Jeanwan Kang, MD, Hassan Albadawi, MD, Virendra I. Patel, MD, Thomas A

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Apolipoprotein E–/– mice have delayed skeletal muscle healing after hind limb ischemia–reperfusion Jeanwan Kang, MD, Hassan Albadawi, MD, Virendra I. Patel, MD, Thomas A. Abbruzzese, MD, Jin-Hyung Yoo, MS, W. Gerald Austen, MD, Michael T. Watkins, MD Journal of Vascular Surgery Volume 48, Issue 3, Pages 701-708 (September 2008) DOI: 10.1016/j.jvs.2008.04.006 Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

Fig 1 Skeletal muscle morphology. (a and b) Masson's trichrome and (c and d) hematoxylin and eosin stains at original magnification ×200 magnification (Bar, 100 μm). a, Gastrocnemius muscle from wild-type (Wt) mice after 1 day of reperfusion (DR). b, Gastrocnemius muscle from apolipoprotein E deficient (ApoE–/–) mice after 1DR. There is no difference in the level of initial injury between Wt and ApoE–/– mice. Both have rounded fibers that have lost their polygonal shape (circle), interstitial edema, and infiltrating leukocytes (short arrows). c, Gastrocnemius muscle from Wt mice after 14DR. d, Gastrocnemius muscle from ApoE–/– mice after 14DR. At 14DR, there is a significant difference in skeletal muscle morphology between Wt and ApoE–/– mice. Most Wt muscle fibers are mature with their characteristic polygonal shape, larger cross-sectional size, and peripherally placed nuclei (long arrow). The ApoE–/– mice have many immature fibers that are smaller and have centrally placed nuclei (oval). Journal of Vascular Surgery 2008 48, 701-708DOI: (10.1016/j.jvs.2008.04.006) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

Fig 2 Skeletal muscle fiber counts. a, There was no difference in percent of injured fibers between wild-type (Wt; C57BL/6) and apolipoprotein E deficient (ApoE–/–) mice at 1 day of reperfusion (DR). b, There was a higher number of immature skeletal muscle fibers in ApoE–/– mice at 14DR (Wt 48% ± 6% injured fibers vs ApoE–/– 42% ± 5% injured fibers; *P = .444). Error bars represent the standard error of the mean. Journal of Vascular Surgery 2008 48, 701-708DOI: (10.1016/j.jvs.2008.04.006) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

Fig 3 Quantitative analysis of Western blots: MyoD and myogenin from reperfused skeletal muscle at 7 and 14 days of reperfusion (DR) in wild-type (Wt; C57BL/6) and apolipoprotein E deficient (ApoE–/–) mice expressed in arbitrary units (AU). a, There was no difference in MyoD levels at 7DR or 14DR. b, Myogenin expression was significantly lower in the ApoE–/– mice at 7DR (Wt, 13.9 ± 1.7 AU vs ApoE–/– 7.7 ± 1.2 AU; *P = .014). There was no difference in myogenin expression at 14DR. Error bars represent the standard error of the mean. Journal of Vascular Surgery 2008 48, 701-708DOI: (10.1016/j.jvs.2008.04.006) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

Fig 4 Skeletal muscle myeloperoxidase (MPO), macrophage inflammatory protein-2 (MIP-2), monocyte chemotactic protein-1 (MCP-1), and osteopontin (OPN) levels in sham wild-type (Wt; C57BL/6) and apolipoprotein E deficient (ApoE–/–) mice and in mice after unilateral hind limb ischemia, followed by 1, 7, or 14 days of reperfusion (DR). a, Tissue MPO levels were significantly lower in the ApoE–/– mice at 7DR (Wt 8.1 ± 0.7 ng/mg vs ApoE–/– 5.3 ± 0.9 ng/mg; *P = .043). There was no difference at 1 or 14DR. b, There was no difference in tissue MIP-2 levels between Wt and ApoE–/– mice at 1, 7, or 14DR. There was a significant difference in tissue MIP-2 levels between Wt and ApoE–/– sham mice (Wt 0.11 ± 0.03 pg/mg vs ApoE–/– 0.20 ± 0.02 pg/mg; **P = .040). c, There was a significant difference in tissue MCP-1 levels at 14DR (Wt 15.7 ± 1.7 pg/mg vs ApoE–/– 38.5 ± 3.9 pg/mg; ± P < .0001). There was no difference at 1 or 7DR. d, There was no difference in tissue OPN levels at any time points. Error bars represent the standard error of the mean. Journal of Vascular Surgery 2008 48, 701-708DOI: (10.1016/j.jvs.2008.04.006) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions