We demonstrate, using a novel model of severe limb ischemia that aged mice maintain the ability to undergo angiogenesis, perhaps to a greater degree than young adult mice; however, reduced blood flow and impaired function in aged mice correlates with reduced arteriogenesis, i.e. large vessel collateral formation. These results demonstrate the importance of arteriogenesis in the development of, and potential therapies for, limb ischemia.
Our model of severe acute limb ischemia uses ligation of the common iliac artery and vein to induce ischemia. Since the internal iliac artery, which supplies the pelvis, is the only major branch between the femoral and common iliac arteries, it is not surprising that young adult mice regain blood flow to a similar degree and with similar kinetics as the more severe femoral limb ischemia models ().15, 20, 33
However, ligation of the iliac artery is technically simpler to perform than excision of the femoral, popliteal, and saphenous arteries and reduces the confounding variable of a large wound in the ischemic limb. The utility of this model is clear as it shows that aged mice have reduced blood flow and function in the ischemic leg compared to young adult mice (), similar to previous results reported in aged rats.21
We used mice with the C57Bl/6 background for comparison to other reports and knockout mice, as well as availability of aged C57Bl/6 mice, rather than the more ischemia-susceptible DBA/1J strain.34
Additional studies that directly compare this model to other severe ones will help clarify the degrees of ischemia that can be achieved; however, our use of iliac vein ligation to increase the severity of the ischemia may prevent direct comparison with other models of ischemia that do not perform concomitant venous ligation. In addition, the effect of venous ligation may be different in young adult and aged mice. Our limiting the examination period to two weeks postoperatively, rather than 3–4 weeks, reflects the poor postoperative survival of aged mice. As such we limited our studies to the minimal possible to detect differences between young adult and aged mice; it is possible that differences in timing, such as in EPC recruitment or incorporation into tissue, may account for our findings, and that these differences might be minimized over time.
Nevertheless, similar preservation of distal tissue in the ischemic limbs of young adult and aged mice was surprising and contrary to the study hypothesis (); we interpret limb preservation in aged mice to be accounted for, at least partially, by preservation of angiogenesis in aged mice ( and ). Our finding of increased angiogenesis in aged mice is also surprising and contrary to the hypothesis of this study, as well as a previous report of decreased angiogenesis after femoral/saphenous artery resection in aged rabbits and mice.18
On the other hand, our finding of increased angiogenesis in aged mice is consistent with higher serum VEGF levels in aged animals with limb ischemia compared to younger animals.21
It is possible that this discrepancy suggests that our iliac artery ligation model is not as severely ischemic as the arterial excision model. However, we believe that other studies performing surgery in the ischemic leg are confounded by the unilateral healing surgical wound in the ischemic leg, possibly diverting EPC away from the distal foot into the more proximal wound.17, 26, 30
A recent study has demonstrated that EPC homing to the ischemic leg is dependent on phosphorylation of Akt, induced by VEGF and stromal derived growth factor (SDF)-1, both of which can be secreted by a healing wound.30, 35
The source of these EPC is controversial but it is possible that they are derived from the bone marrow, as suggested by several other studies.13, 17, 26, 30
Our finding that aged mice have diminished ability to form large vessel collaterals was demonstrated by CT angiography as well as direct angiography (). Although direct angiography confirmed the results of CT angiography, we note that direct angiography was less sensitive than CT angiography and was only able to detect more proximal collaterals, near the area of iliac ligation. In addition, we noted greater numbers of doubly-actin-positive/lectin-positive arterioles in the distal limb muscles of the ischemic limbs of aged animals compared to those in younger animals (data not shown), although the few number of doubly-positive cells in the field is subject to sampling error precluding reliable statistical analysis. Diminished capability for arteriogenesis is consistent with the functional outcome of reduced blood flow and function in ischemic legs of aged mice () and is also consistent with the increased angiogenesis in aged mice compared to younger mice as a potential compensatory mechanism (). Arteriogenesis, dilation of preexisting arterioles to increase functional blood flow, is distinct from microvascular angiogenesis as well as embryonic vasculogenesis.27, 36–38
Limb ischemia in both human patients and animal models stimulates VEGF and other angiogenic cytokines, such as stem cell factor, SDF-1, angiopoietin-1 and -2, and PLGF; however these factors, after stimulating c-kit, CXCR4, tie-2, and VEGFR-1 and -2 receptors, stimulate angiogenesis that is insufficient to overcome atherosclerotic, or animals models of, limb ischemia.36
The clinical observation of robust collateral formation allowing asymptomatic limb perfusion and function in many patients confirms the importance of arteriogenesis as a necessary component of revascularization.27
It is not surprising that trials of therapeutic “angiogenesis” are now focusing on factors that stimulate multiple pathways, including factors that stimulate arteriogenesis, such as monocyte chemoattractant protein, colony-stimulating factors, and cell-based approaches.37, 39
These approaches capitalize on the observations that circulating cells, perhaps monocytes or stem cells, may release multiple factors that stimulate multiple processes including angiogenesis and arteriogenesis.38
In summary, we developed a novel model of severe limb ischemia that does not create a large unilateral surgical wound in the ischemic leg. This model demonstrates that aged mice have deficient arteriogenesis that, in spite of robust angiogenesis, results in diminished limb blood flow and function. These results suggest that strategies to simulate arteriogenesis may complement those that increase angiogenesis, and may result in improved relief of ischemia.