Apolipoprotein E-/- mice have delayed skeletal muscle healing after hind limb ischemia-reperfusion.
BACKGROUND
Classic studies of limb ischemia-reperfusion injury have been performed using young healthy mice. However, patients with peripheral vascular disease are older and often exhibit metabolic derangements that may delay healing after revascularization. Mice with genetic deletion of apolipoprotein E (ApoE(-/-)) have been used as a model in various experimental scenarios of hypercholesterolemia. These experiments evaluated the inflammatory response and changes in skeletal muscle morphology during the acute and chronic phases of limb ischemia-reperfusion injury in aged ApoE(-/-) mice.


METHODS
Age-matched ApoE(-/-) and wild-type (Wt) mice underwent 1.5 hours of unilateral hind limb ischemia, followed by 1, 7, or 14 days of reperfusion (DR). Histologic analysis of skeletal muscle fiber injury was assessed at 1DR. Morphologic evidence of muscular fiber maturation was assessed at 14DR. Levels of MyoD and myogenin, markers of skeletal muscle differentiation, were assessed at 7 and 14DR using Western blots. Markers of inflammation, including myeloperoxidase, macrophage inflammatory protein-2 (MIP-2), monocyte chemotactic protein-1 (MCP-1), and osteopontin, were assayed using enzyme-linked immunosorbent assay and chemokine (C-C motif) receptor 2 (CCR2) using Western blots at 1, 7, and 14DR. After 1DR, tissue adenosine 5'-triphosphate (ATP) levels were measured to assess metabolic activity. Unpaired t test and Mann-Whitney test were used for comparisons.


RESULTS
Histologic evaluation of skeletal muscle after 1DR showed no difference in the degree of injury between Wt and ApoE(-/-) mice. However, at 14DR, ApoE(-/-) mice had higher percentage of immature muscle fibers than Wt mice. Myogenin level was lower in the ApoE(-/-) mice at 7DR. Injured skeletal muscle of ApoE(-/-) mice had lower levels of myeloperoxidase than Wt mice at 7 DR and higher levels of MCP-1 at 14DR. There was no difference in the levels of tissue ATP, MIP-2, osteopontin, or CCR2 at all experimental intervals.


CONCLUSIONS
Although there was no difference between the injured muscle of Wt and ApoE(-/-) mice during the acute phase of reperfusion, ApoE(-/-) mice showed delay in skeletal muscle healing during the chronic phase of reperfusion. This lag in muscle regeneration was associated with lower levels of myogenin at 7DR and an increased level of MCP-1 at 14DR in the ApoE(-/-) mice. The delay in skeletal muscle healing in the ApoE(-/-) mice may have broader implications for poor tissue healing and functional recovery in elderly patients who have vascular risk factors such as hypercholesterolemia.
Department of Surgery, Division of Vascular and Endovascular Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA.
Jeanwan
J
Kang
Hassan
H
Albadawi
Virendra I
VI
Patel
Thomas A
TA
Abbruzzese
Jin-Hyung
JH
Yoo
W Gerald
WG
Austen
Michael T
MT
Watkins
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001057:Apolipoproteins E / Q000172:deficiency* / Q000235:genetics; D015153:Blotting, Western; D018932:Chemokine CCL2 / Q000378:metabolism; D054426:Chemokine CXCL2 / Q000378:metabolism; D002784:Cholesterol / Q000097:blood; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006614:Hindlimb; D006937:Hypercholesterolemia / Q000150:complications / Q000378:metabolism / Q000503:physiopathology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018482:Muscle, Skeletal / Q000098:blood supply / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D017570:MyoD Protein / Q000378:metabolism; D018008:Myogenin / Q000378:metabolism; D053495:Osteopontin / Q000378:metabolism; D009195:Peroxidase / Q000378:metabolism; D054390:Receptors, CCR2 / Q000378:metabolism; D015427:Reperfusion Injury / Q000150:complications / Q000378:metabolism / Q000503:physiopathology*; D013997:Time Factors; D014945:Wound Healing
D001057:Apolipoproteins E; C491768:Ccl2 protein, mouse; C515335:Ccr2 protein, mouse; D018932:Chemokine CCL2; D054426:Chemokine CXCL2; C404915:Cxcl2 protein, mouse; D017570:MyoD Protein; C057363:MyoD1 myogenic differentiation protein; C503801:Myog protein, mouse; D018008:Myogenin; D054390:Receptors, CCR2; C506896:Spp1 protein, mouse; D053495:Osteopontin; D000255:Adenosine Triphosphate; D002784:Cholesterol; D009195:Peroxidase