Periodic Reporting for period 1 - B3M (Breaking into the brain- basement membranes and the perivascular niche)
Período documentado: 2022-08-01 hasta 2025-01-31
In neuroinflammation leukocytes reside for several days in the perivascular niche of cerebral blood vessels - defined by the basal surface of the endothelium, the endothelial basement membrane (BM) and an outer parenchymal BM with associated astrocyte endfeet (Fig. 1) - a poorly studied site but of utmost fundamental and clinical relevance. This site harbours genetically distinct resident cell populations, the function of which are unclear. BMs define the perivascular niche and the sealed nature of this compartment in an unknown manner. B3M uses several approaches to recreate this perivascular niche in vitro or to mimic biochemical and/or mechanical aspects of this site, with the aim of better understanding its function in neuroinflammation.
Endothelial cells derived from induced pluripotent stem cells (iPSC), expressing brain characteristics, are seeded into microchannels in a novel dextran-hydrogel with tuneable adhesive and stiffness properties to most correctly mimic the in vivo environmental properties and to recapitulated the in vivo spatial arrangement of cells and BMs. The system permits perfusion with immune cells and live imaging.
Parallel ex vivo and synthetic approaches further break down the complexity of this site into discrete steps and, using multiscale imaging of new split-cre transgenic mice, we track, target and profile perivascular cells lacking receptors to key BM components. Studies to date on leukocyte entry into the brain focus on endothelial properties or immune cell behaviour with little consideration of the 3D relationship between cellular and BM barriers and their functional interdependence. Our unique knowledge on extracellular matrix structure/function of cerebral vessels and leukocyte migration into the brain, permits identification of key elements of the perivascular niche and how they can be mimicked in vitro and targeted in vivo. B3M’s cross-disciplinary approach deciphers cellular and molecular events occurring after leukocyte penetration of the endothelium in the perivascular niche, shedding light on this poorly studied aspect of inflammation.
Endothelial cells derived from induced pluripotent stem cells (iPSC), expressing brain characteristics, are seeded into microchannels in a novel dextran-hydrogel with tuneable adhesive and stiffness properties to most correctly mimic the in vivo environmental properties and to recapitulated the in vivo spatial arrangement of cells and BMs. The system permits perfusion with immune cells and live imaging.
Parallel ex vivo and synthetic approaches further break down the complexity of this site into discrete steps and, using multiscale imaging of new split-cre transgenic mice, we track, target and profile perivascular cells lacking receptors to key BM components. Studies to date on leukocyte entry into the brain focus on endothelial properties or immune cell behaviour with little consideration of the 3D relationship between cellular and BM barriers and their functional interdependence. Our unique knowledge on extracellular matrix structure/function of cerebral vessels and leukocyte migration into the brain, permits identification of key elements of the perivascular niche and how they can be mimicked in vitro and targeted in vivo. B3M’s cross-disciplinary approach deciphers cellular and molecular events occurring after leukocyte penetration of the endothelium in the perivascular niche, shedding light on this poorly studied aspect of inflammation.
The main aims of B3M are (1) the establishment of a 3D model of brain blood vessels that resemble those in vivo both in terms of cellular and BM layers and which are in an environment that resembles the (low) stiffness of the brain parenchyma, and (2) to dissect the steps in leukocyte extravasation using a series of novel in vitro set ups, that will allow precise investigation of steps that would otherwise remain difficult to assess (events occurring after penetration of the endothelium and before entry into the inflamed brain).
The project comprises 5 WPs, all of which have commenced and are progressing according to plan. WP1 focuses on development of a 3D model of cerebral microvessels using a novel dextran-hydrogel system, and has to date established the conditions needed for both formation of an endothelial monolayer with a basal BM and for spreading and polarization of astrocytes (Fig. 2). A protocol for iPSC differentiation to brain-like endothelial cells has been established, which differs to others to date in that it maintains the endothelial molecular profile but promotes the tight junctional interactions and barrier formation, typical of brain endothelium. This now permits testing of iPSC derived brain-like endothelial cells in the 3D dextran hydrogels together with astrocytes and pericytes or conditioned medium thereof.
WP2 and WP3 address the mechanical aspects of the subendothelial niche and penetration of the BM, respectively. A breakthrough has been the development of the first in vitro system for visualisation of leukocyte migration across excised BMs. This now permits precise investigation of both leukocytes and the BM during the extravasation processes and dissection of factors such as tension across the BM, receptors on the leukocytes, involvement of proteases, and signalling cascades in leukocytes required for penetration of the BM.
Parallel in vivo analyses have addressed the role of vascular BMs on the maintenance of perivascular myeloid populations, which have recently received considerable attention but the function of which remains unclear. Our data provide new information demonstrating that interactions between perivascular laminins myeloid precursor affect their development and the seeding of the meningeal and perivascular niches, and that they are required for the long term survival of perivascular macrophages.
The project comprises 5 WPs, all of which have commenced and are progressing according to plan. WP1 focuses on development of a 3D model of cerebral microvessels using a novel dextran-hydrogel system, and has to date established the conditions needed for both formation of an endothelial monolayer with a basal BM and for spreading and polarization of astrocytes (Fig. 2). A protocol for iPSC differentiation to brain-like endothelial cells has been established, which differs to others to date in that it maintains the endothelial molecular profile but promotes the tight junctional interactions and barrier formation, typical of brain endothelium. This now permits testing of iPSC derived brain-like endothelial cells in the 3D dextran hydrogels together with astrocytes and pericytes or conditioned medium thereof.
WP2 and WP3 address the mechanical aspects of the subendothelial niche and penetration of the BM, respectively. A breakthrough has been the development of the first in vitro system for visualisation of leukocyte migration across excised BMs. This now permits precise investigation of both leukocytes and the BM during the extravasation processes and dissection of factors such as tension across the BM, receptors on the leukocytes, involvement of proteases, and signalling cascades in leukocytes required for penetration of the BM.
Parallel in vivo analyses have addressed the role of vascular BMs on the maintenance of perivascular myeloid populations, which have recently received considerable attention but the function of which remains unclear. Our data provide new information demonstrating that interactions between perivascular laminins myeloid precursor affect their development and the seeding of the meningeal and perivascular niches, and that they are required for the long term survival of perivascular macrophages.
The two major developments to date have been (1) establishment of endothelial tubes in 3D (in DexVs gels) that resemble brain microvessels, and (2) development of an in vitro system that now permits live imaging of leukocyte migration across excised mesenteric BMs. Both of these achievements provide new and powerful tools that permit addressing scientific questions that were previously not possible.
The challenge still remains to achieve the double BM structure shown in Fig. 1 in vitro in the DexVs hydrogels. While 3D endothelial tubes with an underlying endothelial BM have been achieved and is already a major improvement on systems available to date, the second parenchymal BM remains difficult to generate. Ongoing experiments testing the effects of perivascular cells or conditioned media thereof on the endothelial cell/astrocyte cocultures in hydrogels will be critical in defining whether this will be achievable within the funding period. However, even if not successful, these experiments are likely to provide new information on cell-cell interactions and/or soluble factors critical for establishing the tight barrier properties typical of brain endothelium. Such information is important both for drug delivery into the brain, e.g. in the case of tumours or inflammation, but for understanding how to restore barrier function when it is compromised, as occurs after stroke.
The incubation chamber enclosing a clamp system for fixing excised mesenteric BM that we have developed in WP3 is the first system that permits both control and measurement of the tension across the BM required for leukocyte transmigration and, importantly, permits live imaging of leukocytes migrating through the BM over hours (because of control CO2 and temperature). It is therefore a valuable instrument which now permits precise investigation of the age-old question of how leukocytes migrate across BMs; it will permit investigation of characteristics of the BM per se and of receptors and signalling pathways in leukocytes that control this process. Such information is fundamental to understanding leukocyte extravasation processes both in the brain and other tissues, and our instrument provides a unique mode of deciphering and manipulating critical factors. This is likely to identify new drug targets for control of inflammatory processes.
The challenge still remains to achieve the double BM structure shown in Fig. 1 in vitro in the DexVs hydrogels. While 3D endothelial tubes with an underlying endothelial BM have been achieved and is already a major improvement on systems available to date, the second parenchymal BM remains difficult to generate. Ongoing experiments testing the effects of perivascular cells or conditioned media thereof on the endothelial cell/astrocyte cocultures in hydrogels will be critical in defining whether this will be achievable within the funding period. However, even if not successful, these experiments are likely to provide new information on cell-cell interactions and/or soluble factors critical for establishing the tight barrier properties typical of brain endothelium. Such information is important both for drug delivery into the brain, e.g. in the case of tumours or inflammation, but for understanding how to restore barrier function when it is compromised, as occurs after stroke.
The incubation chamber enclosing a clamp system for fixing excised mesenteric BM that we have developed in WP3 is the first system that permits both control and measurement of the tension across the BM required for leukocyte transmigration and, importantly, permits live imaging of leukocytes migrating through the BM over hours (because of control CO2 and temperature). It is therefore a valuable instrument which now permits precise investigation of the age-old question of how leukocytes migrate across BMs; it will permit investigation of characteristics of the BM per se and of receptors and signalling pathways in leukocytes that control this process. Such information is fundamental to understanding leukocyte extravasation processes both in the brain and other tissues, and our instrument provides a unique mode of deciphering and manipulating critical factors. This is likely to identify new drug targets for control of inflammatory processes.