Establishing And Maintaining The Blood-Brain Barrier: Epigenetic And Signaling Determinants

Reviewer #1 (Public Review):

The blood-brain barrier separates neural tissue from blood-borne factors and is important for maintaining central nervous system health and function. Endothelial cells are the site of the barrier. These cells exhibit unique features relative to peripheral endothelium and a unique pattern of gene expression. There remains much to be learned about how the transcriptome of brain endothelial cells is established in development and maintained throughout life.

The manuscript by Sadanandan, Thomas et al. investigates this question by examining transcriptional and epigenetic changes in brain endothelial cells in embryonic and adult mice. Changes in transcript levels and histone marks for various BBB-relevant transcripts, including Cldn5, Mfsd2a and Zic3 were observed between E13.5 and adult mice. To perform these experiments, endothelial cells were isolated from E13.5 and adult mice, then cultured in vitro, then sequenced. This approach is problematic. It is well-established that brain endothelial cells rapidly lose their organotypic features in culture (https://elifesciences.org/articles/51276). Indeed, one of the primary genes investigated in this study, Cldn1, exhibits very low expression at the transcript level in vivo but is strongly upregulated in cultured ECs.

(https://elifesciences.org/articles/36187 ; https://markfsabbagh.shinyapps.io/vectrdb/)

This undermines the conclusions of the study.

An additional concern is that for many experiments, siRNA knockdowns are performed without validation of the efficacy of the knockdown.

Some experiments in the paper are promising, however. For example, the knockout of HDAC2 in endothelial cells resulting in BBB leakage was striking. Investigating the mechanisms underlying this phenotype in vivo could yield important insights.

Reviewer #2 (Public Review):

Sadanandan et al describe their studies in mice of HDAC and Polycomb function in the context of vascular endothelial cell (EC) gene expression relevant to the blood-brain barrier, (BBB). This topic is of interest because the BBB gene expression program represents an interesting and important vascular diversification mechanism. From an applied point of view, modifying this program could have therapeutic benefits in situations where BBB function is compromised.

The study involves comparing the transcriptomes of cultured CNS ECs at E13 and adult stages and then perturbing EC gene expression pharmacologically in cell culture (with HDAC and Polycomb inhibitors) and genetically in vivo by EC-specific conditional KO of HDAC2 and Polycomb component EZH2.

This reviewer has several critiques of the study.

First, based on published data, the effect of culturing CNS ECs is likely to have profound effects on their differentiation, especially as related to their CNS-specific phenotypes. Related to this, the authors do not state how long the cells were cultured.

Second, the use of qPCR assays for quantifying ChIP and transcript levels is inferior to ChIPseq and RNAseq. Whole genome methods, such as ChIPseq, permit a level of quality assessment that is not possible with qPCR methods. The authors should use whole genome NextGen sequencing approaches, show the alignment of reads to the genome from replicate experiments, and quantitatively analyze the technical quality of the data.

Third, the observation that pharmacologic inhibitor experiments and conditional KO experiments targeting HDAC2 and the Polycomb complex perturb EC gene expression or BBB integrity, respectively, is not particularly surprising as these proteins have broad roles in epigenetic regulation in a wide variety of cell types.

Author Response

We believe that these findings make a significant contribution to the field of CNS endothelial cell biology and blood-brain barrier. We thank you for your time and consideration.

Reviewers' 1 and 2 concern on endothelial cells (ECs) transcription changes on culture.

We would like to express our gratitude to the reviewers for their critical comments. We are pleased to address the concerns raised by performing FACS sorting of the CNS ECs from E-13.5 and adult brain. However, it is important to note that both E-13.5 ECs and adult ECs were cultured in the same media. It is worth mentioning that this work was initiated in 2017, whereas the article mentioned by Reviewer 1 was published in 2020. We went through a series of standardization steps before identifying the Corning endothelial cell culture media (Cat#355054) with 2% FCS as the optimal medium for preserving EC identity in culture. Conversely, if PromoCell media (C-22110) is used, a decrease in the Wnt pathway can be observed, and the use of 5% FCS enhances the Wnt pathway in E-13.5 ECs. The article mentioned by Reviewer 1 (https://elifesciences.org/articles/51276) did not take these differences in culture media into account. Additionally, we did not employ puromycin for obtaining pure ECs, and the ECs were cultured for a maximum of 8 days. Our in vitro study serves as a model for identifying the epigenetic regulators HDAC2 and PRC2 as controllers of BBB gene transcription, which is subsequently validated in an in vivo model.

Reviewer-1 Comment 2- An additional concern is that for many experiments, siRNA knockdowns are performed without validation of the efficacy of the knockdown

In the revised version of this manuscript, we will include validation results to demonstrate the effectiveness of siRNA knockdown experiments.

Reviewer-1 Comment 3- Some experiments in the paper are promising, however. For example, the knockout of HDAC2 in endothelial cells resulting in BBB leakage was striking. Investigating the mechanisms underlying this phenotype in vivo could yield important insights.

We appreciate your positive comment. The in vivo HDAC2 knockout experiment will serve as a validation of our in vitro findings, indicating that the epigenetic regulator HDAC2 can control the expression of endothelial cell (EC) genes involved in angiogenesis, blood-brain barrier (BBB) formation, and maturation. We are actively working on this model, and we plan to publish additional molecular data on epigenetically regulated CNS vascular development and maintenance in our future publications.

Reviewer 2 Comment-2 The use of qPCR assays for quantifying ChIP and transcript levels is inferior to ChIPseq and RNAseq. Whole genome methods, such as ChIPseq, permit a level of quality assessment that is not possible with qPCR methods. The authors should use whole genome NextGen sequencing approaches, show the alignment of reads to the genome from replicate experiments, and quantitatively analyze the technical quality of the data.

We appreciate the reviewer's comment. While it is true that whole-genome methods such as ChIP-seq and RNA-seq provide comprehensive and high-throughput analysis compared to qPCR assays, it would be incorrect to consider qPCR as inferior. qPCR assays offer advantages in terms of sensitivity, specificity, validation, confirmation, and targeted analysis. We agree that performing a comprehensive analysis of HDAC2 and PRC2 targeted endothelial cell (EC) genes is important. We are currently in the process of generating this data, and as soon as it is complete, we will publish it accordingly.

Reviewer 2 Comment-3 Third, the observation that pharmacologic inhibitor experiments and conditional KO experiments targeting HDAC2 and the Polycomb complex perturb EC gene expression or BBB integrity, respectively, is not particularly surprising as these proteins have broad roles in epigenetic regulation in a wide variety of cell types.

We appreciate the comments from the reviewers. Our results provide valuable insights into the specific epigenetic mechanisms that regulate BBB genes It is important to recognize that different cell types possess stage-specific distinct epigenetic landscapes and regulatory mechanisms. Rather than having broad roles across diverse cell types, it is more likely that HDAC2 (eventhough there are several other class and subtypes of HDACs) and the Polycomb complex exhibit specific functions within the context of EC gene expression or BBB integrity.

Moreover, the significance of our findings is enhanced by the fact that epigenetic modifications are often reversible with the assistance of epigenetic regulators. This makes them promising targets for BBB modulation. Targeting epigenetic regulators can have a widespread impact, as these mechanisms regulate numerous genes that collectively have the potential to promote the vascular repair.

A practical advantage is that FDA-approved HDAC2 inhibitors, as well as PRC2 inhibitors (such as those mentioned in clinical trials NCT03211988 and NCT02601950, are already available. This facilitates the repurposing of drugs and expedites their potential for clinical translation.

Please note: illustrations of Fig-1, 4 and 6 are created using Biorender.com, license purchased by Spiros Blackburn. This will be added to the Acknowledgments.