RESUMO
The blood-brain barrier (BBB) acquires unique properties to regulate neuronal function during development. The formation of the BBB, which occurs in tandem with angiogenesis, is directed by the Wnt/ß-catenin signaling pathway. Yet the exact molecular interplay remains elusive. Our study reveals the G protein-coupled receptor GPR126 as a critical target of canonical Wnt signaling, essential for the development of the BBB's distinctive vascular characteristics and its functional integrity. Endothelial cell-specific deletion of the Gpr126 gene in mice induced aberrant vascular morphogenesis, resulting in disrupted BBB organization. Simultaneously, heightened transcytosis in vitro compromised barrier integrity, resulting in enhanced vascular permeability. Mechanistically, GPR126 enhanced endothelial cell migration, pivotal for angiogenesis, acting through an interaction between LRP1 and ß1 integrin, thereby balancing the levels of ß1 integrin activation and recycling. Overall, we identified GPR126 as a specifier of an organotypic vascular structure, which sustained angiogenesis and guaranteed the acquisition of the BBB properties during development.
Assuntos
Barreira Hematoencefálica , Integrina beta1 , Receptores Acoplados a Proteínas G , Animais , Camundongos , Barreira Hematoencefálica/metabolismo , Permeabilidade Capilar , Movimento Celular , Células Endoteliais/metabolismo , Integrina beta1/metabolismo , Integrina beta1/genética , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Camundongos Knockout , Neovascularização Fisiológica , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Via de Sinalização Wnt , Masculino , FemininoRESUMO
Recently, therapies utilizing extracellular vesicles (EVs) derived from mesenchymal stromal/stem cells (MSCs) have begun to show promise in clinical trials. However, EV therapeutic potential varies with MSC tissue source and in vitro expansion through passaging. To find the optimal MSC source for clinically translatable EV-derived therapies, this study aims to compare the angiogenic and immunomodulatory potentials and the protein and miRNA cargo compositions of EVs isolated from the two most common clinical sources of adult MSCs, bone marrow and adipose tissue, across different passage numbers. Primary bone marrow-derived MSCs (BMSCs) and adipose-derived MSCs (ASCs) were isolated from adult female Lewis rats and expanded in vitro to the indicated passage numbers (P2, P4, and P8). EVs were isolated from the culture medium of P2, P4, and P8 BMSCs and ASCs and characterized for EV size, number, surface markers, protein content, and morphology. EVs isolated from different tissue sources showed different EV yields per cell, EV sizes, and protein yield per EV. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of proteomics data and miRNA seq data identified key proteins and pathways associated with differences between BMSC-EVs and ASC-EVs, as well as differences due to passage number. In vitro tube formation assays employing human umbilical vein endothelial cells suggested that both tissue source and passage number had significant effects on the angiogenic capacity of EVs. With or without lipopolysaccharide (LPS) stimulation, EVs more significantly impacted expression of M2-macrophage genes (IL-10, Arg1, TGFß) than M1-macrophage genes (IL-6, NOS2, TNFα). By correlating the proteomics analyses with the miRNA seq analysis and differences observed in our in vitro immunomodulatory, angiogenic, and proliferation assays, this study highlights the trade-offs that may be necessary in selecting the optimal MSC source for development of clinical EV therapies.
Assuntos
Vesículas Extracelulares , Células-Tronco Mesenquimais , MicroRNAs , Ratos Endogâmicos Lew , Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , MicroRNAs/genética , Animais , Feminino , Ratos , Tecido Adiposo/metabolismo , Tecido Adiposo/citologia , Neovascularização Fisiológica , Imunomodulação , Humanos , Células Cultivadas , Proliferação de Células , Células da Medula Óssea/metabolismoRESUMO
Endothelial cell physiology is governed by its unique microenvironment at the interface between blood and tissue. A major contributor to the endothelial biophysical environment is blood hydrostatic pressure, which in mechanical terms applies isotropic compressive stress on the cells. While other mechanical factors, such as shear stress and circumferential stretch, have been extensively studied, little is known about the role of hydrostatic pressure in the regulation of endothelial cell behavior. Here we show that hydrostatic pressure triggers partial and transient endothelial-to-mesenchymal transition in endothelial monolayers of different vascular beds. Values mimicking microvascular pressure environments promote proliferative and migratory behavior and impair barrier properties that are characteristic of a mesenchymal transition, resulting in increased sprouting angiogenesis in 3D organotypic model systems ex vivo and in vitro. Mechanistically, this response is linked to differential cadherin expression at the adherens junctions, and to an increased YAP expression, nuclear localization, and transcriptional activity. Inhibition of YAP transcriptional activity prevents pressure-induced sprouting angiogenesis. Together, this work establishes hydrostatic pressure as a key modulator of endothelial homeostasis and as a crucial component of the endothelial mechanical niche.
Assuntos
Junções Aderentes , Pressão Hidrostática , Neovascularização Fisiológica , Transdução de Sinais , Proteínas de Sinalização YAP , Animais , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Junções Aderentes/metabolismo , Caderinas/metabolismo , Caderinas/genética , Movimento Celular , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Sinalização YAP/metabolismoRESUMO
Osteogenesis is tightly coupled with angiogenesis spatiotemporally. Previous studies have demonstrated that type H blood vessel formed by endothelial cells with high expression of CD31 and Emcn (CD31hi Emcnhi ECs) play a crucial role in bone regeneration. The mechanism of the molecular communication around CD31hi Emcnhi ECs and bone mesenchymal stem cells (BMSCs) in the osteogenic microenvironment is unclear. This study indicates that exosomes from bone mesenchymal stem cells with 7 days osteogenic differentiation (7D-BMSCs-exo) may promote CD31hi Emcnhi ECs angiogenesis, which was verified by tube formation assay, qRT-PCR, Western blot, immunofluorescence staining and µCT assays etc. in vitro and in vivo. Furthermore, by exosomal miRNA microarray and WGCNA assays, we identified downregulated miR-150-5p as the most relative hub gene coupling osteogenic differentiation and type H blood vessel angiogenesis. With bioinformatics assays, dual luciferase reporter experiments, qRT-PCR and Western blot assays, SOX2(SRY-Box Transcription Factor 2) was confirmed as a novel downstream target gene of miR-150-5p in exosomes, which might be a pivotal mechanism regulating CD31hi Emcnhi ECs formation. Additionally, JC-1 immunofluorescence staining, Western blot and seahorse assay results showed that the overexpression of SOX2 could shift metabolic reprogramming from oxidative phosphorylation (OXPHOS) to glycolysis to enhance the CD31hi Emcnhi ECs formation. The PI3k/Akt signaling pathway might play a key role in this process. In summary, BMSCs in osteogenic differentiation might secrete exosomes with low miR-150-5p expression to induce type H blood vessel formation by mediating SOX2 overexpression in ECs. These findings might reveal a molecular mechanism of osteogenesis coupled with type H blood vessel angiogenesis in the osteogenic microenvironment and provide a new therapeutic target or cell-free remedy for osteogenesis impaired diseases.
Assuntos
Diferenciação Celular , Células Endoteliais , Exossomos , Células-Tronco Mesenquimais , MicroRNAs , Neovascularização Fisiológica , Osteogênese , MicroRNAs/genética , MicroRNAs/metabolismo , Exossomos/metabolismo , Osteogênese/genética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Diferenciação Celular/genética , Neovascularização Fisiológica/genética , Animais , Células Endoteliais/metabolismo , Células Endoteliais/citologia , Camundongos , Humanos , Células Cultivadas , Transdução de Sinais , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXB1/genética , Reprogramação Metabólica , AngiogêneseRESUMO
Bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exos) have been shown to promote angiogenesis after ischemic stroke, in which microRNAs (miRs) are believed to play an important role in exosome-mediated therapeutic effects, though the mechanism is still not clear. In this study, a series of molecular biological and cellular assays, both in vitro and in vivo, were performed to elucidate the role of exosomal miR-486 in angiogenesis following cerebral ischemic and its molecular mechanisms. Our results revealed that BMSC-Exos significantly improved neurological function and increased microvessel density in ischemic stroke rats. In vitro assays showed that BMSC-Exos promoted the proliferation, migration, and tube formation ability of oxygen-glucose deprivation/reoxygenation (OGD/R) injured rat brain microvascular endothelial cells (RBMECs). Importantly, BMSC-Exos increased the expression of miR-486 and phosphorylated protein kinase B (p-Akt) and down-regulated the protein level of phosphatase and tensin homolog (PTEN) in vivo and in vitro. Mechanistic studies demonstrated that transfection with miR-486 mimic enhanced RBMECs angiogenesis and increased p-Akt expression, while inhibited PTEN expression. On the other hand, the miR-486 inhibitor induced an opposite effect, which could be blocked by PTEN siRNA. It was thus concluded that exosomal miR-486 from BMSCs may enhance the functional recovery by promoting angiogenesis following cerebral ischemic injury, which might be related to its regulation of the PTEN/Akt pathway.
Assuntos
Exossomos , Células-Tronco Mesenquimais , MicroRNAs , Neovascularização Fisiológica , PTEN Fosfo-Hidrolase , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Animais , PTEN Fosfo-Hidrolase/metabolismo , PTEN Fosfo-Hidrolase/genética , MicroRNAs/metabolismo , MicroRNAs/genética , Exossomos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Masculino , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Ratos Sprague-Dawley , Células Endoteliais/metabolismo , Proliferação de Células , Movimento Celular , Modelos Animais de Doenças , AngiogêneseRESUMO
Pressure ulcers (PU) are caused by persistent long-term pressure, which compromises the integrity of the epidermis, dermis, and subcutaneous adipose tissue layer by layer, making it difficult to heal. Platelet products such as platelet lysate (PL) can promote tissue regeneration by secreting numerous growth factors based on clinical studies on skin wound healing. However, the components of PL are difficult to retain in wounds. Gelatin methacrylate (GelMA) is a photopolymerizable hydrogel that has lately emerged as a promising material for tissue engineering and regenerative medicine. The PL liquid was extracted, flow cytometrically detected for CD41a markers, and evenly dispersed in the GelMA hydrogel to produce a surplus growth factor hydrogel system (PL@GM). The microstructure of the hydrogel system was observed under a scanning electron microscope, and its sustained release efficiency and biological safety were tested in vitro. Cell viability and migration of human dermal fibroblasts, and tube formation assays of human umbilical vein endothelial cells were applied to evaluate the ability of PL to promote wound healing and regeneration in vitro. Real-time polymerase chain reaction (PCR) and western blot analyses were performed to elucidate the skin regeneration mechanism of PL. We verified PL's therapeutic effectiveness and histological analysis on the PU model. PL promoted cell viability, migration, wound healing and angiogenesis in vitro. Real-time PCR and western blot indicated PL suppressed inflammation and promoted collagen I synthesis by activating STAT3. PL@GM hydrogel system demonstrated optimal biocompatibility and favorable effects on essential cells for wound healing. PL@GM also significantly stimulated PU healing, skin regeneration, and the formation of subcutaneous collagen and blood vessels. PL@GM could accelerate PU healing by promoting fibroblasts to migrate and secrete collagen and endothelial cells to vascularize. PL@GM promises to be an effective and convenient treatment modality for PU, like chronic wound treatment.
Assuntos
Angiogênese , Plaquetas , Gelatina , Metacrilatos , Úlcera por Pressão , Pele , Cicatrização , Animais , Humanos , Camundongos , Angiogênese/efeitos dos fármacos , Plaquetas/metabolismo , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Gelatina/química , Gelatina/farmacologia , Células Endoteliais da Veia Umbilical Humana , Hidrogéis/química , Metacrilatos/química , Metacrilatos/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Úlcera por Pressão/terapia , Regeneração/efeitos dos fármacos , Pele/irrigação sanguínea , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/patologia , Fator de Transcrição STAT3/metabolismo , Cicatrização/efeitos dos fármacosRESUMO
Estrogens regulate numerous physiological and pathological processes, including wide-ranging effects in wound healing. The effects of estrogens are mediated through multiple estrogen receptors (ERs), including the classical nuclear ERs (ERα and ER ß ), that typically regulate gene expression, and the 7-transmembrane G protein-coupled estrogen receptor (GPER), that predominantly mediates rapid "non-genomic" signaling. Estrogen modulates the expression of various genes involved in epidermal function and regeneration, inflammation, matrix production, and protease inhibition, all critical to wound healing. Our previous work demonstrated improved myocutaneous wound healing in female mice compared to male mice. In the current study, we employed male and female GPER knockout mice to investigate the role of this estrogen receptor in wound revascularization and tissue viability. Using a murine myocutaneous flap model of graded ischemia, we measured real-time flap perfusion via laser speckle perfusion imaging. We conducted histologic and immunohistochemical analyses to assess skin and muscle viability, microvascular density and vessel morphology. Our results demonstrate that GPER is crucial in wound healing, mediating effects that are both dependent and independent of sex. Lack of GPER expression is associated with increased skin necrosis, reduced flap perfusion and altered vessel morphology. These findings contribute to understanding GPER signaling in wound healing and suggest possible therapeutic opportunities by targeting GPER.
Assuntos
Camundongos Knockout , Neovascularização Fisiológica , Receptores de Estrogênio , Receptores Acoplados a Proteínas G , Cicatrização , Animais , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores de Estrogênio/metabolismo , Receptores de Estrogênio/genética , Masculino , Camundongos , Feminino , Pele/metabolismo , Pele/irrigação sanguínea , Isquemia/metabolismo , Retalhos CirúrgicosRESUMO
Vascular endothelial growth factor receptor (VEGFR)-2 is a key switch for angiogenesis, which is observed in various human diseases. In this study, a novel system for advanced prime editing (PE), termed PE6h, is developed, consisting of dual lentiviral vectors: (1) a clustered regularly interspaced palindromic repeat-associated protein 9 (H840A) nickase fused with reverse transcriptase and an enhanced PE guide RNA and (2) a dominant negative (DN) MutL homolog 1 gene with nicking guide RNA. PE6h was used to edit VEGFR2 (c.18315T>A, 50.8%) to generate a premature stop codon (TAG from AAG), resulting in the production of DN-VEGFR2 (787 aa) in human retinal microvascular endothelial cells (HRECs). DN-VEGFR2 impeded VEGF-induced phosphorylation of VEGFR2, Akt, and extracellular signal-regulated kinase-1/2 and tube formation in PE6h-edited HRECs in vitro. Overall, our results highlight the potential of PE6h to inhibit angiogenesis in vivo.
Assuntos
Células Endoteliais , Edição de Genes , Receptor 2 de Fatores de Crescimento do Endotélio Vascular , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Humanos , Edição de Genes/métodos , Células Endoteliais/metabolismo , Neovascularização Fisiológica , Sistemas CRISPR-Cas , Fosforilação , RNA Guia de Sistemas CRISPR-Cas , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Neovascularização Patológica/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Proteína 9 Associada à CRISPR/genética , Retina/metabolismo , Vetores Genéticos , AngiogêneseRESUMO
Catalpol, a natural iridoid glycoside, has potential therapeutic benefits, including anti-inflammatory and neuroprotective effects. Investigating catalpol's role in angiogenesis is critical for understanding its potential therapeutic applications, particularly in diseases where modulating angiogenesis is beneficial. This study investigates catalpol's influence on angiogenesis and its mechanisms, combining network pharmacology and in vitro experiments. The target genes corresponding to the catalpol were analyzed by SwissTargetPrediction. Then angiogenesis-related targets were acquired from databases like GeneCards. Subsequently, the Database for Annotation, Visualization and Integrated Discovery was employed for Gene Ontology and pathway analysis, while Cytoscape visualized protein interactions. The effect of catalpol on viability and angiogenesis of HUVECs was further examined using Cell Counting Kit-8 and angiogenesis assays. RT-qPCR and western blot were applied to check the expression of angiogenesis-related proteins. Totally, 312 target genes of catalpol and 823 angiogenesis-related targets were obtained with 56 common targets leading to PPI network analysis, highlighting hub genes (AKT1, EGFR, STAT3, MAPK3, and CASP3). These hub genes were mainly enriched in lipid and atherosclerosis pathway and EGFR-related pathway. The in vitro experimental results showed that catalpol achieved a concentration-dependent increase in HUVECs viability. Catalpol also promoted the migration and angiogenesis of HUVECs and up-regulated the expression of EGFR. EGFR knockdown inhibited the effect of catalpol on HUVECs. Catalpol promotes angiogenesis in HUVECs by upregulating EGFR and angiogenesis-related proteins, indicating its potential therapeutic application in vascular-related diseases.
Assuntos
Células Endoteliais da Veia Umbilical Humana , Glucosídeos Iridoides , Farmacologia em Rede , Humanos , Glucosídeos Iridoides/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Mapas de Interação de Proteínas/efeitos dos fármacos , Receptores ErbB/metabolismo , Receptores ErbB/genética , Transdução de Sinais/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , AngiogêneseRESUMO
BACKGROUND: In the repair of massive tissue defects using expanded large skin flaps, the incidence of complications increases with the size of the expanded area. Currently, stem cell therapy has limitations to solve this problem. We hypothesized that conditioned medium of adipose-derived stem cells (ADSC-CM) collected following mechanical pretreatment can assist skin expansion. METHODS: Rat aortic endothelial cells and fibroblasts were cultured with ADSC-CM collected under 0%, 10%, 12%, and 15% stretching force. Ten-milliliter cylindrical soft tissue expanders were subcutaneously implanted into the backs of 36 Sprague-Dawley rats. The 0% and 10% stretch groups were injected with ADSC-CM collected under 0% and 10% stretching force, respectively, while the control group was not injected. After 3, 7, 14, and 30 days of expansion, expanded skin tissue was harvested for staining and qPCR analyses. RESULTS: Endothelial cells had the best lumen formation and highest migration rate, and fibroblasts secreted the most collagen upon culture with ADSC-CM collected under 10% stretching force. The skin expansion rate was significantly increased in the 10% stretch group. After 7 days of expansion, the number of blood vessels in the expanded area, expression of the angiogenesis-associated proteins vascular endothelial growth factor, basic fibroblast growth factor, and hepatocyte growth factor, and collagen deposition were significantly increased in the 10% stretch group. CONCLUSIONS: The optimal mechanical force upregulates specific paracrine proteins in ADSCs to increase angiogenesis and collagen secretion, and thereby promote skin regeneration and expansion. This study provides a new auxiliary method to expand large skin flaps.
Assuntos
Tecido Adiposo , Comunicação Parácrina , Ratos Sprague-Dawley , Pele , Animais , Ratos , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Pele/metabolismo , Fibroblastos/metabolismo , Fibroblastos/citologia , Células Endoteliais/metabolismo , Células Endoteliais/citologia , Meios de Cultivo Condicionados/farmacologia , Expansão de Tecido/métodos , Masculino , Células-Tronco/metabolismo , Células-Tronco/citologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Células Cultivadas , Neovascularização Fisiológica , Estresse MecânicoRESUMO
Converging evidence indicates that extra-embryonic yolk sac is the source of both macrophages and endothelial cells in adult mouse tissues. Prevailing views are that these embryonically derived cells are maintained after birth by proliferative self-renewal in their differentiated states. Here we identify clonogenic endothelial-macrophage (EndoMac) progenitor cells in the adventitia of embryonic and postnatal mouse aorta, that are independent of Flt3-mediated bone marrow hematopoiesis and derive from an early embryonic CX3CR1+ and CSF1R+ source. These bipotent progenitors are proliferative and vasculogenic, contributing to adventitial neovascularization and formation of perfused blood vessels after transfer into ischemic tissue. We establish a regulatory role for angiotensin II, which enhances their clonogenic and differentiation properties and rapidly stimulates their proliferative expansion in vivo. Our findings demonstrate that embryonically derived EndoMac progenitors participate in local vasculogenic responses in the aortic wall by contributing to the expansion of endothelial cells and macrophages postnatally.
Assuntos
Aorta , Macrófagos , Animais , Macrófagos/citologia , Macrófagos/metabolismo , Aorta/citologia , Camundongos , Receptor 1 de Quimiocina CX3C/metabolismo , Receptor 1 de Quimiocina CX3C/genética , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Diferenciação Celular , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Angiotensina II , Proliferação de Células , Células-Tronco/citologia , Células-Tronco/metabolismo , Camundongos Endogâmicos C57BL , Feminino , Neovascularização Fisiológica , Receptores de Quimiocinas/metabolismo , Receptores de Quimiocinas/genética , Masculino , Hematopoese/fisiologia , Tirosina Quinase 3 Semelhante a fmsRESUMO
Pulsed electromagnetic field (PEMF) therapy has been extensively investigated in clinical studies for the treatment of angiogenesis-related diseases. However, there is a lack of research on the impact of PEMFs on energy metabolism and mitochondrial dynamics during angiogenesis. The present study included tube formation and CCK-8 assays. A Seahorse assay was conducted to analyze energy metabolism, and mitochondrial membrane potential assays, mitochondrial imaging, and reactive oxygen species assays were used to measure changes in mitochondrial structure and function in human umbilical vein endothelial cells (HUVECs) exposed to PEMFs. Real-time polymerase chain reaction was used to analyze the mRNA expression levels of antioxidants, glycolytic pathway-related genes, and genes associated with mitochondrial fission and fusion. The tube formation assay demonstrated a significantly greater tube network in the PEMF group compared to the control group. The glycolysis and mitochondrial stress tests revealed that PEMFs promoted a shift in the energy metabolism pattern of HUVECs from oxidative phosphorylation to aerobic glycolysis. Mitochondrial imaging revealed a wire-like mitochondrial morphology in the control group, and treatment with PEMFs led to shorter and more granular mitochondria. Our major findings indicate that exposure to PEMFs accelerates angiogenesis in HUVECs, likely by inducing energy metabolism reprogramming and mitochondrial fission.
Assuntos
Campos Eletromagnéticos , Metabolismo Energético , Células Endoteliais da Veia Umbilical Humana , Dinâmica Mitocondrial , Neovascularização Fisiológica , Humanos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Glicólise , Potencial da Membrana Mitocondrial , Reprogramação Metabólica , AngiogêneseRESUMO
Nitric oxide (NO) regulates vascular homeostasis and plays a key role in revascularization and angiogenesis. The endothelial nitric oxide synthase (eNOS) enzyme catalyzes NO production in endothelial cells. Overexpression of the eNOS gene has been implicated in pathologies with dysfunctional angiogenic processes, such as cancer. Therefore, modulating eNOS gene expression using small interfering RNAs (siRNAs) represents a viable strategy for antitumor therapy. siRNAs are highly specific to the target gene, thus reducing off-target effects. Given the widespread distribution of endothelium and the crucial physiological role of eNOS, localized delivery of nucleic acid to the affected area is essential. Therefore, the development of an efficient eNOS-siRNA delivery carrier capable of controlled release is imperative for targeting specific vascular regions, particularly those associated with tumor vascular growth. Thus, this study aims to utilize ultrasound-mediated microbubble destruction (UMMD) technology with cationic microbubbles loaded with eNOS-siRNA to enhance transfection efficiency and improve siRNA delivery, thereby preventing sprouting angiogenesis. The efficiency of eNOS-siRNA transfection facilitated by UMMD was assessed using bEnd.3 cells. Synthesis of nitric oxide and eNOS protein expression were also evaluated. The silencing of eNOS gene in a model of angiogenesis was assayed using the rat aortic ring assay. The results showed that from 6 to 24 h, the transfection of fluorescent siRNA with UMMD was twice as high as that of lipofection. Moreover, transfection of eNOS-siRNA with UMMD enhanced the knockdown level (65.40 ± 4.50%) compared to lipofectamine (40 ± 1.70%). Silencing of eNOS gene with UMMD required less amount of eNOS-siRNA (42 ng) to decrease the level of eNOS protein expression (52.30 ± 0.08%) to the same extent as 79 ng of eNOS-siRNA using lipofectamine (56.30 ± 0.10%). NO production assisted by UMMD was reduced by 81% compared to 67% reduction transfecting with lipofectamine. This diminished NO production led to higher attenuation of aortic ring outgrowth. Three-fold reduction compared to lipofectamine transfection. In conclusion, we propose the combination of eNOS-siRNA and UMMD as an efficient, safe, non-viral nucleic acid transfection strategy for inhibition of tumor progression.
Assuntos
Aorta , Microbolhas , Óxido Nítrico Sintase Tipo III , Óxido Nítrico , RNA Interferente Pequeno , Transfecção , Animais , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico Sintase Tipo III/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Transfecção/métodos , Aorta/metabolismo , Óxido Nítrico/metabolismo , Camundongos , Masculino , Linhagem Celular , Neovascularização Fisiológica/genéticaRESUMO
Macrophages regulate angiogenesis, repair, conduction, and homeostasis in heart tissue. Landau et al.1 demonstrate that incorporating primitive macrophages into engineered heart tissues significantly promotes long-term vascularization and cardiac maturation. This advance demonstrates the importance of resident immune-vascular microenvironments in cardiac tissue engineering, marking an important step forward for heart-on-chip technologies.
Assuntos
Macrófagos , Neovascularização Fisiológica , Engenharia Tecidual , Engenharia Tecidual/métodos , Macrófagos/metabolismo , Macrófagos/citologia , Humanos , Animais , Miocárdio/citologia , Coração/fisiologiaRESUMO
Direct reprogramming provides a novel breakthrough for generating functional endothelial cells (ECs) without the need for intermediate stem or progenitor states, offering a promising resource for cardiovascular research and treatment. ETV2 is a key transcription factor that has been identified as a pioneering factor for specifying endothelial lineage. Achieving precise ETV2 induction is essential for effective endothelial reprogramming, and maintaining the reprogrammed cellular phenotype relies on a specific combination of growth factors and small molecules. Thus, we hereby provide a straightforward and comprehensive protocol for generating two distinct types of reprogrammed ECs (rECs) from human dermal fibroblasts (HDFs). Early rECs demonstrate a robust neovascularization property but lack the mature EC phenotype, while late rECs exhibit phenotypical similarity to human postnatal ECs and have a neovascularization capacity similar to early rECs. Both cell types can be derived from human somatic source cells, making them suitable for personalized disease investigations, drug discovery, and disease therapy.
Assuntos
Técnicas de Cultura de Células , Reprogramação Celular , Células Endoteliais , Fibroblastos , Humanos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Técnicas de Cultura de Células/métodos , Células Cultivadas , Neovascularização Fisiológica , Diferenciação Celular , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Técnicas de Reprogramação Celular/métodosRESUMO
MicroRNAs (miRs) regulate physiological and pathological processes, including ischemia-induced angiogenesis and neovascularization. They can be transferred between cells by extracellular vesicles (EVs). However, the specific miRs that are packaged in EVs released from skeletal muscles, and how this process is modulated by ischemia, remain to be determined. We used a mouse model of hindlimb ischemia and next generation sequencing (NGS) to perform a complete profiling of miR expression and determine the effect of ischemia in skeletal muscles, and in EVs of different sizes (microvesicles (MVs) and exosomes) released from these muscles. Ischemia significantly modulated miR expression in whole muscles and EVs, increasing the levels of several miRs that can have pro-angiogenic effects (angiomiRs). We found that specific angiomiRs are selectively enriched in MVs and/or exosomes in response to ischemia. In silico approaches indicate that these miRs modulate pathways that play key roles in angiogenesis and neovascularization, including HIF1/VEGF signaling, regulation of actin cytoskeleton and focal adhesion, NOTCH, PI3K/AKT, RAS/MAPK, JAK/STAT, TGFb/SMAD signaling and the NO/cGMP/PKG pathway. Thus, we show for the first time that angiomiRs are selectively enriched in MVs and exosomes released from ischemic muscles. These angiomiRs could be targeted in order to improve the angiogenic function of EVs for potential novel therapeutic applications in patients with severe ischemic vascular diseases.
Assuntos
Vesículas Extracelulares , Isquemia , MicroRNAs , Músculo Esquelético , Neovascularização Fisiológica , Animais , Músculo Esquelético/metabolismo , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/patologia , Vesículas Extracelulares/metabolismo , MicroRNAs/metabolismo , MicroRNAs/genética , Isquemia/metabolismo , Isquemia/patologia , Camundongos , Membro Posterior/irrigação sanguínea , Membro Posterior/patologia , Camundongos Endogâmicos C57BL , Transdução de Sinais , Masculino , Exossomos/metabolismo , Neovascularização Patológica/metabolismo , AngiogêneseRESUMO
Capillary plexus cultivation is crucial in tissue engineering and regenerative medicine. Theoretical simulations have been conducted to supplement the expensive experimental works. However, the mechanisms connecting mechanical and chemical stimuli remained undefined, and the functions of the different VEGF forms in the culture environment were still unclear. In this paper, we developed a hybrid model for simulating short-term in vitro capillary incubations. We used the Cellular Potts model to predict individual cell migration, morphology change, and continuum mechanics to quantify biogel deformation and VEGF transport dynamics. By bridging the mechanical regulation and chemical stimulation in the model, the results showed good agreement between the predicted network topology and experiments, in which elongated cells connected, forming the network cords and round cells gathered, creating cobblestone-like aggregates. The results revealed that the capillary-like networks could develop in high integrity only when the mechanical and chemical couplings worked adequately, with the cell morphology and haptotaxis driven by the soluble and bound forms of VEGF, respectively, functioning simultaneously.
Assuntos
Capilares , Simulação por Computador , Fator A de Crescimento do Endotélio Vascular , Fator A de Crescimento do Endotélio Vascular/metabolismo , Capilares/metabolismo , Humanos , Movimento Celular/fisiologia , Modelos Biológicos , Biologia Computacional , Neovascularização Fisiológica/fisiologia , Engenharia Tecidual/métodosRESUMO
Background/aim: In practice, waiting 2-3 weeks for interpolation flaps pedicle division result in certain morbidities and discomfort for patient. The division time of flap pedicle depends on neovascularization from the recipient bed and includes wound healing stages. We aimed to investigate the effect of recombinant human epidermal growth factor (rhEGF) on the flap viability during early pedicle division. Materials and methods: Thirty-six rats were allocated to two main groups as control and study. A cranial based flap measuring 5 × 5 cm was elevated from the back, including all layers of the skin. While the cranial half of the defect was primarily closed, the flap was inset into the distal half. In the study group, a single dose of 20 µg EGF was injected into the recipient site and wound edges before the flap inset. The control group received no treatment. Each main group was divided into three subgroups based on pedicle division time of 8, 11 and 14 days. After pedicle division, each flap was monitored and photographed for 7 days, and histopathological samples were collected. Viable and necrotic areas were compared, and flaps were examined histopathologically. Results: The necrosis area in the study group on the 11th day was significantly lower than that in the control group. The fibroblastic activity, granulation tissue and neovascularization on the 8th day, the granulation tissue level on the 11th day, and the neovascularization level on the 14th day were significantly higher in the study groups. Conclusion: Following the application of EGF, the necrosis area decreased within the study group. Histopathological assessments revealed a statistically significant increase in parameters related to granulation tissue and fibroblastic activity, notably neovascularization, across all subgroups within the study. It was concluded that the use of EGF positively affected the neovascularization, and flaps could be divided earlier.
Assuntos
Fator de Crescimento Epidérmico , Neovascularização Fisiológica , Proteínas Recombinantes , Retalhos Cirúrgicos , Animais , Fator de Crescimento Epidérmico/farmacologia , Fator de Crescimento Epidérmico/administração & dosagem , Retalhos Cirúrgicos/irrigação sanguínea , Ratos , Neovascularização Fisiológica/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/administração & dosagem , Cicatrização/efeitos dos fármacos , Humanos , Masculino , Ratos Sprague-DawleyAssuntos
Receptor do Peptídeo Semelhante ao Glucagon 1 , Regeneração , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Animais , Humanos , Regeneração/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Transdução de SinaisRESUMO
The functional maturation of the pituitary gland requires adequate cell differentiation and vascular network formation. Although spatiotemporal signaling and transcription factors are known to govern pituitary development, the involvement of primary cilia, nonmoving hair-like organelles, remains unclear. In this study, we uncovered the contribution of primary cilia to cell-type determination and vascular network formation during pituitary development. Homozygous knockout mice lacking a ciliary kinase, Dyrk2-/-, exhibit abnormalities in ciliary structure and pituitary hypoplasia, accompanied by varying degrees of failure in differentiation among all types of hormone-producing cells in the anterior lobe. Aberrations in cell differentiation in Dyrk2-/- mice arise from a decrease in the expression of crucial transcription factors, Lhx4, Lhx3, and Prop1, resulting from the inactivity of Hedgehog (Hh) signaling during the early stages of development. Furthermore, the loss of Dyrk2 results in vascular system abnormalities during the middle to late stages of development. Mechanistically, transcriptome analyses revealed the downregulation of vitronectin-integrin αvß3-VEGFR2 signaling, essential for orchestrating vascular development. Collectively, our findings demonstrate that primary cilia play a pivotal role as critical regulators of cell survival, cell determination, and angiogenesis during pituitary gland development through the activation of Hh signaling. These findings expand our understanding of the potential link between pituitary dysfunction in human disorders and ciliopathies.