Vascular Endothelial Growth Factor Prevents Endothelial-to-Mesenchymal Transition in Hypertrophy - PubMed (original) (raw)

Vascular Endothelial Growth Factor Prevents Endothelial-to-Mesenchymal Transition in Hypertrophy

Ben M-W Illigens et al. Ann Thorac Surg. 2017 Sep.

Abstract

Background: In hypertrophy, progressive loss of function caused by impaired diastolic compliance correlates with advancing cardiac fibrosis. Endothelial cells contribute to this process through endothelial-to-mesenchymal transition (EndMT) resulting from inductive signals such as transforming growth factor (TGF-β). Vascular endothelial growth factor (VEGF) has proven effective in preserving systolic function and delaying the onset of failure. In this study, we hypothesize that VEGF inhibits EndMT and prevents cardiac fibrosis, thereby preserving diastolic function.

Methods: The descending aorta was banded in newborn rabbits. At 4 and 6 weeks, hypertrophied animals were treated with intrapericardial VEGF protein and compared with controls (n = 7 per group). Weekly transthoracic echocardiography measured peak systolic stress. At 7 weeks, diastolic stiffness was determined through pressure-volume curves, fibrosis by Masson trichrome stain and hydroxyproline assay, EndMT by immunohistochemistry, and activation of TGF-β and SMAD2/3 by quantitative real-time polymerase chain reaction.

Results: Peak systolic stress was preserved during the entire observation period, and diastolic compliance was maintained in treated animals (hypertrophied: 20 ± 1 vs treated: 11 ± 3 and controls: 12 ± 2; p < 0.05). Collagen was significantly higher in the hypertrophied group by Masson trichrome (hypertrophied: 3.1 ± 0.9 vs treated: 1.8 ± 0.6) and by hydroxyproline assay (hypertrophied: 2.8 ± 0.6 vs treated: 1.4 ± 0.4; p < 0.05). Fluorescent immunostaining showed active EndMT in the hypertrophied group but significantly less in treated hearts, which was directly associated with a significant increase in TGF-β/SMAD-2 messenger RNA expression.

Conclusions: EndMT contributes to cardiac fibrosis in hypertrophied hearts. VEGF treatment inhibits EndMT and prevents the deposition of collagen that leads to myocardial stiffness through TGF-β/SMAD-dependent activation. This presents a therapeutic opportunity to prevent diastolic failure and preserve cardiac function in pressure-loaded hearts.

Copyright © 2017 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1

Peak systolic stress and diastolic compliance: (A) Peak systolic stress was determined by weekly echocardiography over the entire observation period. Normalization of wall stress as adaptive mechanism to compensate for increased pressure-loading is preserved in VEGF-treated hypertrophied hearts compared to untreated hearts (n=7/group; *p<0.05 versus VEGF-treated and control). (B) Pressure volume curves were obtained on arrested isolated perfused hearts. Passive stiffness of the myocardium, determined in arrested hearts, is significantly higher in untreated hypertrophied hearts corresponding with a higher amount of collagen deposition in the extracellular matrix (n=7/group; *p<0.05 versus VEGF-treated and control).

Figure 2

Figure 2

Myocardial fibrosis determined by hydroxyproline assay: (A) Total amount of collagen is significantly increased in untreated hypertrophied hearts compared to VEGF-treated and control hearts (n=7/group; *p<0.05). (B) Soluble collagen is significantly increased in untreated hypertrophied hearts compared to VEGF-treated and control hearts (n=7/group; *p<0.05). (C) Cross-linked collagen is significantly increased in untreated hypertrophied hearts compared to VEGF-treated and control hearts (n=7/group; *p<0.05).

Figure 3

Figure 3

Histological assessment of fibrosis: (A) As indicated by the representative histological sections stained with Masson’s Trichrome, collagen deposition (blue) is significantly higher in untreated hypertrophied hearts rather than VEGF-treated and control hearts (n=7/group; *p=0.001). (B) Total amount of collagen was analyzed by light microscopy and summary of data is depicted here (n=7/group; *p<0.05 versus VEGF-treated and control).

Figure 4

Figure 4

Endothelial-to-mesenchymal transition (EndMT): (A) Representative immunohistochemical sections of EndMT in untreated hypertrophied hearts and VEGF treated hypertrophied hearts are shown. EndMT is identified by double-labeling of cells with CD31, an endothelial cell marker in red, and FSP-1, a fibroblast marker in green. (B) Data summary shows cells undergoing EndMT (cells merged for CD31 and FSP-1) expressed per total number of CD31 positive cells. There are significantly less endothelial cells undergoing EndMT in VEGF treated hypertrophied hearts comparable to control hearts compared to untreated hypertrophied hearts (n=4/group; p=0.001 VEGF-treated and control versus untreated hypertrophy). (C) Representative immunohistochemical sections of EndMT in untreated hypertrophied hearts and VEGF treated hypertrophied hearts are shown. EndMT is identified by double-labeling nuclei of CD31 positive cells in red with pSMAD2/3 in green (indicated by white arrows). (D) Data summary shows cells undergoing EndMT (CD31 positive cells with merged nuclei) expressed per total number of CD31 positive cells. These results support the extent of EndMT (n=4/group; p=0.001).

Figure 5

Figure 5

TGF-β pathway activation via qRT-PCR: TGF-β and SMAD2 are significantly up-regulated in untreated hypertrophied hearts which coincides with EndMT. Data are expressed as fold-change from control (n=6/group; *p=0.01).

Comment in

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