ABSTRACT
NG2+ pericytes, as the possible precursor cells of mesenchymal stem cells (MSCs), have drawn attention due to their ability to differentiate into odontoblasts. Cav1.2 is involved in the differentiation process of stem cells, but its role in the differentiation of NG2+ pericytes is not clear. The aim of the present study was to examine the role of Cav1.2 in the differentiation of NG2+ pericytes into odontoblasts. NG2+ pericytes were obtained from human dental pulp cells by magnetic-activated cell sorting. During the odontogenic differentiation of NG2+ pericytes, the effects of the Cav1.2 inhibitors, nimodipine and Cav1.2 knockdown shRNA, were analyzed by real-time polymerase chain reaction and alizarin red staining. NG2CreERT2/Rosa26-GFP lineage-tracing mice were established to further investigate the roles of NG2+ pericytes and Cav1.2 in incisor self-repair after injury in vivo. At 10 min, 1 day, and 3 days after pulp injuries in transgenic mice, NG2-GFP+ and Cav1.2 immunofluorescence co-staining was performed on the incisors. Nimodipine treatment and Cav1.2 knockdown showed similar inhibition of calcium nodule formation and mRNA levels of osteogenic markers (DSPP, DMP1, and Runx2, p < 0.05). NG2+ pericytes migrated from their inherent perivascular location to the odontoblast layers after pulp injury. Cav1.2 showed a similar response pattern as NG2+ pericytes and gradually returned to normal levels. In addition, many co-stained areas of Cav1.2 and NG2+ pericytes, both near the perivascular and odontoblast layers, were observed. These results indicate that Cav1.2 played a vital role in the odontogenic differentiation of NG2+ pericytes, and that it might be closely linked to the NG2+ pericytes-mediated repair of dental pulp injury in vivo.
Subject(s)
Extracellular Matrix Proteins , Pericytes , Mice , Humans , Animals , Pericytes/chemistry , Nimodipine , Dental Pulp , Cell Differentiation , Odontoblasts , Cells, CulturedABSTRACT
BACKGROUND: The pathways that control protein transport across the blood-brain barrier (BBB) remain poorly characterized. Despite great advances in recapitulating the human BBB in vitro, current models are not suitable for systematic analysis of the molecular mechanisms of antibody transport. The gaps in our mechanistic understanding of antibody transcytosis hinder new therapeutic delivery strategy development. METHODS: We applied a novel bioengineering approach to generate human BBB organoids by the self-assembly of astrocytes, pericytes and brain endothelial cells with unprecedented throughput and reproducibility using micro patterned hydrogels. We designed a semi-automated and scalable imaging assay to measure receptor-mediated transcytosis of antibodies. Finally, we developed a workflow to use CRISPR/Cas9 gene editing in BBB organoid arrays to knock out regulators of endocytosis specifically in brain endothelial cells in order to dissect the molecular mechanisms of receptor-mediated transcytosis. RESULTS: BBB organoid arrays allowed the simultaneous growth of more than 3000 homogenous organoids per individual experiment in a highly reproducible manner. BBB organoid arrays showed low permeability to macromolecules and prevented transport of human non-targeting antibodies. In contrast, a monovalent antibody targeting the human transferrin receptor underwent dose- and time-dependent transcytosis in organoids. Using CRISPR/Cas9 gene editing in BBB organoid arrays, we showed that clathrin, but not caveolin, is required for transferrin receptor-dependent transcytosis. CONCLUSIONS: Human BBB organoid arrays are a robust high-throughput platform that can be used to discover new mechanisms of receptor-mediated antibody transcytosis. The implementation of this platform during early stages of drug discovery can accelerate the development of new brain delivery technologies.
Subject(s)
Antibodies/metabolism , Bioengineering/methods , Blood-Brain Barrier/metabolism , Organoids/metabolism , Receptors, Transferrin/metabolism , Transcytosis/physiology , Animals , Antibodies/analysis , Astrocytes/chemistry , Astrocytes/metabolism , Blood-Brain Barrier/chemistry , Blood-Brain Barrier/cytology , Cells, Cultured , Coculture Techniques , Endothelial Cells/chemistry , Endothelial Cells/metabolism , Humans , Organoids/chemistry , Organoids/cytology , Pericytes/chemistry , Pericytes/metabolism , Receptors, Transferrin/analysisABSTRACT
Pericytes are a unique class of mural cells essential for angiogenesis, maintenance of the vasculature and are key players in microvascular pathology. However, their diversity and specific roles are poorly understood, limiting our insight into vascular physiology and the ability to develop effective therapies. Here, in the mouse retina, a tractable model of the CNS, we evaluated distinct classes of mural cells along the vascular tree for both structural characterization and physiological manipulation of blood flow. To accomplish this, we first tested three inducible mural cell-specific mouse lines using a sensitive Ai14 reporter and tamoxifen application either by a systemic injection, or by local administration in the form of eye drops. The specificity and pattern of cre activation varied significantly across the three lines, under either the PDGFRß or NG2 promoter (Pdgfrß-CreRha, Pdgfrß-CreCsln, and Cspg4-Cre). In particular, a mouse line with Cre under the NG2 promoter resulted in sparse TdTomato labeling of mural cells, allowing for an unambiguous characterization of anatomical features of individual sphincter cells and capillary pericytes. Furthermore, in one PDGFRß line, we found that focal eye drop application of tamoxifen led to an exclusive Cre-activation in pericytes, without affecting arterial mural cells. We then used this approach to boost capillary blood flow by selective expression of Halorhodopsin, a highly precise hyperpolarizing optogenetic actuator. The ability to exclusively target capillary pericytes may prove a precise and potentially powerful tool to treat microcirculation deficits, a common pathology in numerous diseases.
Subject(s)
Blood Flow Velocity/physiology , Capillaries/physiology , Pericytes/physiology , Regional Blood Flow/physiology , Retina/physiology , Administration, Ophthalmic , Animals , Blood Flow Velocity/drug effects , Capillaries/chemistry , Capillaries/drug effects , Mice , Mice, Transgenic , Microscopy, Fluorescence, Multiphoton/methods , Pericytes/chemistry , Pericytes/drug effects , Regional Blood Flow/drug effects , Retina/chemistry , Retina/cytology , Retina/drug effects , Tamoxifen/administration & dosageABSTRACT
Two cases of myopericytosis combined with pericytoma originating within the lung are reported. These are rare pulmonary tumors. The differential diagnosis for hemangiopericytoma and pericytic tumors with glomus elements is discussed. Both myopericytic lesions mimic other lesions, which are more commonly seen in the lung. Based on the expression of vascular growth factor receptors 2 and 3, an antiangiogenic therapy was suggested for the patient with the myopericytoma. A treatment with an angiogenesis inhibitor resulted in a regression of the tumor, but not the precursor lesion. Probably a more specific therapy using tyrosine kinase inhibitors for VEGFR2/3 might better control these myopericytic proliferations.
Subject(s)
Lung Neoplasms/pathology , Lung/pathology , Myopericytoma/pathology , Pericytes/pathology , Precancerous Conditions/pathology , Adult , Aged, 80 and over , Angiogenesis Inhibitors/therapeutic use , Biomarkers, Tumor/analysis , Female , Humans , Lung/chemistry , Lung/diagnostic imaging , Lung/drug effects , Lung Neoplasms/chemistry , Lung Neoplasms/diagnostic imaging , Lung Neoplasms/drug therapy , Myopericytoma/chemistry , Myopericytoma/diagnostic imaging , Myopericytoma/drug therapy , Pericytes/chemistry , Pericytes/drug effects , Precancerous Conditions/diagnostic imaging , Precancerous Conditions/drug therapy , Precancerous Conditions/metabolism , Treatment Outcome , Vascular Endothelial Growth Factor Receptor-2/analysis , Vascular Endothelial Growth Factor Receptor-3/analysisABSTRACT
Pericyte, a kind of pluripotent cell, may regulate the irrigation flow and permeability of microcirculation. Pericytes are similar to the smooth muscle cells, which express several kinds of contractile proteins and have contractility. The dysfunction of pericytes is related to many microvascular diseases, including hypoxia, hypertension, diabetic retinopathy, fibrosis, inflammation, Alzheimer's disease, multiple sclerosis, and tumor formation. For a long time, their existence and function have been neglected. The distribution, structure, biomarker, related signaling pathways as well as the roles of pericytes on vascular diseases will be introduced in this review.
Subject(s)
Pericytes , Research , Contractile Proteins/metabolism , Humans , Microcirculation , Pericytes/chemistry , Pericytes/pathology , Pericytes/physiology , Vascular Diseases/etiologyABSTRACT
An impairment of amyloid ß-peptide (Aß) clearance is suggested to play a key role in the pathogenesis of sporadic Alzheimer's disease (AD). Amyloid degradation is mediated by various mechanisms including fragmentation by enzymes like neprilysin, matrix metalloproteinases (MMPs) and a recently identified amyloidolytic activity of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1). BACE1 cleavage of Aß40 and Aß42 results in the formation of a common Aß34 intermediate which was found elevated in cerebrospinal fluid levels of patients at the earliest disease stages. To further investigate the role of Aß34 as a marker for amyloid clearance in AD, we performed a systematic and comprehensive analysis of Aß34 immunoreactivity in hippocampal and cortical post-mortem brain tissue from AD patients and non-demented elderly individuals. In early Braak stages, Aß34 was predominantly detectable in a subset of brain capillaries associated with pericytes, while in later disease stages, in clinically diagnosed AD, this pericyte-associated Aß34 immunoreactivity was largely lost. Aß34 was also detected in isolated human cortical microvessels associated with brain pericytes and its levels correlated with Aß40, but not with Aß42 levels. Moreover, a significantly decreased Aß34/Aß40 ratio was observed in microvessels from AD patients in comparison to non-demented controls suggesting a reduced proteolytic degradation of Aß40 to Aß34 in AD. In line with the hypothesis that pericytes at the neurovascular unit are major producers of Aß34, biochemical studies in cultured human primary pericytes revealed a time and dose dependent increase of Aß34 levels upon treatment with recombinant Aß40 peptides while Aß34 production was impaired when Aß40 uptake was reduced or BACE1 activity was inhibited. Collectively, our findings indicate that Aß34 is generated by a novel BACE1-mediated Aß clearance pathway in pericytes of brain capillaries. As amyloid clearance is significantly reduced in AD, impairment of this pathway might be a major driver of the pathogenesis in sporadic AD.
Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Brain/metabolism , Capillaries/metabolism , Peptide Fragments/metabolism , Pericytes/metabolism , Proteolysis , Aged , Aged, 80 and over , Alzheimer Disease/pathology , Amyloid beta-Peptides/analysis , Brain/pathology , Capillaries/chemistry , Capillaries/pathology , Cells, Cultured , Female , Humans , Male , Peptide Fragments/analysis , Pericytes/chemistry , Pericytes/pathologyABSTRACT
The study of cerebral microvessels is becoming increasingly important in a wide variety of conditions, such as stroke, sepsis, traumatic brain injury and neurodegenerative diseases. However, the molecular mechanisms underlying cerebral microvascular dysfunction in these conditions are largely unknown. The molecular characterization of cerebral microvessels in experimental disease models has been hindered by the lack of a standardized method to reproducibly isolate intact cerebral microvessels with consistent cellular compositions and without the use of enzymatic digestion, which causes undesirable molecular and metabolic changes. Herein, we describe an optimized protocol for microvessel isolation from mouse brain cortex that yields microvessel fragments with consistent populations of discrete blood-brain barrier (BBB) components (endothelial cells, pericytes and astrocyte end feet) while retaining high RNA integrity and protein post-translational modifications (e.g., phosphorylation). We demonstrate that this protocol allows the quantification of changes in gene expression in a disease model (stroke) and the activation of signaling pathways in mice subjected to drug administration in vivo. We also describe the isolation of genomic DNA (gDNA) and bisulfite treatment for the assessment of DNA methylation, as well as the optimization of chromatin extraction and shearing from cortical microvessels. This optimized protocol and the described applications should improve the understanding of the molecular mechanisms governing cerebral microvascular dysfunction, which may help in the development of novel therapies for stroke and other neurologic conditions.
Subject(s)
Brain/blood supply , Microvessels/cytology , Animals , Astrocytes/chemistry , Astrocytes/cytology , Blood-Brain Barrier/chemistry , Blood-Brain Barrier/cytology , Brain Chemistry , DNA/analysis , Endothelial Cells/chemistry , Endothelial Cells/cytology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microvessels/chemistry , Pericytes/chemistry , Pericytes/cytology , RNA/analysisABSTRACT
OBJECTIVE: To investigate the role of microRNA-15b in diabetic retinopathy and its underlying mechanism. MATERIALS AND METHODS: Diabetes rat model was established by streptozotocin injection. The mRNA expression of microRNA-15b in retinal capillary endothelial cells and pericytes of diabetic rats was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The mRNA and protein expressions of vascular endothelial growth factor A (VEGFA) were detected by qRT-PCR and Western blot, respectively. MicroRNA-15b mimics or inhibitor were transfected into retinal capillary endothelial cells and pericytes of diabetic rats, respectively. The mRNA expressions of microRNA-15b and VEGFA were detected by qRT-PCR. Cell counting kit-8 (CCK-8) assay was used to detect the proliferation of capillary endothelial cells and pericytes. Dual-Luciferase reporter gene assay was conducted to verify the binding condition of microRNA-15b and VEGFA. RNA immunoprecipitation (RIP) assay was performed to determine whether microRNA-15b could bind to AGO2. Rescue experiments were finally carried out by detecting the proliferation of retinal capillary endothelial cells and pericytes after downregulation or overexpression of microRNA-15b and VEGFA. RESULTS: The expression of microRNA-15b decreased, whereas VEGFA expression increased in retinal capillary endothelial cells and pericytes of diabetic rats. High expression of microRNA-15b in retinal capillary endothelial cells and pericytes resulted in VEGFA down-regulation and decreased proliferation. RIP assay results indicated that microRNA-15b could interact with AGO2. Additionally, Dual-Luciferase reporter gene assay showed that VEGFA is a direct target gene of microRNA-15b. VEGFA overexpression could reverse the inhibited proliferation of retinal capillary endothelial cells and pericytes induced by microRNA-15b overexpression. Similarly, VEGFA knockdown could reverse the effect of the low expression of microRNA-15b on the proliferation of retinal capillary endothelial cells and pericytes. CONCLUSIONS: Low expression of microRNA-15b in retinal capillary endothelial cells and pericytes of diabetic rats promotes the development of diabetic retinopathy by up-regulating VEGFA.
Subject(s)
Diabetes Mellitus, Experimental/complications , Diabetic Retinopathy/genetics , MicroRNAs/genetics , Pericytes/cytology , Retinal Vessels/cytology , Vascular Endothelial Growth Factor A/genetics , Animals , Cell Proliferation , Cells, Cultured , Diabetes Mellitus, Experimental/chemically induced , Diabetes Mellitus, Experimental/genetics , Diabetes Mellitus, Experimental/metabolism , Diabetic Retinopathy/metabolism , Disease Models, Animal , Down-Regulation , Endothelial Cells/chemistry , Endothelial Cells/cytology , Pericytes/chemistry , Rats , Retinal Vessels/chemistry , Streptozocin , Up-Regulation , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Aim: Pericytes maintain homeostatic functions in the blood-brain barrier. N6-methyladenosine (m6A) is critical for various biological processes, but the role of mRNA m6A methylation in hypertension has not been fully elucidated. Methods: The m6A methylation levels of Wistar Kyoto rat pericytes and spontaneously hypertensive rat pericytes were detected via m6A high-throughput sequencing. Results: The m6A methylations were more enriched in the coding sequence region, 3'UTR and 5'UTR of mRNAs, with the m6A motifs being relatively conserved across the different conditions investigated. The average m6A abundance of spontaneously hypertensive rat pericytes exhibited global reductions in the pericytes. Conclusion: This study revealed the m6A landscapes and identified an epitranscriptomic mechanism during the development of mammalian hypertension.
Subject(s)
Adenosine/analogs & derivatives , Epigenomics/methods , Gene Expression Profiling/methods , Gene Regulatory Networks , Hypertension/genetics , Pericytes/chemistry , 3' Untranslated Regions , 5' Untranslated Regions , Adenosine/chemistry , Animals , Disease Models, Animal , Gene Expression Regulation , High-Throughput Nucleotide Sequencing , Male , Methylation , Organ Specificity , Rats , Rats, Inbred SHR , Rats, Inbred WKYABSTRACT
The delivery of anticancer drugs is hampered by tumor vessels with abnormal structure and function, which requires that vessel normalization be mediated by pharmaceutics. The current strategies for vessel normalization focus on direct modulation of endothelial cells (ECs), which frequently affect vessels in normal tissues. Modulating EC-supporting cells, such as pericytes (PCs), is a new direction. Here, we produced a fusion protein, Z-TNFα, by fusing the platelet-derived growth factor receptor ß (PDGFRß)- antagonistic affibody ZPDGFRß to tumor necrosis factor α (TNFα). Owing to the affinity of fused ZPDGFRß for PDGFRß, Z-TNFα binds PDGFRß+ PCs but not PDGFRß- ECs. Low-dose (1⯵g/mouse) Z-TNFα treatment remodeled the tumor vessels, thus reducing vessel permeability and increasing vessel perfusion. As a result, the Z-TNFα treatment improved the delivery of doxorubicin (DOX) and enhanced its antitumor effect, indicating that Z-TNFα induced normalization of tumor vessels. Mechanically, the tumor vessel normalization mediated by Z-TNFα might be attributed to the reduction of vascular endothelial growth factor (VEGF) secretion by PCs and the elevated expression of intercellular cell adhesion molecule-1 (ICAM-1) in PCs, which might suppress the proliferation and migration of ECs and simultaneously trigger interaction between perivascular macrophages and PCs. These results demonstrated that tumor-associated PCs could be considered novel target cells for vessel normalization, and Z-TNFα might be developed as a potential tool for antitumor combination therapy.
Subject(s)
Antineoplastic Agents/chemistry , Doxorubicin/chemistry , Drug Carriers/chemistry , Pericytes/chemistry , Receptor, Platelet-Derived Growth Factor beta/chemistry , Recombinant Fusion Proteins/chemistry , Tumor Necrosis Factor-alpha/chemistry , Animals , Antineoplastic Agents/pharmacology , Biological Transport , Cell Line, Tumor , Combinatorial Chemistry Techniques/methods , Doxorubicin/pharmacology , Drug Liberation , Endothelial Cells/drug effects , Female , Gene Expression Regulation/drug effects , Humans , Intercellular Adhesion Molecule-1/genetics , Intercellular Adhesion Molecule-1/metabolism , Mice , Mice, Inbred ICR , Neoplasms, Experimental , Protein Kinase Inhibitors/metabolism , Receptor, Platelet-Derived Growth Factor beta/metabolism , Recombinant Fusion Proteins/metabolism , Signal Transduction/drug effects , Treatment Outcome , Tumor Necrosis Factor-alpha/metabolism , Vascular Endothelial Growth Factor AABSTRACT
Functional hyperemia, a regional increase of blood flow triggered by local neural activation, is used to map brain activity in health and disease. However, the spatial-temporal dynamics of functional hyperemia remain unclear. Two-photon imaging of the entire vascular arbor in NG2-creERT2;GCaMP6f mice shows that local synaptic activation, measured via oligodendrocyte precursor cell (OPC) Ca2+ signaling, generates a synchronous Ca2+ drop in pericytes and smooth muscle cells (SMCs) enwrapping all upstream vessels feeding the activated synapses. Surprisingly, the onset timing, direction, and amplitude of vessel diameter and blood velocity changes vary dramatically from juxta-synaptic capillaries back to the pial arteriole. These results establish a precise spatial-temporal sequence of vascular changes triggered by neural activity and essential for the interpretation of blood-flow-based imaging techniques such as BOLD-fMRI.
Subject(s)
Brain/blood supply , Brain/physiology , Hyperemia/physiopathology , Pia Mater/blood supply , Pia Mater/physiology , Synapses/physiology , Animals , Brain Chemistry/physiology , Hyperemia/diagnosis , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Confocal/methods , Muscle, Smooth, Vascular/chemistry , Muscle, Smooth, Vascular/physiology , Pericytes/chemistry , Pericytes/physiology , Pia Mater/chemistry , Synapses/chemistryABSTRACT
Follistatin like-1 (Fstl1) is a secreted glycoprotein and can be up-regulated by TGF-ß1. To better study the function of Fstl1 in lung development, we examined Fstl1 expression in the developing lung, in a cell type specific manner, using a tamoxifen inducible Fstl1-reporter mouse strain. Our results show that Fstl1 is ubiquitously expressed at saccular stage in the developing lung. At E18.5, Fstl1 expression is robust in most type of mesenchymal cells, including airway smooth muscle cells surrounding airways, vascular smooth muscle cells, endothelial cells, and vascular pericytes from blood vessel, but not PDGFRα+ fibroblasts in the distal alveolar sacs. Meanwhile, relative weak and sporadic signals of Fstl1 expression are observed in epithelium, including a subgroup of club cells in proximal airways and a few type II alveolar epithelial cells in distal airways. Our data help to understand the critical role of Fstl1 in lung development and lung disease pathogenesis.
Subject(s)
Cells/metabolism , Follistatin-Related Proteins/metabolism , Lung/embryology , Animals , Embryo, Mammalian , Endothelial Cells/chemistry , Endothelial Cells/metabolism , Mesenchymal Stem Cells/chemistry , Mesenchymal Stem Cells/metabolism , Mice , Myocytes, Smooth Muscle/chemistry , Myocytes, Smooth Muscle/metabolism , Pericytes/chemistry , Pericytes/metabolism , Respiratory System/cytology , Tissue DistributionABSTRACT
Telocytes (TCs) are stromal cells with telopodes, which represent long, thin, moniliform cell processes; however, this morphological feature alone is insufficient to define a cell type. Specific markers of lymphatic endothelial cells (LECs), such as Prox-1, podoplanin (D2-40) or LYVE-1, are not usually tested in TCs. We thus aimed at performing a study in light microscopy to evaluate whether or not LECs could be mistaken for TCs. Therefore we used CD34, α-smooth muscle actin and D2-40 for an immunohistochemical study on archived paraffin-embedded samples of uterine leiomyoma. Lymphatic vessels were identified by the expression of D2-40, but on the microscopic slides, false spindle-shaped TCs appearances either corresponded to collapsed lymphatic lumina or were determined by grazing longitudinal cuts of lymphatics. It is therefore mandatory to check the expression of lymphatic markers in telocyte-like cells and, moreover, to carefully examine the bidimensional cuts in order to avoid false results.
Subject(s)
Membrane Glycoproteins/chemistry , Telocytes/pathology , Telopodes/pathology , Actins/analysis , Antigens, CD34/analysis , Biomarkers , Female , Humans , Immunohistochemistry , Leiomyoma/chemistry , Leiomyoma/pathology , Lymphatic System/chemistry , Lymphatic System/pathology , Middle Aged , Paraffin Embedding , Pericytes/chemistry , Telocytes/chemistry , Telopodes/chemistry , Uterine Neoplasms/chemistry , Uterine Neoplasms/pathologyABSTRACT
BACKGROUND: Cerebrovascular dysfunction and inflammation occur in epilepsy. Here we asked whether pericytes, a pivotal cellular component of brain capillaries, undergo pathological modifications during experimental epileptogenesis and in human epilepsy. We evaluated whether pro-inflammatory cytokines, present in the brain during seizures, contribute to pericyte morphological modifications. METHODS: In vivo, unilateral intra-hippocampal kainic acid (KA) injections were performed in NG2DsRed/C57BL6 mice to induce status epilepticus (SE), epileptogenesis, and spontaneous recurrent seizures (SRS). NG2DsRed mice were used to visualize pericytes during seizure progression. The effect triggered by recombinant IL-1ß, TNFα, or IL-6 on pericytes was evaluated in NG2DsRed hippocampal slices and in human-derived cell culture. Human brain specimens obtained from temporal lobe epilepsy (TLE) with or without sclerosis (HS) and focal cortical dysplasia (FCD-IIb) were evaluated for pericyte-microglial cerebrovascular assembly. RESULTS: A disarray of NG2DsRed+ pericyte soma and ramifications was found 72â¯h post-SE and 1â¯week post-SE (epileptogenesis) in the hippocampus. Pericyte modifications topographically overlapped with IBA1+ microglia clustering around the capillaries with cases of pericytes lodged within the microglial cells. Microglial clustering around the NG2DsRed pericytes lingered at SRS. Pericyte proliferation (Ki67+) occurred 72â¯h post-SE and during epileptogenesis and returned towards control levels at SRS. Human epileptic brain tissues showed pericyte-microglia assemblies with IBA1/HLA microglial cells outlining the capillary wall in TLE-HS and FCD-IIb specimens. Inflammatory mediators contributed to pericyte modifications, in particular IL-1ß elicited pericyte morphological changes and pericyte-microglia clustering in NG2DsRed hippocampal slices. Modifications also occurred when pro-inflammatory cytokines were added to an in vitro culture of pericytes. CONCLUSIONS: These results indicate the occurrence of pericytosis during seizures and introduce a pericyte-microglial mediated mechanism of blood-brain barrier dysfunction in epilepsy.
Subject(s)
Cerebrovascular Circulation/physiology , Disease Progression , Inflammation Mediators/metabolism , Microglia/metabolism , Pericytes/metabolism , Seizures/metabolism , Adolescent , Adult , Animals , Blood-Brain Barrier/chemistry , Blood-Brain Barrier/metabolism , Blood-Brain Barrier/physiopathology , Cells, Cultured , Child , Child, Preschool , Female , Hippocampus/blood supply , Hippocampus/chemistry , Hippocampus/metabolism , Hippocampus/physiopathology , Humans , Infant , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microglia/chemistry , Pericytes/chemistry , Seizures/physiopathologyABSTRACT
The pericytic (perivascular myoid cell) family of tumors is a distinctive group of mesenchymal neoplasms encountered in superficial sites and only rarely seen in viscera. The pericytic family subtends a spectrum of lesions, namely, glomus tumors and variants; myopericytoma, including myofibroma; and angioleiomyoma. In light of the contemporary classification of pericytic lesions, we identified and reviewed 17 cases of renal pericytic tumors from the files of 6 referral centers. These tumors presented over an age range of 17 to 76 years (mean 46.7, median 53), with essentially equal male-female ratio. History of hypertension (available in 11 patients) was noted in 7 (64%), which persisted even after surgical resection, including in 2 younger patients (17 and 30 years). The tumors (1.7-11.0 cm) included glomus tumors (n=11); glomangiomyoma (n=1); glomus tumor with atypical features (n=1); and angioleiomyoma (n=1), as well as tumors showing features overlapping pericytic tumor subtypes (n=3). The histomorphology observed in these renal examples closely resembled that of their soft tissue counterparts, a subset with symplastic changes and atypical features, and pericytic immunophenotype. Despite large size and deep site, no progression was identified during a median of 7 months follow-up (1-62 months). In context of prior reported experience, our series identifies a wide morphologic spectrum, including lesions presenting composite morphologies. Taken with the experience of others, our series further corroborates that malignant behavior is rare, and that criteria associated with aggression among soft tissue pericytic tumors may not be predictive for those in the kidney.
Subject(s)
Angiomyoma/pathology , Glomus Tumor/pathology , Kidney Neoplasms/pathology , Pericytes/pathology , Adolescent , Adult , Aged , Angiomyoma/chemistry , Angiomyoma/complications , Angiomyoma/surgery , Biomarkers, Tumor/analysis , Biopsy , Blood Pressure , Female , Glomus Tumor/chemistry , Glomus Tumor/complications , Glomus Tumor/surgery , Humans , Hypertension/etiology , Hypertension/physiopathology , Immunohistochemistry , Kidney Neoplasms/chemistry , Kidney Neoplasms/complications , Kidney Neoplasms/surgery , Male , Middle Aged , Nephrectomy , Pericytes/chemistry , Time Factors , Treatment Outcome , Tumor Burden , United States , Young AdultABSTRACT
Our aims were to identify pericyte-specific markers for the analysis of formalin-fixed, paraffin-embedded human tissue samples, and to characterize perivascular myoid cell neoplasms phenotypically. Previously identified pericyte markers failed to distinguish pericytes from other cellular types, such as vascular smooth muscle cells (vSMCs) and fibroblasts, in immunohistochemistry analysis. However, we compared gene expression profiles between pericytes, vSMCs, and fibroblasts, and performed human skin vasculature immunohistochemistry analysis, which led to the identification of myosin 1B (MYO1B) as a novel pericyte marker. Afterward, we investigated the expression levels of MYO1B and h-caldesmon (h-CD) in perivascular myoid cell neoplasms, angioleiomyomas (n=28), glomus tumors (n=23), and myopericytomas (n=3). Angioleiomyomas were shown to contain MYO1B-negative and h-CD-positive (MYO1B-hCD+) tumor cells, with vSMC features. Glomus tumors were predominantly composed of the MYO1B+hCD+ tumor cells, with the intermediate features between pericytes and vSMCs, whereas MYO1B+hCD- tumor cells with pericytic features and/or the MYO1B-hCD+ tumor cells with vSMC features were frequently found in these tumors. The perivascular concentric pattern of 2 myopericytoma cases was composed of MYO1B+hCD+ tumor cells, whereas that of one myopericytoma contained MYO1B-hCD+ tumor cells. These results indicate that the ability to distinguish between these cell types may allow us to understand the differentiation and origin of perivascular myoid cell neoplasms. This is the first study to identify cell properties of perivascular myoid cell neoplasms by using a pericyte-specific marker with considerably lower expression in vSMCs and fibroblasts.
Subject(s)
Angiomyoma/chemistry , Biomarkers, Tumor/analysis , Glomus Tumor/chemistry , Myofibroma/chemistry , Myosin Type I/analysis , Pericytes/chemistry , Soft Tissue Neoplasms/chemistry , Adult , Aged , Angiomyoma/genetics , Angiomyoma/pathology , Animals , Biomarkers, Tumor/genetics , Biopsy , Calmodulin-Binding Proteins/analysis , Cell Differentiation , Cell Lineage , Female , Fibroblasts/chemistry , Fluorescent Antibody Technique , Gene Expression Profiling , Glomus Tumor/genetics , Glomus Tumor/pathology , Humans , Male , Mice, Inbred C57BL , Mice, Transgenic , Middle Aged , Myocytes, Smooth Muscle/chemistry , Myocytes, Smooth Muscle/pathology , Myofibroma/genetics , Myofibroma/pathology , Myosin Type I/genetics , Pericytes/pathology , Phenotype , Skin/chemistry , Soft Tissue Neoplasms/genetics , Soft Tissue Neoplasms/pathologyABSTRACT
OBJECTIVE: The purpose of this paper was to study the electrophysiological properties and the type of potassium channels on cell membrane in the stria vascularis pericytes in cochlear of guinea pig. METHODS: Firstly examined the expression of the stria vascularis pericytes by desmin, a marker of pericytes, in cochlear of guinea pig with immunofluorescent method. Using whole-cell patch clamp recording techniques to observe electrophysiological properties in the cochlear pericytes in stria vascularis of guinea pig. RESULTS: Pericytes were predominately distributed in the capillaries of cochlea.The average membrane capacitance, resistance, and potential of a single pericyte in stria vascularis were(5.9±0.3)pF, (2.2±0.3)GΩ and (-30.9±1.2)mV, respectively by using patch clamp technique. In addition, the average current density of cochlear pericyte was voltage-sensitive (Vh from 0 to + 60 mV, in 20 mV steps). The pericytes exhibited outward current and this property could be blocked by TEA (tetraethylammonium) 1 mmol/L, a large-conductance calcium-activated potassium channel(BKCa)inhibitor and 4-AP (4-aminopyridine) 1 mmol/L, a voltage-dependent K(+) channels(KV) channel blocker. TEA blocked the outward current from (296.2±35.9)pA to (163.7±16.8)pA and 4-AP blocked the outward current from (248.7±39.8)pA to (158.0±38.0)pA. CONCLUSION: These results suggest that pericytes in stria vascularis have BKCa and KV channels.
Subject(s)
Cochlea/physiology , Pericytes/physiology , Potassium Channels/analysis , Stria Vascularis/cytology , 4-Aminopyridine/pharmacology , Animals , Electrophysiological Phenomena/drug effects , Guinea Pigs , Patch-Clamp Techniques , Pericytes/chemistry , Pericytes/drug effects , Potassium Channel Blockers/pharmacology , Potassium Channels, Calcium-Activated/analysis , Stria Vascularis/chemistry , Tetraethylammonium/pharmacologyABSTRACT
Pericytes are modified smooth muscle cells that closely enwrap small blood vessels, regulating and supporting the microvasculature through direct endothelial contact. Pericytes demonstrate a distinct immunohistochemical profile, including expression of smooth muscle actin, CD146, platelet-derived growth factor receptor ß, and regulator of G-protein signaling 5. Previously, pericyte-related antigens have been observed to be present among a group of soft tissue tumors with a perivascular growth pattern, including glomus tumor, myopericytoma, and angioleiomyoma. Similarly, malignant tumor cells have been shown to have a pericyte-like immunoprofile when present in a perivascular location, seen in malignant melanoma, glioblastoma, and adenocarcinoma. Here, we examine well-differentiated liposarcoma specimens, which showed some element of perivascular areas with the appearance of smooth muscle (n = 7 tumors). Immunohistochemical staining was performed for pericyte antigens, including smooth muscle actin, CD146, platelet-derived growth factor receptor ß, and regulator of G-protein signaling 5. Results showed consistent pericytic marker expression among liposarcoma tumor cells within a perivascular distribution. MDM2 immunohistochemistry and fluorescence in situ hybridization for MDM2 revealed that these perivascular cells were of tumor origin (7/7 tumors), whereas double immunohistochemical detection for CD31/CD146 ruled out an endothelial cell contribution. These findings further support the concept of pericytic mimicry, already established in diverse malignancies, and its presence in well-differentiated liposarcoma. The extent to which pericytic mimicry has prognostic significance in liposarcoma is as yet unknown.
Subject(s)
Cell Differentiation , Lipoma/pathology , Liposarcoma/pathology , Molecular Mimicry , Pericytes/pathology , Actins/analysis , Adult , Aged , Biomarkers, Tumor/analysis , Biopsy , CD146 Antigen/analysis , Female , Humans , Immunohistochemistry , In Situ Hybridization, Fluorescence , Lipoma/chemistry , Lipoma/genetics , Liposarcoma/chemistry , Liposarcoma/genetics , Male , Middle Aged , Pericytes/chemistry , Phenotype , Proto-Oncogene Proteins c-mdm2/analysis , Proto-Oncogene Proteins c-mdm2/genetics , RGS Proteins/analysis , Receptor, Platelet-Derived Growth Factor beta/analysis , Retrospective StudiesABSTRACT
The aim of the present research was to trace CD34+ stromal fibroblastic cells (CD34+ SFCs) in the palatal connective tissue harvested for muco-gingival surgical procedures and in granulation tissues from periodontal pockets using immunohistochemical and transmission electron microscopy. Immunohistochemical analysis targeted the presence of three antigens: CD31, α-smooth muscle actin (α-SMA), and CD34. In the palate, CD31 staining revealed a colored inner ring of the vessels representing the endothelium, α-SMA+ was located in the medial layer of the vasculature, and CD34 was intensely expressed by endothelial cells and artery adventitial cells (considered to be CD34+ SFCs). Granulation tissue showed the same pattern for CD31+ and α-SMA, but a different staining pattern for CD34. Ultrastructural examination of the palatal tissue highlighted perivascular cells with fibroblast-like characteristics and pericytes in close spatial relationship to endothelial cells. The ultrastructural evaluation of granulation tissue sections confirmed the presence of neovasculature and the inflammatory nature of this tissue. The present study traced the presence of CD34+ SFCs and of pericytes in the palatal connective tissue thus highlighting once more its intrinsic regenerative capabilities. The clinical and systemic factors triggering mobilization and influencing the fate of local CD34+SCFs and other progenitors are issues to be further investigated.
Subject(s)
Antigens, CD34/analysis , Fibroblasts/physiology , Gingiva/physiology , Granulation Tissue/growth & development , Mouth Mucosa/physiology , Palate/physiology , Regeneration , Fibroblasts/chemistry , Gingiva/cytology , Humans , Immunohistochemistry , Microscopy, Electron, Transmission , Mouth Mucosa/cytology , Palate/cytology , Pericytes/chemistry , Pericytes/physiologyABSTRACT
Müller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling Müller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms. There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5 has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label Müller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.