Cardiovascular Research

Cardiovascular Research 2001 49(1):177-188; doi:10.1016/S0008-6363(00)00210-8
© 2001 by European Society of Cardiology

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Copyright © 2000, European Society of Cardiology

Transient down-regulation of L-type Ca²⁺ channel and dystrophin expression after balloon injury in rat aortic cells

Jean-François Quignard^a, Marie-Cécile Harricane^b, Claudine Ménard^a, Philippe Lory^a, Joël Nargeot^a, Loïc Capron^c, Dominique Mornet^d and Sylvain Richard^a,*

^aIGH, CNRS UPR 1142, 141 rue de la Cardonille, 34396 Montpellier Cedex 5, France
^bCRBM, CNRS UPR 1086, Université de Montpellier I, 1919 route de Mende, 34293 Montpellier Cedex 5, France
^cUniversité de Paris VI, service de médecine interne, l'Hôtel-Dieu, 75181 Paris Cedex 04, France
^dINSERM U 128, IFR 24, 1919 route de Mende, 34293 Montpellier Cedex 5, France

^* Corresponding author. Correspondence address: INSERM U-390, Hôpital Arnaud de Villeneuve, F-34295 Montpellier Cedex 5, France. Tel.: +33-467-415-244; fax: +33-467-415-242 srichard{at}montp.inserm.fr

Received 19 May 2000; accepted 15 August 2000

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
Objective: Migration and proliferation of arterial smooth muscle cells are critical responses during restenosis after balloon angioplasty. We investigated the changes in the expression of Ca²⁺ channels and dystrophin, two determinants of contraction, after balloon injury of rat aortas. Methods: Proliferation and migration of aortic myocytes were triggered in vivo by the passage of an inflated balloon catheter in the aortas of 12-week-old male Wistar rats. We used the whole-cell patch clamp technique to investigate Ba²⁺ currents (I_Ba) through Ca²⁺ channels in single cells freshly isolated from media and neointima at various times after injury (days 2, 7, 15, 30 and 45). Results: No T-type Ca²⁺ channel current was recorded in any cell at any time. In contrast, a dihydropyridine (DHP)-sensitive L-type I_Bawas recorded consistently in the media of intact aorta. After aortic injury, I_Ba decreased dramatically (at days 2 and 7) but recovered over time to reach normal amplitude on days 30 and 45. In the neointima, I_Ba was absent on day 15 but also increased gradually over time as observed at days 30 and 45. The use of a specific antibody directed against the L-type Ca²⁺ channel _1C subunit showed, both by immunostaining and by Western blotting, no expression of the Ca²⁺ channel protein on day 15. Parallel immunodetection of dystrophin showed that this marker of the contractile phenotype of SMCs was also not detectable at this stage in neointimal cells. Both proteins were re-expressed at days 45 and 63. Balloon injury induces a transient down-regulation of I_Ba in arterial cells. Conclusions: Cell dedifferentiation and proliferation in vivo abolish the expression of L-type Ca²⁺ channels and dystrophin in neointimal cells. These changes may be critical in the regulation of Ca²⁺ homeostasis and, thereby, contraction of the arterial SMCs during restenosis following angioplasty.

KEYWORDS Ca²⁺: calcium; [Ca²⁺]_i, cytosolic free calcium concentration; DHP, dihydropyridine; SMCs, smooth muscle cells; FITC, fluorescein isothiocyanate; SDS, sodium dodecyl sulfate; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid h-CaD: heavy-molecular-weight caldesmon; l-CaD, low-molecular-weight caldesmon; PMSF, phenyl methyl sulfonyl fluoride; CsOH, caesium hydroxide; TBS–T, Tris–HCl buffered saline–Tween; EGTA, ethylene glycol-bis (β-aminoethylether)N,N,N',N'-tetraacetic acid; BSA, bovine serum albumin; ATP, adenosine-5'-triphosphate; GTP, guanosine-5'-triphosphate

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
The development and maintenance of contractile tone of normal arterial smooth muscle cells (SMCs) is controlled by the concentration of free cytosolic calcium ([Ca²⁺]_i) [1,2]. Voltage-gated Ca²⁺ channels constitute a major route for the Ca²⁺ influx required to induce an increase in [Ca²⁺]_i in SMCs [2,3]. Consequently, they are key targets in the control of vascular tone by antihypertensive and vasodilating drugs [4,5]. Two types of Ca²⁺ channels are distinguished by their electrophysiological and pharmacological properties in arterial SMCs: (i) dihydropyridine (DHP)-sensitive L-type Ca²⁺ channels expressed in all types of SMCs, including human coronary SMCs, and involved in the excitation–contraction coupling [4–6]; and (ii) T-type Ca²⁺ channels that are expressed mostly in primary cultured SMCs (e.g. from rat aorta and human coronary artery) suggesting that the T-type currents may be involved in another function (other functions) than contraction such as cell cycling and proliferation [6–9].

Dystrophin is a membrane-associated cytoskeletal protein which is also involved in the regulation of [Ca²⁺]_i and contraction [10]. Its deficiency leads to Duchenne muscular dystrophy [11]. Its lack in myotubes from mdx mice (X-linked muscular dystrophy) has been associated with elevated [Ca²⁺]_i and dysregulation of Ca²⁺ entry through Ca²⁺ permeable stretch-regulated ion channels [12,13]. In arterial SMCs, dystrophin (or its isoforms) is abundant and may provide mechanical reinforcement of the sarcolemma contributing to maintain membrane integrity during cycles of contraction and relaxation [14–17]. Interestingly, dystrophin is not expressed in dedifferentiated cells. Its expression rather parallels contractility in primary cultured rat aortic myocytes which suggests that dystrophin is a phenotypic marker of cell differentiation [18,19].

SMCs in normal adult arteries are differentiated, contractile and quiescent. However, migration and proliferation of arterial SMCs are critical responses during restenosis after balloon angioplasty of coronary arteries and during the development of primary atherosclerotic lesions, resulting in neointimal growth and narrowing of the vessel lumen [20]. The SMCs migrate from the arterial media into the intima where they replicate and synthesise important amounts of extracellular material [21–23]. Therefore, migration and proliferation of SMCs contribute to thickening and remodelling of the vessel wall. They also occur during experimental situations such as during neointima formation after balloon injury [24].

In order to understand the mechanisms of restenosis and to set preventive therapy, it is important to study membrane associated key proteins involved in the control of [Ca²⁺]_i and contraction. Phenotypic modulation of arterial SMCs, which occurs after balloon injury, is indeed likely to modify the properties or the level of expression of these proteins. A major implication is that the pharmacological profile of a modulated cell may be quite different from that of a normal cell. In the present work, we have investigated the presence, nature and functional properties of the Ca²⁺ channels and the presence of dystrophin at various times after injury using an experimental model of rat aortic injury with a balloon catheter [25]. We used the whole-cell patch-clamp method to investigate Ca²⁺-channel currents in freshly isolated SMCs from both the media and resulting neointima. Immunochemical studies of Ca²⁺ channels and dystrophin were also performed in parallel to investigate the expression of these proteins.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
2.1 Aortic injury
The investigation conforms with the guide for the care and use of laboratory animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996) and the French Ministry of Agriculture (authorisation No. 004814). Three batches of 15 adult rats (12-week-old male Wistar Kyoto rats, Iffa-Credo, l'Arbresle, France) were used. Animals were anaesthetised with 150 mg/kg of ketamine hydrochloride injected intraperitoneally. A deflated balloon embolectomic catheter (Fogarty size 2F; American Edwards Laboratories, Santa Ana, CA, USA) was introduced into the aorta via the left common carotid artery down to the level of the renal arteries. The balloon was then inflated with 50 µl of distilled water and the catheter was withdrawn slowly. More water (40–60 µl) was injected into the balloon after the passage of the diaphragm had been felt, when the calibre of the aorta becomes slightly larger, and the catheter was pulled up to the origin of the left common carotid artery. The balloon was passed in the aorta under the same conditions three times. After removal of the catheter, the left carotid artery was doubly ligated, and the incision was closed with surgical staples. This type of hard injury strongly stimulates DNA-synthesis by the intima media; the mitotic reaction is at its peak 2–3 days after catheterisation and declines progressively thereafter, while SMCs migrate and divide in the intima. A neointima begins to develop on the 7th day, and becomes macroscopically distinguishable from the underlying media around the 14th day, when DNA synthesis has almost declined to that of the control level in uninjured aorta [26,27]. We studied uninjured aortas (control) and aortas collected 2, 7, 15, 30, 45 and 63 days after injury. Immediately after killing the animal using an overdose of ketamine, thoracic aorta was aseptically opened longitudinally, and dissected in sterile phosphate buffer saline medium and (when appropriate, i.e. from 15 days after injury, and later on) neointima layers were identified based on their topological localisation and visual aspect. Using two pairs of fine forceps, under a magnifying glass, the intima thickening was separated from the media, and the media from the adventitia. The two separated tissues were processed in parallel for different protocols as described below. The neonatal aortic medias were dissected from 1-day-old rats.

2.2 Electrophysiology
Neointima strips and media chips were separately incubated overnight in papain at 4°C and dispersed the next morning. A high percentage (80%) of viable elongated cells was observed. Cells were used immediately for electrophysiological studies. Whole-cell recordings were performed at 20–22°C for 4 h after the dispersion in conditions optimised to isolate Ca²⁺ channel currents [9]. We used Ba²⁺ instead of Ca²⁺ as the charge carrier through Ca²⁺ channels for two reasons. First, Ba²⁺ is more permeable and, therefore, Ba²⁺ currents (I_Ba) are much larger than the Ca²⁺ currents which helps to resolve small currents. Second, T- and L-type currents are more easily distinguished by their kinetics of inactivation. The recording pipettes (3–5 M) were filled with (mmol/l): CsCl 130, EGTA 10, HEPES 25, Mg-ATP 3, Mg-GTP 0.5, glucose 10, succinic acid 5, aspartic acid 5. The bathing solution contained (mmol/l): CsCl 120, Ba(OH)₂ 20, HEPES 10, 4-aminopyridine 5, glucose 10. For both solutions pH was adjusted to 7.3 with methane sulfonic acid; and osmolarity was 300–310 mOsm. The voltage-clamp circuit and the multiple microcapillary perfusion system for application of the DHP agonist [(±)Bay K 8644] and antagonist (nicardipine) were as described previously [9]. After seal formation (resistance ranging between 1 and 20 G and membrane disruption, series resistance (estimated from the decay of the capacitive transients) were typically 2–3 times the pipette resistance (<4 M) and were electronically compensated by >80%. Capacitive transient and linear leakage currents were subtracted using a 4 subpulse (P/–4) to resolve small inward currents. Peak current amplitudes were measured by difference with the baseline current. Cell surface of the myocytes was estimated by measuring their capacitance (determined by integrating the capacitive transient current) in order to compare densities, rather than amplitudes, of the currents. Current density was obtained by dividing current amplitude by cell capacity (pA/pF). Results are expressed as mean±SEM. We used unpaired t-test with Welch's correction to determine the significance of the observed differences between groups of values with two-sided P value and unequal variances. Results were considered as not significant (ns) with P<0.05, significant (*) with 0.01<P<0.05, very significant (**) with 0.001<P<0.01 and extremely significant (***) with P<0.001.

2.3 Antibodies
A monoclonal 12G9 antibody against dystrophin, previously described, and a polyclonal antibody, pA1C, directed against the Ca²⁺ channel _1C subunit, which is characterised elsewhere, were used [28,29]. The polyclonal smooth muscle actin antibody directed against this protein was a gift of Dr. V. Hanin [30]. Monoclonal smooth muscle actin, vimentin and desmin antibodies were purchased from Sigma.

2.4 Immunoblot detections
Three animals were used in each experiment. Crude protein extracts from aortic tissue were obtained as follows: media or neointima were homogenised in buffer containing 5% SDS, 0.1% saponin, or 0.1% triton, 5% β-mercaptoethanol, 1 mmol/l PMSF, 0.1 mg/ml soybean trypsin inhibitor, 0.1 mg/ml leupeptine, 1 mmol/l iodoacetamide, 15% glycerol, 0.001% bromophenol and 50 mmol/l Tris–HCl, pH 9.0. A 50-µl volume of each sample was denatured by boiling for 2 min, and loaded on 8% SDS polyacrylamide gels for dystrophin and _1C Ca²⁺ channel subunit analyses. To compare proteins extraction carefully, we equilibrated each time the different samples by analysing smooth muscle actin content. After overnight electrotransfer (30 V, 100 mA) in buffer (25 mmol/l Tris–HCl, 192 mmol/l glycine, 0.1% SDS and 20% methanol, pH 8.3), nitrocellulose membranes (0.2 mm) were blocked with 3% BSA (bovine serum albumin) dissolved in TBS–T buffer (10 mmol/l Tris HCl, 150 mmol/l NaCl, 0.05% Tween 20, pH 8) for 30 min at room temperature. Blots were incubated for 1 h with specific monoclonal or polyclonal antibodies at room temperature (20–22°C). Using a secondary specific goat anti-mouse or goat anti-rabbit antibody coupled to phosphatase alkaline (1/5000 dilution, Jackson), the protein band was visualised with p-nitroblue tetrazolium, and 5-bromo-4-chloro-3-indolylphosphate substrate. The following proteins were always used as molecular-weight markers, myosin (199 kDa), β-galactosidase (120 kDa), and ovalbumin (48 kDa) from Bio-Rad. Dystrophin was identified as a single band at around 400 kDa and the _1C Ca²⁺ channel subunit as a 195-kDa protein band as described before [29,31,32].

2.5 Immunofluorescence
Fragments of injured aorta were fixed either directly by freezing in isopentane precooled with nitrogen or with 3.5% formaldehyde–PBS at 4°C, incubated in 30% sucrose then frozen in isopentane. Frozen sections (7 µm) were fixed in acetone and incubated with the respective antibodies. Primary antibodies were incubated for 1 h at room temperature in a humid chamber. The slides were then washed in PBS, washed again in PBS, pH 9, and finally in PBS, as described before [33]. The sections were then incubated for 1 h with a fluorescent CY3-anti-mouse (Chemicon, dilution 1/500) or goat anti-rabbit FITC conjugated antibody (Sigma, dilution 1/80), washed as above, once in PBS containing 0.3% Eriochrome black to mask the autofluorescence of elastin and mounted in Moviol.

2.6 Electron microscopy
Newborn and adult rat aortas were fixed with 3% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, post-fixed 1 h at 4°C in 1% osmium tetroxide in 0.1 M cacodylate, pH 6.5 and, after 1 h impregnation in 2% aqueous uranyl acetate, embedded in Epon. Ultra-thin sections were stained with uranyl acetate and lead citrate and examined at 100 kV with a Jeol 2000EX electron microscope.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
3.1 Ca²⁺ channel currents in normal adult aortic myocytes
In SMCs freshly isolated from the media of control animals, we recorded only a slowly inactivating high-voltage activated Ca²⁺ channel current (I_Ba). Fig. 1A shows the typical waveform of this current recorded at +10 mV. Fig. 1B shows the complete current/voltage relationship of I_Ba. Typically, I_Ba activated between –30 and –20 mV and peaked around +10 mV. This current, which we have characterised in detail before [9], is generated by a DHP-sensitive L-type Ca²⁺ channel. Indeed, I_Ba was blocked by nicardipine employed at 5 µmol/l (data not shown). I_Ba was markedly enhanced following application of a saturating concentration of the potent DHP agonist Bay K 8644 (1 µmol/l) which is highly specific of L-type Ca²⁺ channels (Fig. 1A). In addition to inducing a large increase of peak current amplitude, Bay K 8644 promoted a marked leftward shift (–10 mV) of both the threshold and maximal peak current amplitude (Fig. 1B). In all experiments, the amplitude of I_Ba was assessed routinely at +10 mV. Bay K 8644 was employed in order to amplify (or reveal) basal currents that were in the limit of detection. No low-voltage-activated T-type current was detected in any of the 45 cells studied.

View larger version (12K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Ca2+-channel currents in a myocyte freshly isolated from the intact media.(A) Typical L-type IBa recorded at +10 mV in absence (1, ) and presence (2, ) of the DHP agonist Bay K 8644 (1 µmol/l). IBa was activated from a holding potential of –100 mV as shown in the upper diagram. (B) complete current–voltage relationship drawn from the cell shown in (A) absence (1, ) and presence (2, ) of Bay K 8644.

 
3.2 Ca²⁺ channel currents after injury
We investigated the Ca²⁺ channel currents in single SMCs freshly isolated from both the media and the neointima (when appropriate; see Methods) at various times after injury. In order to compare densities, rather than amplitudes, of current, cell surface of neointimal and medial cells was estimated by measuring their capacitance. Averaged values ranged between 25 and 35 pF with no statistical difference between the various subgroups of cells recorded at various times after injury. We found a marked decrease in the averaged density of I_Ba recorded in the medial cells during the first days after injury (Fig. 2A). This decrease occurred immediately, as observed on day-2, and was maximal on day 7. The density of I_Ba fell drastically to almost zero but recovered normal amplitude at days 30 and 45. At day 2, 41% of cells expressed no basal current at all. This percentage increased to 65% at day 7. No morphological difference was obvious. However, the averaged density of I_Ba remained significantly lower in the group of cells exhibiting basal I_Ba at day 2 and day 7 (data not shown) than in the population of cells exhibiting basal I_Ba from uninjured medias. Despite the use of Bay K 8644 to help the resolution of small currents, a similar transient decrease of I_Ba was observed (Fig. 2B). In addition, Bay K 8644 had no effect in cells with no basal current.

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Ca2+-channel currents in myocytes freshly isolated from the media at various times after balloon injury. (A) Mean density of IBa peak amplitude (recorded at +10 mV) as observed between 2 and 45 days after injury. Control cells were isolated from intact medias. (B) Same representation as in A but current density was measured in the presence of Bay K 8644 (1 µmol/l). The numbers of cells recorded for each stage is given in parenthesis.

 
Typical I_Ba recorded in cells freshly isolated from the neointima formed 15 days and later after balloon injury are shown in Fig. 3A. Neointimal cells were not investigated before day 15 because no neointima was readily detected or the amount of material was too small to be reliably identified and accurately dissected. Fig. 3B shows the evolution of the averaged current density in neointimal cells as observed in absence and presence of Bay K 8644 at days 15, 30 and 45 after the injury. At day 15, no I_Ba was recorded even when using Bay K 8644 (Fig. 3A and B) suggesting that the L-type Ca²⁺ channels were absent. However, we observed a gradual appearance of I_Ba over time on days 30 and 45 (Fig. 3A and B). These currents were markedly increased by Bay K 8644 (Fig. 3Abc,Bb). Their waveform, current/voltage relationship and sensitivity to DHPs confirmed that they were generated by L-type Ca²⁺ channels. Forty-five days after injury, the amplitude of I_Ba in neointimal cells was largely recovered but not to the same extent as in the medial cells (Fig. 3B as compared to Fig. 2). It is important to note again that no T-type current was experimentally detected at any stage either in medial cells (n = 70) or in intimal cells (n = 58). The lack of agonist for T-type Ca²⁺ channels did not allow to amplify potential currents as described with for the Bay K 8644 on small L-type currents. Nevertheless, if it exists, such a level of current (<5 pA; density <0.3 pA/pF) would reflect a very small number of functional Ca²⁺ channels and a very small contribution to Ca²⁺ entry.

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Ca2+-channel currents in cells freshly isolated from the neointima formed in adult rat aortas after balloon injury. (A) Representative IBa recorded in absence (1) and presence (2) of Bay K 8644 (1 µmol/l) on day 15 (a), day 30 (b) and day 45 (c) after injury. (B) Mean density of IBa peak amplitude (recorded at +10 mV) as observed between 15 and 45 days after injury in absence (a) and presence (b) of Bay K 8644 (1 µmol/l). No intima was available for study prior to day 15.The numbers of cells recorded for each stage is given in parenthesis.

 
3.3 Western blot analyses
The alteration of Ca²⁺ channel currents, demonstrated by electrophysiological approach, could be interpreted as reflecting absence of the Ca²⁺ channel protein or alternatively presence of a non-functional protein in intimal cells 15 days after aortic injury. Therefore, a polyclonal antibody which identifies the _1C subunit of cardiac L-type Ca²⁺ channel [29] was used to explore this issue using Western blot analysis and immunocytochemistry. We conducted combined experiments in parallel using an antibody directed against dystrophin because this protein is a good marker of SMC differentiation and contractile phenotype. Western blot analyses of Ca²⁺ channel _1C subunit and dystrophin in SMC crude protein extracts from aortic samples are presented in Fig. 4. In agreement with our previous study [18], dystrophin (D) was identified as a 400-kDa single protein band in all crude protein samples corresponding to specific extraction from aortic media. The Ca²⁺ channel _1C subunit (C) was also detected in parallel as a 195-kDa protein band in the media on all days analysed. To evaluate the presence or absence for both proteins, we compared at each time the injured media and the normal untouched media. The immunostaining pattern appeared almost similar on day-15 (Fig. 4, lanes 2 and 3). Neither dystrophin nor the Ca²⁺ channel _1C subunit were detected in the neointima crude protein extraction on day 15 (Fig. 4, first panel, lanes 1), but they were detected in the media (Fig. 4, first panel, lanes 2). However, both proteins were detected in parallel on day 45 and day 63 after injury. There was a lower abundance for both proteins in the neointima protein extracts (lanes 1) as compared to the media protein extracts (lanes 2) on day 45 (Fig. 4, second panel). This difference was not obvious anymore on day 63 (Fig. 4, last panel).

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Western blot analysis of crude protein extracts from aortic samples. Immunoblot detections with specific antibodies are presented for dystrophin (D) and Ca2+ channel 1C subunit (C). At 15, 45 and 63 days after aortic injury, samples corresponding to crude membrane extracts from neointima (lanes 1) and media (lanes 2) of injured arteries, and from the normal untouched media (lane 3) were analysed with specific antibodies (see Methods). A protein band detected with a molecular weight of 400 kDa corresponded to dystrophin and a protein band detected with a molecular weight of 195 kDa was related to Ca2+ channel 1C subunit. In each lane, separation of the same extracts on a gradient SDS–PAGE stained with Coomassie brilliant blue enabled estimation of the actin content.

 
3.4 Detection of _1C Ca²⁺ channel and dystrophin
Dystrophin was detected in the media of uninjured aorta (Fig. 5A), but absent from the neointima on day 15 (Fig. 5B) and again detected in neointima on day 45 (Fig. 5C). In parallel to dystrophin detection, the expression of contractile and cytoskeletal proteins smooth muscle actin (Fig. 5D), desmin and vimentin were examined (data not shown). The presence of actin and vimentin was confirmed in both the neointima and media of all aortas while only about half of the SMCs contained desmin. These later results agree with previous cytoskeletal studies [34]. Expression of the Ca²⁺ channel _1C subunit was also detected in the media of normal arteries (Fig. 5E). In contrast, it was absent in the neointima on day 15 (Fig. 5F). It was present again in neointima on day 45 (Fig. 5G). When the first antibody was omitted, only a slight autofluorescence attributable to elastin was observable (Fig. 5H).

View larger version (69K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Immunofluorescence labelling of 10 µm aortic cross-sections for immunodetection of dystrophin (A–C) and Ca2+ channel 1C subunit (E–G) in normal (A,E) and injured adult aortas on day 15 (B,F) and day 45 (C,G) days after injury. On day 15, intimal thickening was not stained for dystrophin (B) and for Ca2+ channel 1C subunit (F). In contrast, both the neointima and the media from non injured and injured aortas (on day 45 after injury) were positive for dystrophin and Ca2+ channel 1C subunit (C,G). On day 45 both intima and media cells were positive for smooth muscle actin (D). Only elastin showed slight autofluorescence when the first antibody was omitted (H). Bar=50 µm.

 
3.5 Electron microscopy
We next examined the phenotypic state of intimal VSMCs through morphologic criteria on day 15 and day 45. Electron microscopic observations of aortic sections at low magnification showed that the endothelium edging the vessel lumen was present only in the untouched aorta (Fig. 6A). In contrast, the presence of a thick neointima was observed on day 15 (Fig. 6B) and day 45 (Fig. 6C) after the balloon injury. The phenotypic state of the cells was more evident at higher magnification (Fig. 7). For comparison, the aortas from newborn rats (1 day postpartum) were also used as an vivo model because their SMCs are known to be in a synthetic phenotype. We also verified, in agreement with previous results [35], by Western blot analysis of crude protein extracts that neonatal aorta contained only low-molecular-weight caldesmon (l-Cad) while adult aorta showed presence of high-molecular-weight caldesmon (h-Cad) (data not shown).

View larger version (112K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Electron microscopy observation of a section next to the lumen of the aorta from an untouched media in a control rat (A), and on day 15 (B), and day 45 after arterial injury (C). On both days 15 and 45, neointima cells are numerous with abundant extracellular matrix, while endothelial cells are not observed as compared to control aorta. E, endothelium; L, vessel lumen; *, neointima. Bar=0.5 µm.

 

View larger version (155K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Electron micrographs of rat aortic SMCs in newborn media (A), normal adult media (C) and adult neointima as observed 15 days (B) and 45 days (D) after injury. Cell cytoplasm in newborn media (A) and in adult neointima at day 15 (B) contained a well-developed endoplasmic reticulum. Microfilaments are barely visible whereas they become the major cytoplasmic component in cells of normal adult media (C) and in neointima 45 days after injury (D). rER, rough endoplasmic reticulum; m, mitochondria; r, ribosomes; mf, microfilaments. Bar=0.5 µm.

 
SMCs from both newborn media (Fig. 7A) and 15-day neointima (Fig. 7B) display a synthetic phenotype, with abundant rough endoplasmic reticulum (rER) while the cytoplasm of adult media cells filled with numerous microfilaments (Fig. 7C). This is in agreement with the respective synthetic and contractile phenotype already described in atherosclerotic plaques [25]. Forty-five days after arterial injury, the neointima was still thick, endothelium continuity was not re-established. The cytoplasm of the SMCs still contained some rER but was filled again principally with numerous microfilaments (Fig. 7D) attesting phenotypic modulation over time after injury and partial recovery towards a more differentiated state.

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
This study provides information at the functional and molecular levels about changes in the expression of Ca²⁺ channels during the aortic response to injury with a balloon catheter. The findings are: (i) L-type Ca²⁺ channel currents are transiently decreased during the first days after injury in medial cells; (ii) this decrease is more severe in neointimal cells and recovery is delayed; (iii) the complete loss of Ca²⁺ channel activity in the neointima reflects a down-regulation of the _1C subunit; (iv) there is a concomitant down-regulation of dystrophin, a marker of cell contractile phenotype; (v) the reappearance of these two proteins parallels cell redifferentiation several weeks after the injury; and (vi) there is no evidence for the presence of T-type Ca²⁺ channel currents at any stage.

Neointima formation, which characterises arterial remodelling after balloon injury, involves several steps [21]. Basically, medial cells dedifferentiate and start to proliferate very early and actively, i.e, 24 h after the injury [36,37]. Maximal proliferative activity is reached after 2–3 days. The second wave of cell activity involves migration of SMCs to form the neointima which expands between day 7 and day 14–21. Low-grade replication is the third wave which can last for weeks or months [36,37]. After maximal thickening has been reached cells tend to redifferentiate [36–38]. These properties were retrieved in our study. Ultrastructural studies showed the presence of a thick neointima at day 15 and SMCs had a synthetic phenotype. Consistently, dystrophin, a marker of the contractile phenotype, was absent. At day 45, the neointima was still present but the cells exhibited a cytoplasm filled with myofilaments and, then, immunological detection revealed the presence of dystrophin both in neointima and in media.

Our results show that balloon injury induces rapidly a dramatic decrease in the density of the L-type Ca²⁺ channel current, involved in the excitation–contraction coupling, in medial cells over a period of time which corresponds to the proliferative ‘first wave’ of SMCs activity. This decrease is transient and the currents start to recover normal amplitude after 4 weeks, i.e. probably after proliferation has ceased. A modulation of I_Ba also takes place in intimal SMCs. Although we were not able to investigate cells at stages earlier than 15 days after injury (owing to the absence or thinness of the neointima), we observed a complete disappearance of the currents at this stage. This was in sharp contrast with the partial recovery that had already occurred in medial cells. Another difference is that recovery in current amplitude was markedly delayed in the neointimal cells. This delay is probably related to phenotypic differences between medial and neointimal cells. The latter can replicate for weeks or months after the injury, which could be related to their prolonged decrease of I_Ba. Immunofluorescence detection and Western blotting demonstrated that the lack of I_Ba in neointimal cells on day 15 after the balloon injury is related to the absence of the Ca²⁺ channel _1C subunit in the membrane. However, this protein reappeared in the neointima several weeks after the balloon injury. Immunofluorescence detection and Western blotting using a monoclonal antibody directed specifically against dystrophin from smooth muscle gave converging results showing a complete decrease of dystrophin in the neointimal cells on day 15. Therefore, our data strongly suggest that the neointimal cells lack both the L-type Ca²⁺ channel and dystrophin in relation to their dedifferentiated replicative state.

There is a parallel between the re-expression of the Ca²⁺ channel currents, dystrophin and cell differentiation. The reappearance of the Ca²⁺ channel _1C subunit follows cell redifferentiation which occurs during the evolution of intimal thickening after arterial injury. It has been previously demonstrated before that expression of dystrophin in vitro is associated with recovery or maintenance of contractility [18,19]. Interestingly, similar correlation between cell phenotype and lack of Ca²⁺ currents has been done in the aortic SMCs of newborn rats. These myocytes are by several criteria still relatively undifferentiated and lack some of the contractile proteins that characterise mature SMCs as confirmed by ultrastructural observations [39]. They express neither Ca²⁺ currents [40] nor dystrophin (unpublished observation). Acquisition of the L-type Ca²⁺ current parallels that of contractility during early development [41]. These results are in line with previous reports that the repertoire of various proteins expressed at early developmental stages is re-expressed during injury (e.g. actin, myosin, osteopontin) [42].

Several studies have described the structural/functional modifications that the activated medial and neointimal SMCs undergo as a result of endothelial injury [34–39]. However, our study shows modulation of the Ca²⁺ channel function. We demonstrate that this modulation reflects a variation in the expression of _1C, the main Ca²⁺ channel subunit (and not of an auxiliary subunit like the β-subunit for example) which is required for Ca²⁺ influx and is, therefore, a major regulator of Ca²⁺ homeostasis. Interestingly, dystrophin is also closely associated with regulation of contraction in arterial SMCs. In fact, two homologous proteins: dystrophin and utrophin (or DRP1) have been detected in vessels [11,43]. Their function is still unclear. However, both proteins are codistributed in small arteries and this has been related to a mechanical function provided by dystrophin while presence of utrophin alone in small veins was presumed to be associated with an architectural function in this tissue [44]. It is well established that a dystrophin-deficient muscle is more exposed to membrane rupture and subsequent muscle necrosis during muscle contraction and tension development [45]. This indicates that dystrophin has an important structural role in maintaining membrane integrity. However, a recent hypothesis regarding a pathophysiological link between the absence of dystrophin and some calcium leakage activity in dystrophic muscles [13,46,47]suggests that dystrophin has a role in aggregating ion channels [48].

During the past years, we and others have suggested that expression of T-type Ca²⁺ channels is associated with the proliferative activity of SMCs in vitro, i.e. in cultured aortic cells [5,9]. In the present study, we find no evidence for the presence of T-type Ca²⁺ channels in SMCs that are naturally proliferative in vivo, a finding which is consistent with the lack of Ca²⁺-channel currents also observed in aortic SMCs freshly isolated from neonatal rats (1-day postpartum) [40]. The discrepancy between proliferative SMCs in vitro and in vivo holds also true for L-type Ca²⁺ channels. Aortic SMCs freshly isolated from both neonatal and injured adult tissues lack L-type I_Ba. In contrast, despite differences in terms of density between the various phases of cell cycle, L-type I_Ba is always observed in proliferative cells in vitro [8,9]. Interestingly, SMCs isolated from either newborn animals or neointimas of injured arteries and grown in primary culture, express both T- and L-type I_Ba after only 3–4 days in primary culture (unpublished data) whereas no I_Ba can be recorded in myocytes freshly isolated from tissues at the corresponding comparable age. This observation indicates that the cells have the potential to express Ca²⁺ channels in culture conditions. Therefore, proliferative cells in vivo and in vitro are not identical with respects to Ca²⁺ channel expression which may depend critically upon cellular environment and particular factors (e.g. serum concentration and nature of mitogenic factors). The effect of cell culture on Ca²⁺ channels expression, and presumably other proteins, should definitely be taken into consideration, when studied in relation with proliferative activity and extrapolated to in vivo (pathophysiology). Our results suggest that T- and L-type Ca²⁺ channels are not necessary for aortic cell proliferation in vivo. The significance of the present results for pathophysiology and therapy remains to be clarified. It is however, tempting to suggest that our results could explain why Ca²⁺ antagonists are unable to interfere with some proliferative stimuli and have only modest antiatherogenic effects in patients with coronary artery disease [49]. They may also explain why Ca²⁺ antagonists reduce somewhat but do not prevent new coronary lesions and why they do not protect significantly against restenosis after coronary angioplasty [50]. It is possible that some of the benefits on vascular VSMCs reactivity, that are often observed at relatively high concentrations, are unrelated to Ca²⁺ channel antagonism [51].

	5 Conclusions

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
The present study brings new insights into the function and the regulation of membrane-associated proteins during the phenotypic modulation characterising arterial response to injury. Taken together, our results suggest that there is a concomitant decreasing effect on expression of dystrophin and of L-type Ca²⁺ channels that are crucial in the development and maintenance of contraction. The disappearance of voltage-gated Ca²⁺ entry results probably in a marked alteration of intracellular Ca²⁺ handling. One implication is that drugs like Ca²⁺ antagonists which fail to affect post-angioplasty restenosis have probably no specific receptor to target during the first weeks after arterial injury.

Time for primary review 32 days.

	Acknowledgements

 
We thank Lisa Matthews for her help with iconography and Dr. G. Dayanithi for critical reading of the manuscript. This work was supported by Fondation pour la Recherche Médicale and Fondation de France (97003982 to SR). This work was also supported in part by grants from INSERM, CNRS and the Association Française contre les Myopathies (AFM).

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 

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