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Cardiovascular Research 2003 60(3):654-663; doi:10.1016/j.cardiores.2003.08.015
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Copyright © 2003, European Society of Cardiology

Stenosis progression after surgical injury in Milan hypertensive rat carotid arteries

Amalia Forte*,a, Salvatore Espositob, Marisa De Feoc, Umberto Galderisia, Cesare Quartoc, Ferdinando Espositoa, Attilio Renzullid, Liberato Berrinoa, Marilena Cipollaroa, Lucio Agozzinob, Maurizio Cotrufoc, Francesco Rossia and Antonino Cascinoa

aExcellence Research Center for Cardiovascular Diseases, Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, Second University of Naples, Via Costantinopoli, 16, 80138 Naples, Italy
bDepartment of Public Medicine, Second University of Naples, Naples, Italy
cDepartment of Cardiothoracic Sciences, Second University of Naples, Naples, Italy
dUnit of Cardiac Surgery, University Magna Graecia, Catanzaro, Italy

*Corresponding author. Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, Second University of Naples, Via Costantinopoli, 16, 80138 Naples, Italy. Tel.: +39-81-5665879; fax: +39-81-5667547. Email address: amalia.forte{at}unina2.it

Received 18 June 2003; revised 15 August 2003; accepted 29 August 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Background: Milan hypertensive rats (MHS) are characterised by an increase in renal sodium reabsorption mainly related to adducin mutations. Interest in this model relies on the genetic link between adducin polymorphisms and primary hypertension, observed also in a subset of patients. Objectives: To investigate the molecular and morphological events involved in carotid stenosis and triggered by surgery in MHS model. Methods: Stenosis was induced through arteriotomy. At different times after injury, the expression profiles of various gene families were investigated by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), while histological techniques were used to follow morphometric and morphological changes. Apoptotic nuclei were revealed by terminal deoxynucleotidyl transferase-dUTP nick end labelling (TUNEL). Results: mRNAs coding for transcription factors c-jun, c-fos and c-myc were rapidly induced by injury. Analysis of apoptosis-related genes revealed a decrease of the Bcl-2/Bax ratio 4 h after vascular trauma (P<0.05), followed by a recovery of antiapoptotic factors 24 and 48 h later. ETA and receptor mRNAs decreased after the injury and were replaced by ETB and AT2 mRNAs. Both ETA and AT1 turned to basal level 48 h after injury. Expression profiles of chatepsins B and D were also determined. A marked neoadventitia led to maximal 60±9% lumen reduction (P<0.05) 30 days after surgery. Media substitution by fibrotic and granulomatous tissue was also evident. Maximal 47±2% apoptotic nuclei were detected 48 h after the injury (P<0.05). Conclusions: The injury applied to MHS carotids induces negative remodelling and a limited apoptotic reaction. These findings could arise from the balancing among proliferative factors, apoptosis-related molecules and relaxant anti-proliferative receptors, all stimulated by the injury.

KEYWORDS Apoptosis; Remodelling; Hypertension; Gene expression; Restenosis; Vascular injury


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Despite the improving results associated with percutaneous interventional revascularization procedures, arterial restenosis remains the "Achilles heel" of intravascular mechanical interventions. Recent follow-up studies have documented restenosis rates to be 30–50%, depending on the kind of vessel injury [1]. The vascular biology of restenosis is complex and not yet well understood; this may explain the lack of effective therapy for preventing restenosis in clinical settings.

Hypertension is one of the most relevant risk factors for the occurrence of restenosis in patients [2,3]. The induction of arterial stenosis in animal models genetically affected by hypertension can provide useful information for a better comprehension of this pathophysiological phenomenon and lead to the identification of putative therapeutic targets.

The animal model investigated in our experiments is the Milan hypertensive rat strain (MHS) obtained by Bianchi et al. in 1974. This strain develops hypertension because of a primary increase in renal tubular Na+ reabsorption, genetically linked to missense mutations within the genes coding for adducin, a heterodimeric cytoskeleton protein composed of related but nonidentical subunits ({alpha}, β or {gamma}). Adducin interacts directly and specifically with the Na+-K+-ATPase, stimulating its activity [4]. The mutant "hypertensive" adducin stimulates the rat renal Na+-K+-ATPase with a higher affinity with respect to wild-type adducin. It can also reduce the rate of Na+-K+-ATPase internalization.

{alpha}-Adducin polymorphisms (G460W, S586C) are genetically linked with essential hypertension also in humans [5]. Since different mutations in rat and human adducin produce similar alterations in renal sodium handling, MHS is a valid model to study the reactions of primary hypertensive patients that are carriers of adducin mutations. In this context, MHS has already been used to test the effectiveness of the antihypertensive drug PST 2238 [6]. Other mutations have also been described in MHS [7–12]. Interest in MHS model also related the findings of Balkestein et al. [13], demonstrating that large artery stiffness is relevant in patients carrier of adducin mutations, when associated to aldosterone synthase polymorphisms.

Since no data were available about arterial stenosis induction in the MHS model, we applied on MHS common carotids a surgical injury involving the full arterial wall thickness, and then investigated, at different times after injury, the expression profile of different gene families, the morphometric and morphological changes as well as the incidence of apoptosis.

Explored gene families have been selected on the basis of their involvement in different events triggered by arterial trauma, such as cell proliferation, extracellular matrix production, SMC contraction, cell migration and apoptosis.

c-myc, c-jun and c-fos are transcription factors belonging to the early expression gene family. The induction of these molecules is important for cell-cycle entry from quiescence and subsequent progression of SMCs to mitosis. c-myc gene codes for a member of basic helix-loop-helix leucine zipper (bHLHZip) family [14]. c-Fos and c-Jun proteins combine to form stable AP-1 heterodimers, which bind to AP-1 consensus sequences present in numerous genes associated with cell proliferative response and extracellular matrix production [15].

Endothelins (ETs) are 21 amino acid endothelium-derived peptides. ET-1 is the physiologically most significant member of the family and regulates vasoconstriction, cell proliferation, tissue remodelling and growth factor release/expression [16]. Two distinct G protein-coupled receptor subtypes, named ETA and ETB, mediate the ET-1 effects.

Angiotensin II (Ang II) is a vasoactive octapeptide produced by the renin–angiotensin system (RAS) that modulates vascular growth, matrix accumulation, inflammation and SMC contraction and migration [17]. Most of the effects of Ang II are exerted through AT1 subtype receptors, while AT2 receptors are decreased soon after birth in most adult organs, including the vessels [18]. AT2 receptors can be re-expressed in pathological conditions and can counteract the AT1-mediated effects.

The cathepsin protease family consists of at least 12 known members, constitutively produced in a variety of tissues. Cathepsins have several properties suggesting their contribution to arterial remodelling. Some cathepsins have been demonstrated to have potent elastolytic or collagenolytic activity, contributing to cell migration [19]. They also have roles in mediating cell death in alternative noncaspase forms of apoptosis [20].

Bcl-2 family is the most prominent gene group involved in cell viability, governed at the molecular level by a balance between proapototic and antiapoptotic signals. Bcl-2 family members promoting cell survival include Bcl-2 and the long form of Bcl-X (Bcl-XL), whereas Bax and the short form of Bcl-X (Bcl-XS) promote apoptosis.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusions
 References
 
2.1. Animals
Studies were carried out on 12-week-old male MHS rats (300–310 g) kindly provided by Prassis-Sigma Tau Research Institute, Italy. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The Animal Care and Use Committee of the Second University of Naples approved all protocols related to this research. Rats were acclimatized and quarantined for at least 1 week before undergoing surgical injury, and were housed at constant temperature (21±1 °C) and relative humidity (60%) under a regular light/dark schedule (light 7.00 a.m. to 7.00 p.m.). Food and water were freely available.

2.2. Vascular injury
The surgical injury model we applied on MHS rat common carotid was performed as already published [21]. Briefly, a plastic Scanlon clamp for coronary artery bypass grafting was placed for 10 s on the carotid artery in order to cause a crushing lesion to the vessel. At the same point where the clamp was applied, a 0.5 mm longitudinal incision was performed on the full thickness of the carotid artery. The incision did not cross to the other side of the vessel. Hemostasis was obtained with a single adventitial 8.0-gauge polypropylene stitch. Once bleeding stopped, the carotid artery was carefully examined and blood pulsation was checked distally to the incision.

2.3. RNA extraction
Total RNA was extracted from left carotid segments marked by the polypropylene stitch applied during the surgery taken 1 (n = 4), 2 (n = 4), 4 (n = 4), 24 (n = 4) and 48 h (n = 4) after injury as well as from uninjured carotids (n = 5), using the RNeasy Kit (Qiagen, Hilden, Germany) according to manufacturer's instructions.

2.4. Semi-quantitative RT-PCR
c-Myc, c-Jun, c-Fos, AT1, AT2, ETA, ETB, Cathepsin D, Cathepsin B, Bcl-2, Bax {alpha}, Bcl-xL/S and GAPDH mRNAs were amplified by reverse transcription-polymerase chain reaction (RT-PCR) on total RNA extracted from surgically injured carotids, as well as from uninjured carotids. The OLIGO 4.05 software (W. Rychlik copyright, 1992) designed primer pairs for PCR amplification (Table 1) on the basis of rat gene sequences reported in the Genbank database. All primers contained 50–60% G/C.


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  		Table 1 PCR primers and sizes of RT-PCR products

 
The optimal annealing temperature for all the primer pairs was 55 °C. RT-PCR amplifications and agarose gel electrophoresis were performed as published elsewhere [22]. Densitometric analysis of RT-PCR product levels was assessed by the Molecular Analyst software associated with Gel Doc 1000 (Bio-Rad, Hercules, CA, USA). The density values of RT-PCR products were normalized with respect to endogenous gapdh product levels. Moreover, the RT-PCR products obtained from injured carotids were normalized with respect to gene expression basal levels detected in untreated carotids.

Each PCR was repeated at least three times. The number of cycles varied according to the expression level of the target gene. An appropriate number of cycles were determined to ensure that PCR was taking place in the linear range, in order to guarantee a proportional relationship between input RNA and densitometric readout.

2.5. Histological analysis
Carotid arteries were taken 2 (n = 3), 7 (n = 4), 14 (n = 6), 21 (n = 5) and 30 days (n = 4) after injury. Rats were anaesthetized and carotids were dissected free from the surrounding tissues. Through thoracotomy, the left ventricle of the beating heart was cannulated with a blunt syringe. The syringe was held in place by a ligature in the ascending aorta. The vessels were perfused at physiological pressure first with saline until the effluent was clear, and then perfusion fixed with 4% buffered (pH 7) formaldehyde. An incision in the right atrium served as outflow tract. Before switching to formaldehyde, the descending thoracic aorta was clamped. Tissue samples were taken 20 min after perfusion. Samples were further fixed in 4% formaldehyde o.n., dehydrated and finally embedded in paraffin. Cross-sections (5 µm) were stained with hematoxylin–orcein for elastic fiber analysis.

For each injured carotid, at least 60 serial cross-sections were observed under a light microscope at 20 x magnification; image screening and photography were performed using a Leica IM 1000 System (Leica, Wetzlar, Germany).

The sections of injured carotids showing maximal remodelling and proliferative phenomena were identified and further analyzed.

Lumen and medial areas were measured using the Leica IM 1000 software (Leica, Heerbrugg, Switzerland). The former was defined as the area enclosed by internal elastic lamina, while the latter was defined as the area enclosed between the external and internal elastic laminae.

In order to reduce individual rat variability, the lumen and medial areas of each treated carotid were normalized with respect to the contralateral uninjured carotid. For each contralateral uninjured carotid, at least 10 sections were analyzed and the average media and lumen areas were calculated. Measurements were performed by two independent observers.

2.6. TUNEL
Assay was performed on carotid arteries taken 4 h (n = 4), 2 days (n = 3), 7 days (n = 4) and 14 days (n = 3) after injury. The 4% formaldehyde-fixed sections (5 µm) were deparaffinized and rehydrated. The tissue was permeabilized with 20 µg/ml proteinase K (Roche Diagnostics, Indianapolis, IN, USA) for 30 min. Endogenous peroxidase activity was blocked with 3% H2O2 for 10 min. Terminal deoxynucleotidyl transferase enzyme and dUTP conjugated to fluorescein were added to the tissue sections according to the manufacturer's specifications (Roche). After end-labeling for 1 h at 37 °C, sections were washed in PBS and incubated with anti-fluorescein antibody conjugated with horseradish peroxidase complex, rinsed in PBS and stained with DAB substrate (Roche). Nuclei were counterstained with hematoxylin. Specimen image screening and photography were performed using a Leica IM 1000 System. Three sections were analyzed for each carotid. The percentages of apoptotic nuclei were calculated by determining the number of hematoxylin-stained nuclei positive for terminal deoxynucleotidyl transferase-dUTP nick end labelling (TUNEL) staining. When observed with a 40 x objective, cells showing morphological features characteristic of apoptosis in addition to positive TUNEL reaction were considered to be apoptotic. Nonspecific cytoplasmatic staining without nuclear involvement was considered negative. A number of randomly selected slides were quantified for TUNEL staining by two independent observers to assess interobserver variation.

2.7. Statistical analysis
All results are expressed as means±S.E.M. Student's t-test was used to evaluate the morphometric and molecular differences between the uninjured and the injured carotids at different times after surgery. P<0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusions
 References
 
3.1. RT-PCR analysis
3.1.1. Transcription factors
Fig. 1a histogram shows that c-Myc mRNA is undetectable in carotids at basal levels, but is rapidly induced by vascular injury, reaching a maximal increase 2 and 4 h after surgery.


Figure 1
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  		Fig. 1 Densitometric analysis of RT-PCR products obtained on total RNA extracted at different times from MHS injured and uninjured carotids. All measurements were normalized with respect to endogenous GAPDH levels. The values are expressed as relative increases over the normalized uninjured control. Data are presented as the mean±S.E.M. *P<0.05 vs. uninjured carotids. mRNA expression levels of (a) c-myc; (b) c-jun and c-fos.

 
The expression profiles in Fig. 1b show that both c-Jun and c-Fos mRNAs reach maximal expression 1 h after injury, followed by a quick return to basal levels.

3.1.2. ET-1 receptors
RT-PCR analysis (Fig. 2a) indicates that the ETA mRNA gradually decreases after surgery, reaching a minimum level at 4 h, with a 21-fold reduction (P<0.05). Conversely, the ETB mRNA is undetectable at basal levels, but gradually increases 2 h after carotid injury, reaching a peak at 24 h.


Figure 2
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  		Fig. 2 Densitometric analysis of RT-PCR products obtained on total RNA extracted at different times from MHS injured and uninjured carotids. All measurements were normalized with respect to endogenous GAPDH levels. The values are expressed as relative increases over the normalized uninjured control. Data are presented as the mean±S.E.M. *P<0.05 vs. uninjured carotids. mRNA expression levels of (a) ET-1 ETA and ETB receptors; (b) Ang II AT1 and AT2 receptors; (c) Cathepsins B and D.

 
3.1.3. Ang II receptors
The RT-PCR results we obtained indicate a gradual decrease of AT1 mRNA, with a maximal 5.2-fold reduction 24 h after surgery (P<0.05), followed by a return to basal levels 48 h later (Fig. 2b). At the same time, we detected a slight increase of AT2 receptor mRNA, which showed a maximal 2.4-fold increase 4 h after MHS carotid injury (P<0.05), followed by a gradual return to basal levels.

3.1.4. Cathepsins B and D
The expression profiles reported in Fig. 2c indicate a rapid decrease of cathepsin D mRNA levels, reaching a maximal 5-fold reduction 24 h after carotid wall injury (P<0.05), followed to an increase to basal levels at 48 h. This profile is quite different from that of cathepsin B, characterised by a significant marked increase 1, 24 and 48 h after surgery.

3.1.5. Apoptosis-related genes
The antiapoptotic factor Bcl-2 showed a 2.4- and 4-fold decrease 2 and 4 h after surgery, respectively (P<0.05), while the antiapoptotic factor Bcl-XL was unaffected by the vessel trauma. The proapoptotic factor Bcl-XS showed a significant 6-fold decrease 48 h after carotid injury (P<0.05). The proapoptotic factor Bax {alpha} showed a significant decrease 2, 4 and 48 h after surgery, with a maximum 3.9-fold reduction 2 h after carotid injury (P<0.05). As a result, only the Bcl-2/Bax {alpha} ratio showed a slight 1.5-fold decrease 4 h after surgery (P<0.05), while both Bcl-2/Bax {alpha} and Bcl-XL/Bcl-XS ratios (Fig. 3) showed a maximal increase 48 h after MHS carotid injury (1.94- and 4.7-fold, respectively) (P<0.05).


Figure 3
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  		Fig. 3 Densitometric analysis of Bcl-2/Bax {alpha} and Bcl-XL/Bcl-XS ratios obtained by RT-PCR on total RNA extracted at different times from MHS injured and uninjured carotids. All measurements were normalized with respect to endogenous GAPDH levels. The values are expressed as relative increases over the normalized uninjured control. Data are presented as the mean±S.E.M. *P<0.05 vs. uninjured carotids.

 
3.2. Histological analysis
Histological analysis performed at different times (up to 30 days) on carotid cross-sections stained with hematoxylin–orcein method for elastic fibers showed morphological changes in injured carotids mainly in the adventitia and in the media.

In adventitia, pooling of extracellular matrix (composed of amorphous material, elastic and reticular fibers) and cellular proliferation (granulomatous tissue) were observed close to the polypropylene stitch; this process caused lumen reduction for compression ab-estrinseco of the artery.

In a number of injured carotids, foreign body giant cells, derived by the fusion of macrophages and characterised by multiple nuclei often arranged in the periphery of the cell, were also detected and were probably related to the application of polypropylene stitch after incision. Moreover, gradual proliferation of SMCs, fibroblasts and elastic fibers with disruption and fragmentation of elastin laminae was also observed in the media (Fig. 4b–f).


Figure 4
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  		Fig. 4 Representative cross-sections of MHS carotids (hematoxylin–orcein staining, 10 x magnification). (a) Uninjured carotid; injured carotid harvested (b) 48 h; (c) 7 days; (d) 14 days; (e) 21 days; (f) 30 days after surgery.

 
Morphometric analysis (Fig. 5a) indicates a progressive lumen reduction in injured carotids, with a maximal 60±9% narrowing 30 days after the surgery (P<0.05).


Figure 5
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  		Fig. 5 (a) Morphometric analysis of MHS carotid lumen area at different times after surgical injury. All measurements were normalized with respect to the contralateral uninjured carotid for each rat. (b) Quantification of TUNEL-positive nuclei in MHS carotid cross-sections. Percentage of apoptotic nuclei was calculated by determining the number of hematoxylin-stained nuclei that were positive for TUNEL staining. Data are presented as the mean±S.E.M. *P<0.05 vs. uninjured carotids.

 
The correct measurement of lumen and media area in carotids harvested 48 h after surgery was not possible since the break through the full wall thickness was not yet reconstituted by granulomatous tissue and induced a collapse of the arterial wall with consequent alteration of the structure (Fig. 4b) during vessel inclusion in paraffin.

3.3. TUNEL staining
The effect of surgical injury on vascular cell apoptosis has been investigated both at early and late time points through the TUNEL assay, which detects endonuclease-induced DNA fragmentation, considered as a marker of apoptosis.

Few or no TUNEL-positive nuclei were detectable in the uninjured control carotids (Fig. 6a). Few positive cells were detectable in intima layer 4 h after surgery, mainly localized close to the injury site. Apoptotic cells were maximal (47±2%) both in intima and media 48 h after vascular trauma (P<0.05) (Figs. 5b and 6c)Go, showing a circumferential distribution. This suggests a recruitment of all SMCs into apoptosis. A gradual decrease of TUNEL-positive cells was measured 7 and 14 days after the surgery (Figs. 5b and 6d–e)Go.


Figure 6
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  		Fig. 6 Representative cross-sections of MHS carotids submitted to TUNEL assay (hematoxylin counterstaining, 40 x magnification). Arrows indicate representative TUNEL-positive nuclei. (a) Uninjured carotid; injured carotid harvested (b) 4 h; (c) 48 h; (d) 7 days; (e) 14 days after surgery.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusions
 References
 
4.1. Molecular analysis
mRNA analysis revealed that the vascular trauma stimulated the synthesis of transcription factors c-jun, c-fos and c-myc during the acute phase after carotid injury (Fig. 1a and b), indicating the shift of SMCs from contractile to proliferative phenotype. The early increase of c-myc, c-Jun and c-Fos we detected after carotid surgery is partially consistent with other observations reported after carotid angioplasty [15,23] and vein graft [24]. Slight differences in timing of gene activation are probably related to the kind of injury, to the rat strain and to the target vessel. The c-myc expression profile induced by vascular trauma in MHS (Fig. 1a) is similar to the pattern we previously detected in SHR carotids submitted to the same kind of injury [25], both showing higher activation rate in comparison to normotensive WKY rats [21].

As far as ET-1 receptors, RT-PCR data indicate a progressive replacement of ETA receptor mRNA with ETB mRNA during the acute phase after injury. Both ETA and ETB mRNAs show a relevant decrease 48 h after the surgery (Fig. 2a). ETA receptors are prevalently located on SMCs and mediate SMC contraction and proliferation. ETB receptors can play antagonistic roles depending on cellular localisation. They mediate ET-1 clearance and relaxation when expressed on endothelial cells, while they stimulate apoptosis and vasoconstriction when expressed on SMCs. Studies about the role of ET-1 receptors during restenosis progression result in contrasting data, depending on animal models, kind of injury, target vessels and molecular techniques applied. For example, Porter et al. [26] demonstrated that in cultured human vein grafts, ETB receptor plays an important role as mediator of hyperplasia. Conversely, Dashwood et al. [27] reported that only the ETA receptor mediates neointima formation in pig coronaries submitted to balloon angioplasty. Thus, the role of ET-1 receptor subtypes in restenosis is still unclear.

The AT1 mRNA expression profile obtained in MHS-injured carotids is similar to that we obtained in SHR [25] and shows substantial differences in comparison to WKY rats [21], in which a gradual increase of AT1 mRNA was evident after injury. This data could indicate a different regulation of vascular RAS in hypertensive strains with respect to normotensive rats. In particular, it could be related to a negative feedback of AT1 mRNA induced by an excessive local expression of Ang II in hypertensive rats after injury, according to a mechanism described by Nickenig et al. [28]. AT2 mRNA is also down-regulated by excessive Ang II, but with a different and delayed mechanism in comparison to AT1 [29], that could explain the decrease of AT2 receptor mRNA 48 h after surgery.

No literature data are currently available about tissue RAS in MHS. Preliminary RT-PCR data we obtained indicate that AT1 mRNA basal levels in MHS carotids are significantly higher than in SHR and WKY rats (Forte et al., unpublished data).

AT1 and AT2 receptors are known to play antagonistic roles under the Ang II stimulation in pathological conditions. More precisely, AT1 mediates vasoconstriction, cell proliferation, hypertrophy and vascular matrix expansion. Conversely, AT2 promotes vasodilation, growth inhibition and apoptosis [17]. AT2 molecules are rapidly decreased after birth; nevertheless, we were able to detect AT2 mRNA at basal levels in adult MHS carotids (Fig. 2b). AT2 is re-expressed in pathological situations such as restenosis induced by vascular injury, in which it seems to elicit growth inhibition of SMCs [30]. Nevertheless, some reports suggest an AT2 receptor involvement in neointima formation [30]. Thus, the relative importance of Ang II receptor subtypes in arterial stenosis induced by injury is still controversial.

Among the numerous members of the cathepsin family, we focused our attention on cathepsins B and D for various reasons. They are both involved in the vascular RAS, contributing to the local production of Ang II [31]. It has also been demonstrated that cathepsin D is more expressed in SHR than in WKY vascular cells [32], and that the activity of cathepsin B increased with advancing hypertensive rat age, indicating their possible involvement in hypertensive vascular changes [33]. Finally, our choice is related to the link of cathepsins B and D with apoptosis [20]. In particular, cathepsin D acts as apoptosis promoter, while cathepsin B exerts an opposing effect.

Considering the multiple roles played by cathepsins, the significance of expression profiles we obtained should be further investigated. In this context, we could speculate that the decrease of cathepsin D and the contemporary increase of cathepsin B mRNA (Fig. 2c) might contribute to the inhibition of SMC apoptosis stimulated by vascular injury, in concordance with the parallel increase of Bcl-2/Bax {alpha} and Bcl-XL/Bcl-XS ratios detected 24 and 48 h after surgery (Fig. 3).

4.2. Histological analysis
MHS has never been used for arterial surgical injury neither for balloon angioplasty, and consequently, no literature data are currently available about morphological changes during carotid stenosis in this model.

The arteriotomy model we applied on MHS rat carotid stimulates media substitution by fibrous tissue at the injury site and the formation of neoadventitia, affecting arterial remodelling via mechanical and synthetic properties (Fig. 4). These data further differentiate MHS and SHR from WKY rats submitted to the same kind of injury [21,25], in which we also detected neointima formation. Our observations are supported by other studies showing that strains with a different genetic background differ in vascular cell apoptosis and in the amount of neointimal production after angioplasty [34]. Nevertheless, MHS, SHR and WKY rat strains showed a similar 60% lumen stenosis 30 days after surgical injury (data not shown), regardless of the hypertensive condition and/or of the different genetic background. This could indicate that both the effect of hypertension in exacerbating the response of the vessel and the genetic differences among the rat strains are overridden by the arteriotomy model of vascular injury we applied.

Vascular occlusions after clinical intervention have been generally attributed to neointima hyperplasia, while the role of adventitia has been largely ignored in the past. Only recently, it has been demonstrated the key role that adventitia plays in the development of negative remodelling following angioplasty and artery graft. Matrix accumulation, vasospasm and constriction of the entire vessel by elastin and collagen fibers also play important roles in ultimate vessel patency.

4.3. Surgery-induced apoptosis
SMC apoptosis has been documented in numerous animal models of acute vascular injury, but not in MHS. Our molecular data indicate that MHS vascular cells appear less prone to programmed cell death in comparison with WKY rats submitted to the same kind of vascular injury [21], in which we observed a gradual decrease of the Bcl-2/Bax {alpha} ratio up to 48 h after the injury. Conversely, the profile of Bcl-2/Bax {alpha} ratio obtained in MHS was similar to the pattern we detected in SHR [25].

The maximal 47±2% of apoptotic nuclei we detected by TUNEL 48 h after surgery (Fig. 5b) is probably the consequence of the decreased Bcl-2/Bax {alpha} ratio observed 4 h after trauma (Fig. 3). Thereafter, the remaining vascular cells activated a mechanism of survival through the activation of antiapoptotic genes Bcl-2 and Bcl-xL (Fig. 3), and of cathepsin B (Fig. 2c). These molecules probably contrasted the proapototic effect of the AT2 receptor, whose mRNA increased soon after surgical injury (Fig. 2b).

The timing of cell apoptosis after vascular injury has been investigated through the TUNEL assay in many different animal models and vessels, generating a large amount of heterogeneous data. For example, 70% of apoptotic nuclei has been detected in carotid media 30 min after angioplasty in rats [35]. Other investigators demonstrated that apoptosis occurs in neointima from 7 to 30 days after angioplasty [36].

Some authors clearly demonstrated the important influence of the kind of vascular trauma both on apoptosis and cell proliferation in different models [37]. This observation implies that our TUNEL data can only be partially compared to other published data, since the surgical injury model we applied on rat common carotid is radically different from balloon angioplasty and could induce a different apoptotic reaction. Nevertheless, the apoptotic reaction we observed is delayed and time-limited in comparison to the above-mentioned models, and the percentage of apoptotic nuclei is less relevant than in other researches. Finally, papers based on balloon angioplasty models cannot report the amount of TUNEL-positive nuclei in the intima, since it is damaged and removed by the inflated balloon. We were able to detect apoptotic nuclei in the carotid intima since 4 h after the surgery (Figs. 5b and 6b)Go, suggesting their particular sensitivity to apoptotic stimulus represented by vascular injury.

A number of studies have documented an imbalance between proliferation and apoptosis in SMCs from genetically hypertensive animals or humans. For example, it has been demonstrated that SMCs from SHR exhibit enhanced proliferation and apoptosis in comparison to SMCs from the normotensive WKY strain [38], while other researchers demonstrated that the incidence of apoptosis decreases in young SHR [39]. Hamet et al. [40] detected an increase of apoptosis in SHR along with the development of hypertension, followed by a decrease below the levels observed in WKY after the age of 24 weeks.

The deregulated equilibrium between SMC proliferation and apoptosis in hypertensive rats could be the basis of their vascular hypertrophy, a primary structural change that contributes to the development of hypertension.


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
References
 
Three major conclusions can be derived from the results of the present study.

First, MHS carotids react to surgical injury with a marked negative remodelling mainly due to adventitial phenomena, rather than with neointima formation.

Second, vascular cells in MHS-injured carotids are activated to proliferate and, when compared to normotensive strain, appear less prone to a rapid apoptotic reaction induced by surgery. Nevertheless, the early expression of ETB and AT2 receptors, involved in vessel wall relaxation and inhibition of cell proliferation, could indicate a tendency of MHS-injured carotids to reduce excessive constrictive remodelling. Variations in cathepsin B and D mRNA can also have a role in apoptosis, local RAS regulation and in vascular remodelling.

Third, comparison of MHS molecular and morphometric data with our previous observations in other strains [21,25] indicates that MHS and SHR share similar gene expression profiles in response to vascular injury in comparison to WKY rats. This difference may be attributable to hypertension. Conversely, all examined strains react to arteriotomy with a comparable carotid lumen stenosis.

Further studies will be required to elucidate the biological meanings of the results about ET-1 and Ang II receptors, as well as about the role of cathepsins in MHS surgically induced stenosis. Moreover, both the systemic and the local RAS should be further analyzed in this interesting model of hypertension.


   Acknowledgements
 
This study was partially supported by MURST/CNR "Biomolecole per la salute umana" L.95/95, P.O.P. 1994/1999, sottoprogramma 5-misura 5.4 azione 5.4.2 annualità 1997 "Farmaci antiipertensivi", by Regione Campania, L. 41, annualità 2000 "DNA Antisenso e Farmaci Antiipertensivi", L. 41, annualità 2000 "Terapia genica della restenosi" and by MIUR 2001 "Centro di Eccellenza per le Malattie Cardiovascolari".

We are grateful to Prassis-Sigma Tau Research Institute (Settimo Milanese, Milan, Italy), and in particular to Dr. Patrizia Ferrari and Prof. Giuseppe Bianchi, for providing us with MHS.


   Notes
 
Time for primary review 27 days


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

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