ARTICLE |
Correspondence to: Nikolaos G. Frangogiannis, Section of Cardiovascular Sciences, Baylor College of Medicine, One Baylor Plaza M/S F-602, Houston, TX 77030. E-mail: ngf@bcm.tmc.edu
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Summary |
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Myocardial infarction (MI) is associated with an angiogenic response, critical for healing and cardiac repair. Using a canine model of myocardial ischemia and reperfusion, we examined the structural characteristics of the evolving microvasculature in healing MI. After 7 days of reperfusion, the infarcted territory was rich in capillaries and contained enlarged, pericyte-poor "mother vessels" and endothelial bridges. During scar maturation arteriolar density in the infarct increased, and a higher percentage of microvessels acquired a pericyte coat (60.4 ± 6.94% after 28 days of reperfusion vs 30.17 ± 3.65% after 7 days of reperfusion; p<0.05). The microvascular endothelium in the early stages of healing showed intense CD31/PECAM-1 and CD146/Mel-CAM immunoreactivity but weak staining with the Griffonia simplicifolia lectin I (GS-I). In contrast, after 28 days of reperfusion, most infarct microvessels demonstrated significant lectin binding. Our findings suggest that the infarct microvasculature undergoes a transition from an early phase of intense angiogenic activity to a maturation stage associated with pericyte recruitment and formation of a muscular coat. In addition, in the endothelium of infarct microvessels CD31 and CD146 expression appears to precede that of the specific sugar groups that bind the GS-I lectin. Understanding of the mechanisms underlying the formation and remodeling of the microvasculature after MI may be important in designing therapeutic interventions to optimize cardiac repair. (J Histochem Cytochem 49:7179, 2002)
Key Words:
myocardial infarction, lectin, CD31, CD146, endothelium, angiogenesis, pericyte, -smooth muscle actin, healing
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Introduction |
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Angiogenesis follows a well-orchestrated sequence of events leading to a phase of vascular maturation and remodeling (vß3 (
vß5 (
Effective wound healing is dependent on the formation of a microvascular network capable of supplying the healing infarct with oxygen and nutrients (
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Materials and Methods |
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Ischemia/reperfusion Protocols
Healthy mongrel dogs (1525 kg) of either sex were surgically instrumented as previously described (1 cm in thickness. The posterior papillary muscle and the posterior free wall were identified. Tissue samples were isolated from infarcted or normally perfused myocardium based on visual inspection. Myocardial segments were fixed in B*5 fixative (
Immunohistochemistry
For histological study of cardiac tissue, sections taken from endocardium to epicardium were fixed in B*5 fixative and embedded in paraffin. Sequential 35 µm sections were cut by microtomy. Immunostaining was performed using the ELITE mouse kit (Vector Laboratories; Burlingame CA) as previously described (
The following primary monoclonal antibodies were used for immunohistochemistry: anti-CD31 monoclonal antibody (-smooth muscle actin antibody (
-smooth muscle actin developed with the alkaline phosphatase substrate kit I (Vector). Dual stained slides were coverslipped without counterstaining.
Lectin Histochemistry
Lectin histochemistry was performed to identify endothelial cells using the Griffonia simplicifolia lectin I (GS-I) as previously described (
Quantitative Analysis and Statistics
At least two ischemic and two control samples were studied from each experiment. Sections stained using dual immunohistochemistry combining peroxidase-based labeling for CD31 with alkaline phosphatase-based staining for -smooth muscle actin were photographed with a Leaf MicroLumina digital camera (resolution 300 dpi) mounted on a Zeiss Axioskop microscope. Ten different fields from each control and ischemic sample were analyzed. The percentage of microvessels coated with an
-smooth muscle actin-positive pericyte coat was calculated in the healing infarcts after 7, 14, and 28 days of reperfusion. In addition, the number of arterioles (microvessels fully coated with a muscular coat and with a minor axis >15 µm) was counted in control and fibrotic areas, and expressed as arterioles/mm2 (arteriolar density). Statistical analysis was performed using ANOVA followed by t-test corrected for multiple comparisons (StudentNewmanKeuls). Significance was set at p<0.05.
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Results |
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Morphological Characteristics of Infarct Microvessels
Immunohistochemical staining for CD31 identified the vascular endothelium in control canine myocardium and in the healing MIs. The infarcted territory was highly vascular after 714 days of reperfusion (Fig 1A). Infarct microvessels were predominantly capillaries, however the presence of enlarged, pericyte-poor "mother vessels" (
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Pericyte Coating During Maturation of Myocardial Scars
Staining for -smooth muscle actin identified myofibroblasts and pericytes in the control myocardium and in the healing infarcts. In control areas,
-smooth muscle actin staining was almost exclusively localized in the arteriolar media (Fig 2A). Using dual immunohistochemical staining, we found that in the control canine myocardium less than 1% of the capillaries had an
-smooth muscle actin-positive pericyte coat (Fig 2A). Healing infarcts showed
-smooth muscle actin immunoreactivity in myofibroblast-like cells and perivascular cells (Fig 2B) as previously described (
-smooth muscle actin-positive pericytes (30.17 ± 3.65% in infarcts after 7 days of reperfusion vs 60.4 ± 6.93% in infarcts after 1 hr of ischemia and 28 days of reperfusion; p<0.05) (Fig 2C and Fig 3A). As the percentage of uncoated microvessels decreased, a significant increase in arteriolar density was noted (35.725 ± 14.05 arterioles/mm2 in infarcts from the 28-day reperfusion group vs 19.11 ± 4.62 arterioles/mm2 in infarcts from the 7-day reperfusion group; p<0.05) (Fig 3B).
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Infarct Neovessels Express CD31 and CD146 but Show Weak Staining with GS-I Histochemistry
Serial sections from the canine myocardium were stained for CD31 (PECAM-1), CD146 (Mel-CAM/MUC18), and the GS-I lectin (Fig 4). All three methods were effective in identifying microvessels in control canine myocardium. However, lectin histochemistry gave stronger capillary staining (Fig 4B). In contrast, CD31 staining was much more intense in cardiac arterioles and venules, whereas capillaries showed weaker labeling (Fig 4A). CD146 immunoreactivity was found in endothelial and vascular smooth muscle cells as previously described (Fig 4C). After 1 hr of ischemia and 7 days of reperfusion, the infarct microvessels demonstrated intense staining for CD31 (Fig 5A and Fig 5B) but weak binding with the GS-I lectin (Fig 5C and Fig 5D). CD31 staining was more intense in microvessels of the healing infarct than in control myocardium from the same sections, and this appeared to be the most effective technique in identifying arterioles, venules, and capillaries in the infarcted areas. CD146 immunoreactivity was noted in many microvascular endothelial cells and pericytes of the healing infarct (Fig 5E and Fig 5F). In contrast, after 28 days of reperfusion most of the microvessels in the mature scar showed significant staining for the lectin (Fig 6). These findings suggest that, in MI neovessels, PECAM-1 expression may precede that of the specific sugar groups that bind the GS-I lectin.
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Discussion |
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Wound healing is associated with an angiogenic response, which is critical for scar formation. An increase in hypoxia-inducible factor-1 (HIF-1) is an early response to myocardial ischemia or infarction (-inducible protein (IP)-10 is noted and may inhibit neovessel formation until the wound is debrided and a provisional fibrin-based matrix is formed (
Structural Properties of the Infarct Microvasculature
Angiogenic microvessels demonstrate significant differences in their architecture compared with normal quiescent vessels.
Healing MIs contain a large number of capillaries in the early stages of healing (-smooth muscle actin-positive pericyte coat were noted. In contrast, maturation of the healing infarct was associated with decreased capillarity (
The dynamic structural changes in the infarct microvasculature may have a significant impact on the repair mechanisms after MI. Mother vessels may evolve into muscular arteries and veins, improving blood supply to the injured myocardium, allowing more effective healing, and decreasing infarct extension and ventricular remodeling. Understanding the mechanisms responsible for the formation and maturation of these vascular structures may lead to effective therapeutic strategies aimed at optimizing cardiac repair.
Endothelial Cells in the Early Healing Phase Express CD31 and CD146 but Do Not Show GS-I Lectin Staining
Lectins are glycoproteins, derived from either plant or animal sources, that bind specific carbohydrate moieties of cell surface glycoproteins (
Our studies also examined the use of CD146 staining to identify the microvasculature in the canine heart. CD146 is a transmembrane glycoprotein constitutively expressed on endothelial cells (
Conclusions
Vessel growth and maturation are critical for scar formation and cardiac repair. This process represents an important therapeutic target in our efforts to improve post-infarction cardiac recovery (
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Acknowledgments |
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Supported by NIH Grant HL-42550, the DeBakey Heart Center, and a grant from the Methodist Hospital Foundation (NGF).
We wish to thank Peggy Jackson, Alida Evans, Alejandro Tumang, Stephanie Butcher, and Kathryn Masterman for outstanding technical assistance and Sharon Malinowski and Connie Mata for editorial assistance with the manuscript.
Received for publication April 11, 2001; accepted August 15, 2001.
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