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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 6, 2001 as doi:10.1096/fj.00-0745fje. |
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Section of Cardiovascular Sciences, Department of Medicine and the DeBakey Heart center,
* Section of Leukocyte Biology, Department of Pediatrics; Baylor College of Medicine, Houston, Texas 77030, USA
2Correspondence: Section of Cardiovascular Sciences, Department of Medicine, Baylor College of Medicine, One Baylor Plaza, M/S F-602, Houston TX 77030, USA. E-mail: ngf{at}bcm.tmc.edu
SPECIFIC AIMS
Chemokines are chemotactic cytokines important in regulating angiogenesis. CXC chemokines containing the ELR motif, such as interleukin 8 (IL-8), are potent angiogenic factors, whereas CXC chemokines that lack the ELR motif, such as interferon-inducible protein 10 (IP-10), are potent angiostatic factors. Regulation of angiogenesis is dependent on the net biological balance between expression of angiogenic and angiostatic molecules in the injured tissue. In this study, we hypothesized that changes in the level of expression of the angiostatic CXC chemokine IP-10 have an important role in regulating neovessel formation after myocardial infarction.
PRINCIPAL FINDINGS
1. IP-10 mRNA induction after experimental canine myocardial
infarction
We used a canine model of circumflex coronary artery occlusion for
1 h, followed by reperfusion intervals ranging from 1 h to 28
days. We demonstrated a marked induction of IP-10 mRNA in the
reperfused infarct after 1 h of coronary occlusion and 1 h of
reperfusion. IP-10 mRNA expression remained high for 3 h
(P<0.01, n=4) after reperfusion of the canine
myocardium and was not detected at levels above controls after 10 h or 24 h of reperfusion (Fig. 1
). No significant IP-10 mRNA up-regulation was noted after reperfusion
intervals ranging from 24 h to 28 days.
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In situ hybridization experiments indicated that IP-10 message was localized in endothelial cells of small venules in the ischemic myocardial segments after 1 h of coronary occlusion and 1 h of reperfusion. Immunohistochemical studies localized IP-10 protein in venular endothelial cells of ischemic and not control myocardial segments. In the ischemic and reperfused canine myocardium, IP-10 protein expression was first noted after 1 h and disappeared after 24 h of reperfusion.
2. Angiogenesis in the infarcted canine myocardium
Using histochemical and immunohistochemical techniques, we studied
histological evidence of angiogenesis in reperfused canine myocardial
infarcts. Our studies examined the time course of formation of a
provisional matrix network, followed by endothelial cell proliferation
and neovessel formation. Extravasation of fibrin was noted after 3–5 h
of reperfusion and provided the substrate for an organized provisional
matrix network after 24–168 h of reperfusion. We noted expression of
the proliferating cell nuclear antigen (PCNA) in endothelial cells,
only in the infarcted myocardium, for the first time after 24 h of
reperfusion. PCNA-positive endothelial cells were more frequent after
72 h of reperfusion. In contrast, no cellular proliferation was
noted in the first 5 h after reperfusion.
3. Tumor growth factor ß (TGF-ß) and not IL-10 inhibits
TNF-
-mediated IP-10 synthesis in canine venular endothelial cells
Previous studies have demonstrated that IL-10 down-regulates
endotoxin-mediated IP-10 synthesis in macrophages. In addition, TGF-ß
suppresses chemokine expression in various cell types. We hypothesized
that IL-10 and TGF-ß, both released in the reperfused infarct, may
selectively inhibit IP-10 synthesis by venular endothelial cells.
TNF- and LPS induced a robust expression of IP-10 and IL-8 mRNA in
isolated canine jugular vein endothelial Cells (CJVEC) after 6 h
of incubation. Addition of TGF-ß, and not IL-10, suppressed TNF-
and LPS-mediated IP-10 up-regulation. Low concentration (1 ng/ml) of
TGF-ß was as effective as higher concentrations (10–100 ng/ml) in
down-regulating TNF--mediated IP-10 induction. In contrast, TGF-ß
had no effect in regulating TNF--mediated IL-8 expression in
isolated CJVEC (Fig. 2
).
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CONCLUSIONS AND SIGNIFICANCE
The healing myocardium demonstrates significant proliferative and metabolic activity, especially as myofibroblasts infiltrate the ischemic areas and deposit extracellular matrix components. Obviously, new vessel formation is crucial for supplying the healing scar with oxygen and nutrients in order to sustain metabolism.
Angiogenesis is dependent on a complex interaction between extracellular matrix, endothelial cells, and pericytes in response to an imbalance in the presence of angiogenic as compared to angiostatic factors in the local environment. The best-described angiogenic factors are vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Recently, members of the CXC chemokine family were reported to have a role in the regulation of angiogenesis. CXC chemokines behave as either angiogenic or angiostatic, depending on the presence of the ‘ELR’ motif. ELR-positive CXC chemokines such as IL-8 are potent angiogenic factors, inducing both in vitro endothelial chemotaxis and in vivo corneal neovascularization. In contrast, the ELR-negative chemokines, such as IP-10, demonstrate robust angiostatic effects in the presence of IL-8 or basic FGF.
Myocardial infarction is associated with an early release of angiogenic
factors in the injured areas. Numerous investigations have indicated
that VEGF, IL-8 and bFGF are rapidly induced in the ischemic
myocardium. Our study demonstrates a marked transient up-regulation of
the angiostatic CXC chemokine IP-10 in ischemic and reperfused
myocardial infarcts. IP-10 mRNA expression is down-regulated after
24 h of reperfusion, whereas IL-8 message levels remain high. We
suggest that suppression of IP-10 expression may be an important factor
in regulating angiogenesis by shifting the biological balance toward a
preponderance of angiogenic factors, allowing unopposed bFGF-, IL-8-,
and VEGF-mediated activity (Fig. 3
).
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The early transient induction of IP-10 in the ischemic myocardium may serve to delay the onset of the angiogenic process until the injured myocardium has been cleared from dead cells and debris by infiltrating phagocytes, and a fibrin-rich provisional matrix is formed to support ingrowth of new blood vessels. This hypothesis is supported by the fact that IP-10 synthesis is confined to the venular endothelium, where angiogenesis is actually initiated. Studies in a variety of pathological processes involving angiogenesis have demonstrated that VEGF-mediated microvascular hyperpermeability and the introduction of a provisional plasma-derived matrix precede the onset of endothelial cell division and new blood vessel formation, and create a milieu that favors fibroblast and endothelial cell migration. Our experiments demonstrated fibrin extravasation 3–5 h after reperfusion of the ischemic myocardium, followed by the formation of a well-organized provisional matrix. Twenty-four hours after reperfusion, PCNA-positive endothelial cells were noted in the healing myocardium and became more numerous at later stages of scar formation. This sequence of cellular events suggests that endothelial cell proliferation and subsequent neovessel formation is delayed until the wound is débrided and a provisional matrix is formed, despite the presence of angiogenic factors in the injured myocardium. Transient expression of the angiostatic chemokine IP-10 may be important in preventing premature neovascularization during the early phase of cardiac repair while the fibrin-based matrix necessary to support angiogenesis is formed.
IP-10 mRNA and protein were localized in the microvascular endothelium
of ischemic myocardial segments after experimental myocardial
infarction. In vitro experiments demonstrated that TNF-, which is
released early after myocardial ischemia, markedly up-regulates IP-10
expression in canine endothelial cells. To investigate the mechanisms
of IP-10 down-regulation after 24 h of reperfusion, we studied the
effects of IL-10 and TGF-ß, both present in the ischemic myocardium,
in regulating cytokine-induced IP-10 expression. IL-10 is up-regulated
in lymphocytes infiltrating the infarcted myocardium, appearing first
after 5 h of reperfusion and peaking after 4–5 days of
reperfusion. Recent studies suggested that IL-10 suppresses endotoxin-
and interferon-mediated IP-10 expression in mononuclear cells.
Bioactive TGF-ß is present in the ischemic myocardium 3 h after
reperfusion and may be released from preformed stores after myocardial
injury modulating the inflammatory response. Our current experiments
demonstrated that TGF-ß and not IL-10 is capable of suppressing
TNF--mediated IP-10 up-regulation in canine endothelial cells. Both
IL-10 and TGF-ß failed to suppress TNF--induced IL-8 expression.
The role of TGF-ß in regulating angiogenesis remains controversial. Previous studies suggest contradictory effects in neovessel formation, depending on the dose. Our data indicate a new indirect role for TGF-ß in stimulating angiogenesis through the selective inhibition of the angiostatic chemokine IP-10. In addition, TGF-ß potently up-regulates endothelial cell and smooth muscle cell bFGF and VEGF expression, enhancing angiogenic factor activity.
Angiogenesis after myocardial infarction is a complex, dynamic process dependent on the balance in expression of angiogenic and angiostatic factors. Early induction of VEGF causes vascular hyperpermeability and extravasation of plasma proteins, forming a matrix that favors mesenchymal cell migration. These events precede and accompany endothelial cell proliferation and neovessel sprouting. Induction of angiostatic factors such as IP-10 in the first few hours after injury may be important in inhibiting premature neovessel formation until the appropriate supportive matrix is present. Subsequently, TGF-ß-mediated down-regulation of IP-10 expression may be crucial to ensuring unopposed angiogenic factor activity, allowing optimal healing and scar formation.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0745fje ; to
cite this article, use FASEB J. (April 5, 2001) 10.1096/fj.00-0745fje 
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