Transcription Profiling of Platelet-Derived Growth Factor-B-Deficient Mouse Embryos Identifies RGS5 as a Novel Marker for Pericytes and Vascular Smooth Muscle Cells
Cecilia BondjersMattias KalénMats HellströmStefan ScheidlAlexandra AbramssonOliver RennerPer LindahlHyeseon ChoJohn H. KehrlChrister Betsholtz
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Pericyte
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Pericytes reside in capillary beds where they share a basement membrane with endothelial cells and regulate their function. However, little is known about embryonic pericyte development, in part, due to lack of specific molecular markers and genetic tools. Here, we applied single cell RNA-sequencing (scRNA-seq) of platelet derived growth factor beta (pdgfrb)-positive cells to molecularly characterize pericytes in zebrafish larvae. scRNA-seq revealed zebrafish cells expressing mouse pericyte gene orthologs, and comparison with bulk RNA-seq from wild-type and pdgfrb mutant larvae further refined a pericyte gene set. Subsequent integration with mouse pericyte scRNA-seq profiles revealed a core set of conserved pericyte genes. Using transgenic reporter lines, we validated pericyte expression of two genes identified in our analysis: NDUFA4 mitochondrial complex associated like 2a (ndufa4l2a), and potassium voltage-gated channel, Isk-related family, member 4 (kcne4). Both reporter lines exhibited pericyte expression in multiple anatomical locations, and kcne4 was also detected in a subset of vascular smooth muscle cells. Thus, our integrated molecular analysis revealed a molecular profile for zebrafish pericytes and allowed us to develop new tools to observe these cells in vivo.
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ABSTRACT Vascular networks comprise endothelial cells and mural cells, which include pericytes and smooth muscle cells. To elucidate the mechanisms controlling mural cell recruitment during development and tissue regeneration, we studied zebrafish caudal fin arteries. Mural cells colonizing arteries proximal to the body wrapped around them, whereas those in more distal regions extended protrusions along the proximo-distal vascular axis. Both cell populations expressed platelet-derived growth factor receptor β (pdgfrb) and the smooth muscle cell marker myosin heavy chain 11a (myh11a). Most wrapping cells in proximal locations additionally expressed actin alpha2, smooth muscle (acta2). Loss of Pdgfrb signalling specifically decreased mural cell numbers at the vascular front. Using lineage tracing, we demonstrate that precursor cells located in periarterial regions and expressing Pgdfrb can give rise to mural cells. Studying tissue regeneration, we did not find evidence that newly formed mural cells were derived from pre-existing cells. Together, our findings reveal conserved roles for Pdgfrb signalling in development and regeneration, and suggest a limited capacity of mural cells to self-renew or contribute to other cell types during tissue regeneration.
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Abstract In the CNS, pericytes are important for maintaining the blood–brain barrier (BBB) and for controlling blood flow. Recently, several methods were suggested for the differentiation of human pluripotent stem cells (hPSCs) into brain mural cells, specifically pericytes or vascular smooth muscle cells (vSMCs). Unfortunately, identifying the pericytes from among such hPSC-derived mural cells has been challenging. This is due both to the lack of pericyte-specific markers and to the loss of defining anatomical information inherent to culture conditions. We therefore asked whether NeuroTrace 500/525, a newly developed dye that shows cell-specific uptake into pericytes in the mouse brain, can help identify human induced pluripotent stem cell (hiPSC)-derived brain pericyte-like cells. First, we found that NeuroTrace 500/525 specifically stains primary cultured human brain pericytes, confirming its specificity in vitro. Second, we found that NeuroTrace 500/525 specifically labels hiPSC-derived pericyte-like cells, but not endothelial cells or vSMCs derived from the same hiPSCs. Last, we found that neuroectoderm-derived vSMCs, which have pericyte-like features, also take up NeuroTrace 500/525. These data indicate NeuroTrace 500/525 is useful for identifying pericyte-like cells among hiPSC-derived brain mural cells.
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Arteriovenous malformations (AVMs) are high-flow lesions directly connecting arteries and veins. In the brain, AVM rupture can cause seizures, stroke, and death. Patients with AVMs exhibit reduced coverage of the vessels by pericytes, the mural cells of microvascular capillaries; however, the mechanism underlying this pericyte reduction and its association with AVM pathogenesis remains unknown. Notch signaling has been proposed to regulate critical pericyte functions. We hypothesized that Notch signaling in pericytes is crucial to maintain pericyte homeostasis and prevent AVM formation. We inhibited Notch signaling specifically in perivascular cells and analyzed the vasculature of these mice. The retinal vessels of mice with deficient perivascular Notch signaling developed severe AVMs, together with a significant reduction in pericytes and vascular smooth muscle cells (vSMC) in the arteries, while vSMCs were increased in the veins. Vascular malformations and pericyte loss were also observed in the forebrain of embryonic mice deficient for perivascular Notch signaling. Moreover, the loss of Notch signaling in pericytes downregulated Pdgfrb levels and increased pericyte apoptosis, pointing to a critical role for Notch in pericyte survival. Overall, our findings reveal a mechanism of AVM formation and highlight the Notch signaling pathway as an essential mediator in this process.
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The amyloid cascade hypothesis has been the most prevalent theory so far to explain the molecular mechanisms underlying the pathogenesis and progression of Alzheimer's disease (AD). In parallel, substantial evidence indicates contribution of cerebrovascular dysfunction to development of AD in the early stage of the disease. Cerebral amyloid angiopathy (CAA) is a cerebrovascular disorder characterized by beta-amyloid (Ab) accumulation in the walls of small and medium vessels in the brain and meninges. Nearly 90 percent of AD patients show CAA pathology linked to vascular dysfunction, eventually leading to hemorrhage lesions in the brain. Pericytes in the blood-brain barrier (BBB) are crucial for stabilizing the vasculature structure. Substantial pericyte loss has been reported in AD, which correlates with the BBB breakdown and decline of cognitive functions. However, the exact role of pericytes in the AD-associated vascular dysfunction and AD progression remain unclear.To investigate whether AD pericytes contribute to BBB impairment, we differentiated pericytes from induced pluripotent stem cell (iPSC) lines generated from two APPswe mutant and two healthy control individuals. These generated pericytes were characterized with qPCR or immunostaining to ensure the expression of classic pericyte markers, such as PDGFRb, a-SMA, and LAMA2. Then, pericytes were co-cultured with control iPSC-derived astrocytes to test the interaction of pericytes with another key cell type in BBB.We found that the GFAP immunoreactivity was increased in those astrocytes co-cultured with APPswe mutant pericytes, indicating activation of astrocytes. We hypothesized that astrocyte activation is triggered by Aβ deposition. Hence, we collected medium from pericyte monocultures after seven days of cultivation and detected by ELISA approx. 64.8 (±23.5) pg/ml of Aβ42 secreted by APPswe pericytes. In control cultures Aβ42 remained undetectable.These results indicate that APPswe pericytes are capable of producing toxic amounts of Aβ42, which could potentially contribute to CAA and affect other surrounding cell types. This is a novel concept since CAA is believed to be caused primarily by neuron-derived Aβ42.
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ABSTRACT 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. 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 mutants provide an important model for definitive investigation of mural cells during early embryonic stages without confounding secondary effects from circulatory defects. Summary statement Genetic analysis in zebrafish demonstrates the conserved role of Pdgfb/Pdgfrb signaling in pericyte and vascular smooth muscle cell formation during vascular development in vertebrates.
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Normal blood microvessels are lined by pericytes, which contribute to microvessel development and stability through mechanisms that are poorly understood. Pericyte deficiency has been implicated in the pathogenesis of microvascular abnormalities associated with diabetes and tumors. However, the unambiguous identification of pericytes is still a problem because of cellular heterogeneity and few available molecular markers. Here we describe an approach to identify pericyte markers based on transcription profiling of pericyte-deficient brain microvessels isolated from platelet-derived growth factor (PDGF-B)-/- and PDGF beta receptor (PDGFRbeta)-/- mouse mutants. The approach was validated by the identification of known pericyte markers among the most down-regulated genes in PDGF-B-/- and PDGFRbeta-/- microvessels. Of candidates for novel pericyte markers, we selected ATP-sensitive potassium-channel Kir6.1 (also known as Kcnj8) and sulfonylurea receptor 2, (SUR2, also known as Abcc9), both part of the same channel complex, as well as delta homologue 1 (DLK1) for in situ hybridization, which demonstrated their specific expression in brain pericytes of mouse embryos. We also show that Kir6.1 is highly expressed in pericytes in brain but undetectable in pericytes in skin and heart. The three new brain pericyte markers are signaling molecules implicated in ion transport and intercellular signaling, potentially opening new windows on pericyte function in brain microvessels.
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Glioblastoma, as the most malignant type, is characterized by robust angiogenesis. In previous study, periostin is one of the highest enriched extracellular matrix components. The latest study pointed that the origin of periostin was glioma initiating cell and associated with glioma progression and prognosis. While, the continuous staining pattern of periostin in tissues which indicated a pericyte-like phenotype did not go parallel with the glioma initiating cell origin. We aim to confirm pericyte is the major source of periostin as well as its functions in pericyte in the angiogenesis of glioblastoma. To this aim, with the support of TCGA and CGGA database, immunostaining and western blot was used to define periostin expressing cells. Further, knocking down periostin in pericyte was followed by 3D-tube formation assay, migration assay to assess the POSTN-mediated pericyte function. Periostin expressing cells majorly displayed the phenotype of mural cells with co-expression of PDGFRB, a well-known pericyte marker. Cells with periostin knockdown displayed a diminished vascular formation when co-cultured with endothelial cells in 3D-tube formation assay. Mechanistically, pericyte with periostin knockdown expressed lower abundance of pro-angiogenic factors mainly via the PDGFB-PDGFRB axis and further affected the activation of intracellular JNK pathway. In addition, the migration of pericyte was attenuated by periostin knockdown mainly via the aberrant modulation of focal adhesion kinase together with the insufficient expression of vinculin. Periositn, as expressed by pericyte, is essential in mediating glioma angiogenesis through various molecular mechanisms. Targeting periostin or its related pathways would be valuable in developing novel therapeutic regimens for GBM patients.
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Pericytes are one of the key players in the neurovascular unit and are thereby involved in regulation of influx and efflux of substances across the blood brain barrier (BBB). Pericytes also serve as vessel-stabilizing cells and play a major role in angiogenesis. Additionally they have phagocytic properties and are thus involved in clearance of neurotoxic substances. Recent studies have demonstrated a loss of pericytes in AD patients, a finding that potentially can underlie the dysfunctional BBB and microbleeds commonly found in AD patients. The pericyte loss, and thereby the loss of clearance mechanisms, may also contribute to the AD characteristic accumulation of amyloid beta (Aβ)1-42. The aim of this study was to reinforce previous studies by investigating pericytic NG2 expression in relation to Aβ1-42 plaque load in AD patients. NG2 is a transmembrane proteoglycan vital for migration, proliferation and differentiation of pericytes and a role for NG2 in angiogenesis has been demonstrated. Hippocampus sections from n=4 postmortem verified diagnosed AD patients and n=5 non-demented elders were immunohistologically stained against NG2 and Aβ1-42. Alterations in NG2 immunoreactivity and pericyte morphology were semi-quantitatively scored. The staining against NG2 clearly visualized round pericyte cell bodies attached to vessels. The number of round pericytes decreased in AD patients with high Aβ1-42 plaque load and was replaced by pericytes with densed and shrinked cellbodies. Additionally, the pericyte NG2 staining in AD patients with high Aβ1-42 plaque load was weaker compared to non-demented controls. Our findings support previous studies demonstrating alterations in the pericyte population in presence of AD pathology.
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