Myoglobin Overexpression Inhibits Reperfusion in the Ischemic Mouse Hindlimb through Impaired Angiogenesis but Not Arteriogenesis

Adaptive vascular remodeling in response to arterial occlusion takes the form of capillary growth (angiogenesis) and outward remodeling of pre-existing collateral arteries (arteriogenesis). However, the relative contributions of angiogenesis and arteriogenesis toward the overall reperfusion response are both highly debated and poorly understood. Here, we tested the hypothesis that myoglobin overexpressing transgenic mice (MbTg+) exhibit impaired angiogenesis in the setting of normal arteriogenesis in response to femoral artery ligation, and thereby serve as a model for disconnecting these two vascular growth processes. After femoral artery ligation, MbTg+ mice were characterized by delayed distal limb reperfusion (by laser Doppler perfusion imaging), decreased foot use, and impaired distal limb muscle angiogenesis in both glycolytic and oxidative muscle fiber regions at day 7. Substantial arteriogenesis occurred in the primary collaterals supplying the ischemic limb in both wild-type and MbTg+ mice; however, there were no significant differences between groups, indicating that myoglobin overexpression does not affect arteriogenesis. Together, these results uniquely demonstrate that functional collateral arteriogenesis alone is not necessarily sufficient for adequate reperfusion after arterial occlusion. Angiogenesis is a key component of an effective reperfusion response, and clinical strategies that target both angiogenesis and arteriogenesis could yield the most efficacious treatments for peripheral arterial disease. Adaptive vascular remodeling in response to arterial occlusion takes the form of capillary growth (angiogenesis) and outward remodeling of pre-existing collateral arteries (arteriogenesis). However, the relative contributions of angiogenesis and arteriogenesis toward the overall reperfusion response are both highly debated and poorly understood. Here, we tested the hypothesis that myoglobin overexpressing transgenic mice (MbTg+) exhibit impaired angiogenesis in the setting of normal arteriogenesis in response to femoral artery ligation, and thereby serve as a model for disconnecting these two vascular growth processes. After femoral artery ligation, MbTg+ mice were characterized by delayed distal limb reperfusion (by laser Doppler perfusion imaging), decreased foot use, and impaired distal limb muscle angiogenesis in both glycolytic and oxidative muscle fiber regions at day 7. Substantial arteriogenesis occurred in the primary collaterals supplying the ischemic limb in both wild-type and MbTg+ mice; however, there were no significant differences between groups, indicating that myoglobin overexpression does not affect arteriogenesis. Together, these results uniquely demonstrate that functional collateral arteriogenesis alone is not necessarily sufficient for adequate reperfusion after arterial occlusion. Angiogenesis is a key component of an effective reperfusion response, and clinical strategies that target both angiogenesis and arteriogenesis could yield the most efficacious treatments for peripheral arterial disease. Peripheral arterial disease (PAD) is caused by atherosclerosis and is characterized by the progressive and often complete occlusion of large- and medium-size arteries at sites other than the heart. PAD most often occurs in the lower limbs, with progressive PAD leading to the debilitating consequences of intermittent claudication and critical limb ischemia. Given the high prevalence (>20% of those >65 years of age1Norgren L. Hiatt W.R. Dormandy J.A. Nehler M.R. Harris K.A. Fowkes F.G. Inter-Society Consensus for the Management of Peripheral Arterial Disease (TASC II).J Vasc Surg. 2007; 45: S5-S67Abstract Full Text Full Text PDF PubMed Scopus (4617) Google Scholar) and economic impact ($4.4 billion estimated treatment costs2Hirsch A.T. Hartman L. Town R.J. Virnig B.A. National health care costs of peripheral arterial disease in the Medicare population.Vasc Med. 2008; 13: 209-215Crossref PubMed Scopus (284) Google Scholar) of PAD, along with few therapeutic options, there is a critical need for developing new therapeutic modalities. One promising approach entails stimulating adaptive vascular remodeling to enhance perfusion around occlusions. To date, however, trials using this approach have largely failed. An improper understanding of the balance of angiogenesis versus arteriogenesis has been cited as a reason for many of these failures.3Van Royen N. Piek J.J. Schaper W. Fulton W.F. A critical review of clinical arteriogenesis research.J Am Coll Cardiol. 2009; 55: 17-25Crossref PubMed Scopus (92) Google Scholar, 4Chilian W.M. Penn M.S. Pung Y.F. Dong F. Mayorga M. Ohanyan V. Logan S. Yin L. Coronary collateral growth–back to the future.J Mol Cell Cardiol. 2012; 52: 905-911Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 5Schirmer S.H. van Nooijen F.C. Piek J.J. van Royen N. Stimulation of collateral artery growth: travelling further down the road to clinical application.Heart. 2009; 95: 191-197Crossref PubMed Scopus (92) Google Scholar Adaptive vascular remodeling to arterial occlusion(s) can be broken down into two aspects. First, in ischemic tissues downstream of an arterial occlusion, capillaries grow from existing vessels via angiogenesis, expanding blood flow distribution throughout the ischemic tissue. In contrast, collateral arteries around the occlusion are stimulated to undergo structural lumenal expansion (ie, arteriogenesis) that allows for greater in-flow into the distal, ischemic tissue. Therapeutic clinical trials have largely focused on only one process (either angiogenesis or arteriogenesis).3Van Royen N. Piek J.J. Schaper W. Fulton W.F. A critical review of clinical arteriogenesis research.J Am Coll Cardiol. 2009; 55: 17-25Crossref PubMed Scopus (92) Google Scholar, 4Chilian W.M. Penn M.S. Pung Y.F. Dong F. Mayorga M. Ohanyan V. Logan S. Yin L. Coronary collateral growth–back to the future.J Mol Cell Cardiol. 2012; 52: 905-911Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 5Schirmer S.H. van Nooijen F.C. Piek J.J. van Royen N. Stimulation of collateral artery growth: travelling further down the road to clinical application.Heart. 2009; 95: 191-197Crossref PubMed Scopus (92) Google Scholar The most direct examples of an unbalanced approach come from the early and prominent failures of many large clinical trials using predominantly angiogenic factors (eg, vascular endothelial growth factor and hypoxia inducible factor 1-α) to induce angiogenesis.6Creager M.A. Olin J.W. Belch J.J.F. Moneta G.L. Henry T.D. Rajagopalan S. Annex B.H. Hiatt W.R. Effect of hypoxia-inducible factor-1alpha gene therapy on walking performance in patients with intermittent claudication.Circulation. 2011; 124: 1765-1773Crossref PubMed Scopus (110) Google Scholar, 7Kastrup J. Jørgensen E. Rück A. Tägil K. Glogar D. Ruzyllo W. Bøtker H.E. Dudek D. Drvota V. Hesse B. Thuesen L. Blomberg P. Gyöngyösi M. Sylvén C. 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Jørgensen E. Wang Y. Thune J.J. Nilsson J.C. Søndergaard L. Johnsen H.E. Køber L. Grande P. Kastrup J. Stem cell mobilization induced by subcutaneous granulocyte-colony stimulating factor to improve cardiac regeneration after acute ST-elevation myocardial infarction: result of the double-blind, randomized, placebo-controlled stem cells in myocardial infarction.Circulation. 2006; 113: 1983-1992Crossref PubMed Scopus (327) Google Scholar, 10Subramaniyam V. Waller E.K. Murrow J.R. Manatunga A. Lonial S. Kasirajan K. Sutcliffe D. Harris W. Taylor W.R. Alexander R.W. Quyyumi A.A. Bone marrow mobilization with granulocyte macrophage colony-stimulating factor improves endothelial dysfunction and exercise capacity in patients with peripheral arterial disease.Am Heart J. 2009; 158: 53-60Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 11Simons M. Annex B.H. Laham R.J. Kleiman N. Henry T. Dauerman H. Udelson J.E. Gervino E.V. Pike M. Whitehouse M.J. Moon T. Chronos N.A. Pharmacological treatment of coronary artery disease with recombinant fibroblast growth factor-2: double-blind, randomized, controlled clinical trial.Circulation. 2002; 105: 788-793Crossref PubMed Scopus (596) Google Scholar, 12Kusumanto Y.H. van Weel V. Mulder N.H. Smit A.J. van den Dungen J.J.A.M. Hooymans J.M.M. Sluiter W.J. Tio R.A. Quax P.H.A. Gans R.O.B. Dullaart R.P.F. Hospers G.A.P. Treatment with intramuscular vascular endothelial growth factor gene compared with placebo for patients with diabetes mellitus and critical limb ischemia: a double-blind randomized trial.Hum Gene Ther. 2006; 17: 683-691Crossref PubMed Scopus (267) Google Scholar A more fruitful strategy was recently hinted at in a study by West et al,13West A.M. Anderson J.D. Epstein F.H. Meyer C.H. Hagspiel K.D. Berr S.S. Harthun N.L. Weltman A.L. Annex B.H. Kramer C.M. Percutaneous intervention in peripheral artery disease improves calf muscle phosphocreatine recovery kinetics: a pilot study.Vasc Med. 2012; 17: 3-9Crossref PubMed Scopus (21) Google Scholar which suggested that even in the presence of increased perfusion pressure to the distal tissue after a percutaneous intervention, revascularization is unable to restore microvascular perfusion in PAD patients. This suggests that microvascular perfusion impairments must be addressed for full functional recovery. Moreover, strategies that do not change large vessel occlusion but alter angiogenesis can be clinically beneficial. In summary, these findings suggest the need to better understand how angiogenesis and arteriogenesis work together to improve reperfusion after arterial occlusion. Data demonstrating how angiogenesis and arteriogenesis separately contribute to reperfusion after arterial occlusion could outline how targeting both aspects of neovascularization could improve therapy. However, being able to how angiogenesis and arteriogenesis contribute to reperfusion after ischemic both a that does not impact arteriogenesis and angiogenesis, and for separately angiogenesis and arteriogenesis. overexpression in muscle serve as a that has an on tissue reperfusion but has on arteriogenesis and muscle myoglobin overexpression has been to angiogenesis and reperfusion in a severe S. M. Y. A.J. Annex B.H. overexpression of myoglobin angiogenesis after Vasc 2008; PubMed Scopus (21) Google Scholar The impaired angiogenesis was to from the myoglobin as a for S. M. Y. A.J. Annex B.H. overexpression of myoglobin angiogenesis after Vasc 2008; PubMed Scopus (21) Google Scholar, A. J. a of A. PubMed Scopus Google Scholar which in the of key and angiogenic factors M. L. E. S. P. F. angiogenesis in and endothelial cell growth and in by PubMed Scopus Google Scholar, T. T. M. C. H. C. M. D. 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R.S. in PubMed Scopus Google Scholar of the myoglobin was in the oxidative of the calf muscle with in the glycolytic regions However, by day after the was in both glycolytic and oxidative muscle and The of study is that the in capillary growth the distal ischemic after is sufficient to limb and even in the setting of normal arteriogenesis. this on key of from the response to in MbTg+ First, we the in reperfusion capacity of MbTg+ that was S. M. Y. A.J. Annex B.H. overexpression of myoglobin angiogenesis after Vasc 2008; PubMed Scopus (21) Google Scholar In the model chosen the more distal femoral arterial for full perfusion recovery to by after MbTg+ mice decreased reperfusion this ischemic the ischemic decreased the of functional tissue and of reperfusion also for of potential differences in the arteriogenic capacity the two arteriogenic capacity was that the in perfusion capacity in MbTg+ mice could not be to impaired arteriogenesis. MbTg+ mice a in their angiogenic as by capillary to muscle fiber at day which to the of significant perfusion are with MbTg+ mice decreased angiogenesis in the distal muscle at to S. M. Y. A.J. Annex B.H. overexpression of myoglobin angiogenesis after Vasc 2008; PubMed Scopus (21) Google Scholar The of angiogenic in MbTg+ mice was more than S. M. Y. A.J. Annex B.H. overexpression of myoglobin angiogenesis after Vasc 2008; PubMed Scopus (21) Google however, this was not as we a severe ischemic capillary differences were not after the impact of impaired angiogenesis is to the decreased perfusion recovery in MbTg+ mice the after angiogenesis is more J.C. J.D. Hartman J.J. in the between capillary and PubMed Scopus Google Scholar Together, these that microvascular in capillary growth the distal ischemic is sufficient to limb and reperfusion in arteriogenesis. how arteriogenesis and angiogenesis contribute to the vascular reperfusion response is critical for the approach for therapeutic have of the relative of the collateral versus remodeling of the distal to the decreased of the vascular downstream of an arterial occlusion. However, those that have that arteriogenesis and the expansion of the pre-existing collateral are the to the by the occlusion of a artery et M.A. M.R. M. A. M. and of vascular to peripheral artery 17: PubMed Scopus Google A is in the the collateral growth at after with the large in perfusion the However, there is a perfusion the MbTg+ and perfusion to no further of arteriogenesis. this does not with the that arteriogenesis is the most of to the distal D. T. A. S. C. T. 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W. of in endothelial cells is for the Cell 2009; PubMed Scopus Google Scholar However, a of the no on collateral remodeling that could the in perfusion or increased tissue M.R. J. M.A. perfusion in and mice does not result from impaired collateral Heart 2009; PubMed Scopus Google Scholar However, the in angiogenesis in mice sufficient for the in reperfusion P. W. of in endothelial cells is for the Cell 2009; PubMed Scopus Google Scholar A can be in the One is the of and The in arteriogenesis with a of van Royen N. J.E. E. J. M. S. M. C.K. Piek J.J. via PubMed Scopus Google Scholar or arteriogenic with J.C. J. A.L. R.J. therapeutic arteriogenesis via the of 2008; PubMed Scopus Google Scholar a or at of on angiogenesis alone in response to Y. J.E. J. L. 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Management of patients with peripheral artery disease of and A of the of Heart on PubMed Scopus Google Scholar is and with angiogenic expansion the ischemic R.J. J.D. Hiatt W.R. Annex B.H. Angiogenesis in muscle in in peripheral artery disease Vasc 2011; PubMed Scopus Google Scholar, J.D. Hiatt W.R. Annex B.H. between muscle capillary and blood flow with capacity in peripheral artery 2011; PubMed Scopus Google Scholar these findings suggest even microvascular remodeling does not contribute to the flow into the ischemic tissue et M.A. M.R. M. A. M. and of vascular to peripheral artery 17: PubMed Scopus Google microvascular and capillary remodeling can a significant in the functional reperfusion the distal tissue of PAD patients. demonstrating that a in angiogenesis alone can contribute to the reperfusion process the most model to study the response to arterial occlusion, we to the between at perfusion the of arteriogenesis or these suggest that to functional recovery and a reperfusion response, both a functional arteriogenesis and angiogenesis clinical strategies that target both of vascular remodeling yield the therapeutic for with Doppler perfusion of mice from and myoglobin overexpressing transgenic mice (MbTg+) for of foot of perfusion are by with the on the of the with of vascular the oxidative and glycolytic regions of and after and after and were to muscle fiber and presence of (ie, relative to capillaries and