Preclinical Assessment of Atorvastatin for Treatment of Endocardial Fibroelastosis

Figures

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  Figure 1. Fibrogenic Activation in HEECs Is Significantly Inhibited by Atorvastatin and by Exposure to Flow at the Protein Level (A) Representative immunohistochemical images double-stained with CD31 (red) and ⍺-smooth muscle actin (⍺-SMA) (green) for unstimulated and transforming growth factor-β– stimulated (TGF-β-stim) healthy endocardial endothelial cells (HEECs) under no-flow conditions. (B) Atorvastatin treated HEECs display significantly less ⍺-SMA compared to untreated cells. Quantitative analysis of ⍺-SMA and collagen expression was evaluated in unstimulated (C,D) and TGF-β-stim cells (E,F) via immunohistochemistry. (C) Atorvastatin significantly decreased ⍺-SMA expression in unstimulated HEECs under no-flow conditions (P < 0.001). Exposure to flow further reduced ⍺-SMA expression (P < 0.001). (D) Flow, but not atorvastatin, significantly reduced collagen expression in HEECs (no flow, untreated vs flow, untreated, P < 0.001; no flow + atorvastatin vs flow, untreated, P < 0.001). (E) Atorvastatin significantly decreased ⍺-SMA expression and TGF-β-stim HEECs under no-flow conditions (P < 0.001). Exposure to flow further reduced ⍺-SMA expression (P < 0.001). (F) Atorvastatin under…

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  Figure 2. Fibrogenic Activation of HEECs Is Significantly Inhibited on Transcription Level by Atorvastatin and by Exposure to Flow Gene expression of Acta2 (A,F), Smad2 (B,G), Col1A1 (C,H), Klf2 (D,I), and Snai2 (E,J) was quantified via quantitative real-time polymerase chain reaction for HEECs under no flow (± atorvastatin) and flow (± atorvastatin) in unstimulated (A-E) and TGF-β-stim (F-J) HEECs. Atorvastatin treatment and exposure to flow inhibited Acta2 and Snai2 expression in both unstimulated ([A] no flow, untreated vs no flow + atorvastatin, P < 0.001; flow, untreated vs flow + atorvastatin, P = 0.001; no flow, untreated vs flow, untreated, P < 0.001; and [B] no flow, untreated vs no flow + atorvastatin, P < 0.001; no flow, untreated vs flow, untreated, P < 0.001) and TGF-β-stim HEECs ([F] no flow, untreated vs no flow + atorvastatin, P < 0.001; no flow, untreated vs flow, untreated, P < 0.001; and [G] no flow, untreated vs no flow + atorvastatin, P = 0.043, n = 6; no flow, untreated vs flow, untreated, P < 0.001). In unstimulated HEECs, this was accompanied by atorvastatin and flow-dependent down-regulation of Smad2 ([E] no flow, untreated vs no flow + atorvastatin, P < 0.001; no…

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  Figure 3. Fibrogenic Activation of EdEECs Is Significantly Inhibited on Transcription Level by Atorvastatin Gene expression of ACTA2 (B,G), SMAD2 (A,F), COL1A1 (C,H), and KLF2 (E) was quantified via quantitative real-time polymerase chain reaction under no flow (± atorvastatin) and flow (± atorvastatin) conditions in endocardial fibroelastosis (EFE)-derived endocardial endothelial cells (EdEECs). Atorvastatin therapy inhibited ACTA2 expression under both no-flow and flow conditions ([B] no-flow, untreated vs no-flow + atorvastatin, P = 0.027; and [G] flow, untreated vs flow + atorvastatin, P = 0.013). The effect of atorvastatin was associated with a significant increase in KLF2 ([E] analysis of variance with Tukey multiple comparison test; no flow, untreated vs no flow + atorvastatin, P = 0.008; no flow, untreated vs flow, untreated, P = 0.003; and no flow, untreated vs flow + atorvastatin, P = 0.015) and under flow exposure also with a decrease in the downstream intermediate of KLF2, SMAD2 ([F] flow, untreated vs flow + atorvastatin, P = 0.010). The pathway is shown in D. No changes were observed in collagen expression. Flow cytometry experiments were performed on cells derived from 12…

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  Figure 4. Left Ventricular Endocardial Fibroelastosis Significantly Decreased in Atorvastatin-Treated Rat Hearts (A) Representative images of untreated/vehicle-treated rat hearts with a continuous layer of EFE throughout the LV, as indicated by Masson’s trichrome staining. Fibrogenic activation of endocardial endothelial cells was confirmed by immunohistochemical double staining for VE-cadherin (red) and αSMA (green) throughout the EFE layer, which infiltrates the underlying myocardium. (B) Atorvastatin-treated rat hearts show thin patches of EFE without infiltration into the myocardium. (C) Bar graphs summarize EFE thickness (n = 8/7). The endocardial layer of each heart was measured using 12 fields of vision with a clockwise pattern within the center of the LV. Significantly less EFE in atorvastatin-compared to untreated/vehicle-treated hearts (P < 0.001). (D) Representative western blots for collagen type I cytosolic protein expression (n = 6/7) and (E) quantification via densitometry. Densitometry measurements were assessed for equal protein loading through cyclophilin B loading and were expressed as changes compared to the untreated/vehicle-treated group. Atorvastatin-treated rat…

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  Figure 5. The Expression of pSMAD2/3 Was Significantly Higher in the Untreated and Vehicle-Treated Rat Hearts (A) Immunohistochemical double-staining with CD-31 (red) and pSMAD2/3 (green) reveals the expression of pSMAD2/3 in endocardial cell nuclei, which is decreased in atorvastatin-treated hearts. (B) SMAD2/3 cytosolic protein expression is presented through representative western blots (n = 6/7), and (D) quantified by densitometry. SMAD2/3 was less expressed in atorvastatin-treated rat hearts (P = 0.0334); (C) pSMAD2 and pSMAD3 nuclear protein expression was evaluated via western blotting (n = 3/3) and quantified by densitometry (E, F). Densitometry measurements were assessed for equal protein loading through cyclophilin B for cytosolic proteins and histone H3 for nuclear proteins, and all measurements were expressed as changes relative to the untreated/vehicle-treated group. pSMAD2 was less in nuclear protein expression of Atorvastatin treated rat hearts (P < 0.001), whereas pSMAD3 was equally expressed in the comparison groups (P = 0.90). Complete unedited gels displayed in supplemental files. Graphs represent the mean ± SEM. ∗∗P < 0.01, ∗∗∗P < 0.001, ns: not significant, two-sided…

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  Figure 6. Slug/Snail Is Highly Expressed in the Nuclei of Endocardial Endothelial Cells Undergoing Fibrogenic Activation Suggestive of EndMT Increased Slug/Snail expression in a (A) representative immunohistochemical images double-stained with CD-31 (red) and Slug/Snail (green) when compared to (B) atorvastatin-treated endocardial endothelial cell nuclei. (C) qRT-PCR gene expression for Slug/Snail was significantly downregulated in atorvastatin-treated rat hearts (P = 0.028) (n = 8/7) and (D) KLF2 was upregulated (P = 0.022; n = 7/6). Data are presented as fold changes normalized to native neonatal rat hearts for Slug/Snail or untreated/vehicle in Klf2. Graphs represent the mean ± SEM. ∗P < 0.05, two-sided t-test or Wilcoxon rank-sum test were performed as applicable following test for normality. White arrows indicate high expression of EndMT transcription factor Slug/Snail. CD31 = cluster of differentiation 31; DAPI = 4’,6-diamidino-2-phenylindole; Klf2 = Krüppel-like factor 2; LV = left ventricle.

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  Figure 7. Heterotopic Rat Heart Transplantation and Echocardiographic Imaging to Confirm Donor Graft Viability (A) Clamping of the recipients’ infrarenal abdominal aorta (white arrow) with an incision for arterial anastomosis and the recipient's IVC (black arrow). (B) Arterial anastomosis of the transplanted non-perfused donor neonatal heart. (C) Viable donor organ after clamp opening with coronary artery perfusion. (D) Decreased left ventricular filling in diastole owing to flow stagnation within the LV. (E) Patent arterial (red arrow) anastomosis and retrograde filling of the donor ascending aorta into the coronary arteries. The blue arrow indicates venous anastomosis between the pulmonary trunk and the recipient's IVC. (F) Development of circumferential endocardial fibroelastosis within the LV of an untreated rat heart on postoperative day 14, as indicated by white arrows. Ao = aorta of the donor graft; IVC = inferior vena cava; LV = left ventricle.