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The Role of Platelet-Derived Growth Factor Signaling in Healing Myocardial Infarcts

Pawel Zymek, MD^_*, Marcin Bujak, MD^_*, Khaled Chatila, MD^_*, Anna Cieslak, MS^_*, Geeta Thakker, PhD, Mark L. Entman, MD, FACC^_* and Nikolaos G. Frangogiannis, MD, FACC^_*,_*

^_* Section of Cardiovascular Sciences, the DeBakey Heart Center, Baylor College of Medicine, and the Methodist Hospital, Houston, Texas
Section of Atherosclerosis, the DeBakey Heart Center, Baylor College of Medicine, and the Methodist Hospital, Houston, Texas.

View larger version (161K): [in this window] [in a new window] [Download PPT slide]   Figure 1 The microvascular network in reperfused mouse infarcts is identified by CD31 immunohistochemistry. (A) CD31 staining of control heart labels the myocardial vasculature. (B) Serial section with omission of the primary antibody serves as a negative control. (C) After 72 h of reperfusion, infarct granulation tissue contains many CD31-positive endothelial cells (arrowheads). (D) After 7 days of reperfusion, the infarcted area contains relatively few capillaries (arrows) and a significant number of coated vessels (arrowheads). Counterstained with eosin (magnification 200x).

View larger version (142K): [in this window] [in a new window] [Download PPT slide]   Figure 2 The -smooth muscle actin (SMA) immunohistochemistry in reperfused murine infarcts. (A) In control murine hearts, -SMA staining identifies the vascular media (arrowheads). (B) Serial section stained with omission of the primary antibody serves as a negative control. (C) After 72 h of reperfusion, -SMA immunohistochemistry is predominantly localized in spindle-shaped myofibroblasts (arrows). Relatively few coated vessels are noted (arrowheads). (D) After 7 days of reperfusion, the infarct vasculature matures through the recruitment of mural cells by infarct neovessels (arrowheads). Counterstained with eosin (magnification 200x).

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   Figure 3 Assessment of mRNA expression of platelet-derived growth factor receptor (PDGFR)- (A), platelet-derived growth factor (PDGF)-A chain (B), PDGFR-ß (C), and PDGF-B chain (D) in mouse infarcts using quantitative real-time polymerase chain reaction. (A) The PDGFR- mRNA expression in the heart peaked after 6 h of reperfusion (**p < 0.01 vs. sham). (B) The PDGF-A mRNA expression also increased after 6 h of reperfusion; however, the difference with sham hearts did not reach statistical significance (p = 0.08). (C and D) The PDGFR-ß (C) and PDGF-B (D) mRNA levels show a similar time course, peaking after 7 days of reperfusion (*p < 0.05 vs. sham, **p < 0.01 vs. sham).

View larger version (54K): [in this window] [in a new window] [Download PPT slide]   Figure 4 Activation of the platelet-derived growth factor receptor (PDGFR)-ß signaling pathway in healing murine myocardial infarcts was detected using immunohistochemical staining with an antibody to phosphorylated PDGFR-ß (pPDGFR-ß). (A) Noninfarcted areas showed minimal staining for pPDGFR-ß. (B) After 7 days of reperfusion, perivascular and mononuclear-like cells (arrowheads) with intense immunoreactivity to pPDGFR-ß were found in the infarcted myocardium. (C) Negative control with omission of the primary antibody shows no staining. Counterstained with eosin (magnification 200x).

View larger version (125K): [in this window] [in a new window] [Download PPT slide]   Figure 5 (A) Platelet-derived growth factor receptor (PDGFR)- and PDGFR-ß inhibition decreased collagen deposition in the infarcted myocardium. (A) After 7 days of reperfusion, Sirius red staining identified a well-organized collagen network in the infarcted heart from animals treated with control rat immunoglobulin (Ig)G. (B) Mice treated with anti–PDGFR- antibody (APA5) had attenuated collagen deposition in the infarcted myocardium. (C) Mice injected with anti–PDGFR-ß antibody (APB5) showed decreased and disorganized collagen deposition. Note the presence of dilated vessels (arrows). (D) Quantitative assessment of collagen deposition in the infarcted heart (*p < 0.05 vs. IgG control group). Magnification 400x.

View larger version (118K): [in this window] [in a new window] [Download PPT slide]   Figure 6 Platelet-derived growth factor receptor (PDGFR)-ß inhibition resulted in impaired vascular maturation and enhanced capillary density in the healing infarct. (A) CD31 immunohistochemistry identified the infarct vasculature in immunoglobulin IgG-treated mice (magnification 200x). (B) Numerous coated vessels were noted in the infarct after 7 days of reperfusion (arrows). (C to F) The PDGFR-ß inhibition with the neutralizing antibody APB5 resulted in impaired vascular maturation and in formation of dilated, disorganized, irregularly-shaped vascular structures (arrows). (C, magnification 200x; D to F, magnification 400x). (G to I) Quantitative analysis of total microvascular density (G), density of uncoated vessels (H), and density of coated vessels (I) in the infarct. The PDGFR-ß inhibition significantly increased microvascular density (**p < 0.01 vs. IgG-treated animals) (G) and the number of uncoated vessels (**p < 0.01 vs. IgG-injected mice) (H), but decreased the density of coated vessels (I) in the infarcted area (*p < 0.05 vs. IgG-treated mice). In contrast, PDGFR- inhibition did not affect vascular maturation. Other abbreviations as in Figure 5.

View larger version (64K): [in this window] [in a new window] [Download PPT slide]   Figure 7 Hemorrhagic foci in PDGFR-ß antibody-treated infarcts. (A) After 7 days of reperfusion, immunoglobulin IgG-treated mice show a well-formed wound with significant deposition of extracellular matrix and no red blood cell extravasation. (B) The PDGFR-ß inhibition with the neutralizing antibody APB5 resulted in extensive extravasation of red blood cells in the infarct (arrows). (C) In contrast, PDGFR- inhibition with the neutralizing antibody APA5 did not induce hemorrhagic areas in the infarct. (D) Quantitative analysis of macrophage density in the infarcted myocardium. The PDGFR- and PDGFR-ß inhibition resulted in enhanced macrophage density in the infarcted myocardium (**p < 0.01 vs. IgG-treated animals). (E) In contrast, neutrophil density was low in the mature scar after 7 days of reperfusion, and not significantly different between groups (magnification 400x). Abbreviations as in Figure 5.