Fibroblast growth factor signals regulate a wave of Hedgehog activation that is essential for coronary vascular development

Abstract

Myocardial infarction and ischemic heart disease are the leading cause of death in the industrial world. Therapies employed for treating these diseases are aimed at promoting increased blood flow to cardiac tissue. Pharmacological induction of new coronary growth has recently been explored, however, clinical trials with known proangiogenic factors have been disappointing. To identify novel therapeutic targets, we have explored signaling pathways that govern embryonic coronary development. Using a combination of genetically engineered mice and an organ culture system, we identified novel roles for fibroblast growth factor (FGF) and Hedgehog (HH) signaling in coronary vascular development. We show that FGF signals promote coronary growth indirectly by signaling to the cardiomyoblast through redundant function of Fgfr1 and Fgfr2. Myocardial FGF signaling triggers a wave of HH activation that is essential for vascular endothelial growth factor (Vegf)-A, Vegf-B, Vegf-C, and angiopoietin-2 (Ang2) expression. We demonstrate that HH is necessary for coronary vascular development and activation of HH signaling is sufficient to promote coronary growth and to rescue coronary defects due to loss of FGF signaling. These studies implicate HH signaling as an essential regulator of coronary vascular development and as a potential therapeutic target for coronary neovascularization. Consistent with this, activation of HH signaling in the adult heart leads to an increase in coronary vessel density.

Figure 1.

Spatiotemporal development of coronary blood vessels. (A–C) Whole-mount PECAM staining of E11.5, E12.5, and E13.5 hearts. (A) At E11.5 coronary vessels emerge from the atrial–ventricular groove (arrow). (B,C) Between E12.5 and E13.5, coronary vessels extend in a wave-like pattern emanating from the atrial–ventricular and interventricular groove and extend toward the ventricular apices. (D–F) Histology at E11.5, E12.5, and E13.5 demonstrating formation of the subepicardial mesenchyme at E12.5 (E) and vessel-like structures within the subepicardial space at E13.5 (F). Green arrowheads denote location of the subepicardial mesenchyme. (G–I) Histological sections of PECAM-stained hearts demonstrating the presence of blood vessels growing within the subepicardial space (asterisk) and within the myocardial wall (black arrowhead). The asterisk and arrow mark the position of the most distal subepicardial and intramyocardial blood vessel, respectively. Open arrowhead denotes endocardial PECAM staining. Magnification: A–C, 25×; D–E,G–H, 400×; F,I, 200×. Bar in A and corresponding arrow in D represent orientation and position of histological sections. (B) Base of ventricle; (A) apex of ventricle.

Figure 2.

Fgf9 regulates coronary vascular development. (A,B) Whole-mount PECAM staining of E13.5 control (A) and Fgf9^−/− (B) hearts. Fgf9^−/− hearts display defects in growth of the coronary plexus. (C) Quantitation of the percentage of the ventricle covered by blood vessels in control and Fgf9^−/− hearts. Asterisk indicates statistically significant difference compared with controls (p < 0.01). (D–I) Histology of control (D–F) and Fgf9^−/− (G–I) hearts demonstrating defects in formation of the subepicardial mesenchyme (green arrowheads) in Fgf9^−/− (H) compared with controls (E) at E12.5. PECAM-stained section of control (F) and Fgf9^−/− (I) hearts showing failure of both subepicardial (asterisk) and intramyocardial (black arrowhead) vessel growth at E13.5. The asterisk and arrowhead mark the position of the most distal subepicardial and intramyocardial blood vessel, respectively. Open arrowhead denotes endocardial PECAM staining. Magnification: A,C, 25×; D,E,G,H, 400×; F,I, 200×. Bar in A and corresponding arrow in D represent orientation and position of histological sections. (B) Base of ventricle; (A) apex of ventricle.

Figure 3.

FGF signaling to endothelial cells is not required for coronary vascular development. (A–D) Endothelial cell targeting with Tie1-cre. PECAM staining at E13.5 of control (A) and Fgfr1/2 ^Tie1-cre DCKO (B) hearts demonstrating formation of a normal vascular plexus in Fgfr1/2^Tie1-cre DCKO hearts. ROSA26-LacZ staining of Fgfr1/2^Tie1-cre DCKO hearts at E13.5 (C) and E16.5 (D) reveals that Cre-mediated recombination has occurred in endothelial cells. (E–H) Endothelial cell targeting with Flk1-cre. PECAM staining of control (E) and Fgfr1/2^Flk1-cre DCKO (F) hearts demonstrating formation of a normal vascular plexus in Fgfr1/2^Flk1-cre DCKO hearts. ROSA26-LacZ staining of Fgfr1/2^Flk1-cre DCKO hearts at E13.5 (G) and E16.5 (H) reveals that Cre-mediated recombination has occurred in endothelial cells. Magnification: A,B,E,F, 25×; C,D,G,H, 400×.

Figure 4.

FGF signaling to the cardiomyoblast is essential for coronary vascular development. (A–F) Whole-mount PECAM staining of control (A–C) and Fgfr1/2^Mlc2v-cre DCKO hearts (D–F). At E11.5, coronary vessels emerge from the atrial–ventricular groove (arrow) of both control (A) and Fgfr1/2^Mlc2v-cre DCKO (D) hearts. At E12.5 the vascular plexus begins to extend toward the ventricular apices in control hearts (B) but is delayed in Fgfr1/2^Mlc2v-cre DCKO (E) hearts. By E13.5, control hearts (C) contain a vascular plexus that encases the entire ventricle, while Fgfr1/2^Mlc2v-cre DCKO hearts (F) contain a vascular plexus that fails to cover the ventricle. (G–L) Histology of control (G–I) and Fgfr1/2^Mlc2v-cre DCKO (J–L) hearts demonstrating defects in formation of the subepicardial mesenchyme (green arrowheads) in Fgfr1/2^Mlc2v-cre DCKO hearts (K) compared with controls (H) at E12.5. PECAM-stained section of control (I) and Fgfr1/2^Mlc2v-cre DCKO (L) hearts showing defects in both subepicardial (asterisk) and intramyocardial (black arrowhead) vessel growth at E13.5. The asterisk and black arrowhead mark the position of the most distal subepicardial and intramyocardial blood vessel, respectively. The open arrowhead denotes endocardial PECAM staining. (M) Quantitation of the percentage of the ventricle covered by blood vessels at E13.5 in control and Fgfr1/2^Mlc2v-cre DCKO hearts. Asterisk indicates statistically significant differences compared with controls (p < 0.01). Magnification: A–F, 25×; G–L, 400×. Bar in A and corresponding arrow in G represent orientation and position of histological sections. (B) Base of ventricle; (A) apex of ventricle.

Figure 5.

FGF signaling to the cardiomyoblast is necessary for Vegf and Angiopoietin expression. (A–C) Whole-mount in situ hybridization for Vegfr-1 (A), Vegfr-2 (B), and Vegfr-3 (C) at E13.5. Vegfr-1 and Vegfr-2 are expressed in coronary endothelial cells, while Vegfr-3 is expressed uniformly within the atrial–ventricular groove. Cryosections reveal Vegfr-1 and Vegfr-2 expression in both subepicardial (asterisk) and intramyocardial (arrowhead) blood vessels and Vegfr-3 expression in epicardial cells (open arrowhead). (D–F) Wave-like progression of Vegf-A expression emanating from the atrial–ventricular and interventricular groove at E12.5 (D) and extending to cover the ventricle by E13.5 (E) in control hearts. (F) Cryosection revealing Vegf-A expression in the myocardium. Open arrowhead denotes lack of staining in the epicardium and subepicardial mesenchyme. (J,K) Vegf-A expression is decreased in Fgfr1/2^Mlc2v-cre DCKO hearts at E12.5 (J) and E13.5 (K) and fails to cover the ventricle at E13.5 compared with controls (shown in E). (G–I) Wave-like progression of Vegf-B expression emanating from the atrial–ventricular and interventricular groove at E12.5 (G) and extending to cover the ventricle by E13.5 (H) in control hearts. (I) Cryosection revealing Vegf-B expression in the myocardium. Open arrowhead denotes lack of staining in the epicardium and subepicardial mesenchyme. (L,M) Vegf-B expression is decreased in Fgfr1/2^Mlc2v-cre DCKO hearts at E12.5 (L) and E13.5 (M) and fails to cover the ventricle at E13.5 compared with controls (shown in H). (N,O) Wave-like progression of Vegf-C expression emanating from the atrial–ventricular and interventricular groove at E12.5 (N) and extending to cover the ventricle by E13.5 (O) in control hearts. (P) Cryosection revealing Vegf-C expression in the perivascular cells (arrowhead). (T,U) Vegf-C expression is decreased in Fgfr1/2^Mlc2v-cre DCKO hearts at E12.5 (T) and E13.5 (U) and fails to cover the ventricle at E13.5 compared with controls (shown in O). (Q,R) Vegf-D is expressed in the atrial–ventricular groove at E12.5 (Q) and E13.5 (R) in control hearts. (S) Cryosections reveal Vegf-D expression in myocardial cells. (V,W) Vegf-D expression is unchanged in Fgfr1/2^Mlc2v-cre DCKO hearts at both E12.5 (V) and E13.5 (W). (X) qRT–PCR analysis of control and Fgfr1/2^Mlc2v-cre DCKO hearts demonstrating decreases in Ang-2, Vegf-A, Vegf-B, and Vegf-C expression. Asterisk represents statistically significant difference compared with controls (p < 0.01). All whole-mount specimens were photographed at 25× magnification and cryosections were photographed at 400× magnification.

Figure 6.

FGF signaling to the cardiomyoblast controls a wave of HH signaling. (A–F) Whole-mount in situ hybridization for Shh. Shh is expressed in the atrial–ventricular groove at E12.5 (A) and E13.5 (B). At E12.5 (C) Shh expression is nearly absent from Fgfr1/2^Mlc2v-cre DCKO hearts but begins to be expressed by E13.5 (D). (E) qRT–PCR demonstrating Shh mRNA decrease in Fgfr1/2^Mlc2v-cre DCKO hearts (p < 0.01). (F) Cryosections of A reveal Shh expression in the epicardium. (G) Immunohistochemistry showing SHH protein expression in the epicardium (red). (Blue) PECAM; (green) cardiac actin. (H–O) Whole-mount in situ hybridization for Ptc1 demonstrating a wave-like progression of Ptc1 expression between E12.5 and E13.5. Ptc1 is expressed in the atrial–ventricular and interventricular groove at E12.5 (H) and extends to cover the ventricle by E13.5 (I). Ptc1 expression is decreased compared with controls at both E12.5 (J) and E13.5 (K) in Fgfr1/2^Mlc2v-cre DCKO hearts. Ptc1 expression also fails to progress in a wave-like fashion and does not cover the ventricle at E13.5 in Fgfr1/2^Mlc2v-cre DCKO hearts. Cryosections of H reveal Ptc1 expression in the cardiomyoblasts (L, arrow) and perivascular cells (N, arrowhead). Immunohistochemistry showing PTC1 protein expression (red) in the myocardium and perivascular cells. (M) Cryosection showing cardiac actin and PECAM expression. (O) Same section as M, but showing PTC1 and PECAM expression. (Blue) PECAM; (green) cardiac actin. White arrowheads indicate perivascular cells positive for PTC1 (O) but lacking cardiac actin staining (M). All whole-mount specimens were photographed at 25× magnification and cryosections were photographed at 400× magnification.

Figure 7.

FGF–HH pathway regulates coronary vascular development and VEGF expression. (A–C) Treatment of heart slices with either FGF9 or SHH led to induction of Vegf-A expression compared with BSA controls. (D–F) Addition of the HH inhibitor, cyclopamine, ablated the ability of either FGF9 or SHH protein to induce Vegf-A expression compared with BSA controls. Vegf-A expression was detected by LacZ staining for a VEGFA-LacZ genetrap. (G–L) Treatment of control (G–I) or Fgfr1/2^Mlc2v-cre DCKO (J–L) heart slices with BSA, FGF9 and SHH protein. In response to FGF9 protein, control hearts (H) showed robust increases in Vegf-A expression, while Fgfr1/2^Mlc2v-cre DCKO (K) hearts showed no induction of Vegf-A expression compared with BSA control. In contrast, SHH treatment of both control (I) and Fgfr1/2^Mlc2v-cre DCKO (L) hearts led to robust increases in Vegf-A expression. Vegf-A expression was assayed by in situ hybridization. (M–P) PECAM staining of whole hearts cultured for 24 (M) or 48 (N) h, demonstrating that coronary vessels grow in a wave-like fashion. (O–P) Cyclopamine treatment abolishes coronary vessel development. (Q–T) In situ hybridization showing that cyclopamine treatment suppresses Vegf-A expression at both 24 h (S) and 48 h (T) compared with controls (Q,R). (U–Y) PECAM staining indicating that coronary defects due to cyclopamine treatment (V) can be rescued by cotreatment with VEGF-A₁₆₅ and ANG2 (Y, arrow). Treatment with ANG2 (W) or VEGF-A165 (X) only, was unable to rescue coronary development in cyclopamine-treated hearts. Hearts were cultured for 48 h. All explants were photographed at 32× magnification.

Figure 8.

Activation of HH signaling in the embryonic myocardium increases subepicardial blood vessel growth. (A,B) LacZ staining of control (A) and GLI2*/Mlc2v-Cre (B) hearts at E12.5. Reduction of LacZ staining in GLI2*/Mlc2v-Cre hearts demonstrates efficient Cre-mediated recombination of the transgene throughout the ventricular myocardium. (C–F) PECAM staining of control (C,E) and GLI2*/Mlc2v-Cre (D,F) hearts. Both control (C) and GLI2*/Mlc2v-Cre (D) hearts contain a normally patterned coronary plexus at E13.5. High magnification reveals that GLI2*/Mlc2v-Cre hearts (F) have a denser coronary plexus than controls (E). (G,H) Histological sections of PECAM-stained control (G) and GLI2*/Mlc2v-Cre (H) hearts reveals increased number of subepicardial blood vessels. (I) Quantitation of blood vessel number/20× field demonstrated statistically significant increases in subepicardial but not intramyocardial blood vessels (p < 0.01). (J,K) In situ hybridization for Ptc1 expression showing increased expression in GLI2*/Mlc2v-Cre hearts (K) compared with controls (J). (L,M) In situ hybridization for Vegf-A expression showing increased expression in GLI2*/Mlc2v-Cre hearts (M) compared with controls (L). (N) qRT–PCR analysis of control and GLI2*/Mlc2v-Cre hearts revealing significant increases in Ang2, Ptc1, and Vegf-A expression. Asterisk indicates statistically significant differences compared with controls (p < 0.01). All whole-mount specimens except E and F were photographed at 25×; E and F were photographed at 90× magnification. Histological sections were photographed at 400× magnification.

Figure 9.

Activation of HH signaling rescues coronary defects in hearts lacking myocardial FGF signaling. (A–C) PECAM staining of control (A), Fgfr1/2^Mlc2v-cre DCKO (B), and Fgfr1/2^Mlc2v-cre;GLI2* hearts (C) at E13.5. Control hearts contained a vascular plexus that encased the entire ventricle, while Fgfr1/2^Mlc2v-cre DCKO hearts contained a vascular plexus that failed to enclose the ventricle. Fgfr1/2^Mlc2v-cre;GLI2* hearts contained a vascular plexus that, like controls, covered the ventricle. (D–F) In situ hybridization for Vegf-A in control (D), Fgfr1/2^Mlc2v-cre DCKO (E), and Fgfr1/2^Mlc2v-cre;GLI2* hearts (F). Fgfr1/2^Mlc2v-cre DCKO hearts displayed decreased and restricted Vegf-A expression, while Fgfr1/2^Mlc2v-cre;GLI2* hearts displayed Vegf-A expression indistinguishable from controls. (G–I) Histological sections of E13.5 PECAM-stained hearts. Fgfr1/2^Mlc2v-cre DCKO hearts (H) had decreased subepicardial (asterisk) and intramyocardial (black arrowhead) blood vessels compared with controls (G). (I) Fgfr1/2^Mlc2v-cre;Gli2* hearts displayed rescued subepicardial (asterisk) but not intramyocardial (black arrowhead) blood vessel development. The asterisk and black arrowhead mark the position of the most distal subepicardial and intramyocardial blood vessel, respectively. Open arrowhead denotes endocardial PECAM staining. (J) Quantitation of the percent of the ventricle covered by blood vessels at E13.5 demonstrating that only Fgfr1/2^Mlc2v-cre DCKO hearts had statistically significant differences from controls. Asterisk indicates a statistically significant difference (p < 0.01). (K) qRT–PCR analysis confirming that Fgfr1/2^Mlc2v-cre;GLI2* hearts contain wild-type levels of Vegf-A. Asterisk indicates a statistically significant difference (p < 0.01) compared with both control and Fgfr1/2^Mlc2v-cre;GLI2* hearts. Bar in A and corresponding arrow in G represent orientation and position of histological sections. (B) Base of ventricle; (A) apex of ventricle. All whole-mount specimens were photographed at 25×. Histological sections were photographed at 400× magnification.

Figure 10.

Activation of HH signaling in the adult heart increases coronary vessel density. (A,B) Histological sections of control (A) and GLI2*/αMHC-ER-Cre (B) hearts after 5 d of transgene induction showing hypercellularity in the interstitial space (green arrow). (C,D) Immunofluorescent PECAM staining of control (C) and GLI2*/αMHC-ER-Cre (D) hearts revealing increased coronary vessel density in the interstitial space. (E,F) Streptavidin-HRP staining (arrows) following intravascular injection of biotinylated Tomato Lectin into control (E) and GLI2*/αMHC-ER-Cre (F) animals demonstrates that HH-induced blood vessels are connected to the systemic vasculature. (G) Quantitation of increased coronary vessel number by qRT–PCR demonstrating that GLI2*/αMHC-ER-Cre hearts have significantly elevated levels of Pecam and Vegfr-2 expression. (H) qRT–PCR indicating increased Ang2, Ptc1, and Vegf-A expression in GLI2*/αMHC-ER-Cre hearts. Asterisk indicates statistically significant differences compared with controls (p < 0.01). Histological sections were photographed at 400× magnification.