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Am J Physiol Heart Circ Physiol 290: H1307-H1310, 2006. First published October 7, 2005; doi:10.1152/ajpheart.00196.2005
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Vascular injury response in mice is dependent on genetic background

Jürgen R. Sindermann,1,2,3 Christiane Köbbert,2,3 Adriane Skaletz-Rorowski,2,3 Günter Breithardt,2,3 Gabriele Plenz,2,3,4 and Keith L. March1

1Krannert Institute of Cardiology and Indiana Center for Vascular Biology and Medicine, Indiana University Medical Center, Indianapolis, Indiana; 2Leibniz Institute for Arteriosclerosis Research; 3Department of Cardiology and Angiology; and 4Department of Thoracic and Cardiovascular Surgery, University of Münster, Münster, Germany

Submitted 28 February 2005 ; accepted in final form 14 September 2005


   ABSTRACT
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 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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Mouse models are employed to unravel the pathophysiology of vascular restenosis. Although much effort has been spent on how to apply an adequate arterial injury, the influence of the genetic background of mice has not yet received sufficient consideration. The study presented herein was designed to demonstrate the influence of the mouse strain on vascular injury response. Mice of a defined background (50% 129 strain and 50% DBA strain) were backcrossed into either the 129 strain or the DBA strain. Male offspring were subjected to a femoral artery injury model by applying an electric current. Morphometric analysis revealed that backcrossing into the 129 strain resulted in a significant (P < 0.001) 17-fold increase in neointima formation (n = 17 mice) compared with backcrossing into the DBA strain (n = 19). The values of neointima area were 9.18 x 103 ± 2.13 x 103 and 0.54 x 103 ± 0.39 x 103 µm2, respectively. In conjunction, the vessel wall area was enhanced by 1.8-fold (P < 0.001). In contrast, no significant differences were found for the areas of the lumen and the tunica media. Similarly, a significant increase in neointima formation was also found for mice of pure 129 strain compared with pure DBA strain. The results underline the importance of the genetic background for studies on vascular injury response. Furthermore, because the mouse genome of the various strains is well defined, serial testing of the genetic background of mice will provide candidate genes and/or genetic modifiers controlling vascular injury response.

mouse strains; smooth muscle; neointima

THE RESPONSE OF THE ARTERIAL vessel wall to processes such as balloon angioplasty and arteriosclerosis resulting in vascular (re)stenosis involves smooth muscle cell proliferation and changes in vessel wall mass (1, 10, 12). The latter is referred to as vascular remodeling, which is distinguished between negative, constrictive remodeling and a positive, adaptive remodeling that can be found in early stages of atherosclerosis (6, 9, 13). Because the mechanisms of these processes are still partially unknown, animal models are an important means to unravel the pathophysiology of vascular (re)stenosis and to develop therapeutic strategies. Several vascular injury models have been used to induce neointima formation in various species (4, 5, 14). Special interest is paid to mouse models because they are most commonly used for targeted gene approaches. Although much effort has been spent on the technical development of injury models best resembling the clinical situation, the importance of the genetic background of mice for the outcome of vascular injury models has not yet received sufficient consideration. However, comprehensive genetic information can be obtained from the mouse genome even with regard to the various mouse strains. Several injury models in mice have been established, among which the perivascular delivery of an electric current is an effective model for arterial neointima formation and reendothelialization (2, 15). Although the electric injury does not reflect the typical injury such as balloon dilation applied in the clinical situation, the vascular wound healing in the mouse artery resembles certain patterns of the restenotic process in patients (2). Preliminary data from our laboratory suggested that the extent of neointima formation in the electric injury model is considerably dependent on the mouse strain. Initial experience suggested that the 129 mouse strain, a strain playing a pivotal role in generating knockout mouse models, shows a strong response to vascular injury. To test this hypothesis, we designed a setting in which mice of a mixed background (50% 129 strain and 50% DBA strain) were backcrossed into either the DBA strain or the 129 strain and offspring were subjected to electric injury of the femoral artery.


   EXPERIMENTAL PROCEDURES
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All manipulations were performed according to National Institutes of Health and were evaluated and approved by the Institutional Animal Use and Care Committee of the Indiana University Medical Center, Indianapolis. Mice of a mixed background defined as 50% 129/J strain and 50% DBA/2J strain were backcrossed into either the DBA/2J or the 129/J strain (Jackson Laboratories). Adult male offspring were included for a perivascular electric injury model, which was used as a modification of the model described by Carmeliet et al. (2). Briefly, the animals were anesthetized with 2.5% Avertin (0.015 ml/g body wt ip) for survival surgery. The left femoral artery was exposed through an incision in the groin, and an electric current of 600 µA was delivered for ~2 s point-by-point through the tips of a bipolar forceps over a total distance of ~3 mm. This electric injury model was shown to result in adequate neointima formation within 2 wk after the procedure (2). For our studies, the animals were allowed to recover for 3 wk before they were euthanized by an overdose of halothane and prepared for histological analysis. To allow perfusion fixation, the chest was opened immediately after death, and the heart was punctured with a 25-gauge cannula for the infusion of PBS under physiological pressure. The blood was drained through an incision of the inferior vena cava. After 5 min, PBS was substituted by 10% buffered formaldehyde and the mice were perfused for an additional 5 min before dissection and overnight postfixation with 10% buffered formaldehyde. For histological analysis, tissues were dehydrated through various concentrations of ethanol and embedded in paraffin by using standard methods. Sections were rehydrated and visualized with Van Gieson stain, hematoxylin and eosin staining, and picrosirius red staining by standard methods. Hematoxylin and eosin stainings were used for the evaluation of smooth muscle cell density in the noninstrumented femoral arteries of mice of pure DBA and pure 129 strain. Immunohistochemistry for CD3-positive cells (monoclonal CD3 antibody, clone number CD3–12; Linaris, Wertheim-Bettingen, Germany) and periodic acid Schiff reaction (PAS staining) were performed for the evaluation of inflammatory cell density, which was judged by counting microscopic fields. Morphometric analysis was performed on Van Gieson stainings by measuring the circumference of the external elastic lamina, internal elastic lamina, and the luminal border. Areas were calculated from circumference measurements by assuming a circular structure under in vivo conditions. For instrumented left femoral arteries and for noninstrumented right femoral arteries, the values were obtained from five sections per vessel and animal. Statistical analysis was performed by Student's t-test. Data are given as means ± SE of the animals per group.


   RESULTS AND DISCUSSION
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Mice of the pure DBA strain and the pure 129 strain did not show obvious differences in femoral artery dimensions (lumen and vessel wall area). Nevertheless, cellular density in the tunica media of noninstrumented femoral arteries was 1.2-fold higher for mice of the 129 strain. Mice of the pure DBA strain revealed 4.67 x 10–3 ± 0.29 x 10–3 cells/µm2, whereas mice of the pure 129 strain displayed a cell density of 5.76 x 10–3 ± 0.23 x 10–3 cells/µm2 (n = 10 in each group; P < 0.01). Mice of a mixed background (50% DBA strain and 50% 129 strain) were backcrossed into either DBA strain or 129 strain. Male offspring of those backcrossed into DBA and those backcrossed into the 129 strain did not show significant differences in the lumen area and vessel wall area (tunica media) of noninstrumented femoral arteries as shown in Table 1. In contrast, femoral arteries treated with the perivascular electric injury showed significant differences in response to injury between the groups. As shown in Fig. 1, neointima formation was significantly (P < 0.001) enhanced for male offspring of mice backcrossed into the 129 strain compared with offspring of those backcrossed into the DBA strain. The values were 9.18 x 103 ± 2.13 x 103 and 0.54 x 103 ± 0.39 x 103 µm2, respectively, which resembles a 17-fold increase in neointima formation for the offspring of mice backcrossed into the 129 strain. In conjunction, these mice displayed a significant (P < 0.001) 1.8-fold increase in total vessel wall area with values of 24.66 x 103 ± 0.26 x 103 vs. 13.80 x 103 ± 0.79 x 103 µm2 for the male offspring of mice backcrossed into the DBA strain. Figure 2, A–H, depicts typical femoral cross sections of both groups illustrating the differences in neointima formation. In contrast, there were no significant differences in the area of the tunica media and the lumen of injured femoral arteries between the groups. Furthermore, no obvious differences were found for the collagen fibers in response to injury (Fig. 2, G–H). To extend our studies, we also analyzed mice of a pure 129 strain and those of a pure DBA strain. In conjunction with the other results, we found a significant increase in neointima formation for the 129 strain with values of 4.15 x 103 ± 1.28 x 103 vs. 0.49 x 103 ± 0.19 x 103 µm2 for the DBA strain (P < 0.01). Even for the pure DBA strain, neointima was present at a low level.


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  		Table 1. Cross-sectional dimensions of noninstrumented right femoral arteries

 

Figure 1
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  		Fig. 1. Morphometric analysis of cross-sectional vessel areas after electric injury of the femoral artery. Mice of a 50% 129 and 50% DBA background were backcrossed into either the 129 strain or DBA strain. Male offspring were subjected to perivascular electric injury of the femoral artery as described in EXPERIMENTAL PROCEDURES. Animals were allowed to recover for 3 wk; perfusion fixation was performed by using 10% buffered formaldehyde. Morphometric analysis was performed on Van Gieson stains of the instrumented left femoral artery. Areas were calculated from circumference measurements by assuming a circular structure under in vivo conditions. Data were obtained from 5 sections per vessel and animal. Histogram represents means ± SE of male offspring of mice backcrossed into the 129 strain (n = 17) and male offspring of those backcrossed into the DBA strain (n = 19). n.s., Not significant.

 

Figure 2
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  		Fig. 2. Comparative displays of typical femoral cross sections after electric injury. Mice of a 50% 129 and 50% DBA background were backcrossed into either the 129 strain or the DBA strain, and male offspring were subjected to electric injury of the left femoral artery. Mice were allowed to recover for 3 wk and were prepared for histological analysis including Van Gieson staining (A–C and E) hematoxylin and eosin staining (D and F), and picrosirius red staining (G and H). N, neointima; M, media; A, adventitia. Original magnifications: A and B, x100; C–H, x400.

 
With regard to potential mechanisms accounting for the enhanced vascular injury response of mice backcrossed into the 129 strain, we studied the invasion of inflammatory cells by CD3 staining and PAS staining. Immunohistochemistry for CD3-positive cells in the adventitia of injured femoral arteries revealed a significant 2.3-fold increase for mice that were backcrossed into the 129 strain compared with those backcrossed into the DBA strain (Fig. 3). This finding was further supported by a significantly 2.1-fold-enhanced density of PAS-positive cells in the femoral adventitia of mice backcrossed into the 129 strain, thereby mirroring the enhanced injury response. The enhanced inflammatory response to injury plus the enhanced smooth muscle cell density of the noninstrumented vessel wall of the 129 strain will have to be discussed as potential strain-dependent mechanisms contributing to the enhanced neointima formation.


Figure 3
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  		Fig. 3. Adventitial CD3-positive cell density after electric injury of the femoral artery. Mice of a 50% 129 and 50% DBA background were backcrossed into either the 129 strain or the DBA strain. Three weeks after electric injury, male offspring were prepared for histological analysis to determine the density of CD3-positive lymphocytes in the adventitia. Histogram represents means ± SE of male offspring of mice backcrossed into the DBA strain (n = 19) and male offspring of those backcrossed into the 129 strain (n = 17).

 
Despite the significant increase in total vessel wall area, the offspring of mice backcrossed into the 129 strain displayed a significantly 1.3-fold-enlarged lumen of injured femoral arteries compared with noninjured contralateral controls (Fig. 4), indicating that adaptive arterial remodeling in the presence of enhanced neointima formation responded to the perivascular electric injury. This remodeling response was in line with the first description of the perivascular electric injury by Carmeliet et al. (2), who described the neointima formation along with the increase in lumen area of injured femoral arteries in mice of a 50% C57Bl6 and 50% 129 mixed genetic background. With a view to our data and those of Carmeliet et al. (2), the adaptive arterial remodeling response appears to be attributable to the perivascular electric injury model rather than to the genetic background of mice because Harmon et al. (7) described a constrictive remodeling response of 129/SvJ mice and DBA/2J mice in response to ligation of the common carotid artery. However, our data clearly demonstrate that the genetic background of mice has a considerable influence on the extent of neointima formation in response to vascular injury. Although much effort has been spent on optimizing technical aspects of injury models and on studying mitogenic or endothelial factors (3, 8, 11, 16), it may even be hypothesized that an optimal genetic background of mice may be necessary to unravel a significant influence of a certain factor. In our studies, we used mice of a mixed background that were backcrossed into either the 129 strain or the DBA strain. The use of mice with a mixed background is common practice, and our data reveal the considerable influence of a shift in the genetic background on the outcome of a study. This issue is of great importance for animal study designs, but it also opens the field of genetic analyses. Because the mouse genome of the various strains is well defined, serial testing of the genetic background of mice will provide candidate genes and/or genetic modifiers controlling vascular injury response.


Figure 4
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  		Fig. 4. Morphometric comparison of lumen area of instrumented and noninstrumented femoral arteries. Mice of a 50% 129 and 50% DBA background were backcrossed into the 129 strain, and male offspring were analyzed with regard to the lumen area of the injured left femoral artery and noninjured contralateral femoral artery. Procedures were performed as described in Fig. 1. Histogram represents means ± SE of male offspring of mice backcrossed into the 129 strain (n = 17).

 

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 ABSTRACT
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 RESULTS AND DISCUSSION
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This work has been supported in part by an Innovative Medical Research grant and Interdisziplinäres Zentrum für Klinische Forschung grant (project A10) of the University of Münster.


   ACKNOWLEDGMENTS
 
We thank Christa Möllmann for excellent technical support.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. R. Sindermann, Dept. of Cardiology and Angiology, Univ. of Münster, Domagkstrasse 3, D-48149 Münster, Germany (e-mail: sinderm{at}uni-muenster.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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This Article
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290/3/H1307    most recent
00196.2005v1
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