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REPORTS
1Krannert Institute of Cardiology and Indiana Center for Vascular Biology and Medicine, Indiana University Medical Center, Indianapolis, Indiana 46202; and 2Institute for Arteriosclerosis Research, 3Department of Cardiology and Angiology, 4Department of Nephrology, and 5Department of Thoracic and Cardiovascular Surgery, University of Münster, 48149 Münster, Germany
Submitted 21 January 2003 ; accepted in final form 23 April 2003
ABSTRACT
Recent studies have revealed the role of the pRb family members pRb and
p130 in the response to vascular injury. We evaluated the arterial injury
response in the absence of p107, a protein that shares a high degree of
homology with the injury-controlling p130. Carotid artery ligation and
perivascular electric injury of the femoral artery were applied to p107
knockout (p107 –/–) mice, and morphometric analysis was performed
3 wk after ligation and electric injury. Arterial vessels of p107
–/– mice were indistinguishable from controls under basal
conditions. After carotid artery ligation the p107 –/– mice
(n = 7) did not display an enhanced ligation response compared with
controls (n = 9), which was studied over a distance of 
450 µm
proximal and 200 µm distal from the ligation site, with regard to
vessel wall area, neointima area, and lumen area. Corresponding with this,
morphometric data obtained from the perivascular electric injury of the
femoral artery confirmed the lack of enhanced ligation and injury response in
the absence of p107. We conclude that the pRb family member p107 is not a key
regulator in vascular injury response. These data, in conjunction with
previously reported results, indicate that the control of vascular injury
response is not a redundant feature of pRb proteins but primarily specific for
p130. Further studies on functional domains of p130 and p107 will help to
resolve the pathways in vascular injury response.
pRb family; smooth muscle; neointima
Recent studies have successfully revealed the inhibition of intimal thickening after balloon angioplasty in porcine coronary arteries by targeting pRb and other regulators of the cell cycle (8). Cytostatic gene therapy with a constitutively active form of the retinoblastoma gene product and p130 gene transfer revealed a significant decrease in SMC proliferation and neointima formation in a rat carotid and porcine femoral artery model of restenosis (4, 5). The role of p107 in SMC proliferation and differentiation is widely unknown. Studies on rat L6 myoblasts revealed that only the hyperphosphorylated form of p107 is present at mitosis and that as cells enter the G1 phase p107 is rapidly dephosphorylated. During differentiation, L6 myoblasts show strongly increased p130 levels, whereas p107 levels are reduced, although both proteins become predominantly hypophosphorylated in terminally differentiated cells (16). A similar upregulation of p130 was found for differentiating mouse C2 myoblasts (3). In general, complexes with the transcription factors E2F in cycling cells appear to be dominated by p107 and pRb, whereas those complexes in differentiated and/or quiescent cells contain p130 and pRb (16). However, data may vary with the cell type, as adipogenic differentiation was associated with a transient increase in p107 (9).
The important question arises of whether the control of cell cycle progression during vascular injury response is a specific effect exerted by certain proteins of the pRb family, e.g., p130, or rather an nonspecific effect found for all pRb proteins. To illuminate this question, we analyzed whether the lack of p107, a pRb family member that shares highly conserved domains with p130 (16), would also result in enhanced injury response.
EXPERIMENTAL PROCEDURES
The p107 knockout (p107 –/–) mice, kindly provided by Tyler Jacks (Massachusetts Institute of Technology, Cambridge, MA) (6, 14), were finally bred into a DBA background (Jackson Laboratories) and selected as littermates comprising p107 –/– mice and p107 +/+ controls. All manipulations were performed according to National Institutes of Health and institutional animal care and use guidelines. Genotypic status of the mice was assessed by PCR screening for the presence of the neogene.
The carotid artery ligation model was performed as a modification of the
model published by Kumar et al.
(12). Briefly, the animals
were anesthetized with 2.5% Avertin (0.015 ml/g body wt ip). The right common
carotid artery was exposed through a small midline incision in the neck and
ligated by a 6-0 propylene suture 2 mm proximal from the carotid
bifurcation. In addition, mice were treated by a perivascular electric injury
derived from the model of Carmeliet et al.
(2). Briefly, under anesthesia
(see above), the left femoral artery was exposed through an incision in the
groin and an electrical current of 600 µA was delivered for 2 s point
by point through the tips of a bipolar forceps over a total distance of 4
mm. The animals were allowed to recover for 3 wk and were then killed by an
overdose of halothane and perfusion fixed by infusion of PBS and 10% buffered
formaldehyde under physiological pressure.
Morphometric analysis was performed on Van Gieson stains by measuring the circumference of the external elastic lamina, the internal elastic lamina, and the luminal border. Areas were calculated from circumference measurements assuming a circular structure under in vivo conditions or by direct area measurement when appropriate. For vessels under basal conditions, the values were obtained from at least three sections per vessel and animal. Statistical analysis was performed by Student's t-test for independent samples. The ligated carotid arteries were analyzed by repeated-measures ANOVA in which sections within a vessel were treated as a repeated measure and the two types of mice as the grouping factor. In the case of missing values of a certain section because of the preparation procedure, data were inserted by using the average of the values on either side. For femoral arteries treated with the perivascular electric injury, five sections from within the lesion were measured per animal. Vessels with obvious thrombosis due to the procedures were excluded from the analysis.
Staining for proliferative cell nuclear antigen (PCNA) (Dako, Carpinteria, CA) was performed on a representative section proximal from the ligation of each carotid artery and used a monoclonal antibody (diluted 1:300) with standard methods. The proportion of PCNA-positive nuclei was evaluated by counting at least three representative microscopic fields per artery, and statistical analysis was performed by Student's t-test for independent samples. Data are given as means ± SE.
RESULTS AND DISCUSSION
The p107 –/– mice were overtly indistinguishable from p107 +/+ control mice and presented average body weights of 24.46 ± 1.16 g compared with 27.39 ± 1.14 g for controls [not significant (NS)]. To evaluate arterial vessel dimensions under basal conditions in the absence of arterial injury, we performed morphometric analysis of the thoracic aorta, left common carotid artery, and right femoral artery. All vessels studied were indistinguishable for both p107 –/– and control mice (Table 1). In correspondence with regular vessel development there were no microscopic alterations, no spontaneous neointima formation, and no spotted thickening of the vessel walls.
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After carotid artery ligation, the p107 –/– mice (n =
7) did not display an enhanced ligation response compared with control mice
(n = 9). This was evaluated over a distance of 450 µm
proximal and also 200 µm distal from the ligation site. As depicted in
Fig. 1A, there was no
evidence for ligation-induced increased total vessel wall area (tunica media +
neointima). The neointima area was almost identical for both groups, as shown
in Fig. 1B. In
addition, the lumen area was comparable for both groups, with P
values of 0.90 proximal and 0.48 distal from the ligation site. In
correspondence, PCNA-positive cells in the vessel wall of ligated carotid
arteries revealed no significant differences between the groups. The
proportion of PCNA-positive cells was almost negligible in the tunica media,
with <2.5% for both groups (NS; P = 0.27), and the neointima
featured values of 19.58 ± 4.91% for p107 –/– and 12.01
± 2.86% for control mice (NS; P = 0.19).
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To test these findings in a different model and a different vessel, we analyzed the influence of perivascular electric injury on the femoral artery. In correspondence with the data obtained from carotid artery ligation, both the p107 –/– mice (n = 8) and control mice (n = 6) revealed comparable values for the femoral total vessel wall area and the lumen area. In addition, the neointima area was also not statistically different for p107 –/– and control mice (Fig. 2).
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This study presents a p107 knockout mouse model demonstrating regular vessel geometry under basal conditions and no enhanced vascular response after arterial ligation or perivascular electric injury. The p107 is a member of the pRb family, a family of cell cycle inhibitors exerting their effects on the cell cycle at least by controlling the activity of E2F transcription factors (10, 11, 16). Proteins of the pRb family such as pRb itself and p130 were recently shown to play pivotal roles in the response to arterial injury in spontaneously hypertensive rats (7) and in mice (18). The present study was initiated to evaluate the role of p107 in vascular ligation and electric injury response. In light of the high degree of homology with p130, especially in the "pocket" domains that bind to other cell cycle regulators, we hypothesized that p107 would also function as a key regulator in our model. Comparable to our findings for p130 –/– mice, the p107 –/– mice did not show any vascular alterations under basal conditions. In contrast to p130 –/– mice, however, the lack of p107 did not result in enhanced vascular ligation response but rather showed neointima formation and vessel wall area comparable to those of control mice. As the studies on p130 and p107 knockout mice may not be directly comparable because of, for example, differences in the genetic background of the two mouse lines, we used another model, perivascular electric injury of the femoral artery, to further evaluate the general validity of our data for p107 –/– mice. Although this model was less susceptible to inducing neointima than the ligation model, it did at least support the essentially comparable finding that the loss of p107 was not associated with enhanced injury or ligation response. Nevertheless, it must be acknowledged that our findings may only be specific for the ligation model or the perivascular electric injury, with potentially limited relevance for other models and species. Furthermore, we cannot exclude that compensatory mechanisms may conceal an underlying early effect. However, we did not intend to study the time course of ligation or electric injury response but rather focused on an advanced stage of response that would be of primary interest from a clinical standpoint.
The findings have several implications. SMC cycle regulation after vascular injury appears to be no redundant effect of all proteins of the pRb family but rather a specific effect found at least for p130, as published recently (7, 18). The present results and previous studies give rise to the hypothesis that the regulation of injury response by pRb and p130 is not only controlled by the binding to other cell cycle regulators through the conserved pocket domains as generally supposed but also by other, not yet defined factors. This provides a new aspect of vascular injury response. Further studies on p130-specific pathways and investigations on functional domains and secondary protein structure of p130 with a focus on proliferation and differentiation are required to illuminate the pathophysiology of injury response. These approaches will pave the way for new and specific therapeutic paradigms.
ACKNOWLEDGMENTS
We acknowledge the support of Tyler Jacks for providing the p107 knockout mice, and we thank Tatjana Walker for excellent technical support.
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FOOTNOTES |
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FOOTNOTES
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.
REFERENCES
This article has been cited by other articles:
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  J. R. Sindermann, C. Kobbert, A. Skaletz-Rorowski, G. Breithardt, G. Plenz, and K. L. March Vascular injury response in mice is dependent on genetic background Am J Physiol Heart Circ Physiol, March 1, 2006; 290(3): H1307 - H1310. [Abstract] [Full Text] [PDF]
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