University of Groningen Endothelial plasticity in fibrosis and epigenetics as a therapeutic target Hulshoff, Melanie

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University of Groningen

Endothelial plasticity in fibrosis and epigenetics as a therapeutic target

Hulshoff, Melanie

DOI:

10.33612/diss.146265795

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Publication date: 2020

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Hulshoff, M. (2020). Endothelial plasticity in fibrosis and epigenetics as a therapeutic target. University of Groningen. https://doi.org/10.33612/diss.146265795

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546222-L-bw-Hulshoff 546222-L-bw-Hulshoff 546222-L-bw-Hulshoff 546222-L-bw-Hulshoff Processed on: 27-10-2020 Processed on: 27-10-2020 Processed on: 27-10-2020

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OUTCOME OF THIS THESIS

Fibrosis is an underlying cause of chronic illnesses such as heart failure and chronic kidney disease [1]. As of yet, no effective treatment for fibrosis is available. In part I of this thesis, we gained insight into the (epi)genetic regulation of endothelial plasticity, as an underlying cause of fibrosis. In part II, we established proof-of-concept to perform (epi)genetic targeting in experimental fibrosis. In this chapter, we discuss the future challenges for (1) the field of EndMT and (2) for the therapeutic targeting of (epi)genetics in fibrosis that originate from this thesis.

FUTURE GOALS TO IMPROVE OUR UNDERSTANDING OF ENDMT

The field of EndMT research is an emerging and exciting field since it provides opportunities for future (epi)genetic therapeutic applications in several diseasese such as organ fibrosis and cardiovascular disease. Nevertheless, several challenges, open questions and limitations concering EndMT need to be overcome in the future.

First, standardization of in vitro models of EndMT is key to unraveling the underlying (epi)genetic mechanisms of EndMT. So far, in vitro models of EndMT are inconsistent because of the use of different types of media and inducers, which affect the extent and phenotype of EndMT, and making it difficult to compare individual studies to each other [2]. The same holds for different time points of stimulation (e.g. 2 hours versus 12 days) and the use of both mouse and human cell lines. Moreover, one study showed that passage number as well as culturing versus freshly isolated endothelial cells affects the non-coding RNA expression [3]. This highlights that non-coding RNA expression is highly adaptable and that proper in vivo validation is key. It is safe to speculate that the same holds for other epigenetic modifiers such as DNA promoter methylation and histone modifications. In my opinion, it would be key to develop consortia with researchers which focus on endothelial plasticity with the aim to achieve a consensus about standardization of in vitro EndMT protocols. We, as EndMT researchers, would largely benefit from this agreement, since we can then focus

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on unraveling EndMT under standard conditions and from there move towards a broader understanding of EndMT. Also, the role of epigenetics and the underlying regulatory mechanisms is most established in cancer where a similar process to EndMT is present: epithelial-to-mesenchymal transition (EMT). Therefore, I believe that close collaboration between EndMT and EMT researchers is key to improve our understanding of endothelial/epithelial plasticity.

Second, our understanding of EndMT is limited by the lack of in vivo models which allow specific inhibition or depletion of EndMT. This restricts our understanding of the causative role that EndMT has in disease pathology. From my perspective, after the identification of EndMT inhibitors in standardized assays, these could then also be applied to develop proper in vivo models of EndMT. Proper standardization of in vitro models and development of in vivo models might also help to explain why different studies came to different conclusions with respect to the extent of EndMT in different in vivo contexts (eg transverse aortic constriction versus ascending aortic constriction) [4,5]. Future stage-specific mapping strategies as well as the recent development of the single-cell sequencing technology would potentially help on this part (even though it is currently limited by its sequencing depth).

Third, EndMT is not a process that all endothelial cells go through and most end-stage studies report on heterogenous cell populations containing EndMT-derived myofibroblasts and EndMT-resistant endothelial cells. Therefore, an open question about EndMT is why certain endothelial cells undergo EndMT whereas others are resistant to EndMT. This might be explained by clonality (which directs endothelial cells from the same clone towards EndMT-prone or EndMT-resistant). The recent established Rainbow system might help to answer this question. The Rainbow system enables to mark different cells and their progeny with distinct fluorescent colors thereby allowing to trace cellular expansion in vivo [6,7]. When driven under an endothelial-specific promoter, the Rainbow system could identify individual endothelial cells and their progeny. This endothelial-specific Rainbow system, in combination with mesenchymal marker analysis, might help to examine whether certain clones are more prone towards EndMT than others. This might help us to identify novel therapeutic strategies aimed at EndMT-prone and/or EndMT-resistant cells.

Future research and perhaps the discovery of novel technologies, will potentially help to overcome the current challenges and limitations concerning

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EndMT. This will help us to reach a better understanding of the (epi)genetic

regulation of EndMT, as an underlying cause of fibrosis.

CURRENT CHALLENGES TO TARGET (EPI)GENETICS IN FIBROSIS

(Epi)genetic targeting to combat fibrosis has exciting opportunities for future use in the clinic. Nevertheless, several questions and challenges concerning (epi)genetic targeting in the context of fibrosis need to be addressed.

One of the questions that need to be answered before performing (epi)genetic targeting to combat fibrosis, is whether to perform cell-specific or general treatment. Cell-specific treatment has most likely the advantage that it is has less adverse effects since it is specifically focused on one cell type. However, since endothelial-derived myofibroblast-like cells and (myo)fibroblasts are believed to express the same markers and exhibit common mechanisms to secrete extracellular matrix, general treatment could result in targeting two aspects of fibrosis at the same time which I believe might potentially have a stronger and pleiothropic effect on the disease pathology.

Concerning the targeting of cell types, it is important to note the matter of balance. Fibroblasts are not only detrimental but also exert beneficial effects (e.g. during initial insults). The same holds for (partial) EndMT which contributes to the formation of new vessels upon ischemia [8,9]. Therefore, I believe that future strategies should be directed towards elimination of some, but definitely not all (myo)fibroblasts and cells that are prone to undergo EndMT, which should be a point of consideration for each future therapy that targets fibrosis.

When focusing specifically on EndMT, it is likely that we have to make a choice which cells we want to target: the cells actively undergoing EndMT or the endothelial cells that have already fully transformed into myofibroblast-like cells. I personally believe that the cells that actively undergo EndMT represent the most targetable population to combat EndMT since (1) they are unique in their phenotype and (2) patients with chronic illnesses most likely have ongoing EndMT as part of the chronic disease pathology. However, gene signatures and marker expression of the active stage of EndMT remain largely unknown which remains a challenge for now.

Also, we have to make a decision whether we want to direct cells that undergo EndMT into a so-called mesenchymal-to-endothelial transition (MEndT)

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to reverse their phenotype. It has been recently described that cardiac fibroblasts adopt an endothelial cell phenotype upon ischemic cardiac injury [10]. However, it is unclear until now whether MEndT also occurs during chronic disease states (such as heart failure) and whether (full) reversal of EndMT via MEndT is possible. Even though MEndT might be beneficial, we can also speculate that when we force EndMT-derived myofibroblast-like cells into MEndT, they could become more prone to (again) undergo EndMT at a later time point. Therefore, future knowledge about how both EndMT and fibrosis are regulated will hopefully guide us towards the right direction.

The lack of effective therapies for fibrosis also suggests that the current experimental models are not efficient enough to translate the in vivo findings to the patient. Concerning EndMT, it is important to mention that there is a lack of robust human data on EndMT [2]. Nevertheless, almost all factors that induce EndMT via epigenetic mechanisms have also been described in the human circulation [11]. This emphasizes the future potential of therapeutically targeting epigenetic modulators in order to combat EndMT-associated pathologies.

The platforms to perform (epi)genetic targeting in the context of fibrosis are already available. The recent discovery of the CRISPR/Cas system allows gene-specific (epi)genetic targeting when deactivated (nuclease deficient) Cas9 is fused to different epigenetic adaptor domains. This, together with a so-called guiding RNA can result in gene-specific epigenetic editing. Currently, there are two major limitations to the CRISPR/Cas system which need to be overcome before performing epigenetic targeting in fibrosis. The first limitation is the off-target effects of the CRISPR/Cas system. The discovery of the so-called high-fidelity Cas9 has significantly improved the off-target effects that were associated with the Cas9 technology [12]. Nevertheless, screening and understanding of potential off-target effects is still key for future therapeutic use of the CRISPR/Cas technology. It is not only the CRISPR/Cas technology in itself which has potential off-target effects, but the same holds when targeting epigenetic modifiers (in particular for non-coding RNAs, but also for chromatin-modifying complexes). Therefore, future research needs to be directed to understand the network of non-coding RNAs and other epigenetic modifiers, to prevent adverse effects when performing epigenetic targeting.

Another aspect which needs to considered for future use of the CRISPR/Cas technology for fibrosis treatment is the delivery method. Adeno associated virus

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(AAV) is considered to be the safest virus so far and therefore often used in

experimental models of fibrosis [13]. However it is safe to speculate that any virus, in particular in combination with a bacteria-derived Cas protein, could have potential disastrous effects in patients. Therefore, future research on alternatives to viral delivery of the CRISPR/Cas technology (eg synthetic vectors) is key for future use of this technology in the clinic.

CONCLUDING REMARKS

To conclude, (epi)genetic regulation of EndMT represents a highly promising but yet insufficiently explored field with the potential for future (epi)genetic targeting of EndMT-associated diseases such as fibrosis.

The field of EndMT as well as (epi)genetic targeting in fibrosis is currently challenged by several limitations and open questions. Future research dedicated to solving these limitations is key to develop (epi)genetic targeting tools which are compatible with the clinic.

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REFERENCES

1. Rockey DC, Bell PD and Hill JA. Fibrosis-a common pathway to organ injury and failure. N Engl J Med 372, 1138-1149 (2015)

2. Kovacic JC, Dimmeler S and Harvey RP et al. Endothelial to mesenchymal transition in cardiovascular disease: JACC state-of-the-art review. J Am Coll Cardiol 73(2), 190-209 (2019)

3. Kuosmanen SM, Kansanen E and Sihvola V et al. MicroRNA Profiling Reveals Distinct Profiles for Tissue-Derived and Cultured Endothelial Cells. Scientific reports 7,10943 (2017)

4. Zeisberg EM, Tarnavski O and Zeisberg M et al. R. Endothelial-to-mesenchymal transition contributes to cardiac fibrosis. Nat Med. 13, 952–961 (2007)

5. Moore-Morris T, Guimarães-Camboa N, Banerjee I, et al. Resident fibroblast lineages mediate pressure overload-induced cardiac fibrosis. J Clin Invest 124, 2921–2934 (2014) 6. Sereti KI, Nguyen NB and Kamran P et al. Analysis of cardiomyocyte clonal expansion

during mouse heart development and injury. Nat Comm 9(1), 754 (2018)

7. Rinkevich Y, Lindau P and Ueno H et al. Germ and lineage restricted stem/progenitors regenerate the mouse digit tip. Nature 476(7361), 409-13 (2011)

8. Manavski Y, Lucas T and Glaser SF et al. Clonal expansion of endothelial cells contributes to ischemia-induced neovascularization. Circ Res. 122, 670–677 (2018)

9. Welch-Reardon KM, Wu N and Hughes CC. A role for partial endothelial-mesenchymal transitions in angiogenesis? Arterioscler Thromb Vasc Biol. 35, 303–308 (2015)

10. Ubil E, Duan J and Pillai IC et al. Mesenchymal-endothelial transition contributes to cardiac neovascularization. Nature 514(7524), 585-90 (2014)

11. Hulshoff MS, Xu X and Krenning G et al. Epigenetic regulation of endothelial-to-mesenchymal transition in chronic heart disease. Arterioscler Thromb Vasc Biol. 38(9), 1986-1996 (2018)

12. Vakulskas CA, Dever DP and Rettig GR et al. A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in heamtopoeietic stem and progenitor cells. Nat Med. 24(8), 1216-1224 (2018)

13. Naso MF, Tomkowicz B and Perry WL et al. Adeno-associated virus (AAV) as a vector for gene therapy. BioDrugs 31(4), 317-334 (2017)

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