A Nonantibiotic Chemically Modified Tetracycline (CMT-3) Inhibits Intimal Thickening
2003; Elsevier BV; Volume: 163; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)63512-2
ISSN1525-2191
AutoresMuzharul M. Islam, Christopher Franco, David W. Courtman, Michelle P. Bendeck,
Tópico(s)Reconstructive Surgery and Microvascular Techniques
ResumoRecent research has shown that the tetracycline antibiotics are pluripotent drugs that inhibit the activity of matrix metalloproteinases (MMPs) and affect many cellular functions including proliferation, migration, and matrix remodeling. We have shown that doxycycline inhibits MMP activity and intimal thickening after injury of the rat carotid artery, however we do not know whether these effects are because of the antibiotic, anti-MMP, or other actions of doxycycline. Recently, chemically modified tetracyclines have been synthesized that lack antibiotic activity but retain anti-MMP activity (CMT-3), or lack both antibiotic and anti-MMP activity (CMT-5). In the current study we have assessed the effects of treatment with CMT-3 or CMT-5 on intimal thickening after balloon catheter injury of the rat carotid artery. Rats were treated by oral gavage with 15 mg/kg/day CMT-3 or CMT-5. CMT-3 significantly reduced smooth muscle cell (SMC) proliferation in both the medial and intimal layers of the injured rat carotid artery compared to CMT-5. Furthermore, CMT-3 inhibited SMC migration from the media to the intima by 86% at 4 days after injury. CMT-3 also decreased MMP-2 activity. Finally, we found that CMT-3 treatment resulted in a significant reduction in intimal cross-sectional area from 0.23 ± 0.01 mm2 in the CMT-5 control group to 0.19 ± 0.01 mm2. There was also a reduction in elastin and collagen accumulation within the intima. We conclude that CMT-3 attenuated intimal thickening after arterial injury by inhibiting SMC proliferation, migration and MMP activity, and accumulation of extracellular matrix. The inhibitory effects of CMT-3 were independent of the antibiotic properties, but were dependent on the anti-MMP activity of the tetracycline family. Recent research has shown that the tetracycline antibiotics are pluripotent drugs that inhibit the activity of matrix metalloproteinases (MMPs) and affect many cellular functions including proliferation, migration, and matrix remodeling. We have shown that doxycycline inhibits MMP activity and intimal thickening after injury of the rat carotid artery, however we do not know whether these effects are because of the antibiotic, anti-MMP, or other actions of doxycycline. Recently, chemically modified tetracyclines have been synthesized that lack antibiotic activity but retain anti-MMP activity (CMT-3), or lack both antibiotic and anti-MMP activity (CMT-5). In the current study we have assessed the effects of treatment with CMT-3 or CMT-5 on intimal thickening after balloon catheter injury of the rat carotid artery. Rats were treated by oral gavage with 15 mg/kg/day CMT-3 or CMT-5. CMT-3 significantly reduced smooth muscle cell (SMC) proliferation in both the medial and intimal layers of the injured rat carotid artery compared to CMT-5. Furthermore, CMT-3 inhibited SMC migration from the media to the intima by 86% at 4 days after injury. CMT-3 also decreased MMP-2 activity. Finally, we found that CMT-3 treatment resulted in a significant reduction in intimal cross-sectional area from 0.23 ± 0.01 mm2 in the CMT-5 control group to 0.19 ± 0.01 mm2. There was also a reduction in elastin and collagen accumulation within the intima. We conclude that CMT-3 attenuated intimal thickening after arterial injury by inhibiting SMC proliferation, migration and MMP activity, and accumulation of extracellular matrix. The inhibitory effects of CMT-3 were independent of the antibiotic properties, but were dependent on the anti-MMP activity of the tetracycline family. The tetracyclines function as antibiotics by inhibiting bacterial protein synthesis,1Nelson MW Chemical and biological dynamics of tetracyclines.Adv Dent Res. 1998; 12: 5-11Crossref PubMed Scopus (81) Google Scholar but recent research has shown that they are pluripotent drugs that affect many functions in mammalian cells. Tetracyclines are potent inhibitors of the matrix metalloproteinase (MMP) family of enzymes,2Golub LM Lee HM Ryan ME Giannobile WV Payne J Sorsa T Tetracyclines inhibit connective tissue breakdown by multiple non-antimicrobial mechanisms.Adv Dent Res. 1998; 12: 12-26Crossref PubMed Scopus (548) Google Scholar and they have been used to reduce tissue degradation in periodontal disease3Ryan ME Ramamurthy S Golub LM Matrix metalloproteinases and their inhibition in periodontal treatment.Curr Opin Periodontol. 1996; 3: 85-96PubMed Google Scholar and arthritis.4Greenwald R Treatment of destructive arthritic disorders with MMP inhibitors.Ann NY Acad Sci. 1994; 732: 181-198Crossref PubMed Scopus (72) Google Scholar Doxycycline, a tetracycline derivative, has been used experimentally to inhibit matrix degradation during abdominal aortic aneurysm formation,5Petrinec D Liao S Holmes DR Reilly JM Parks WC Thompson RW Doxycycline inhibition of aneurysmal degeneration in an elastase-induced rat model of abdominal aortic aneurysm: preservation of aortic elastin associated with suppressed production of 92 kD gelatinase.J Vasc Surg. 1996; 23: 336-346Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 6Curci JA Petrinec D Liao S Golub LM Thompson RW Pharmacologic suppression of experimental abdominal aortic aneurysms: a comparison of doxycycline and four chemically modified tetracyclines.J Vasc Surg. 1998; 28: 1082-1093Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 7Boyle JR McDermott E Crowther M Wills AD Bell PR Thompson MM Doxycycline inhibits elastin degradation and reduces metalloproteinase activity in a model of aneurysmal disease.J Vasc Surg. 1998; 27: 354-361Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 8Prall AK Longo GM Mayhan WG Waltke EA Fleckten B Thompson RW Baxter BT Doxycycline in patients with abdominal aortic aneurysms and in mice: comparison of serum levels and effect on aneurysm growth in mice.J Vasc Surg. 2002; 35: 923-929Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar and recent clinical studies have investigated the use of doxycycline to limit aneurysm growth.9Thompson RW Baxter BT MMP inhibition in abdominal aortic aneurysms. Rationale for a prospective randomized clinical trial.Ann NY Acad Sci. 1999; 878: 159-178Crossref PubMed Scopus (171) Google Scholar, 10Franklin IJ Harley SL Greenhalgh RM Powell JT Uptake of tetracycline by aortic aneurysm wall and its effect on inflammation and proteolysis.Br J Surg. 1999; 86: 771-775Crossref PubMed Scopus (44) Google Scholar, 11Curci JA Mao D Bohner DG Allen BT Rubin BG Reilly JM Sicard GA Thompson RW Preoperative treatment with doxycycline reduces aortic wall expression and activation of matrix metalloproteinases in patients with abdominal aortic aneurysms.J Vasc Surg. 2000; 31: 325-342Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 12Mosorin M Juvonen J Biancari F Satta J Surcel HM Leinonen M Saikku P Juvonen T Use of doxycycline to decrease the growth rate of abdominal aortic aneurysms: a randomized, double-blind, placebo-controlled pilot study.J Vasc Surg. 2001; 34: 606-610Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 13Baxter BT Pearce WH Waltke EA Littooy FN Hallett Jr, JW Kent KC Upchurch Jr, GR Chaikof EL Mills JL Fleckten B Longo GM Lee JK Thompson RW Prolonged administration of doxycycline in patients with small asymptomatic abdominal aortic aneurysms: report of a prospective (phase II) multicenter study.J Vasc Surg. 2002; 36: 1-12Abstract Full Text PDF PubMed Scopus (290) Google ScholarTetracyclines also inhibit cell proliferation, cell migration, and synthesis of the extracellular matrix in a variety of cell types studied in culture.14Guerin C Laterra J Masnyk T Golub LM Brem H Selective endothelial growth inhibition by tetracyclines that inhibit collagenase.Biochem Biophys Res Commun. 1992; 188: 740-745Crossref PubMed Scopus (56) Google Scholar, 15Fife RS Sledge Jr, GW Effects of doxycycline on in vitro growth, migration, and gelatinase activity of breast carcinoma cells.J Lab Clin Med. 1995; 125: 407-411PubMed Google Scholar, 16Gilbertson-Beadling S Powers EA Stamp-Cole M Scott PS Wallace TL Copeland J Petzold G Mitchell M Ledbetter S Poorman R The tetracycline analogs minocycline and doxycycline inhibit angiogenesis in vitro by a non-metalloproteinase-dependent mechanism.Cancer Chemother Pharmacol. 1995; 36: 418-424Crossref PubMed Scopus (107) Google Scholar, 17Seftor RE Seftor EA De LJ Kleiner DE Leferson J Stetler-Stevenson WG McNamara TF Golub LM Hendrix MJ Chemically modified tetracyclines inhibit human melanoma cell invasion and metastasis.Clin Exp Metastasis. 1998; 16: 217-225Crossref PubMed Scopus (140) Google Scholar, 18Meng Q Xu J Goldberg ID Rosen EM Greenwald RA Fan S Influence of chemically modified tetracyclines on proliferation, invasion and migration properties of MDA-MB-468 human breast cancer cells.Clin Exp Metastasis. 2000; 18: 139-146Crossref PubMed Scopus (23) Google Scholar, 19Davies SR Cole AA Schmid TM Doxycycline inhibits type X collagen synthesis in avian hypertrophic chondrocyte cultures.J Biol Chem. 1996; 271: 25966-25970Crossref PubMed Scopus (14) Google Scholar, 20Lokeshwar BL MMP inhibition in prostate cancer.Ann NY Acad Sci. 1999; 878: 271-289Crossref PubMed Scopus (147) Google Scholar, 21TeKoppele JM Beekman B Verzijl N Koopman JL DeGroot J Bank RA Doxycycline inhibits collagen synthesis by differentiated articular chondrocytes.Adv Dent Res. 1998; 12: 63-67Crossref PubMed Google Scholar Smooth muscle cell (SMC) proliferation, migration, and matrix synthesis contribute to the neointimal thickening observed in atherosclerosis, restenosis, and vein graft disease. Recently we tested doxycycline using an in vivo model of balloon catheter injury to the rat carotid artery, and showed that doxycycline inhibited SMC proliferation and migration, which led to an attenuation of intimal thickening.22Bendeck MP Conte M Zhang M Nili N Strauss BH Farwell SM Doxycycline modulates smooth muscle cell growth, migration and matrix remodeling after arterial injury.Am J Pathol. 2002; 160: 1089-1095Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar Furthermore, Loftus and colleagues23Loftus IM Porter K Peterson M Boyle J London NJ Bell PR Thompson MM MMP inhibition reduces intimal hyperplasia in a human vein graft stenosis model.Ann NY Acad Sci. 1999; 878: 547-550Crossref PubMed Scopus (24) Google Scholar have shown that treatment with doxycycline reduces intimal thickening in vein grafts placed in organ culture. Taken together, these studies suggest that tetracyclines may be useful in the treatment of intimal thickening. However, given the multiplicity of effects, we do not know whether the antibiotic, anti-MMP, or other actions of doxycycline were responsible for the inhibition of intimal growth.In the current study we use two chemically modified derivatives of tetracycline CMT-3 and CMT-5. CMT-3 (COL-3) is produced by deletion of the dimethylamino group from carbon 4 in the A ring of tetracycline, which abolishes the antibiotic activity but not the anti-MMP activity of the molecule. Further modification by replacement of the carbon 11 carbonyl oxygen and the carbon 12 hydroxyl groups with nitrogen, abolishes the anti-MMP activity, giving rise to CMT-5 (COL-5), which is neither antibiotic nor anti-MMP.24Golub LM Suomalainen K Sorsa T Host modulation with tetracyclines and their chemically modified analogues.Curr Opin Dent. 1992; 2: 80-90PubMed Google Scholar Our purpose was to compare the effects of CMT-3 and CMT-5 on intimal thickening using the rat carotid artery injury model.Materials and MethodsSurgeryAnimal experiments were performed according to the guidelines of the Canada Council on Animal Care. Male Sprague-Dawley rats (Charles River, Constant, Quebec, Canada) weighing 375 to 415 g were used. Rats were anesthetized by intraperitoneal injection of 4.6 mg/kg xylazine (Rompum; Bayer Inc., Etobicoke, Ontario, Canada) and 70 mg/kg ketamine (Ketaset; Ayerst Veterinarian Laboratories, Guelph, Ontario, Canada). Balloon catheter denudation of the left common carotid artery was performed as described previously.25Bendeck MP Zempo N Clowes AW Galardy RE Reidy MA Smooth muscle cell migration and matrix metalloproteinase expression after arterial injury in the rat.Circ Res. 1994; 75: 539-545Crossref PubMed Scopus (538) Google Scholar CMT-3 (6-demethyl-6-deoxy-4-dedimethylamino tetracycline) and CMT-5 (a pyrazole derivative) were provided by CollaGenex Pharmaceuticals Inc., Newton, PA. The CMTs were administered daily by oral gavage at a dose of 15 mg/kg/day, starting 24 hours before surgery. This dose was chosen based on previous studies that found it to be the minimum effective dose abolishing MMP activity in the rat aorta in aneurysm studies.6Curci JA Petrinec D Liao S Golub LM Thompson RW Pharmacologic suppression of experimental abdominal aortic aneurysms: a comparison of doxycycline and four chemically modified tetracyclines.J Vasc Surg. 1998; 28: 1082-1093Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar The CMTs were suspended at a concentration of 20 mg/ml in a solution of 1% carboxymethylcellulose containing 0.02% methyl 4-hydroxybenzoate, 0.01% propyl 4-hydroxybenzoate, 0.5% ethanol and 1% N-methyl-2-pyrrolidone. The rats were sacrificed at various time points after injury, chosen as follows based on previous studies elucidating the kinetics of the injury response. SMC proliferation was measured in the media (2 and 7 days) and intima (7 days).26Clowes AW Reidy MA Clowes MM Kinetics of cellular proliferation after arterial injury. I. Smooth muscle growth in the absence of endothelium.Lab Invest. 1983; 49: 327-333PubMed Google Scholar Migration of cells from media to the intima and MMP activity were measured at 4 days.25Bendeck MP Zempo N Clowes AW Galardy RE Reidy MA Smooth muscle cell migration and matrix metalloproteinase expression after arterial injury in the rat.Circ Res. 1994; 75: 539-545Crossref PubMed Scopus (538) Google Scholar Development of the neointima and accumulation of matrix were assessed at 14 days after injury. To label cells entering S phase in the 2- and 7-day groups, the rats were injected subcutaneously with three doses of 10 mg of 5-bromo-2′-deoxyuridine (BrdU; Boehringer Mannheim Corp., Montreal, Quebec, Canada) at 17, 8, and 1 hour before sacrifice. Rats were killed by an intraperitoneal injection of T61 (Invert Canada Ltd., Whitby, Ontario, Canada), then the carotids were perfusion fixed for 4 minutes with 4% paraformaldehyde in phosphate-buffered saline at a pressure of 120 mmHg. Paraffin-embedded sections were cut at positions 1-cm and 2-cm downstream of the origin of the common carotid artery and used for morphometric analysis.Histomorphometry and Matrix StainingSMC replication rates in the media and intima were measured 2 and 7 days after injury by immunostaining carotid cross-sections for BrdU and determining the percentage of BrdU-labeled cells present as previously described.27Lindner V Olson NE Clowes AW Reidy MA Inhibition of smooth muscle cell proliferation in injured rat arteries. Interaction of heparin with basic fibroblast growth factor.J Clin Invest. 1992; 90: 2044-2049Crossref PubMed Scopus (148) Google Scholar SMC apoptosis rates in the media at 2 days after injury were measured by terminal dUTP nick-end labeling of cross-sections using the Apoptag kit (Intergen, Purchase, NY) according to manufacturer's directions, and counting the number of terminal dUTP nick-end labeling positive SMCs within the media of each arterial section. At the 4-day time point, a 1-cm length was excised from the middle of the common carotid artery and used for assessment of SMC migration into the intima as previously described.28Nikkari ST Jarvelainen HT Wight TN Ferguson M Clowes AW Smooth muscle cell expression of extracellular matrix genes after arterial injury.Am J Pathol. 1994; 144: 1348-1356PubMed Google Scholar Briefly, intimal cells on the surface of the fixed common carotid artery segments were immunostained with an antibody against histone H1 (mAb 1276; Chemicon, Temecula, CA). The number of intimal cell nuclei per square mm of surface area was counted by light microscopy. The migration assay takes advantage of the fact that the first SMCs appear in the intima 3 to 4 days after injury; it takes ∼24 hours for the cells to progress through the cell cycle, so the cells are counted before going through a round of replication.Images of the cross-sections were obtained using a Nikon E600 microscope (Nikon, Mississauga, Ontario, Canada), digitized using a digital camera (model C4742-95-12NRB; Hamamatsu, Inc.) and analyzed using a computer-assisted morphometric analysis system (Simple; C Imaging Systems, Mars, PA). Measurements of intimal and medial areas (14 days after injury) were made as follows. Lumen area was determined by tracing around the inside edge of the vessel to determine the circumference, and calculating the lumen area assuming a circular geometry. Intimal area was measured as the area encompassed by the internal elastic lamina minus the lumen area (in this case lumen area was determined by tracing around the inside edge of the vessel and calculating the area inside). Medial area was measured as the area encompassed by the external elastic lamina minus the area encompassed by the internal elastic lamina (including lumen area). SMC density in the media and the intima was calculated by counting the total number of SMC nuclei within the layer on a cross-section, and dividing it by the cross-sectional area of that vessel layer.Cross-sections taken 14 days after injury were also stained with Movat's pentachrome or picrosirius red dye (PSR) to examine elastin and collagen within the matrix. To measure fibrillar collagen, an orientation-independent birefringence imaging system, LC-PolScope, (CRI, Woburn, MA) was used to perform quantitative polarization microscopy on PSR-stained sections.29Madibally SV Solomon V Mitchell RN Van De WL Yarmush ML Toner M Influence of insulin therapy on burn wound healing in rats.J Surg Res. 2003; 109: 92-100Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 30Oldenbourg R A new view on polarization microscopy.Nature. 1996; 381: 811-812Crossref PubMed Scopus (180) Google Scholar The system consists of liquid crystal filters, a polarization algorithm and a digital image processor. In PolScope images the brightness of each pixel is proportional to the birefringence retardance of the object. Using these images, specimen anisotropy (retardance and azimuth) were determined at all points simultaneously permitting measurements of the orientation and degree of organization of collagen fibers. Four rats per group were used for this analysis.ZymogramsSeven rats were used to measure the activity of MMP-2 and MMP-9 in the carotid artery as previously described.25Bendeck MP Zempo N Clowes AW Galardy RE Reidy MA Smooth muscle cell migration and matrix metalloproteinase expression after arterial injury in the rat.Circ Res. 1994; 75: 539-545Crossref PubMed Scopus (538) Google Scholar Extracts of individual carotid arteries were prepared, total protein content for each carotid was measured, and samples each containing 20 μg of total protein were subject to electrophoresis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels that contained 0.1% gelatin as a substrate for MMP digestion. After electrophoresis, the gels were incubated 16 hours, then stained with Coomassie Blue, and MMP activity was evident as cleared bands of substrate lysis. The MMPs were identified by their molecular weights and inhibition by ethylenediaminetetraacetic acid or phenanthroline. Activity was quantitated by scanning densitometric analysis using Scion Imaging Software (Scion Corp., Frederick, MD)Statistical AnalysisValues are expressed as mean ± SEM. Group means for CMT-3 versus CMT-5 treatment were compared by the two-tailed Student's t-test for independent samples.ResultsThere were no significant differences in body weight between CMT-3- and CMT-5-treated rats.SMC ProliferationPrevious studies have shown that medial SMC replication peaks at 2 days after balloon injury of the rat carotid artery, whereas intimal SMC replication peaks at 7 days. We found that medial SMC replication was significantly reduced by CMT-3 treatment at 2 and 7 days after injury, compared to CMT-5-treated rats (Figure 1A). At 2 days after injury, medial proliferation in the CMT-5-treated rats was 5.40 ± 0.31% compared to 3.04 ± 0.14% in the CMT-3-treated rats. At 7 days after arterial injury, medial SMC proliferation was reduced from 2.70 ± 0.05% in CMT-5-treated rats, to 1.96 ± 0.67% in the CMT-3-treated group. We measured intimal SMC replication at 7 days after balloon injury. CMT-3 treatment reduced intimal SMC proliferation from 22.6 ± 0.9% to 15.2 ± 1.1% at 7 days of balloon catheter injury (Figure 1B). There was no significant difference in the percentage of apoptotic SMCs in the media between the CMT-3- and the CMT-5-treated rats at 2 days after injury (data not shown).SMC MigrationSMC migration from the media to the intima was measured at 4 days after injury. The first SMCs appeared in the intima 3 to 4 days after balloon injury, and it takes ∼24 hours for the SMCs to complete a cycle of cell division, therefore this assay measured migration before the cells were able to proliferate within the intima. There were far fewer cells on the intimal surface of CMT-3-treated rats compared to CMT-5-treated rats at 4 days after injury (Figure 2A). The number of intimal SMCs was 143 ± 16 cells/mm2 in CMT-5-treated rats compared to 20 ± 2 cells/mm2 in CMT-3-treated rats (Figure 2B).Figure 2.A: En face photomicrographs showing SMCs in the intima at 4 days after carotid injury. B: Quantitative analysis of SMC migration. Filled bars represent values from CMT-5-treated control rats, and open bars values from CMT-3-treated rats. Values are mean ± SEM; the number of rats in each group is indicated at the bottom of the bar. *, The value measured in the CMT-3 group was significantly less than the CMT-5 group. Scale bar, 100 μm (A).View Large Image Figure ViewerDownload Hi-res image Download (PPT)MMP ActivityGelatin zymograms were used to assess MMP activity in the balloon-injured rat carotids. MMPs are secreted in a latent zymogen form, but the zymogen appears active on gels because sodium dodecyl sulfate present in the gel partially denatures the MMP exposing the active site. Four major bands with molecular weights of 88, 70, 68, and 62 kd were visible on zymograms containing carotid extracts from CMT-5-treated rats (Figure 3A). We have previously shown that these lytic bands correspond to active MMP-9 (88 kd), the zymogen form of MMP-2 (70 kd), and active MMP-2 (62 kd), respectively.25Bendeck MP Zempo N Clowes AW Galardy RE Reidy MA Smooth muscle cell migration and matrix metalloproteinase expression after arterial injury in the rat.Circ Res. 1994; 75: 539-545Crossref PubMed Scopus (538) Google Scholar The 68-kd band probably represents an intermediate activation product of MMP-2. In the arterial extracts from the CMT-3-treated rats, the intensity of all the MMP-2 bands was decreased compared to the CMT-5-treated rats (control) (Figure 3A). Densitometric analysis of the zymogram confirmed that in the CMT-3-treated group MMP-2 zymogen was reduced to 83%, and MMP-2 active was reduced to 52% of values in the CMT-5 group (Figure 3B). MMP-9 active was not affected by CMT-3 treatment.Figure 3.A: Gelatin zymogram showing the activity of MMP-9 active (88 kd) and MMP-2 (70-kd latent and 62-kd active) in carotid arteries from CMT-5- and CMT-3-treated rats 4 days after balloon catheter injury. Individual carotid extracts were prepared and 20 μg of total protein from each was run in each well. B: Scanning densitometry was performed on zymogram gels, and the values for each MMP band were expressed as a percentage of the value obtained for the CMT-5 group. Four samples were averaged for the CMT-5 group and three for the CMT-3 group to obtain these measurements. Filled bars represent values from CMT-5-treated control rats, and open bars values from CMT-3-treated rats.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Vessel Wall MorphometryTreatment with CMT-3 for 14 days after balloon catheter injury resulted in a marked decrease in intimal thickening when compared to the CMT-5-treated animals (Figure 4A). Intimal area measured on vessel cross-sections was 0.23 ± 0.01 mm2 in the CMT-5 group compared to 0.19 ± 0.01 mm2 in the CMT-3 group (Figure 4B). By contrast, there was no significant change in medial area between the two groups (Figure 4C). Lumen area was increased in the CMT-3 group; lumen area measured 0.23 ± 0.02 mm2 in the CMT-3 group versus 0.16 ± 0.01 mm2 in the CMT-5 group (Figure 4D). Two weeks after injury, intimal SMC number was significantly decreased from 2673 ± 80 in CMT-5-treated rats to 1954 ± 83 SMCs in CMT-3-treated rats (Figure 5A). Intimal SMC density was not different between the CMT-3 and CMT-5 groups (Figure 5B). Medial SMC number was not different between the two groups, nor was medial SMC density (Figure 5, C and D). There were no significant differences in internal elastic lamina or external elastic lamina perimeter between the two groups (data not shown) indicating that CMT-3 treatment did not alter the remodeling of vessel diameter.Figure 4.A: Photomicrographs of carotid cross-sections taken 14 days after balloon injury. Cross-sectional areas of the intima (B), media (C), and lumen (D) in carotid arteries from CMT-5 (filled bars)- and CMT-3 (open bars)-treated rats measured at 14 days after injury. Values are mean ± SEM; the number of rats in each group is indicated at the bottom of the bar. *, The value measured in the CMT-3 group was significantly different from the CMT-5 group. Scale bar, 100 μm (A).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5.SMC number in the intima (A) and media (C), and SMC density per unit area in the intima (B) and media (D). Filled bars represent values from CMT-5-treated control rats, and open bars values from CMT-3-treated rats. Values are mean ± SEM; the number of rats in each group is indicated at the bottom of the bar. *, The value measured in the CMT-3 group was significantly less than the CMT-5 group.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Collagen and Elastin AccumulationVessel cross-sections stained with PSR for collagen were visualized using transmitted light microscopy to determine the amount and the localization of collagen in the aortic wall (Figure 6, A and B). Decreased collagen staining was evident in the carotids from CMT-3-treated rats, especially in the intimal layer. The sections were also analyzed using quantitative polarized light microscopy to assess the content of collagen fibrils. PSR stains all collagen, however only collagen assembled into fibrils is birefringent and therefore visible by polarized light microscopy. Dense fibers of collagen were evident within the intimal layer of CMT-5 carotid arteries at 14 days after injury (Figure 6C). By contrast, there was less collagen apparent in the intima of CMT-3-treated rats (Figure 6D). Retardance values calculated across each section are shown underneath the photographs, and the results demonstrate abundant collagen fibers in the sublumenal intima of the CMT-5-treated rats, but not the CMT-3 rats. The PolScope was also used to assess fiber orientation, and this was measured and displayed on an azimuth map (Figure 6, E and F). This demonstrated a dense band of collagen fibers oriented in parallel within the innermost layer of the intima in the CMT-5-treated rats (Figure 6E), whereas the CMT-3-treated rats lacked this inner band of collagen fibers (Figure 6F). Average retardance values calculated from this inner layer of the intima, revealed a decrease in average retardance from 6.5 ± 1.9 in the CMT-5 group to 3.7 ± 0.8 in the CMT-3 group, indicating a decrease in collagen fiber content in the intima of the CMT-3-treated rats (n = 4 per group). A decrease in intimal elastin accumulation was also evident in the CMT-3-treated rats on visualization of sections stained with Movat's pentachrome [Figure 6, G (CMT-5) and H (CMT-3)].Figure 6.Photomicrographs of carotid cross-sections taken 14 days after injury from CMT-5 (A, C, E, G)- or CMT-3 (B, D, F, H)-treated rats. A and B: Sections stained with PSR for collagen visualized by transmitted light microscopy. C and D: PSR-stained sections visualized under polarized light microscopy and analyzed with a PolScope to show retardance because of birefringent collagen fibers. Retardance values calculated across the section are shown underneath. E and F: Pseudocolor images (azimuth map) representing birefringence slow axis orientation (ie, collagen fiber orientation). Color-coding scheme is seen at bottom right of each image, and color represents collagen orientation, while intensity represents magnitude. G and H: Sections stained with Movat's pentachrome and visualized by transmitted light microscopy to show elastin. Position of vessel lumen is indicated by L. Scale bars, 100
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