Abnormal Flow Conditions Promote Endocardial Fibroelastosis Via Endothelial-to-Mesenchymal Transition, Which Is Responsive to Losartan Treatment

Figures

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  Figure 1. Neonatal Heterotopic Heart Transplantation With Different Left Ventricular Flow Conditions for EFE Modeling (A) Schema of flow in the left ventricle (LV) after neonatal heterotopic heart transplantation: native heart loaded/normal flow, unloaded/static flow, and regurgitant flow. (B) On day 7, native, loaded/normal flow, unloaded/static flow, and regurgitant flow models were examined with hematoxylin and eosin (H&E) staining that demonstrate a large area of fibrosis shown with the unloaded and regurgitant flow models. (C) Masson trichrome staining shows increased collagen deposition along the endocardium (yellow arrowheads) with the unloaded and regurgitant flow models (D) Immunofluorescence staining with 4’,6-diamidino-2-phenylindole (DAPI) (blue), CD31 (green), and αSMA (red) shows endocardial endothelial cells in the unloaded and regurgitant flow models undergoing endothelial-to-mesenchymal transition, denoted by co-expression of CD31 and αSMA (white arrowheads), whereas native and loaded models do not. α-SMA = alpha-smooth muscle actin; Ao = aortic; EFE = endocardial fibroelastosis; IVC = inferior vena cava; LA = left atrium; PA = pulmonary artery; RA = right atrium; RV =…

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  Figure 2. Absence of Normal Blood Flow is Associated With EFE-Like Remodeling Through EndoMT in Endocardium (A) Area of fibrosis in unloaded and regurgitant flow groups is found to be statistically increased compared to the loaded groups. (B) EFE tissue from human patient is surgically removed. H&E, Masson trichrome, and immunofluorescence staining with DAPI (blue), CD31 (green), and αSMA (red) show increased fibrosis and endothelial-to-mesenchymal transition (EndoMT). (C) Real-time quantitative polymerase chain reaction (RT qPCR) data show increased EndoMT mRNA (αSMA, TGFβ1, SNAIL, and SLUG) in unloaded and aortic regurgitation (AR) groups when compared to loaded and native hearts. (D) Schema of the TGFβ pathway shows SMAD cascade activation and upregulation of genes associated with EndoMT. (E) Western blot analysis shows increased SNAIL/SLUG protein in unloaded and AR groups when compared to loaded and native groups. GAPDH = glyceraldehyde 3-phosphate dehydrogenase; TGF = transforming growth factor; VE = vascular endothelial; other abbreviations as in Figure 1.

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  Figure 3. Treatment of Neonatal Transplanted Heart With Losartan Decreases EndoMT and Ameliorates EFE (A) Schema of losartan action on TGFβ production. (B) Area of fibrosis measurements shows statistically decreased fibrosis with losartan treatment. (C) H&E, Masson trichrome, and immunofluorescence staining with DAPI (blue), CD31 (green), αSMA (red), staining shows decreased endocardial fibroelastosis and EndoMT with losartan treatment. (D) RT qPCR shows decreased mRNA associated with EndoMT (α-SMA, TGF-β1, SNAIL, and SLUG) in the unloaded heart with losartan treatment. (E) Western blot analysis shows decreased SNAIL/SLUG protein in losartan treated groups denoting decreased EndoMT. Abbreviations as in Figures 1 and 2.

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  Figure 4. Examining HUEECs Isolated From LV in Response to Different Flow Conditions Fluorescence-activated cell sorting (FACS) analysis shows high levels of CD31 and low levels of PDGFR in isolated human endocardial endothelial cells (HUEECs). (B) Immunofluorescence staining shows increased expression of endocardial-specific marker NFATC1 in HUEECs when compared to human umbilical vein endothelial cells (HUVECs). (C) Monolayer of HUEECs under 5 dyne/cm2 shear stress shows cobblestone architecture and more arrangement in the direction of flow. (D) Measurement of cellular orientation angles shows that HUEECs subjected to laminar shear stress (LSS) have a narrow range of angles that align with flow, whereas HUEECs under static conditions have a wide range of angles consistent with disorientation. (E) Principle component analysis shows that samples cluster together by application of static or LSS conditions on PC3. (F) Volcano plot shows differential expressed genes enriched in static or LSS samples (G) Gene set enrichment analysis shows that Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of fluid response to shear stress are enriched in LSS compared to static groups. Heatmaps show…

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  Figure 5. Notch Signaling Pathway is Related to Flow-Triggered EFE Remodeling (A) RNA sequence analysis of HUEECs show increase NOTCH1 signaling with static conditions versus LSS. (B) NOTCH1 staining in the native, loaded, unloaded, and regurgitant flow models show increased NOTCH1 staining with unloaded/static flow and regurgitant flow groups. (C) NOTCH1 staining is found to be decreased with losartan treatment. (D) Western blot staining shows increased NOTCH1 protein expression in unloaded and AR samples, consistent with NOTCH1 staining. Similarly, losartan treatment decreases NOTCH1 expression. Abbreviations as in Figures 1, 2, and 4.