RESUMEN
OBJECTIVE: The glucocorticoid receptor (GR) is a member of the nuclear receptor family that controls key biological processes in the cardiovascular system and has recently been shown to modulate Wnt signaling in endothelial cells. Wnt/ß-catenin signaling has been demonstrated to be crucial in the process of angiogenesis. In the current study, we studied whether GR could regulate angiogenesis via the Wnt/ß-catenin pathway. APPROACH AND RESULTSA: Key components of the Wnt/ß-catenin pathway were evaluated using quantitative PCR and Western blot in the presence or absence of GR. Enhanced angiogenesis was found in GR deficiency in vitro and confirmed with cell viability assays, proliferation assays and tube formation assays. Consistent with these in vitro findings, endothelial cell-specific GR loss GR in vivo promoted angiogenesis in both a hind limb ischemia model and sponge implantation assay. Results were further verified in a novel mouse model lacking endothelial LRP5/6, a key receptor in canonical Wnt signaling, and showed substantially suppressed angiogenesis using these same in vitro and in vivo assays. To further investigate the mechanism of GR regulation of Wnt signaling, autophagy flux was investigated in endothelial cells by visualizing auto phagolysosomes as well as by assessing P62 degradation and LC3B conversion. Results indicated that potentiated autophagy flux participated in GR-Wnt regulation. CONCLUSIONS: Lack of endothelial GR triggers autophagy flux, leads to activation of Wnt/ß-catenin signaling and promotes angiogenesis. There may also be a synergistic interaction between autophagy and Wnt/ß-catenin signaling.
Asunto(s)
Neovascularización Fisiológica , Receptores de Glucocorticoides/deficiencia , Regulación hacia Arriba , Vía de Señalización Wnt , beta Catenina/metabolismo , Animales , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones , Ratones Noqueados , Receptores de Glucocorticoides/metabolismo , beta Catenina/genéticaRESUMEN
BACKGROUND: Vascular smooth muscle cells (SMCs) synthesize extracellular matrix (ECM) that contributes to tissue remodeling after revascularization interventions. The cytokine transforming growth factor ß (TGF-ß) is induced on tissue injury and regulates tissue remodeling and wound healing, but dysregulated signaling results in excess ECM deposition and fibrosis. The LIM (Lin11, Isl-1 & Mec-3) domain protein LIM domain only 7 (LMO7) is a TGF-ß1 target gene in hepatoma cells, but its role in vascular physiology and fibrosis is unknown. METHODS: We use carotid ligation and femoral artery denudation models in mice with global or inducible smooth muscle-specific deletion of LMO7, and knockout, knockdown, overexpression, and mutagenesis approaches in mouse and human SMC, and human arteriovenous fistula and cardiac allograft vasculopathy samples to assess the role of LMO7 in neointima and fibrosis. RESULTS: We demonstrate that LMO7 is induced postinjury and by TGF-ß in SMC in vitro. Global or SMC-specific LMO7 deletion enhanced neointimal formation, TGF-ß signaling, ECM deposition, and proliferation in vascular injury models. LMO7 loss of function in human and mouse SMC enhanced ECM protein expression at baseline and after TGF-ß treatment. TGF-ß neutralization or receptor antagonism prevented the exacerbated neointimal formation and ECM synthesis conferred by loss of LMO7. Notably, loss of LMO7 coordinately amplified TGF-ß signaling by inducing expression of Tgfb1 mRNA, TGF-ß protein, αv and ß3 integrins that promote activation of latent TGF-ß, and downstream effectors SMAD3 phosphorylation and connective tissue growth factor. Mechanistically, the LMO7 LIM domain interacts with activator protein 1 transcription factor subunits c-FOS and c-JUN and promotes their ubiquitination and degradation, disrupting activator protein 1-dependent TGF-ß autoinduction. Importantly, preliminary studies suggest that LMO7 is upregulated in human intimal hyperplastic arteriovenous fistula and cardiac allograft vasculopathy samples, and inversely correlates with SMAD3 phosphorylation in cardiac allograft vasculopathy. CONCLUSIONS: LMO7 is induced by TGF-ß and serves to limit vascular fibrotic responses through negative feedback regulation of the TGF-ß pathway. This mechanism has important implications for intimal hyperplasia, wound healing, and fibrotic diseases.
Asunto(s)
Proteínas con Dominio LIM/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Remodelación Vascular , Lesiones del Sistema Vascular/metabolismo , Animales , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Retroalimentación Fisiológica , Fibrosis , Hiperplasia , Integrina alfaVbeta3/metabolismo , Proteínas con Dominio LIM/deficiencia , Proteínas con Dominio LIM/genética , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/lesiones , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Neointima , Transducción de Señal , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta1/genética , Lesiones del Sistema Vascular/genética , Lesiones del Sistema Vascular/patologíaRESUMEN
Human-induced pluripotent stem cell-derived vascular smooth muscle cells (hiPSC-VSMCs) with proangiogenic properties have huge therapeutic potential. While hiPSC-VSMCs have already been utilized for wound healing using a biomimetic collagen scaffold, an in situ forming hydrogel mimicking the native environment of skin offers the promise of hiPSC-VSMC mediated repair and regeneration. Herein, the impact of a collagen type-I-hyaluronic acid (HA) in situ hydrogel cross-linked using a polyethylene glycol-based cross-linker on hiPSC-VSMCs viability and proangiogenic paracrine secretion was investigated. Our study demonstrated increases in cell viability, maintenance of phenotype and proangiogenic growth factor secretion, and proangiogenic activity in response to the conditioned medium. The optimally cross-linked and functionalized collagen type-I/HA hydrogel system developed in this study shows promise as an in situ hiPSC-VSMC carrier system for wound regeneration.
Asunto(s)
Colágeno/química , Ácido Hialurónico/química , Hidrogeles/química , Células Madre Pluripotentes Inducidas/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citologíaRESUMEN
OBJECTIVE: The importance of PI3K/Akt signaling in the vasculature has been demonstrated in several models, as global loss of Akt1 results in impaired postnatal ischemia- and VEGF-induced angiogenesis. The ubiquitous expression of Akt1, however, raises the possibility of cell-type-dependent Akt1-driven actions, thereby necessitating tissue-specific characterization. APPROACH AND RESULTS: Herein, we used an inducible, endothelial-specific Akt1-deleted adult mouse model (Akt1iECKO) to characterize the endothelial cell autonomous functions of Akt1 in the vascular system. Endothelial-targeted ablation of Akt1 reduces eNOS (endothelial nitric oxide synthase) phosphorylation and promotes both increased vascular contractility in isolated vessels and elevated diastolic blood pressures throughout the diurnal cycle in vivo. Furthermore, Akt1iECKO mice subject to the hindlimb ischemia model display impaired blood flow and decreased arteriogenesis. CONCLUSIONS: Endothelial Akt1 signaling is necessary for ischemic resolution post-injury and likely reflects the consequence of NO insufficiency critical for vascular repair.
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Aorta Torácica/enzimología , Células Endoteliales/enzimología , Isquemia/enzimología , Músculo Esquelético/irrigación sanguínea , Neovascularización Fisiológica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Vasoconstricción , Animales , Velocidad del Flujo Sanguíneo , Presión Sanguínea , Modelos Animales de Enfermedad , Miembro Posterior , Isquemia/genética , Isquemia/patología , Isquemia/fisiopatología , Masculino , Ratones Noqueados , Músculo Esquelético/enzimología , Músculo Esquelético/patología , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/deficiencia , Proteínas Proto-Oncogénicas c-akt/genética , Flujo Sanguíneo Regional , Transducción de SeñalRESUMEN
Interactions between collagenous extracellular matrices and von Willebrand factor (VWF) are critical for hemostasis and thrombosis. In the present study, we investigated the contribution of an extracellular matrix (ECM) abnormality to the bleeding diathesis in thrombospondin-2 (TSP2) knockout (KO) mice. First, we performed adoptive bone marrow transplantation and observed that introduction of wild-type (WT) marrow into lethally irradiated TSP2 KO mice did not rescue the bleeding diathesis. However, platelets in transplanted mice displayed an inherent aggregation defect, which complicated interpretation. Second, we performed interposition of arterial segments denuded of endothelium. Denuded TSP2 KO arteries grafted into WT mice remained patent in vivo. In contrast, WT grafts underwent thrombosis and were completely occluded within 24 to 48 hours. The nonthrombogenic property of the TSP2 KO ECM was confirmed in vitro by exposing platelets to TSP2 KO dermal fibroblast (DF)-derived ECM. To further probe the effect of TSP2 deficiency, ECM production and deposition by WT and TSP2 KO DFs was analyzed via polymerase chain reaction, immunofluorescence, and scanning electron microscopy and showed similar patterns. In addition, atomic force microscopy (AFM) analysis of WT and TSP2 KO ECM did not reveal differences in stiffness. In contrast, reduced VWF accumulation on TSP2 KO ECM was observed when matrices were subjected to plasma under physiological flow. AFM utilizing VWF-coated 2-µm beads confirmed the weak binding to TSP2 KO ECM, providing a mechanistic explanation for the lack of thrombus formation. Therefore, our studies show that ECM assembly is critical for interaction of collagen with VWF and subsequent thrombogenic responses.
Asunto(s)
Plaquetas/patología , Adhesión Celular/fisiología , Fibroblastos/patología , Trombosis/patología , Trombospondinas/fisiología , Factor de von Willebrand/metabolismo , Animales , Plaquetas/metabolismo , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Fibroblastos/metabolismo , Hemostasis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Adhesividad Plaquetaria , Trombosis/metabolismoRESUMEN
OBJECTIVE: It is widely accepted that the presence of a glycosaminoglycan-rich glycocalyx is essential for endothelialized vasculature health; in fact, a damaged or impaired glycocalyx has been demonstrated in many vascular diseases. Currently, there are no methods that characterize glycocalyx functionality, thus limiting investigators' ability to assess the role of the glycocalyx in vascular health. APPROACH AND RESULTS: We have developed novel, easy-to-use, in vitro assays that directly quantify live endothelialized surface's functional heparin weights and their anticoagulant capacity to inactivate Factor Xa and thrombin. Using our assays, we characterized 2 commonly used vascular models: native rat aorta and cultured human umbilical vein endothelial cell monolayer. We determined heparin contents to be ≈10 000 ng/cm(2) on the native aorta and ≈10-fold lower on cultured human umbilical vein endothelial cells. Interestingly, human umbilical vein endothelial cells demonstrated a 5-fold lower anticoagulation capacity in inactivating both Factor Xa and thrombin relative to native aortas. We verified the validity and accuracy of the novel assays developed in this work using liquid chromatography-mass spectrometry analysis. CONCLUSIONS: Our assays are of high relevance in the vascular community because they can be used to establish the antithrombogenic capacity of many different types of surfaces such as vascular grafts and transplants. This work will also advance the capacity for glycocalyx-targeting therapeutics development to treat damaged vasculatures.
Asunto(s)
Aorta Torácica/metabolismo , Bioensayo/métodos , Coagulación Sanguínea , Factor Xa/metabolismo , Glicocálix/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Trombina/metabolismo , Animales , Antitrombinas/metabolismo , Aorta Torácica/ultraestructura , Células Cultivadas , Cromatografía Liquida , Glicocálix/ultraestructura , Heparina/metabolismo , Heparitina Sulfato/metabolismo , Células Endoteliales de la Vena Umbilical Humana/ultraestructura , Masculino , Espectrometría de Masas , Microscopía Electrónica de Transmisión , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
The PI3K/Akt pathway is necessary for several key endothelial cell (EC) functions, including cell growth, migration, survival, and vascular tone. However, existing literature supports the idea that Akt can be either pro- or antiangiogenic, possibly due to compensation by multiple isoforms in the EC when a single isoform is deleted. Thus, biochemical, genetic, and proteomic studies were conducted to examine isoform-substrate specificity for Akt1 vs. Akt2. In vitro, Akt1 preferentially phosphorylates endothelial nitric oxide synthase (eNOS) and promotes NO release, whereas nonphysiological overexpression of Akt2 can bypass the loss of Akt1. Conditional deletion of Akt1 in the EC, in the absence or presence of Akt2, retards retinal angiogenesis, implying that Akt1 exerts a nonredundant function during physiological angiogenesis. Finally, proteomic analysis of Akt substrates isolated from Akt1- or Akt2-deficient ECs documents that phosphorylation of multiple Akt substrates regulating angiogenic signaling is reduced in Akt1-deficient, but not Akt2-deficient, ECs, including eNOS and Forkhead box proteins. Therefore, Akt1 promotes angiogenesis largely due to phosphorylation and regulation of important downstream effectors that promote aspects of angiogenic signaling.
Asunto(s)
Endotelio Vascular/metabolismo , Neovascularización Fisiológica/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Vasos Retinianos/metabolismo , Animales , Línea Celular Transformada , Pulmón/irrigación sanguínea , Pulmón/citología , Ratones , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo III/metabolismo , Compuestos de Mostaza Nitrogenada/metabolismo , Fosforilación/fisiología , Proteómica , Proteínas Proto-Oncogénicas c-akt/genética , Retina/patología , Vasos Retinianos/patología , Transducción de Señal/fisiología , Especificidad por SustratoRESUMEN
Vascular remodeling is essential for tissue repair and is regulated by multiple factors, including thrombospondin-2 (TSP2) and hypoxia/VEGF-induced activation of Akt. In contrast to TSP2 knock-out (KO) mice, Akt1 KO mice have elevated TSP2 expression and delayed tissue repair. To investigate the contribution of increased TSP2 to Akt1 KO mice phenotypes, we generated Akt1/TSP2 double KO (DKO) mice. Full-thickness excisional wounds in DKO mice healed at an accelerated rate when compared with Akt1 KO mice. Isolated dermal Akt1 KO fibroblasts expressed increased TSP2 and displayed altered morphology and defects in migration and adhesion. These defects were rescued in DKO fibroblasts or after TSP2 knockdown. Conversely, the addition of exogenous TSP2 to WT cells induced cell morphology and migration rates that were similar to those of Akt1 KO cells. Akt1 KO fibroblasts displayed reduced adhesion to fibronectin with manganese stimulation when compared with WT and DKO cells, revealing an Akt1-dependent role for TSP2 in regulating integrin-mediated adhesions; however, this effect was not due to changes in ß1 integrin surface expression or activation. Consistent with these results, Akt1 KO fibroblasts displayed reduced Rac1 activation that was dependent upon expression of TSP2 and could be rescued by a constitutively active Rac mutant. Our observations show that repression of TSP2 expression is a critical aspect of Akt1 function in tissue repair.
Asunto(s)
Fibroblastos/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Piel/metabolismo , Trombospondinas/genética , Heridas no Penetrantes/genética , Animales , Movimiento Celular , Fibroblastos/patología , Regulación de la Expresión Génica , Prueba de Complementación Genética , Integrina beta1/genética , Integrina beta1/metabolismo , Ratones , Ratones Noqueados , Neuropéptidos/genética , Neuropéptidos/metabolismo , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/deficiencia , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Piel/lesiones , Piel/patología , Trombospondinas/deficiencia , Cicatrización de Heridas/genética , Heridas no Penetrantes/metabolismo , Heridas no Penetrantes/patología , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Angiopoietin-2 (Ang2) is an extracellular protein and one of the principal ligands of Tie2 receptor that is involved in the regulation of vascular integrity, quiescence, and inflammation. The mode of secretion of Ang2 has never been established, however. Here, we provide evidence that Ang2 is secreted from endothelial cells via exosomes and that this process is inhibited by the PI3K/Akt/endothelial nitric oxide synthase (eNOS) signaling pathway, whereas it is positively regulated by the syndecan-4/syntenin pathway. Vascular defects in Akt1 null mice arise, in part, because of excessive Ang2 secretion and can be rescued by the syndecan-4 knock-out that reduces extracellular Ang2 levels. This novel mechanism connects three critical signaling pathways: angiopoietin/Tie2, PI3K/Akt/eNOS, and syndecan/syntenin, which play important roles in vascular growth and stabilization.
Asunto(s)
Angiopoyetina 2/metabolismo , Células Endoteliales/metabolismo , Exosomas/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/fisiología , Sindecano-4/metabolismo , Sinteninas/metabolismo , Angiopoyetina 2/genética , Animales , Células Cultivadas , Células Endoteliales/citología , Exosomas/genética , Ratones , Ratones Noqueados , Neovascularización Fisiológica/fisiología , Óxido Nítrico Sintasa de Tipo III/genética , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Sindecano-4/genética , Sinteninas/genéticaRESUMEN
BACKGROUND: Numerous proteins and small leucine-rich proteoglycans (SLRPs) make up the composition of the extracellular matrix (ECM). Assembly of individual fibrillar components in the ECM, such as collagen, elastin, and fibronectin, is understood at the molecular level. In contrast, the incorporation of non-fibrillar components and their functions in the ECM are not fully understood. SCOPE OF REVIEW: This review will focus on the role of the matricellular protein thrombospondin (TSP) 2 in ECM assembly. Based on findings in TSP2-null mice and in vitro studies, we describe the participation of TSP2 in ECM assembly, cell-ECM interactions, and modulation of the levels of matrix metalloproteinases (MMPs). MAJOR CONCLUSIONS: Evidence summarized in this review suggests that TSP2 can influence collagen fibrillogenesis without being an integral component of fibrils. Altered ECM assembly and excessive breakdown of ECM can have both positive and negative consequences including increased angiogenesis during tissue repair and compromised cardiac tissue integrity, respectively. GENERAL SIGNIFICANCE: Proper ECM assembly is critical for maintaining cell functions and providing structural support. Lack of TSP2 is associated with increased angiogenesis, in part, due to altered endothelial cell-ECM interactions. Therefore, minor changes in ECM composition can have profound effects on cell and tissue function. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.
Asunto(s)
Matriz Extracelular/metabolismo , Trombospondinas/metabolismo , Animales , Colágeno/metabolismo , Modelos Animales de Enfermedad , Matriz Extracelular/ultraestructura , Humanos , Fenotipo , Trombospondinas/deficiencia , Ingeniería de TejidosRESUMEN
BACKGROUND: Preterm premature rupture of membranes remains a major complication after fetoscopic laser surgery (FLS) for twin-twin transfusion syndrome (TTTS). We studied the histologic changes of fetal membranes post-FLS and investigated a possible impact of amniotic fluid (AF) dilution. METHODS: Fetal membranes of 31 pregnancies that underwent FLS for TTTS were investigated histologically at delivery at different sites: trocar site of recipient sac and at distance, donor sac, and inter-twin membrane. RESULTS: The trocar insertion site on the recipient sac showed no signs of histologic hallmarks of healing. Wide-spread alteration in collagen organization and higher apoptotic index in the amnion of the recipient sac which were absent in donor's and reference membranes. To explain the mechanisms, we analyzed the AF composition of recipient sacs from TTTS pregnancies vs. GA-matched healthy singleton controls and found glucose, protein and lactate dehydrogenase activity were all significantly lower in TTTS sacs consistent with over-dilution of recipient's AF (~2-fold). In-vitro exposure of healthy amniochorion to analogous dilutional stress conditions recapitulated the histologic changes and induced apoptosis and autophagy. CONCLUSION: Alteration in structural integrity of the recipient's amniochorion, possibly in response to dilution stress, along with ineffective repair mechanisms may explain the increased incidence of preterm birth post-FLS.
Asunto(s)
Membranas Extraembrionarias/patología , Transfusión Feto-Fetal/fisiopatología , Transfusión Feto-Fetal/cirugía , Fetoscopía/efectos adversos , Fetoscopía/métodos , Adulto , Líquido Amniótico/química , Apoptosis , Autofagia , Femenino , Rotura Prematura de Membranas Fetales/etiología , Rotura Prematura de Membranas Fetales/prevención & control , Edad Gestacional , Glucosa/análisis , Humanos , Inmunohistoquímica , L-Lactato Deshidrogenasa/metabolismo , Rayos Láser , Embarazo , Resultado del Embarazo , Nacimiento Prematuro , GemelosRESUMEN
Implantation of biomaterials in vascularized tissues elicits the sequential engagement of molecular and cellular elements that constitute the foreign body response. Initial events include the non-specific adsorption of proteins to the biomaterial surface that render it adhesive for cells such as neutrophils and macrophages. The latter undergo unique activation and in some cases undergo cell-cell fusion to form foreign body giant cells that contribute to implant damage and fibrotic encapsulation. In this review, we discuss the molecular events that contribute to macrophage activation and fusion with a focus on the role of the inflammasome, signaling pathways such as JAK/STAT and NF-κB, and the putative involvement of micro RNAs in the regulation of these processes.
Asunto(s)
Materiales Biocompatibles/efectos adversos , Fibroblastos/efectos de los fármacos , Reacción a Cuerpo Extraño/inmunología , Células Gigantes de Cuerpo Extraño/efectos de los fármacos , Activación de Macrófagos/efectos de los fármacos , Fibroblastos/inmunología , Fibroblastos/patología , Fibrosis , Reacción a Cuerpo Extraño/etiología , Reacción a Cuerpo Extraño/patología , Regulación de la Expresión Génica , Células Gigantes de Cuerpo Extraño/inmunología , Células Gigantes de Cuerpo Extraño/patología , Humanos , Inflamasomas/efectos de los fármacos , Inflamasomas/inmunología , Quinasas Janus/genética , Quinasas Janus/inmunología , MicroARNs/genética , MicroARNs/inmunología , FN-kappa B/genética , FN-kappa B/inmunología , Prótesis e Implantes/efectos adversos , Unión Proteica/efectos de los fármacos , Factores de Transcripción STAT/genética , Factores de Transcripción STAT/inmunología , Transducción de SeñalRESUMEN
Injury- and ischemia-induced angiogenesis is critical for tissue repair and requires nitric oxide (NO) derived from endothelial nitric oxide synthase (eNOS). We present evidence that NO induces angiogenesis by modulating the level of the angiogenesis inhibitor thrombospondin 2 (TSP2). TSP2 levels were higher than WT in eNOS KO tissues in hind-limb ischemia and cutaneous wounds. In vitro studies confirmed that NO represses TSP2 promoter activity. Moreover, double-eNOS/TSP2 KO mice were generated and found to rescue the phenotype of eNOS KO mice. Studies in mice with knock-in constitutively active or inactive eNOS on the Akt-1 KO background showed that eNOS activity correlates with TSP2 levels. Our observations of NO-mediated regulation of angiogenesis via the suppression of TSP2 expression provide a description of improved eNOS KO phenotype by means other than restoring NO signaling.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Óxido Nítrico Sintasa de Tipo III/metabolismo , Trombospondinas/biosíntesis , Animales , Matriz Extracelular/metabolismo , Células HEK293 , Humanos , Isquemia , Ratones , Ratones Noqueados , Células 3T3 NIH , Neovascularización Patológica , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/genética , Transducción de Señal , Piel/patología , Trombospondinas/genéticaRESUMEN
Implantation of biomaterials and devices into soft tissues leads to the development of the foreign body response (FBR), which can interfere with implant function and eventually lead to failure. The FBR consists of overlapping acute and persistent inflammatory phases coupled with collagenous encapsulation and currently there are no therapeutic options. Initiation of the FBR involves macrophage activation, proceeding to giant cell formation, fibroblast activation, and collagen matrix deposition. Despite the recognition of this sequence of events, the molecular pathways required for the FBR have not been elucidated. We have identified that the acute inflammatory response to biomaterials requires nucleotide-binding domain and leucine-rich repeat-containing 3 (Nlrp3), apoptosis-associated speck-like protein containing CARD (Asc), and caspase-1, as well as plasma membrane cholesterol, and Syk signaling. Full development of the FBR is dependent on Asc and caspase-1, but not Nlrp3. The common antiinflammatory drug aspirin can reduce inflammasome activation and significantly reduce the FBR. Taken together, these findings expand the role of the inflammasome from one of sensing damage associated molecular patterns (DAMPs) to sensing all particulate matter irrespective of size. In addition, implication of the inflammasome in biomaterial recognition identifies key pathways, which can be targeted to limit the FBR.
Asunto(s)
Materiales Biocompatibles/efectos adversos , Caspasa 1/metabolismo , Proteínas del Citoesqueleto/metabolismo , Reacción a Cuerpo Extraño/patología , Inflamasomas/metabolismo , Inflamación/patología , Administración Oral , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Aspirina/administración & dosificación , Aspirina/efectos adversos , Proteínas Adaptadoras de Señalización CARD , Proteínas de Unión al Calcio/metabolismo , Proteínas Portadoras/metabolismo , Análisis por Conglomerados , Reacción a Cuerpo Extraño/complicaciones , Reacción a Cuerpo Extraño/enzimología , Reacción a Cuerpo Extraño/inmunología , Células Gigantes/efectos de los fármacos , Células Gigantes/inmunología , Células Gigantes/patología , Inflamación/complicaciones , Inflamación/enzimología , Inflamación/inmunología , Interleucina-1beta/biosíntesis , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/patología , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Microesferas , Proteína con Dominio Pirina 3 de la Familia NLR , Polimetil Metacrilato/efectos adversosRESUMEN
Bone extracellular matrix (ECM) has been shown to mimic aspects of the tissue's complex microenvironment, suggesting its potential role in promoting bone repair. However, current ECM-based therapies suffer from limitations such as inefficient scale-up, lack of mechanical integrity, and sub-optimal efficacy. Here, we fabricated hydrogels from decellularized ECM (dECM) from wild type (WT) and thrombospondin-2 knockout (TSP2KO) mouse bones. TSP2KO bone ECM hydrogel was found to have distinct mechanical properties and collagen fibril assembly from WT. Furthermore, TSP2KO hydrogel promoted mesenchymal stem cell (MSC) attachment, spreading, and invasion in vitro. Similarly, it promoted formation of tube-like structures by human umbilical vein endothelial cells (HUVECs). When applied to a murine calvarial defect model, TSP2KO hydrogel enhanced repair, in part, due to increased angiogenesis. Our study suggests the pro-angiogenic therapeutic potential of TSP2KO bone ECM hydrogel in bone repair. STATEMENT OF SIGNIFICANCE: The study describes the first successful preparation of a novel hydrogel made from decellularized mouse bones. Bones from wild-type mice and mice lacking thrombospondin 2 (TSP2) were used to fabricate the gels. Hydrogels from TSP2KO bones have unique characteristics in structure and biomechanics. These gels interacted well with cells in vitro and helped repair damaged bone in a mouse model. Therefore, TSP2KO bone-derived hydrogel has translational potential for accelerating repair of bone defects that are otherwise difficult to heal. This study not only creates a new material with promise for healing, but also validates tunability of native biomaterials by genetic engineering.
RESUMEN
Volumetric muscle loss (VML) represents a clinical challenge due to the limited regenerative capacity of skeletal muscle. Most often, it results in scar tissue formation and loss of function, which cannot be prevented by current therapies. Decellularized extracellular matrix (DEM) has emerged as a native biomaterial for the enhancement of tissue repair. Here, we report the generation and characterization of hydrogels derived from DEM prepared from WT or thrombospondin (TSP)-2 null muscle tissue. TSP2-null hydrogels, when compared to WT, displayed altered architecture, protein composition, and biomechanical properties and allowed enhanced invasion of C2C12 myocytes and chord formation by endothelial cells. They also displayed enhanced cell invasion, innervation, and angiogenesis following subcutaneous implantation. To evaluate their regenerative capacity, WT or TSP2 null hydrogels were used to treat VML injury to tibialis anterior muscles and the latter induced greater recruitment of repair cells, innervation, and blood vessel formation and reduced inflammation. Taken together, these observations indicate that TSP2-null hydrogels enhance angiogenesis and promote muscle repair in a VML model.
Asunto(s)
Células Endoteliales , Hidrogeles , Hidrogeles/farmacología , Angiogénesis , Matriz Extracelular/metabolismo , Músculo Esquelético , NeurogénesisRESUMEN
HuR is a member of the Drosophila Elav protein family that binds mRNA degradation sequences and prevents RNase-mediated degradation. Such HuR-mediated mRNA stabilization, which is stimulated by integrin engagement and is controlled at the level of HuR nuclear export, is critically involved in T-cell cytokine production. However, HuR's role in macrophage soluble factor production, in particular in response to angiogenic stimuli, has not yet been established. We show that the labile transcripts that encode vascular endothelial growth factor and matrix metalloproteinase-9 are stabilized when murine macrophages adhere to the ß(2) integrin ligand intercellular adhesion molecule-1. This mRNA stabilization response was absent in bone marrow-derived macrophages obtained from conditional macrophage-specific HuR knockout mice. The microvascular angiogenic response to an inflammatory stimulus (ie, subcutaneous polyvinyl alcohol sponge implantation) was markedly diminished in these macrophage HuR knockout mice despite the equal levels of macrophage localization to those observed in littermate wild-type controls. Furthermore, blood flow recovery and ischemic muscle neovascularization after femoral artery ligation were impaired in the conditional macrophage-specific HuR knockout mice. These results demonstrate that dynamic effects on mRNA, mediated by the RNA-binding and RNA-stabilizing protein HuR, are required for macrophage production of angiogenic factors, which play critical roles in the neovascular responses to a variety of stimuli, including tissue ischemia.
Asunto(s)
Inductores de la Angiogénesis/metabolismo , Antígenos CD18/fisiología , Proteínas ELAV/fisiología , Macrófagos/metabolismo , Neovascularización Patológica/metabolismo , Animales , Adhesión Celular/fisiología , Células Cultivadas , Modelos Animales de Enfermedad , Proteínas ELAV/deficiencia , Proteínas ELAV/genética , Regulación de la Expresión Génica/fisiología , Técnicas de Inactivación de Genes , Miembro Posterior/irrigación sanguínea , Inflamación/complicaciones , Isquemia/genética , Isquemia/fisiopatología , Ratones , Ratones Noqueados , Músculo Esquelético/irrigación sanguínea , Neovascularización Patológica/etiología , Neovascularización Patológica/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodosRESUMEN
Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are the earliest tissue-engineered vascular grafts (TEVGs) to be used clinically. These TEVGs transform into living blood vessels in vivo, with an endothelial cell (EC) lining invested by smooth muscle cells (SMCs); however, the process by which this occurs is unclear. To test if the seeded BMCs differentiate into the mature vascular cells of the neovessel, we implanted an immunodeficient mouse recipient with human BMC (hBMC)-seeded scaffolds. As in humans, TEVGs implanted in a mouse host as venous interposition grafts gradually transformed into living blood vessels over a 6-month time course. Seeded hBMCs, however, were no longer detectable within a few days of implantation. Instead, scaffolds were initially repopulated by mouse monocytes and subsequently repopulated by mouse SMCs and ECs. Seeded BMCs secreted significant amounts of monocyte chemoattractant protein-1 and increased early monocyte recruitment. These findings suggest TEVGs transform into functional neovessels via an inflammatory process of vascular remodeling.
Asunto(s)
Implantación de Prótesis Vascular/métodos , Prótesis Vascular , Vasos Sanguíneos/fisiopatología , Ingeniería de Tejidos/métodos , Animales , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patología , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/ultraestructura , Técnicas de Cultivo de Célula , Diferenciación Celular , Células Cultivadas , Quimiocina CCL2/metabolismo , Humanos , Inmunohistoquímica , Inflamación/fisiopatología , Ratones , Ratones SCID , Microscopía Electrónica de Rastreo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Andamios del Tejido , Trasplante HeterólogoRESUMEN
Homotypic cell fusion occurs in several cell types including macrophages in the formation of foreign body giant cells. Previously, monocyte chemoattractant protein-1 (MCP-1) was demonstrated to be required for foreign body giant cell formation in the foreign body response. The present study investigated the fusion defect in MCP-1-null macrophages by implanting biomaterials intraperitoneally in wild-type and MCP-1-null mice and monitoring the macrophage response at 12 hours to 4 weeks. MCP-1-null mice exhibited reduced accumulation and fusion of macrophages on implants, which was associated with attenuation of the foreign body response. Consistent with previous in vitro findings, the level of matrix metalloproteinase-9 (MMP-9) was reduced in MCP-1-null macrophages adherent to implants. In contrast, CCR2 expression was unaffected. In vitro studies revealed reduced tumor necrosis factor-α (TNF-α) production and abnormal subcellular redistribution of E-cadherin and ß-catenin during fusion in MCP-1-null macrophages. Exogenous TNF-α caused an increase in the production of MMP-9 and rescued the fusion defect. Addition of GM6001 (MMP inhibitor) or NSC23766 (Rac1 inhibitor) indicated two distinct induction pathways, one for E-cadherin/ß-catenin and one for MCP-1, TNF-α, and MMP-9. Considered together, these observations demonstrate that induction of E-cadherin/ß-catenin is not sufficient for fusion in the absence of MCP-1 or the downstream mediators TNF-α and MMP-9. Moreover, attenuation of the foreign body response in intraperitoneal implants in MCP-1-null mice demonstrates that the process depends on tissue-specific factors.
Asunto(s)
Cadherinas/metabolismo , Quimiocina CCL2/metabolismo , Células Gigantes de Cuerpo Extraño/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Western Blotting , Adhesión Celular/fisiología , Quimiotaxis de Leucocito/fisiología , Ensayo de Inmunoadsorción Enzimática , Inmunohistoquímica , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Microscopía Electrónica de Transmisión , Reacción en Cadena de la PolimerasaRESUMEN
Thrombospondin (TSP)-2-null mice have an altered brain foreign body response (FBR) characterized by increases in inflammation, extracellular matrix deposition, and leakage of the blood-brain barrier (BBB). In the present study, we investigated the role of TSP-2 in BBB repair during the brain FBR to mixed cellulose ester filters implanted in the cortex of wild-type (WT) and TSP-2-null mice for 2 days to 8 weeks. Histological and immunohistochemical analysis revealed enhanced and prolonged neuroinflammation in TSP-2-null mice up to 8 weeks after implantation. In addition, recovery of the BBB was compromised and was associated with increased gelatinolytic activity and low levels of collagen type IV in the basement membranes of TSP-2-null blood vessels. An analysis of protein extracts from implantation sites revealed elevated levels of matrix metalloproteinase (MMP)-2 and MMP-9 in TSP-2-null brains. TSP-2-null astrocytes secreted higher levels of both MMPs in vitro compared with their WT counterparts. Furthermore, TSP-2-null astrocytes were deficient in supporting the recovery of barrier function in WT endothelial cells. Finally, Western blot analysis of astrocytes and brain endothelial cells revealed TSP-2 expression only in the former. Taken together, our observations suggest that astrocyte-derived TSP-2 is critical for the maintenance of physiological MMP-2 and MMP-9 levels during the FBR and contributes to the repair of the BBB.