Endothelial-specific Loss of IFT88 Promotes Endothelial-to-Mesenchymal Transition and Exacerbates Bleomycin-induced Pulmonary Fibrosis

Shweta Singh,Hannah Bazinet,Jean-Francois Desjardins,David Michels,Hao H Chen,Krishna K Singh,Kim A Connelly,Antoinette Bugyei-Twum,Zongyi Liu,Pratiek N Matkar,Jameela Joseph,Mohammad Qadura,Howard Leong-Poi,Lillian Esene,Mehroz Ehsan,Mohamed Adam,Hien Nguyen,Elizabeth Mebrahtu

doi:10.1038/s41598-020-61292-9

Shweta Singh, Hannah Bazinet + Show 16 more

Open Access

https://doi.org/10.1038/s41598-020-61292-9

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Abstract

Intraflagellar transport protein 88 (Ift88) is required for ciliogenesis and shear stress-induced dissolution of cilia in embryonic endothelial cells coincides with endothelial-to-mesenchymal transition (EndMT) in the developing heart. EndMT is also suggested to underlie heart and lung fibrosis, however, the mechanism linking endothelial Ift88, its effect on EndMT and organ fibrosis remains mainly unexplored. We silenced Ift88 in endothelial cells (ECs) in vitro and generated endothelial cell-specific Ift88-knockout mice (Ift88endo) in vivo to evaluate EndMT and its contribution towards organ fibrosis, respectively. Ift88-silencing in ECs led to mesenchymal cells-like changes in endothelial cells. The expression level of the endothelial markers (CD31, Tie-2 and VE-cadherin) were significantly reduced with a concomitant increase in the expression level of mesenchymal markers (αSMA, N-Cadherin and FSP-1) in Ift88-silenced ECs. Increased EndMT was associated with increased expression of profibrotic Collagen I expression and increased proliferation in Ift88-silenced ECs. Loss of Ift88 in ECs was further associated with increased expression of Sonic Hedgehog signaling effectors. In vivo, endothelial cells isolated from the heart and lung of Ift88endo mice demonstrated loss of Ift88 expression in the endothelium. The Ift88endo mice were born in expected Mendelian ratios without any adverse cardiac phenotypes at baseline. Cardiac and pulmonary endothelial cells isolated from the Ift88endo mice demonstrated signs of EndMT and bleomycin treatment exacerbated pulmonary fibrosis in Ift88endo mice. Pressure overload stress in the form of aortic banding did not reveal a significant difference in cardiac fibrosis between Ift88endo mice and control mice. Our findings demonstrate a novel association between endothelial cilia with EndMT and cell proliferation and also show that loss of endothelial cilia-associated increase in EndMT contributes specifically towards pulmonary fibrosis.

Highlights

Intraflagellar transport protein 88 (Ift88) is required for ciliogenesis and shear stress-induced dissolution of cilia in embryonic endothelial cells coincides with endothelial-to-mesenchymal transition (EndMT) in the developing heart
It is important to note that physiologically endothelial cilia is not a permanent structure and embryonic endothelial cells (ECs) which are exposed to high shear stress, temporarily dissolve their cilia and this process coincides with transforming growth factor-β (Tgfβ)-induced endothelial-to-mesenchymal transition (EndMT)[7]
Our EndMT marker analysis data demonstrated a significant reduction in the expression level of endothelial markers CD31, Tie-2 and VE-Cadherin with a concomitant increase in the expression level of mesenchymal markers αSMA, N-Cadherin and FSP-1 at both the transcript (Fig. 1D) and protein (Fig. 1E) levels in the siIft88-transfected in comparison to scrambled control-transfected HUVECs

Summary

Introduction

Intraflagellar transport protein 88 (Ift88) is required for ciliogenesis and shear stress-induced dissolution of cilia in embryonic endothelial cells coincides with endothelial-to-mesenchymal transition (EndMT) in the developing heart. Our findings demonstrate a novel association between endothelial cilia with EndMT and cell proliferation and show that loss of endothelial cilia-associated increase in EndMT contributes towards pulmonary fibrosis. In the setting of loss of endothelial cilia, the contribution of EndMT towards fibroblasts accumulation, and cardiac and pulmonary fibrosis remains unknown. Our data demonstrate that loss of endothelial Ift[88] leads to EndMT and increased proliferation in vitro, and exacerbates bleomycin-induced pulmonary fibrosis in vivo. Our data indicates that loss of endothelial cilia associated EndMT does not contribute towards pressure-overload induced cardiac fibrosis, supporting studies where EndMT was demonstrated not to participate in cardiac fibrosis[18]

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