Electrode cuff-induced changes in DNA and PDGF gene expression in the rat carotid artery

Atherosclerosir. 100 (1993) 103-112 0 1993 Elsevier Scientific Publishers Printed and Published in Ireland

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Electrode cuff-induced changes in DNA and PDGF gene expression in the rat carotid artery Glenda E. Bilder, Charles J. Kasiewski, Robert J. Costello, Thomas G. Hodge and Mark H. Perrone RhBne-Poulenc Rorer Central Research, 500 Arcola Road, Collegeville, PA 19426 (USA) (Received 17 July, 1992) (Revised, received 24 December, 1992) (Accepted 4 January, 1993)

Low current (0.25, 3 mA) stimulation through a miniature electrode cuff encased around the carotid artery of the rat was used to induce intimal hyperplasia, an important feature of the atherosclerotic plaque and a phenomenon limiting the long term success of angioplasty. Compared to contralateral unstimulated arteries, 1l- 14 days of daily transmural stimulation of cuffed arteries (20 min period) significantly increased the amount of extracted DNA (diphenylamine calorimetric assay). Low current (0.25 mA) was as effective as 3 mA in producing an increase in extractable DNA. The cuff alone without applied current also stimulated an increase in DNA content but to a smaller degree than in arteries receiving current. Infusion of a calcium channel antagonist, diltiazem, at a dose which achieved therapeutic drug levels, significantly reduced the amount of electrode cuff-induced DNA content but had no effect on the increase in DNA induced by the presence of the cuff without applied current. Gene expression of PDGF-A chain, PDGF-B chain and PDGF-beta receptor (or) (Northern analysis of extracted carotid RNA) increased within 4 h after electrical stimulation with 3 mA. Lower current (0.25 mA) and the presence of the cuff also enhanced PDGF gene expression but with a delayed onset of several days. The pattern of gene expression for PDGF ligands and or during the 11-14 days of stimulation differed, but each remained above contralateral control levels. It is concluded that the continued coexpression of PDGF and one of its receptors may contribute to induced hyperplastic changes.

Key words: Vascular DNA; Platelet-derived growth factor; Intimal hyperplasia; Diltiazem; Electrode cuff; PDGF gene expression; Calcium channel antagonists

Correspondence to: Dr. Glenda Bilder, 500 Arcola Road, Collegeville, PA 19426, USA. Tel.: 215-454-5088; Fax: 215-454-5658.

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Introduction A diverse and complex mixture of vascular and immune cells, lipids and extracellular matrix material are found in the human atherosclerotic plaque [1,2]. Among these plaque components, the intimal smooth muscle cell (SMC) is a consistent and predominant feature [3]. These cells are thought to be derived from medial SMC [4] or resident intimal SMC [5,6]. It is postulated from results of epidemiological and animal studies that subtle vascular injury induced by oxidized lipids, increased vascular tone, toxins, altered arterial wall shear stress or hypoxia initiate a cascade of events which encourage migration and/or proliferation of SMC in the subendothelial space [1,7-lo]. Cellular mediators which promote and maintain SMC migration and proliferation following vascular injury have not been clearly defined. Plateletderived growth factor (PDGF), a potent mitogen and chemoattractant for SMC, is one of several postulated factors [ 111. Results of immunocytochemical and in situ hybridization of human endarterectomy sections show an increase in the PDGF receptor (p-subunit) and the mRNA for the A and B chains of PDGF in mesenchymalappearing intimal smooth muscle cells [ 12,131. Since PDGF is secreted by vascular cells (SMC, macrophages, endothelial cells) in culture [ 14- 161 and phenotypic modulation influences the expression of the alpha and beta PDGF receptors [ 171, PDGF may influence intimal hyperplasia through autocrine/paracrine mechanisms [ 111. To initiate investigations into the possible factors promoting the growth of intimal SMC of the atherosclerotic plaque, SMC proliferation was induced in the rat by application of transmural electrical stimulation through an implanted miniature electrode cuff. A similar model has previously been shown to induce intimal hyperplasia in the rabbit [18]. Unique to this model is the induction of changes in endothelium permeability without overt denudation [ 191. Subtle alterations in endothelial function are thought to be a necessary first event in development of atherosclerotic plaques 111. Since this model has not been previously characterized in the rat, confirmatory experiments were done to demonstrate histologically and

biochemically the presence of electrode cuffinduced intimal hyperplasia. The effect of diltiazem, a calcium channel antagonist, on electrically-induced DNA changes in the rat was also studied since the rabbit model is sensitive to this class of drugs [19]. The main focus of these studies, however, was to determine whether PDGF might play a role in intimal SMC proliferation in this model by measuring mRNA expression of the PDGF A and B chains and the PDGF beta receptor subunit (or). Materials and Methods Animals

Sprague-Dawley male rats (300-350 g) were purchased from Charles River (Taconic Farms) and used throughout. The research animals used in this study were housed and cared for following the procedures described in the NIH Guide for the Care and Use of Research Animals. The research protocol was reviewed and approved by the RPR-Animal Care and Use Committee, as required by the Animal Welfare Act. Electrode cuff

Intimal smooth muscle cell proliferation was induced in the carotid artery by implantation of a miniature silastic gold electrode cuff according to methods modified from Betz and Scholte [ 181. The implanted cuff was connected by silver-coated copper wires (Cooner Wire Co., Chatsworth, CA) to a microplug (Microtech Inc., West Chester, PA) attached to the skull. Through leads from a Grass Stimulator and constant current converter to the microplug, arteries received DC current (lo-15 Hz, 0.25-3 mA) transmurally for a 20 min period once daily for 1-14 days. depending on the study. In some animals, electrode cuffs were implanted but no current was applied. Cuffs were fashioned from medical silastic tubing (0.062 inches internal diameter x 0.095 inches outer diameter, Dow Corning Corp., Midland, MI). Two gold leads (24 carat, 32 gauge) were placed within the lumen of the silastic tube, and attached to opposite walls by wrapping excess to outside. Final cuff length was 5 mm. Leads were attached to the gold by Duratec Solder (60/40 resin core, SPC Technology, Bensenville, IL) coated

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with silastic adhesive (Dow Corning Medical Grade, Midland, MI). Cuffs were autoclaved prior to implantation. Cuff implantation

Rats were anesthetized with sodium pentobarbital (50 mg/kg i.p.) Carotid arteries, exposed through a midline neck incision, were carefully dissected free of adherent tissue. The silastic cuff was cut lengthwise and placed around the left carotid artery. The contralateral right artery, treated similarly, but not encased with a cuff, served as a sham control. The cuff was closed with a single suture; lead wires were anchored to the adjacent muscle by 5-O silk and the free ends pulled over the shoulder muscles to an opening midway between the ears. Leads were soldered to a microplug at this time and the pod was anchored to the skull with stainless steel screws and fast acting acrylic. The skin was drawn tight in a pursestring fashion (2.0 silk) around the acrylic. Evaluation

of vascular injury

Vascular changes induced by transmural stimulation were evaluated for: (a) histological presence of intimal hyperplasia, (b) change in extracted DNA content, and (c) gene expression of the PDGF A chain (PDGF-A) and B chain (PDGF-B) and PDGF receptor+ subunit (PDGF-fir). For histological evaluation, sham and cuffed arteries were removed from anesthetized rats after 14 days of stimulation, placed in buffered formalin, embedded in paraffin and processed for Movat Pentachrome staining. To determine changes in DNA content, sham and cuffed arteries were removed, dissected free of adventitia, blotted, and cut to equal lengths (10 mm). In pilot studies, postmortem contracture between cuffed and uncuffed arteries did not exceed 10%. Arteries were flash frozen in liquid nitrogen and kept at -70°C until use. Arteries (10 mm) were homogenized in 6% perchloric acid and extracted as described [20]. DNA was quantitated by diphenylamine calorimetric assay [21]. Standard curves were generated with purified calf thymus DNA. For evaluation of gene expression, carotid arteries were removed 4 h after the last period of

stimulation, cleaned as above and cut lengthwise and denuded of endothelial cells by gentle rubbing with a cotton swab. Arteries from 3-4 rats were pooled and homogenized in guanidine isothiocyanate (4 M) fl-mercaptoethanol (0.1 M)-sodium acetate (0.025 M), using the Tekmar tissumizer. Gel separation, transfer, hybridization and washing conditions were as before [22]. Membranes were hybridized with [32P]CTP nicktranslated cDNAs: v-sis (Oncor), PDGF-A chain cDNA of Dl clone, a 1.3-kb EcoRI and Hind111 digest of PUC13 [23] and PDGF+r cDNA, a 2. lkb PstI digest of PVI based on published sequence [24]. After exposure of membranes to Kodak XAR-2 film for 3-5 days, autoradiographs were quantitated by laser densitometry (LKB). Equality of transfer of sample RNA was determined by densitometry of the negative of membranes containing ethidium bromide-treated RNA. Density of probed RNA was expressed relative to ethidium bromide 28s ribosomal RNA. Normalization to a housekeeping gene, /3-actin, was inappropriate since this mRNA was found to change with induction of vascular changes ([25], unpublished observation). Diltiazem

infusion

pumps (Alza 2002) containing Osmotic diltiazem or saline vehicle were placed in the peritoneal cavity during implantation of the electrode cuff. Diltiazem was infused at rates of 0.7 and 6.9 &kg per h for 11 or 14 days. The plasma concentration of diltiazem was determined in a separate experiment in rats (n = 3) implanted for 14 days with osmotic pumps containing the highest infusion dose of diltiazem (6.9 &kg per h). Blood samples were withdrawn into a solution containing (final concentration in blood): aprotinin (200 pg/ml), EDTA (1.7 r&ml), soybean trypsin inhibitor (40 &ml), and centrifuged 10 min (100 x g). Plasma samples were evaluated by HPLC analysis for diltiazem and desacetyl diltiazem by Pharmaco Analytical Laboratory (Richmond, VA). Materials

Diltiazem was purchased from Sigma, St. Louis, MO. 32P-CTP and [3H]thymidine are products of Amersham (Arlington Heights, IL) and DuPont

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Company (Wilmington, DE), respectively. v-sis cDNA was obtained from Oncor (Gaithersburg, MD). PDGF-A chain and PDGF-/3 receptor cDNA were prepared from plasmids kindly supplied by Drs. C.-H. Heldin and V. Masakowski, respectively. Statistics

For statistical analysis of extracted DNA, the uncuffed DNA was subtracted from the cuffed DNA. Extracted DNA (cuff - no cuff) at the three current levels (0.0 mA, 0.25 mA and 3 mA) and the comparison of the infused groups (saline and diltiazem) with respect to extracted DNA were analyzed by means of a one-way analysis of variance. Other comparisons where necessary were made with a paired t-test.

f

Results Vascular changes induced by electrode cuff in rats

To evaluate the change in vascular DNA induced in this model, the change in vascular DNA concentration was determined. The presence of the cuff alone produced a 2-fold increase in extracted DNA per millimeter artery. With electrical stimulation, DNA concentration increased 4.5fold over the contralateral artery. This increase at 0.25 and 3 mA was significantly greater (P < 0.05) than that produced by the cuff alone (Fig. 1). Low current (0.25 mA) was as effective as 3 mA in producing an increase in extractable DNA. Intimal SMC proliferation was not observed in uncuffed contralateral carotids (Fig. 2a). Application of 1 or 3 mA of current to the cuffed left carotid artery of the rat induced intimal hyperplasia (Fig. 2b,c). However, the presence of the cuff in the absence of applied current produced variable effects on subendothelial SMC proliferation, ranging from no effect or in some rats, intimal hyperplasia of variable degree. Effect of diltiazem infusion on vascular DNA

In arteries

stimulated

with 0.25 or 3 mA,

Fig. 1. Effect of electrically-induced vascular injury on extracted DNA of carotid arteries. Rats were electrically stimulated once daily for 14 days at indicated currents. DNA (mean f S.E.M., n = 6) was extracted and quantitated by diphenylamine calorimetric assay as described in Methods.

diltiazem (6.85 &kg per h) significantly (P < 0.02) inhibited the increase in DNA concentration (Table 1). A lower dose of diltiazem (0.7 pg/kg per h) also inhibited 3 mA-induced increase in DNA. Diltiazem infusion however did not inhibit the increase in DNA by cuff alone (0 mA). The plasma concentrations of diltiazem and its metabolite achieved by 6.9 &kg per h infusion rate were: (@ml): diltiazem, 112 f 48 (n = 3), desacetyldiltiazem, 30.4 f 0.1 (n = 3). Gene expression

To evaluate a possible role of PDGF in this model of intimal hyperplasia, mRNA was prepared from arteries at 1 and 2 days after cuff

Fig. 2. Movat pentachrome stained cross-section (x 66) of rat carotid arteries with and without transmural electrical stimulation. Rats were implanted with electrode cuffs and stimulated for 14 days as described. Arterial sections: (A) right carotid, cuff; (B) left carotid, 1 mA, moderate intimal SMC proliferation; (C) left carotid, 3 mA, marked intimal SMC proliferation.

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TABLE 1

implantation. Contralateral uncuffed arteries exhibited low levels of PDGF-A mRNA (3.0, 2.4-2.6 kb) and low levels of PDGF-/3r mRNA (5.3 kb); PDGF-B chain mRNA was undetectable. Electrical stimulation (l-3 mA) induced PDGF-B chain and PDGF-or mRNA expression. PDGF-A mRNA was marginally increased by 3 mA (Fig. 3). The time course of change of PDGF gene expression with electrical stimulation was determined in a second series of experiments. PDGF-B mRNA remained elevated throughout the two weeks of electrical stimulation (Fig. 4). In contrast, PDGF-A mRNA, primarily (2.4 kb), increased steadily with 3 mA electrical stimulation but dissipated by day 14 (Fig. 4). Lower current (0.25 mA) and the cuff alone also stimulated A-chain expression but compared with 3 mA onset this effect was delayed and remained elevated. PDGF-fir gene expression was continuously elevated above control (uncuffed contralateral) values but relative expression cycled during the 14 day study (Fig. 5). As with PDGF-A chain expression, PDGF-@r mRNA was increased markedly with 3 mA (day 1). The cuff and low current, however, enhanced receptor expression only at later times.

EFFECT OF DILTIAZEM ON EXTRACTED VASCULAR DNA Rats were electrically stimulated for 20 mitt daily for 14 days at currents below. DNA was extracted and quantitated by calorimetric assay. Extracted DNA (mean f S.E.M.) represents DNA of stimulated artery minus unstimulated contralateral control. Diltiazem was administered by implanted Alza minipump. n, number of animals; D&H, diltiazem infusion at calculated rate of 6.85 &kg per h; Dil-L, diltiazem infusion at calculated rate of 0.7 &kg per h. Treatment

n

Increase in extracted DNA (gg/mm artery)

Saline Dil-H

6 4

0.48 f 0.10 0.53 * 0.19

0.25 mA Saline Dil-H

6 6

0.83 * 0.10 0.40 f 0.09

6 4 2

0.89 f 0.18 0.39 f 0.07 0.27 f 0.03

OmA

3mA

Saline Dil-L Dil-H

PDGF-B I

PDGF-A Cl

ia s

ux

5

’

I

PDG F-a, 1

I U-J

’

’

z

=

’

-28s

’

4.3 kb -’ -

28s

18s

Fig. 3. Northern analysis of early expression of PDGF-B chain, PDGF-A chain, PDGF-fl receptor mRNA. Total RNA from electrically stimulated rats was prepared as described in Methods. Total RNA (3 pg) was applied to each lane. mRNA of PDGF-B chain (3.5 kb), PDGF-A chain (2.6, 2.1 kb) and PDGF-f.3 receptor (5.3 kb) was identified from ribosomal bands and lambda Hind111 DNA ladder. Total RNA from HUVEC and U205 cells were used as positive markers for PDGF-B chain and PDGF-A chain mRNA, respectively. Negatives of membranes containing ethidium bromide-stained mRNA are shown below the hybridized mRNA. S represents electrically stimulated carotid; U represents right carotid without cuff.

109

PDGF A-CHAIN

3

PDGF B-CHAIN

9 Time

11

2

(Days)

cl OmA

7 Time

H

0.25 mA

mm

14

(Days)

3mA

Fig. 4. Time course of PDGF-A chain and B-chain expression with electrical stimulation. Total RNA from electrically stimulated rats was prepared as described in Methods. Total RNA (3 pg) was applied to each lane and hybridized with either v-sis cDNA or A-chain cDNA. Graph represents densitometric analysis averaged from 2 experiments, normalized to relative density of ethidium bromide bands and expressed as percentage of maximal density.

PDGF p-RECEPTOR

Fig. 5. Time course of PDGF-P receptor gene expression with electrical stimulation. Total RNA of the PDGF from electrically stimulated rats was prepared as described in Methods. Total RNA (3 bg) from 3 rats was applied to each lane and hybridized with or cDNA. Graph represents densitometric analysis averaged from 2 experiments, normalized to relative density of ethidium bromide values and expressed as percentage of maximal response.

110

Discussion We have used transmural electrical stimulation applied daily over an 1 l- 14 day period to induce intimal hyperplasia in the rat carotid artery. The presence of cuff alone (0 mA) increased vascular DNA concentration and this effect was additionally enhanced by applied currents, 0.25 and 3 mA (Fig. 1, Table 1). Histological findings paralleled those of induced DNA changes. Intimal hyperplasia was observed in some but not all cuffed, non-stimulated arteries (0 mA); more prominent degrees of intimal hyperplasia, however, occurred with electrical stimulation (Fig. 2). Intimal hyperplasia induced by transmural current in the rat has not previously been reported. Although modified, our methods are based on a similar electrode cuff model in the rabbit [ 181. Qualitative similarities are evident between the two species. For example, in rabbits, DNA proliferation, as determined histologically by an increase in subendothelial SMC layers, increased throughout the first 2 weeks of stimulation, achieving within the initial 2 weeks of stimulation, 90% of a 4-week hyperplastic response [26]. However, in contrast to our findings in the rat in which electrical stimulation induced a measurable and reproducible increase (4-fold) in DNA content after 14 days of stimulation, quantitation of extracted DNA in the rabbit yielded only a marginal (12%) increase in stimulated arteries compared with contralateral controls [27]. Differences in medial thickness of the two species and protocols of extraction and quantitation may account for this discrepancy. Our findings show that electrically-induced intimal hyperplasia, as measured by an increase in extracted DNA content, is inhibited by continuous administration of diltiazem. The mechanism by which diltiazem inhibits electrode cuff increase in vascular DNA content is unknown. The effectiveness of diltiazem in this model may result from many factors such as preservation of endothelial impermeability as shown with flunarizine treatment in the electrode cuffed rabbit model [191, smooth muscle cell antimitogenic effects as demonstrated for some calcium antagonists in cultured cells [28], prevention of platelet aggregation and degranulation as demonstrated in vitro [291

and prevention of calcium fluxes and redistribution of membrane receptors, two of many events induced in cells by current-induced electrical fields [30-321. A major goal of this study was to determine whether PDGF gene expression was influenced by electrode cuff injury and whether the patterns of gene expression might implicate PDGF as a possible mediator in this type of vascular remodeling. Compared with the uninjured contralateral artery, electrical stimulation after l-2 days at 3 mA rapidly increased the relative concentrations of PDGF-B chain and PDGF-/3r mRNA and marginally affected A-chain expression (Fig. 3). PDGF-/3r mRNA levels, however, declined with continued stimulation, PDGF-B chain remained elevated and A-chain mRNA gradually increased (Figs. 4, 5). It is noted that these changes represent concentration levels of transcripts. PDGF exists as one of at least three isoforms comprised of homodimers or heterodimers of similar, nonidentical chains, termed A and B [ 1I]. In order to activate the tyrosine kinase activity intrinsic to the PDGF receptor, PDGF ligands dimerize two PDGF receptor subunits, alpha receptor (or) or or in an isoform-specific manner [33]. Since a compatible isoform/subunit combination is the or and the BB homodimer, early and continuous coexpression of mRNA for these proteins suggests that in this model a condition favorable for SMC mitogenesis may exist. That coexpression of the PDGF receptor and an appropriate ligand forms an autocrine/paracrine network underlying SMC migration/proliferation, has been proposed [ 111. Low current stimulation and the cuff alone also enhance PDGF and PDGF receptor gene expression but with delayed onset. Since 0.25 mA is as effective as 3 mA in increasing vascular DNA concentration, it is suggested that whereas both reach the same end point (elevated DNA content) at 14 days, the rate at which this occurs is possibly more rapid for the 3 mA stimulus. Measurement of the rate of increase in DNA concentration with the electrode cuff would help to resolve this issue. In summary, we have shown in a small laboratory animal that a localized region of intimal hyperplasia can be generated by electrode cuff stimulation. Measurable increases in DNA concentration occur after 2 weeks of daily application

Ill

of current. These changes are inhibited by infusion of a therapeutic dose of diltiazem. This model also shows early and continuous gene expression of PDGF A and B chain and PDGF-fi receptor. This model may therefore be useful for evaluation of potentially anti-atherogenic drugs which target inhibition of SMC proliferation/migration components of the atherosclerotic plaque. Whether diltiazem inhibits PDGF gene expression is an important future study.

II 12

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Acknowledgments 15

The authors appreciate the assistance provided by Dr. E. Betz and Mr. J. Nielands on the production and use of the electrode cuff. We acknowledge the technical contribution of Mr. J. Krawiec and expert secretarial skills of Ms. R. Ratkiewicz.

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