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1.
Nat Commun ; 12(1): 1096, 2021 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-33597545

RESUMO

The thymus' key function in the immune system is to provide the necessary environment for the development of diverse and self-tolerant T lymphocytes. While recent evidence suggests that the thymic stroma is comprised of more functionally distinct subpopulations than previously appreciated, the extent of this cellular heterogeneity in the human thymus is not well understood. Here we use single-cell RNA sequencing to comprehensively profile the human thymic stroma across multiple stages of life. Mesenchyme, pericytes and endothelial cells are identified as potential key regulators of thymic epithelial cell differentiation and thymocyte migration. In-depth analyses of epithelial cells reveal the presence of ionocytes as a medullary population, while the expression of tissue-specific antigens is mapped to different subsets of epithelial cells. This work thus provides important insight on how the diversity of thymic cells is established, and how this heterogeneity contributes to the induction of immune tolerance in humans.


Assuntos
Células Epiteliais/metabolismo , Perfilação da Expressão Gênica/métodos , Heterogeneidade Genética , Análise de Célula Única/métodos , Timo/metabolismo , Adulto , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Pericitos/citologia , Pericitos/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo , Timócitos/citologia , Timócitos/metabolismo , Timo/citologia , Timo/embriologia
2.
Methods Mol Biol ; 2235: 27-35, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576968

RESUMO

Pericytes are mural cells closely associated with endothelial cells in capillaries and microvessels. They are precursors of mesenchymal stem/stromal cells that have historically been retrospectively characterized in culture. We established a protocol, described in this chapter, to characterize and isolate pericytes from multiple human organs by flow cytometry and fluorescence-activated cell sorting. This prospective purification of pericytes brings us a step forward in the development of strategies for their use in the clinic.


Assuntos
Citometria de Fluxo/métodos , Pericitos/citologia , Pericitos/transplante , Capilares/citologia , Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Células Cultivadas , Células Endoteliais/citologia , Humanos , Células-Tronco Mesenquimais/citologia , Microvasos/citologia , Pericitos/metabolismo , Fenótipo
3.
Methods Mol Biol ; 2235: 37-45, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576969

RESUMO

Pericytes are found in all vascularized organs and are defined anatomically as perivascular cells that closely surround endothelial cells in capillaries and microvessels and are embedded within the same basement membrane. They have been shown to have diverse physiological and pathological functions including regulation of blood pressure, and tissue regeneration and scarring. Fundamental to understanding the role these cells play in these diverse processes is the ability to accurately identify and localize them in vivo. To do this, we have developed multicolor immunohistochemistry protocols described in this chapter.


Assuntos
Imuno-Histoquímica/métodos , Pericitos/citologia , Pericitos/transplante , Capilares/citologia , Diferenciação Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/citologia , Humanos , Microvasos/citologia , Pericitos/metabolismo , Fenótipo
4.
Methods Mol Biol ; 2235: 47-59, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576970

RESUMO

We report the use of self-assembled peptide (F2/S) hydrogels and cellular metabolomics to identify a number of innate molecules that are integral to the metabolic processes which drive cellular differentiation of multipotent pericyte stem cells. The culture system relies solely on substrate mechanics to induce differentiation in the absence of traditional differentiation media and therefore is a non-invasive approach to assessing cellular behavior at the molecular level and identifying key metabolites in this process. This novel approach demonstrates that simple metabolites can provide an alternative means to direct stem cell differentiation and that biomaterials can be used to identify them simply and quickly.


Assuntos
Metabolômica/métodos , Pericitos/citologia , Pericitos/transplante , Animais , Materiais Biocompatíveis/metabolismo , Capilares/citologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Células Endoteliais/citologia , Humanos , Hidrogéis/química , Microvasos/citologia , Células-Tronco Multipotentes/efeitos dos fármacos , Peptídeos/química , Pericitos/metabolismo , Fenótipo
5.
Methods Mol Biol ; 2235: 119-125, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576973

RESUMO

Human pluripotent stem cells (hPSCs), either embryonic or induced, offer a plentiful platform for derivation of multiple cell types. Pericytes, generated from hPSCs, are multipotent precursors with vasculogenic features that exhibit high proliferation capability in long-term cultures. Administration of hPSC-pericytes into ischemic murine hind limb is associated with therapeutic angiogenesis and attenuation of muscle wasting. Here, we describe the protocol for derivation of large numbers of pericytes from spontaneously differentiating hPSC-embryoid bodies.


Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Pericitos/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Pericitos/citologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo
6.
Methods Mol Biol ; 2235: 61-87, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576971

RESUMO

The goal of lineage tracing is to understand body formation over time by discovering which cells are the progeny of a specific, identified, ancestral progenitor. Subsidiary questions include unequivocal identification of what they have become, how many descendants develop, whether they live or die, and where they are located in the tissue or body at the end of the window examined. A classical approach in experimental embryology, lineage tracing continues to be used in developmental biology and stem cell and cancer research, wherever cellular potential and behavior need to be studied in multiple dimensions, of which one is time. Each technical approach has its advantages and drawbacks. This chapter, with some previously unpublished data, will concentrate nonexclusively on the use of interspecies chimeras to explore the origins of perivascular (or mural) cells, of which those adjacent to the vascular endothelium are termed pericytes for this purpose. These studies laid the groundwork for our understanding that pericytes derive from progenitor mesenchymal pools of multiple origins in the vertebrate embryo, some of which persist into adulthood. The results obtained through xenografting, like in the methodology described here, complement those obtained through genetic lineage-tracing techniques within a given species.


Assuntos
Linhagem da Célula/fisiologia , Pericitos/citologia , Transplante Heterólogo/métodos , Animais , Ontologias Biológicas , Diferenciação Celular , Linhagem da Célula/genética , Embrião de Galinha , Quimera/genética , Quimera/fisiologia , Endotélio Vascular , Células Germinativas , Humanos , Pericitos/metabolismo , Células-Tronco
7.
Methods Mol Biol ; 2235: 89-117, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576972

RESUMO

The brain's high energy requirements drive the need for close coupling of local neuronal activity to blood supply. Capillaries have been shown to dilate before arterioles in response to sensory stimulation, pointing to a key role for microvascular pericytes in mediating cerebrovascular dynamics. However, many aspects of these cells' function remain unknown and even controversial, from their identification, to the mechanism and regulation of their contractility in physiology and disease. Investigating how pericytes regulate vascular diameter is therefore likely to be the subject of many future experiments. Here we provide protocols for three different techniques (ex vivo slice imaging, in vivo imaging, and immunohistochemistry) that are highly valuable for performing such experiments.


Assuntos
Circulação Cerebrovascular/fisiologia , Imagem Óptica/métodos , Pericitos/metabolismo , Animais , Arteríolas/fisiopatologia , Encéfalo/fisiopatologia , Isquemia Encefálica/fisiopatologia , Capilares/fisiopatologia , Humanos , Neurônios/metabolismo , Acoplamento Neurovascular/fisiologia , Pericitos/citologia
8.
Methods Mol Biol ; 2235: 127-137, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576974

RESUMO

Human pericytes are a perivascular cell population with mesenchymal stem cell properties, present in all vascularized tissues. Human pericytes have a distinct immunoprofile, which may be leveraged for purposes of cell purification. Adipose tissue is the most commonly used cell source for human pericyte derivation. Pericytes can be isolated by FACS (fluorescence-activated cell sorting), most commonly procured from liposuction aspirates. Pericytes have clonal multilineage differentiation potential, and their potential utility for bone regeneration has been described across multiple animal models. The following review will discuss in vivo methods for assessing the bone-forming potential of purified pericytes. Potential models include (1) mouse intramuscular implantation, (2) mouse calvarial defect implantation, and (3) rat spinal fusion models. In addition, the presented surgical protocols may be used for the in vivo analysis of other osteoprogenitor cell types.


Assuntos
Células da Medula Óssea/metabolismo , Pericitos/metabolismo , Engenharia Tecidual/métodos , Tecido Adiposo/citologia , Animais , Células da Medula Óssea/citologia , Regeneração Óssea/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular , Separação Celular/métodos , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Osteogênese/fisiologia , Pericitos/citologia , Ratos
9.
Methods Mol Biol ; 2235: 155-167, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576976

RESUMO

Mesoangioblasts (MABs) are vessel-associated stem cells that express pericyte markers and are originally isolated from the embryonic dorsal aorta. From postnatal small vessels of skeletal muscle and heart, it is possible to isolate cells with similar characteristics to embryonic MABs. Adult MABs have the capacity to self-renew and to differentiate into cell types of mesodermal lineages upon proper culture conditions. To date, the origin of MABs and the relationship with other muscle stem cells are still debated. Recently, in a phase I-II clinical trial, intra-arterial HLA-matched MABs were proved to be relatively safe. Novel information on MAB pure populations is desirable, and implementation of their therapeutic potential is mandatory to approach efficacy in MAB-based treatments. This chapter provides an overview of the current techniques for isolation and characterization of rodent, canine, human, and equine adult MABs.


Assuntos
Diferenciação Celular/fisiologia , Separação Celular/métodos , Pericitos/citologia , Animais , Aorta/citologia , Cães , Cavalos , Humanos , Mesoderma/citologia , Camundongos , Desenvolvimento Muscular , Músculo Esquelético/citologia , Mioblastos/citologia , Pericitos/fisiologia , Ratos , Células-Tronco/citologia
10.
Methods Mol Biol ; 2206: 57-66, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32754811

RESUMO

The construction of vascular networks is essential for developing functional organ/tissue constructs in terms of oxygen and nutrient supply. Although recent advances in microfluidic techniques have allowed for the construction of microvascular networks using microfluidic devices, their structures cannot be maintained for extended periods of time due to a lack of perivascular cells. To construct long-lasting microvascular networks, it is important that perivascular cells are present to provide structural support to vessels, because in vivo microvessels are covered by perivascular cells and stabilized. Here, we describe a microfluidic cell culture platform for the construction of microvascular networks with supportive perivascular cells. Our results showed that microvascular networks covered by pericyte-like perivascular cells formed in a microfluidic device and their structures were maintained for at least 3 weeks in vitro.


Assuntos
Células Endoteliais da Veia Umbilical Humana/citologia , Células-Tronco Mesenquimais/citologia , Microfluídica/métodos , Células Cultivadas , Técnicas de Cocultura/métodos , Humanos , Microvasos/citologia , Pericitos/citologia
11.
Methods Mol Biol ; 2206: 143-150, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32754816

RESUMO

Pericytes are integral part of neurovascular unit and play a role in the maintenance of blood-brain barrier integrity, angiogenesis, and cerebral blood flow regulation. Despite their important functional roles, a univocal phenotypic identification is still emerging also for the lack of a "pan-pericyte" marker. In the present study, we describe in detail the method for performing fluorescence immunohistochemistry on thick free-floating sections from human fetal brain in high resolution laser confocal microscopy. This method enables to obtain three-dimensional images of pericytes and provides insights about their distribution and localization in the microvessels of human developing brain.


Assuntos
Encéfalo/irrigação sanguínea , Microscopia Confocal/métodos , Microvasos/citologia , Pericitos/citologia , Barreira Hematoencefálica/citologia , Circulação Cerebrovascular/fisiologia , Humanos , Imageamento Tridimensional/métodos , Imuno-Histoquímica/métodos , Neovascularização Fisiológica/fisiologia
12.
Nature ; 585(7823): 91-95, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32788726

RESUMO

Signalling between cells of the neurovascular unit, or neurovascular coupling, is essential to match local blood flow with neuronal activity. Pericytes interact with endothelial cells and extend processes that wrap capillaries, covering up to 90% of their surface area1,2. Pericytes are candidates to regulate microcirculatory blood flow because they are strategically positioned along capillaries, contain contractile proteins and respond rapidly to neuronal stimulation3,4, but whether they synchronize microvascular dynamics and neurovascular coupling within a capillary network was unknown. Here we identify nanotube-like processes that connect two bona fide pericytes on separate capillary systems, forming a functional network in the mouse retina, which we named interpericyte tunnelling nanotubes (IP-TNTs). We provide evidence that these (i) have an open-ended proximal side and a closed-ended terminal (end-foot) that connects with distal pericyte processes via gap junctions, (ii) carry organelles including mitochondria, which can travel along these processes, and (iii) serve as a conduit for intercellular Ca2+ waves, thus mediating communication between pericytes. Using two-photon microscope live imaging, we demonstrate that retinal pericytes rely on IP-TNTs to control local neurovascular coupling and coordinate light-evoked responses between adjacent capillaries. IP-TNT damage following ablation or ischaemia disrupts intercellular Ca2+ waves, impairing blood flow regulation and neurovascular coupling. Notably, pharmacological blockade of Ca2+ influx preserves IP-TNTs, rescues light-evoked capillary responses and restores blood flow after reperfusion. Our study thus defines IP-TNTs and characterizes their critical role in regulating neurovascular coupling in the living retina under both physiological and pathological conditions.


Assuntos
Nanotubos , Acoplamento Neurovascular , Pericitos/metabolismo , Animais , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Cálcio/metabolismo , Sinalização do Cálcio , Capilares/fisiopatologia , Capilares/efeitos da radiação , Comunicação Celular , Feminino , Junções Comunicantes/metabolismo , Hemodinâmica , Masculino , Camundongos , Mitocôndrias/metabolismo , Acoplamento Neurovascular/fisiologia , Pericitos/citologia , Pericitos/patologia , Retina/citologia , Retina/patologia
13.
Nat Commun ; 11(1): 3953, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769974

RESUMO

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.


Assuntos
Diferenciação Celular , Fibroblastos/fisiologia , Células-Tronco Mesenquimais/fisiologia , Miócitos de Músculo Liso/fisiologia , Pericitos/fisiologia , Animais , Separação Celular , Vasos Coronários/citologia , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Citometria de Fluxo , Intestinos/irrigação sanguínea , Intestinos/citologia , Masculino , Camundongos , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/citologia , Músculo Liso Vascular/citologia , Miocárdio/citologia , Miócitos de Músculo Liso/citologia , Pericitos/citologia , RNA-Seq , Análise de Célula Única , Bexiga Urinária/irrigação sanguínea , Bexiga Urinária/citologia
14.
J Vis Exp ; (159)2020 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-32538913

RESUMO

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte function is not fully known. Pericytes have been suggested to be involved in capillary development, regulation of endothelial barrier tightness and trancytosis activity, regulation of capillary tone and to play crucial roles in certain brain pathologies. Pericytes are challenging to investigate in the intact brain due to the difficulties in visualizing processes in the brain parenchyma, as well as the close proximity to the other cells of the NVU. The present protocol describes a method for isolation and culture of primary bovine brain capillary pericytes and their following usage in calcium imaging studies, where effects of agonists involved in brain signaling and pathologies can be investigated. Cortical capillary fragments are allowed to attach to the bottom of culture flasks and, after 6 days, endothelial cells and pericytes have grown out from the capillary fragments. The endothelial cells are removed by gentle trypsinization and pericytes are cultured for 5 additional days before passaging. Isolated pericytes are seeded in 96-well culture plates and loaded with the calcium indicator dye (Fura-2 acetoxymethyl (AM)) to allow for measurements of intracellular calcium levels in a plate reader setup. Alternatively, pericytes are seeded on coverslips and mounted in cell chambers. Following loading with the calcium indicator (Cal-520 AM), calcium live-imaging can be performed using confocal microscopy at an excitation wavelength of 488 nm and emission wavelength of 510-520 nm. The method described here has been used to obtain the first intracellular calcium measurements from primary brain capillary pericytes, demonstrating that pericytes are stimulated via ATP and are able to contract in vitro.


Assuntos
Cálcio/análise , Capilares/citologia , Técnicas de Cultura de Células , Pericitos/citologia , Animais , Encéfalo , Bovinos , Separação Celular , Citosol/química , Microscopia Confocal
15.
Cell Physiol Biochem ; 54(2): 271-286, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32233339

RESUMO

BACKGROUND/AIMS: Pericytes (PCs) are multipotent vascular precursors that play a critical physiological role in the development and maintenance of blood vessel integrity. In this study, we aim to characterize PCs isolated from human abdominal adipose tissue and develop an integration-free induced pluripotent stem cells (iPSCs) using episomal vectors. METHODS: The ultrastructure of adipose tissue-derived PCs was determined using scanning and transmission electron microscopy. The expression of mesenchymal stem cells (MSCs) and pericyte markers were examined using flow cytometry and PCR analysis. PCs were induced to adipogenic, osteogenic and myogenic lineages, and their angiogenic potential was determined using tube formation assay. We further established pericyte reprogramming protocol using episomal vectors. RESULTS: Our data showed that human adipose tissue-derived PCs uniformly expressed MSCs, CD105 and CD73, and PCs markers, desmin, and alpha smooth muscle actin (α-SMA), while lacked the expression of HLA-DR and the hematopoietic markers CD34, CD11b and CD45. Ultrastructure analysis showed typical internal structure for the PCs with a characteristic prominent eccentric nuclei and cytoplasmic invaginations forming a caveolar system. Functional analysis showed efficient differentiation into adipocytes, osteocytes, and myocyte-like cells. Adipose tissue-derived PCs showed angiogenic potential using tube-forming assay. To determine further application of these cells for personalized therapy, we reprogrammed PCs into induced pluripotent stem cells (iPSCs) using episomal vectors. Reprogrammed cells gradually lost their fusiform shape, acquired the epithelial cell morphology and formed colonies. Furthermore, reprogrammed cells successfully expressed the pluripotency markers OCT4, Nanog, SSEA-4, and ß-catenin, an early reprogramming marker. CONCLUSION: The accessibility and abundance of human fat supports the application of adipose derived PCs as a novel and promising source of cell therapy and regenerative medicine.


Assuntos
Tecido Adiposo/citologia , Técnicas de Reprogramação Celular/métodos , Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Pericitos/citologia , 5'-Nucleotidase/metabolismo , Actinas/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Tecido Adiposo/ultraestrutura , Linhagem da Célula , Células Cultivadas , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Desmina/metabolismo , Endoglina/metabolismo , Citometria de Fluxo , Proteínas Ligadas por GPI/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Células Musculares/citologia , Células Musculares/metabolismo , Desenvolvimento Muscular/genética , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Osteócitos/citologia , Osteócitos/metabolismo , Osteogênese/genética , Pericitos/metabolismo , Pericitos/ultraestrutura , Antígenos Embrionários Estágio-Específicos/metabolismo , beta Catenina/metabolismo
16.
Bull Exp Biol Med ; 168(3): 334-340, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31940128

RESUMO

The changes in endothelial progenitor cells and progenitor cells of angiogenesis, pericytes and smooth muscle cells, were studied in female C57BL/6 mice with a combination of metabolic impairments induced by injections of sodium glutamate and lung emphysema modeled by the administration of cigarette smoke extract. It was observed that sodium glutamate significantly enhances pathological changes in the lungs (inflammation and lung emphysema) induced by the administration of cigarette smoke extract. Recruiting of endothelial progenitor cells (CD45-CD31+CD34+ and CD31+CD34+CD146-) and progenitor cells of angiogenesis (CD45-CD117+CD309+) was registered in the injured lungs. Angiogenesis impairment induced by combined exposure is related to altered migration of pericytes (CD31-CD34-CD146+) and smooth muscle cells (CD31-CD34+CD146+) in emphysema-like enlarged lung tissue.


Assuntos
Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Pericitos/citologia , Pericitos/metabolismo , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/patologia , Animais , Antígenos CD34/metabolismo , Antígeno CD146/metabolismo , Fumar Cigarros/efeitos adversos , Células Progenitoras Endoteliais/citologia , Células Progenitoras Endoteliais/efeitos dos fármacos , Células Progenitoras Endoteliais/metabolismo , Feminino , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/efeitos dos fármacos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo
17.
Int J Mol Sci ; 20(24)2019 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-31861092

RESUMO

Pericytes are branched cells located in the wall of capillary blood vessels that are found throughout the body, embedded within the microvascular basement membrane and wrapping endothelial cells, with which they establish a strong physical contact. Pericytes regulate angiogenesis, vessel stabilization, and contribute to the formation of both the blood-brain and blood-retina barriers by Angiopoietin-1/Tie-2, platelet derived growth factor (PDGF) and transforming growth factor (TGF) signaling pathways, regulating pericyte-endothelial cell communication. Human pericytes that have been cultured for a long period give rise to multilineage progenitor cells and exhibit mesenchymal stem cell (MSC) features. We focused our attention on the roles of pericytes in brain and ocular diseases. In particular, pericyte involvement in brain ischemia, brain tumors, diabetic retinopathy, and uveal melanoma is described. Several molecules, such as adenosine and nitric oxide, are responsible for pericyte shrinkage during ischemia-reperfusion. Anti-inflammatory molecules, such as IL-10, TGFß, and MHC-II, which are increased in glioblastoma-activated pericytes, are responsible for tumor growth. As regards the eye, pericytes play a role not only in ocular vessel stabilization, but also as a stem cell niche that contributes to regenerative processes in diabetic retinopathy. Moreover, pericytes participate in melanoma cell extravasation and the genetic ablation of the PDGF receptor reduces the number of pericytes and aberrant tumor microvessel formation with important implications for therapy efficacy. Thanks to their MSC features, pericytes could be considered excellent candidates to promote nervous tissue repair and for regenerative medicine.


Assuntos
Encéfalo/fisiologia , Microvasos/fisiologia , Pericitos/fisiologia , Regeneração/fisiologia , Retina/fisiologia , Vasos Retinianos/fisiologia , Animais , Barreira Hematoencefálica/fisiologia , Barreira Hematorretiniana/fisiologia , Encéfalo/irrigação sanguínea , Humanos , Microvasos/citologia , Pericitos/citologia
18.
Metabolism ; 101: 153998, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31666193

RESUMO

BACKGROUND: The incidence of growth hormone deficiency (GHD) in adamantinomatous craniopharyngioma (aCP) is significantly higher than in other sellar region tumors, but the possible mechanism is still elusive. A high level of inflammatory responses is another feature of aCP. We investigated the internal connection between interleukin-1α (IL-1α) and GHD, while focusing on its biological activities in pituitary fibrosis. MATERIALS AND METHODS: To diagnosis of GHD, the Body Mass Index (BMI), Insulin Like Growth Factor-1(IGF-1) and peak growth hormone (GH) values after insulin stimulation test of 15 aCP patients were recorded. Histological staining was performed on the aCP samples. Levels of 9 proinflammatory cytokines in tumor tissue and cell supernatant were detected using Millipore bead arrays. The effect of IL-1α on GH secretion was evaluated in vivo and in vitro. Western blot, qRT-PCR and cell functional assays were used to explore the potential mechanism through which IL-1α acts on GH secretion. The stereotactic ALZET osmotic pump technique was used to simulate aCP secretion of proinflammatory cytokines in rats. Recombinant IL-1α (rrIL-1α) and conditioned media (CM) prepared from the supernatant of aCP cells was infused directly into the intra-sellar at a rate of 1 µl/h over 28 days, and then the effects of IL-1α treatment on pathological changes of pituitary gland and GH secretion were measured. To further confirm whether IL-1α affects GH secretion through IL-1R1, an IL-1R1 blocker (IL-1R1a, 10 mg/kg body weight, once daily) was administered subcutaneously from the first day until day 28. RESULTS: There was a significant positive correlation between pituitary fibrosis and GHD (rS = 0.756, P = 0.001). A number of cytokines, in particular IL-1α, interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1), were elevated in tumor tissue and cell supernatant. Only IL-1α showed a significant difference between the GHD group and the No-GHD group (P < 0.001, F = 6.251 in tumor tissue; P = 0.003, F = 1.529 in cell supernatant). IL-1α significantly reduced GH secretion in coculture of GH3 and pericytes. The activation of pericytes induced by IL-1α was mediated by the IL-1R1 signaling pathway. In vivo, IL-1α induces pituitary fibrosis, further leading to a decreased level of GH. This pathological change was antagonized by IL-1R1a. CONCLUSION: This study found that the cross talk between aCP cells and stroma cells in the pituitary, i.e. pericytes, is an essential factor in the formation of GHD, and we propose that neutralization of IL-1α signaling might be a potential therapy for GHD in aCP.


Assuntos
Comunicação Celular , Craniofaringioma/patologia , Hormônio do Crescimento Humano/deficiência , Interleucina-1alfa/farmacologia , Pericitos/efeitos dos fármacos , Adulto , Animais , Craniofaringioma/etiologia , Citocinas/metabolismo , Feminino , Fibrose , Hormônio do Crescimento Humano/efeitos dos fármacos , Hormônio do Crescimento Humano/metabolismo , Humanos , Inflamação , Masculino , Pericitos/citologia , Hipófise/metabolismo , Hipófise/patologia , Ratos
19.
Sci Rep ; 9(1): 15586, 2019 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-31666598

RESUMO

Microvascular homeostasis is strictly regulated, requiring close interaction between endothelial cells and pericytes. Here, we aimed to improve our understanding of how microvascular crosstalk affects pericytes. Human-derived pericytes, cultured in absence, or presence of human endothelial cells, were studied by RNA sequencing. Compared with mono-cultured pericytes, a total of 6704 genes were differentially expressed in co-cultured pericytes. Direct endothelial contact induced transcriptome profiles associated with pericyte maturation, suppression of extracellular matrix production, proliferation, and morphological adaptation. In vitro studies confirmed enhanced pericyte proliferation mediated by endothelial-derived PDGFB and pericyte-derived HB-EGF and FGF2. Endothelial-induced PLXNA2 and ACTR3 upregulation also triggered pericyte morphological adaptation. Pathway analysis predicted a key role for TGFß signaling in endothelial-induced pericyte differentiation, whereas the effect of signaling via gap- and adherens junctions was limited. We demonstrate that endothelial cells have a major impact on the transcriptional profile of pericytes, regulating endothelial-induced maturation, proliferation, and suppression of ECM production.


Assuntos
Diferenciação Celular/genética , Células Endoteliais/citologia , Perfilação da Expressão Gênica , Microvasos/citologia , Pericitos/citologia , Junções Aderentes/metabolismo , Matriz Extracelular/metabolismo , Junções Comunicantes/metabolismo , Humanos , Transdução de Sinais/genética
20.
Sci Rep ; 9(1): 15037, 2019 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-31636275

RESUMO

Exogenous androgen replacement is used to treat symptoms associated with low testosterone in males. However, adverse cardiovascular risk and negative fertility impacts impel development of alternative approaches to restore/maintain Leydig cell (LC) androgen production. Stem Leydig cell (SLC) transplantation shows promise in this regard however, practicality of SLC isolation/transplantation impede clinical translation. Multipotent human adipose-derived perivascular stem cells (hAd-PSCs) represent an attractive extragonadal stem cell source for regenerative therapies in the testis but their therapeutic potential in this context is unexplored. We asked whether hAd-PSCs could be converted into Leydig-like cells and determined their capacity to promote regeneration in LC-ablated rat testes. Exposure of hAd-PSCs to differentiation-inducing factors in vitro upregulated steroidogenic genes but did not fully induce LC differentiation. In vivo, no difference in LC-regeneration was noted between Sham and hAd-PSC-transplanted rats. Interestingly, Cyp17a1 expression increased in hAd-PSC-transplanted testes compared to intact vehicle controls and the luteinising hormone/testosterone ratio returned to Vehicle control levels which was not the case in EDS + Sham animals. Notably, hAd-PSCs were undetectable one-month after transplantation suggesting this effect is likely mediated via paracrine mechanisms during the initial stages of regeneration; either directly by interacting with regenerating LCs, or through indirect interactions with trophic macrophages.


Assuntos
Tecido Adiposo/citologia , Linhagem da Célula , Células Intersticiais do Testículo/citologia , Pericitos/citologia , Regeneração , Esteroides/metabolismo , Animais , Contagem de Células , Células Cultivadas , Regulação da Expressão Gênica , Hormônios/sangue , Humanos , Masculino , Tamanho do Órgão , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Endogâmicos WKY , Testículo/anatomia & histologia , Testículo/citologia
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