Temporal Exposure of Cryptic Collagen Epitopes within Ischemic Muscle during Hindlimb Reperfusion
2005; Elsevier BV; Volume: 167; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)61222-9
ISSN1525-2191
AutoresPaul J. Gagne, Nikita Tihonov, Xialou Li, Joseph Glaser, Jhenrong Qiao, Michael J. Silberstein, Herman Yee, Elizabeth Gagne, Peter C. Brooks,
Tópico(s)Peripheral Artery Disease Management
ResumoChronic 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. 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. Severe peripheral arterial occlusive disease is a devastating condition that results in gangrene, chronic ulceration, rest pain, and amputation.1Lewis CD Peripheral arterial disease of the lower extremity.J Cardiovasc Nurs. 2001; : 45-63Crossref PubMed Scopus (11) Google Scholar Current therapeutic options are limited for successful treatment of peripheral arterial occlusive disease-induced limb-threatening arterial insufficiency. Therapeutic angiogenesis remains a promising alternative, although current approaches using growth factors have failed to conclusively yield long-term, clinically significant improvements in blood flow. Therefore, a better understanding of the cellular and molecular mechanisms regulating revascularization and reperfusion after ischemic injury is of great importance. Angiogenesis is the proliferation of new capillaries via sprouting from existing vessels or through bridging and intussusception of existing capillaries.2Carmeliet P Mechanisms of angiogenesis and arteriogenesis.Nat Med. 2000; 6: 389-395Crossref PubMed Scopus (3560) Google Scholar, 3Risau W Mechanisms of angiogenesis.Nature. 1997; 386: 671-674Crossref PubMed Scopus (4930) Google Scholar This complex physiological process likely contributes, in part, to ischemic limb reperfusion.4Hershey JC Baskin EP Glass JD Hartman HA Gilberto DB Rogers IT Cook JJ Revascularization in the rabbit hindlimb: dissociation between capillary sprouting and arteriogenesis.Cardiovasc Res. 2001; 49: 618-625Crossref PubMed Scopus (152) Google Scholar, 5Heilmann C Beyersdorf F Lutter G Collateral growth: cells arrive at the construction site.Cardiovasc Surg. 2002; 10: 570-578Crossref PubMed Scopus (28) Google Scholar, 6Wahlberg E Angiogenesis and arteriogenesis in limb ischemia.J Vasc Surg. 2003; 38: 198-203Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar A second, distinct process that also contributes to enhanced blood flow following ischemia is arteriogenesis. Arteriogenesis is the process whereby latent but existing bypasses, known as collateral vessels, are activated in response to limb ischemia caused by occlusion of a main axial artery.7Helisch A Schaper W Arteriogenesis: the development and growth of collateral arteries.Microcirculation. 2003; 10: 83-97Crossref PubMed Google Scholar, 8Schaper W Buschmann I Arteriogenesis, the good and bad of it.Cardiovasc Res. 1999; 43: 835-837Crossref PubMed Scopus (83) Google Scholar Although a number of stimulators of both angiogenesis and arteriogenesis have been evaluated clinically for the treatment of ischemic diseases, failed human trials have emphasized the shortcomings in our understanding of spontaneous revascularization.9Isner JM Baumgartner I Rauh G Schainfeld R Blair R Manor O Razvi S Symes JF Treatment of thromboangiitis obliterans (Buerger's disease) by intramuscular gene transfer of vascular endothelial growth factor: preliminary clinical results.J Vasc Surg. 1998; : 964-973Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar, 10Baumgartner I Pieczek A Manor O Blair R Kearney M Walsh K Isner JM Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia.Circulation. 1998; : 1114-1123Crossref PubMed Scopus (1084) Google Scholar Thus, a more fundamental understanding of the molecular mechanisms controlling reperfusion is of paramount importance for overcoming this clinical problem. Previous studies have documented the critical importance of growth factors, growth factor receptors, and various proteolytic enzymes in creating a permissive microenvironment for blood vessel growth.2Carmeliet P Mechanisms of angiogenesis and arteriogenesis.Nat Med. 2000; 6: 389-395Crossref PubMed Scopus (3560) Google Scholar, 11Silvestre JS Mallat Z Tamarat R Duriez M Tedgui A Levy BI Regulation of matrix metalloproteinase activity in ischemic tissue by interleukin-10: role in ischemia-induced angiogenesis in mice hindlimb.Circ Res. 2000; 89: 259-264Crossref Scopus (94) Google Scholar, 12Holash J Maisonpierre PC Compton D Boland P Alexander CR Zagzag D Yancopoulos GD Wiegand SJ Vessel cooption, regression and growth in tumors mediated by angiopoietins and VEGF.Science. 1999; 284: 1994-1998Crossref PubMed Scopus (1940) Google Scholar, 13Ingber DE Mechanical signaling and the cellular response to extracellular matrix in angiogenesis and cardiovascular physiology.Circ Res. 2002; 91: 877-887Crossref PubMed Scopus (541) Google Scholar Interestingly, studies have provided evidence that the extracellular matrix (ECM) plays an important role in regulating angiogenesis.13Ingber DE Mechanical signaling and the cellular response to extracellular matrix in angiogenesis and cardiovascular physiology.Circ Res. 2002; 91: 877-887Crossref PubMed Scopus (541) Google Scholar, 14Xu J Rodriguez D Petitclerc E Kim JJ Hangai M Yuen SM Davis GE Brooks PC Proteolytic exposure of a cryptic site within collagen type IV is required for angiogenesis and tumor growth in vivo.J Cell Biol. 2001; 154: 1069-1079Crossref PubMed Scopus (407) Google Scholar, 15Franco CD Hou G Bendeck MP Collagens, integrins, and the discoidin domain receptors in arterial occlusive disease.Trends Cardiovasc Med. 2002; 12: 143-148Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 16Schwartz MA Integrin signaling revisited.Trends Cell Biol. 2001; 11: 466-470Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar In particular, proteolytic remodeling of genetically distinct forms of collagen can expose cryptic regulatory elements that are normally inaccessible to cells.14Xu J Rodriguez D Petitclerc E Kim JJ Hangai M Yuen SM Davis GE Brooks PC Proteolytic exposure of a cryptic site within collagen type IV is required for angiogenesis and tumor growth in vivo.J Cell Biol. 2001; 154: 1069-1079Crossref PubMed Scopus (407) Google Scholar, 17Davis GE Affinity of integrins for damaged extracellular matrix: avb3 binds to denatured collagen type 1 through RGD sites.Biochem Biophys Res Commun. 1992; 182: 1025-1031Crossref PubMed Scopus (303) Google Scholar, 18Hangai M Kitaya N Xu J Chan CK Kim JJ Werb Z Ryan SJ Brooks PC Matrix metalloproteinase-9-dependent exposure of a cryptic migratory control site in collagen is required before retinal angiogenesis.Am J Pathol. 2002; 161: 1429-1437Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 19Xu J Rodriguez D Kim JJ Brooks PC Generation of monoclonal antibodies to cryptic collagen sites by using subtractive immunization.Hybridoma. 2000; : 375-385Crossref PubMed Scopus (49) Google Scholar Cellular interaction with these cryptic elements may initiate unique signaling cascades required for new blood vessel growth.14Xu J Rodriguez D Petitclerc E Kim JJ Hangai M Yuen SM Davis GE Brooks PC Proteolytic exposure of a cryptic site within collagen type IV is required for angiogenesis and tumor growth in vivo.J Cell Biol. 2001; 154: 1069-1079Crossref PubMed Scopus (407) Google Scholar, 18Hangai M Kitaya N Xu J Chan CK Kim JJ Werb Z Ryan SJ Brooks PC Matrix metalloproteinase-9-dependent exposure of a cryptic migratory control site in collagen is required before retinal angiogenesis.Am J Pathol. 2002; 161: 1429-1437Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar In fact, a monoclonal antibody (mAb) directed to a cryptic regulatory site within collagen type IV (HUIV26) potently inhibits new blood vessel growth in multiple in vivo models. Interestingly, recent studies have defined a second cryptic site (HUI77) shown to be present within a variety of distinct forms of collagen, including collagen types I to V.19Xu J Rodriguez D Kim JJ Brooks PC Generation of monoclonal antibodies to cryptic collagen sites by using subtractive immunization.Hybridoma. 2000; : 375-385Crossref PubMed Scopus (49) Google Scholar Exposure of this unique cryptic epitope has been detected within the basement membrane of angiogenic blood vessels and within the interstitial matrix of malignant tumors. Little, if any, HU177 has been detected within normal tissues.19Xu J Rodriguez D Kim JJ Brooks PC Generation of monoclonal antibodies to cryptic collagen sites by using subtractive immunization.Hybridoma. 2000; : 375-385Crossref PubMed Scopus (49) Google Scholar Importantly, exposure of these cryptic sites can be modulated by proteolysis as well as radiation.20Brooks PC Roth JM Lymberis SC DeWyngaert K Broek D Formenti SC Ionizing radiation modulates the exposure of the HUIV26 cryptic epitope within collagen type IV during angiogenesis.Int J Radiat Oncol Biol Phys. 2002; : 1194-1201Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar In this regard, members of the matrix metalloproteinase (MMP) family such as MMP-2 and MMP-9 have been shown to specifically cleave triple helical collagen-IV, the predominant form of collagen found in vascular basement membranes.21Mignatti P Rifkin D Plasminogen activators and matrix metalloproteinases in angiogenesis.Enzyme Protein. 1996; 49: 117-137Crossref PubMed Scopus (294) Google Scholar, 22Galis ZS Khatri JJ Matrix metalloproteinases in vascular remodeling and atherogenesis: the good, the bad, and the ugly.Circ Res. 2002; 90: 251-262Crossref PubMed Scopus (1703) Google Scholar Because MMP-mediated remodeling of the collagenous microenvironment contributes to angiogenesis, it is likely that ECM remodeling also contributes to revascularization and reperfusion of ischemic tissues. Although studies have examined the expression and functional significance of MMPs in ischemic tissues,23Muhs BE Plitas G Delgado Y Ianus I Shaw JP Adelman MA Lamparello P Shamamian P Gagne P Temporal expression and activation of matrix metalloproteinases-2, -9, and membrane type 1-matrix metalloproteinase following acute hindlimb ischemia.J Surg Res. 2003; 111: 8-15Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 24Johnson C Sung H Lessner S Fini M Galis ZS Matrix metalloproteinase-9 is required for adequate angiogenic revascularization of ischemic tissues.Circ Res. 2004; 94: 262-268Crossref PubMed Scopus (165) Google Scholar little is known concerning the exposure of cryptic epitopes during this process. Previous studies have established that successful murine limb revascularization occurs over a time course of 30 to 35 days after induction of severe ischemia in a murine hindlimb model.25Couffinhal T Silver M Zheng LP Kearney M Witzenbichler B Isner JM Mouse model of angiogenesis.Am J Pathol. 1998; 152: 1667-1679PubMed Google Scholar Collateral vessel enlargement (arteriogenesis) and angiogenesis are thought to contribute to the revascularization observed within this model.4Hershey JC Baskin EP Glass JD Hartman HA Gilberto DB Rogers IT Cook JJ Revascularization in the rabbit hindlimb: dissociation between capillary sprouting and arteriogenesis.Cardiovasc Res. 2001; 49: 618-625Crossref PubMed Scopus (152) Google Scholar, 25Couffinhal T Silver M Zheng LP Kearney M Witzenbichler B Isner JM Mouse model of angiogenesis.Am J Pathol. 1998; 152: 1667-1679PubMed Google Scholar Therefore, to gain a more complete understanding of the mechanisms that regulate reperfusion of ischemic limbs, an analysis of the biochemical and structural changes occurring within the ECM of ischemic skeletal muscle was carried out. In particular, the functional and temporal expression of specific MMPs and their respective endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs), were examined within the ischemic hindlimb. Here, we provide evidence, for the first time, that the unique cryptic collagen epitope, HU177, is specifically exposed within ischemic muscle tissue in a time-dependent manner and may be dependent, in part, on the availability of MMP-9. MMP-2 and MMP-9 levels (active and total) in muscle lysates were measured using Biotrak Activity assay systems (Amersham Biosciences, Piscataway, NJ). Purified MMP-2 and MMP-9 (Amersham Biosciences) were used as controls. Enzyme-linked immunosorbent assay (ELISA) kits (Amersham Biosciences) were used to measure TIMP-1 and TIMP-2 levels in muscle lysates. Purified TIMP-1 and TIMP-2 (Amersham Biosciences) were used as controls and to derive standard curves. Muscle fragments were embedded in OCT compound (Tissue Tek; Sakura Finetek USA, Inc., Torrance, CA) for the preparation of frozen sections. Development of the mAb HU177 through subtractive immunization has been previously described.19Xu J Rodriguez D Kim JJ Brooks PC Generation of monoclonal antibodies to cryptic collagen sites by using subtractive immunization.Hybridoma. 2000; : 375-385Crossref PubMed Scopus (49) Google Scholar mAb QH2b is a humanized version of the mAb HU177 and was provided as a gift by Cancervax (Carlsbad, CA). mAb QH2B was used to stain for the HU177 cryptic collagen epitope on frozen sections of muscle. A goat anti-human IgG horseradish peroxidase (HRP) conjugate (BioSource, Camarillo, CA) was the secondary antibody used in the mAb QH2B staining experiments. Peroxidase Substrate kit (Vector Laboratories, Inc., Burlingame, CA) was used to develop the HRP in the immunohistochemical staining experiments. Harris hematoxylin (EMD Biosciences, Inc., La Jolla, CA) was used to counterstain the frozen sections to better define histology. Permount (Fischer Scientific, Fair Lawn, NJ) was used to fix slips on the frozen sections. Rat anti-mouse monoclonal CD31 antibody (Pharmingen, Franklin Lakes, NJ) was used for the costaining experiments with mAb QH2B. Goat anti-rat biotin-conjugated antibody (Pharmingen) and alkaline phosphatase-labeled streptavidin (Ventana Medical Systems, Tucson, AZ) were used to label the mAb CD31 on tissue sections. HRP-labeled goat anti-human antibody (Sigma-Aldrich, St. Louis, MO) was used to label the mAb QH2B in the costaining experiments. Several different mouse strains were used in this study. FVB/N mice (Taconic Farms, Germantown, NY) 6 to 8 weeks of age and weighing 20 to 30 g were used for all experiments involving quantification of MMP-2, MMP-9, TIMP-1, TIMP-2, and serial measurement of HU177 cryptic site exposure. MMP-9 null mice in a 129SvEv background were kindly provided by Drs. Robert Senior and Michael Shipley (Washington University, St. Louis, MO).26Vu TH Shipley JM Bergers G Berger JE Helms JA Hanahan D Shapiro SD Senior RM Werb Z MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes.Cell. 1998; : 411-422Abstract Full Text Full Text PDF PubMed Scopus (1527) Google Scholar Backbreeding of the MMP-9 null mice was performed periodically to prevent genetic drift. 129SvEv mice (Taconic Farms) were used as background-matched control animals in all experiments involving MMP-9 null mice. Protocols were approved by New York University School of Medicine's Institutional Animal Care and Use Committee. The animals were anesthetized with 0.12 to 0.15 ml of an anesthesia cocktail [1.5 ml of ketamine (100 mg/ml; Fort Dodge Animal Health, Fort Dodge, IA); 0.5 ml of acepromazine (10 mg/ml; Boehringer Ingelheim, St. Joseph, MO); and 1.5 ml of xylazine (20 mg/ml; Ben Venue Laboratories, Inc., Bedford, OH) in 8.5 ml of phosphate buffered saline (PBS)] intraperitoneally before the surgical procedure. Postoperatively, the animals were warmed, administered 3.0 ml of sterile 0.9% normal saline solution (Abbott Laboratories, Chicago, IL) to correct iatrogenic volume depletion, and closely monitored. Operative intervention was performed to create unilateral hindlimb ischemia in the mice. Exposure was obtained by performing a skin incision in the right groin. The neurovascular bundle was located, and the femoral artery was then identified and dissected free. The femoral artery, in the most proximal portion of the hindlimb, was ligated and then divided. The skin was closed using interrupted 6-0 nylon sutures. To document changes in blood flow preoperatively, postoperatively, and during the recovery period, laser Doppler imaging was used. Laser Doppler imaging (LDI) to determine limb blood flow has been described previously.25Couffinhal T Silver M Zheng LP Kearney M Witzenbichler B Isner JM Mouse model of angiogenesis.Am J Pathol. 1998; 152: 1667-1679PubMed Google Scholar Briefly, the lower torso and both hindlimbs were scanned with a Moor LDI VR laser Doppler (Moor Instruments Limited, Wilmington, DE) in a raster pattern for detection of a Doppler shift from the moving blood in the microvasculature of the skin and the underlying arterioles and venules. Blood flow was represented in a color-coded digital image and reported numerically, using proprietary Moor LDI Perfusion Measurement software (v3.09; Moor Instruments Limited). A black-and-white digital image was simultaneously obtained, and both color-coded and black-and-white images were stored on a laptop computer (Winbook Z1; Winbook Computer Corporation, Hilliard, OH). Whole-limb blood flow was determined using Moor LDI Image Processing software (v3.09; Moor Instruments Limited). To compute whole-limb blood flow, the entire hindlimb was digitally outlined based on anatomical landmarks in the image. The Image Processing software was then used to calculate the mean flow of blood within the designated area. Blood flow in the ischemic limb was indexed against that within the nonischemic limb. The nonischemic limb served as an internal control to account for any variability in blood flow due to extraneous factors such as systemic hemodynamic factors, animal surface vasoreactivity, and external environmental changes such as ambient temperature. As part of the measurement procedure, animals were anesthetized, as described above, before LDI. LDI was performed before and immediately after femoral artery ligation to document the level of arterial insufficiency rendered. Reperfusion studies were performed through serial LDI measurements at defined time points. Immediately after harvesting, 30- to 50-mg fragments of hindlimb calf muscle were snap-frozen on dry ice and then stored at −80°C until the protein was extracted. Muscle fragments were placed in 1 ml of extraction buffer on ice containing 50 mmol/L Tris-HCl (pH 7.5), 300 mmol/L NaCl, and 1% Triton X-100. The tissue was minced and homogenized on ice, using a PCU 11 Polytron (Kinematica Ag, Littau-Lucerne, Switzerland). The specimens were centrifuged at 10,000 × g at 4°C for 20 minutes, and the supernatant was decanted and filtered using a 50-μm filter column (Fisher Scientific, Pittsburgh, PA). All muscle lysate specimens were then stored at −80°C until assayed. Bioactivity assays were performed using Biotrak's activity assay system (Amersham Biosciences) according to the manufacturer's protocol. Briefly, muscle lysate samples were placed in 96-microtitre well plates coated with anti-MMP-2 (100 μl/well). The plates were incubated overnight at 4°C. The following day, the plates were washed four times with washing buffer. P-Aminophenylmercuric acetate was added to samples from which the "total" MMP-2 (ie, total latent and active MMP-2) was measured. Buffer alone was added to samples prepared for measuring "active" (ie, endogenous active MMP-2) levels of MMP-2. Detection agent was then added to all wells (50 μl/well), and the plate was read at 405 nm (t = 0 minutes) and again after a 3-hour incubation at 37°C. The same procedure was followed to evaluate the amount of endogenous active MMP-9 and the total MMP-9. Purified MMP-2 and MMP-9 (Amersham Biosciences) were used, as positive and negative controls. Standard curves generated from known quantities of MMP-2 and MMP-9 were used to calculate the concentration of active MMP-2 or MMP-9 in these samples. Commercially available ELISA kits (Amersham Biosciences) were used to measure TIMP-1 and TIMP-2 levels in muscle lysates according to the manufacturer's instructions. Briefly, standards and samples were incubated in microtitre wells coated with anti-TIMP-1 and anti-TIMP-2 antibody, respectively. Peroxidase-labeled antibody directed to the respective TIMPs was added to each well. The amount of peroxidase was determined by the addition of a TMB substrate followed by addition of 100 μl of 1 mol/L sulfuric acid. The plates were read at 450 nm. Standard curves were generated from known quantities of TIMP-1 and TIMP-2 (Amersham Biosciences) to calculate the concentration of TIMP-1 and TIMP-2 in the samples. Calf muscle from ischemic and nonischemic limbs was harvested, and 3 × 3 mm2 muscle sections were imbedded in OCT compound (Tissue Tek; Sakura Finetek USA, Inc.). The blocks were snap frozen and stored at −80°C until sectioned. Four-micrometer sections were cut on a Microtome Cryostat (Microm HM 505E; Microm International GmbH Robert/Boschster, Walldorf, Germany) and fixed in a 50:50 solution of methanol (VWR International, West Chester, PA) and acetone (Fischer Scientific, Fair Lawn, NJ) for 3 minutes at room temperature. The sections were then dried for 90 to 120 minutes at room temperature and washed with a 0.05 mol/L ethylenediamine tetraacetic acid/PBS solution. Each section was then treated with 3% hydrogen peroxide for 10 minutes to reduce endogenous peroxidase activity, washed as above, and then blocked with 1% bovine serum albumin (BSA) (Sigma-Aldrich, Inc.) for 1 hour at room temperature. The sections were then washed twice with the same ethylenediamine tetraacetic acid/PBS solution and incubated with mAb QH2B (100 μg/ml in 1% BSA) (Cancervax) at 4°C overnight. The sections were then washed with PBS, blocked with 1% BSA for 30 minutes, and then washed twice with PBS all at room temperature. Goat anti-human IgG HRP conjugate (BioSource) was diluted in 1% BSA (1:1000) and was applied to each section for 30 minutes at room temperature. The slides were washed twice with PBS, and then DAB was applied for 1 to 3 minutes (Peroxidase Substrate kit, Vector Laboratories, Inc.). The slides were washed with PBS and counterstained with Harris hematoxylin (EMD Biosciences, Inc.). The sections were then dehydrated through graded alcohols (ie, 70 to 100% ethanol), preserved with xylene, and then mounted with Permount (Fischer Scientific). Stained sections were analyzed on an Olympus BX51 microscope (Olympus Corporation, Tokyo, Japan), and representative areas were digitally photographed with a Sony DXC-S500 (Sony Corporation, Tokyo, Japan) high-resolution digital camera. The images were stored on a Power Macintosh G4 computer (Apple Computer, Cupertino, CA). The images were analyzed and scored for staining intensity with Adobe Photoshop 6.0 (Adobe Systems, Inc., San Jose, CA) by two blinded observers. Briefly, total pixel number for each image was measured, and the background pixels (eg, white and non-muscle) were then subtracted from the total pixel number to determine the pixel count representing total muscle tissue. Staining was then measured, the number of pixels representing positively stained tissue was compared with the pixel count representing total muscle tissue, and a percentage was calculated (www.musc.edu/cando/earthkam/measure/Measuring.html). Muscle sections from three animals at each time point were examined, and three to five representative microscopic fields per animal were analyzed. Frozen sections were cut and dried as described above. Each section was treated with 3% hydrogen peroxide for 10 minutes to reduce endogenous peroxidase activity and then washed with Tris-HCl (Ventana Medical Systems). Using an automated immunohistochemical slide-staining system (NexES, Ventana Medical Systems) co-staining for CD31 and HU177 was accomplished. Staining for CD31 was carried out using rat anti-mouse monoclonal CD31 antibody (Pharmingen) followed by incubation with goat anti-rat biotin-conjugated antibody (Pharmingen) and alkaline phosphatase-labeled streptavidin (Ventana Medical Systems). For detection of the HU177 cryptic epitope, tissue was incubated with mAb QH2B followed by incubation with HRP-labeled goat anti-human antibody (Sigma-Aldrich). All slides were then manually counterstained with Harris hematoxylin and mounted as described above. Unpaired t-test with Welch correction was performed using GraphPad InStat version 3.0b for Macintosh (GraphPad Software, San Diego, CA). P < 0.05 was considered significant. Error bars in all figures represent the SEM. A more in-depth understanding of the molecular alterations occurring within the vascular microenvironment of ischemic skeletal muscle is of great importance to the development of more effective approaches for treating arterial insufficiency. To study specific molecular changes in the ECM of ischemic skeletal muscle, a murine model of hindlimb ischemia was used.25Couffinhal T Silver M Zheng LP Kearney M Witzenbichler B Isner JM Mouse model of angiogenesis.Am J Pathol. 1998; 152: 1667-1679PubMed Google Scholar As shown in Figure 1, proximal ligation of the femoral artery in the murine hindlimb leads to an acute 80% decrease in arterial blood flow within 24 hours of surgery compared with the normal contralateral hindlimb. Beginning approximately 48 hours later, an active phase of recovery begins with an increase in blood flow observed in the ischemic limb that ultimately plateaus with blood flow stable at approximately 66% of the contralateral non-ischemic limb. Blood flow in the calf of the ischemic limb remains relatively stable from 14 to 28 days after injury (Figure 1). Importantly, little if any change in this stabilized blood flow was observed throughout an 8-week time period after arterial ligation (data not shown). These findings are consistent with previously reported results25Couffinhal T Silver M Zheng LP Kearney M Witzenbichler B Isner JM Mouse model of angiogenesis.Am J Pathol. 1998; 152: 1667-1679PubMed Google Scholar and suggest that while blood flow returns, complete recovery to the level observed before the injury fails to occur. Previous studies have suggested that angiogenesis and arteriogenesis may contribute to the reperfusion observed in ischemic limb models.4Hershey JC Baskin EP Glass JD Hartman HA Gilberto DB Rogers IT Cook JJ Revascularization in the rabbit hindlimb: dissociation between capillary sprouting and arteriogenesis.Cardiovasc Res. 2001; 49: 618-625Crossref PubMed Scopus (152) Google Scholar Moreover, studies also suggest that MMPs play crucial roles in angiogenesis and arteriogenesis.22Galis ZS Khatri JJ Matrix metalloproteinases in vascular remodeling and atherogenesis: the good, the bad, and the ugly.Circ Res. 2002; 90: 251-262Crossref PubMed Scopus (1703) Google Scholar, 27Carmeli E Moas M Reznick A Coleman R Matrix metalloproteinases and skeletal muscle: a brief review.Muscle Nerve. 2004; 29: 191-197Crossref PubMed Scopus (188) Google Scholar To this end, we analyzed the temporal levels and functional activity of MMP-2 and MMP-9 within is
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