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Accelerated coronary angiogenesis by vegfr1-knockout endocardial cells.

Zhang Z, Zhou B - PLoS ONE (2013)

Bottom Line: We also revealed by qPCR analysis that expression of genes involved in the Vegf-Notch pathway was augmented in the Vegfr1- hearts.We further showed that inhibition of Notch signaling blocked the formation of coronary plexuses by the ventricular endocardial cells.These results establish that Vegfr1 produced in the endocardium negatively regulates embryonic coronary angiogenesis, possibly by limiting the Vegf-Notch signaling.

View Article: PubMed Central - PubMed

Affiliation: The State Key Laboratory of Biotherapy, West China Medical School of Sichuan University, Chengdu, Sichuan, China.

ABSTRACT
During mouse heart development, ventricular endocardial cells give rise to the coronary arteries by angiogenesis. Myocardially-derived vascular endothelial growth factor-a (Vegfa) regulates embryonic coronary angiogenesis through vascular endothelial growth factor-receptor 2 (Vegfr2) expressed in the endocardium. In this study, we investigated the role of endocardially-produced soluble Vegfr1 (sVegfr1) in the coronary angiogenesis. We deleted sVegfr1 in the endocardium of the developing mouse heart and found that this deletion resulted in a precocious formation of coronary plexuses. Using an ex vivo coronary angiogenesis assay, we showed that the Vegfr1- ventricular endocardial cells underwent excessive angiogenesis and generated extensive endothelial tubular networks. We also revealed by qPCR analysis that expression of genes involved in the Vegf-Notch pathway was augmented in the Vegfr1- hearts. We further showed that inhibition of Notch signaling blocked the formation of coronary plexuses by the ventricular endocardial cells. These results establish that Vegfr1 produced in the endocardium negatively regulates embryonic coronary angiogenesis, possibly by limiting the Vegf-Notch signaling.

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Disruption of Vegfr1 in the endocardium results in the precocious and abnormal coronary plexuses.A–H, Wholemount Pecam1 immunostainings of E11.5 hearts show the newly formed coronary plexuses in the ventricles. Note greatly increased formation of these primitive vessels by the R1 CKO hearts (B, D, F, H) compared to the control hearts (A, C, E, G). I, Statistic analysis showing the increase is significant. n  = 7 hearts for each group, *p<0.05, error bars = SD. J–O, Immunostained frontal sections of E11.5 ventricles with Pecam1 antibodies showing that the precocious coronary plexuses (red staining) in the R1 CKO ventricles are dilated and resemble the primitive blood islands (K, M, O; indicated by arrowheads and asterisks), which are not present in the control hearts (J, L, N). Dash lines and schematic diagrams showing these early-formed abnormal coronary plexuses within the myocardium of the R1 CKO hearts. ep, epicardium; myo, myocardium; oft, outflow track; lv/rv, left/right ventricle; tb, trabeculae; scale bar  = 50 µm.
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pone-0070570-g002: Disruption of Vegfr1 in the endocardium results in the precocious and abnormal coronary plexuses.A–H, Wholemount Pecam1 immunostainings of E11.5 hearts show the newly formed coronary plexuses in the ventricles. Note greatly increased formation of these primitive vessels by the R1 CKO hearts (B, D, F, H) compared to the control hearts (A, C, E, G). I, Statistic analysis showing the increase is significant. n  = 7 hearts for each group, *p<0.05, error bars = SD. J–O, Immunostained frontal sections of E11.5 ventricles with Pecam1 antibodies showing that the precocious coronary plexuses (red staining) in the R1 CKO ventricles are dilated and resemble the primitive blood islands (K, M, O; indicated by arrowheads and asterisks), which are not present in the control hearts (J, L, N). Dash lines and schematic diagrams showing these early-formed abnormal coronary plexuses within the myocardium of the R1 CKO hearts. ep, epicardium; myo, myocardium; oft, outflow track; lv/rv, left/right ventricle; tb, trabeculae; scale bar  = 50 µm.

Mentions: Embryonic coronary angiogenesis in mice takes place between E11.5 and E12.5. To determine the role of endocardially-produced Vegfr1 in this process, we disrupted Vegfr1 in the endocardium by using the Nfatc1Cre and Vegfr1f/f mice. The Rosa26 Cre reporter line, R26fslacZ[37], was used to validate the endocardial-specific deletion, which showed the Cre-mediated lacZ expression present only in the endocardium of E12.5 heart (Fig. 1A, 1B). Further, semi-quantitative PCR analysis showed expression of sVegfr1 was completely abolished in the E11.5 R1 CKO hearts, whereas its expression outside the heart was not affected (Fig. 1C), thus confirming the tissue specificity and efficacy of sVegfr1 deletion. The deletion was not embryonic lethal; however, it resulted in abnormal primitive coronary vessels and thin myocardium (Fig. 1D). Specifically, wholemount Pecam1 staining revealed that the primitive coronary plexuses developed precociously in the R1 CKO hearts at E11.5 (Fig. 2A-H). Quantitative analysis confirmed that, while an average of 15 early vessel-forming sites was seen in the control heart at this stage, this number was significantly increased by 3-fold in the R1 CKO heart (Fig. 2I). Close examination of Pecam1-stainined sections showed that these precociously formed vessels by the Vegfr1- endocardial cells were abnormally dilated; their lumens directly attached to the ventricular endocardium and penetrated through the ventricular myocardium into the subepicardial space (Fig. 2J, 2L, 2N vs. 2K, 2M, 2O).


Accelerated coronary angiogenesis by vegfr1-knockout endocardial cells.

Zhang Z, Zhou B - PLoS ONE (2013)

Disruption of Vegfr1 in the endocardium results in the precocious and abnormal coronary plexuses.A–H, Wholemount Pecam1 immunostainings of E11.5 hearts show the newly formed coronary plexuses in the ventricles. Note greatly increased formation of these primitive vessels by the R1 CKO hearts (B, D, F, H) compared to the control hearts (A, C, E, G). I, Statistic analysis showing the increase is significant. n  = 7 hearts for each group, *p<0.05, error bars = SD. J–O, Immunostained frontal sections of E11.5 ventricles with Pecam1 antibodies showing that the precocious coronary plexuses (red staining) in the R1 CKO ventricles are dilated and resemble the primitive blood islands (K, M, O; indicated by arrowheads and asterisks), which are not present in the control hearts (J, L, N). Dash lines and schematic diagrams showing these early-formed abnormal coronary plexuses within the myocardium of the R1 CKO hearts. ep, epicardium; myo, myocardium; oft, outflow track; lv/rv, left/right ventricle; tb, trabeculae; scale bar  = 50 µm.
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pone-0070570-g002: Disruption of Vegfr1 in the endocardium results in the precocious and abnormal coronary plexuses.A–H, Wholemount Pecam1 immunostainings of E11.5 hearts show the newly formed coronary plexuses in the ventricles. Note greatly increased formation of these primitive vessels by the R1 CKO hearts (B, D, F, H) compared to the control hearts (A, C, E, G). I, Statistic analysis showing the increase is significant. n  = 7 hearts for each group, *p<0.05, error bars = SD. J–O, Immunostained frontal sections of E11.5 ventricles with Pecam1 antibodies showing that the precocious coronary plexuses (red staining) in the R1 CKO ventricles are dilated and resemble the primitive blood islands (K, M, O; indicated by arrowheads and asterisks), which are not present in the control hearts (J, L, N). Dash lines and schematic diagrams showing these early-formed abnormal coronary plexuses within the myocardium of the R1 CKO hearts. ep, epicardium; myo, myocardium; oft, outflow track; lv/rv, left/right ventricle; tb, trabeculae; scale bar  = 50 µm.
Mentions: Embryonic coronary angiogenesis in mice takes place between E11.5 and E12.5. To determine the role of endocardially-produced Vegfr1 in this process, we disrupted Vegfr1 in the endocardium by using the Nfatc1Cre and Vegfr1f/f mice. The Rosa26 Cre reporter line, R26fslacZ[37], was used to validate the endocardial-specific deletion, which showed the Cre-mediated lacZ expression present only in the endocardium of E12.5 heart (Fig. 1A, 1B). Further, semi-quantitative PCR analysis showed expression of sVegfr1 was completely abolished in the E11.5 R1 CKO hearts, whereas its expression outside the heart was not affected (Fig. 1C), thus confirming the tissue specificity and efficacy of sVegfr1 deletion. The deletion was not embryonic lethal; however, it resulted in abnormal primitive coronary vessels and thin myocardium (Fig. 1D). Specifically, wholemount Pecam1 staining revealed that the primitive coronary plexuses developed precociously in the R1 CKO hearts at E11.5 (Fig. 2A-H). Quantitative analysis confirmed that, while an average of 15 early vessel-forming sites was seen in the control heart at this stage, this number was significantly increased by 3-fold in the R1 CKO heart (Fig. 2I). Close examination of Pecam1-stainined sections showed that these precociously formed vessels by the Vegfr1- endocardial cells were abnormally dilated; their lumens directly attached to the ventricular endocardium and penetrated through the ventricular myocardium into the subepicardial space (Fig. 2J, 2L, 2N vs. 2K, 2M, 2O).

Bottom Line: We also revealed by qPCR analysis that expression of genes involved in the Vegf-Notch pathway was augmented in the Vegfr1- hearts.We further showed that inhibition of Notch signaling blocked the formation of coronary plexuses by the ventricular endocardial cells.These results establish that Vegfr1 produced in the endocardium negatively regulates embryonic coronary angiogenesis, possibly by limiting the Vegf-Notch signaling.

View Article: PubMed Central - PubMed

Affiliation: The State Key Laboratory of Biotherapy, West China Medical School of Sichuan University, Chengdu, Sichuan, China.

ABSTRACT
During mouse heart development, ventricular endocardial cells give rise to the coronary arteries by angiogenesis. Myocardially-derived vascular endothelial growth factor-a (Vegfa) regulates embryonic coronary angiogenesis through vascular endothelial growth factor-receptor 2 (Vegfr2) expressed in the endocardium. In this study, we investigated the role of endocardially-produced soluble Vegfr1 (sVegfr1) in the coronary angiogenesis. We deleted sVegfr1 in the endocardium of the developing mouse heart and found that this deletion resulted in a precocious formation of coronary plexuses. Using an ex vivo coronary angiogenesis assay, we showed that the Vegfr1- ventricular endocardial cells underwent excessive angiogenesis and generated extensive endothelial tubular networks. We also revealed by qPCR analysis that expression of genes involved in the Vegf-Notch pathway was augmented in the Vegfr1- hearts. We further showed that inhibition of Notch signaling blocked the formation of coronary plexuses by the ventricular endocardial cells. These results establish that Vegfr1 produced in the endocardium negatively regulates embryonic coronary angiogenesis, possibly by limiting the Vegf-Notch signaling.

Show MeSH
Related in: MedlinePlus