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. 2005 Nov;167(5):1349-59.
doi: 10.1016/S0002-9440(10)61222-9.

Temporal exposure of cryptic collagen epitopes within ischemic muscle during hindlimb reperfusion

Paul J Gagne  1 , Nikita Tihonov, Xialou Li, Joseph Glaser, Jhenrong Qiao, Michael Silberstein, Herman Yee, Elizabeth Gagne, Peter Brooks
Affiliations

Temporal exposure of cryptic collagen epitopes within ischemic muscle during hindlimb reperfusion

Paul J Gagne et al. Am J Pathol. 2005 Nov.

Abstract

Chronic limb-threatening ischemia is a devastating disease with limited surgical options. However, inducing controlled angiogenesis and enhancing reperfusion holds therapeutic promise. To gain a better understanding of the mechanisms that contribute to limb reperfusion, we examined the temporal biochemical and structural changes occurring within the extracellular matrix of ischemic skeletal muscle. Both the latent and active forms of MMP-2 and -9 significantly increased during the active phase of limb reperfusion. Moreover, small but significant alterations in tissue inhibitors of metalloproteinase levels also occurred during a similar time course, consistent with a net increase in extracellular matrix remodeling. This temporal increase in MMP activity coincided with enhanced exposure of the unique HU177 cryptic collagen epitope. Although the HUIV26 cryptic collagen epitope has been implicated in angiogenesis, little is known concerning such epitopes within ischemic muscle tissue. Here, we provide the first evidence that a functionally distinct cryptic collagen epitope (HU177) is temporally exposed in ischemic muscle tissue during the active phase of reperfusion. Interestingly, the exposure of the HU177 epitope was greatly diminished in MMP-9 null mice, corresponding with significantly reduced limb reperfusion. Therefore, the regulated exposure of a unique cryptic collagen epitope within ischemic muscle suggests an important role for collagen remodeling during the active phase of ischemic limb reperfusion.

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Figures

Figure 1
Figure 1
Laser Doppler imaging of ischemic hindlimb after surgical ligation of the femoral artery (ie, hindlimb ischemia). Wild-type mice 6 to 8 weeks of age underwent surgical ligation of the femoral artery. Blood flow was assessed by laser Doppler imaging before ligation (day 0) and at days 1, 3, 7, 14, 21, and 28 after surgery. Blood flow in the ischemic calf is expressed as a ratio of the blood flow in the contralateral nonischemic limb. Data represent an average value for all animals at each time point (n = 7). Blood flow decreases to 20% after arterial ligation and then recovers to 66%. Error bars represent the SEM.
Figure 2
Figure 2
Effect of ischemia over time on MMP-2 and MMP-9 levels both total and active for 30 days after onset of hindlimb ischemia. MMP-2 and -9 levels were assessed by quantitative bioactivity assays of muscle tissue taken from the ischemic and nonischemic limbs of wild-type mice on days 0 (pre-op), 1, 3, 7, 14, and 30. For both MMP-9 and MMP-2, total levels increased markedly within 3 days and then diminished by 30 days after surgery compared with nonischemic calf muscle. Active MMP-9 and MMP-2 remain markedly elevated in the ischemic calf muscle compared with the nonischemic muscle between 3 and 14 days after surgery when reperfusion by LDI was most marked. A: Total levels of MMP-2 including both the latent and the active forms in the ischemic and nonischemic muscle tissue (n = 8) at each time point. The error bars in the nonischemic data are narrow and obscured by the associated data point. B: Level of active MMP-2 present in the ischemic and nonischemic muscle tissue (n = 8) at each time point. The error bars for the nonischemic data do not exist because active MMP-2 was not detectable in the nonischemic calf muscle. C: Represents the total level of MMP-9, including both the latent form and the active forms, in the ischemic and nonischemic muscle tissue (n = 8) at each time point. D: Represents the level of active MMP-9 present in the ischemic and nonischemic muscle tissue (n = 8) at each time point. For A through D the asterisk denotes a significant difference (P < 0.05) between the ischemic and the contralateral nonischemic limb at the same time point. Error bars represent the SEM.
Figure 3
Figure 3
Effect of ischemia over time on TIMP-1 and TIMP-2 levels for 30 days after onset of hindlimb ischemia. ELISAs were used to measure total TIMP levels in the muscle tissue of ischemic and nonischemic limbs of wild-type mice on days 0 (pre-op), 1, 3, 7, 14, and 30 after ischemia. A: TIMP-1 levels measured by ELISAs. Each time point represents an average value (n = 8). The asterisk indicates a significant difference (P < 0.05) between the ischemic and the contralateral nonischemic muscle tissue at the same time point. TIMP-1 levels increased at day 7 at the same time that total MMP-9 levels decreased. B: TIMP-2 levels measured by ELISA. Each time point represents an average value (n = 8). There was no significant difference in levels between ischemic and nonischemic limbs at any time point.
Figure 4
Figure 4
Location and time course of HU177 cryptic collagen epitope exposure after onset of hindlimb ischemia. A: Frozen sections (×40) of ischemic muscle harvested at days 0 (pre-op), 1, 3, 7, 14, and 30 days were stained for the HU177 cryptic collagen epitope using mAb QH2B. Sections were counterstained with horseradish peroxidase for detection of antibody. B: Frozen sections (×40) of ischemic muscle harvested at day 7 were costained for the cryptic collagen epitope, HU177, and the endothelial cell marker, CD31. HU177 is denoted with the brown horseradish peroxidase staining, whereas CD31 is indicated by the red alkaline phosphatase staining. The solid arrows indicate CD31 staining of blood vessel endothelial cells in the ECM between ischemic skeletal muscle cells. The arrowheads indicate HU177 staining in the ECM, which colocalizes with and surrounds CD31+ endothelial cells. C: Quantification of HU177 staining was performed using digital images of stained sections. Sections were analyzed by blinded observers using Adobe Photoshop. The number of mAb QH2B-stained pixels in each image was determined and expressed as a ratio of the total number of pixels of muscle tissue present in each slide. Each time point is the average value obtained from analyzing three to five representative microscopic fields from muscle sections from animals at each time point (n = 3) for HU177 staining. HU177 exposure parallels increased MMP-9 and MMP-2 activity and limb reperfusion. The asterisk denotes a significant difference (P < 0.05) between nonischemic tissue at day 0 (preoperative) and ischemic tissue at days 7 and 30.
Figure 5
Figure 5
Comparison of blood flow between MMP-9 null and wild-type mice for 14 days after onset of hindlimb ischemia. 129SvEv mice (wild-type) and MMP-9 null mice underwent surgical ligation of the femoral artery. Blood flow in the calf/foot was assessed by laser Doppler imaging before ligation (day 0) and at days 1, 3, 5, and 14 after surgery. Blood flow in the ischemic limb was expressed as a ratio of the blood flow in the contralateral nonischemic limb. Blood flow in the distal limb of the MMP-9 null mice was significantly diminished, compared with the wild-type controls, during the first 2 weeks after induction of ischemia. Each time point represents the average value for all animals at each time point (n ≥ 9). The asterisk denotes a significant difference (P < 0.05) between ischemic limb reperfusion in wild-type versus MMP-9 null mice.
Figure 6
Figure 6
A comparison of HU177 staining of ischemic calf muscle tissue in MMP-9 null mice versus wild-type mice. A: Frozen sections (×40) of ischemic muscle tissue harvested 7 days after onset of ischemia were stained with mAb QH2B for the cryptic collagen epitope HU177. Sections were counterstained with horseradish peroxidase for detection of antibody. The ECM of the ischemic skeletal muscle of the MMP-9 null mice exhibit markedly less HU177 cryptic site exposure. B: Digital images were analyzed by a blinded observer using Adobe Photoshop. The number of pixels stained was determined and expressed as a ratio of the total number of pixels of muscle tissue present in each slide. Muscle sections from the ischemic calf of wild-type (WT) (n = 3) and MMP-9 null (n = 3) mice were examined, and four representative microscopic fields per animal were analyzed. Each data point is the average value obtained from analyzing these representative microscopic fields. Error bars represent the SEM.
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