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The role of PDGF-B in brain blood vessels
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology. (Christer Betsholtz)
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The development of blood vessels is dependent on several molecular cues to form properly. A functional PDGF-B/PDGFR-b signaling is paramount for the investment of mural cells, that provide with support, to the developing vasculature. Mutations in PDGFB and PDGFRB are linked to PFBC, an age-dependent neurodegenerative condition manifested by vessel associated calcifications in the brain. The overall aim of the work presented in here was to investigate PFBC related calcifications and analyze the effects of impaired PDGF-B/PDGFR-b signaling on the formation of brain calcifications in different mouse models.

In paper I, we functionally analyzed PFBC-related PDGFB and PDGFRB mutations in vitro. While all PDGFB mutations lead to abolished protein function, PDGFRB mutations have more diverse consequences. We also show that reduced Pdgfb and Pdgfrb levels are insufficient for the formation of brain calcifications in several mouse strains. Moreover, region-specific susceptibility factors seem to reside in PFBC pathogenesis that are distinct from pericyte coverage and BBB deficiency.

In paper II, we described the molecular composition and cellular association of calcified nodules that develop in two mouse models of PFBC, Pdgfbret/ret and Slc20a2-/- mice. We show that the nodules are composed of pro- and anti-mineralization proteins and that they are in direct association with astrocytes and microglia

In paper III, we analyzed the effects of EC-specific ablation of PDGF-B in adult brain vasculature.  We report a substantial decrease of pericyte coverage and altered VSMC morphology and that this phenotype is inadequate to trigger the formation of calcifications or affect BBB integrity.

The aim of paper IV was to molecularly define the adult mouse brain vasculature by taking advantage of the scRNAseq technique. Here, we describe a gradual change in expression profile along the arteriovenous axis: ECs present a continuum along the axis while mural cell expression profile is punctuated. 

In summary, this thesis present detailed description of calcifications formed in mouse models of PFBC and address the role of impaired PDGF-B/PDGFR-b signaling for the formation of nodules in mice. Furthermore, the scRNaseq analysis performed on healthy adult brain vasculature has paved the way for future analysis in mouse models of PFBC.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. , p. 48
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1556
Keywords [en]
PFBC, PDGFB, PDGFRB, Brain vasculature, Mural cells
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-380088ISBN: 978-91-513-0607-0 (print)OAI: oai:DiVA.org:uu-380088DiVA, id: diva2:1298528
Public defence
2019-05-15, Rudbecksalen, Dag Hammarskjölds väg 20, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2019-04-18 Created: 2019-03-24 Last updated: 2019-06-18
List of papers
1. Functional Characterization of Germline Mutations in PDGFB and PDGFRB in Primary Familial Brain Calcification
Open this publication in new window or tab >>Functional Characterization of Germline Mutations in PDGFB and PDGFRB in Primary Familial Brain Calcification
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2015 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 11, article id e0143407Article in journal (Refereed) Published
Abstract [en]

Primary Familial Brain Calcification (PFBC), a neurodegenerative disease characterized by progressive pericapillary calcifications, has recently been linked to heterozygous mutations in PDGFB and PDGFRB genes. Here, we functionally analyzed several of these mutations in vitro. All six analyzed PDGFB mutations led to complete loss of PDGF-B function either through abolished protein synthesis or through defective binding and/or stimulation of PDGF-R beta. The three analyzed PDGFRB mutations had more diverse consequences. Whereas PDGF-R beta autophosphorylation was almost totally abolished in the PDGFRB L658P mutation, the two sporadic PDGFRB mutations R987W and E1071V caused reductions in protein levels and specific changes in the intensity and kinetics of PLC. activation, respectively. Since at least some of the PDGFB mutations were predicted to act through haploinsufficiency, we explored the consequences of reduced Pdgfb or Pdgfrb transcript and protein levels in mice. Heterozygous Pdgfb or Pdgfrb knockouts, as well as double Pdgfb(+/-); Pdgfrb(+/-) mice did not develop brain calcification, nor did Pdgfrb(redeye/redeye) mice, which show a 90% reduction of PDGFR beta protein levels. In contrast, Pdgfb(ret/ret) mice, which have altered tissue distribution of PDGF-B protein due to loss of a proteoglycan binding motif, developed brain calcifications. We also determined pericyte coverage in calcification-prone and non-calcification-prone brain regions in Pdgfb(ret/ret) mice. Surprisingly and contrary to our hypothesis, we found that the calcification-prone brain regions in Pdgfb(ret/ret) mice model had a higher pericyte coverage and a more intact blood-brain barrier (BBB) compared to non-calcification-prone brain regions. While our findings provide clear evidence that loss-of-function mutations in PDGFB or PDGFRB cause PFBC, they also demonstrate species differences in the threshold levels of PDGF-B/PDGF-R beta signaling that protect against small-vessel calcification in the brain. They further implicate region-specific susceptibility factor(s) in PFBC pathogenesis that are distinct from pericyte and BBB deficiency.

National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-272285 (URN)10.1371/journal.pone.0143407 (DOI)000365853900111 ()
Funder
EU, European Research CouncilSwedish Research CouncilSwedish Cancer SocietyKnut and Alice Wallenberg Foundation
Available from: 2016-01-15 Created: 2016-01-13 Last updated: 2019-03-24Bibliographically approved
2. Astrocyte – microglial association and matrix deposition define common pathogenic events in primary familial brain calcification
Open this publication in new window or tab >>Astrocyte – microglial association and matrix deposition define common pathogenic events in primary familial brain calcification
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Primary Familial Brain Calcification (PFBC) is an age-dependent and rare neurodegenerative disorder characterized by microvascular calcium phosphate deposits in deep brain regions. Known genetic causes of PFBC include loss-of-function mutations in genes involved in either of three processes - platelet-derived growth factor (PDGF) signalling, phosphate homeostasis or protein glycosylation - with unclear molecular links. To provide insight into the pathogenesis of PFBC, we analysed murine models of PFBC for two of these processes, Pdgfbret­­/ret and Slc20a2-/- mice, with regard to the structure, molecular composition, development and distribution of perivascular calcified nodules. Analyses by transmission electron microscopy and immunofluorescence revealed a multi-layered nodular ultrastructure and the direct contact between nodules and reactive astrocytes and microglia in both models. However, whereas Pdgfbret/ret mice developed large, solitary, smooth-surfaced nodules, Slc20a2-/- deposits were multi-lobulated and occurred in clusters. Nodular distribution in the brain also differed between the two mutants. Proteomic analysis and immunofluorescence revealed a common molecular composition of the nodules, involving proteins implicated in bone homeostasis, but also proteins not previously linked to tissue mineralization. Pericyte coverage and blood-brain barrier integrity were both intact in Slc20a2-/- mice, suggesting that these two features, both compromised in Pdgfbret/ret mice, are likely not the causal triggers of PFBC pathogenesis. Instead, gene expression and spatial correlations point to astrocytes as culprit cells in PFBC. 

National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-380085 (URN)
Available from: 2019-03-24 Created: 2019-03-24 Last updated: 2019-03-24
3. Endothelium-derived PDGF-B is essential for adult brain pericyte and vascular smooth muscle cell maintenance
Open this publication in new window or tab >>Endothelium-derived PDGF-B is essential for adult brain pericyte and vascular smooth muscle cell maintenance
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(English)Manuscript (preprint) (Other academic)
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-380087 (URN)
Note

Platelet-derived growth factor B (PDGF-B) released from endothelial cells is indispensable for mural cell proliferation and recruitment to the developing vasculature during embryonic and postnatal growth. However, it is not known whether endothelial PDGF-B is also important for the maintenance of mural cell coverage and function in the fully developed adult brain vasculature. Here, we deleted Pdgfb in an endothelial-specific manner in 2-months-old mice and analyzed the effects at 4, 12 or 18 months of age. We demonstrate that endothelial PDGF-B is crucial for the maintenance of full pericyte and vascular smooth muscle cell coverage in the adult vasculature. At 12-18 months of age, Pdgfb-deleted mice showed ≈50% decrease in pericyte:endothelial cell ratio, a similar reduction in longitudinal capillary coverage by pericyte processes and >70% loss of pericyte marker expression. Also the vascular smooth muscle cell (VSMC) compartment was affected, albeit less extensively compared to the pericytes. Surprisingly, the adult-induced loss of PDGF-B did not result in significant disruption of the blood-brain barrier function, or in the formation of vascular calcifications.  

Available from: 2019-03-24 Created: 2019-03-24 Last updated: 2019-03-24
4. A molecular atlas of cell types and zonation in the brain vasculature
Open this publication in new window or tab >>A molecular atlas of cell types and zonation in the brain vasculature
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2018 (English)In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 554, no 7693, p. 475-480Article in journal (Refereed) Published
Abstract [en]

Cerebrovascular disease is the third most common cause of death in developed countries, but our understanding of the cells that compose the cerebral vasculature is limited. Here, using vascular single-cell transcriptomics, we provide molecular definitions for the principal types of blood vascular and vessel-associated cells in the adult mouse brain. We uncover the transcriptional basis of the gradual phenotypic change (zonation) along the arteriovenous axis and reveal unexpected cell type differences: a seamless continuum for endothelial cells versus a punctuated continuum for mural cells. We also provide insight into pericyte organotypicity and define a population of perivascular fibroblast-like cells that are present on all vessel types except capillaries. Our work illustrates the power of single-cell transcriptomics to decode the higher organizational principles of a tissue and may provide the initial chapter in a molecular encyclopaedia of the mammalian vasculature.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2018
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-349342 (URN)10.1038/nature25739 (DOI)000425597400036 ()29443965 (PubMedID)
Funder
AstraZenecaSwedish Research Council, 2015-00550, K2014-64X-20097-09-5EU, European Research Council, AdG294556Swedish Cancer Society, 150735, CAN 2016/271Knut and Alice Wallenberg Foundation, 2015.0030The Swedish Brain Foundation
Available from: 2018-04-26 Created: 2018-04-26 Last updated: 2019-03-24Bibliographically approved

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