Visualization of vascular mural cells in developing brain using genetically labeled transgenic reporter mice

The establishment of a fully functional blood vascular system requires elaborate angiogenic and vascular maturation events in order to fulfill organ-specific anatomical and physiological needs. Although vascular mural cells, i.e. pericytes and vascular smooth muscle cells, are known to play fundamental roles during these processes, their characteristics during vascular development remain incompletely understood. In this report, we utilized transgenic reporter mice in which mural cells are genetically labeled to examine developing vascular mural cells in the central nervous system (CNS). We found platelet-derived growth factor receptor β gene ( Pdgfrb)-driven EGFP reporter expression as a suitable marker for vascular mural cells at the earliest stages of mouse brain vascularization. Furthermore, the combination of Pdgfrb and NG2 gene (Cspg4) driven reporter expression increased the specificity of brain vascular mural cell labeling at later stages. The expression of other known pericyte markers revealed time-, region- and marker-specific patterns, suggesting heterogeneity in mural cell maturation. We conclude that transgenic reporter mice provide an important tool to explore the development of CNS pericytes in health and disease.

Download Full-text

Mural Cells: Potential Therapeutic Targets to Bridge Cardiovascular Disease and Neurodegeneration

Cells ◽

10.3390/cells10030593 ◽

2021 ◽

Vol 10 (3) ◽

pp. 593

Author(s):

Alexander Lin ◽

Niridu Jude Peiris ◽

Harkirat Dhaliwal ◽

Maria Hakim ◽

Weizhen Li ◽

...

Keyword(s):

Smooth Muscle ◽

Neurodegenerative Diseases ◽

Structural Integrity ◽

Vascular System ◽

Vascular Wall ◽

Cell Plasticity ◽

Mural Cell ◽

Mural Cells ◽

Heterogenous Population

Mural cells collectively refer to the smooth muscle cells and pericytes of the vasculature. This heterogenous population of cells play a crucial role in the regulation of blood pressure, distribution, and the structural integrity of the vascular wall. As such, dysfunction of mural cells can lead to the pathogenesis and progression of a number of diseases pertaining to the vascular system. Cardiovascular diseases, particularly atherosclerosis, are perhaps the most well-described mural cell-centric case. For instance, atherosclerotic plaques are most often described as being composed of a proliferative smooth muscle cap accompanied by a necrotic core. More recently, the role of dysfunctional mural cells in neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease, is being recognized. In this review, we begin with an exploration of the mechanisms underlying atherosclerosis and neurodegenerative diseases, such as mural cell plasticity. Next, we highlight a selection of signaling pathways (PDGF, Notch and inflammatory signaling) that are conserved across both diseases. We propose that conserved mural cell signaling mechanisms can be exploited for the identification or development of dual-pronged therapeutics that impart both cardio- and neuroprotective qualities.

Download Full-text

Regenerating vascular mural cells in zebrafish fin blood vessels are not derived from pre-existing ones and differentially require pdgfrb signaling for their development

10.1101/2021.03.27.437334 ◽

2021 ◽

Author(s):

Arndt F Siekmann ◽

Elvin Vincent Leonard ◽

Ricardo Figueroa ◽

Jeroen Bussmann ◽

Julio D Amigo ◽

...

Keyword(s):

Endothelial Cells ◽

Smooth Muscle ◽

Tissue Regeneration ◽

Growth Factor Receptor ◽

Cell Types ◽

Lineage Tracing ◽

The Body ◽

Mural Cell ◽

Caudal Fin ◽

Mural Cells

Vascular networks are comprised of endothelial cells and mural cells, which include pericytes and smooth muscle cells. It is well established that new endothelial cells are derived from pre-existing ones during the angiogenic phase of blood vessel growth. By contrast, mural cell ontogeny is less clear with an ongoing debate whether mural cells possess mesenchymal stem cell properties. To elucidate the mechanisms controlling mural cell recruitment during development and tissue regeneration, we studied the formation of zebrafish caudal fin arteries. Mural cells showed morphological heterogeneity: cells colonizing arteries proximal to the body wrapped around them, while those in more distal regions extended protrusions along the proximo-distal vascular axis. Despite these differences, both cell populations expressed platelet-derived growth factor receptor beta (Pdgfrb) and the smooth muscle cell marker myosin heavy chain 11a (Myh11a). Loss of Pdgfrb signalling during development or tissue regeneration resulted in a substantial decrease in mural cells at the vascular front, while those proximal to the body were less affected. Using lineage tracing, we demonstrate that precursor cells located in periarterial regions of the caudal fin and expressing Pgdfrb can give rise to mural cells, while in regeneration newly formed mural cells were not derived from pre-existing ones. Together, our findings reveal conserved roles for pdgfrb signalling in development and regeneration, while at the same time illustrating a limited capacity of mural cells to self-renew or contribute to other cell types during tissue regeneration.

Download Full-text

Validation of Specific and Reliable Genetic Tools to Identify, Label, and Target Cardiac Pericytes in Mice

Journal of the American Heart Association ◽

10.1161/jaha.121.023171 ◽

2021 ◽

Author(s):

Linda Alex ◽

Izabela Tuleta ◽

Venugopal Harikrishnan ◽

Nikolaos G. Frangogiannis

Keyword(s):

Large Population ◽

Growth Factor Receptor ◽

Cell Types ◽

Lineage Tracing ◽

Matricellular Proteins ◽

Mural Cells ◽

Reporter Mice ◽

Fibrillar Collagens ◽

Dual Reporter ◽

Genes Encoding

Background In the myocardium, pericytes are often confused with other interstitial cell types, such as fibroblasts. The lack of well‐characterized and specific tools for identification, lineage tracing, and conditional targeting of myocardial pericytes has hampered studies on their role in heart disease. In the current study, we characterize and validate specific and reliable strategies for labeling and targeting of cardiac pericytes. Methods and Results Using the neuron‐glial antigen 2 (NG2) DsRed reporter line, we identified a large population of NG2+ periendothelial cells in mouse atria, ventricles, and valves. To examine possible overlap of NG2+ mural cells with fibroblasts, we generated NG2 DsRed ; platelet‐derived growth factor receptor (PDGFR) α EGFP pericyte/fibroblast dual reporter mice. Myocardial NG2+ pericytes and PDGFRα+ fibroblasts were identified as nonoverlapping cellular populations with distinct transcriptional signatures. PDGFRα+ fibroblasts expressed high levels of fibrillar collagens, matrix metalloproteinases, tissue inhibitor of metalloproteinases, and genes encoding matricellular proteins, whereas NG2+ pericytes expressed high levels of Pdgfrb , Adamts1 , and Vtn . To validate the specificity of pericyte Cre drivers, we crossed these lines with PDGFRα EGFP fibroblast reporter mice. The constitutive NG2 Cre driver did not specifically track mural cells, labeling many cardiomyocytes. However, the inducible NG2 CreER driver specifically traced vascular mural cells in the ventricle and in the aorta, without significant labeling of PDGFRα+ fibroblasts. In contrast, the inducible PDGFRβ CreER line labeled not only mural cells but also the majority of cardiac and aortic fibroblasts. Conclusions Fibroblasts and pericytes are topographically and transcriptomically distinct populations of cardiac interstitial cells. The inducible NG2 CreER driver optimally targets cardiac pericytes; in contrast, the inducible PDGFRβ CreER line lacks specificity.

Download Full-text

Development of vascular myogenic responses in zebrafish

10.1101/713248 ◽

2019 ◽

Cited By ~ 1

Author(s):

Nabila Bahrami ◽

Sarah J. Childs

Keyword(s):

Vascular Tone ◽

Vascular System ◽

Cardiac Contractility ◽

Mural Cell ◽

Pharmacological Agents ◽

Genetic Ablation ◽

Mural Cells ◽

Ep4 Receptor ◽

Contraction And Relaxation

ABSTRACTThe vascular system is placed under enormous stress at the onset of cardiac contractility and blood flow. Nascent blood vessel tubes initially consist of a thin endothelial wall and rapidly acquire support from mural cells (pericytes and vascular smooth muscle cells; vSMCs). Following their association with vessels, mural cells acquire vasoactive ability (contraction and relaxation). However, we have little information as to when this vasoactivity first develops, and the extent to which each mural cell type contributes to vascular tone regulation during development. For the first time in an in vivo system, we highlight the dynamic changes in mural cell vasoactivity during development. We assess mural cell vasoactivity in the early zebrafish cerebral vasculature in response to pharmacological agents. We determine that pericyte-covered vessels constrict and dilate at 4 days post fertilization (dpf) but not at 6 dpf. The prostaglandin EP4 receptor contributes to pericyte-covered vessel dilation at 4 dpf. In contrast, vSMC-covered vessels constrict but do not dilate at 4 dpf. At 6 dpf, vSMC-covered vessels continue to constrict but only dilate from a pre-constricted state. Using genetic ablation, we demonstrate that mural cell contraction and relaxation is an active response by pericytes and vSMCs. Thus, we show that both pericytes and vSMCs have the ability to regulate cerebral vascular tone but at different stages of development. Pericytes are involved in regulating vessel diameters prior to the maturation of the vSMCs. Once vSMCs mature, pericytes are no longer active, and only vSMCs regulate vascular tone in the developing embryonic brain of zebrafish. The onset of vasoactivity of vSMCs corresponds to the development of increased neuronal activity and neurovascular coupling.

Download Full-text

Molecular and Circulating Biomarkers of Brain Tumors

International Journal of Molecular Sciences ◽

10.3390/ijms22137039 ◽

2021 ◽

Vol 22 (13) ◽

pp. 7039

Author(s):

Wojciech Jelski ◽

Barbara Mroczko

Keyword(s):

Brain Tumors ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Extracellular Vesicles ◽

Dna Methyltransferase ◽

Growth Factor Receptor ◽

Response To Treatment ◽

Liquid Biopsies ◽

Methylguanine Dna Methyltransferase ◽

The Central Nervous System

Brain tumors are the most common malignant primary intracranial tumors of the central nervous system. They are often recognized too late for successful therapy. Minimally invasive methods are needed to establish a diagnosis or monitor the response to treatment of CNS tumors. Brain tumors release molecular information into the circulation. Liquid biopsies collect and analyze tumor components in body fluids, and there is an increasing interest in the investigation of liquid biopsies as a substitute for tumor tissue. Tumor-derived biomarkers include nucleic acids, proteins, and tumor-derived extracellular vesicles that accumulate in blood or cerebrospinal fluid. In recent years, circulating tumor cells have also been identified in the blood of glioblastoma patients. In this review of the literature, the authors highlight the significance, regulation, and prevalence of molecular biomarkers such as O6-methylguanine-DNA methyltransferase, epidermal growth factor receptor, and isocitrate dehydrogenase. Herein, we critically review the available literature on plasma circulating tumor cells (CTCs), cell-free tumors (ctDNAs), circulating cell-free microRNAs (cfmiRNAs), and circulating extracellular vesicles (EVs) for the diagnosis and monitoring of brain tumor. Currently available markers have significant limitations.While much research has been conductedon these markers, there is still a significant amount that we do not yet understand, which may account for some conflicting reports in the literature.

Download Full-text

Bioengineered Livers: A New Tool for Drug Testing and a Promising Solution to Meet the Growing Demand for Donor Organs

European Surgical Research ◽

10.1159/000446211 ◽

2016 ◽

Vol 57 (3-4) ◽

pp. 224-239 ◽

Cited By ~ 9

Author(s):

Franziska Mußbach ◽

Utz Settmacher ◽

Olaf Dirsch ◽

Chichi Xie ◽

Uta Dahmen

Keyword(s):

Drug Testing ◽

Vascular System ◽

Observation Time ◽

Heterotopic Transplantation ◽

Physiological Models ◽

Innovative Strategy ◽

Pharmacological Research ◽

Organ Specific ◽

Static Conditions ◽

Donor Organs

Background: Organ engineering is a new innovative strategy to cope with two problems: the need for physiological models for pharmacological research and donor organs for transplantation. A functional scaffold is generated from explanted organs by removing all cells (decellularization) by perfusing the organ with ionic or nonionic detergents via the vascular system. Subsequently the acellular scaffold is reseeded with organ-specific cells (repopulation) to generate a functional organ. Summary: This review gives an overview of the state of the art describing the decellularization process, the subsequent quality assessment, the repopulation techniques and the functional assessment. It emphasizes the use of scaffolds as matrix for culturing human liver cells for drug testing. Further, it highlights the techniques for transplanting these engineered scaffolds in allogeneic or xenogeneic animals in order to test their biocompatibility and use as organ grafts. Key Messages: The first issue is the so-called decellularization, which is best explored and resulted in a multitude of different protocols. The most promising approach seems to be the combination of pulsatile perfusion of the liver with Triton X-100 and SDS via hepatic artery and portal vein. Widely accepted parameters of quality control include the quantitative assessment of the DNA content and the visualization of eventually remaining nuclei confirmed by HE staining. Investigations regarding the composition of the extracellular matrix focused on histological determination of laminin, collagen, fibronectin and elastin and remained qualitatively. Repopulation is the second issue which is addressed. Selection of the most suitable cell type is a highly controversial topic. Currently, the highest potential is seen for progenitor and stem cells. Cells are infused into the scaffold and cultured under static conditions or in a bioreactor allowing dynamic perfusion of the scaffold. The quality of repopulation is mainly assessed by routine histology and basic functional assays. These promising results prompted to consider the use of a liver scaffold repopulated with human cells for pharmacological research. Transplantation of the (repopulated) scaffold is the third topic which is not yet widely addressed. Few studies report the heterotopic transplantation of repopulated liver tissue without vascular anastomosis. Even fewer studies deal with the heterotopic transplantation of a scaffold or a repopulated liver lobe. However, observation time was still limited to hours, and long-term graft survival has not been reported yet. These exciting results emphasize the potential of this new and promising strategy to create physiological models for pharmacological research and to generate liver grafts for the transplant community to treat organ failure. However, the scientific need for further development in the field of liver engineering is still tremendous.

Download Full-text

Integrative analysis of the human brain mural cell transcriptome

Journal of Cerebral Blood Flow & Metabolism ◽

10.1177/0271678x211013700 ◽

2021 ◽

pp. 0271678X2110137

Author(s):

Benjamin D Gastfriend ◽

Koji L Foreman ◽

Moriah E Katt ◽

Sean P Palecek ◽

Eric V Shusta

Keyword(s):

Human Brain ◽

Cell Function ◽

In Vitro Models ◽

Molecular Characteristics ◽

Mural Cell ◽

Mural Cells ◽

Brain Pericytes ◽

Mouse Species ◽

Cell Transcriptome

Brain mural cells, including pericytes and vascular smooth muscle cells, are important for vascular development, blood-brain barrier function, and neurovascular coupling, but the molecular characteristics of human brain mural cells are incompletely characterized. Single cell RNA-sequencing (scRNA-seq) is increasingly being applied to assess cellular diversity in the human brain, but the scarcity of mural cells in whole brain samples has limited their molecular profiling. Here, we leverage the combined power of multiple independent human brain scRNA-seq datasets to build a transcriptomic database of human brain mural cells. We use this combined dataset to determine human-mouse species differences in mural cell transcriptomes, culture-induced dedifferentiation of human brain pericytes, and human mural cell organotypicity, with several key findings validated by RNA fluorescence in situ hybridization. Together, this work improves knowledge regarding the molecular constituents of human brain mural cells, serves as a resource for hypothesis generation in understanding brain mural cell function, and will facilitate comparative evaluation of animal and in vitro models.

Download Full-text

Abstract 120: Pericytes Contribute To Myofibroblast Expansion And Vascular Maturation In The Infarcted Heart

Circulation Research ◽

10.1161/res.129.suppl_1.120 ◽

2021 ◽

Vol 129 (Suppl_1) ◽

Author(s):

Linda Alex ◽

Kai Su ◽

Izabela Tuleta ◽

Nikolaos G Frangogiannis

Keyword(s):

Lineage Tracing ◽

Cytokine Gene Expression ◽

Infarct Zone ◽

Mural Cell ◽

Infarcted Myocardium ◽

Reporter Mice ◽

Infarct Healing ◽

Subsequent Activation ◽

Vascular Maturation

Infarct healing is dependent on recruitment of inflammatory leukocytes and subsequent activation of myofibroblasts (MF) and neovessel formation, ultimately resulting in formation of a highly vascularized collagen-enriched scar. Though the heart has an abundant population of periendothelial pericytes, its role in wound healing upon myocardial infarction (MI) has not been studied. We hypothesized that in the infarcted myocardium, pericytes may become activated, contributing to inflammatory, fibrotic and angiogenic responses. We used pericyte/fibroblast reporter mice (NG2 DsRed ;PDGFRα GFP ), lineage tracing studies and in vitro approaches to study the fate and role of pericytes in the infarcted myocardium. In normal hearts, NG2+/PDGFRα- pericytes and PDGFRα+/NG2- fibroblasts had distinct transcriptomic profiles. Pericytes expressed mural genes like Acta2 , Pdgfrb and low amounts of extracellular matrix (ECM) genes, whereas fibroblasts synthesized collagens, Timp2/3 and matricellular genes. 7 days post-MI, expansion of the NG2+ population in the infarct zone was associated with emergence of non-mural NG2+/αSMA+ cells with MF characteristics. FACS-sorted NG2+/PDGFRα- cells from 7-day infarcts expressed higher levels of collagens when compared to NG2+/PDGFRα- cells from normal hearts. Infarct pericytes had high integrin and MMP14 expression, reflecting an activated migratory phenotype. Lineage tracing using NG2CreER TM ;Rosa tdTomato ;PDGFRα GFP mice showed that 5.7%±1.04 of PDGFRα+ fibroblasts and 10.49%±2.73 of infarct MFs were derived from NG2+ lineage. Pericyte-derived fibroblasts exhibited higher ECM gene synthesis, in comparison to fibroblasts from non-pericyte origin, while pericyte-derived mural cells showed accentuated inflammatory cytokine gene expression. Immunostaining showed pericytes actively contribute to vascular maturation, forming a mural cell coat enwrapping infarct neovessels. In vitro, TGFβ induced integrins, collagens and MMPs in human pericytes, similar to the changes observed in infarct pericytes. Taken together, our evidences show that after MI, pericytes become activated and contribute to repair by undergoing conversion to a subset of myofibroblasts and by coating infarct neovessels.

Download Full-text

Abstract TP475: Effect of Udenafil, a Phosphodiesterase-5 Inhibitor, on Angiogenesis in a Cav-1 Deficient Moyamoya Disease Model

Stroke ◽

10.1161/str.51.suppl_1.tp475 ◽

2020 ◽

Vol 51 (Suppl_1) ◽

Author(s):

Hyung Jun Kim ◽

Oh Young Bang ◽

In-Young Baek ◽

Jae-Hwan Kim ◽

Ye Sel Kim ◽

...

Keyword(s):

Moyamoya Disease ◽

Growth Factor Receptor ◽

Guanosine Monophosphate ◽

Therapeutic Effects ◽

Mural Cell ◽

Phosphodiesterase 5 Inhibitor ◽

Vessel Maturation ◽

Phosphodiesterase 5

Background: Although pathogenic mechanisms of moyamoya disease remain unknown, recent studies suggest that it is a caveolae disease. This study evaluated the effect of udenafil, a phosphodiesterase-5 inhibitor, on vessel maturation in in vitro and in vivo moyamoya disease models. Methods: Angiogenesis and vessel maturation were assessed in in vitro models, caveolin-1 (Cav-1) knockdown human umbilical vessel endothelial cells (HUVECs) and coronary artery smooth muscle cells (CASMCs), and in in vivo model of bilateral internal carotid artery occlusion (bICAo). Udenafil was administered (1,3,10 and 30 μM) in cell culture conditions, and functional studies (migration and tube formation assay) were performed and vessel maturation factors and cyclic guanosine monophosphate (cGMP) accumulation were measured. Udenafil (3 and 10 mg/kg) was orally administered once daily for 4 weeks in bICAo rat model, and histological analysis for angiogenesis and vessel maturation was performed. Results: Udenafil increased vessel formation in both Cav-1 knockdown HUVEC and bICAo models without increased migration/proliferation of HUVECs and CASMCs. Udenafil increased CD31+ vessel density and NG2/Col4+ mural cell density in bICAo models. Cav-1 knockdown inhibited accumulation of cGMP, and udenafil treatment restored cGMP levels in Cav-1 knockdown HUVEC models. Vessel maturation factors (angiopoietin-1 and platelet-derived growth factor receptor-β) and angiogenic factors (endothelial nitric oxide synthase) were increased after treatment with udenafil in vitro . Conclusion: Our results indicate that udenafil reversed cellular levels of cGMP related to Cav-1 deficiency and induced angiogenesis and vessel maturation. Further studies are warranted to confirm the therapeutic effects of this strategy in moyamoya disease.

Download Full-text

Abstract MP123: The Fate and Role of Pericytes in Repair and Remodeling of the Infarcted Heart

Circulation Research ◽

10.1161/res.127.suppl_1.mp123 ◽

2020 ◽

Vol 127 (Suppl_1) ◽

Author(s):

Linda Alex ◽

Ya Su ◽

Nikolaos G Frangogiannis

Keyword(s):

Lineage Tracing ◽

Mrna Levels ◽

Infarct Zone ◽

Subsequent Formation ◽

Mural Cells ◽

Infarcted Heart ◽

Reporter Mice ◽

Ng2 Cells ◽

Migratory Phenotype

Repair of the infarcted heart is dependent on inflammation-driven activation of myofibroblasts (MFs) and subsequent formation of a scar. Though pericytes have been implicated in injury-associated fibroblast activation in several organs, their potential role in cardiac repair and fibrosis has not been studied. We hypothesized that myocardial infarction (MI) may induce pericyte activation, contributing to repair through pericyte to MF conversion, secretion of fibrogenic mediators, or regulation of angiogenesis. In order to test the hypothesis, we generated pericyte/fibroblast reporter mice (NG2 DsRed ;PDGFRα GFP ). In normal myocardium, NG2 labeled peri-endothelial mural cells that coexpressed PDGFRβ, whereas PDGFRα identified interstitial cells with fibroblast characteristics. Pericytes and fibroblasts had distinct transcriptomic profiles: NG2+/PDGFRα- pericytes expressed αSMA and low amounts of extracellular matrix (ECM) genes, whereas PDGFRα+/NG2- fibroblasts synthesized collagens. Pericyte rarefaction was noted in the necrotic core 3 days after non-reperfused MI. 3-7 days post MI, expansion of the NG2+ population in the infarct zone was associated with emergence of non-mural NG2+/αSMA+ cells with MF characteristics. FACS-sorted NG2+/PDGFRα- cells from 7-day infarcts expressed higher levels of ColIα2 (7.2±1.0-fold) and ColIIIα1 (8.9±1.14-fold), when compared to NG2+/PDGFRα- cells from normal hearts. NG2+ cells had high mRNA levels of integrins α1, αV, β1, and β5, and of MMP14, reflecting an activated migratory phenotype. To examine whether expression of ECM genes by infarct pericytes is due to fibroblast conversion, we did lineage tracing studies using NG2CreER TM ;Rosa tdTomato mice bred with the PDGFRα GFP line for reliable fibroblast identification. 7 days post MI, 5.7%±1.04 of PDGFRα+ fibroblasts were derived from NG2+ cells. Also, αSMA staining showed that 10.49%±2.73 of infarct MFs were derived from NG2+ lineage. The majority of mural cells wrapping neovessels were derived from NG2+ cells, suggesting a role for resident pericytes in infarct angiogenesis. In conclusion, upon MI, pericytes become activated and contribute to repair by undergoing conversion to a subset of myofibroblasts and by coating infarct neovessels.

Download Full-text