RESUMEN
The retina transforms light into electrical signals, which are sent to the brain via the optic nerve to form our visual perception. This complex signal processing is performed by the retinal neuron and requires a significant amount of energy. Since neurons are unable to store energy, they must obtain glucose and oxygen from the bloodstream to produce energy to match metabolic needs. This process is called neurovascular coupling (NVC), and it is based on a precise mechanism that is not totally understood. The discovery of fine tubular processes termed tunnelling nanotubes (TNTs) set a new type of cell-to-cell communication. TNTs are extensions of the cellular membrane that allow the transfer of material between connected cells. Recently, they have been reported in the brain and retina of living mice, where they connect pericytes, which are vascular mural cells that regulate vessel diameter. Accordingly, these TNTs were termed interpericyte tunnelling nanotubes (IPTNTs), which showed a vital role in blood delivery and NVC. In this chapter, we review the involvement of TNTs in NVC and discuss their implications in retinal neurodegeneration.
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Comunicación Celular , Retina , Animales , Humanos , Retina/fisiología , Comunicación Celular/fisiología , Pericitos/fisiología , Nanotubos , Ratones , Acoplamiento Neurovascular/fisiología , Vasos Retinianos/fisiología , Estructuras de la Membrana CelularRESUMEN
Diabetic retinopathy is a common yet severe complication of diabetes mellitus and is the leading cause of blindness in middle-aged adults. After years of poorly managed hyperglycemia, complications begin as non-proliferative diabetic retinopathy but can then progress into the proliferative stage marked by neovascularization of the retina. Multiple pathologic mechanisms caused by chronic hyperglycemia damage the retinal vasculature leading to pericyte drop out and the progression of the disease. This review outlines the major pathways of pathogenesis in diabetic retinopathy, highlighting the protective role pericytes play in preserving the blood-retinal barrier. Given the loss of this cell line is a defining feature of the disease, ways in which to prevent pericyte dropout within retinal vasculature is discussed, targeting various pathogenesis pathways of diabetic retinopathy.
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Barrera Hematorretinal , Retinopatía Diabética , Pericitos , Retinopatía Diabética/patología , Retinopatía Diabética/metabolismo , Pericitos/metabolismo , Pericitos/patología , Humanos , Barrera Hematorretinal/metabolismo , Animales , Vasos Retinianos/patología , Vasos Retinianos/metabolismoRESUMEN
BACKGROUND: Cell-based strategies are being explored as a therapeutic option for muscular dystrophies, using a variety of cell types from different origin and with different characteristics. Primary pericytes are multifunctional cells found in the capillary bed that exhibit stem cell-like and myogenic regenerative properties. This unique combination allows them to be applied systemically, presenting a promising opportunity for body-wide muscle regeneration. We previously reported the successful isolation of ALP+ pericytes from skeletal muscle of patients with myotonic dystrophy type 1 (DM1). These pericytes maintained normal growth parameters and myogenic characteristics in vitro despite the presence of nuclear (CUG)n RNA foci, the cellular hallmark of DM1. Here, we examined the behaviour of DM1 pericytes during myogenic differentiation. METHODS: DMPK (CTG)n repeat lengths in patient pericytes were assessed using small pool PCR, to be able to relate variation in myogenic properties and disease hallmarks to repeat expansion. Pericytes from unaffected controls and DM1 patients were cultured under differentiating conditions in vitro. In addition, the pericytes were grown in co-cultures with myoblasts to examine their regenerative capacity by forming hybrid myotubes. Finally, the effect of pericyte fusion on DM1 disease hallmarks was investigated. RESULTS: Small pool PCR analysis revealed the presence of somatic mosaicism in pericyte cell pools. Upon differentiation to myotubes, DMPK expression was upregulated, leading to an increase in nuclear foci sequestering MBNL1 protein. Remarkably, despite the manifestation of these disease biomarkers, patient-derived pericytes demonstrated myogenic potential in co-culture experiments comparable to unaffected pericytes and myoblasts. However, only the unaffected pericytes improved the disease hallmarks in hybrid myotubes. From 20% onwards, the fraction of unaffected nuclei in myotubes positively correlated with a reduction of the number of RNA foci and an increase in the amount of free MBNL1. CONCLUSIONS: Fusion of only a limited number of unaffected myogenic precursors to DM1 myotubes already ameliorates cellular disease hallmarks, offering promise for the development of cell transplantation strategies to lower disease burden.
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Diferenciación Celular , Fibras Musculares Esqueléticas , Distrofia Miotónica , Proteína Quinasa de Distrofia Miotónica , Pericitos , Humanos , Distrofia Miotónica/metabolismo , Distrofia Miotónica/genética , Distrofia Miotónica/terapia , Distrofia Miotónica/patología , Fibras Musculares Esqueléticas/metabolismo , Pericitos/metabolismo , Proteína Quinasa de Distrofia Miotónica/genética , Proteína Quinasa de Distrofia Miotónica/metabolismo , Mioblastos/metabolismo , Mioblastos/citología , Desarrollo de Músculos , Células Cultivadas , Masculino , Adulto , Femenino , Técnicas de Cocultivo , Persona de Mediana Edad , Fusión CelularRESUMEN
The pericyte coverage of microvessels is altered in metabolic diseases, but the mechanisms regulating pericyte-endothelial cell communication remain unclear. This study investigated the formation and function of pericyte tunneling nanotubes (TNTs) and their impact on endothelial cell metabolism. TNTs were analyzed in vitro in retinas and co-cultures of pericytes and endothelial cells. Using mass spectrometry, the influence of pericytes on endothelial cell metabolism was examined. TNTs were present in the murine retina, and although diabetes was associated with a decrease in pericyte coverage, TNTs were longer. In vitro, pericytes formed TNTs in the presence of PDGF, extending toward endothelial cells and facilitating mitochondrial transport from pericytes to endothelial cells. In experiments with mitochondria-depleted endothelial cells displaying defective TCA cycle metabolism, pericytes restored the mitochondrial network and metabolism. 19,20-Dihydroxydocosapentaenoic acid (19,20-DHDP), known to disrupt pericyte-endothelial cell junctions, prevented TNT formation and metabolic rescue in mitochondria-depleted endothelial cells. 19,20-DHDP also caused significant changes in the protein composition of pericyte-endothelial cell junctions and involved pathways related to phosphatidylinositol 3-kinase, PDGF receptor, and RhoA signaling. Pericyte TNTs contact endothelial cells and support mitochondrial transfer, influencing metabolism. This protective mechanism is disrupted by 19,20-DHDP, a fatty acid mediator linked to diabetic retinopathy.
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Comunicación Celular , Ácidos Docosahexaenoicos , Células Endoteliales , Pericitos , Pericitos/metabolismo , Animales , Células Endoteliales/metabolismo , Ácidos Docosahexaenoicos/farmacología , Ácidos Docosahexaenoicos/metabolismo , Ratones , Mitocondrias/metabolismo , Humanos , Ratones Endogámicos C57BL , Técnicas de Cocultivo , Retina/metabolismo , Retina/citología , Nanotubos/química , Estructuras de la Membrana CelularRESUMEN
Alzheimer's disease (AD) is the most common cause of dementia, characterized by memory loss, cognitive decline, personality changes, and various neurological symptoms. The role of blood-brain barrier (BBB) injury, extracellular matrix (ECM) abnormalities, and oligodendrocytes (ODCs) dysfunction in AD has gained increasing attention, yet the detailed pathogenesis remains elusive. This study integrates single-cell sequencing of AD patients' cerebrovascular system with a genome-wide association analysis. It aims to elucidate the associations and potential mechanisms behind pericytes injury, ECM disorder, and ODCs dysfunction in AD pathogenesis. Finally, we identified that abnormalities in the pericyte PI3K-AKT-FOXO signaling pathway may be involved in the pathogenic process of AD. This comprehensive approach sheds new light on the complex etiology of AD and opens avenues for advanced research into its pathogenesis and therapeutic strategies.
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Enfermedad de Alzheimer , Barrera Hematoencefálica , Estudio de Asociación del Genoma Completo , Pericitos , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/etiología , Humanos , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Pericitos/patología , Pericitos/metabolismo , Transducción de Señal , Oligodendroglía/metabolismo , Oligodendroglía/patología , Matriz Extracelular/metabolismo , Microvasos/patología , Microvasos/metabolismo , Análisis de la Célula Individual , Femenino , Masculino , Fosfatidilinositol 3-Quinasas/metabolismoRESUMEN
Pericytes are versatile cells integral to the blood vessel walls of the microcirculation, where they exhibit specific stem cell traits. They are essential in modulating blood flow, ensuring vascular permeability, and maintaining homeostasis and are involved in the tissue repair process. The human endometrium is a unique and complex tissue that serves as a natural scar-free healing model with its cyclical repair and regeneration process every month. The regulation of pericytes has gained increasing attention due to their involvement in various physiological and pathological processes. However, endometrial pericytes are less well studied compared to the pericytes in other organs. This review aims to provide a comprehensive overview of endometrial pericytes, with a focus on elucidating their physiological function and potential implications in uterine disorders.
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Endometrio , Pericitos , Enfermedades Uterinas , Humanos , Pericitos/metabolismo , Endometrio/metabolismo , Endometrio/fisiología , Femenino , Enfermedades Uterinas/patología , Enfermedades Uterinas/fisiopatologíaRESUMEN
BACKGROUND: TGF (transforming growth factor)-ß pathway is central to blood-brain barrier development as it regulates cross talk between pericytes and endothelial cells. Murine embryos lacking TGFß receptor Alk5 (activin receptor-like kinase 5) in brain pericytes (mutants) display endothelial cell hyperproliferation, abnormal vessel morphology, and gross germinal matrix hemorrhage-intraventricular hemorrhage (GMH-IVH), leading to perinatal lethality. Mechanisms underlying how ALK5 signaling in pericytes noncell autonomously regulates endothelial cell behavior remain elusive. METHODS: Transcriptomic analysis of human brain pericytes with ALK5 silencing identified differential gene expression. Brain vascular cells isolated from mutant embryonic mice with GMH-IVH and preterm human IVH brain samples were utilized for target validation. Finally, pharmacological and genetic inhibition was used to study the therapeutic effects on GMH-IVH pathology. RESULTS: Herein, we establish that the TGFß/ALK5 pathway robustly represses ANGPT2 (angiopoietin-2) in pericytes via epigenetic remodeling. TGFß-driven SMAD (suppressor of mothers against decapentaplegic) 3/4 associates with TGIF1 (TGFß-induced factor homeobox 1) and HDAC (histone deacetylase) 5 to form a corepressor complex at the Angpt2 promoter, resulting in promoter deacetylation and gene repression. Moreover, murine and human germinal matrix vessels display increased ANGPT2 expression during GMH-IVH. Isolation of vascular cells from murine germinal matrix identifies pericytes as a cellular source of excessive ANGPT2. In addition, mutant endothelial cells exhibit higher phosphorylated TIE2 (tyrosine protein kinase receptor). Pharmacological or genetic inhibition of ANGPT2 in mutants improves germinal matrix vessel morphology and attenuates GMH pathogenesis. Importantly, genetic ablation of Angpt2 in mutant pericytes prevents perinatal lethality, prolonging survival. CONCLUSIONS: This study demonstrates that TGFß-mediated ANGPT2 repression in pericytes is critical for maintaining blood-brain barrier integrity and identifies pericyte-derived ANGPT2 as an important pathological target for GMH-IVH.
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Angiopoyetina 2 , Pericitos , Factor de Crecimiento Transformador beta , Pericitos/metabolismo , Pericitos/patología , Animales , Ratones , Humanos , Angiopoyetina 2/metabolismo , Angiopoyetina 2/genética , Factor de Crecimiento Transformador beta/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta/genética , Hemorragia Cerebral/metabolismo , Hemorragia Cerebral/patología , Hemorragia Cerebral/genética , Transducción de Señal/fisiología , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/genética , Células Endoteliales/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Diabetes mediates endothelial dysfunction and increases the risk of Alzheimer's disease and related dementias. Diabetes also dysregulates the ET system. ET-1-mediated constriction of brain microvascular pericytes (BMVPCs) has been shown to contribute to brain hypoperfusion. Cellular senescence, a process that arrests the proliferation of harmful cells and instigates phenotypical changes and proinflammatory responses in endothelial cells that impact their survival and function. Thus, we hypothesized that ET-1 mediates BMVPC senescence and phenotypical changes in diabetes-like conditions. Human BMVPCs were incubated in diabetes-like conditions with or without ET-1 (1 µmol/L) for 3 and 7 days. Hydrogen peroxide (100 µmol/L H2O2) was used as a positive control for senescence and to mimic ischemic conditions. Cells were stained for senescence-associated ß-galactosidase or processed for immunoblotting and quantitative real-time PCR analyses. In additional experiments, cells were stimulated with ET-1 in the presence or absence of ETA receptor antagonist BQ-123 (20 µmol/L) or ETB receptor antagonist BQ-788 (20 µmol/L). ET-1 stimulation increased ß-galactosidase accumulation which was prevented by BQ-123. ET-1 also increased traditional senescence marker p16 protein and pericyte-specific senescence markers, TGFB1i1, PP1CA, and IGFBP7. Furthermore, ET-1 stimulated contractile protein α-SMA and microglial marker ostepontin in high glucose suggesting a shift toward an ensheathing or microglia-like phenotype. In conclusion, ET-1 triggers senescence, alters ETA and ETB receptors, and causes phenotypical changes in BMVPCs under diabetes-like conditions. These in vitro findings need to be further studied in vivo to establish the role of ETA receptors in the progression of pericyte senescence and phenotypical changes in VCID.
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Encéfalo , Senescencia Celular , Endotelina-1 , Pericitos , Receptor de Endotelina A , Humanos , Encéfalo/metabolismo , Encéfalo/patología , Células Cultivadas , Senescencia Celular/efectos de los fármacos , Diabetes Mellitus/metabolismo , Endotelina-1/metabolismo , Endotelina-1/farmacología , Pericitos/metabolismo , Pericitos/efectos de los fármacos , Pericitos/patología , Fenotipo , Receptor de Endotelina A/metabolismo , Receptor de Endotelina A/genéticaRESUMEN
Traumatic brain injury impairs brain function through various mechanisms. Recent studies have shown that alterations in pericytes in various diseases affect neurovascular function, but the effects of TBI on hippocampal pericytes remain unclear. Here, we investigated the effects of RAGE activation on pericytes after TBI using male C57BL/6 J mice. Hippocampal samples were collected at different time points within 7 days after TBI, the expression of PDGFR-ß, NG2 and the HMGB1-S100B/RAGE signaling pathway was assessed by Western blotting, and the integrity of the hippocampal BBB at different time points was measured by immunofluorescence. RAGE-associated BBB damage in hippocampal pericytes occurred early after cortical impact. By culturing primary mouse brain microvascular pericytes, we determined the different effects of HMGB1-S100B on pericyte RAGE. To investigate whether RAGE blockade could protect neurological function after TBI, we reproduced the process of CCI by administering FPS-ZM1 to RAGE-/- mice. TEM images and BBB damage-related assays showed that inhibition of RAGE resulted in a significant improvement in the number of hippocampal vascular basement membranes and tight junctions and a reduction in perivascular oedema compared with those in the untreated group. In contrast, mouse behavioural testing and doublecortin staining indicated that targeting the HMGB1-S100B/RAGE axis after CCI could protect neurological function by reducing pericyte-associated BBB damage. In conclusion, the present study provides experimental evidence for the strong correlation between the pericyte HMGB1-S100B/RAGE axis and NVU damage in the hippocampus at the early stage of TBI and further demonstrates that pericyte RAGE serves as an important target for the protection of neurological function after TBI.
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Barrera Hematoencefálica , Lesiones Traumáticas del Encéfalo , Hipocampo , Ratones Endogámicos C57BL , Pericitos , Receptor para Productos Finales de Glicación Avanzada , Animales , Pericitos/metabolismo , Pericitos/patología , Lesiones Traumáticas del Encéfalo/patología , Lesiones Traumáticas del Encéfalo/metabolismo , Ratones , Masculino , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Hipocampo/metabolismo , Hipocampo/patología , Barrera Hematoencefálica/patología , Barrera Hematoencefálica/metabolismo , Ratones Noqueados , Proteína HMGB1/metabolismo , Subunidad beta de la Proteína de Unión al Calcio S100/metabolismo , BenzamidasRESUMEN
To investigate the sex-dependent differentiation of Sox10 cells and their response to pathological conditions such as lipopolysaccharide (LPS) exposure or ischemia, we utilized Sox10 Cre-ERT2, tdTomato mice. Tamoxifen administration induced the expression of red fluorescent protein (RFP) in these cells, facilitating their subsequent tracking and analysis after LPS injection and ischemia via immunofluorescence staining. Propidium iodide (PI) was injected to label necrotic cells following LPS administration. We found that the conversion of Sox10 cells to pericytes in female mice was significantly higher than in male mice, especially in those exposed to LPS. After LPS injection, the number of PI+ necrotic cells were significantly greater in females than in males. Moreover, RFP+ cells did not co-localize with glial fibrillary acidic protein (GFAP) or cluster of differentiation 11b (CD11b). Similarly, after brain ischemia, RFP+ cells did not express cluster of differentiation 13 (CD13), neuronal nuclei (NeuN), GFAP, or ionised calcium binding adaptor molecule 1 (Iba-1). These findings indicate that the conversion of Sox10 cells to pericytes following LPS exposure is sex-dependent, with neither male nor female groups showing differentiation into other cell types after LPS exposure or under ischemic conditions. The differences in LPS-induced necrosis of pericytes between sexes may explain the variations in the conversion of Sox10 cells to pericytes in both sexes.
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Lipopolisacáridos , Oligodendroglía , Pericitos , Factores de Transcripción SOXE , Animales , Femenino , Masculino , Pericitos/metabolismo , Pericitos/efectos de los fármacos , Ratones , Factores de Transcripción SOXE/metabolismo , Oligodendroglía/metabolismo , Oligodendroglía/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Caracteres Sexuales , Factores Sexuales , Ratones TransgénicosRESUMEN
The microvascular system consists of two cell types: endothelial and mural (pericytes and vascular smooth muscle cells; VSMCs) cells. Communication between endothelial and mural cells plays a pivotal role in the maintenance of vascular homeostasis; however, in vivo molecular and cellular mechanisms underlying mural cell development remain unclear. In this study, we found that macrophages played a crucial role in TGFß-dependent pericyte-to-VSMC differentiation during retinal vasculature development. In mice with constitutively active Foxo1 overexpression, substantial accumulation of TGFß1-producing macrophages and pericytes around the angiogenic front region was observed. Additionally, the TGFß-SMAD pathway was activated in pericytes adjacent to macrophages, resulting in excess ectopic α-smooth muscle actin-positive VSMCs. Furthermore, we identified endothelial SEMA3C as an attractant for macrophages. In vivo neutralization of SEMA3C rescued macrophage accumulation and ectopic VSMC phenotypes in the mice, as well as drug-induced macrophage depletion. Therefore, macrophages play an important physiological role in VSMC development via the FOXO1-SEMA3C pathway.
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Proteína Forkhead Box O1 , Macrófagos , Músculo Liso Vascular , Miocitos del Músculo Liso , Semaforinas , Animales , Macrófagos/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/citología , Ratones , Semaforinas/metabolismo , Semaforinas/genética , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O1/genética , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/citología , Pericitos/metabolismo , Pericitos/citología , Diferenciación Celular , Transducción de Señal , Vasos Retinianos/metabolismo , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción Forkhead/genética , Factor de Crecimiento Transformador beta1/metabolismo , Ratones Endogámicos C57BLRESUMEN
INTRODUCTION: Pericytes wrap microvessels and interact with endothelial cells to regulate vascular growth. Though pericyte dropout has been reported in pathological human placentae and mouse models of placental pathology, there has been limited investigation of the role and function of placental pericytes in vascular health and pathology. This study aimed to investigate the angiogenic potential of human placental pericytes relative to other villous cell populations. METHODS: Primary human placental pericytes, human umbilical vein endothelial cells (HUVEC), and BeWo cells ( ± 20 µM forskolin) were cultured in 1 % O2 or ambient air, followed by analysis of secreted angiogenic factors (ELISA). Additionally, the placental pericytes and HUVECs were co-cultured in a 3D sprouting assay to assess the capacity of pericytes to contribute to vascular sprouts. RESULTS: 1 % O2 affected secretion of angiogenic factors in placental pericytes, HUVECs, and syncytialized BeWo cells. Specifically, in placental pericytes, angiopoietin-1 (ANG1) and soluble fms-like tyrosine kinase-1 (sFLT1) were decreased, while vascular endothelial growth factor (VEGF) was increased. In HUVECS, matrix metalloproteinase-2 (MMP2), VEGF, angiopoietin-2 (ANG2), platelet-derived growth factor beta (PDGFB), placental growth factor (PlGF), and sFLT1 were increased. In syncytialized BeWo cells, VEGF, MMP2, PDGFB, PlGF, and sFLT1 secretion were increased. Placental pericytes and HUVECS colocalized to vessel sprouts in the 3-D sprouting assay. DISCUSSION: Hypoxic conditions altered placental pericyte, endothelial, and syncytialized BeWo secretion of angiogenic factors. We speculate that pericyte dropout and, by extension, the loss of pericyte-derived angiogenic factors in hypoxic conditions may contribute to compromised fetal vascular development observed in placental pathologies.
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Células Endoteliales de la Vena Umbilical Humana , Pericitos , Placenta , Pericitos/metabolismo , Humanos , Femenino , Embarazo , Placenta/metabolismo , Placenta/irrigación sanguínea , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Neovascularización Fisiológica/fisiología , Transducción de Señal/fisiología , Hipoxia de la Célula/fisiología , Técnicas de Cocultivo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Células Cultivadas , Hipoxia/metabolismoRESUMEN
Diabetic retinopathy (DR) is the leading cause of vision loss and blindness among working-age adults. Pericyte loss is an early pathological feature of DR. Under hyperglycemic conditions, reactive oxygen species (ROS) production increases, leading to oxidative stress and subsequent mitochondrial dysfunction and apoptosis. Dysfunctional pericyte can cause retinal vascular leakage, obliteration, and neovascularization. Glutaredoxin 2 (Grx2) is a mitochondrial glutathione-dependent oxidoreductase which protects cells against oxidative insults by safeguarding mitochondrial function. Whether Grx2 plays a protective role in diabetes-induced microvascular dysfunction remains unclear. Our findings revealed that diabetes-related stress reduced Grx2 expression in pericytes, but not in endothelial cells. Grx2 knock-in ameliorated diabetes-induced microvascular dysfunction in vivo DR models. Decreased Grx2 expression led to significant pericyte apoptosis, and pericyte dysfunction, namely reduced pericyte recruitment towards endothelial cells and increased endothelial cell permeability. Conversely, upregulating Grx2 reversed these effects. Furthermore, Grx2 regulated pericyte apoptosis by modulating complex I activity, which is crucial for pericyte mitochondrial function. Overall, our study uncovered a novel mechanism whereby high glucose inhibited Grx2 expression in vivo and in vitro. Grx2 downregulation exacerbated pericyte apoptosis, pericyte dysfunction, and retinal vascular dysfunction by inactivating complex I and mediating mitochondrial dysfunction in pericytes.
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Apoptosis , Diabetes Mellitus Experimental , Retinopatía Diabética , Regulación hacia Abajo , Glutarredoxinas , Pericitos , Vasos Retinianos , Pericitos/metabolismo , Pericitos/patología , Animales , Glutarredoxinas/metabolismo , Glutarredoxinas/genética , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Vasos Retinianos/patología , Vasos Retinianos/metabolismo , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Masculino , Células Cultivadas , Permeabilidad Capilar , Western BlottingRESUMEN
Diabetic retinopathy, a leading cause of vision impairment, is marked by microvascular complications in the retina, including pericyte loss, a key indicator of early-stage disease. This study explores the therapeutic potential of exosomes derived from immortalized adipose-mesenchymal stem cells differentiated into pericyte-like cells in restoring the function of mouse retinal microvascular endothelial cells damaged by high glucose conditions, thereby contributing to the understanding of early diabetic retinopathy intervention strategies. To induce immortalized adipose-mesenchymal stem cells differentiation into pericyte-like cells, the study employed pericyte growth supplement. And confirmed the success of cell differentiation through the detection of α-smooth muscle actin and neural/glial antigen 2 expression by Western blot and immunofluorescence. Exosomes were isolated from the culture supernatant of immortalized adipose-mesenchymal stem cells using ultracentrifugation and characterized through Western blot for exosomal markers (CD9, CD81, and TSG101), transmission electron microscopy, and nanoparticle tracking analysis. Their influence on mouse retinal microvascular endothelial cells under high glucose stress was assessed through various functional assays. Findings revealed that exosomes, especially those from pericyte-like immortalized adipose-mesenchymal stem cells, were efficiently internalized by retinal microvascular endothelial cells and effectively counteracted high glucose-induced apoptosis. These exosomes also mitigated the rise in reactive oxygen species levels and suppressed the migratory and angiogenic properties of retinal microvascular endothelial cells, as demonstrated by Transwell and tube formation assays, respectively. Furthermore, they preserved endothelial barrier function, reducing hyperglycemia-induced permeability. At the molecular level, qRT-PCR analysis showed that exosome treatment modulated the expression of critical genes involved in angiogenesis (VEGF-A, ANG2, MMP9), inflammation (IL-1ß, TNF-α), gap junction communication (CX43), and cytoskeletal regulation (ROCK1), with the most prominent effects seen with exosomes from pericyte-like immortalized adipose-mesenchymal stem cells. High glucose increased the expression of pro-angiogenic and pro-inflammatory markers, which were effectively normalized post-exosome treatment. In conclusion, this research highlights the reparative capacity of exosomes secreted by pericyte-like differentiated immortalized adipose-mesenchymal stem cells in reversing the detrimental effects of high glucose on retinal microvascular endothelial cells. By reducing apoptosis, oxidative stress, inflammation, and abnormal angiogenic behavior, these exosomes present a promising avenue for therapeutic intervention in early diabetic retinopathy. Future studies can focus on elucidating the precise molecular mechanisms and exploring their translational potential in vivo.
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Western Blotting , Diferenciación Celular , Retinopatía Diabética , Exosomas , Glucosa , Células Madre Mesenquimatosas , Pericitos , Vasos Retinianos , Exosomas/metabolismo , Pericitos/metabolismo , Animales , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Ratones , Vasos Retinianos/citología , Vasos Retinianos/metabolismo , Glucosa/farmacología , Retinopatía Diabética/metabolismo , Células Cultivadas , Tejido Adiposo/citología , Tejido Adiposo/metabolismo , Endotelio Vascular/metabolismo , Endotelio Vascular/citología , Microscopía Electrónica de Transmisión , Ratones Endogámicos C57BL , Apoptosis , Células Endoteliales/metabolismoRESUMEN
Endomucin (EMCN) is a 261 amino acid transmembrane glycoprotein that is highly expressed by venous and capillary endothelial cells where it plays a role in VEGF-mediated angiogenesis and regulation of immune cell recruitment. However, it is better known as a histological marker, where it has become widespread due to the commercial availability of high-quality antibodies that work under a wide range of conditions and in many tissues. The specificity of EMCN staining has been well-validated in retinal vessels, but while it has been used extensively as a marker in other tissues of the eye, including the choroid, the pattern of expression has not been described in detail. Here, in addition to endothelial expression in the choriocapillaris and deeper vascular layers, we characterize a population of EMCN-positive perivascular cells in the mouse choroid that did not co-localize with cells expressing other endothelial markers such as PECAM1 or PODXL. To confirm that these cells represented a new population of EMCN-expressing stromal cells, we then performed single cell RNA sequencing in choroids from adult wild-type mice. Analysis of this new dataset confirmed that, in addition to endothelial cells, Emcn mRNA expression was present in choroidal pericytes and a subset of fibroblasts, but not vascular smooth muscle cells. Besides Emcn, no known endothelial gene expression was detected in these cell populations, confirming that they did not represent endothelial-stromal doublets, a common technical artifact in single cell RNA seq datasets. Instead, choroidal Emcn-expressing fibroblasts exhibited high levels of chemokine and interferon signaling genes, while Emcn-negative fibroblasts were enriched in genes encoding extracellular matrix proteins. Emcn expressing fibroblasts were also detected in published datasets from mouse brain and human choroid, suggesting that stromal Emcn expression was not unique to the choroid and was evolutionarily conserved. Together, these findings highlight unique fibroblast and pericyte populations in the choroid and provide new context for the role of EMCN in the eye.
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Coroides , Ratones Endogámicos C57BL , Pericitos , Animales , Coroides/metabolismo , Coroides/irrigación sanguínea , Ratones , Humanos , Pericitos/metabolismo , Células Endoteliales/metabolismo , Regulación de la Expresión Génica/fisiología , ARN Mensajero/genética , Fibroblastos/metabolismo , Masculino , Endotelio Vascular/metabolismoRESUMEN
OBJECTIVES: Diabetes is closely linked to hearing loss, yet the exact mechanisms remain unclear. Cochlear stria vascularis and pericytes (PCs) are crucial for hearing. This study investigates whether high glucose induces apoptosis in the cochlear stria vascularis and pericytes via elevated ROS levels due to oxidative stress, impacting hearing loss. METHODS: We established a type II diabetes model in C57BL/6J mice and used auditory brainstem response (ABR), Evans blue staining, HE staining, immunohistochemistry, and immunofluorescence to observe changes in hearing, blood-labyrinth barrier (BLB) permeability, stria vascularis morphology, and apoptosis protein expression. Primary cultured stria vascularis pericytes were subjected to high glucose, and apoptosis levels were assessed using flow cytometry, Annexin V-FITC, Hoechst 33342 staining, Western blot, Mitosox, and JC-1 probes. RESULTS: Diabetic mice showed decreased hearing thresholds, reduced stria vascularis density, increased oxidative stress, cell apoptosis, and decreased antioxidant levels. High glucose exposure increased apoptosis and ROS content in pericytes, while mitochondrial membrane potential decreased, with AIF and cytochrome C (CytC) released from mitochondria to the cytoplasm. Adding oxidative scavengers reduced AIF and CytC release, decreasing pericyte apoptosis. DISCUSSION: Hyperglycemia may induce mitochondrial apoptosis of cochlear stria vascularis pericytes through oxidative stress.
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Factor Inductor de la Apoptosis , Apoptosis , Citocromos c , Hiperglucemia , Ratones Endogámicos C57BL , Mitocondrias , Estrés Oxidativo , Pericitos , Proteínas Proto-Oncogénicas c-bcl-2 , Especies Reactivas de Oxígeno , Estría Vascular , Animales , Pericitos/metabolismo , Pericitos/efectos de los fármacos , Pericitos/patología , Estría Vascular/metabolismo , Estría Vascular/patología , Ratones , Especies Reactivas de Oxígeno/metabolismo , Mitocondrias/metabolismo , Citocromos c/metabolismo , Factor Inductor de la Apoptosis/metabolismo , Hiperglucemia/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Masculino , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Cóclea/metabolismo , Cóclea/patologíaRESUMEN
The combination of lineage tracing and immunohistochemistry has helped to identify subpopulations and fate of hepatic stellate cells (HSC) in murine liver. HSC are sinusoidal pericytes that act as myofibroblast precursors after liver injury. Single cell RNA sequencing approaches have recently helped to differentiate central and portal HSC. A specific Cre line to lineage trace portal HSC has not yet been described. We used three Cre lines (Lrat-Cre, PDGFRß-CreERT2 and SMMHC-CreERT2) known to label mesenchymal cells including HSC in combination with a tdTomato-expressing reporter. All three Cre lines labeled populations of HSC as well as smooth muscle cells (SMC). Using the SMMHC-CreERT2, we identified a subtype of HSC in the periportal area of the hepatic lobule (termed zone 1-HSC). We lineage traced tdTomato-expressing zone 1-HSC over 1 year, described fibrotic behavior in two fibrosis models and investigated their possible role during fibrosis. This HSC subtype resides in zone 1 under healthy conditions; however, zonation is disrupted in preclinical models of liver fibrosis (CCl4 and MASH). Zone 1-HSC do not transform into αSMA-expressing myofibroblasts. Rather, they participate in sinusoidal capillarization. We describe a novel subtype of HSC restricted to zone 1 under physiological conditions and its possible function after liver injury. In contrast to the accepted notion, this HSC subtype does not transform into αSMA-positive myofibroblasts; rather, zone 1-HSC adopt properties of capillary pericytes, thereby participating in sinusoidal capillarization.
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Células Estrelladas Hepáticas , Cirrosis Hepática , Miofibroblastos , Animales , Células Estrelladas Hepáticas/metabolismo , Células Estrelladas Hepáticas/patología , Miofibroblastos/metabolismo , Miofibroblastos/patología , Ratones , Cirrosis Hepática/patología , Cirrosis Hepática/metabolismo , Hígado/patología , Hígado/metabolismo , Pericitos/metabolismo , Pericitos/patología , Linaje de la Célula , Masculino , Diferenciación Celular , Modelos Animales de Enfermedad , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Excessive pericyte coverage promotes tumor growth, and a downregulation may solve this dilemma. Due to the double-edged sword role of vascular pericytes in tumor microenvironment (TME), indiscriminately decreasing pericyte coverage by imatinib causes poor treatment outcomes. Here, we optimized the use of imatinib in a colorectal cancer (CRC) model in high pericyte-coverage status, and revealed the value of multiparametric magnetic resonance imaging (mpMRI) at 9.4T in monitoring treatment-related changes in pericyte coverage and the TME. METHODS: CRC xenograft models were evaluated by histological vascular characterizations and mpMRI. Mice with the highest pericyte coverage were treated with imatinib or saline; then, vascular characterizations, tumor apoptosis and HIF-1α level were analyzed histologically, and alterations in the expression of Bcl-2/bax pathway were assessed through qPCR. The effects of imatinib were monitored by dynamic contrast-enhanced (DCE)-, diffusion-weighted imaging (DWI)- and amide proton transfer chemical exchange saturation transfer (APT CEST)-MRI at 9.4T. RESULTS: The DCE- parameters provided a good histologic match the tumor vascular characterizations. In the high pericyte coverage status, imatinib exhibited significant tumor growth inhibition, necrosis increase and pericyte coverage downregulation, and these changes were accompanied by increased vessel permeability, decreased microvessel density (MVD), increased tumor apoptosis and altered gene expression of apoptosis-related Bcl-2/bax pathway. Strategically, a 4-day imatinib effectively decreased pericyte coverage and HIF-1α level, and continuous treatment led to a less marked decrease in pericyte coverage and re-elevated HIF-1α level. Correlation analysis confirmed the feasibility of using mpMRI parameters to monitor imatinib treatment, with DCE-derived Ve and Ktrans being most correlated with pericyte coverage, Ve with vessel permeability, AUC with microvessel density (MVD), DWI-derived ADC with tumor apoptosis, and APT CEST-derived MTRasym at 1 µT with HIF-1α. CONCLUSIONS: These results provided an optimized imatinib regimen to achieve decreasing pericyte coverage and HIF-1α level in the high pericyte-coverage CRC model, and offered an ultrahigh-field multiparametric MRI approach for monitoring pericyte coverage and dynamics response of the TME to treatment.
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Apoptosis , Neoplasias Colorrectales , Subunidad alfa del Factor 1 Inducible por Hipoxia , Mesilato de Imatinib , Imágenes de Resonancia Magnética Multiparamétrica , Pericitos , Mesilato de Imatinib/farmacología , Mesilato de Imatinib/uso terapéutico , Animales , Pericitos/metabolismo , Pericitos/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/diagnóstico por imagen , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Línea Celular Tumoral , Apoptosis/efectos de los fármacos , Humanos , Ratones Desnudos , Microambiente Tumoral/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) serves as a pro-angiogenic factor; however, there is to our knowledge currently no reported research on the relationship between HB-EGF and diabetic erectile dysfunction (ED). AIM: In this study we aimed to determine whether HB-EGF can improve the erectile function of streptozotocin-induced diabetic mice and to explore the related mechanisms. METHODS: Eight-week-old male C57BL/6 mice were used for diabetes induction. Diabetes mellitus (DM) was induced by low-dose injections of streptozotocin (50 mg/kg) for 5 consecutive days. Eight weeks after streptozotocin injections, DM was determined by measuring blood glucose and body weight. Diabetic mice were treated with two intracavernous administrations of phosphate-buffered saline (20 µL) or various doses of HB-EGF (days -3 and 0; 1, 5, and 10 µg in 20 µL of phosphate-buffered saline). The angiogenesis effect of HB-EGF was confirmed by tube formation and migration assays in mouse cavernous endothelial cells and mouse cavernous pericytes under high-glucose conditions. Erectile function was measured by electrical stimulation of the cavernous nerve, as well as histological examination and Western blot analysis for mechanism assessment. OUTCOMES: In vitro angiogenesis, cell proliferation, in vivo intracavernous pressure, neurovascular regeneration, cavernous permeability, and survival signaling were the outcomes measured. RESULTS: Expression of HB-EGF was reduced under diabetic conditions. Exogenous HB-EGF induced angiogenesis in mouse cavernous endothelial cells and mouse cavernous pericytes under high-glucose conditions. Erectile function was decreased in the DM group, whereas administration of HB-EGF resulted in a significant improvement of erectile function (91% of the age-matched control group) in association with increased neurovascular content, including cavernous endothelial cells, pericytes, and neuronal cells. Histological and Western blot analyses revealed a significant increase in the permeability of the corpus cavernosum in DM mice, which was attenuated by HB-EGF treatment. The protein expression of phospho-Akt Ser473 and phosphorylated endothelial nitric oxide synthase Ser1177 increased after HB-EGF treatment. CLINICAL IMPLICATIONS: The use of HB-EGF may be an effective strategy to treat ED associated with DM or other neurovascular diseases. STRENGTHS AND LIMITATIONS: Similarly to other pro-angiogenic factors, HB-EGF has dual roles in vascular and neuronal development. Our study focused on broadly evaluating the role of HB-EGF in diabetic ED. In view of the properties of HB-EGF as an angiogenic factor, its dose concentration should be strictly controlled to avoid potential side effects. CONCLUSION: In the diabetic ED mouse model in this study erectile function was improved by HB-EGF, which may provide new treatment strategies for patients with ED who do not respond to phosphodiesterase 5 Inhibitors.