Brain Endothelial Cells in Contrary to the Aortic Do Not Transport but Degrade Low-Density Lipoproteins via Both LDLR and ALK1

Round 1

Reviewer 1 Report

The article is devoted to an important problem of modern medicine – the metabolism and transport of LDL (LDL). This is evidenced by a large number of publications devoted to the violation of cholesterol metabolism. There are no publicly available publications that would fully disclose the differences in the transport and metabolism of LDL in the aortic endothelium and the endothelium of peripheral vessels of the brain, as well as the role of various transport systems of associated molecules in this. Therefore, this publication is relevant and timely.

The authors have done a lot of work, which allowed us to conclude that the endothelial cells of the aorta cleave a very small amount of LDL and transcellular most of it, and the endothelial cells of the peripheral vessels of the brain decompose, but do not transport LDL. In addition, the authors proved that the LDLR-clathrin pathway leads to degradation of LDL in endothelial cells of any type. ALK1, which promotes transcellular LDL transport in aortic endothelial cells, also further limits LDL degradation in brain endothelial cells. SR-BI and caveolin-1, which also promote the absorption and transport of LDL into the aortic endothelial cells, do not limit either the binding or association of LDL with brain endothelial cells.

The study was carried out at a high technical level using the latest research methods. This made it possible to use various criteria in substantiating the conclusions. The reliability of the obtained results is achieved by an adequately constructed design of the experiment, adequate statistical processing of the obtained scientific data.

The article is quite well illustrated. The drawings are complete and informative. They allow unambiguously interpreting the received data.

The discussion is structured logically. The discussed provisions logically follow from the results obtained and the cited literature data.

 

Remarks

1. In order to increase the appeal to the publication and its citation, it is advisable to provide schemes of LDL transport and metabolism in the aortic endothelium and endothelium of peripheral vessels of the brain, showing the revealed differences.

The remark is of a recommendatory nature that does not reduce the value of the work performed. Their implementation remains at the discretion of the authors.

Recommended for printing.

Author Response

Accordingly to the reviewer suggestion, we added a graphical abstract to the revised manuscript. 

For the Graphical Abstract please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

This manuscript describes a interesting set of studies comparing cultured peripheral vascular (aortic) endothelial cells (ECs) and brain-derived ECs for their ability to uptake, transcytose, or degrade low-density lipoproteins.  The authors conclude that peripheral and brain ECs differ in that, while cells from either vascular bed type can take up labeled LDL, degradation of LDL predominates over transcytosis in brain ECs , whereas aortic ECs effectively traffic LDL across a confluent monolayer.  These findings are an important contribution to our understanding of the mechanisms that limit LDL trafficking into the brain and further implicate a set of proteins potentially vital for preventing atherosclerosis in the brain and whose roles maybe be altered in, for example, dementia.

The paper is well written, and the experimental methods are thoroughly described. 

Comment: The authors include an admirably candid paragraph identifying limitations of their study, focusing on the use of monocultures of brain ECs, i.e., without astrocytes whose presence may alter the ECs' capabilities for LDL disposition.  It would be helpful to supplement this acknowledgement of limitations with a comment on the possible impact of culture conditions (cells driven into proliferation by VEGF and FGF in the BulletKits) on the expression and operation of the LDL trafficking 'machinery.'

 

Author Response

Although both hAEC and hCMEC/D3 were grown in VEGF and FGF containing medium, the LDL binding, association and transport experiments were performed in medium lacking growth factors. All experiments were also performed in non-proliferating confluent cells. Further, our group previously reported that although VEGF treatment promotes HDL interaction to hAEC, VEGF does not alter LDL binding, association and transport through these cells (Velagapudi 2017). According to these observations, we believe the presence of VEGF or FGF in both endothelial cells growth media does not represent a limitation to the current study.

 

Reference:

1. Velagapudi et al, VEGF-A Regulates cellular localization of SR-BI as well as transendothelial transport of HDL but not LDL. Atheriosclerosis, Thrombosis and Vascular Biology, 2017.