Integrative studies of brain pericytes and their involvement in glioma and COVID-19 Oudenaarden, Clara

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LUND UNIVERSITY PO Box 117 221 00 Lund +46 46-222 00 00

Integrative studies of brain pericytes and their involvement in glioma and COVID-19

Oudenaarden, Clara

2022

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Oudenaarden, C. (2022). Integrative studies of brain pericytes and their involvement in glioma and COVID-19.

[Doctoral Thesis (compilation), Department of Laboratory Medicine]. Lund University, Faculty of Medicine.

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Integrative studies of brain pericytes and their involvement in glioma and COVID-19

CLARA OUDENAARDEN

DEPARTMENT OF LABORATORY MEDICINE | FACULTY OF MEDICINE | LUND UNIVERSITY

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Department of Laboratory Medicine Lund University, Faculty of Medicine Doctoral Dissertation Series 2022:20 ISBN 978-91-8021-181-9

ISSN 1652-8220 ₉⁷⁸⁹¹⁸⁰

211819NORDIC SWAN ECOLABEL 3041 0903Printed by Media-Tryck, Lund 2022

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Integrative studies of brain pericytes and their involvement in glioma and COVID-19

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Integrative studies of brain pericytes and their involvement in glioma and COVID-19

Clara R. L. Oudenaarden

DOCTORAL DISSERTATION

by due permission of the Faculty of Medicine, Lund University, Sweden.

To be defended at Sharience, Spark Building.

Friday, 11^th of February 2022 at 09:00 h.

Faculty opponent Dr. Annika Keller

Division of Neurosurgery, University Hospital of Zurich

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Organization LUND UNIVERSITY

Document name Doctoral dissertation Faculty of Medicine

Department of Laboratory Medicine

Date of issue 11^th February 2022 Author: Clara Oudenaarden Sponsoring organization: n/a

Title and subtitle: Integrative studies of brain pericytes and their involvement in glioma and COVID-19 Abstract

Glioblastoma is an aggressive and incurable grade 4 brain tumour. Despite maximum treatment efforts, recurrence invariable arises mainly due to the infiltrative nature of individual tumour cells, a highly proliferative vasculature, and the presumed existence of glioblastoma stem-like cells (GSCs). These features all contribute to treatment resistance and are in some way linked to pericytes. Pericytes are multi-functional vascular mural cells that are embedded within the vascular basement membrane of the microvasculature. In addition to providing blood vessel stability, pericytes act as and interact with immune cells, and can regulate blood flow. In glioblastoma, pericytes are loosely attached to the vasculature resulting in a lack of contact-inhibition to the underlying endothelium, causing excessive endothelial cell proliferation and sprouting. In addition, infiltrating glioblastoma cells have been observed migrating along the vascular basement membrane into the surrounding brain parenchyma. Thirdly, pericytes are an integral component of the perivascular niche (PVN), a safe haven for GSCs. Despite this key position of pericytes in glioblastoma, their significance in the glioblastoma microenvironment remains relatively obscure.

This thesis focuses on elucidating the role of pericytes in glioblastoma. Without making assumptions if pericytes are promoting or suppressing tumour development, we perform integrative studies on brain pericytes in glioblastoma and explore their heterogeneity. In paper 1 we analysed gene expression profiles of pericytes in glioblastoma patients and physiological brain tissue. Gene signatures that were enriched in glioblastoma pericytes were associated with a worse overall survival when compared to signatures that did not show any significant difference.

In paper 2 we performed single-cell transcriptome analysis on perivascular cells that were in silico isolated from mouse glioblastoma tissue. We annotated two pericyte subsets, as well as perivascular fibroblasts and smooth muscle cells. Transcriptomics on the complete murine glioblastoma tissue was conducted in paper 3. In this paper, tumours from pericyte-poor mice were compared with those from mice with a normal pericyte coverage, with the aim to elucidate the effect of pericytes on tumour cells and other components of the glioblastoma microenvironment.

Finally, the current COVID-19 pandemic directed us to investigate the role of pericytes in neurological symptomatology in COVID-19 patients. We provide indisputable evidence that in the brain the SARS-CoV-2 entry receptor ACE2 is exclusively expressed on brain pericytes and is patient-specific. Moreover, COVID-19 patients that expressed high levels of ACE2 in brain pericytes suffered from severe neurological affliction prior to decease.

Key words: Pericytes, glioblastoma, glioma, scRNA-seq, COVID-19, blood vessels, tumour microenvironment Classification system and/or index terms (if any): NA

Supplementary bibliographical information: NA Language: English ISSN and key title: 1652-8220

Lund University, Faculty of Medicine Doctoral Dissertation series 2022:20 ISBN: 978-91-8021-181-9

Recipient’s notes: NA Number of pages 90 Price: NA

Security classification: NA

I, the undersigned, being the copyright owner of the abstract of the above-mentioned dissertation, hereby grant to all reference sources permission to publish and disseminate the abstract of the above-mentioned dissertation.

Signature Date 2022-01-03

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Integrative studies of brain pericytes and their involvement in glioma and COVID-19

Clara R. L. Oudenaarden

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Cover: The extensive pericyte coverage of the brain vasculature in and by Clara Oudenaarden

Copyright pp 1-90 Clara Oudenaarden

Paper 1 © by the authors (published in Molecular Oncology, 2021) Paper 2 © by the authors (manuscript unpublished)

Paper 3 © by the authors (manuscript unpublished)

Paper 4 © by the authors (published in International Journal of Molecular Sciences, 2021)

Faculty of Medicine

Department of Laboratory Medicine ISBN 978-91-8021-181-9

ISSN 1652-8220

Printed in Sweden by Media-Tryck, Lund University Lund 2022

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Strongly wrapping arms Embracing vasculature Perfect pericyte

“Crescit scribendo scribendi studium”

Desiderius Erasmus Roterodamus

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Papers included in the thesis

Paper 1

Upregulated functional gene expression programmes in tumour pericytes mark progression in patients with low-grade glioma (2021) Oudenaarden, C., Sjölund, J., Pietras, K., Molecular Oncology.

Paper 2

Dissociation of murine high-grade glioma with the objective of single-pericyte sequencing, Oudenaarden C., Bolivar P., Braun S., Sjölund, J., Pietras, K. – Manuscript

Paper 3

Pericyte deprivation remodels the tumor microenvironment and promotes tumor growth in a high-grade glioma mouse model, Braun, S.^∗, Oudenaarden, C.^∗, Bolivar, P.^∗, Sjölund, J., Cordero, E., Möller, C., Pietras, K. – Manuscript

∗ Authors contributed equally to the work

Paper 4

Infection of brain pericytes underlying neuropathology of COVID-19 patients (2021) Bocci, M.^∗, Oudenaarden, C.^∗, Sàenz-Sardà, X., Simrén, J., Edén, A., Sjölund, J., Möller, C., Gisslén, M., Zetterberg, H., Englund, E., Pietras, K.

International Journal of Molecular Sciences, 22 (21).

∗ Authors contributed equally to the work

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List of abbreviations

αSMA Alpha smooth muscle actin

ACE2 Angiotensin converting enzyme 2

Ang1 Angiopoietin 1

ANPEP Amino peptidase

BBB Blood-brain barrier

bFGF Basic fibroblast growth factor

BTB Blood-tumour barrier

CCL CC Chemokine ligand

COVID-19 Coronavirus disease 2019

CXCL Chemokine (C-X-C) motif ligand

CSPG4 Chondroitin sulphate proteoglycan 4

DNA Deoxyribose nucleic acid

ECM Extracellular matrix

EGFR Epidermal growth factor receptor

FOXO1 Forkhead box protein O1

GBM Glioblastoma multiforme

G-CIMP Glioma CpG island methylator phenotype

GJA Gap junction protein alpha

GSC Glioblastoma stem-like cell

HGG High-grade glioma

HSPG Heparin sulphate proteoglycan

ICAM-1 Intercellular adhesion molecule 1

IDH Isocitrate dehydrogenase

IL Interleukin

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LGG Low-grade glioma

MCAM Melanoma cell adhesion molecule

MGMT O⁶-methylguanine DNA methyltransferase

MMP Matrix metalloprotease

NF1 Neurofibromatosis type 1

NG2 Neural/glial antigen 2

NVU Neurovascular unit

Olig2 Oligodendrocyte transcription factor 2 PDGF(R) Platelet-derived growth factor (receptor)

PEG Pericyte-enriched gene

PTEN Phosphatase and tensin homolog

PVF Perivascular fibroblast

PVN Perivascular niche

RGS5 Regulator of G-protein signalling 5

RNA Ribonucleic acid

SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2 scRNA-seq Single-cell RNA sequencing

VSMC Vascular smooth muscle cell

TCGA The Cancer Genome Atlas

TERT Telomerase reverse transcriptase

TNF-α Tumour necrosis factor alpha

TGF Transforming growth factor

Tie2 Tyrosine kinase with immunoglobulin-like and EGF- like domains 2

TMZ Temozolomide

TP53 Tumour protein 53

UMAP Uniform Manifold Approximation and Projection VCAM-1 Vascular cell adhesion molecule 1

VEGF(R) Vascular endothelial growth factor (receptor)

WHO World Health Organisation

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Acknowledgements

First and foremost, I am extremely thankful to Kristian. Thank you super much for giving me the opportunity to complete my PhD education under your wings. I am really happy I could explore the fascinating glioblastoma pericytes with your expertise in tumour biology. Thank you for always making time for me, despite your engagement in many scientific and family activities. During the past 6.5 years you have shared your profound knowledge with me, from demonstrations in the mouse house on how to isolate the pancreas, to explanatory drawings that you effortlessly sketched during our meetings. I appreciate you gave me the opportunity to visit both local and distant conferences and courses to expand my knowledge. Any communication with you has always been very encouraging and kept the spirit high during my extended PhD. Like the Roman author Syrus wrote “Malum est consilium, quod mutari non potest”, translated as “Bad is the plan that cannot be changed”, this was very applicable to my PhD. However, your optimistic, yet realistic, approach of guiding projects has benefitted my PhD greatly, acknowledging when it was time to move on. I also appreciate you let me explore on my own to learn from mistakes that I might encounter, thank you, I learned a lot!

Thank you for taking the group to nice local restaurants for a Christmas dinner or on utflykter to combine science with the beautiful Skåne nature (utile dulci!). Thank you, Kristian, I am grateful to have had you as my supervisor.

Sebastian, on my first day at work you waited for me at the Medicon Village entrance, and with the exception of your parental leave, I think we have spend every other day together in the lab. We shared our common interest for the almighty brain tumour pericytes, but mainly we shared a lot of laughter. For instance, during the many hours in the virus lab, patiently injecting mice the whole day, or that time when we by accident tossed Niks -80°C box behind the freezer, I don't know why, but we couldn't stop laughing. Thank you for letting me taste your special tea mixes during our one-on-one pericyte discussions and for being sincerely happy for me when I got to know Andrés (after all, this was step one in operation 'parental leave').

Thank you for being such an amazing colleague and friend, I could not have wished for a better brain companion during these past years. Literally actually, because due to auto-correct you are saved in my phone as Sebastian Brain!

Dear Sophie. Where to start? I think I can truly call you more my friend than my colleague. Sharing the office with you from day one has brought us very close together. Many Monday mornings we reported our weekend experiences to each

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other, but we also understood when it was time to get back to work. I could always count on you, last-minute corrections on my Swedish popular science summary, a forgotten debit card or towel, or just a listening ear, you were there for me! We started as storage-refill buddies, grew from occasional lunch buddies to frequent lunch buddies, (our significant others even became biking buddies), and in the end we even made it to gym buddies, who would have thought that?! Time will tell if we even become bread-making buddies one day, but I am sure we will be buddies in some way :)

Jonas, my other great office mate! It is impressive how much you know, scientifically and not-scientifically, and impressive how many papers you can fit on your desk. Thank you for working together with me on my first author paper and providing all the missing pieces in no time. Your constructive advice on all (!) my manuscripts and suggestions for cool restaurants to visit in Malmö was much appreciated :).

Matteo, thank you for the many times you have made me laugh with your expressive stories, or smile when I tasted again one of your delicious bakes. I was lucky to work more closely with you on the COVID-19 project in my last year. I learned a lot from your expertise on many procedures that we used and from your exquisite scientific writing skills. We shared many frustrations by going through many revisions and manuscript versions, and took turns in the long protocols, but we made it in the end! Thank you for always being such a considerate colleague, for accompanying me to the IKEA when I just arrived, all the nice gifts over the years, and being so kind to me from the first day.

Paulina, muchísimas gracias for all the work you have contributed to the single-cell project! Sebastian and I could not have done it without you. Thank you for your patience when presenting the latest data and explaining so clearly how you did the analyses. Thank you for teaching me enough R skills to produce any additional feature plots myself when I found an interesting gene, as you will see in the ‘present investigation’, I made good use of it!

Thank you to all my other group members, it was a pleasure to have worked with all of you. Mikkel, you were a very pleasant group mate! It was always interesting to discuss stainings with you, one convinced of fibroblasts while the other was sure they were pericytes. Thank you for the funny moments in the pub of Umeå, outdrinking Sophie and me as a proper Dane and ordering tequila shots for us when Sophie didn’t notice. Steven, I think one of your first days was the PhD defence of Eliane, now 5 years later, one of your last days will be mine. You were a really nice colleague, you have a great sense of humour, and are a perfect pub quiz teammate.

Charlène, although you joined our lab from a distance, you effortlessly blended right into to the group. Thank you for all your thoughts on my dataset and the nice lunches when you could visit Lund for a few days. Ryu, it was really nice to hear when you chose the free desk in our office to be yours. I thought Sophie, Jonas, and

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I made a good impression on you when we first met, but then I found out the desk fitted best for your two screens and printer. That was a bummer! Anyway it was very nice to have you around as my office mate and thank you for being so patient when you showed me around for the western blot protocol! Jessica, we share the love for Ferrero Rocher! It was very nice to have you as the latest PhD recruit to the KP group. Thank you for being a nice company at lunch and in the lab. Carmen, actually, you are our latest recruit! I am happy you could join our lab. Thank you for all your efforts on keeping the confocal microscope in a good shape! Sara, you were the asset to the lab we needed! I admire you for how long you can sit on the back of a motorcycle! Mehrnaz, you already stole my heart when you came for the interview and wore a DNA helix necklace! It was very nice to have you in the group the past years, to share our frustrations on endless submissions or share Sebastians Rittersport chocolate after he was called away for another sneezing kid. Kristin, thank you very much for all the administrative support. You fixed that refund despite an unclear scan of a receipt and stayed polite after another missing delivery note.

The group would be a mess without you! Thank you for the nice conversations when you drove me back home after lab outings, and for the preparations of my dissertation.

Many thanks to the past members of the KP group. Eugenia, Nik, Eliane, and Micha, you were all there when I came to the group and all of you contributed a lot to making me feel welcome in the lab and at home in Sweden. Eugenia, a special acknowledgement to you for all your contributions on the animal experiments. Your patience, expertise, and down to earth attitude were very helpful when practicing new animal procedures. Nik, thank you for your recipes, both for scientific buffers and on a culinary level. Eliane, thank you for being very kind to me and preparing me mentally for a rocky road on pericytes. Micha, thank you for a lot of fun! You have a creative mind, and it was entertaining to work with you on lab movies or Christmas parties! On a scientific point, thank you for guiding me on my first steps in the bioinformatic landscape and accompanying me with animal procedures. Ewa, it was very nice to have you as my colleague for a few years. We had a lot of fun during the IVBM in Helsinki, especially when we found the deserted Heidi’s Bier bar!

Besides the support of my group members, this thesis could not have been established without the help of many others. At the TCR department I specifically want to acknowledge Margareta and Christina, for always managing the department with a big smile. No matter which machine was broken, beeping, or smoking (!), you found a way to fix it. Both of you were a very pleasant lunch company and an excellent way to practice my Swedish. Christina, a special thanks to all the sections and stainings you contributed to my projects! Vasiliki and Gjendine, it was very nice to have you as my colleagues and friends during my PhD. We all went through a similar timeline throughout our PhD, but eventually you caught up with me, your recently published theses have not left my side the past

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weeks! Thank you for a lot of fun in and outside the lab; lunches, quizzes, Christmas parties, spex, and many other fun occasions. Thanks for listening to all my frustrations and, Gjendine, for providing chocolate cookies at the right moment or interrupting with ‘Echt!?’ somehow always at the right time. Vasiliki, we shared our interest for the tumour brain, our discussions on protocols or procedures were very helpful to me, as well as the talks about neck and back exercises! Håkan and Ramin, you were a very pleasant nearby office company. Ramin, thank you for always greeting me when arriving and leaving from your office, no matter if you were in a rush. Håkan thank you for the help with my Swedish homework, unwillingly borrowing your chairs, and serving as the best Santa of TCR. Besides, you two acted as an excellent Sinterklaas and Zwarte Piet together! Wonde, thank you for all your help with the IVIS and chasing mice. Renée, it was very gezellig to have you around in the lab as a fellow Dutch. Thank you for making me feel welcome in Sweden by inviting me to good dinners and board game nights. Pia, thank you for managing the BioCARE and LUCC events and always taking time to drop by my office for a chat.

Göran, thank you for holding the important responsibility of acting as my co- supervisor. You fulfilled the role of co-supervisor perfectly, stepping in if needed (which was not) and being readily available when it was necessary.

I am grateful to all other members of TCR for contributing either scientifically or mentally to this thesis. With many of you I have interacted in some way, often by working on spex or TCR activities (thank you to everyone for the great teamwork putting together these nice ‘events’ every time!), but also going to glioma-related conferences (Elinn), playing tennis (Etienne, Kamila, Arthur), spontaneous chats about football (Javan and Christian) or Pokémon (Christian). I am very fortunate that I can say that many of my colleagues became my friends and that I enjoyed going to work every day thanks to all of you.

Outside the department I want to extend my sincere gratitude to Elisabet and Xavi.

Thank you both for the pleasant collaboration on the COVID-19 project, for providing us with precious patient samples, sharing your knowledge and experiences with us from a different perspective, and for giving me the chance to go through the sample preparations with my own hands. To my Göteborg co-authors on the COVID-19 paper, Joel, Arvid, Magnus, and Henrik, I appreciate you contributed to our project with your patient data and the time you took to comment on our manuscript. Thanks to Teia and Zhi from the FACS core facility, and Anna from the Multipark facility. Your experienced courses have been a tremendous help to my understanding of flow cytometry results and planning my experiments. Anna, specifically thanks to you for spending many hours with my samples, far beyond regular working hours we endlessly calibrated streams and unclogged the nozzle to achieve full plates for sequencing. Thank you for giving me a spot in the precious BD courses as well. David and Johan, thank for your time and sharing your

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thoughts regarding our unexpected sequencing results. Gunilla from Mediatryck thank you for your patient and thorough handling of the printing of this thesis.

I also want to thank Sophie + Fredrik, Andrés, and my dad, who, as native speakers contributed greatly to the Swedish, Spanish, and Dutch popular science summaries, respectively.

I want to thank my opponent Annika and the committee members Bjarne, Anja, and Karin. I have not yet met any of you, but I am very glad that such brain, tumour, and pericyte experts agreed to the important positions of opponent and committee members. I hope we can all meet in person soon and discuss our favourite subject!

Emma and Sofie, it was sad for us that you left TCR, but I am happy to see you back as chair and vice-chair! Thank you for the pleasant discussions and fika moments the past years.

From outside the country, I want to thank Mariona for welcoming me to her lab in Barcelona when I was on holiday, and giving me the chance to learn from Ana who patiently and skillfully showed me the tricks of retina isolation for our short-term collaboration on ALK5 in pericytes.

From outside science, I would like to recognize the contribution of Math Osseforth, my former Latin and ancient Greek teacher. Thank you for your translation of the Erasmus quote and your talent for conscientious research to find its source (Figure S1).

From outside everything, but at the same time very close to me, I would like to thank my dear parents and ‘little’ brother for their support and believe in me to go abroad, for visiting me up north whenever they could, and sending Dutch delicacies when they couldn’t. A special thanks to my dad for his thorough proofreading of my thesis, I am stepping in your footsteps now with the publication of my own book!

Finally, Andrés, you are my favourite pericyte subset, unique in your kind, always wrapping your strong arms around me after a long workday. I am happy I had you by side during my PhD.

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Popular science summary

Tumours do not only consist out of tumour cells. There are many other components that make up a tumour, such as blood vessels, immune cells, and collagen fibres, which are collectively referred to as the tumour microenvironment. Tumour cells communicate with all parts of this microenvironment, and this contributes to tumour growth and their ability to spread and resist therapy. This thesis describes the significance of an intriguing cell type in this tumour microenvironment, the pericyte, in the most aggressive form of adult brain tumours, glioblastoma.

A person who gets diagnosed with glioblastoma today will have to undergo surgery, followed by radiation and chemotherapy, yet, this intense treatment is not enough to save the life of the patient. Glioblastoma is difficult to treat because it grows fast into the surrounding healthy brain, and the infiltrating glioblastoma cells are very hard to reach by any treatment without damaging normal brain cells. Another feature that makes glioblastoma so deadly is the presence of many unfunctional, leaky blood vessels, which cannot properly deliver oxygen and nutrients to the tumour cells.

This defective vasculature causes an environment that is low in oxygen (hypoxic environment), and cells in the middle of the tumour will not be able to survive, eventually dying in a process called necrosis. The tumour cells try to escape their fate and move away from the hypoxic environment. These escaping cells appear under the microscope as a ring of cells that are tightly packed together. The combination of this ‘necrotic centre’ with the ring of migrating cells around it, is called ‘pseudopalisading necrosis’, and is a hallmark of glioblastoma.

Blood vessels consist out of a wall of endothelial cells and are wrapped tightly by insulating pericytes. However, the walls of tumour blood vessels contain holes because the endothelial cells are not properly connected and the pericytes are loosely attached to the vessel wall. Tumour cells instruct themselves to the formation of these disrupted vessels by communicating with the existing blood vessel network and stimulate to their expansion, a process called tumour angiogenesis. Strategies have been developed against endothelial cells and to stop tumour cells from developing their own vessels. However, despite being a highly vascularized tumour, in glioblastoma this approach has been without success.

In this thesis our focus was on the pericytes, the other cell type that is part of the blood vessels. Besides being crucial for blood vessel integrity, pericytes interact

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with immune cells in the blood stream and even perform immune functions themselves, both in healthy and in tumour brain. Plus, in glioblastoma, pericytes have been described to be an essential part of the perivascular niche, a specific environment that supports cancer stem cells, which often contribute to therapy resistance. Despite the important position that pericytes hold in the glioblastoma microenvironment, this cell type is relatively understudied and not fully understood.

Therefore, this thesis aimed to shed light on the significance of pericyte in glioblastoma. In paper 1, we compare the gene expression from pericytes between healthy brain and glioblastoma patients, and discover that those pericytes are quite different. Most importantly, we show that certain genes enriched in pericytes are associated with a poorer survival in glioblastoma patients. In paper 2, we investigate a way to isolate pericytes from mouse brain material and in paper 3 we use those results to construct a detailed genetic map of mouse glioblastoma and its microenvironment. We are still advancing to understand this genetic puzzle and future analysis will have to clarify the complex interactions that are going on between pericytes, tumour cells, and other parts of the microenvironment. Finally, in paper 4, we were fascinated by the expression of the protein ACE2 in pericytes.

ACE2 was discovered as the gateway for SARS-CoV-2, the virus that causes COVID-19. We demonstrate that in the human brain ACE2 is patient-specific and is exclusively expressed by pericytes. Strikingly, COVID-19 patients with high levels of ACE2 in brain pericytes experienced severe neurological symptoms.

Thereby this study indicates a possible role of ACE2-positive pericytes in the development of neurological symptoms in COVID-19 patients.

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Populairwetenschappelijke samenvatting

Een tumor bestaat uit meer dan alleen kankercellen. De omgeving van de tumorcellen bestaat onder andere uit bloedvaten, immuuncellen en bindweefsel, en gezamenlijk vormen zij het micromilieu van de tumor.

Tumorcellen communiceren met alle onderdelen van dit micromilieu en dit draagt bij aan tumorgroei, uitzaaiingen en resistentie tegen behandelingen. Dit proefschrift beschrijft de rol van de pericyte, een intrigerend celtype in het tumor micro-milieu, in de meest agressieve vorm van hersentumoren bij volwassenen, het glioblastoom.

Een patiënt die anno nu de diagnose glioblastoom krijgt, wordt geopereerd en bestraald en krijgt chemotherapie. Die intensieve behandeling is echter niet voldoende om het leven van de patiënt te redden. Glioblastoom is moeilijk te behandelen, omdat het snel groeit binnen het omliggende, gezonde hersenweefsel.

De infiltrerende glioblastoomcellen zijn daardoor bij een operatie of een andere behandeling zeer moeilijk te bereiken zonder de gezonde hersencellen te beschadigen. Een glioblastoom is bovendien vaak dodelijk door de aanwezigheid van veel niet-functionele, lekkende bloedvaten, die de tumorcellen niet goed van zuurstof en voedingsstoffen voorzien. Dit defecte bloedvatennetwerk veroorzaakt een zuurstofarme omgeving (hypoxische omgeving) en cellen in het centrum van de tumor kunnen dit niet overleven. Zij zullen uiteindelijk sterven in een proces dat necrose wordt genoemd. De tumorcellen proberen aan hun lot te ontsnappen door de hypoxische omgeving te verlaten. Deze ontsnappende cellen zien er onder de microscoop uit als een ring van cellen die dicht op elkaar gepakt zitten. De combinatie van dit ‘necrotische centrum’ met de ring van migrerende cellen, wordt

‘pseudopaliserende necrose’ genoemd en een karakteristiek kenmerk van glioblastoom.

Bloedvaten hebben een wand van endotheelcellen die stevig zijn omhuld door isolerende pericyten. De wanden van tumorbloedvaten bevatten echter scheurtjes, doordat de endotheelcellen niet goed met elkaar zijn verbonden en de pericyten slechts losjes aan de vaatwand zijn bevestigd. Door te communiceren met het bestaande bloedvatennetwerk instrueren tumorcellen zelf tot de vorming van deze verstoorde bloedvaten en stimuleren hun uitgroei, in een proces dat tumorangiogenese wordt genoemd. Er zijn strategieën ontwikkeld tegen endotheelcellen om te voorkomen dat tumorcellen hun eigen bloedvaten

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ontwikkelen. Ondanks dat glioblastoom een tumor is met veel bloedvaten, heeft deze aanpak niet tot het beoogde succes geleid.

In dit proefschrift lag onze focus op de pericyten, het andere celtype dat deel uitmaakt van de bloedvaten. Behalve dat ze cruciaal zijn voor de integriteit van bloedvaten, communiceren pericyten ook met immuuncellen in de bloedbaan en voeren ze zelf immuunfuncties uit, zowel in gezonde hersenen als in een hersentumor. Bovendien vormen pericyten bij glioblastoom een essentieel onderdeel van de perivasculaire niche, een specifieke omgeving die kankerstamcellen ondersteunt en die vaak de oorzaak is van resistentie. Ondanks de belangrijke positie die pericyten in het micromilieu van glioblastoom innemen, is dit celtype relatief weinig bestudeerd en niet volledig begrepen.

Dit proefschrift is bedoeld om licht te werpen op de betekenis van pericyten in glioblastoom. In artikel 1 vergelijken we de genexpressie van pericyten in gezonde hersenen met die in glioblastoompatiënten, en ontdekken we dat die pericyten aanzienlijk verschillen. Het belangrijkste dat we in dit artikel aantonen is dat bepaalde genen die sterk tot expressie komen in pericyten geassocieerd zijn met een mindere kans op overleving bij glioblastoompatiënten. In artikel 2 onderzoeken we een manier om pericyten te isoleren uit muizenhersenen en in artikel 3 gebruiken we die resultaten om een gedetailleerde genetische kaart te construeren van muisglioblastoom en de daarbij behorende micro-omgeving. We zijn nog steeds bezig om deze genetische puzzel te begrijpen en vervolgonderzoek zal de complexe interacties tussen pericyten, tumorcellen en andere delen van het micromilieu moeten ophelderen. Ten slotte waren we in artikel 4 gefascineerd door de expressie van het eiwit ACE2 in pericyten. ACE2 werd ontdekt als de toegangspoort voor SARS-CoV-2, het virus dat COVID-19 veroorzaakt. We laten zien dat ACE2 in het menselijk brein patiëntspecifiek is en uitsluitend tot expressie komt in pericyten.

Bovendien zien we dat COVID-19 patiënten waarbij ACE2-positive pericyten in de hersenen werden aangetroffen, opmerkelijk zware neurologische symptomen ondervonden voordat ze overleden. Hiermee leveren we bewijs voor een mogelijke rol van ACE2-positieve pericyten bij de ontwikkeling van neurologische symptomen bij COVID-19-patiënten.

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Populärvetenskaplig sammanfattning

Tumörer består av mer än bara tumörceller. En tumör består till exempel av blodkärl, immunceller och kollagenfibrer, som tillsammans kallas tumörens mikromiljö. Tumörceller kommunicerar med alla delar av denna mikromiljö, vilket bidrar till tumörtillväxt och förmågan att sprida sig och motstå terapi.

Denna avhandling beskriver betydelsen av en spännande celltyp i tumörens mikromiljö, pericyten, hos den mest aggressiva formen av hjärntumörer hos vuxna, glioblastom.

Friska blodkärl består av en vägg av endotelceller, tätt omslutna av ett skyddande yttre lager av pericyter. Förutom att vara avgörande för blodkärlens stabilitet, interagerar pericyter med immunceller i blodomloppet och utför också immunfunktioner själva, både i frisk hjärnvävnad och i tumörvävnad. I tumörblodkärl är pericyterna däremot bara löst fästa vid endotelcellerna som dessutom inte sitter ihop ordentligt, utan bildar ett läckande blodkärl. Tumörceller styr bildandet av dessa störda kärl genom att kommunicera med det befintliga blodkärlsnätverket och stimulera till dess expansion, en process som kallas tumörangiogenes.

En person som idag får diagnosen glioblastom kommer att behöva genomgå operation, följt av strålning och kemoterapi. Tyvärr är denna intensiva behandling inte tillräcklig för att rädda patientens liv. Glioblastom är svår att behandla eftersom den snabbt växer in i den omgivande friska hjärnvävnaden. De infiltrerande glioblastomcellerna är mycket svåra att nå med behandling utan att skada normala hjärnceller. En annan egenskap som gör glioblastom så dödlig är förekomsten av många ofärdiga och läckande blodkärl, vilket kan relateras till pericyterna, som inte kan leverera tillräckligt med syre och näringsämnen till tumörcellerna. Denna defekta kärlstruktur orsakar en miljö som är syrefattig (hypoxisk miljö). Celler i centrum av tumören överlever inte i denna miljö och dör så småningom i en process som kallas nekros. Tumörcellerna gör dock sitt bästa att fly sitt öde genom att röra sig (migrera) bort från den hypoxiska miljön. Dessa migrerande celler syns under mikroskopet som en ring av tätt packade celler. Kombinationen av en nekrotisk mitt med ringen av migrerande celler runt omkring kallas pseudopaliserande nekros.

Förekomsten av denna struktur i ett patientprov tyder på en mer aggressivt växande tumör.

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I den här avhandlingen har vi fokuserat på pericyterna. I glioblastom har pericyter visat sig vara en viktig del av den perivaskulära nischen, en specifik miljö som stöder cancerstamceller vilka ofta bidrar till terapiresistens. Trots den viktiga position som pericyter har i glioblastommikromiljön är denna celltyp relativt understuderad och inte helt förstådd.

Denna avhandling syftar till att beskriva och reda ut betydelsen av pericyter vid glioblastom. I delarbete 1 jämförde vi genuttrycket hos pericyter från frisk hjärnvävnad och glioblastomvävnad, och upptäckte att dessa pericyter är väldigt olika. Mer specifikt visade vi att vissa gener med högt uttryck i pericyter är associerade med en sämre överlevnad hos glioblastompatienter. I delarbete 2 undersökte vi ett sätt att isolera pericyter från mushjärna och i delarbete 3 använde vi dessa resultat för att konstruera en detaljerad genetisk karta över musglioblastom och dess mikromiljö. Vi försöker fortfarande att förstå detta genetiska pussel och ytterligare forskning behövs för att belysa de komplexa interaktioner som pågår mellan pericyter, tumörceller och andra delar av mikromiljön. Slutligen, i delarbete 4, studerade vi uttrycket av proteinet ACE2 i pericyter. ACE2 har visat sig vara ingångsporten för SARS-CoV-2, viruset som orsakar Covid-19. Vi visar att ACE2 i den mänskliga hjärnan uteslutande uttrycks av pericyter och är patientspecifik.

Dessutom ser vi att Covid-19-patienter hos vilka ACE2-positiva pericyter hittades i hjärnan upplevde anmärkningsvärt allvarliga neurologiska symtom innan de dog.

Våra resultat pekar på en möjlig roll för ACE2-positiva pericyter i utvecklingen av neurologiska symtom hos Covid-19-patienter.

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Resumen de divulgación científica

Los tumores no solo están formados por células tumorales. Hay muchos otros componentes que forman un tumor, como los vasos sanguíneos, las células inmunitarias y las fibras de colágeno, que se denominan colectivamente microambiente tumoral. Las células tumorales se comunican con todas las partes de este microambiente y esto contribuye al crecimiento del tumor y su capacidad para propagarse y resistir la terapia. Esta tesis describe la importancia de un tipo de célula intrigante en este microambiente tumoral, el pericito, en la forma más agresiva de tumores cerebrales adultos, el glioblastoma.

Una persona que sea diagnosticada con glioblastoma el día de hoy, tendrá que someterse a una cirugía, seguida de radiación y quimioterapia; sin embargo, este tratamiento intenso no es suficiente para salvar la vida de paciente. La chingadera del glioblastoma es difícil de tratar porque crece rápidamente en el cerebro sano circundante y las células del glioblastoma infiltradas son muy difíciles de alcanzar con cualquier tratamiento sin dañar las células cerebrales normales. Otra característica que hace que el glioblastoma sea tan mortal es la presencia de muchos vasos sanguíneos defectuosos que tienen fugas y no son funcionales, que no pueden suministrar oxígeno y nutrientes de manera adecuada a las células tumorales. Esta vasculatura defectuosa causa un ambiente con poco oxígeno (ambiente hipóxico), y las células en el medio del tumor no podrán sobrevivir y eventualmente morirán en un proceso llamado necrosis. Las células tumorales intentan escapar de su destino y alejarse del ambiente hipóxico. Estas células que escapan aparecen bajo el microscopio como un anillo de células que están muy juntas. La combinación de este "centro necrótico" con el anillo de células migratorias a su alrededor forman una estructura que se denomina "necrosis pseudopalisante". La presencia de esta estructura en un paciente indica un tumor de crecimiento más agresivo.

Los vasos sanguíneos consisten en una pared de células endoteliales y están envueltos firmemente por pericitos aislantes. Sin embargo, las paredes de los vasos sanguíneos del tumor contienen orificios debido a que las células endoteliales no están conectadas correctamente y los pericitos están adheridos de manera suelta a la pared del vaso. Curiosamente, las células tumorales también instruyen para la formación de estos vasos rotos comunicándose con la red de vasos sanguíneos existente y estimulan su expansión, en un proceso llamado angiogénesis tumoral. Se han desarrollado estrategias contra las células endoteliales y para evitar que las

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células tumorales desarrollen sus propios vasos. Sin embargo, a pesar de ser un tumor muy vascularizado, en el glioblastoma este método no ha tenido éxito.

En esta tesis, nuestro enfoque se centró en los pericitos, el otro tipo de célula que forma parte de los vasos sanguíneos. Además de ser cruciales para la integridad de los vasos sanguíneos, los pericitos interactúan con las células inmunes en el torrente sanguíneo e incluso realizan funciones inmunes ellos mismos, tanto en el cerebro sano como en el tumor. Además, en el glioblastoma, se ha descrito que los pericitos son una parte esencial del nicho perivascular, un entorno específico que apoya a las células madre cancerosas, que a menudo contribuyen a la resistencia a la terapia. A pesar de la importante posición que ocupan los pericitos en el microambiente del glioblastoma, este tipo de células está relativamente poco estudiado y no se comprende completamente.

Por lo tanto, esta tesis tuvo como objetivo arrojar luz sobre la importancia del pericito en el glioblastoma. En el artículo 1, comparamos la expresión génica de los pericitos entre el cerebro sano y los pacientes con glioblastoma, y descubrimos que esos pericitos son bastante diferentes. Más importante aún, mostramos que ciertos genes enriquecidos en pericitos se asocian con una menor supervivencia en pacientes con glioblastoma. En el artículo 2, investigamos una forma de aislar pericitos del material cerebral de ratón y en el artículo 3 usamos esos resultados para construir un mapa genético detallado del glioblastoma de ratón y su microambiente. Todavía estamos avanzando para comprender este rompecabezas genético y los análisis futuros deberán aclarar las complejas interacciones que están ocurriendo entre los pericitos, las células tumorales y otras partes del microambiente. Finalmente, en el artículo 4, nos fascinó la presencia de la proteína ACE2 en los pericitos. El ACE2 fue descubierto como la puerta de entrada para el SARS-CoV-2, el virus que causa el COVID-19. Demostramos que en el cerebro humano, la presencia del ACE2 es específica por paciente y se expresa exclusivamente por pericitos. Además, vemos que los pacientes con COVID-19 en los que se encontraron pericitos ACE2 positivos en el cerebro experimentaron síntomas neurológicos notablemente graves antes de morir. Con este estudio, proporcionamos evidencia de un posible papel de los pericitos positivos para ACE2 en el desarrollo de síntomas neurológicos en pacientes con COVID-19.

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Abstract

Glioblastoma is an aggressive and incurable grade 4 brain tumour. Despite maximum treatment efforts, recurrence invariable arises mainly due to the infiltrative nature of individual tumour cells, a highly proliferative vasculature, and the presumed existence of glioblastoma stem-like cells (GSCs). These features all contribute to treatment resistance and are in some way linked to pericytes. Pericytes are multi-functional vascular mural cells that are embedded within the vascular basement membrane of the microvasculature. In addition to providing blood vessel stability, pericytes act as and interact with immune cells, and can regulate blood flow. In glioblastoma, pericytes are loosely attached to the vasculature resulting in a lack of contact-inhibition to the underlying endothelium, causing excessive endothelial cell proliferation and sprouting. In addition, infiltrating glioblastoma cells have been observed migrating along the vascular basement membrane into the surrounding brain parenchyma.

Thirdly, pericytes are an integral component of the perivascular niche (PVN), a safe haven for GSCs. Despite this key position of pericytes in glioblastoma, their significance in the glioblastoma microenvironment remains relatively obscure.

This thesis focuses on elucidating the role of pericytes in glioblastoma. Without making assumptions if pericytes are promoting or suppressing tumour development, we perform integrative studies on brain pericytes in glioblastoma and explore their heterogeneity. In paper 1 we analysed gene expression profiles of pericytes in glioblastoma patients and physiological brain tissue. Gene signatures that were enriched in glioblastoma pericytes were associated with a worse overall survival when compared to signatures that did not show any significant difference. In paper 2 we performed single-cell transcriptome analysis on perivascular cells that were in silico isolated from mouse glioblastoma tissue. We annotated two pericyte subsets, as well as perivascular fibroblasts and VSMCs. Transcriptomics on the complete murine glioblastoma tissue was conducted in paper 3. In this paper, tumours from pericyte-poor mice were compared with those from mice with a normal pericyte coverage, with the aim to elucidate the effect of pericytes on tumour cells and other components of the glioblastoma microenvironment.

Finally, the current COVID-19 pandemic directed us to investigate the role of pericytes in neurological symptomatology in COVID-19 patients. We provide indisputable evidence that in the brain the SARS-CoV-2 entry receptor ACE2 is exclusively expressed on brain pericytes and is patient-specific. Moreover, COVID- 19 patients that expressed high levels of ACE2 in brain pericytes suffered from severe neurological affliction prior to decease.

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Background

Blood and lymph vessels compose indispensable, conveying networks of the human body fluids. Where the blood circulatory system supplies vital nutrients and oxygen to nourish every tissue, the lymph vessels drain the excessive interstitial fluid back to the blood stream and accommodate immune cell trafficking (Bautch and Caron, 2015; Karpanen and Alitalo, 2008). The exchange of these gases and nutrients takes place in the smallest vessels of the circulatory system, the capillaries.

The length of the human blood vessels is extensive, with a collective length in a single human of more than 160,000 km they would be able to encircle the earth four times (Aird, 2005). This corresponds to a network of approximately 10¹² endothelial cells, the cells making up the inner lining of all blood vessel walls, but with a mere total weight of 100 gram (Jaffe, 1987).

New blood vessels can arise through two main processes. Early in human embryonic development, around day 18, blood vessels originate de novo from the mesoderm through a process called vasculogenesis, in which angioblasts differentiate into endothelial cells and form the primary vascular tree (Gerecht-Nir et al., 2004; Risau and Flamme, 1995). Subsequently, during the successive process called angiogenesis, differentiated endothelial cells sprout from existing vessels and expand the present vessel network (Risau, 1997). From here on, vasculogenesis and angiogenesis occur simultaneously to expand the vascular system (Bautch and Caron, 2015). In addition, lymph vessels originate from venous endothelial cells, later in embryonic development around week 5 (Gerecht-Nir et al., 2004).

In vertebrates, the larger blood vessels (arteries and veins) consist out of three layers; the tunica intima, the tunica media, and the tunica adventitia (Mazurek et al., 2017). The innermost tunica intima consists of a single layer of endothelial cells which line the lumen of the vessel wall and are in contact with the blood. This internal layer is surrounded by several layers of circumferentially arranged vascular smooth muscle cells (VSMCs) that together make up the tunica media. The tunica adventitia is the outermost, fibrous layer of collagenous matrix with herein embedded fibroblasts, immune cells, and progenitor cells (Majesky et al., 2012;

Mazurek et al., 2017). In smaller vessels, such as capillaries and pre- and post- capillary vessels (arterioles and venules, respectively), the monolayer of endothelial cells is enveloped by a discontinuous layer of pericytes instead of VSMCs (Cleaver and Melton, 2003). Collectively, pericytes and VSMCs are referred to as mural cells.

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The term pericytes was coined in 1923 by Karl Wilhelm Zimmermann, referring to a cell (‘cyte’) that lies around (‘peri’) the capillary. However, 50 years earlier these cells were already observed independently by Carl Joseph Eberth (1871) and Charles-Marie Benjamin Rouget (1873) (Eberth, 1871; Rouget, 1873) (Figure S2 and S3). Eberth realised spindle-shaped adventitia cells lining the capillaries of adult frogs, and Rouget described peculiar, branched cells which move in regular, interrupted rows along the capillaries and grip them with their numerous appendages (Eberth, 1871; Rouget, 1873; Zimmermann, 1923). Already at that time, Rouget postulated that these cells act as contractile elements. Furthermore, the Danish professor August Krogh was awarded the Nobel Prize in Physiology or Medicine in 1920, for his discovery of the ‘capillary motor regulating mechanism’ (Krogh, 1919). At that time not knowing which elusive cell type performed this process, this mechanism, among many others, can now be ascribed to the intriguing pericytes (Grubb et al., 2020).

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Chapter 1 – Pericytes

Since the introduction of the term pericytes in 1923, their definition has been debated extensively regarding their function and marker expression, however, changed barely when focusing on location and morphology. Currently, a pericyte is defined as a cell with a protruding cell body that encapsulates the microvascular endothelial cells and is embedded in the vascular basement membrane (Dessalles et al., 2021). They have elongated, finger-like projections that embrace the capillary and are in contact with multiple endothelial cells (see back-side image) (Hirschi and D'Amore, 1996). The bulging cell body (soma) contains the nucleus surrounded by little cytoplasm. From here extend slender, primary processes with a diameter of only 0.05-0.4 μm arranged along the longitudinal vessel axis and are mainly built up of cytoskeletal elements (Bruns and Palade, 1968). Secondary processes branch from the primary ones and are transversely oriented, wrapping around the vessel circumference (Hartmann et al., 2015).

Pericytes are distributed frequently along the microvessels, but have been primarily observed to be present at endothelial cell branch points (Armulik et al., 2011).

Despite their regular presence and long extruding processes, pericytes are not in constant physical contact with each other. Opposed to VSMCs, they form a discontinuous layer, and their probing processes avoid contact, even retracting after reaching a process from a neighbouring pericyte (Berthiaume et al., 2018).

The traditional pericyte that contains an extruding soma (bump-on-log morphology) is still recognised as such for the capillary pericytes specifically. In addition, a continuum of distinct morphologies for pericytes along the microvasculature has been identified (Figure 1) (Grant et al., 2019). On pre-capillaries, there are so-called

‘ensheathing pericytes’ with short primary processes and longer secondary processes that completely encircle the capillary. Along the mid-capillary there are

‘thin-stranded pericytes’ with vastly long primary processes and extra short secondary processes that only partially encircle the vessel (Grant et al., 2019).

Thirdly, mesh pericytes are located on the post-capillaries and have a fractal-like organisation without the typical primary and secondary branching pattern (Grant et al., 2019; Hartmann et al., 2015). Recently, pre-capillary sphincters have been denoted as a type of pericytes at the first branching capillaries. Those pericytes have more circumferential processes around the capillaries and less longitudinal processes (Grubb et al., 2020).

Although pericytes and VSMCs are often mistaken for one another due their association with the vasculature, their morphology is profoundly different. The characteristic bump-on-a-log morphology of pericytes separates it from the more flattened cell body of VSMCs. In addition, VSMCs extend on a much shorter distance along a vessel, ∼20 µm, as compared to lengths of ∼40, 100, and 150 µm for ensheathing, mesh, and thin-stranded pericytes, respectively (Grant et al., 2019).

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Figure 1. Continuum of pericyte morphology along microvessels.

In addition to an expansion of terminology on pericyte morphology, over the years pericytes have been ascribed multiple functions and interactive players. The next section describes the sophisticated signalling pathways delegated and received by pericytes to and from their surroundings, and the significant roles these pathways and interactions include.

Function, interactive players, and signalling pathways

Following pericyte discovery, the knowledge about their functions has elaborated immensely. From simple, scaffolding cells, pericytes transformed to pivotal, multifaceted cells that play an essential role in development, stability, integrity, and remodelling of blood vessels (Gerhardt and Betsholtz, 2003). Furthermore, these multi-functional cells regulate capillary blood flow, vascular permeability, and inflammatory responses (Armulik et al., 2011).

Vessel development, stability, and integrity

The primary cellular associates of pericytes are the endothelial cells. The proportion of pericytes to endothelial cells is tissue-dependent, with ratios varying from 1:1 in the retina and central nervous system (CNS) to 1:10 in the lung, and 1:100 in striated muscle (Shepro and Morel, 1993). Tight interaction between those two vascular cell types is crucial for vessel stability and integrity, as well as development of a mature microvasculature. Pericytes and endothelial cells are in direct physical contact with each other via several ways: peg-socket interdigitations, adhesion plaques, and gap junctions (Dessalles et al., 2021).

The intimate peg-socket connections are a very characteristic way of surface contact between pericytes and the underlying endothelium. They are micron-sized pericyte

‘fingers’ (pegs), that penetrate into invaginations (sockets) in the endothelial cells, and pericytes possibly use this contact to pull on the endothelial cells (Braverman and Sibley, 1990; Caruso et al., 2009). Adherence junctions consist out of N- cadherin interactions, and adherence plaques are fibronectin patches that anchor the pericyte to the endothelial cells (Gerhardt et al., 1999; Gerhardt et al., 2000). Gap

Arteriole Pre-capillary Capillary Post-capillary Venule

Thin-stranded pericyte

Mesh pericyte Ensheathing

pericyte

VSMC Mesh pericyte VSMC

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junctions are specialized channels formed by connexxin 43 proteins (Cx43; also known as gap-junction protein alpha 1 (GJA1)) that connect the cytoplasm of pericytes with that of endothelial cells and allow for exchange of small molecules and ions (Cuevas et al., 1984; Fujimoto, 1995).

This intricate, physical contact between pericytes and endothelial cells is required for blood vessel maturation and stabilisation. Once pericytes make a stable interaction with endothelial cells they inhibit their proliferation and stimulate enforcement of inter-endothelial cell adherence junctions consisting out of VE- cadherin (Kruse et al., 2019; Orlidge and D'Amore, 1987). In addition, upon cell- cell contact, a tight adhesion establishes between Notch3 on pericytes with Jagged- 1 (Jag-1) on endothelial cells, which suppresses pericyte proliferation and promotes quiescence (Liu et al., 2009).

Besides physical contact with the endothelium, the pericytes have a tight connection with the non-cellular matrix, the vascular basement membrane. Pericytes are surrounded by this basement membrane, with exception of the previously described numerous focal points where they are in contact with endothelial cells. The microvascular basement membrane constitutes out of two major extracellular matrix components, collagen IV and laminin, which are linked together by heparin- sulphate proteoglycans (HSPGs) and nidogens (Nid1 and Nid2) (Leclech et al., 2020; Timpl, 1989). Formation of the vascular basement membrane is dependent on interaction of pericytes and endothelial cells. When pericytes are recruited around developing microvessels, extracellular deposition of collagen IV, fibronectin, HSPGs, and nidogens initiates assembly of the basement membrane (Stratman et al., 2009; Stratman et al., 2010). In addition, pericytes themselves contribute to the vascular basement membrane by deposition of collagen IV, fibronectin, and laminins (Jeon et al., 1996; Mandarino et al., 1993).

Pericyte-endothelial cell signalling

In addition to the physical interactions, pericytes and endothelial cells signal to each other through paracrine signalling that affects their growth and development positively and negatively (Figure 2). The major paracrine signalling is through the family of platelet-derived growth factors (PDGFs) and their receptors (PDGFRs).

During ongoing angiogenesis, the sprouting, tip endothelial cells release the dimer PDGF-BB protein into the surrounding extra cellular matrix (ECM), which is a major growth factor for the pericytes (Hellstrom et al., 1999). Specifically, a retention motif on the C-terminal of PDGF-BB binds with HSPGs in the ECM (Lindblom et al., 2003; Ostman et al., 1991). As a result, the nearby PDGFR-β- expressing pericytes sense the retained PDGF-BB protein and subsequently are attracted to the sprouting vessel (Lindblom et al., 2003). This PDGF-BB/PDGFR-β communication is essential for proper blood vessel formation and development of an organism, since complete knock-out of either Pdgfb or Pdgfrb during

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embryogenesis, results in a non-viable outcome due to excessive haemorrhaging (Leveen et al., 1994; Lindahl et al., 1997; Soriano, 1994).

Besides PDGF-BB/PDGFR-β signalling, there are additional signalling factors that stimulate blood vessel growth, such as basic fibroblast growth factor (bFGF) (Nakamura et al., 2016) and vascular endothelial growth factors (VEGFs) (Darland et al., 2003). Especially VEGF-A and its binding to VEGFR-2 on endothelial cells are essential for vasculogenesis and angiogenesis; knock-out of either the ligand or the receptor results in embryonic lethality due to severe vasculature defects (Carmeliet et al., 1996; Shalaby et al., 1995). VEGF-A exists in four different isoforms that differ from each other in their affinity to bind HSPGs (Patel-Hett and D'Amore, 2011). Gradients of the smaller, more soluble VEGF-A isoforms guide migration of sprouting endothelial tip cells that express high levels of VEGFR-2.

The larger isoforms with higher HSPG affinity accumulate in the ECM and their concentration determines proliferation of sprout stalks (Gerhardt et al., 2003; Patel- Hett and D'Amore, 2011).

Where the PDGF-BB/PDGFR-β and VEGF-A/VEGFR-2 signalling stimulate blood vessel growth, there are several counteracting pathways that inhibit endothelial cell proliferation, stimulate quiescence, and induce vessel stabilisation. The major pathway through which pericytes negatively affect endothelial cell growth is TGF- β signalling. TGF-β is involved in suppressing endothelial cell proliferation. The latent, inactive form of TGF-β is activated when pericytes and endothelial cells make direct contact with each other, after which the latter gets inhibited in its proliferation by activated TGF-β (Antonelli-Orlidge et al., 1989). Similarly, reciprocal signalling from endothelial cell-derived TGF-β inhibits proliferation of pericytes (Yan and Sage, 1998). Concurrently with their differentiation and in response to TGF-β, pericytes secrete an isoform of VEGF-A that is retained in the ECM, thereby locally stimulating endothelial cell survival and promoting vessel stability (Darland et al., 2003). In addition, pericytes have been proposed to express the VEGF receptor VEGFR-1, which acts as a ligand-trap of VEGFs (Cao et al., 2010). Binding of VEGF-A to this receptor sequesters it from the endothelial- expressed VEGFR-2, hence, preventing initiation of angiogenesis in mature and quiescent blood vessels (Eilken et al., 2017; Fong et al., 1995). Thus, both pericytes and endothelial cells secrete TGF-β and express the corresponding receptors, which makes this a complex signalling pathway where both cell types are interdependent on one another.

In addition, two TGF-β receptors, activin-like kinase (ALK) 1 and 5, exert opposing cellular effects (Goumans et al., 2003; Goumans et al., 2002). Both receptors, as well as their TGF-β ligand, are essential for embryonic development, since deletion of either three genes results in embryonic lethality due to defective angiogenesis and impaired mural cell recruitment and differentiation (Dickson et al., 1995; Larsson et al., 2001; Oh et al., 2000). Endothelial cells express both ALK1 and ALK5, but

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activation of ALK1 upon binding of TGF-β promotes cell proliferation and migration through Smad1/5, and activation of ALK5 stimulates vessel maturation through Smad2/3 (Goumans et al., 2002). ALK1 has been reported to be exclusive to endothelial cells, but ALK5 is broadly expressed by multiple cell types including pericytes (Aguilera and Brekken, 2014; Seki et al., 2006). Similar to ALK5 activation in the endothelium, ALK5 activation in pericytes stimulates quiescence and vessel stabilisation, partly through the Smad2/3 mediated transcription of FN1 (Aguilera and Brekken, 2014; Gaengel et al., 2009).

Angiopoietins are glycoproteins that interact with their receptors Tie1 and Tie2 to increase vessel stability. Angiopoietin-1 (Ang1) is secreted by pericytes and upon interaction with Tie2 on endothelial cells it maintains vessel integrity. Both proteins are very critical for proper blood vessel formation, since knock-out of either one results in angiogenic deficits and early embryonic lethality (Dumont et al., 1994;

Suri et al., 1996). Upon pericyte-secreted Ang1-binding to Tie2, the latter is phosphorylated and stimulates vessel stabilisation through regulation of transcription factor FOXO1 (Daly et al., 2004). Interestingly, Ang2, the antagonistic counterpart of Ang1, is stored in endothelial cells and swiftly released upon stimulation, after which it competes for Tie2 binding. Ang2 only weakly activates Tie2 and therefore counteracts Ang1 activity (Fiedler et al., 2004; Yuan et al., 2009).

Unexpectedly, it was later discovered that pericytes themselves also express Tie2, although in low levels, and that Tie2 activation in pericytes controls vessel maturation (Teichert et al., 2017).

A detailed balance among the aforementioned players is required for appropriate development of the vascular tree during embryogenesis and during adulthood processes that require blood vessel regeneration, such as wound healing and the female reproductive cycle (Gordon et al., 1995, female).

Figure 2. Pericyte-endothelial interactions.

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Regulating capillary blood flow

Already in 1873 Rouget proposed that pericytes have contractile potential and possibly could mediate blood flow (Rouget, 1873). The contractility has been an ongoing debate (Hill et al., 2015) and is a highly investigated functional property of pericytes. The contractility varies between the type of pericyte and expression levels of alpha smooth muscle actin (αSMA, gene symbol: ACTA2), especially the ensheathing pericytes are accepted to possess contractile capabilities (Dessalles et al., 2021; Nehls and Drenckhahn, 1991).

The expression of contractile proteins, such as αSMA and tropomyosin, in stress fibres of pericytes (Alarcon-Martinez et al., 2018; DeNofrio et al., 1989; Joyce et al., 1985; Wallow and Burnside, 1980) is evidence for the contractile potential of pericytes. Especially, work in retinal and cerebral capillaries has shown contractility of these mural cells (Hall et al., 2014; Hamilton et al., 2010; Kornfield and Newman, 2014; Kureli et al., 2020). In addition, the continuing controversy includes those that agree that pericytes express αSMA, but that VSMCs are the significant cells with contractile capabilities (Fernandez-Klett et al., 2010), and others who disagree with any contractility of pericytes at all (Hill et al., 2015). Recently, it has been shown that pericytes form precapillary sphincters on the junction of the arteriole and the first branching capillary. These pericytes display high levels of αSMA and can alter cerebrovascular flow after constriction (Grubb et al., 2020).

Inflammatory response

Pericytes play many roles in immunoregulation, such as leukocyte trafficking and cytokine secretion (Rustenhoven et al., 2017). Mesh pericytes on post-capillaries have a morphology that is less suitable for contractile functions, and therefore these pericytes are thought to be responsible for regulating the immune functions. For instance, it was demonstrated that neutrophils crawl along venular walls guided by pericyte-expressed intercellular adhesion molecule 1 (ICAM-1) and enter the tissue between adjacent pericytes (Proebstl et al., 2012). Another cell adhesion molecule expressed by pericytes is melanoma cell adhesion molecule (MCAM, also known as CD146) (Crisan et al., 2008) and has been shown to contribute to transmigration (Bardin et al., 2009), and is therefore possibly another mechanism that pericytes could use for leukocyte extravasation. Moreover, pericytes along the mid-capillaries and arterioles have been observed to instruct neutrophils and macrophages to extravasate through the walls of the ensuing post-capillary venules, followed by additional interactions between these migratory innate immune cells and pericytes through ICAM-1 and macrophage migration-inhibitory factor (MIF) (Stark et al., 2013). To facilitate neutrophil extravasation, pericytes remodel the basement membrane around venules and allow for increased inter-pericyte gaps through their relaxation (Wang et al., 2012). In addition, pericytes respond to the pro- inflammatory cytokine interleukin 17 (IL-17) by upregulating other pro-

Integrative studies of brain pericytes and their involvement in glioma and COVID-19 Oudenaarden, Clara

Integrative studies of brain pericytes and their involvement in glioma and COVID-19

Integrative studies of brain pericytes and their involvement in glioma and COVID-19

Integrative studies of brain pericytes and their involvement in glioma and COVID-19

Strongly wrapping arms Embracing vasculature Perfect pericyte

“Crescit scribendo scribendi studium”

Desiderius Erasmus Roterodamus

Table of Contents

Papers included in the thesis

Paper 1

Paper 2

Paper 3

Paper 4

List of abbreviations

Acknowledgements

Popular science summary

Populairwetenschappelijke samenvatting

Populärvetenskaplig sammanfattning

Resumen de divulgación científica

Abstract

Background

Chapter 1 – Pericytes