Publications by authors named "Christer Betsholtz"

190 Publications

Adult-induced genetic ablation distinguishes PDGFB roles in blood-brain barrier maintenance and development.

J Cereb Blood Flow Metab 2021 Oct 25:271678X211056395. Epub 2021 Oct 25.

Department of Immunology, Genetics, and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.

Platelet-derived growth factor B (PDGFB) released from endothelial cells is indispensable for pericyte recruitment during angiogenesis in embryonic and postnatal organ growth. Constitutive genetic loss-of-function of PDGFB leads to pericyte hypoplasia and the formation of a sparse, dilated and venous-shifted brain microvasculature with dysfunctional blood-brain barrier (BBB) in mice, as well as the formation of microvascular calcification in both mice and humans. Endothelial PDGFB is also expressed in the adult quiescent microvasculature, but here its importance is unknown. We show that deletion of in endothelial cells in 2-months-old mice causes a slowly progressing pericyte loss leading, at 12-18 months of age, to ≈50% decrease in endothelial:pericyte cell ratio, ≈60% decrease in pericyte longitudinal capillary coverage and >70% decrease in pericyte marker expression. Similar to constitutive loss of , this correlates with increased BBB permeability. However, in contrast to the constitutive loss of , adult-induced loss does not lead to vessel dilation, impaired arterio-venous zonation or the formation of microvascular calcifications. We conclude that PDFGB expression in quiescent adult microvascular brain endothelium is critical for the maintenance of pericyte coverage and normal BBB function, but that microvessel dilation, rarefaction, arterio-venous skewing and calcification reflect developmental roles of PDGFB.
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http://dx.doi.org/10.1177/0271678X211056395DOI Listing
October 2021

A Switch from Cell-Associated to Soluble PDGF-B Protects against Atherosclerosis, despite Driving Extramedullary Hematopoiesis.

Cells 2021 07 10;10(7). Epub 2021 Jul 10.

Department of Pathology, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University Medical Center, 6229 HX Maastricht, The Netherlands.

Platelet-derived growth factor B (PDGF-B) is a mitogenic, migratory and survival factor. Cell-associated PDGF-B recruits stabilizing pericytes towards blood vessels through retention in extracellular matrix. We hypothesized that the genetic ablation of cell-associated PDGF-B by retention motif deletion would reduce the local availability of PDGF-B, resulting in microvascular pericyte loss, microvascular permeability and exacerbated atherosclerosis. Therefore, mice were fed a high cholesterol diet. Although plaque size was increased in the aortic root of mice, microvessel density and intraplaque hemorrhage were unexpectedly unaffected. Plaque macrophage content was reduced, which is likely attributable to increased apoptosis, as judged by increased TUNEL+ cells in plaques (2.1-fold) and increased macrophage apoptosis upon 7-ketocholesterol or oxidized LDL incubation in vitro. Moreover, plaque collagen content increased independent of mesenchymal cell density. The decreased macrophage matrix metalloproteinase activity could partly explain collagen content. In addition to the beneficial vascular effects, we observed reduced body weight gain related to smaller fat deposition in liver and adipose tissue. While dampening plaque inflammation, paradoxically induced systemic leukocytosis. The increased incorporation of 5-ethynyl-2'-deoxyuridine indicated increased extramedullary hematopoiesis and the increased proliferation of circulating leukocytes. We concluded that confers vascular and metabolic effects, which appeared to be protective against diet-induced cardiovascular burden. These effects were unrelated to arterial mesenchymal cell content or adventitial microvessel density and leakage. In contrast, the deletion drives splenic hematopoiesis and subsequent leukocytosis in hypercholesterolemia.
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http://dx.doi.org/10.3390/cells10071746DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8308020PMC
July 2021

ELTD1-deletion reduces vascular abnormality and improves T-cell recruitment after PD-1 blockade in glioma.

Neuro Oncol 2021 Aug 4. Epub 2021 Aug 4.

Department of Immunology, Genetics and Pathology, The Rudbeck Laboratory, Science for Life Laboratory, Uppsala University, 75185 Uppsala, Sweden.

Background: Tumor vessels in glioma are molecularly and functionally abnormal, contributing to treatment resistance. Proteins differentially expressed in glioma vessels can change vessel phenotype and be targeted for therapy. ELTD1 (Adgrl4) is an orphan member of the adhesion G-protein-coupled receptor family upregulated in glioma vessels, and has been suggested as a potential therapeutic target. However, the role of ELTD1 in regulating vessel function in glioblastoma is poorly understood.

Methods: ELTD1 expression in human gliomas and its association with patient survival was determined using tissue microarrays and public databases. The role of ELTD1 in regulating tumor vessel phenotype was analyzed using orthotopic glioma models and ELTD1 -/- mice. Endothelial cells isolated from murine gliomas were transcriptionally profiled to determine differentially expressed genes and pathways. The consequence of ELTD1-deletion on glioma immunity was determined by treating tumor bearing mice with PD-1-blocking antibodies.

Results: ELTD1 levels were upregulated in human glioma vessels, increased with tumor malignancy, and were associated with poor patient survival. Progression of orthotopic gliomas was not affected by ELTD1-deletion, however, tumor vascular function was improved in ELTD1 -/- mice. Bioinformatic analysis of differentially expressed genes indicated increased inflammatory response and decreased proliferation in tumor endothelium in ELTD1 -/- mice. Consistent with an enhanced inflammatory response, ELTD1-deletion improved T-cell infiltration in GL261-bearing mice after PD-1 checkpoint blockade.

Conclusion: Our data demonstrate that ELTD1 participates in inducing vascular dysfunction in glioma, and suggests that targeting of ELTD1 may normalize the vessels and improve the response to immunotherapy.
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http://dx.doi.org/10.1093/neuonc/noab181DOI Listing
August 2021

Conserved and context-dependent roles for pdgfrb signaling during zebrafish vascular mural cell development.

Dev Biol 2021 Nov 24;479:11-22. Epub 2021 Jul 24.

Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Medical School, Worcester, MA, 01650, United States. Electronic address:

Platelet derived growth factor beta and its receptor, Pdgfrb, play essential roles in the development of vascular mural cells, including pericytes and vascular smooth muscle cells. To determine if this role was conserved in zebrafish, we analyzed pdgfb and pdgfrb mutant lines. Similar to mouse, pdgfb and pdgfrb mutant zebrafish lack brain pericytes and exhibit anatomically selective loss of vascular smooth muscle coverage. Despite these defects, pdgfrb mutant zebrafish did not otherwise exhibit circulatory defects at larval stages. However, beginning at juvenile stages, we observed severe cranial hemorrhage and vessel dilation associated with loss of pericytes and vascular smooth muscle cells in pdgfrb mutants. Similar to mouse, pdgfrb mutant zebrafish also displayed structural defects in the glomerulus, but normal development of hepatic stellate cells. We also noted defective mural cell investment on coronary vessels with concomitant defects in their development. Together, our studies support a conserved requirement for Pdgfrb signaling in mural cells. In addition, these zebrafish mutants provide an important model for definitive investigation of mural cells during early embryonic stages without confounding secondary effects from circulatory defects.
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http://dx.doi.org/10.1016/j.ydbio.2021.06.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8410673PMC
November 2021

Key molecular alterations in endothelial cells in human glioblastoma uncovered through single-cell RNA sequencing.

JCI Insight 2021 Aug 9;6(15). Epub 2021 Aug 9.

Key Laboratory of Ministry of Education for Medicinal Plant Resource and Natural Pharmaceutical Chemistry, National Engineering Laboratory for Resource Developing of Endangered Chinese Crude Drugs in Northwest of China, China-Sweden International Joint Research Center for Brain Diseases, College of Life Sciences, Shaanxi Normal University, Xi'an, China.

Passage of systemically delivered pharmacological agents into the brain is largely blocked by the blood-brain-barrier (BBB), an organotypic specialization of brain endothelial cells (ECs). Tumor vessels in glioblastoma (GBM), the most common malignant brain tumor in humans, are abnormally permeable, but this phenotype is heterogeneous and may differ between the tumor's center and invasive front. Here, through single-cell RNA sequencing (scRNA-seq) of freshly isolated ECs from human glioblastoma and paired tumor peripheral tissues, we have constructed a molecular atlas of human brain ECs providing unprecedented molecular insight into the heterogeneity of the human BBB and its molecular alteration in glioblastoma. We identified 5 distinct EC phenotypes representing different states of EC activation and BBB impairment, and associated with different anatomical locations within and around the tumor. This unique data resource provides key information for designing rational therapeutic regimens and optimizing drug delivery.
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http://dx.doi.org/10.1172/jci.insight.150861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8410070PMC
August 2021

Single-cell RNA sequencing reveals the mesangial identity and species diversity of glomerular cell transcriptomes.

Nat Commun 2021 04 9;12(1):2141. Epub 2021 Apr 9.

Karolinska Institute/AstraZeneca Integrated Cardio Metabolic Center (ICMC), Huddinge, Sweden.

Molecular characterization of the individual cell types in human kidney as well as model organisms are critical in defining organ function and understanding translational aspects of biomedical research. Previous studies have uncovered gene expression profiles of several kidney glomerular cell types, however, important cells, including mesangial (MCs) and glomerular parietal epithelial cells (PECs), are missing or incompletely described, and a systematic comparison between mouse and human kidney is lacking. To this end, we use Smart-seq2 to profile 4332 individual glomerulus-associated cells isolated from human living donor renal biopsies and mouse kidney. The analysis reveals genetic programs for all four glomerular cell types (podocytes, glomerular endothelial cells, MCs and PECs) as well as rare glomerulus-associated macula densa cells. Importantly, we detect heterogeneity in glomerulus-associated Pdgfrb-expressing cells, including bona fide intraglomerular MCs with the functionally active phagocytic molecular machinery, as well as a unique mural cell type located in the central stalk region of the glomerulus tuft. Furthermore, we observe remarkable species differences in the individual gene expression profiles of defined glomerular cell types that highlight translational challenges in the field and provide a guide to design translational studies.
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http://dx.doi.org/10.1038/s41467-021-22331-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8035407PMC
April 2021

A human cell type similar to murine central nervous system perivascular fibroblasts.

Exp Cell Res 2021 05 31;402(2):112576. Epub 2021 Mar 31.

Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden. Electronic address:

The brain vasculature has several specific features, one of them being the blood-brain barrier (BBB), which supports and protects the brain by allowing for the passage of oxygen and nutrients, while at the same time preventing passage of pathogens and toxins. The BBB also prevents efficient delivery of drugs to the brain, e.g. for treatment of brain tumors. In the murine brain, perivascular fibroblasts were recently identified as a novel potential constituent of the BBB. Here we present the existence of human cells that could be the equivalent to the murine brain perivascular fibroblasts. Using RNA sequencing, we show a similar transcriptomic profile of cultured human brain cells and murine perivascular fibroblasts. These data open up a window for new hypotheses on cell types involved in human CNS diseases.
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http://dx.doi.org/10.1016/j.yexcr.2021.112576DOI Listing
May 2021

A novel podocyte protein, R3h domain containing-like, inhibits TGF-β-induced p38 MAPK and regulates the structure of podocytes and glomerular basement membrane.

J Mol Med (Berl) 2021 06 23;99(6):859-876. Epub 2021 Feb 23.

Department of Endocrinology, Hematology, and Gerontology, Chiba University Graduate School of Medicine, Chiba, 260-8670, Japan.

Not only in kidney glomerular physiological function but also glomerular pathology especially in diabetic condition, glomerular podocytes play pivotal roles. Therefore, it is important to increase our knowledge about the genes and proteins expressed in podocytes. Recently, we have identified a novel podocyte-expressed gene, R3h domain containing-like (R3hdml) and analyzed its function in vivo as well as in vitro. Transforming growth factor-β (TGF-β) signaling regulated the expression of R3hdml. And R3hdml inhibited p38 mitogen-activated protein kinase phosphorylation, which was induced by TGF-β, leading to the amelioration of podocyte apoptosis. Furthermore, a lack of R3hdml in mice significantly worsened glomerular function in streptozotocin (STZ)-induced diabetes, while overexpression of R3hdml ameliorated albuminuria in STZ-induced diabetes. Our results surmise that the functional analyses of R3hdml may lead to the development of novel therapeutic strategies for diabetic nephropathy in the future. KEY MESSAGES: • A novel podocyte expressed protein R3h domain containing-like was identified. • R3HDML inhibits podocyte apoptosis by inhibiting TGF-β-mediated p38 MAPK signaling. • Overexpression of R3HDML ameliorates albuminuria in STZ-induced diabetes mice. • R3HDML may prove to be a novel therapeutic strategy for diabetic nephropathy.
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http://dx.doi.org/10.1007/s00109-021-02050-wDOI Listing
June 2021

The infantile myofibromatosis NOTCH3 L1519P mutation leads to hyperactivated ligand-independent Notch signaling and increased PDGFRB expression.

Dis Model Mech 2021 Jan 28. Epub 2021 Jan 28.

Department of Neurobiology, Care Science and Society, Karolinska Institutet, Sweden

Infantile myofibromatosis (IMF) is a benign tumor form characterized by the development of nonmetastatic tumors in skin, bone, muscle and sometimes viscera. Autosomal dominant forms of IMF are caused by mutations in the gene, but a family carrying a L1519P mutation in the gene has also recently been identified. In this report, we address the molecular consequences of the NOTCH3 mutation and the relationship between the NOTCH and PDGFRB signaling in IMF. The NOTCH3 receptor generates enhanced downstream signaling in a ligand-independent manner. Despite the enhanced signaling, the NOTCH3 receptor is absent from the cell surface and instead accumulates in the endoplasmic reticulum. Furthermore, the localization of the NOTCH3 receptor in the bipartite, heterodimeric state is altered, combined with avid secretion of the mutated extracellular domain from the cell. Chloroquine treatment strongly reduces the amount of secreted NOTCH3 extracellular domain and decreases signaling. Finally, NOTCH3 upregulates PDGFRB expression in fibroblasts, supporting a functional link between Notch and PDGF dysregulation in IMF. Collectively, our data define a NOTCH3-PDGFRB axis in IMF, where an IMF-mutated NOTCH3 receptor elevates PDGFRB expression. The functional characterization of a ligand-independent gain-of-function NOTCH3 mutation is important for Notch therapy considerations for IMF, including strategies aimed at altering lysosome function.
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http://dx.doi.org/10.1242/dmm.046300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7927659PMC
January 2021

The Ion Channel and GPCR Toolkit of Brain Capillary Pericytes.

Front Cell Neurosci 2020 18;14:601324. Epub 2020 Dec 18.

Department of Physiology, School of Medicine, University of Maryland, Baltimore, MD, United States.

Brain pericytes reside on the abluminal surface of capillaries, and their processes cover ~90% of the length of the capillary bed. These cells were first described almost 150 years ago (Eberth, 1871; Rouget, 1873) and have been the subject of intense experimental scrutiny in recent years, but their physiological roles remain uncertain and little is known of the complement of signaling elements that they employ to carry out their functions. In this review, we synthesize functional data with single-cell RNAseq screens to explore the ion channel and G protein-coupled receptor (GPCR) toolkit of mesh and thin-strand pericytes of the brain, with the aim of providing a framework for deeper explorations of the molecular mechanisms that govern pericyte physiology. We argue that their complement of channels and receptors ideally positions capillary pericytes to play a central role in adapting blood flow to meet the challenge of satisfying neuronal energy requirements from deep within the capillary bed, by enabling dynamic regulation of their membrane potential to influence the electrical output of the cell. In particular, we outline how genetic and functional evidence suggest an important role for G-coupled GPCRs and ATP-sensitive potassium (K) channels in this context. We put forth a predictive model for long-range hyperpolarizing electrical signaling from pericytes to upstream arterioles, and detail the TRP and Ca channels and G, G, and G signaling processes that counterbalance this. We underscore critical questions that need to be addressed to further advance our understanding of the signaling topology of capillary pericytes, and how this contributes to their physiological roles and their dysfunction in disease.
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http://dx.doi.org/10.3389/fncel.2020.601324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7775489PMC
December 2020

Single-Cell Analysis of Blood-Brain Barrier Response to Pericyte Loss.

Circ Res 2021 02 30;128(4):e46-e62. Epub 2020 Dec 30.

Department of Immunology, Genetics, and Pathology, Rudbeck Laboratory, Uppsala University, Sweden (M.A.M., L.H., S.N., E.V.-L., K.N., B.J., B.L., M.J., M.V., C.B.).

Rationale: Pericytes are capillary mural cells playing a role in stabilizing newly formed blood vessels during development and tissue repair. Loss of pericytes has been described in several brain disorders, and genetically induced pericyte deficiency in the brain leads to increased macromolecular leakage across the blood-brain barrier (BBB). However, the molecular details of the endothelial response to pericyte deficiency remain elusive.

Objective: To map the transcriptional changes in brain endothelial cells resulting from lack of pericyte contact at single-cell level and to correlate them with regional heterogeneities in BBB function and vascular phenotype.

Methods And Results: We reveal transcriptional, morphological, and functional consequences of pericyte absence for brain endothelial cells using a combination of methodologies, including single-cell RNA sequencing, tracer analyses, and immunofluorescent detection of protein expression in pericyte-deficient adult mice. We find that endothelial cells without pericyte contact retain a general BBB-specific gene expression profile, however, they acquire a venous-shifted molecular pattern and become transformed regarding the expression of numerous growth factors and regulatory proteins. Adult brains display ongoing angiogenic sprouting without concomitant cell proliferation providing unique insights into the endothelial tip cell transcriptome. We also reveal heterogeneous modes of pericyte-deficient BBB impairment, where hotspot leakage sites display arteriolar-shifted identity and pinpoint putative BBB regulators. By testing the causal involvement of some of these using reverse genetics, we uncover a reinforcing role for angiopoietin 2 at the BBB.

Conclusions: By elucidating the complexity of endothelial response to pericyte deficiency at cellular resolution, our study provides insight into the importance of brain pericytes for endothelial arterio-venous zonation, angiogenic quiescence, and a limited set of BBB functions. The BBB-reinforcing role of ANGPT2 (angiopoietin 2) is paradoxical given its wider role as TIE2 (TEK receptor tyrosine kinase) receptor antagonist and may suggest a unique and context-dependent function of ANGPT2 in the brain.
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http://dx.doi.org/10.1161/CIRCRESAHA.120.317473DOI Listing
February 2021

VEGF-B Promotes Endocardium-Derived Coronary Vessel Development and Cardiac Regeneration.

Circulation 2021 Jan 18;143(1):65-77. Epub 2020 Nov 18.

Wihuri Research Institute and Translational Cancer Medicine Program, Faculty of Medicine (M.R., I.S., J.P., K.A.H., E.A., R.K., K.A.).

Background: Recent discoveries have indicated that, in the developing heart, sinus venosus and endocardium provide major sources of endothelium for coronary vessel growth that supports the expanding myocardium. Here we set out to study the origin of the coronary vessels that develop in response to vascular endothelial growth factor B (VEGF-B) in the heart and the effect of VEGF-B on recovery from myocardial infarction.

Methods: We used mice and rats expressing a VEGF-B transgene, VEGF-B-gene-deleted mice and rats, apelin-CreERT, and natriuretic peptide receptor 3-CreERT recombinase-mediated genetic cell lineage tracing and viral vector-mediated VEGF-B gene transfer in adult mice. Left anterior descending coronary vessel ligation was performed, and 5-ethynyl-2'-deoxyuridine-mediated proliferating cell cycle labeling; flow cytometry; histological, immunohistochemical, and biochemical methods; single-cell RNA sequencing and subsequent bioinformatic analysis; microcomputed tomography; and fluorescent- and tracer-mediated vascular perfusion imaging analyses were used to study the development and function of the VEGF-B-induced vessels in the heart.

Results: We show that cardiomyocyte overexpression of VEGF-B in mice and rats during development promotes the growth of novel vessels that originate directly from the cardiac ventricles and maintain connection with the coronary vessels in subendocardial myocardium. In adult mice, endothelial proliferation induced by VEGF-B gene transfer was located predominantly in the subendocardial coronary vessels. Furthermore, VEGF-B gene transduction before or concomitantly with ligation of the left anterior descending coronary artery promoted endocardium-derived vessel development into the myocardium and improved cardiac tissue remodeling and cardiac function.

Conclusions: The myocardial VEGF-B transgene promotes the formation of endocardium-derived coronary vessels during development, endothelial proliferation in subendocardial myocardium in adult mice, and structural and functional rescue of cardiac tissue after myocardial infarction. VEGF-B could provide a new therapeutic strategy for cardiac neovascularization after coronary occlusion to rescue the most vulnerable myocardial tissue.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.120.050635DOI Listing
January 2021

Vascular PDGFR-alpha protects against BBB dysfunction after stroke in mice.

Angiogenesis 2021 02 12;24(1):35-46. Epub 2020 Sep 12.

Department of Pathology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, 930-0194, Japan.

Blood-brain barrier (BBB) dysfunction underlies the pathogenesis of many neurological diseases. Platelet-derived growth factor receptor-alpha (PDGFRα) induces hemorrhagic transformation (HT) downstream of tissue plasminogen activator in thrombolytic therapy of acute stroke. Thus, PDGFs are attractive therapeutic targets for BBB dysfunction. In the present study, we examined the role of PDGF signaling in the process of tissue remodeling after middle cerebral arterial occlusion (MCAO) in mice. Firstly, we found that imatinib increased lesion size after permanent MCAO in wild-type mice. Moreover, imatinib-induced HT only when administrated in the subacute phase of MCAO, but not in the acute phase. Secondly, we generated genetically mutated mice (C-KO mice) that showed decreased expression of perivascular PDGFRα. Additionally, transient MCAO experiments were performed in these mice. We found that the ischemic lesion size was not affected; however, the recruitment of PDGFRα/type I collagen-expressing perivascular cells was significantly downregulated, and HT and IgG leakage was augmented only in the subacute phase of stroke in C-KO mice. In both experiments, we found that the expression of tight junction proteins and PDGFRβ-expressing pericyte coverage was not significantly affected in imatinib-treated mice and in C-KO mice. The specific implication of PDGFRα signaling was suggestive of protective effects against BBB dysfunction during the subacute phase of stroke. Vascular TGF-β1 expression was downregulated in both imatinib-treated and C-KO mice, along with sustained levels of MMP9. Therefore, PDGFRα effects may be mediated by TGF-β1 which exerts potent protective effects in the BBB.
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http://dx.doi.org/10.1007/s10456-020-09742-wDOI Listing
February 2021

Single-cell analysis uncovers fibroblast heterogeneity and criteria for fibroblast and mural cell identification and discrimination.

Nat Commun 2020 08 7;11(1):3953. Epub 2020 Aug 7.

Karolinska Institutet/AstraZeneca Integrated Cardio Metabolic Centre, Blickagången 6, SE-14157, Huddinge, Sweden.

Many important cell types in adult vertebrates have a mesenchymal origin, including fibroblasts and vascular mural cells. Although their biological importance is undisputed, the level of mesenchymal cell heterogeneity within and between organs, while appreciated, has not been analyzed in detail. Here, we compare single-cell transcriptional profiles of fibroblasts and vascular mural cells across four murine muscular organs: heart, skeletal muscle, intestine and bladder. We reveal gene expression signatures that demarcate fibroblasts from mural cells and provide molecular signatures for cell subtype identification. We observe striking inter- and intra-organ heterogeneity amongst the fibroblasts, primarily reflecting differences in the expression of extracellular matrix components. Fibroblast subtypes localize to discrete anatomical positions offering novel predictions about physiological function(s) and regulatory signaling circuits. Our data shed new light on the diversity of poorly defined classes of cells and provide a foundation for improved understanding of their roles in physiological and pathological processes.
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http://dx.doi.org/10.1038/s41467-020-17740-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7414220PMC
August 2020

Platelet-Specific PDGFB Ablation Impairs Tumor Vessel Integrity and Promotes Metastasis.

Cancer Res 2020 08 25;80(16):3345-3358. Epub 2020 Jun 25.

Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, Biomedical Center, Uppsala, Sweden.

Platelet-derived growth factor B (PDGFB) plays a crucial role in recruitment of PDGF receptor β-positive pericytes to blood vessels. The endothelium is an essential source of PDGFB in this process. Platelets constitute a major reservoir of PDGFB and are continuously activated in the tumor microenvironment, exposing tumors to the plethora of growth factors contained in platelet granules. Here, we show that tumor vascular function, as well as pericyte coverage is significantly impaired in mice with conditional knockout of PDGFB in platelets. A lack of PDGFB in platelets led to enhanced hypoxia and epithelial-to-mesenchymal transition in the primary tumors, elevated levels of circulating tumor cells, and increased spontaneous metastasis to the liver or lungs in two mouse models. These findings establish a previously unknown role for platelet-derived PDGFB, whereby it promotes and maintains vascular integrity in the tumor microenvironment by contributing to the recruitment of pericytes. SIGNIFICANCE: Conditional knockout of PDGFB in platelets demonstrates its previously unknown role in the maintenance of tumor vascular integrity and host protection against metastasis.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-3533DOI Listing
August 2020

Radiation Triggers a Dynamic Sequence of Transient Microglial Alterations in Juvenile Brain.

Cell Rep 2020 06;31(9):107699

Department of Women's and Children's Health, Karolinska Institutet, 171 64 Stockholm, Sweden; Henan Key Laboratory of Child Brain Injury, The Third Affiliated Hospital and Institute of Neuroscience, Zhengzhou University, Zhengzhou 4500 52, China; Pediatric Oncology, Karolinska University Hospital, 171 64 Stockholm, Sweden. Electronic address:

Cranial irradiation (IR), an effective tool to treat malignant brain tumors, triggers a chronic pro-inflammatory microglial response, at least in the adult brain. Using single-cell and bulk RNA sequencing, combined with histology, we show that the microglial response in the juvenile mouse hippocampus is rapid but returns toward normal within 1 week. The response is characterized by a series of temporally distinct homeostasis-, sensome-, and inflammation-related molecular signatures. We find that a single microglial cell simultaneously upregulates transcripts associated with pro- and anti-inflammatory microglial phenotypes. Finally, we show that juvenile and adult irradiated microglia are already transcriptionally distinct in the early phase after IR. Our results indicate that microglia are involved in the initial stages but may not be responsible for driving long-term inflammation in the juvenile brain.
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http://dx.doi.org/10.1016/j.celrep.2020.107699DOI Listing
June 2020

Specific fibroblast subpopulations and neuronal structures provide local sources of Vegfc-processing components during zebrafish lymphangiogenesis.

Nat Commun 2020 06 1;11(1):2724. Epub 2020 Jun 1.

Institute for Cardiovascular Organogenesis and Regeneration, WWU Münster, Münster, Germany.

Proteolytical processing of the growth factor VEGFC through the concerted activity of CCBE1 and ADAMTS3 is required for lymphatic development to occur. How these factors act together in time and space, and which cell types produce these factors is not understood. Here we assess the function of Adamts3 and the related protease Adamts14 during zebrafish lymphangiogenesis and show both proteins to be able to process Vegfc. Only the simultaneous loss of both protein functions results in lymphatic defects identical to vegfc loss-of-function situations. Cell transplantation experiments demonstrate neuronal structures and/or fibroblasts to constitute cellular sources not only for both proteases but also for Ccbe1 and Vegfc. We further show that this locally restricted Vegfc maturation is needed to trigger normal lymphatic sprouting and directional migration. Our data provide a single-cell resolution model for establishing secretion and processing hubs for Vegfc during developmental lymphangiogenesis.
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http://dx.doi.org/10.1038/s41467-020-16552-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7264274PMC
June 2020

Pericyte dysfunction due to Shb gene deficiency increases B16F10 melanoma lung metastasis.

Int J Cancer 2020 11 30;147(9):2634-2644. Epub 2020 May 30.

Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden.

Intravasation, vascular dissemination and metastasis of malignant tumor cells require their passage through the vascular wall which is commonly composed of pericytes and endothelial cells. We currently decided to investigate the relative contribution of these cell types to B16F10 melanoma metastasis in mice using an experimental model of host Shb gene (Src homology 2 domain-containing protein B) inactivation. Conditional inactivation of Shb in endothelial cells using Cdh5-CreERt2 resulted in decreased tumor growth, reduced vascular leakage, increased hypoxia and no effect on pericyte coverage and lung metastasis. RNAseq of tumor endothelial cells from these mice revealed changes in cellular components such as adherens junctions and focal adhesions by gene ontology analysis that were in line with the observed effects on leakage and junction morphology. Conditional inactivation of Shb in pericytes using Pdgfrb-CreERt2 resulted in decreased pericyte coverage of small tumor vessels with lumen, increased leakage, aberrant platelet-derived growth factor receptor B (PDGFRB) signaling and a higher frequency of lung metastasis without concomitant effects on tumor growth or oxygenation. Flow cytometry failed to reveal immune cell alterations that could explain the metastatic phenotype in this genetic model of Shb deficiency. It is concluded that proper pericyte function plays a significant role in suppressing B16F10 lung metastasis.
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http://dx.doi.org/10.1002/ijc.33110DOI Listing
November 2020

Lack of Flvcr2 impairs brain angiogenesis without affecting the blood-brain barrier.

J Clin Invest 2020 08;130(8):4055-4068

Department of Pediatrics, and.

Fowler syndrome is a rare autosomal recessive brain vascular disorder caused by mutation in FLVCR2 in humans. The disease occurs during a critical period of brain vascular development, is characterized by glomeruloid vasculopathy and hydrocephalus, and is almost invariably prenatally fatal. Here, we sought to gain insights into the process of brain vascularization and the pathogenesis of Fowler syndrome by inactivating Flvcr2 in mice. We showed that Flvcr2 was necessary for angiogenic sprouting in the brain, but surprisingly dispensable for maintaining the blood-brain barrier. Endothelial cells lacking Flvcr2 had altered expression of angiogenic factors, failed to adopt tip cell properties, and displayed reduced sprouting, leading to vascular malformations similar to those seen in humans with Fowler syndrome. Brain hypovascularization was associated with hypoxia and tissue infarction, ultimately causing hydrocephalus and death of mutant animals. Strikingly, despite severe vascular anomalies and brain tissue infarction, the blood-brain barrier was maintained in Flvcr2 mutant mice. Our Fowler syndrome model therefore defined the pathobiology of this disease and provided new insights into brain angiogenesis by showing uncoupling of vessel morphogenesis and blood-brain barrier formation.
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http://dx.doi.org/10.1172/JCI136578DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7410045PMC
August 2020

Lung developmental arrest caused by PDGF-A deletion: consequences for the adult mouse lung.

Am J Physiol Lung Cell Mol Physiol 2020 04 18;318(4):L831-L843. Epub 2020 Mar 18.

Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.

PDGF-A is a key contributor to lung development in mice. Its expression is needed for secondary septation of the alveoli and deletion of the gene leads to abnormally enlarged alveolar air spaces in mice. In humans, the same phenotype is the hallmark of bronchopulmonary dysplasia (BPD), a disease that affects premature babies and may have long lasting consequences in adulthood. So far, the knowledge regarding adult effects of developmental arrest in the lung is limited. This is attributable to few follow-up studies of BPD survivors and lack of good experimental models that could help predict the outcomes of this early age disease for the adult individual. In this study, we used the constitutive lung-specific deletion mouse model to analyze the consequences of developmental lung defects in adult mice. We assessed lung morphology, physiology, cellular content, ECM composition and proteomics data in mature mice, that perinatally exhibited lungs with a BPD-like morphology. Histological and physiological analyses both revealed that enlarged alveolar air spaces remained until adulthood, resulting in higher lung compliance and higher respiratory volume in knockout mice. Still, no or only small differences were seen in cellular, ECM and protein content when comparing knockout and control mice. Taken together, our results indicate that deletion-induced lung developmental arrest has consequences for the adult lung at the morphological and functional level. In addition, these mice can reach adulthood with a BPD-like phenotype, which makes them a robust model to further investigate the pathophysiological progression of the disease and test putative regenerative therapies.
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http://dx.doi.org/10.1152/ajplung.00295.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7191480PMC
April 2020

Sphingosine 1-phosphate-regulated transcriptomes in heterogenous arterial and lymphatic endothelium of the aorta.

Elife 2020 02 24;9. Epub 2020 Feb 24.

Vascular Biology Program, Boston Children's Hospital, Deapartment of Surgery, Harvard Medical School, Boston, United States.

Despite the medical importance of G protein-coupled receptors (GPCRs), in vivo cellular heterogeneity of GPCR signaling and downstream transcriptional responses are not understood. We report the comprehensive characterization of transcriptomes (bulk and single-cell) and chromatin domains regulated by sphingosine 1-phosphate receptor-1 (S1PR1) in adult mouse aortic endothelial cells. First, S1PR1 regulates NFκB and nuclear glucocorticoid receptor pathways to suppress inflammation-related mRNAs. Second, S1PR1 signaling in the heterogenous endothelial cell (EC) subtypes occurs at spatially-distinct areas of the aorta. For example, a transcriptomically distinct arterial EC population at vascular branch points (aEC1) exhibits ligand-independent S1PR1/ß-arrestin coupling. In contrast, circulatory S1P-dependent S1PR1/ß-arrestin coupling was observed in non-branch point aEC2 cells that exhibit an inflammatory gene expression signature. Moreover, S1P/S1PR1 signaling regulates the expression of lymphangiogenic and inflammation-related transcripts in an adventitial lymphatic EC (LEC) population in a ligand-dependent manner. These insights add resolution to existing concepts of endothelial heterogeneity, GPCR signaling and S1P biology.
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http://dx.doi.org/10.7554/eLife.52690DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054001PMC
February 2020

Sphingosine 1-Phosphate Receptor Signaling Establishes AP-1 Gradients to Allow for Retinal Endothelial Cell Specialization.

Dev Cell 2020 03 13;52(6):779-793.e7. Epub 2020 Feb 13.

Vascular Biology Program, Boston Children's Hospital, Department of Surgery, Harvard Medical School, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA; Dana-Farber Cancer Institute, Boston, MA, USA. Electronic address:

Transcriptional mechanisms that drive angiogenesis and organotypic vascular endothelial cell specialization are poorly understood. Here, we show that retinal endothelial sphingosine 1-phosphate receptors (S1PRs), which restrain vascular endothelial growth factor (VEGF)-induced angiogenesis, spatially restrict expression of JunB, a member of the activator protein 1 (AP-1) family of transcription factors (TFs). Mechanistically, VEGF induces JunB expression at the sprouting vascular front while S1PR-dependent vascular endothelial (VE)-cadherin assembly suppresses JunB expression in the nascent vascular network, thus creating a gradient of this TF. Endothelial-specific JunB knockout mice showed diminished expression of neurovascular guidance genes and attenuated retinal vascular network progression. In addition, endothelial S1PR signaling is required for normal expression of β-catenin-dependent genes such as TCF/LEF1 and ZIC3 TFs, transporters, and junctional proteins. These results show that S1PR signaling restricts JunB function to the expanding vascular front, thus creating an AP-1 gradient and enabling organotypic endothelial cell specialization of the vascular network.
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http://dx.doi.org/10.1016/j.devcel.2020.01.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541081PMC
March 2020

Emerging links between cerebrovascular and neurodegenerative diseases-a special role for pericytes.

EMBO Rep 2019 11 16;20(11):e48070. Epub 2019 Oct 16.

Integrated Cardio Metabolic Centre (ICMC), Huddinge, Sweden.

Neurodegenerative and cerebrovascular diseases cause considerable human suffering, and therapy options for these two disease categories are limited or non-existing. It is an emerging notion that neurodegenerative and cerebrovascular diseases are linked in several ways, and in this review, we discuss the current status regarding vascular dysregulation in neurodegenerative disease, and conversely, how cerebrovascular diseases are associated with central nervous system (CNS) degeneration and dysfunction. The emerging links between neurodegenerative and cerebrovascular diseases are reviewed with a particular focus on pericytes-important cells that ensheath the endothelium in the microvasculature and which are pivotal for blood-brain barrier function and cerebral blood flow. Finally, we address how novel molecular and cellular insights into pericytes and other vascular cell types may open new avenues for diagnosis and therapy development for these important diseases.
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http://dx.doi.org/10.15252/embr.201948070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6831996PMC
November 2019

Astrocyte-microglial association and matrix composition are common events in the natural history of primary familial brain calcification.

Brain Pathol 2020 05 10;30(3):446-464. Epub 2019 Oct 10.

Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.

Primary familial brain calcification (PFBC) is an age-dependent and rare neurodegenerative disorder characterized by microvascular calcium phosphate deposits in the 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) signaling, phosphate homeostasis or protein glycosylation-with unclear molecular links. To provide insight into the pathogenesis of PFBC, we analyzed murine models of PFBC for the first two of these processes in Pdgfb 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 that calcified nodules in both of these models have a multilayered ultrastructure and occur in direct contact with reactive astrocytes and microglia. However, whereas nodules in Pdgfb mice were large, solitary and smooth surfaced, the nodules in Slc20a2 mice were multi-lobulated and occurred in clusters. The regional distribution of nodules also differed between the two models. Proteomic analysis and immunofluorescence stainings revealed a common molecular composition of the nodules in the two models, involving proteins implicated in bone homeostasis, but also proteins not previously linked to tissue mineralization. While the brain vasculature of Pdgfb mice has been reported to display reduced pericyte coverage and abnormal permeability, we found that Slc20a2 mice have a normal pericyte coverage and no overtly increased permeability. Thus, lack of pericytes and increase in permeability of the blood-brain barrier are likely not the causal triggers for PFBC pathogenesis. Instead, gene expression and spatial correlations suggest that astrocytes are intimately linked to the calcification process in PFBC.
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http://dx.doi.org/10.1111/bpa.12787DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7317599PMC
May 2020

Formation and metabolism of oxysterols and cholestenoic acids found in the mouse circulation: Lessons learnt from deuterium-enrichment experiments and the CYP46A1 transgenic mouse.

J Steroid Biochem Mol Biol 2019 12 18;195:105475. Epub 2019 Sep 18.

Swansea University Medical School, ILS1 Building, Singleton Park, Swansea SA2 8PP, Wales, UK. Electronic address:

While the presence and abundance of the major oxysterols and cholestenoic acids in the circulation is well established, minor cholesterol metabolites may also have biological importance and be of value to investigate. In this study by observing the metabolism of deuterium-labelled cholesterol in the pdgfb mouse, a mouse model with increased vascular permeability in brain, and by studying the sterol content of plasma from the CYP46A1 transgenic mouse overexpressing the human cholesterol 24S-hydroxylase enzyme we have been able to identify a number of minor cholesterol metabolites found in the circulation, make approximate-quantitative measurements and postulate pathways for their formation. These "proof of principle" data may have relevance when using mouse models to mimic human disease and in respect of the increasing possibility of treating human neurodegenerative diseases with pharmaceuticals designed to enhance the activity of CYP46A1 or by adeno-associated virus delivery of CYP46A1.
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http://dx.doi.org/10.1016/j.jsbmb.2019.105475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6880786PMC
December 2019

R3hdml regulates satellite cell proliferation and differentiation.

EMBO Rep 2019 11 16;20(11):e47957. Epub 2019 Sep 16.

Department of Endocrinology, Hematology, and Gerontology, Chiba University Graduate School of Medicine, Chiba, Japan.

In this study, we identified a previously uncharacterized skeletal satellite cell-secreted protein, R3h domain containing-like (R3hdml). Expression of R3hdml increases during skeletal muscle development and differentiation in mice. Body weight and skeletal muscle mass of R3hdml knockout (KO) mice are lower compared to control mice. Expression levels of cell cycle-related markers, phosphorylation of Akt, and expression of insulin-like growth factor within the skeletal muscle are reduced in R3hdml KO mice compared to control mice. Expression of R3hdml increases during muscle regeneration in response to cardiotoxin (CTX)-induced muscle injury. Recovery of handgrip strength after CTX injection was significantly impaired in R3hdml KO mice, which is rescued by R3hdml. Our results indicate that R3hdml is required for skeletal muscle development, regeneration, and, in particular, satellite cell proliferation and differentiation.
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http://dx.doi.org/10.15252/embr.201947957DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6832012PMC
November 2019

Heterogeneity and plasticity in healthy and atherosclerotic vasculature explored by single-cell sequencing.

Cardiovasc Res 2019 Oct;115(12):1705-1715

Pathology Department, CARIM School for Cardiovascular Diseases, MUMC Maastricht, P. Debyelaan 25, Maastricht, the Netherlands.

Cellular characteristics and their adjustment to a state of disease have become more evident due to recent advances in imaging, fluorescent reporter mice, and whole genome RNA sequencing. The uncovered cellular heterogeneity and/or plasticity potentially complicates experimental studies and clinical applications, as markers derived from whole tissue 'bulk' sequencing is unable to yield a subtype transcriptome and specific markers. Here, we propose definitions on heterogeneity and plasticity, discuss current knowledge thereof in the vasculature and how this may be improved by single-cell sequencing (SCS). SCS is emerging as an emerging technique, enabling researchers to investigate different cell populations in more depth than ever before. Cell selection methods, e.g. flow assisted cell sorting, and the quantity of cells can influence the choice of SCS method. Smart-Seq2 offers sequencing of the complete mRNA molecule on a low quantity of cells, while Drop-seq is possible on large numbers of cells on a more superficial level. SCS has given more insight in heterogeneity in healthy vasculature, where it revealed that zonation is crucial in gene expression profiles among the anatomical axis. In diseased vasculature, this heterogeneity seems even more prominent with discovery of new immune subsets in atherosclerosis as proof. Vascular smooth muscle cells and mesenchymal cells also share these plastic characteristics with the ability to up-regulate markers linked to stem cells, such as Sca-1 or CD34. Current SCS studies show some limitations to the number of replicates, quantity of cells used, or the loss of spatial information. Bioinformatical tools could give some more insight in current datasets, making use of pseudo-time analysis or RNA velocity to investigate cell differentiation or polarization. In this review, we discuss the use of SCS in unravelling heterogeneity in the vasculature, its current limitations and promising future applications.
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http://dx.doi.org/10.1093/cvr/cvz185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6873093PMC
October 2019
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