Endothelial fibroblast growth factor receptor signaling is required for vascular remodeling following cardiac ischemia-reperfusion injury

Abstract

Fibroblast growth factor (FGF) signaling is cardioprotective in various models of myocardial infarction. FGF receptors (FGFRs) are expressed in multiple cell types in the adult heart, but the cell type-specific FGFR signaling that mediates different cardioprotective endpoints is not known. To determine the requirement for FGFR signaling in endothelium in cardiac ischemia-reperfusion injury, we conditionally inactivated the Fgfr1 and Fgfr2 genes in endothelial cells with Tie2-Cre (Tie2-Cre, Fgfr1(f/f), Fgfr2(f/f) DCKO mice). Tie2-Cre, Fgfr1(f/f), Fgfr2(f/f) DCKO mice had normal baseline cardiac morphometry, function, and vessel density. When subjected to closed-chest, regional cardiac ischemia-reperfusion injury, Tie2-Cre, Fgfr1(f/f), Fgfr2(f/f) DCKO mice showed a significantly increased hypokinetic area at 7 days, but not 1 day, after reperfusion. Tie2-Cre, Fgfr1(f/f), Fgfr2(f/f) DCKO mice also showed significantly worsened cardiac function compared with controls at 7 days but not 1 day after reperfusion. Pathophysiological analysis showed significantly decreased vessel density, increased endothelial cell apoptosis, and worsened tissue hypoxia in the peri-infarct area at 7 days following reperfusion. Notably, Tie2-Cre, Fgfr1(f/f), Fgfr2(f/f) DCKO mice showed no impairment in the cardiac hypertrophic response. These data demonstrate an essential role for FGFR1 and FGFR2 in endothelial cells for cardiac functional recovery and vascular remodeling following in vivo cardiac ischemia-reperfusion injury, without affecting the cardiac hypertrophic response. This study suggests the potential for therapeutic benefit from activation of endothelial FGFR pathways following ischemic injury to the heart.

Keywords: cardiac ischemia-reperfusion injury; endothelium; fibroblast growth factor; myocardial infarction; vascular remodeling.

Fig. 1.

Endothelial-specific ablation of Fgfr1 and Fgfr2. A: representative images of mTmG reporter gene expression in adult left ventricle (LV) of control and Tie2-Cre, Fgfr1^f/f, Fgfr2^f/f DCKO mice. GFP expression is seen in an endothelial pattern in Tie2-Cre, Fgfr1^f/f, Fgfr2^f/f DCKO mice, while Tomato expression is observed in nonendothelial cells of Tie2-Cre, Fgfr1^f/f, Fgfr2^f/f DCKO mice and all cells of control hearts. Green: GFP; red: tomato. Scale bars = 50 μm in ×10 magnification images and 25 μm in ×20 magnification images. B: quantitative RT-PCR of FACS-sorted lung endothelial cells showing significantly reduced expression of Fgfr1 and Fgfr2 in Tie2-Cre, Fgfr1^f/f, Fgfr2^f/f DCKO mice compared with controls. *P < 0.01 vs. control; n = 4.

Fig. 2.

Removal of endothelial Fgfr1 and Fgfr2 does not significantly alter left anterior descending artery perfusion territory. A: representative whole mount images of FITC-dextran injected control and Tie2-Cre, Fgfr1^f/f, Fgfr2^f/f DCKO mice showing similar area at risk with left-anterior descending artery ligation. The hypoperfused area is outlined in white dashed lines. Scale bar = 1 mm. B: quantification of FITC-dextran injected hearts showing no significant difference in the hypoperfused area (%LV) (n = 3). C: echocardiography during the ischemic period showing no significant difference in control and Tie2-Cre, Fgfr1^f/f, Fgfr2^f/f DCKO mice for area at risk (AAR) or segmental wall motion score index (SWMSI) (n = 7). D: measurements of cardiac function showed significantly decreased ejection fraction during ischemia for both control and Tie2-Cre, Fgfr1^f/f, Fgfr2^f/f DCKO hearts but no significant difference between genotypes at either time point (#P < 0.01 vs. preischemia; n = 7).

Fig. 3.

Endothelial Fgfr1 and Fgfr2 ablation results in worsened cardiac functional recovery and increased hypokinetic area at 7 but not 1 day after I/R injury. Tie2-Cre, Fgfr1^f/f, Fgfr2^f/f DCKO hearts show reduced ejection fraction (A) and increased hypokinetic area (B) at 7 days but not 1 day after ischemia-reperfusion (I/R) injury. C: representative images of Masson's trichrome stained control and Tie2-Cre, Fgfr1^f/f, Fgfr2^f/f DCKO mice 7 days after I/R injury. Scar size (%LV) is significantly larger in Tie2-Cre, Fgfr1^f/f, Fgfr2^f/f DCKO mice 7 days after I/R injury compared with controls. Scale bar = 1 mm. *P < 0.05 vs. control; #P < 0.05 vs. sham; +P < 0.05 vs. 1-day time point; n = 4–12.

Fig. 4.

Endothelial Fgfr1 and Fgfr2 ablation does not affect CD45-positive cell number after I/R injury. A: representative images of CD45-positive cells in the peri-infarct area of control and Tie2-Cre, Fgfr1^f/f, Fgfr2^f/f DCKO mice after I/R injury (green: CD45; blue: DAPI). Scale bar = 20 μm. B: quantitation of CD45-positive cells showing significantly increased numbers at 3 days and significantly decreased numbers at 7 days after I/R injury compared with 1 day after I/R injury in both control and Tie2-Cre, Fgfr1^f/f, Fgfr2^f/f DCKO hearts. There is no significant difference in CD45-positive cell number between control and Tie2-Cre, Fgfr1^f/f, Fgfr2^f/f DCKO hearts at any time point. #P < 0.01 vs. 1-day time point; n = 4–8.

Fig. 5.

The cardiac hypertrophic response after cardiac I/R injury is not affected by lack of endothelial Fgfr1 and Fgfr2. A: representative images of wheat germ agglutinin (WGA) staining in the peri-infarct area of control and Tie2-Cre, Fgfr1^f/f, Fgfr2^f/f DCKO mice at 3 and 7 days after I/R injury or sham treatment (green: WGA; blue: DAPI). Scale bar = 50 μm. B: after I/R injury, there is a significant increase in cardiomyocyte cross-sectional area in the peri-infarct area, but there is no significant difference in the cardiac hypertrophic response between control and Tie2-Cre, Fgfr1^f/f, Fgfr2^f/f DCKO hearts at any time point. #P < 0.05 vs. sham; n = 4–13.

Fig. 6.

Endothelial Fgfr1 and Fgfr2 are required for vascular remodeling after I/R injury. A: representative images of immunofluorescence for α-smooth muscle actin (red) and nuclei (DAPI: blue) in the peri-infarct area of the LV. Scale bar = 100 μm. B: after I/R injury, there is a significant decrease in the number of vessels in the peri-infarct area and Tie2-Cre, Fgfr1^f/f, Fgfr2^f/f DCKO hearts show decreased vessel density compared with controls at 7 days after I/R injury. C: there are not significant differences in vessel diameter in any treatment group. *P < 0.05 vs. control; #P < 0.05 vs. sham; n = 4–8.

Fig. 7.

Endothelial Fgfr1 and Fgfr2 regulate capillary density during postischemic cardiac remodeling. A: representative images of immunofluorescence for CD31 (red) and nuclei (blue) showing capillaries in the peri-infarct area. Scale bar = 100 μm. B: Tie2-Cre, Fgfr1^f/f, Fgfr2^f/f DCKO hearts have significantly decreased capillary density in the peri-infarct area at 7 days after I/R injury compared with controls. C: no difference in capillary area was observed in any treatment group. *P < 0.05 vs. control; #P < 0.05 vs. sham; n = 4–8.

Fig. 8.

Endothelial Fgfr1 and Fgfr2 mediate endothelial cell survival after cardiac I/R injury. A: representative images of immunofluorescence of CD31 (red) and Terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL)-positive cells (green) in the peri-infarct area. Inset: ×3 magnification showing a TUNEL-positive CD31-positive endothelial cell. Scale bar = 20 μm. B: representative images of immunofluorescence for CD31 (red) and bromodeoxyuridine (BrdU)-positive cells (green) in the peri-infarct area. Tie2-Cre, Fgfr1^f/f, Fgfr2^f/f DCKO hearts have significantly more TUNEL-positive endothelial cells at 3 days after I/R injury (C) but no significant difference in the number of BrdU-positive endothelial cells (D). *P < 0.05 vs. control; n = 5–9.

Fig. 9.

Hypoxyprobe staining scoring template. Representative images are shown to depict the scoring system that was used for blinded assessment of hypoxia. Scale bars = 200 μm in ×10 and 50 μm in ×40.

Fig. 10.

Endothelial Fgfr1 and Fgfr2 ablation results in persistent hypoxia in the peri-infarct area. A: representative images of hypoxyprobe labeling of hypoxia in the peri-infarct region of Tie2-Cre, Fgfr1^f/f, Fgfr2^f/f DCKO and control hearts 3 and 7 days after I/R injury. (brown: hypoxyprobe; blue: hematoxylin). Scale bar = 50 μm. Tie2-Cre, Fgfr1^f/f, Fgfr2^f/f DCKO hearts have a significantly higher average hypoxia score (B) and %hypoxic area (C) in the peri-infarct area of the LV compared with control hearts. *P < 0.05 vs. control; n = 3–4.