathobiological or phenotypically altered states. These antiproliferative actions of fortilin seem to be limited to vascular SMCs, as differences in the extent of endothelial cell regeneration between Ad-fortilin–treated and Ad-luciferase–treated vessels were not detected. Interestingly, when the 14-day data are normalized to area, the diminished neointima of the fortilin-treated animals displayed unchanged cellular density. Acute inhibition of apoptosis in fortilin-transfected injured cells, as discussed below, may eventuate in an increased ability of the remaining viable SMCs to synthesize matrix components. This suggests that fortilin, in addition to its antiproliferative characteristics, may indirectly act to stimulate production of matrix components, elements that are formative and essential to the neointima and to vessel architecture, in phenotypically modified cells. Moreover, fortilin, in addition to its inhibitory effect on cell cycle progression, negatively modulates vascular SMC migration as an additional mechanism controlling neointima development.

Stimulation of apoptosis in injured SMCs has also been shown to regulate injury-induced neointima formation.^2,17,18 In this study, the influence of adenoviral fortilin on balloon-injured arterial SMC apoptosis was evaluated by both TUNEL and light microscopy. Results suggest that fortilin negatively regulates the extent of programmed cell death after arterial injury. These results confirm previously documented antiapoptotic functions of fortilin observed under controlled culture conditions.^1,10 Elevated vascular SMC apoptosis leads to SMC diminution and is implicated in the degeneration of the medial wall,¹⁹ thus contributing to progressive arterial dilatation and aneurysmal formation.^5,20 This pathological degeneration of the medial wall from elevated apoptosis presents an undesirable characteristic of pro-apoptotic therapy aimed at minimizing injury-induced stenosis.^2,3 We present here redundant actions of a multifunctional protein fortilin, capable of reducing neointima formation through powerful antiproliferative and antimigratory actions concomitant with the ability to reduce vascular cell apoptosis and maintain vessel wall integrity.

Complementing the in vivo data cited above, results from cell culture experiments confirm an inhibitory role for fortilin, as cells transfected with Ad-fortilin showed significantly reduced cellular proliferation and markedly inhibited DNA synthesis. Similar to conditions after vascular trauma, fortilin exerted protection against SMC proliferation only in actively proliferating cells; quiescent cell populations were not influenced by fortilin transfection. In an effort to exclude elevated apoptosis as a mechanism contributing to the observed reduction in cell number in the fortilin-treated group, TUNEL analysis was performed and demonstrated that fortilin did not induce programmed cell death. A plausible mechanism for fortilin-induced inhibition of cell proliferation is suggested by results using the human homologue to TCTP, p23. Through the use of immunoprecipitation/localization experiments, TCTP/p23 was shown to exert properties of a tubulin binding protein, similar to that of the microtubule-associated protein MAP-1B, that associates with microtubules in a cell cycle-dependent fashion leading to rearrangement of the microtubule network and increased microtubule mass and stability.²¹ This microtubule stabilizing effect of TCTP/p23 inhibits the onset of mitosis, and TCTP/p23-overexpressing cells display significantly reduced proliferative capacities.

We believe that the apparent paradox of an antiapoptotic factor contributing to a reduced neointimal mass is best explained by the abundance of data described herein that demostrate novel and robust inhibitory actions of fortilin on DNA replication and cell division in injured arteries, along with decreased SMC numbers and reduced DNA replication observed under cell culture conditions. These results strongly propose that the primary mechanism of fortilin is through antiproliferative action, observed under both inimical and eutrophic conditions. In addition, the ability of an apoptosis-modifying factor to sensitize neighboring cells to alter their proliferative capacity has been previously observed and suggested to be dependent on a variety of biochemical responses.⁴

In conclusion, results from this study provide direct new evidence that the recently described protein fortilin exerts significant and redundant vasoprotective functions capable of minimizing the arterial response to injury. Adenoviral fortilin treatment immediately after experimental arterial angioplasty markedly attenuated neointimal development through suppression of SMC proliferation and DNA replication. Fortilin may also exert an antimigratory ability as an additional protective mechanism. Antiapoptotic data indicate that fortilin exerts these effects without compromising vessel wall integrity in the face of vascular trauma. We believe that fortilin represents a powerful new multifunctional protein that provides a potential target for the development of novel therapies aimed at the treatment of occlusive vascular diseases.

	Acknowledgments

 
This research project was supported by National Institutes of Health Grants HL04015 and HL68024, The Roderick Duncan MacDonald General Research Fund at St Luke’s Episcopal Hospital, and by grants from the American Heart Association, The Gillson Longenbaugh Foundation, and The Methodist Hospital Foundation.

	Footnotes

 
Guest editor for this article was Prediman K. Shah, MD, Cedars-Sinai Medical Center, Los Angeles, Calif.

This article originally appeared Online on December 9, 2002 (Circulation. 2002;106:r68-r75).

Received October 17, 2002; accepted November 6, 2002.

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