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In the context of arteriovenous fistula (AVF) failure, local delivery enables the release of higher concentrations of drugs that can suppress neointimal hyperplasia (NIH) while reducing systemic adverse effects. However, the radiolucency of polymeric delivery systems hinders long-term in vivo surveillance of safety and efficacy. We hypothesize that using a radiopaque perivascular wrap to deliver anti-NIH drugs could enhance AVF maturation. Through electrospinning, we fabricated multifunctional perivascular polycaprolactone (PCL) wraps loaded with bismuth nanoparticles (BiNPs) for enhanced radiologic visibility and drugs that can attenuate NIHârosuvastatin (Rosu) and rapamycin (Rapa). The following groups were tested on the AVFs of a total of 24 Sprague-Dawley rats with induced chronic kidney disease: control (i.e., without wrap), PCL-Bi (i.e., wrap with BiNPs), PCL-Bi-Rosu, and PCL-Bi-Rapa. We found that BiNPs significantly improved the wraps' radiopacity without affecting biocompatibility. The drug release profiles of Rosu (hydrophilic drug) and Rapa (hydrophobic drug) differed significantly. Rosu demonstrated a burst release followed by gradual tapering over 8 weeks, while Rapa demonstrated a gradual release similar to that of the hydrophobic BiNPs. In vivo investigations revealed that both drug-loaded wraps can reduce vascular stenosis on ultrasonography and histomorphometry, as well as reduce [18F]Fluorodeoxyglucose uptake on positron emission tomography. Immunohistochemical studies revealed that PCL-Bi-Rosu primarily attenuated endothelial dysfunction and hypoxia in the neointimal layer, while PCL-Bi-Rapa modulated hypoxia, inflammation, and cellular proliferation across the whole outflow vein. In summary, the controlled delivery of drugs with different properties and mechanisms of action against NIH through a multifunctional, radiopaque perivascular wrap can improve imaging and histologic parameters of AVF maturation.
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Bismuto , Ratas Sprague-Dawley , Rosuvastatina Cálcica , Sirolimus , Animales , Ratas , Sirolimus/química , Sirolimus/farmacología , Rosuvastatina Cálcica/química , Rosuvastatina Cálcica/farmacología , Rosuvastatina Cálcica/farmacocinética , Bismuto/química , Bismuto/farmacología , Poliésteres/química , Masculino , Fístula Arteriovenosa/patología , Nanopartículas del Metal/química , Neointima/patología , Nanopartículas/química , Humanos , Liberación de FármacosRESUMEN
Bioresorbable perivascular scaffolds loaded with antiproliferative agents have been shown to enhance arteriovenous fistula (AVF) maturation by inhibiting neointimal hyperplasia (NIH). These scaffolds, which can mimic the three-dimensional architecture of the vascular extracellular matrix, also have an untapped potential for the local delivery of cell therapies against NIH. Hence, an electrospun perivascular scaffold from polycaprolactone (PCL) to support mesenchymal stem cell (MSC) attachment and gradual elution at the AVF's outflow vein is fabricated. Chronic kidney disease (CKD) in Sprague-Dawley rats is induced by performing 5/6th nephrectomy, then AVFs for scaffold application are created. The following groups of CKD rats are compared: no perivascular scaffold (i.e., control), PCL alone, and PCL+MSC scaffold. PCL and PCL+MSC significantly improve ultrasonographic (i.e., luminal diameter, wall-to-lumen ratio, and flow rate) and histologic (i.e., neointima-to-lumen ratio, neointima-to-media ratio) parameters compared to control, with PCL+MSC demonstrating further improvement in these parameters compared to PCL alone. Moreover, only PCL+MSC significantly reduces 18 F-fluorodeoxyglucose uptake on positron emission tomography. These findings suggest that adding MSCs promotes greater luminal expansion and potentially reduces the inflammatory process underlying NIH. The results demonstrate the utility of mechanical support loaded with MSCs at the outflow vein immediately after AVF formation to support maturation by minimizing NIH.
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Fístula Arteriovenosa , Derivación Arteriovenosa Quirúrgica , Células Madre Mesenquimatosas , Insuficiencia Renal Crónica , Ratas , Animales , Hiperplasia/patología , Ratas Sprague-Dawley , Neointima/patología , Implantes Absorbibles , Tomografía Computarizada por Rayos X , Insuficiencia Renal Crónica/terapia , Insuficiencia Renal Crónica/patología , Fístula Arteriovenosa/patología , Células Madre Mesenquimatosas/patología , Andamios del TejidoRESUMEN
Mesenchymal stem cell (MSC)-seeded polymeric perivascular wraps have been shown to enhance arteriovenous fistula (AVF) maturation. However, the wraps' radiolucency makes their placement and integrity difficult to monitor. Through electrospinning, we infused gold nanoparticles (AuNPs) into polycaprolactone (PCL) wraps to improve their radiopacity and tested whether infusion affects the previously reported beneficial effects of the wraps on the AVF's outflow vein. Sprague Dawley rat MSCs were seeded on the surface of the wraps. We then compared the effects of five AVF treatments-no perivascular wrap (i.e., control), PCL wrap, PCL + MSC wrap, PCL-Au wrap, and PCL-Au + MSC wrap-on AVF maturation in a Sprague Dawley rat model of chronic kidney disease (n = 3 per group). Via micro-CT, AuNP-infused wraps demonstrated a significantly higher radiopacity compared to that of the wraps without AuNPs. Wraps with and without AuNPs equally reduced vascular stenoses, as seen via ultrasonography and histomorphometry. In the immunofluorescence analysis, representative MSC-seeded wraps demonstrated reduced neointimal staining for markers of infiltration with smooth muscle cells (α-SMA), inflammatory cells (CD45), and fibroblasts (vimentin) compared to that of the control and wraps without MSCs. In conclusion, AuNP infusion allows in vivo monitoring via micro-CT of MSC-seeded polymeric wraps over time, without compromising the benefits of the wrap for AVF maturation.
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Fístula Arteriovenosa , Células Madre Mesenquimatosas , Nanopartículas del Metal , Ratas , Animales , Oro , Ratas Sprague-Dawley , Implantes Absorbibles , Fístula Arteriovenosa/terapiaRESUMEN
Background: Arteriovenous fistulas (AVFs) are a vital intervention for patients requiring hemodialysis, but they also contribute to overall mortality due to access malfunction. The most common cause of both AVF non-maturation and secondary failure is neointimal hyperplasia (NIH). Absorbable polycaprolactone (PCL) perivascular wraps can address these complications by incorporating drugs to attenuate NIH, such as rosuvastatin (ROSU), and metallic nanoparticles for visualization and device monitoring. Objectives: This study aimed to assess the impacts of gold nanoparticle (AuNP) and ROSU-loaded perivascular wraps on vasculature NIH and AVF maturation and patency in a chronic kidney disease rat model. Methods: Electrospun wraps containing combinations of PCL, AuNP, and ROSU were monitored for in vitro drug elution, nanoparticle release, tensile strength, and cell viability. Perivascular wraps were implanted in chronic kidney disease rats for in vivo ultrasound (US) and micro-computed tomography (mCT) imaging. AVF specimens were collected for histological analyses. Results: No difference in cell line viability was observed in ROSU-containing grafts. In vitro release studies of ROSU and AuNPs correlated with decreasing radiopacity over time on in vivo mCT analysis. The mCT study also demonstrated increased radiopacity in AuNP-loaded wraps compared with PCL and control. The addition of ROSU demonstrated decreased US and histologic measurements of NIH. Conclusions: The reduced NIH seen with ROSU-loading of perivascular wraps suggests a synergistic effect between mechanical support and anti-hyperplasia medication. Furthermore, the addition of AuNPs increased wrap radiopacity. Together, our results show that radiopaque, AuNP-, and ROSU-loaded PCL grafts induce AVF maturation and suppress NIH while facilitating optimal implanted device visualization.
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Background: To address high rates of arteriovenous fistula (AVF) failure, a mesenchymal stem cell (MSC)-seeded polymeric perivascular wrap has been developed to reduce neointimal hyperplasia (NIH) and enhance AVF maturation in a rat model. However, the wrap's radiolucency makes its placement and integrity difficult to monitor. Purpose: In this study, we infused gold nanoparticles (AuNPs) into the polymeric perivascular wrap to improve its radiopacity and tested the effect of infusion on the previously reported beneficial effects of the polymeric wrap on the AVF outflow vein. Materials and Methods: We fabricated a polymeric perivascular wrap made of polycaprolactone (PCL) infused with AuNPs via electrospinning. Sprague-Dawley rat mesenchymal stem cells (MSCs) were seeded on the surface of the wraps. We then compared the effect of five AVF treatments-no perivascular wrap (i.e., control), PCL wrap, PCL+MSC wrap, PCL-Au wrap, and PCL-Au+MSC wrap-on AVF maturation in a Sprague-Dawley rat model of chronic kidney disease (n=3 per group). Statistical significance was defined as p<.05, and one-way analysis of variance was performed using GraphPad Prism software. Results: On micro-CT, AuNP-infused wraps demonstrated significantly higher radiopacity compared to wraps without AuNPs. On ultrasonography, wraps with and without AuNPs equally reduced the wall-to-lumen ratio of the outflow vein, a marker of vascular stenosis. On histomorphometric analysis, wraps with and without AuNPs equally reduced the neointima-to- lumen ratio of the outflow vein, a measure of NIH. On immunofluorescence analysis, representative MSC-seeded wraps demonstrated reduced neointimal staining for markers of smooth muscle cells (α-SMA), inflammatory cells (CD45), and fibroblasts (vimentin) infiltration when compared to control and wraps without MSCs. Conclusion: Gold nanoparticle infusion allows the in vivo monitoring via micro-CT of a mesenchymal stem cell-seeded polymeric wrap over time without compromising the benefits of the wrap on arteriovenous fistula maturation. Summary Statement: Gold nanoparticle infusion enables in vivo monitoring via micro-CT of the placement and integrity over time of mesenchymal stem cell-seeded polymeric wrap supporting arteriovenous fistula maturation. Key Results: Gold nanoparticle (AuNP)-infused perivascular wraps demonstrated higher radiopacity on micro-CT compared with wraps without AuNPs after 8 weeks.AuNP-infused perivascular wraps equally improved the wall-to-lumen ratio of the outflow vein (a marker of vascular stenosis) when compared with wraps without AuNPs, as seen on US.AuNP-infused perivascular wraps equally reduced the neointima-to-lumen ratio of the outflow vein (a measure of neointimal hyperplasia) when compared with wraps without AuNPs, as seen on histomorphometry.
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BACKGROUND: The infiltration of inflammatory cells during a kidney injury stimulates myofibroblast activation leading to kidney fibrosis. Fibroblast-specific protein 1 (FSP-1) positive cells have been reported as either myofibroblasts or monocytes during tissue fibrosis. The functions of FSP-1+ cells that are associated with the development of renal fibrosis and the signaling pathways that regulate FSP-1+ cell activation have not been well defined. METHODS: In mice with unilateral ureteral obstruction (UUO), we characterized FSP-1+ cells and determined the role of the Notch signaling pathway in the activation of bone marrow-derived FSP-1+ cells during kidney fibrosis. RESULTS: In kidneys from mice with UUO, the FSP-1+ cells accumulated significantly in the tubulointerstitial area. By using immunostaining and FSP-1 reporter mice, we found that FSP-1 was co-stained with inflammatory cell markers, but not myofibroblast markers. Results from mice with bone marrow transplantations showed that FSP-1+ cells in obstructed kidneys represent a bone marrow-derived population of inflammatory cells. In cultured FSP-1+ cells, the inhibition of Notch signaling suppressed the activation and cytokine secretion of FSP-1+ cells that were induced by LPS but not by IL-4. The specific KO or blockade of Notch signaling in bone marrow-derived FSP-1+ cells suppressed UUO-induced ECM deposition, the infiltration of FSP-1+ inflammatory cells, and cytokine production. These responses ameliorated myofibroblast accumulation and renal fibrosis in obstructed kidneys. CONCLUSION: Our study reveals that most FSP-1+ cells in obstructed kidneys are activated macrophages that are derived from bone marrow and that Notch signaling activates the production of M1 cytokines in FSP-1+ monocytes/macrophages, which is important for renal inflammation and fibrosis.
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Enfermedades Renales , Obstrucción Ureteral , Animales , Ratones , Médula Ósea/metabolismo , Citocinas/metabolismo , Fibrosis , Riñón/patología , Enfermedades Renales/patología , Proteína de Unión al Calcio S100A4/metabolismo , Obstrucción Ureteral/complicacionesRESUMEN
Aristolochic acid (AA), extracted from Aristolochiaceae plants, plays an essential role in traditional herbal medicines and is used for different diseases. However, AA has been found to be nephrotoxic and is known to cause aristolochic acid nephropathy (AAN). AA-induced acute kidney injury (AKI) is a syndrome in AAN with a high morbidity that manifests mitochondrial damage as a key part of its pathological progression. Melatonin primarily serves as a mitochondria-targeted antioxidant. However, its mitochondrial protective role in AA-induced AKI is barely reported. In this study, mice were administrated 2.5 mg/kg AA to induce AKI. Melatonin reduced the increase in Upro and Scr and attenuated the necrosis and atrophy of renal proximal tubules in mice exposed to AA. Melatonin suppressed ROS generation, MDA levels and iNOS expression and increased SOD activities in vivo and in vitro. Intriguingly, the in vivo study revealed that melatonin decreased mitochondrial fragmentation in renal proximal tubular cells and increased ATP levels in kidney tissues in response to AA. In vitro, melatonin restored the mitochondrial membrane potential (MMP) in NRK-52E and HK-2 cells and led to an elevation in ATP levels. Confocal immunofluorescence data showed that puncta containing Mito-tracker and GFP-LC3A/B were reduced, thereby impeding the mitophagy of tubular epithelial cells. Furthermore, melatonin decreased LC3A/B-II expression and increased p62 expression. The apoptosis of tubular epithelial cells induced by AA was decreased. Therefore, our findings revealed that melatonin could prevent AA-induced AKI by attenuating mitochondrial damage, which may provide a potential therapeutic method for renal AA toxicity.
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BACKGROUND: Arteriovenous fistula (AVF) maturation is a process involving remodeling of venous arm of the AVFs. It is a challenge to balance adaptive AVF remodeling and neointima formation. In this study we temporally controlled Notch activation to promote AVF maturation while avoiding neointima formation. METHODS: Temporal Notch activation was controlled by regulating the expression of Notch transcription factor, RBP-Jκ, or dnMAML1 (dominant negative MAML2) in vascular smooth muscle cells (VSMCs). AVF mouse model was created and VSMC phenotype dynamic changes during AVF remodeling were determined. RESULTS: Activated Notch was found in the nuclei of neointimal VSMCs in AVFs from uremic mice. We found that the VSMCs near the anastomosis became dedifferentiated and activated after AVF creation. These dedifferentiated VSMCs regained smooth muscle contractile markers later during AVF remodeling. However, global or VSMC-specific KO of RBP-Jκ at early stage (before or 1 week after AVF surgery) blocked VSMC differentiation and neointima formation in AVFs. These un-matured AVFs showed less intact endothelium and increased infiltration of inflammatory cells. Consequently, the VSMC fate in the neointima was completely shut down, leading to an un-arterialized AVF. In contrast, KO of RBP-Jκ at late stage (3 weeks after AVF surgery), it could not block neointima formation and vascular stenosis. Inhibition of Notch activation at week 1 or 2, could maintain VSMC contractile markers expression and facilitate AVF maturation. CONCLUSIONS: This work uncovers the molecular and cellular events in each segment of AVF remodeling and found that neither sustained increasing nor blocking of Notch signaling improves AVF maturation. It highlights a novel strategy to improve AVF patency: temporally controlled Notch activation can achieve a balance between adaptive AVF remodeling and neointima formation to improve AVF maturation. TRANSLATIONAL PERSPECTIVE: Adaptive vascular remodeling is required for AVF maturation. The balance of wall thickening of the vein and neointima formation in AVF determines the fate of AVF function. Sustained activation of Notch signaling in VSMCs promotes neointima formation, while deficiency of Notch signaling at early stage during AVF remodeling prevents VSMC accumulation and differentiation from forming a functional AVFs. These responses also delay EC regeneration and impair EC barrier function with increased inflammation leading to failed vascular remodeling of AVFs. Thus, a strategy to temporal regulate Notch activation will improve AVF maturation.
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Fístula Arteriovenosa , Derivación Arteriovenosa Quirúrgica , Animales , Ratones , Neointima , Remodelación Vascular , Miocitos del Músculo LisoRESUMEN
Jumonji domain-containing protein-3 (JMJD3), a histone H3 lysine 27 (H3K27) demethylase, promotes endothelial regeneration, but its function in neointimal hyperplasia (NIH) of arteriovenous fistulas (AVFs) has not been explored. In this study, we examined the contribution of endothelial JMJD3 to NIH of AVFs and the mechanisms underlying JMJD3 expression during kidney failure. We found that endothelial JMJD3 expression was negatively associated with NIH of AVFs in patients with kidney failure. JMJD3 expression in endothelial cells (ECs) was also downregulated in the vasculature of chronic kidney disease (CKD) mice. In addition, specific knockout of endothelial JMJD3 delayed EC regeneration, enhanced endothelial mesenchymal transition, impaired endothelial barrier function as determined by increased Evans blue staining and inflammatory cell infiltration, and accelerated neointima formation in AVFs created by venous end to arterial side anastomosis in CKD mice. Mechanistically, JMJD3 expression was downregulated via binding of transforming growth factor beta 1-mediated Hes family transcription factor Hes1 to its gene promoter. Knockdown of JMJD3 enhanced H3K27 methylation, thereby inhibiting transcriptional activity at promoters of EC markers and reducing migration and proliferation of ECs. Furthermore, knockdown of endothelial JMJD3 decreased endothelial nitric oxide synthase expression and nitric oxide production, leading to the proliferation of vascular smooth muscle cells. In conclusion, we demonstrate that decreased expression of endothelial JMJD3 impairs EC regeneration and function and accelerates neointima formation in AVFs. We propose increasing the expression of endothelial JMJD3 could represent a new strategy for preventing endothelial dysfunction, attenuating NIH, and improving AVF patency in patients with kidney disease.
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Fístula Arteriovenosa , Histona Demetilasas con Dominio de Jumonji/genética , Insuficiencia Renal Crónica , Animales , Fístula Arteriovenosa/genética , Fístula Arteriovenosa/patología , Regulación hacia Abajo , Células Endoteliales/metabolismo , Células Endoteliales/patología , Humanos , Hiperplasia/genética , Hiperplasia/patología , Histona Demetilasas con Dominio de Jumonji/metabolismo , Ratones , Neointima/genéticaRESUMEN
The vascular endothelium is present within metabolic organs and actively regulates energy metabolism. Here we show osteocalcin, recognized as a bone-secreted metabolic hormone, is expressed in mouse primary endothelial cells isolated from heart, lung and liver. In human osteocalcin promoter-driven green fluorescent protein transgenic mice, green fluorescent protein signals are enriched in endothelial cells lining aorta, small vessels and capillaries and abundant in aorta, skeletal muscle and eye of adult mice. The depletion of lipoprotein receptor-related protein 1 induces osteocalcin through a Forkhead box O -dependent pathway in endothelial cells. Whereas depletion of osteocalcin abolishes the glucose-lowering effect of low-density lipoprotein receptor-related protein 1 depletion, osteocalcin treatment normalizes hyperglycemia in multiple mouse models. Mechanistically, osteocalcin receptor-G protein-coupled receptor family C group 6 member A and insulin-like-growth-factor-1 receptor are in the same complex with osteocalcin and required for osteocalcin-promoted insulin signaling pathway. Therefore, our results reveal an endocrine/paracrine role of endothelial cells in regulating insulin sensitivity, which may have therapeutic implications in treating diabetes and insulin resistance through manipulating vascular endothelium.
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Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Glucosa/metabolismo , Hiperglucemia/genética , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Osteocalcina/genética , Animales , Células Endoteliales/patología , Endotelio Vascular/patología , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Regulación de la Expresión Génica , Genes Reporteros , Prueba de Tolerancia a la Glucosa , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Hiperglucemia/metabolismo , Hiperglucemia/patología , Insulina/metabolismo , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/deficiencia , Masculino , Ratones , Ratones Noqueados , Osteoblastos/metabolismo , Osteoblastos/patología , Osteocalcina/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transducción de SeñalRESUMEN
This study aimed to observe the influence of selenium (Se) deficiency on sperm quality and selenoprotein expression in rats. Four-week male Wista rats were randomly divided into three groups: Se-A, Se-L, and Se-D (respectively for Se- adequate, low, and deficient group). After 9 weeks, the rats were sacrificed by anesthesia, with the cauda epididymidis quickly fetched for sperm count, motility, and deformity. Meanwhile the blood, liver, brain, heart, and testis were collected for Se and biochemical analysis. It was found that the rats in Se-D had poor growth, while the Se concentrations in blood, liver, and heart for Se-D decreased significantly, compared with Se-A and Se-L (p < 0.01). But no significant difference was observed in testis and brain and also no statistical significance for sperm count. The sperm motility for Se-A (63.07%) was significantly higher than Se-L (53.91%) and Se-D (54.15%). Deformities were observed in both Se-L and Se-D. Both glutathione peroxidases (GPxs) and selenoprotein-P (SEPP1) levels in plasma and tissues of Se-D were significantly lower than those of Se-A and Se-L (p < 0.01). The SEPP1 levels in heart and brain of Se-L were lower than Se-A (p < 0.01). There was no statistical difference for GPx1 between Se-A and Se-L. The GPx4 level in testis of Se-L was lower than Se-A (p < 0.05). However, the SEPP1 in plasma, liver, testis, and the GPx3 level in plasma of Se-L were higher than those of Se-A (p < 0.05 or p < 0.01). Our results show that dietary Se deficiency could reduce GPx4 and SEPP1 expression in testis, which further influence sperm motility and may cause sperm deformity. Selenoprotein expression in some tissues of Se-L was higher than that of Se-A, but sperm quality and GPx4 expression in testis were not improved for Se-L. Low active pseudoselenoproteins might be synthesized in low-Se condition. The underlying mechanism needs to be further investigated.
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Dieta , Selenio , Selenoproteína P/metabolismo , Motilidad Espermática , Testículo , Animales , Masculino , Fosfolípido Hidroperóxido Glutatión Peroxidasa , Ratas , Selenoproteínas , Espermatozoides , Testículo/enzimologíaRESUMEN
Chronic kidney disease (CKD) induces the failure of arteriovenous fistulas (AVFs) and promotes the differentiation of vascular adventitial GLI1-positive mesenchymal stem cells (GMCs). However, the roles of GMCs in forming neointima in AVFs remain unknown. GMCs isolated from CKD mice showed increased potential capacity of differentiation into myofibroblast-like cells. Increased activation of expression of PDGFRA and hedgehog (HH) signaling were detected in adventitial cells of AVFs from patients with end-stage kidney disease and CKD mice. PDGFRA was translocated and accumulated in early endosome when sonic hedgehog was overexpressed. In endosome, PDGFRA-mediated activation of TGFB1/SMAD signaling promoted the differentiation of GMCs into myofibroblasts, extracellular matrix deposition, and vascular fibrosis. These responses resulted in neointima formation and AVF failure. KO of Pdgfra or inhibition of HH signaling in GMCs suppressed the differentiation of GMCs into myofibroblasts. In vivo, specific KO of Pdgfra inhibited GMC activation and vascular fibrosis, resulting in suppression of neointima formation and improvement of AVF patency despite CKD. Our findings could yield strategies for maintaining AVF functions.
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Fístula Arteriovenosa/patología , Células Madre Mesenquimatosas/patología , Músculo Liso Vascular/patología , Miofibroblastos/patología , Neointima/patología , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/fisiología , Insuficiencia Renal Crónica/complicaciones , Animales , Fístula Arteriovenosa/etiología , Fístula Arteriovenosa/metabolismo , Diferenciación Celular , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/metabolismo , Miofibroblastos/metabolismo , Neointima/etiología , Neointima/metabolismoRESUMEN
Renal fibrosis features exaggerated inflammation, extracellular matrix (ECM) deposition, and peritubular capillary loss. We previously showed that IL-10 stimulates high-molecular weight hyaluronan (HMW-HA) expression by fibroblasts, and we hypothesize that HMW-HA attenuates renal fibrosis by reducing inflammation and ECM remodeling. We studied the effects of IL-10 overexpression on HA production and scarring in mouse models of unilateral ureteral obstruction (UUO) and ischemia/reperfusion (I/R) to investigate whether IL-10 antifibrotic effects are HA dependent. C57BL/6J mice were fed with the HA synthesis inhibitor, 4-methylumbelliferone (4-MU), before UUO. We observed that in vivo injury increased intratubular spaces, ECM deposition, and HA expression at day 7 and onward. IL-10 overexpression reduced renal fibrosis in both models, promoted HMW-HA synthesis and stability in UUO, and regulated cell proliferation in I/R. 4-MU inhibited IL-10-driven antifibrotic effects, indicating that HMW-HA is necessary for cytokine-mediated reduction of fibrosis. We also found that IL-10 induces in vitro HMW-HA production by renal fibroblasts via STAT3-dependent upregulation of HA synthase 2. We propose that IL-10-induced HMW-HA synthesis plays cytoprotective and antifibrotic roles in kidney injury, thereby revealing an effective strategy to attenuate renal fibrosis in obstructive and ischemic pathologies.
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Fibroblastos/metabolismo , Riñón/lesiones , Riñón/metabolismo , Peso Molecular , Animales , Proliferación Celular/efectos de los fármacos , Citocinas/metabolismo , Matriz Extracelular/metabolismo , Inflamación/metabolismo , Riñón/patología , Ratones Endogámicos C57BLRESUMEN
AIMS: It is well-established that endothelial dysfunction promotes activation of vascular smooth muscle cell (VSMC). Whether decreased accumulation of VSMCs affects endothelial regeneration and functions in arteriovenous graft (AVG) remodelling has not been studied. We sought to identify mechanisms by which the Notch ligand, Jagged1, in VSMCs regulates endothelial cell (EC) functions in AVGs. METHODS AND RESULTS: AVGs were created in transgenic mice bearing VSMC-specific knockout (KO) or overexpression of Jagged1. VSMC migration, EC regeneration, and its barrier functions as well as AVG remodelling were evaluated. Jagged1 expression was induced in VSMCs of neointima in the AVGs. Jagged1 KO in VSMCs inhibited the accumulation of extracellular matrix as well as VSMC migration. Fewer α-SMA-positive VSMCs were found in AVGs created in VSMC-specific Jagged1 KO mice (VSMCJagged1 KO mice) vs. in WT mice. Decreased VSMCs in AVGs were associated with deterioration of EC functions. In AVGs created in transgenic mice bearing Jagged1 KO in VSMCs exhibited delayed EC regeneration and impaired EC barrier function. Barrier dysfunction of ECs increased inflammatory cell infiltration and dysregulation of AVG remodelling and arterialization. The increased expression of IL-1ß in macrophages was associated with expression of adhesion markers in ECs in AVGs created in VSMCJagged1 KO mice. In contrast, AVGs created in mice with overexpression of Jagged1 in VSMCs exhibited improved EC regeneration plus decreased macrophage infiltration. This led to AVG remodelling and arterialization. In co-cultures of ECs and VSMCs, Jagged1 deficiency in VSMCs suppressed N-cadherin and integrin ß3 expression in ECs. Inhibition of integrin ß3 activation delayed EC spreading and migration. Notably, Jagged1 overexpression in VSMCs or treatment with recombinant Jagged1 stimulated the expression of N-cadherin and integrin ß3 in ECs. Jagged1-induced responses were blocked by inhibition of Notch signalling. CONCLUSIONS: Jagged1 expression in VSMCs maintains EC barrier functions and blocks infiltration of macrophages. These responses promote remodelling and arterialization of AVGs.
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Derivación Arteriovenosa Quirúrgica/efectos adversos , Comunicación Celular , Proliferación Celular , Células Endoteliales/metabolismo , Proteína Jagged-1/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Repitelización , Animales , Cadherinas/metabolismo , Arteria Carótida Común/metabolismo , Arteria Carótida Común/patología , Arteria Carótida Común/cirugía , Movimiento Celular , Células Cultivadas , Técnicas de Cocultivo , Regulación hacia Abajo , Células Endoteliales/patología , Integrina beta3/metabolismo , Interleucina-1beta/metabolismo , Proteína Jagged-1/genética , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/patología , Músculo Liso Vascular/cirugía , Miocitos del Músculo Liso/patología , Neointima , Transducción de SeñalRESUMEN
Neointima formation is a major contributor to arteriovenous fistula (AVF) failure. We have previously shown that activation of the Notch signaling pathway contributes to neointima formation by promoting the migration of vascular smooth muscle cells (VSMCs) into the venous anastomosis. In the current study we investigated the mechanisms underlying the dedifferentiation and migration of VSMCs, and in particular the role of bone marrow-derived fibroblast specific protein 1 (FSP-1)+ cells, another cell type found in models of vascular injury. Using VSMC-specific reporter mice, we found that most of the VSMCs participating in AVF neointima formation originated from dedifferentiated VSMCs. We also observed infiltration of bone marrow-derived FSP-1+ cells into the arterial anastomosis where they could interact with VSMCs. In vitro, conditioned media from FSP-1+ cells stimulated VSMC proliferation and phenotype switching. Activated Notch signaling transformed FSP-1+ cells into type I macrophages and stimulated secretion of cytokines and growth factors. Pretreatment with a Notch inhibitor or knockout of the canonical downstream factor RBP-Jκ in bone marrow-derived FSP1+ cells decreased FSP1+ cell infiltration into murine AVFs, attenuating VSMC dedifferentiation and neointima formation. Our results suggest that targeting Notch signaling could provide a new therapeutic strategy to improve AVF patency.
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Derivación Arteriovenosa Quirúrgica/efectos adversos , Miocitos del Músculo Liso/patología , Neointima/patología , Receptores Notch/metabolismo , Diálisis Renal/efectos adversos , Animales , Desdiferenciación Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Noqueados , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Neointima/etiología , Neointima/prevención & control , Cultivo Primario de Células , Receptores Notch/antagonistas & inhibidores , Diálisis Renal/métodos , Insuficiencia Renal Crónica/terapia , Proteína de Unión al Calcio S100A4/metabolismo , Transducción de Señal/efectos de los fármacos , Grado de Desobstrucción Vascular/efectos de los fármacosRESUMEN
Objective- Hypoxic pulmonary hypertension (HPH) is characterized by proliferative vascular remodeling. Abnormal pulmonary artery smooth muscle cells proliferation and endothelial dysfunction are the primary cellular bases of vascular remodeling. AQP1 (aquaporin-1) is regulated by oxygen level and has been observed to play a role in the proliferation and migration of pulmonary artery smooth muscle cells. The role of AQP1 in HPH pathogenesis has not been directly determined to date. To determine the possible roles of AQP1 in the pathogenesis of HPH and explore its possible mechanisms. Approach and Results- Aqp1 knockout mice were used, and HPH model was established in this study. Primary pulmonary artery smooth muscle cells, primary mouse lung endothelial cells, and lung tissue sections from HPH model were used. Immunohistochemistry, immunofluorescence and Western blot, cell cycle, apoptosis, and migration analysis were performed in this study. AQP1 expression was upregulated by chronic hypoxia exposure, both in pulmonary artery endothelia and medial smooth muscle layer of mice. Aqp1 deficiency attenuated the elevation of right ventricular systolic pressures and mitigated pulmonary vascular structure remodeling. AQP1 deletion reduced abnormal cell proliferation in pulmonary artery and accompanied with accumulation of HIF (hypoxia-inducible factor). In vitro, Aqp1 deletion reduced hypoxia-induced proliferation, apoptosis resistance, and migration ability of primary cultured pulmonary artery smooth muscle cells and repressed HIF-1α protein stability. Furthermore, Aqp1 deficiency protected lung endothelial cells from apoptosis in response to hypoxic injury. Conclusions- Our data showed that Aqp1 deficiency could attenuate hypoxia-induced vascular remodeling in the development of HPH. AQP1 may be a potential target for pulmonary hypertension treatment.
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Acuaporina 1/fisiología , Hipertensión Pulmonar/etiología , Hipoxia/complicaciones , Animales , Acuaporina 1/genética , Células Cultivadas , Ciclina D1/fisiología , Hipertensión Pulmonar/fisiopatología , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Masculino , Ratones , Ratones Noqueados , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/fisiología , Remodelación VascularRESUMEN
RATIONALE & OBJECTIVE: Vascular access type (arteriovenous fistula [AVF] vs arteriovenous graft [AVG] vs central venous catheter [CVC]) associates with clinical outcomes in patients with end-stage kidney disease undergoing hemodialysis. Whether a similar association exists with outcomes after kidney transplantation is unknown. We hypothesized that AVGs would associate with worse outcomes, perhaps owing to persistent subclinical inflammation. STUDY DESIGN: Retrospective cohort study. SETTING & PARTICIPANTS: Using US registry data merged with electronic health records of a large dialysis organization (2006-2011), we selected patients receiving a first-ever kidney transplant after undergoing more than 30 days of hemodialysis. EXPOSURE: Hemodialysis access used during the patient's last pretransplantation hemodialysis session. OUTCOMES: Patients were followed up from kidney transplantation for all-cause mortality, kidney allograft loss from any cause, and allograft loss not from death. ANALYTICAL APPROACH: Time-to-event analysis including Kaplan-Meier plots and Cox proportional hazards regression estimated cause-specific HRs and 95% CIs. RESULTS: Among 9,291 patients who underwent kidney transplantation between 2006 and 2011, a total of 65.3% used an AVF, 20.4% used an AVG, and 14.3% used a CVC for hemodialysis before transplantation. Multivariable regression models adjusted for demographic variables, comorbid conditions, transplant characteristics, and laboratory parameters identified no independent associations between vascular access type and all-cause mortality (HRAVG, 1.13 [95% CI, 0.97-1.33]; HRCVC, 1.00 [95% CI, 0.83-1.21]). Similarly, AVG and CVC use were not independently associated with all-cause allograft loss compared with AVF use (HRAVG, 1.13 [95% CI, 1.00-1.28]; HRCVC, 1.12 [95% CI, 0.96-1.29]). CVC use was associated with 30% higher risk for allograft loss from causes other than death compared with AVF use (HRCVC, 1.30 [95% CI, 1.06-1.57]), but AVGs were not (HRAVG, 1.17 [95% CI, 0.98-1.39]). LIMITATIONS: Nonrandomized exposure leading to potential residual confounding. CONCLUSIONS: No association was found for AVG use before kidney transplantation with mortality, all-cause allograft loss, and allograft loss from all causes other than death, compared with AVF use. The association of CVC use with allograft loss from causes other than death requires further investigation.
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BACKGROUND/AIMS: Neointimal hyperplasia is responsible for stenosis, which requires corrective vascular surgery, and is also a major morphological feature of many cardiovascular diseases. This hyperplasia involves the endothelial-to-mesenchymal transition (EndMT). We investigated whether integrin ß3 can modulate the EndMT, as well as its underlying mechanism. METHODS: Integrin ß3 was overexpressed or knocked down in human umbilical vein endothelial cells (HUVECs). The expression of endothelial markers and mesenchymal markers was determined by real-time reverse transcription PCR (RT-PCR), immunofluorescence staining, and western blot analysis. Notch signaling pathway components were detected by real-time RT-PCR and western blot analysis. Cell mobility was evaluated by wound-healing, Transwell, and spreading assays. Fibroblast-specific protein 1 (FSP-1) promoter activity was determined by luciferase assay. RESULTS: Transforming growth factor (TGF)-ß1 treatment or integrin ß3 overexpression significantly promoted the EndMT by downregulating VE-cadherin and CD31 and upregulating smooth muscle actin α and FSP-1 in HUVECs, and by enhancing cell migration. Knockdown of integrin ß3 reversed these effects. Notch signaling was activated after TGF-ß1 treatment of HUVECs. Knockdown of integrin ß3 suppressed TGF-ß1-induced Notch activation and expression of the Notch downstream target FSP-1. CONCLUSION: Integrin ß3 may promote the EndMT in HUVECs through activation of the Notch signaling pathway.
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Transición Epitelial-Mesenquimal , Integrina beta3/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Actinas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD/metabolismo , Fístula Arteriovenosa/metabolismo , Fístula Arteriovenosa/patología , Cadherinas/metabolismo , Proteínas de Unión al Calcio/metabolismo , Movimiento Celular/efectos de los fármacos , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Transición Epitelial-Mesenquimal/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Integrina beta3/química , Integrina beta3/genética , Persona de Mediana Edad , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteína de Unión al Calcio S100A4 , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta1/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Intestinal macrophages are critical for gastrointestinal (GI) homeostasis, but our understanding of their role in regulating intestinal motility is incomplete. Here, we report that CX3C chemokine receptor 1-expressing muscularis macrophages (MMs) were required to maintain normal GI motility. MMs expressed the transient receptor potential vanilloid 4 (TRPV4) channel, which senses thermal, mechanical, and chemical cues. Selective pharmacologic inhibition of TRPV4 or conditional deletion of TRPV4 from macrophages decreased intestinal motility and was sufficient to reverse the GI hypermotility that is associated with chemotherapy treatment. Mechanistically, stimulation of MMs via TRPV4 promoted the release of prostaglandin E2 and elicited colon contraction in a paracrine manner via prostaglandin E receptor signaling in intestinal smooth muscle cells without input from the enteric nervous system. Collectively, our data identify TRPV4-expressing MMs as an essential component required for maintaining normal GI motility and provide potential drug targets for GI motility disorders.
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Colon/fisiología , Motilidad Gastrointestinal , Macrófagos/metabolismo , Miocitos del Músculo Liso/metabolismo , Transducción de Señal , Canales Catiónicos TRPV/metabolismo , Animales , Receptor 1 de Quimiocinas CX3C/metabolismo , Colon/fisiopatología , Ciclooxigenasa 1/deficiencia , Ciclooxigenasa 1/metabolismo , Dinoprostona/análisis , Dinoprostona/metabolismo , Femenino , Mucosa Gástrica/citología , Expresión Génica , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Contracción Muscular , Receptores de Prostaglandina E/antagonistas & inhibidores , Receptores de Prostaglandina E/metabolismo , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales Catiónicos TRPV/deficiencia , Canales Catiónicos TRPV/genéticaRESUMEN
Hydrodynamic injection creates a local, high-pressure environment to transfect various tissues with plasmid DNA and other substances. Hydrodynamic tail vein injection, for example, is a well-established method by which the liver can be transfected. This manuscript describes an application of hydrodynamic principles by injection of the mouse kidney directly with plasmid DNA for kidney-specific gene expression. Mice are anesthetized and the kidney is exposed by a flank incision followed by a fast injection of a plasmid DNA-containing solution directly into the renal pelvis. The needle is kept in place for ten seconds and the incision site is sutured. The following day, live animal imaging, Western blot, or immunohistochemistry may be used to assay gene expression, or other assays suited to the transgene of choice are used for detection of the protein of interest. Published methods to prolong gene expression include transposon-mediated transgene integration and cyclophosphamide treatment to inhibit the immune response to the transgene.