Foramen ovale closure is a process of endothelial-to-mesenchymal transition leading to fibrosis

Abstract

Patent foramen ovale (PFO) is an atrial septal deformity present in around 25% of the general population. PFO is associated with major causes of morbidity, including stroke and migraine. PFO appears to be heritable but genes involved in the closure of foramen ovale have not been identified. The aim of this study is to determine molecular pathways and genes that are responsible to the postnatal closure of the foramen ovale. Using Sprague-Dawley rat hearts as a model we analysed the dynamic histological changes and gene expressions at the foramen ovale region between embryonic day 20 and postnatal day 7. We observed a gradual loss of the endothelial marker PECAM1, an upregulation of the mesenchymal marker vimentin and α-smooth muscle actin, the elevation of the transcription factor Snail, and an increase of fibroblast activation protein (FAP) in the foramen ovale region as well as the deposition of collagen-rich connective tissues at the closed foramen ovale, suggesting endothelial-to-mesenchymal transition (EndMT) occurring during foramen ovale closure which leads to fibrosis. In addition, Notch1 and Notch3 receptors, Notch ligand Jagged1 and Notch effector HRT1 were highly expressed in the endocardium of the foramen ovale region during EndMT. Activation of Notch3 alone in an endothelial cell culture model was able to drive EndMT and transform endothelial cells to mesenchymal phenotype. Our data demonstrate for the first time that FO closure is a process of EndMT-mediated fibrosis, and Notch signalling is an important player participating in this process. Elucidation of the molecular mechanisms of the closure of foramen ovale informs the pathogenesis of PFO and may provide potential options for screening and prevention of PFO related conditions.

Figure 1. Selected tissue sections of the FO region showing FO closure in the fetal and neonatal rat heart.

A. Closure and fusion of the FO during E20–P6. B. Immunohistochemical staining for nuclear cell proliferation marker PCNA (brown) at P2. Both PCNA and Control sections were counterstained with haematoxylin showing blue nuclei. Scale bars = 200 µm in A; scale bars = 50 µm in B. RA, right atrium; LA, left atrium; SP, septum primum; SS, septum secundum; FO, foramen ovale.

Figure 2. Evidence of fibrosis during and after FO closure in rat heart.

A. H&E staining of FO region in neonates P4 and P7. Reduced cell density is shown by the arrows. Scale bars = 200. B. FAP mRNA expression during FO closure as determined by qRT-PCR. ***p<0.001, n = 3. C. Evidence of fibrosis in the remainder of FO region in adult rat hearts. a. H&E staining; b. Martius Scarlet Blue (MSB) staining. Scale bars = 500 µm. RA, right atrium; LA, left atrium; SP, septum primum; SS, septum secundum.

Figure 3. Immunohistochemistry staining for Snail in rat heart tissue sections during FO closure.

Positive signals are brown colour by DAB staining. Scale bars = 50 µm. Negative control: sections were stained with secondary antibodies only.

Figure 4. Immunohistochemistry staining for vimentin and PECAM1 in rat heart tissue sections during FO closure.

Double immunefluorescent staining was performed for vimentin (red) and PECAM1 (green). Scale bars = 50 µm. Nuclei were counterstained with DAPI (blue). Arrows indicate cells expressing both PECAM1 and vimentin. Negative control: sections were stained with secondary antibodies only.

Figure 5. Immunohistochemistry staining of Notch pathway proteins during FO closure in rat heart.

Tissue sections of rat FO region were immunestained with Notch1, Notch3, Jagged1 and HRT1 using specific antibodies and visualised by DAB detection kit (brown). All sections were nuclear counterstained with haematoxylin (blue). Scale bars = 200 µm. SP, septum primum; SS, septum secundum. Arrows indicate highly expressed proteins.

Figure 6. The mRNA expression of Notch1, Notch3, Jagged1 and HRT1 in the FO region of rat heart.

Total RNA of FO region was extracted from tissue samples obtained by laser microdissection and subjected to qRT-PCR. Results are presented as relative expression in comparison to E20. **p<0.01, ***p<0.001, n = 3.

Figure 7. Activation of Notch3 in endothelial cells induces nuclear Snail expression. hCAECs were infected by adenovirus-N3ICD (A–C) or adenovirus vector-only (D–F).

48 hours after the infection the cells were double stained for Notch3 (red) and Snail (green). C and F, merged image with DAPI staining of the nuclei. Scale bar = 50 µm.

Figure 8. Activation of Notch3 in endothelial cells induces cellular changes typical of EndMT.

A, Changes in cell morphology 7 days after infection (A-a), and the expression of vimentin and PECAM1 in N3ICD infected cells (A-b and A-c). Arrows in A-b indicate loss of membrane staining of PECAM1 in Notch3 positive cells; and arrows in A-f indicate membrane PECAM1. Arrows in A-c indicate cells exhibiting cell spreading. B, Western blotting for mesenchymal marker (vimentin) and endothelial markers (PECAM1) with β-Actin as a protein loading control. C & D, Quantitation of the protein expressions of PECAM1 (C) and vimentin (D). Data presented as the ratio to β-Actin, mean ± SE. *p<0.05, n = 3. Scale bar = 50 µm.