RESUMEN

Arteriogenesis is an inflammatory driven mechanism, describing the growth of a natural bypass from pre-existing collateral arteries to compensate for an occluded artery. The complement system component C3 is a potent natural inflammatory activator. Here, we investigated its impact on the process of collateral artery growth using C3-deficient (C3 -/-) and wildtype control mice in a murine hindlimb model of arteriogenesis. Induction of arteriogenesis by unilateral femoral artery ligation resulted in decreased perfusion recovery in C3 -/- mice on day 7 as shown by Laser Doppler imaging. Immunofluorescence staining revealed a reduced vascular cell proliferation in C3 -/- mice. Gene expression analysis displayed a significant reduction in monocyte chemoattractant protein-1 (MCP-1) expression in C3 -/- mice. Interestingly, 3 days after induction of arteriogenesis, the number of macrophages (CD68+) recruited to growing collaterals was not affected by C3 deficiency. However, a significant reduction in inflammatory M1-like polarized macrophages (CD68+/MRC1-) was noted. Forced mast cell activation by Compound 48/80 as well as exogenous MCP-1 application rescued the number of M1-like polarized macrophages along with perfusion recovery in C3 -/- mice. In summary, this study demonstrates that complement C3 influences arteriogenesis by mediating MCP-1 expression, which is essential for the induction and enhancement of sterile inflammation.

Asunto(s)

Circulación Colateral , Complemento C3 , Inflamación , Animales , Inflamación/patología , Ratones , Complemento C3/metabolismo , Complemento C3/genética , Quimiocina CCL2/metabolismo , Quimiocina CCL2/genética , Macrófagos/metabolismo , Neovascularización Fisiológica/genética , Ratones Endogámicos C57BL , Miembro Posterior/irrigación sanguínea , Ratones Noqueados , Arteria Femoral/patología , Arterias/crecimiento & desarrollo , Arterias/metabolismo , Masculino , Proliferación Celular , Mastocitos/metabolismo

RESUMEN

Epigenetic regulation is an important entry point to study the pathogenesis of selective fetal growth restriction (sFGR), and an understanding of the role of long noncoding RNAs (lncRNAs) in sFGR is lacking. Our study aimed to investigate the potential role of a lncRNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), in sFGR using molecular biology experiments and gain- or loss-of-function assays. We found that the levels of MALAT1, ERRγ, and HSD17B1 were downregulated and that of miR-424 was upregulated in the placental shares of the smaller twins. Moreover, angiogenesis was impaired in the placental share of the smaller fetus and MALAT1 could regulate the paracrine effects of trophoblasts on endothelium angiogenesis and proliferation by regulating miR-424. In trophoblasts, MALAT1 could competitively bind to miR-424 to regulate the expression of ERRγ and HSD17B1, thus regulating trophoblast invasion and migration. MALAT1 overexpression could decrease apoptosis and promote proliferation, alleviating cell damage induced by hypoxia. Taken together, the downregulation of MALAT1 can reduce the expression of ERRγ and HSD17B1 by competitively binding to miR-424, impairing the proangiogenic effect of trophoblasts, trophoblast invasion and migration, and the ability of trophoblasts to compensate for hypoxia, which may be involved in the pathogenesis of sFGR through various aspects.

Asunto(s)

Movimiento Celular , Proliferación Celular , Retardo del Crecimiento Fetal , MicroARNs , ARN Largo no Codificante , Trofoblastos , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Humanos , Trofoblastos/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Movimiento Celular/genética , Femenino , Retardo del Crecimiento Fetal/genética , Retardo del Crecimiento Fetal/metabolismo , Retardo del Crecimiento Fetal/patología , Embarazo , Proliferación Celular/genética , Receptores de Estrógenos/metabolismo , Receptores de Estrógenos/genética , Apoptosis/genética , Neovascularización Fisiológica/genética , Placenta/metabolismo , Estradiol Deshidrogenasas

RESUMEN

Sterile inflammation after injury is important for tissue restoration. In injured human and mouse tissues, macrophages were recently found to accumulate perivascularly. This study investigates if macrophages adopt a mural cell phenotype important for restoration after ischemic injury. Single-cell RNA sequencing of fate-mapped macrophages from ischemic mouse muscles demonstrates a macrophage-toward-mural cell switch of a subpopulation of macrophages with downregulated myeloid cell genes and upregulated mural cell genes, including PDGFRß. This observation was further strengthened when including unspliced transcripts in the analysis. The macrophage switch was proven functionally relevant, as induction of macrophage-specific PDGFRß deficiency prevented their perivascular macrophage phenotype, impaired vessel maturation and increased vessel leakiness, which ultimately reduced limb function. In conclusion, macrophages in adult ischemic tissue were demonstrated to undergo a cellular program to morphologically, transcriptomically and functionally resemble mural cells while weakening their macrophage identity. The macrophage-to-mural cell-like phenotypic switch is crucial for restoring tissue function and warrants further exploration as a potential target for immunotherapies to enhance healing.

Asunto(s)

Modelos Animales de Enfermedad , Isquemia , Macrófagos , Animales , Macrófagos/metabolismo , Macrófagos/inmunología , Isquemia/metabolismo , Isquemia/patología , Isquemia/genética , Fenotipo , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Músculo Esquelético/lesiones , Cicatrización de Heridas/genética , Cicatrización de Heridas/fisiología , Ratones Endogámicos C57BL , Ratones , Masculino , Miembro Posterior/irrigación sanguínea , Neovascularización Fisiológica/genética , Regulación hacia Arriba , Transcriptoma , Análisis de la Célula Individual , Biomarcadores/metabolismo , Recuperación de la Función , Ratones Noqueados

RESUMEN

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.

Asunto(s)

Diferenciación Celular , Células Endoteliales , Exosomas , Células Madre Mesenquimatosas , MicroARNs , Neovascularización Fisiológica , Osteogénesis , MicroARNs/genética , MicroARNs/metabolismo , Exosomas/metabolismo , Osteogénesis/genética , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Diferenciación Celular/genética , Neovascularización Fisiológica/genética , Animales , Células Endoteliales/metabolismo , Células Endoteliales/citología , Ratones , Humanos , Células Cultivadas , Transducción de Señal , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción SOXB1/genética , Reprogramación Metabólica , Angiogénesis

RESUMEN

BACKGROUND: Bone tissue engineering offers a new approach for the treatment of bone defects, with angiogenesis being critical to the survival and development of tissue-engineered bone. Mineralized osteoblasts (MOBs) have been reported to promote vascular formation by endothelial cells (ECs) through the secretion of exosomes containing a variety of angiogenic factors. The aim of the present study was to investigate the effect of miR-423-5p contained within exosomes derived from MOBs (MOB-Exos) on EC angiogenesis. METHODS: The Cell Counting Kit-8 (CCK-8), scratch wound healing, Transwell migration, and tube formation assays were conducted to assess the in vitro effects of MOB-Exos on EC proliferation, migration, and tubule-forming capabilities. The miR-423-5p level in MOB-Exos was quantified using quantitative polymerase chain reaction (qPCR). Co-culture experiments were used to study the exosomal transport of miR-423-5p and its angiogenic effects. High-throughput sequencing was used to identify differentially expressed genes, and a dual luciferase reporter assay to determine whether CXCL10 was a direct target gene for miR-423-5p. Furthermore, the in vivo effect of MOB-Exos-derived miR-423-5p on angiogenesis was evaluated using a subcutaneous xenograft model. RESULTS: MOB-Exos significantly promoted the in vitro proliferation, migration, and tubule formation of ECs. A high level of miR-423-5p was found in MOB-Exos and promoted the angiogenesis of ECs. The CXCL10 gene was significantly downregulated in ECs upon miR-423-5p mimic transfection. Dual luciferase reporter assay confirmed the direct binding of miR-423-5p to the CXCL10 gene. miR-423-5p derived from MOB-Exos upregulated expression of the vascular markers CD31 and vascular endothelial growth factor (VEGF) in vivo, thus underscoring its angiogenic potential. CONCLUSION: This study found that miR-423-5p derived from MOB-Exos could potentially enhance EC angiogenesis via the regulation of CXCL10. Therefore, exosomes are promising therapeutic candidates for clinical bone defects.

Asunto(s)

Quimiocina CXCL10 , Exosomas , MicroARNs , Neovascularización Fisiológica , Osteoblastos , MicroARNs/genética , MicroARNs/metabolismo , Humanos , Osteoblastos/metabolismo , Osteoblastos/citología , Exosomas/metabolismo , Exosomas/genética , Animales , Neovascularización Fisiológica/genética , Quimiocina CXCL10/metabolismo , Quimiocina CXCL10/genética , Células Endoteliales/metabolismo , Proliferación Celular/genética , Movimiento Celular , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Ratones , Ratones Desnudos , Angiogénesis

RESUMEN

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.

Asunto(s)

Aorta , Microburbujas , Óxido Nítrico Sintasa de Tipo III , Óxido Nítrico , ARN Interferente Pequeño , Transfección , Animales , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico Sintasa de Tipo III/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Transfección/métodos , Aorta/metabolismo , Óxido Nítrico/metabolismo , Ratones , Masculino , Línea Celular , Neovascularización Fisiológica/genética

RESUMEN

This research explores the role of microRNA in senescence of human endothelial progenitor cells (EPCs) induced by replication. Hsa-miR-134-5p was found up-regulated in senescent EPCs where overexpression improved angiogenic activity. Hsa-miR-134-5p, which targeted transforming growth factor ß-activated kinase 1-binding protein 1 (TAB1) gene, down-regulated TAB1 protein, and inhibited phosphorylation of p38 mitogen-activated protein kinase (p38) in hsa-miR-134-5p-overexpressed senescent EPCs. Treatment with siRNA specific to TAB1 (TAB1si) down-regulated TAB1 protein and subsequently inhibited p38 activation in senescent EPCs. Treatment with TAB1si and p38 inhibitor, respectively, showed angiogenic improvement. In parallel, transforming growth factor Beta 1 (TGF-ß1) was down-regulated in hsa-miR-134-5p-overexpressed senescent EPCs and addition of TGF-ß1 suppressed the angiogenic improvement. Analysis of peripheral blood mononuclear cells (PBMCs) disclosed expression levels of hsa-miR-134-5p altered in adult life, reaching a peak before 65 years, and then falling in advanced age. Calculation of the Framingham risk score showed the score inversely correlates with the hsa-miR-134-5p expression level. In summary, hsa-miR-134-5p is involved in the regulation of senescence-related change of angiogenic activity via TAB1-p38 signalling and via TGF-ß1 reduction. Hsa-miR-134-5p has a potential cellular rejuvenation effect in human senescent EPCs. Detection of human PBMC-derived hsa-miR-134-5p predicts cardiovascular risk.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Enfermedades Cardiovasculares , Senescencia Celular , Células Progenitoras Endoteliales , Leucocitos Mononucleares , MicroARNs , Proteínas Quinasas p38 Activadas por Mitógenos , MicroARNs/genética , MicroARNs/metabolismo , Humanos , Células Progenitoras Endoteliales/metabolismo , Senescencia Celular/genética , Leucocitos Mononucleares/metabolismo , Persona de Mediana Edad , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Masculino , Enfermedades Cardiovasculares/genética , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/patología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Femenino , Anciano , Neovascularización Fisiológica/genética , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/genética , Adulto , Factores de Riesgo

RESUMEN

Diabetic foot ulcers, as one of the chronic wounds, are a serious challenge in the global healthcare system which have shown notable growth in recent years. DFU is associated with impairment in various stages of wound healing, including angiogenesis. Aberrant expression of microRNAs (miRNAs) involved in the disruption of the balance between angiogenic and anti-angiogenic factors, plays a crucial role in angiogenesis dysfunction. Alteration in the expression of angiomiRNAs (angiomiRs) have the potential to function as biomarkers in chronic wounds. Additionally, considering the rising importance of therapeutic RNAs, there is potential for utilizing angiomiRs in wound healing to induce angiogenesis. This review aims to explore angiogenesis in chronic wounds and investigate the mechanisms mediated by pro- and anti-angiomiRs in the context of diabetic foot ulcers.

Asunto(s)

Pie Diabético , MicroARNs , Cicatrización de Heridas , MicroARNs/genética , MicroARNs/metabolismo , Pie Diabético/genética , Pie Diabético/metabolismo , Humanos , Cicatrización de Heridas/genética , Animales , Neovascularización Patológica , Neovascularización Fisiológica/genética

RESUMEN

The poor healing characteristics of diabetic foot ulcers are partially attributed to diabetes-induced pro-inflammatory wounds. Our previous study reported that both miR-146a-5p and miR-200b-3p decrease endothelial inflammation in human aortic endothelial cells and db/db diabetic mice. Although miR-146a-5p has been reported to improve diabetic wound healing, the role of miR-200b-3p is not clear. This study compared the roles of these miRNAs in diabetic wound healing. Two 8-mm full-thickness wounds were created in 12-week-old male db/db mice on the left and right back. After surgery, 100 ng miR-146a-5p, miR-200b-3p, or miR-negative control (NC) was injected in each wound. Full-thickness skin samples were harvested from mice at the 14th day for real-time polymerase chain reaction and immunohistochemistry analyses. At the 14th day, the miR-200b-3p group showed better wound healing and greater granulation tissue thickness than the miR-146a-5p group. The miR-200b-3p group showed a significant decrease of IL-6 and IL-1ß gene expression and a significant increase of Col3α1 gene expression compared to those in the miR-NC group. The miR-200b-3p group had the lowest gene expression of TGF-ß1, followed by the miR-146a-5p and miR-NC groups. Our findings suggest that the miR-200b-3p group had better healing characteristics than the other two groups. Immunohistochemical staining revealed that CD68 immunoreactivity was significantly decreased in both the miR-146a-5p and miR-200b-3p groups compared with that in the miR-NC group. In addition, CD31 immunoreactivity was significantly higher in the miR-200b-3p group than in the miR-146a-5p group. In conclusion, these results suggest that miR-200b-3p is more effective than miR-146a-5p in promoting diabetic wound healing through its anti-inflammatory and pro-angiogenic effects.

Asunto(s)

MicroARNs , Cicatrización de Heridas , MicroARNs/genética , MicroARNs/metabolismo , Animales , Cicatrización de Heridas/genética , Masculino , Ratones , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/genética , Pie Diabético/genética , Pie Diabético/metabolismo , Pie Diabético/patología , Neovascularización Fisiológica/genética , Interleucina-6/metabolismo , Interleucina-6/genética , Antígenos de Diferenciación Mielomonocítica/metabolismo , Antígenos de Diferenciación Mielomonocítica/genética , Interleucina-1beta/metabolismo , Interleucina-1beta/genética , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Antígenos CD/genética , Antígenos CD/metabolismo , Piel/metabolismo , Piel/patología , Inflamación/genética , Inflamación/patología , Inflamación/metabolismo , Ratones Endogámicos C57BL , Molécula CD68

RESUMEN

Diabetes mellitus is a major cause of blindness and chronic ulcers in the working-age population worldwide. Wound healing is deeply dependent on neovascularization to restore blood flow. Former research has found that differentially expressed circular RNAs (circRNAs) are associated with hyperglycaemia-induced endothelial cell damage, and hypoxia-pretreated adipose-derived stem cells (ADSCs)-extracellular vesicle (HEV) transplants have a more therapeutic effect to enhance wound healing in diabetic mice by delivery circRNA. The current investigation employed high-throughput sequencing to identify circRNAs that are abnormally expressed between EV and HEV. The regulatory mechanism and predicted targets of one differentially expressed circRNA, circ-IGF1R, were investigated utilizing bioinformatics analyses, luciferase reporter assays, angiogenic differentiation assays, flow cytometric apoptosis analysis and RT-qPCR. Circ-IGF1R expression increased in HEV, and downregulation of circ-IGF1R suppressed and reversed the promotion effect of HEV on angiogenesis in ulcerated tissue. Bioinformatics analyses and luciferase reporter assays confirmed that miR-503-5p was the downstream target of circ-IGF1R, and inhibiting miR-503-5p restored the promotion effect of HEV on angiogenesis after circ-IGF1R silence. The study also found that miR-503-5p can interact with 3'-UTR of both HK2 and VEGFA. Overexpression of HK2 or VEGFA restored the promotion effect of HExo on angiogenesis after circ-IGF1R silence. Overexpression miR-503-5p or silence HK2/VEGFA reversed the protective effect of circ-IGF1R to MLMECs angiogenic differentiation. Overexpression of circ-IGF1R increased the protective effect of HEV on the promotion of wound healing in mice with diabetes. Circ-IGF1R promotes HIF-1α expression through miR-503-5p sponging. Our data demonstrate that circ-IGF1R overexpression EVs from ADSCs suppress high glucose-induced endothelial cell damage by regulating miR-503-5p/HK2/VEGFA axis.

Asunto(s)

Vesículas Extracelulares , MicroARNs , ARN Circular , Receptor IGF Tipo 1 , Factor A de Crecimiento Endotelial Vascular , MicroARNs/genética , MicroARNs/metabolismo , ARN Circular/genética , ARN Circular/metabolismo , Animales , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/trasplante , Ratones , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Receptor IGF Tipo 1/metabolismo , Receptor IGF Tipo 1/genética , Humanos , Células Madre/metabolismo , Masculino , Regulación de la Expresión Génica , Cicatrización de Heridas/genética , Hipoxia de la Célula/genética , Transducción de Señal , Regulación hacia Arriba/genética , Neovascularización Fisiológica/genética

RESUMEN

The placenta, as a "transit station" between mother and fetus, has functions delivering nutrients, excreting metabolic wastes and secreting hormones. A healthy placenta is essential for fetal growth and development while the melatonergic system seems to play a critical physiological role in this organ since melatonin, its synthetic enzymes and receptors are present in the placenta. In current study, Mtnr1a and Mtnr1b knockout mice were constructed to explore the potential roles of melatonergic system played on the placental function and intrauterine growth retardation (IUGR). The result showed that Mtnr1a knockout had little effect on placental function while Mtnr1b knockout reduced placental efficiency and increased IUGR. Considering the extremely high incidence of IURG in sows, the pregnant sows were treated with melatonin. This treatment reduced the incidence of IUGR. All the evidence suggests that the intact melatonergic system in placenta is required for its function. Mechanistical studies uncovered that Mtnr1b knockout increased placental oxidative stress and apoptosis but reduced the angiogenesis. The RNA sequencing combined with histochemistry study identified the reduced angiogenesis and placental vascular density in Mtnr1b knockout mice. These alterations were mediated by the disrupted STAT3/VEGFR2/PI3K/AKT pathway, i.e., Mtnr1b knockout reduced the phosphorylation of STAT3 which is the promotor of VEGFR2. The downregulated VEGFR2 and its downstream elements of PI3K and AKT expressions, then, jeopardizes the angiogenesis and placental development.

Asunto(s)

Retardo del Crecimiento Fetal , Melatonina , Ratones Noqueados , Neovascularización Fisiológica , Placenta , Receptor de Melatonina MT2 , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular , Receptor 2 de Factores de Crecimiento Endotelial Vascular , Animales , Femenino , Embarazo , Placenta/metabolismo , Placenta/irrigación sanguínea , Retardo del Crecimiento Fetal/genética , Retardo del Crecimiento Fetal/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Neovascularización Fisiológica/efectos de los fármacos , Neovascularización Fisiológica/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Melatonina/farmacología , Receptor de Melatonina MT2/genética , Receptor de Melatonina MT2/metabolismo , Ratones , Receptor de Melatonina MT1/genética , Receptor de Melatonina MT1/metabolismo , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/genética , Apoptosis , Ratones Endogámicos C57BL , Estrés Oxidativo , Porcinos , Angiogénesis

RESUMEN

Knowledge of the molecular mechanisms that underlie the regulation of major adaptive responses to an unbalanced oxygen tension is central to understanding tissue homeostasis and disease. Hypoxia-inducible transcription factors (HIFs) coordinate changes in the transcriptome that control these adaptive responses. Here, we focused on the functional role of the transcriptional repressor basic-helix-loop-helix family member e40 (Bhlhe40), which we previously identified in a meta-analysis as one of the most consistently upregulated genes in response to hypoxia across various cell types. We investigated the role of Bhlhe40 in controlling proliferation and angiogenesis using a gene editing strategy in mouse embryonic stem cells (mESCs) that we differentiated in embryoid bodies (EBs). We observed that hypoxia-induced Bhlhe40 expression was compatible with the rapid proliferation of pluripotent mESCs under low oxygen tension. However, in EBs, hypoxia triggered a Bhlhe40-dependent cell cycle arrest in most progenitor cells and endothelial cells within vascular structures. Furthermore, Bhlhe40 knockout increased the basal vascularization of the EBs in normoxia and exacerbated the hypoxia-induced vascularization, supporting a novel role for Bhlhe40 as a negative regulator of blood vessel formation. Our findings implicate Bhlhe40 in mediating key functional adaptive responses to hypoxia, such as proliferation arrest and angiogenesis.

Asunto(s)

Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Hipoxia de la Célula , Proliferación Celular , Cuerpos Embrioides , Células Madre Embrionarias de Ratones , Neovascularización Fisiológica , Animales , Ratones , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Cuerpos Embrioides/metabolismo , Cuerpos Embrioides/citología , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Embrionarias de Ratones/citología , Neovascularización Fisiológica/genética , Diferenciación Celular/genética , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/genética , Células Endoteliales/metabolismo , Angiogénesis

RESUMEN

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.

Asunto(s)

Diferenciación Celular , Cilios , Neovascularización Fisiológica , Hipófisis , Animales , Ratones , Angiogénesis , Cilios/metabolismo , Cilios/fisiología , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Ratones Noqueados , Neovascularización Fisiológica/genética , Neovascularización Fisiológica/fisiología , Hipófisis/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Quinasas DyrK/genética

RESUMEN

AIM: Impaired wound healing in patients with diabetes can develop into nonhealing ulcerations. Because bone marrow mesenchymal stem cells (BMSCs) exosomes can promote wound healing, this study aims to investigate the mechanism of BMSCs-isolated exosomal miR-221-3p in angiogenesis and diabetic wound healing. METHODS: To mimic diabetes in vitro, human umbilical vein endothelial cells (HUVECs) were subjected to high glucose (HG). Exosomes were derived from BMSCs and identified by transmission electron microscopy (TEM), western blot analysis and dynamic light scattering (DLS). The ability to differentiate BMSCs was assessed via Oil red O staining, alkaline phosphatase (ALP) staining and alizarin red staining. The ability to internalise PKH26-labelled exosomes was assessed using confocal microscopy. Migration, cell viability and angiogenesis were tested by scratch, MTT and tube formation assays separately. The miRNA and protein levels were analysed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) or western blotting. The relationship among miR-221-3p, FOXP1 and SPRY1 was determined using the dual-luciferase reporter, ChIP and RIP assays. RESULTS: Exosomal miR-221-3p was successfully isolated from BMSCs and delivered into HUVECs. HG was found to suppress the angiogenesis, cell viability and migration of HUVECs and exosomal miR-221-3p separated from BMSCs inhibited the above phenomenon. FOXP1 could transcriptionally upregulate SPRY1, and the silencing of FOXP1 reversed the HG-stimulated angiogenesis inhibition, cell viability and migration in HUVECs via the downregulation of SPRY1. Meanwhile, miR-221-3p directly targeted FOXP1 and the overexpression of FOXP1 reversed the positive effect of exosomal miR-221-3p on HUVEC angiogenesis. CONCLUSION: Exosomal miR-221-3p isolated from BMSCs promoted angiogenesis in diabetic wounds through the mediation of the FOXP1/SPRY1 axis. Furthermore, the findings of this study can provide new insights into probing strategies against diabetes.

Asunto(s)

Angiogénesis , Factores de Transcripción Forkhead , Células Madre Mesenquimatosas , MicroARNs , Neovascularización Fisiológica , Proteínas Represoras , Cicatrización de Heridas , Humanos , Movimiento Celular/genética , Regulación hacia Abajo , Exosomas/metabolismo , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción Forkhead/genética , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Madre Mesenquimatosas/metabolismo , MicroARNs/metabolismo , MicroARNs/genética , Neovascularización Fisiológica/genética , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Cicatrización de Heridas/genética

RESUMEN

INTRODUCTION: Recurrent pregnancy loss (RPL) is defined as the spontaneous loss of two or more consecutive pregnancies before 20 weeks of gestation, and affects 7.46 % of the Indian population. About 40-50 % of RPL cases are idiopathic making it a therapeutic challenge for clinicians. This study focuses on elucidating the role of hypoxia-associated placental angiogenesis in these idiopathic RPL cases. METHODS: Whole blood and product of conception (POCs) were collected from RPL patients (N = 87) and cases of voluntary abortions (medically terminated pregnancy, MTP; n = 110) as controls with informed consent. Serum separated from whole blood was used to study the ROS-antioxidant status in the cases and controls through colorimetric assays and ELISA. The mRNA extracted from placental tissue samples were used to determine the hypoxic and angiogenic status in cases and controls through real time PCR. Statistical analysis was also carried out to correlate the differential hypoxic status between RPL and MTP cohorts with the expression of angiogenic factors (VEGFA, VEGFR1 and VEGFR2). RESULTS: HIF1α mRNA expression was found to be upregulated in the RPL cases. While the serum levels of H2O2 (p = 0.012), guanine oxides and lipid hydroperoxides (LPO) were increased in the RPL cases, reduced glutathione (GSH) was found to be significantly decreased (p = 0.012). Additionally, AUROC analysis also shows an excellent discriminatory ability of 0.850 for serum H2O2 levels. VEGF-A and VEGF-R1 mRNA expression was also found to be downregulated in the RPL cases compared to MTP. DISCUSSION: This study indicates that increased oxidative stress may lead to aberrations in the VEGF pathway resulting in improper placentation in RPL cases, and subsequently, pregnancy loss.

Asunto(s)

Aborto Habitual , Placenta , Humanos , Femenino , Embarazo , Aborto Habitual/metabolismo , Aborto Habitual/genética , Placenta/metabolismo , Placenta/irrigación sanguínea , Adulto , India , Hipoxia/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Activación Transcripcional , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Estudios de Casos y Controles , Neovascularización Fisiológica/genética , Adulto Joven , Angiogénesis

RESUMEN

BACKGROUND: Peripheral artery disease (PAD) is an ischemic disease with a rising incidence worldwide. The lncRNA H19 (H19) is enriched in endothelial progenitor cells (EPCs), and transplantation of pyroptosis-resistant H19-overexpressed EPCs (oe-H19-EPCs) may promote vasculogenesis and blood flow recovery in PAD, especially with critical limb ischemia (CLI). METHODS: EPCs isolated from human peripheral blood was characterized using immunofluorescence and flow cytometry. Cell proliferation was determined with CCK8 and EdU assays. Cell migration was assessed by Transwell and wound healing assays. The angiogenic potential was evaluated using tube formation assay. The pyroptosis pathway-related protein in EPCs was detected by western blot. The binding sites of H19 and FADD on miR-107 were analyzed using Luciferase assays. In vivo, oe-H19-EPCs were transplanted into a mouse ischemic limb model, and blood flow was detected by laser Doppler imaging. The transcriptional landscape behind the therapeutic effects of oe-H19-EPCs on ischemic limbs were examined with whole transcriptome sequencing. RESULTS: Overexpression of H19 in EPCs led to an increase in proliferation, migration, and tube formation abilities. These effects were mediated through pyroptosis pathway, which is regulated by the H19/miR-107/FADD axis. Transplantation of oe-H19-EPCs in a mouse ischemic limb model promoted vasculogenesis and blood flow recovery. Whole transcriptome sequencing indicated significant activation of vasculogenesis pathway in the ischemic limbs following treatment with oe-H19-EPCs. CONCLUSIONS: Overexpression of H19 increases FADD level by competitively binding to miR-107, leading to enhanced proliferation, migration, vasculogenesis, and inhibition of pyroptosis in EPCs. These effects ultimately promote the recovery of blood flow in CLI.

Asunto(s)

Células Progenitoras Endoteliales , Proteína de Dominio de Muerte Asociada a Fas , Isquemia , MicroARNs , Piroptosis , ARN Largo no Codificante , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Piroptosis/genética , Células Progenitoras Endoteliales/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Isquemia/metabolismo , Isquemia/patología , Isquemia/genética , Humanos , Animales , Ratones , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Proteína de Dominio de Muerte Asociada a Fas/genética , Masculino , Extremidad Inferior/irrigación sanguínea , Extremidad Inferior/patología , Movimiento Celular/genética , Proliferación Celular , Neovascularización Fisiológica/genética , Ratones Endogámicos C57BL , Enfermedad Arterial Periférica/metabolismo , Enfermedad Arterial Periférica/patología , Enfermedad Arterial Periférica/genética , Modelos Animales de Enfermedad

RESUMEN

BACKGROUND: Deep vein thrombosis (DVT) is a common vascular surgical disease caused by the coagulation of blood in the deep veins, and predominantly occur in the lower limbs. Endothelial progenitor cells (EPCs) are multi-functional stem cells, which are precursors of vascular endothelial cells. EPCs have gradually evolved into a promising treatment strategy for promoting deep vein thrombus dissolution and recanalization through the stimulation of various physical and chemical factors. METHODS: In this study, we utilized a mouse DVT model and performed several experiments including qRT-PCR, Western blot, tube formation, wound healing, Transwell assay, immunofluorescence, flow cytometry analysis, and immunoprecipitation to investigate the role of HOXD9 in the function of EPCs cells. The therapeutic effect of EPCs overexpressing HOXD9 on the DVT model and its mechanism were also explored. RESULTS: Overexpression of HOXD9 significantly enhanced the angiogenesis and migration abilities of EPCs, while inhibiting cell apoptosis. Additionally, results indicated that HOXD9 specifically targeted the HRD1 promoter region and regulated the downstream PINK1-mediated mitophagy. Interestingly, intravenous injection of EPCs overexpressing HOXD9 into mice promoted thrombus dissolution and recanalization, significantly decreasing venous thrombosis. CONCLUSIONS: The findings of this study reveal that HOXD9 plays a pivotal role in stimulating vascular formation in endothelial progenitor cells, indicating its potential as a therapeutic target for DVT management.

Asunto(s)

Modelos Animales de Enfermedad , Células Progenitoras Endoteliales , Proteínas de Homeodominio , Mitofagia , Neovascularización Fisiológica , Trombosis de la Vena , Animales , Células Progenitoras Endoteliales/metabolismo , Ratones , Trombosis de la Vena/metabolismo , Trombosis de la Vena/genética , Trombosis de la Vena/terapia , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/genética , Mitofagia/genética , Neovascularización Fisiológica/genética , Movimiento Celular , Masculino , Apoptosis , Humanos , Angiogénesis

RESUMEN

MicroRNAs (miRNAs) are non-coding RNAs involved in the regulation of gene expression associated with cell differentiation, proliferation, adhesion, and important biological functions such as inflammation. miRNAs play roles associated with the pathogenesis of chronic degenerative disorders including cardiovascular diseases. Understanding the influence of miRNAs and their target genes can effectively streamline the identification of key biologically active pathways that are important in the development of vascular grafts through the tissue engineering of blood vessels. To determine miRNA expression levels and identify miRNA target genes and pathways with biological roles in scaffolds that have been repopulated with adipose-derived stem cells (ASCs) generated through tissue engineering for the construction of blood vessels. miRNA quantification assays were performed in triplicate to determine miRNA expression in a total of 20 samples: five controls (natural inferior vena cava), five scaffolds recellularized with ASCs and differentiated into the endothelium (luminal layer), five samples of complete scaffolds seeded with ASCs differentiated into the endothelium (luminal layer) and smooth muscle (extraluminal layer), and five samples of ASC without cell differentiation. Several differentially expressed miRNAs were identified and predicted to modulate target genes with roles in key pathways associated with angiogenesis, vascular system control, and endothelial and smooth muscle regulation, including migration, proliferation, and growth. These findings underscore the involvement of these pathways in the regulatory mechanisms that are essential for vascular scaffold production through tissue engineering. Our research contributes to the knowledge of miRNA-regulated mechanisms, which may impact the design of vascular substitutes, and provide valuable insights for enhancing clinical practice. The molecular pathways regulated by miRNAs in tissue engineering of blood vessels (TEBV) allowed us to elucidate the main phenomena involved in cellular differentiation to constitute a blood vessel, with the main pathways being essential for angiogenesis, cellular differentiation, and differentiation into vascular smooth muscle.

Asunto(s)

Diferenciación Celular , MicroARNs , Ingeniería de Tejidos , Andamios del Tejido , MicroARNs/genética , MicroARNs/metabolismo , Ingeniería de Tejidos/métodos , Humanos , Andamios del Tejido/química , Diferenciación Celular/genética , Tejido Adiposo/metabolismo , Tejido Adiposo/citología , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/crecimiento & desarrollo , Regulación de la Expresión Génica , Neovascularización Fisiológica/genética , Células Madre/metabolismo , Células Madre/citología , Proliferación Celular/genética , Transducción de Señal

RESUMEN

Mitochondria are central to endothelial cell activation and angiogenesis, with the RNA polymerase mitochondrial (POLRMT) serving as a key protein in regulating mitochondrial transcription and oxidative phosphorylation. In our study, we examined the impact of POLRMT on angiogenesis and found that its silencing or knockout (KO) in human umbilical vein endothelial cells (HUVECs) and other endothelial cells resulted in robust anti-angiogenic effects, impeding cell proliferation, migration, and capillary tube formation. Depletion of POLRMT led to impaired mitochondrial function, characterized by mitochondrial depolarization, oxidative stress, lipid oxidation, DNA damage, and reduced ATP production, along with significant apoptosis activation. Conversely, overexpressing POLRMT promoted angiogenic activity in the endothelial cells. In vivo experiments demonstrated that endothelial knockdown of POLRMT, by intravitreous injection of endothelial specific POLRMT shRNA adeno-associated virus, inhibited retinal angiogenesis. In addition, inhibiting POLRMT with a first-in-class inhibitor IMT1 exerted significant anti-angiogenic impact in vitro and in vivo. Significantly elevated expression of POLRMT was observed in the retinal tissues of streptozotocin-induced diabetic retinopathy (DR) mice. POLRMT endothelial knockdown inhibited pathological retinal angiogenesis and mitigated retinal ganglion cell (RGC) degeneration in DR mice. At last, POLRMT expression exhibited a substantial increase in the retinal proliferative membrane tissues of human DR patients. These findings collectively establish the indispensable role of POLRMT in angiogenesis, both in vitro and in vivo.

Asunto(s)

ARN Polimerasas Dirigidas por ADN , Células Endoteliales de la Vena Umbilical Humana , Mitocondrias , Humanos , Animales , Ratones , Mitocondrias/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , Retinopatía Diabética/patología , Retinopatía Diabética/metabolismo , Retinopatía Diabética/genética , Ratones Endogámicos C57BL , Proliferación Celular , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Masculino , Neovascularización Fisiológica/genética , Movimiento Celular , Apoptosis , Angiogénesis

RESUMEN

This study explores the impact of senescence on autocrine C-C motif chemokine ligand 5 (CCL5) in human endothelial progenitor cell (EPCs), addressing the poorly understood decline in number and function of EPCs during ageing. We examined the effects of replication-induced senescence on CCL5/CCL5 receptor (CCR5) signalling and angiogenic activity of EPCs in vitro and in vivo. We also explored microRNAs controlling CCL5 secretion in senescent EPCs, its impact on EPC angiogenic activity, and validated our findings in humans. CCL5 secretion and CCR5 levels in senescent EPCs were reduced, leading to attenuated angiogenic activity. CCL5 enhanced EPC proliferation via the CCR5/AKT/P70S6K axis and increased vascular endothelial growth factor (VEGF) secretion. Up-regulation of miR-409 in senescent EPCs resulted in decreased CCL5 secretion, inhibiting the angiogenic activity, though these negative effects were counteracted by the addition of CCL5 and VEGF. In a mouse hind limb ischemia model, CCL5 improved the angiogenic activity of senescent EPCs. Analysis involving 62 healthy donors revealed a negative association between CCL5 levels, age and Framingham Risk Score. These findings propose CCL5 as a potential biomarker for detection of EPC senescence and cardiovascular risk assessment, suggesting its therapeutic potential for age-related cardiovascular disorders.

Asunto(s)

Senescencia Celular , Quimiocina CCL5 , Células Progenitoras Endoteliales , MicroARNs , Neovascularización Fisiológica , Animales , Humanos , Masculino , Ratones , Angiogénesis , Proliferación Celular , Quimiocina CCL5/metabolismo , Quimiocina CCL5/genética , Regulación hacia Abajo/genética , Células Progenitoras Endoteliales/metabolismo , Células Progenitoras Endoteliales/citología , Isquemia/metabolismo , Isquemia/patología , Isquemia/genética , MicroARNs/genética , MicroARNs/metabolismo , Neovascularización Fisiológica/genética , Receptores CCR5/metabolismo , Receptores CCR5/genética , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética