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ZFYVE21 is an ancient, endosome-associated protein that is highly expressed in endothelial cells (ECs) but whose function(s) in vivo are undefined. Here, we identified ZFYVE21 as an essential regulator of vascular barrier function in the aging kidney. ZFYVE21 levels significantly decline in ECs in aged human and mouse kidneys. To investigate attendant effects, we generated EC-specific Zfyve21-/- reporter mice. These knockout mice developed accelerated aging phenotypes including reduced endothelial nitric oxide (ENOS) activity, failure to thrive, and kidney insufficiency. Kidneys from Zfyve21 EC-/- mice showed interstitial edema and glomerular EC injury. ZFYVE21-mediated phenotypes were not programmed developmentally as loss of ZFYVE21 in ECs during adulthood phenocopied its loss prenatally, and a nitric oxide donor normalized kidney function in adult hosts. Using live cell imaging and human kidney organ cultures, we found that in a GTPase Rab5- and protein kinase Akt-dependent manner, ZFYVE21 reduced vesicular levels of inhibitory caveolin-1 and promoted transfer of Golgi-derived ENOS to a perinuclear Rab5+ vesicular population to functionally sustain ENOS activity. Thus, our work defines a ZFYVE21- mediated trafficking mechanism sustaining ENOS activity and demonstrates the relevance of this pathway for maintaining kidney function with aging.
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Tissue-resident macrophages play important roles in tissue homeostasis and repair. However, how macrophages monitor and maintain tissue integrity is not well understood. The extracellular matrix (ECM) is a key structural and organizational component of all tissues. Here, we find that macrophages sense the mechanical properties of the ECM to regulate a specific tissue repair program. We show that macrophage mechanosensing is mediated by cytoskeletal remodeling and can be performed in three-dimensional environments through a noncanonical, integrin-independent mechanism analogous to amoeboid migration. We find that these cytoskeletal dynamics also integrate biochemical signaling by colony-stimulating factor 1 and ultimately regulate chromatin accessibility to control the mechanosensitive gene expression program. This study identifies an "amoeboid" mode of ECM mechanosensing through which macrophages may regulate tissue repair and fibrosis.
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Matriz Extracelular , Macrófagos , Matriz Extracelular/metabolismo , Macrófagos/metabolismo , Citoesqueleto , Integrinas/metabolismo , Transducción de SeñalRESUMEN
Interactions between endothelial cells (ECs) and mural pericytes (PCs) are critical in maintaining the stability and function of the microvascular wall. Abnormal interactions between these two cell types are a hallmark of progressive fibrotic diseases such as systemic sclerosis (also known as scleroderma). However, the role of PCs in signaling microvascular dysfunction remains underexplored. We hypothesized that integrin-matrix interactions contribute to PC migration from the vascular wall and conversion into interstitial myofibroblasts. Herein, pro-inflammatory tumor necrosis factor α (TNFα) or a fibrotic growth factor [transforming growth factor ß1 (TGF-ß1)] were used to evaluate human PC inflammatory and fibrotic phenotypes by assessing their migration, matrix deposition, integrin expression, and subsequent effects on endothelial dysfunction. Both TNFα and TGF-ß1 treatment altered integrin expression and matrix protein deposition, but only fibrotic TGF-ß1 drove PC migration in an integrin-dependent manner. In addition, integrin-dependent PC migration was correlated to changes in EC angiopoietin-2 levels, a marker of vascular instability. Finally, there was evidence of changes in vascular stability corresponding to disease state in human systemic sclerosis skin. This work shows that TNFα and TGF-ß1 induce changes in PC integrin expression and matrix deposition that facilitate migration and reduce vascular stability, providing evidence that microvascular destabilization can be an early indicator of tissue fibrosis.
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Movimiento Celular , Fibrosis , Integrinas , Pericitos , Esclerodermia Sistémica , Factor de Crecimiento Transformador beta1 , Pericitos/metabolismo , Pericitos/patología , Humanos , Factor de Crecimiento Transformador beta1/metabolismo , Esclerodermia Sistémica/patología , Esclerodermia Sistémica/metabolismo , Integrinas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Microvasos/patología , Microvasos/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/patología , Piel/patología , Piel/metabolismo , Piel/irrigación sanguíneaRESUMEN
The ability to engineer complex multicellular systems has enormous potential to inform our understanding of biological processes and disease and alter the drug development process. Engineering living systems to emulate natural processes or to incorporate new functions relies on a detailed understanding of the biochemical, mechanical, and other cues between cells and between cells and their environment that result in the coordinated action of multicellular systems. On April 3-6, 2022, experts in the field met at the Keystone symposium "Engineering Multicellular Living Systems" to discuss recent advances in understanding how cells cooperate within a multicellular system, as well as recent efforts to engineer systems like organ-on-a-chip models, biological robots, and organoids. Given the similarities and common themes, this meeting was held in conjunction with the symposium "Organoids as Tools for Fundamental Discovery and Translation".
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Ingeniería , Organoides , Humanos , Ingeniería de TejidosRESUMEN
Although delivery of neural stem cell (NSC) as a therapeutic treatment for intracerebral hemorrhage (ICH) provides promise, NSC delivery typically has extremely low survival rates. Here, we investigate endothelial cell (EC) and pericyte (PC) interactions with NSC, where our results demonstrate that EC, and not PC, promote NSC cell proliferation and reduce cytotoxicity under glucose deprivation (GD). Additionally, NSC proliferation was increased upon treatment with EC conditioned media, inhibited with antagonism of VEGFR3. In an NSC + EC co-culture we detected elevated levels of VEGF-C, not seen for NSC cultured alone. Exogenous VEGF-C induced NSC upregulation of VEGFR3, promoted proliferation, and reduced cytotoxicity. Finally, we delivered microbeads containing NSC + EC into a murine ICH cavity, where VEGF-C was increasingly present in the injury site, not seen upon delivery NSC encapsulated alone. These studies demonstrate that EC-secreted VEGF-C may promote NSC survival during injury, enhancing the potential for cell delivery therapies for stroke.
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Células-Madre Neurales , Factor C de Crecimiento Endotelial Vascular , Animales , Diferenciación Celular , Medios de Cultivo Condicionados , Células Endoteliales , RatonesRESUMEN
Obesity and the metabolic disease epidemic has led to an increase in morbidity and mortality. A rise in adipose thermogenic capacity via activation of brown or beige fat is a potential treatment for metabolic diseases. However, an understanding of how local factors control adipocyte fate is limited. Mice with a null mutation in the laminin α4 (LAMA4) gene (KO) exhibit resistance to obesity and enhanced expression of thermogenic fat markers in white adipose tissue (WAT). In this study, changes in WAT extracellular matrix composition in the absence of LAMA4 were evaluated using liquid chromatography/tandem mass spectrometry. KO-mice showed lower levels of collagen 1A1 and 3A1, and integrins α7 (ITA7) and ß1 (ITB1). ITA7-ITB1 and collagen 1A1-3A1 protein levels were lower in brown adipose tissue compared to WAT in wild-type mice. Immunohistochemical staining confirmed lower levels and different spatial distribution of ITA7 in KO-WAT. In culture studies, ITA7 and LAMA4 levels decreased following a 12-day differentiation of adipose-derived stem cells into beige fat, and knock-down of ITA7 during differentiation increased beiging. These results demonstrate that extracellular matrix interactions regulate adipocyte thermogenic capacity and that ITA7 plays a role in beige adipose formation. A better understanding of the mechanisms underlying these interactions can be used to improve systemic energy metabolism and glucose homeostasis.
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Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Integrinas/metabolismo , Termogénesis , Animales , Proteínas de la Matriz Extracelular/genética , Integrinas/genética , Ratones , Ratones NoqueadosRESUMEN
Neuroblasts have a clustered phenotype critical for their unidirectional migration, which in part is dependent on signaling from microvascular endothelial cells (EC) and pericytes (PC). Diffusible signals secreted by vascular cells have been demonstrated to increase survival, proliferation, and differentiation of subventricular zone resident neural stem cells (NSC); however, the signals that promote the necessary initiating step of NSC clustering are undefined. To investigate the role of vascular cells in promoting NSC clustering and directing migration, we created a 3-D hydrogel that mimics the biomechanics, biochemistry, and architectural complexity of brain tissue. We demonstrate that EC, and not PC, have a crucial role in NSC clustering and migration, further verified through microfluidic chamber systems and traction force microscopy. Ablation of the extended NSC aggregate arm halts aggregate movement, suggesting that clustering is a prerequisite for migration. When cultured with EC, NSC clustering occurs and NSC coincidentally increase their expression of N-cadherin, as compared to NSC cultured alone. NSC-presented N-cadherin expression was increased following exposure to EC secreted metalloproteinase-2 (MMP2). We demonstrate that inhibition of MMP2 prevented NSC N-cadherin surface expression and subsequent NSC clustering, even when NSC were in direct contact with EC. Furthermore, with exogenous activation of EGFR, which serves as a downstream activator of N-cadherin cleavage, NSC form clusters. Our results suggest that EC secretion of MMP2 promotes NSC clustering through N-cadherin expression. The insight gained about the mechanisms by which EC promote NSC migration may enhance NSC therapeutic response to sites of injury.
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Cadherinas/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Células-Madre Neurales/metabolismo , Animales , Cadherinas/genética , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Línea Celular , Movimiento Celular/genética , Movimiento Celular/fisiología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Hidrogeles/química , Metaloproteinasa 2 de la Matriz/genética , RatonesRESUMEN
Targeting different cell surface receptors with nanoparticle (NP)-based platforms can result in differential particle binding properties that may impact their localization, bioavailability, and, ultimately, the therapeutic efficacy of an encapsulated payload. Conventional in vitro assays comparing the efficacy of targeted NPs often do not adequately control for these differences in particle-receptor binding, potentially confounding their therapeutic readouts and possibly even limiting their experimental value. In this work, we characterize the conditions under which NPs loaded with Bruton's Tyrosine Kinase (BTK) inhibitor differentially suppress primary B cell activation when targeting either CD19 (internalizing) or B220 (noninternalizing) surface receptors. Surface binding of fluorescently labeled CD19- and B220-targeted NPs was analyzed and quantitatively correlated with the number of bound particles at given treatment concentrations. Using this binding data, suppression of B cell activation was directly compared for differentially targeted (CD19 vs B220) NPs loaded with a BTK inhibitor at a range of particle drug loading concentrations. When NPs were loaded with lower amounts of drug, CD19-mediated internalization demonstrated increased inhibition of B cell proliferation compared with B220 NPs. However, these differences were mitigated when particles were loaded with higher concentrations of BTK inhibitor and B220-mediated "paracrine-like" delivery demonstrated superior suppression of cellular activation when cells were bound to lower overall numbers of NPs. Taken together, these results demonstrate that inhibition of B cell activation can be optimized for NPs targeting either internalizing or noninternalizing surface receptors and that particle internalization is likely not a requisite endpoint when designing particles for delivery of BTK inhibitor to B cells.
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Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Nanopartículas/administración & dosificación , Inhibidores de Proteínas Quinasas/administración & dosificación , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Antígenos CD19/metabolismo , Proliferación Celular/efectos de los fármacos , Femenino , Antígenos Comunes de Leucocito/metabolismo , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Transducción de Señal/efectos de los fármacosRESUMEN
Kidneys are highly vascular organs that despite their relatively small size receive 20% of the cardiac output. The highly intricate, delicately organized structure of renal microcirculation is essential to enable renal function and glomerular filtration rate through the local modulation of renal blood flow and intraglomerular pressure. Not surprisingly, the dysregulation of blood flow within the microvessels (abnormal vasoreactivity), fibrosis driven by disordered vascular-renal cross talk, or the loss of renal microvasculature (rarefaction) is associated with kidney disease. In addition, kidney disease can cause microcirculatory dysfunction in distant organs such as the heart and brain, mediated by mechanisms that remain to be elucidated. The objective of this review is to highlight the role of renal microvasculature in kidney disease. The overview will outline the impetus to study renal microvasculature, the bidirectional relationship between kidney disease and microvascular dysfunction, the key pathways driving microvascular diseases such as vasoreactivity, the cell dynamics coordinating fibrosis, and vessel rarefaction. Finally, we will also briefly highlight new therapies targeting the renal microvasculature to improve renal function.
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Enfermedades Renales , Microcirculación , Fibrosis , Humanos , Riñón/patología , Enfermedades Renales/patología , Microvasos/patologíaRESUMEN
Intracerebral implantation of neural stem cells (NSCs) to treat stroke remains an inefficient process with <5% of injected cells being retained. To improve the retention and distribution of NSCs after a stroke, we investigated the utility of NSCs' encapsulation in polyethylene glycol (PEG) microspheres. We first characterized the impact of the physical properties of different syringes and needles, as well as ejection speed, upon delivery of microspheres to the stroke injured rat brain. A 20 G needle size at a 10 µL/min flow rate achieved the most efficient microsphere ejection. Secondly, we optimized the delivery vehicles for in vivo implantation of PEG microspheres. The suspension of microspheres in extracellular matrix (ECM) hydrogel showed superior retention and distribution in a cortical stroke caused by photothrombosis, as well as in a striatal and cortical cavity ensuing middle cerebral artery occlusion (MCAo). Thirdly, NSCs or NSCs + endothelial cells (ECs) encapsulated into biodegradable microspheres were implanted into a large stroke cavity. Cells in microspheres exhibited a high viability, survived freezing and transport. Implantation of 110 cells/microsphere suspended in ECM hydrogel produced a highly efficient delivery that resulted in the widespread distribution of NSCs in the tissue cavity and damaged peri-infarct tissues. Co-delivery of ECs enhanced the in vivo survival and distribution of â¼1.1 million NSCs. The delivery of NSCs and ECs can be dramatically improved using microsphere encapsulation combined with suspension in ECM hydrogel. These biomaterial innovations are essential to advance clinical efforts to improve the treatment of stroke using intracerebral cell therapy.
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Células Endoteliales/efectos de los fármacos , Hidrogeles/farmacología , Microesferas , Células-Madre Neurales/efectos de los fármacos , Accidente Cerebrovascular/tratamiento farmacológico , Animales , Matriz Extracelular/metabolismo , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Polietilenglicoles/farmacología , Accidente Cerebrovascular/metabolismoRESUMEN
BACKGROUND: Airway eosinophilia is a prominent feature of asthma and chronic rhinosinusitis (CRS), and the endothelium plays a key role in eosinophil trafficking. To date, microRNA-1 (miR-1) is the only microRNA known to be regulated in the lung endothelium in asthma models. OBJECTIVE: We sought to determine the role of endothelial miR-1 in allergic airway inflammation. METHODS: We measured microRNA and mRNA expression using quantitative RT-PCR. We used ovalbumin and house dust mite models of asthma. Endothelium-specific overexpression of miR-1 was achieved through lentiviral vector delivery or induction of a transgene. Tissue eosinophilia was quantified by using Congo red and anti-eosinophil peroxidase staining. We measured eosinophil binding with a Sykes-Moore adhesion chamber. Target recruitment to RNA-induced silencing complex was assessed by using anti-Argonaute2 RNA immunoprecipitation. Surface P-selectin levels were measured by using flow cytometry. RESULTS: Serum miR-1 levels had inverse correlations with sputum eosinophilia, airway obstruction, and number of hospitalizations in asthmatic patients and sinonasal tissue eosinophilia in patients with CRS. IL-13 stimulation decreased miR-1 levels in human lung endothelium. Endothelium-specific overexpression of miR-1 reduced airway eosinophilia and asthma phenotypes in murine models and inhibited IL-13-induced eosinophil binding to endothelial cells. miR-1 recruited P-selectin, thymic stromal lymphopoietin, eotaxin-3, and thrombopoietin receptor to the RNA-induced silencing complex; downregulated these genes in the lung endothelium; and reduced surface P-selectin levels in IL-13-stimulated endothelial cells. In our asthma and CRS cohorts, miR-1 levels correlated inversely with its target genes. CONCLUSION: Endothelial miR-1 regulates eosinophil trafficking in the setting of allergic airway inflammation. miR-1 has therapeutic potential in asthmatic patients and patients with CRS.
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Asma/inmunología , Quimiotaxis de Leucocito/inmunología , MicroARNs/inmunología , MicroARNs/metabolismo , Rinitis Alérgica Perenne/inmunología , Sinusitis/inmunología , Animales , Asma/metabolismo , Asma/patología , Células Endoteliales/metabolismo , Eosinófilos , Humanos , Ratones , Eosinofilia Pulmonar/inmunología , Eosinofilia Pulmonar/metabolismo , Eosinofilia Pulmonar/patología , Rinitis Alérgica Perenne/metabolismo , Rinitis Alérgica Perenne/patología , Sinusitis/metabolismo , Sinusitis/patologíaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Stem cell therapies demonstrate promising results as treatment for neurological disease and injury, owing to their innate ability to enhance endogenous neural tissue repair and promote functional recovery. However, delivery of undifferentiated and viable neuronal stem cells requires an engineered delivery system that promotes integration of transplanted cells into the inflamed and cytotoxic region of damaged tissue. Within the brain, endothelial cells (EC) of the subventricular zone play a critical role in neural stem cell (NSC) maintenance, quiescence and survival. Therefore, here, we describe the use of polyethylene glycol microbeads for the coincident delivery of EC and NSC as a means of enhancing appropriate NSC quiescence and survival during transplantation into the mouse brain. We demonstrate that EC and NSC co-encapsulation maintained NSC quiescence, enhanced NSC viability, and facilitated NSC extravasation in vitro, as compared to NSC encapsulated alone. In addition, co-encapsulated cells delivered to an in vivo non-injury model reduced inflammatory response compared to freely injected NSC. These results suggest the strong potential of a biomimetic engineered niche for NSC delivery into the brain following neurological injury.
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Encapsulación Celular/métodos , Ventrículos Laterales/cirugía , Microesferas , Células-Madre Neurales/trasplante , Trasplante de Células Madre/métodos , Animales , Diferenciación Celular , Línea Celular , Proliferación Celular , Supervivencia Celular , Células Endoteliales/metabolismo , Ventrículos Laterales/metabolismo , Masculino , Metaloproteinasas de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Células-Madre Neurales/fisiología , Neuronas , Polietilenglicoles/química , Recuperación de la Función , Nicho de Células MadreRESUMEN
Dysregulated neutrophil extravasation contributes to the pathogenesis of many inflammatory disorders. Pericytes (PCs) have been implicated in the regulation of neutrophil transmigration, and previous work demonstrates that endothelial cell (EC)-derived signals reduce PC barrier function; however, the signaling mechanisms are unknown. Here, we demonstrate a novel role for EC-derived macrophage migration inhibitory factor (MIF) in inhibiting PC contractility and facilitating neutrophil transmigration. With the use of micro-ELISAs, RNA sequencing, quantitative PCR, and flow cytometry, we found that ECs secrete MIF, and PCs upregulate CD74 in response to TNF-α. We demonstrate that EC-derived MIF decreases PC contractility on 2-dimensional silicone substrates via reduction of phosphorylated myosin light chain. With the use of an in vitro microvascular model of the human EC-PC barrier, we demonstrate that MIF decreases the PC barrier to human neutrophil transmigration by increasing intercellular PC gap formation. For the first time, an EC-specific MIF knockout mouse was used to investigate the effects of selective deletion of EC MIF. In a model of acute lung injury, selective deletion of EC MIF decreases neutrophil infiltration to the bronchoalveolar lavage and tissue and simultaneously decreases PC relaxation by increasing myosin light-chain phosphorylation. We conclude that paracrine signals from EC via MIF decrease PC contraction and enhance PC-regulated neutrophil transmigration.-Pellowe, A. S., Sauler, M., Hou, Y., Merola, J., Liu, R., Calderon, B., Lauridsen, H. M., Harris, M. R., Leng, L., Zhang, Y., Tilstam, P. V., Pober, J. S., Bucala, R., Lee, P. J., Gonzalez, A. L. Endothelial cell-secreted MIF reduces pericyte contractility and enhances neutrophil extravasation.
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Endotelio Vascular/metabolismo , Oxidorreductasas Intramoleculares/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Neutrófilos/citología , Pericitos/citología , Animales , Líquido del Lavado Bronquioalveolar , Células Cultivadas , Endotelio Vascular/citología , Ensayo de Inmunoadsorción Enzimática , Humanos , Oxidorreductasas Intramoleculares/genética , Factores Inhibidores de la Migración de Macrófagos/genética , Ratones , Ratones NoqueadosRESUMEN
PURPOSE OF REVIEW: Neural stem cells (NSCs) have the potential to proliferate and differentiate into functional neurons, heightening their potential use for therapeutic applications. This review explores bioengineered systems which recapitulate NSC niche cell-cell and cell-matrix interactions. RECENT FINDINGS: Delivery of NSCs to the cytotoxic injured brain is limited by low cell survival rates post-transplantation and poor maintenance of native niche bioactive components. The use of biomaterial platforms can mimic in vivo the environment of the two germinal areas of the adult brain in which NSCs thrive. An environmental mimic that includes extracellular proteins and moieties, along with appropriate biomechanical cues has recently demonstrated promising results in enhancing neurogenesis, aiding the production of a bioengineered niche. SUMMARY: Biocomposition, biomechanics, and biostructure can be manipulated through engineered platforms to re-create the biofunctionality of an NSC niche. Upon transplantation and delivery with biomimetic scaffolds, NSCs show potential to promote functional recovery and rebuild neural circuitry post neurological trauma.
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Idiopathic pulmonary fibrosis (IPF) is a fatal disease of unknown etiology characterized by a compositionally and mechanically altered extracellular matrix. Poor understanding of the origin of α-smooth muscle actin (α-SMA) expressing myofibroblasts has hindered curative therapies. Though proposed as a source of myofibroblasts in mammalian tissues, identification of microvascular pericytes (PC) as contributors to α-SMA-expressing populations in human IPF and the mechanisms driving this accumulation remain unexplored. Here, we demonstrate enhanced detection of α-SMA+ cells coexpressing the PC marker neural/glial antigen 2 in the human IPF lung. Isolated human PC cultured on decellularized IPF lung matrices adopt expression of α-SMA, demonstrating that these cells undergo phenotypic transition in response to direct contact with the extracellular matrix (ECM) of the fibrotic human lung. Using potentially novel human lung-conjugated hydrogels with tunable mechanical properties, we decoupled PC responses to matrix composition and stiffness to show that α-SMA+ PC accumulate in a mechanosensitive manner independent of matrix composition. PC activated with TGF-ß1 remodel the normal lung matrix, increasing tissue stiffness to facilitate the emergence of α-SMA+ PC via MKL-1/MTRFA mechanotranduction. Nintedanib, a tyrosine-kinase inhibitor approved for IPF treatment, restores the elastic modulus of fibrotic lung matrices to reverse the α-SMA+ phenotype. This work furthers our understanding of the role that microvascular PC play in the evolution of IPF, describes the creation of an ex vivo platform that advances the study of fibrosis, and presents a potentially novel mode of action for a commonly used antifibrotic therapy that has great relevance for human disease.
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Fibrosis Pulmonar Idiopática/patología , Miofibroblastos/fisiología , Pericitos/fisiología , Actinas/metabolismo , Antígenos/metabolismo , Células Cultivadas , Elasticidad , Inhibidores Enzimáticos/farmacología , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Humanos , Fibrosis Pulmonar Idiopática/metabolismo , Indoles/farmacología , Pulmón/metabolismo , Mecanotransducción Celular/efectos de los fármacos , Mecanotransducción Celular/fisiología , Metaloproteasas/biosíntesis , Miofibroblastos/metabolismo , Pericitos/efectos de los fármacos , Fenotipo , Proteoglicanos/metabolismo , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
RATIONALE: Idiopathic pulmonary fibrosis (IPF) involves the accumulation of α-smooth muscle actin-expressing myofibroblasts arising from interactions with soluble mediators such as transforming growth factor-ß1 (TGF-ß1) and mechanical influences such as local tissue stiffness. Whereas IPF fibroblasts are enriched for aerobic glycolysis and innate immune receptor activation, innate immune ligands related to mitochondrial injury, such as extracellular mitochondrial DNA (mtDNA), have not been identified in IPF. OBJECTIVES: We aimed to define an association between mtDNA and fibroblast responses in IPF. METHODS: We evaluated the response of normal human lung fibroblasts (NHLFs) to stimulation with mtDNA and determined whether the glycolytic reprogramming that occurs in response to TGF-ß1 stimulation and direct contact with stiff substrates, and spontaneously in IPF fibroblasts, is associated with excessive levels of mtDNA. We measured mtDNA concentrations in bronchoalveolar lavage (BAL) from subjects with and without IPF, as well as in plasma samples from two longitudinal IPF cohorts and demographically matched control subjects. MEASUREMENTS AND MAIN RESULTS: Exposure to mtDNA augments α-smooth muscle actin expression in NHLFs. The metabolic changes in NHLFs that are induced by interactions with TGF-ß1 or stiff hydrogels are accompanied by the accumulation of extracellular mtDNA. These findings replicate the spontaneous phenotype of IPF fibroblasts. mtDNA concentrations are increased in IPF BAL and plasma, and in the latter compartment, they display robust associations with disease progression and reduced event-free survival. CONCLUSIONS: These findings demonstrate a previously unrecognized and highly novel connection between metabolic reprogramming, mtDNA, fibroblast activation, and clinical outcomes that provides new insight into IPF.
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ADN Mitocondrial/metabolismo , Fibroblastos/metabolismo , Fibrosis Pulmonar Idiopática/metabolismo , Fibrosis Pulmonar Idiopática/mortalidad , Anciano , Supervivencia sin Enfermedad , Femenino , Humanos , MasculinoRESUMEN
Sweet syndrome (SS) is a prototypical neutrophilic dermatosis, a class of inflammatory diseases marked by elevated levels of tumor necrosis factor (TNF)-α and IL-17A, pathologic neutrophil recruitment, and microvascular remodeling. Histologic analyses of four matrix proteins-collagen I and IV, laminin, and fibronectin-in skin biopsies of patients with SS reveal that the basement membrane of dermal postcapillary venules undergoes changes in structure and composition. Increased neutrophil recruitment in vivo was associated with increases in collagen IV, decreases in laminin, and varied changes in fibronectin. In vitro studies using TNF-α and IL-17A were conducted to dissect basement membrane remodeling. Prolonged dual activation of cultured human pericytes with TNF-α and IL-17A augmented collagen IV production, similar to in vivo remodeling. Co-activation of pericytes with TNF-α and IL-17A also elevated fibronectin levels with little direct effect on laminin. However, the expression of fibronectin- and laminin-specific matrix metalloproteinases (MMPs), particularly MMP-3, was significantly up-regulated. Interactions between pericytes and neutrophils in culture yielded even higher levels of active MMPs, facilitating fibronectin and laminin degradation, and likely contributing to the varied levels of detectable fibronectin and the decreases in laminin observed in vivo. These data indicate that pericyte-neutrophil interactions play a role in mediating microvascular changes in SS and suggest that targeting MMP-3 may be effective in protecting vascular wall integrity.
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Membrana Basal/efectos de los fármacos , Interleucina-17/farmacología , Neutrófilos/metabolismo , Pericitos/efectos de los fármacos , Síndrome de Sweet/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Anciano , Membrana Basal/metabolismo , Membrana Basal/patología , Células Cultivadas , Colágeno Tipo IV/metabolismo , Femenino , Fibronectinas/metabolismo , Humanos , Laminina/metabolismo , Masculino , Metaloproteinasa 3 de la Matriz/metabolismo , Persona de Mediana Edad , Neutrófilos/patología , Pericitos/metabolismo , Pericitos/patología , Síndrome de Sweet/patologíaRESUMEN
The vascular basement membrane-a thin, elastic layer of extracellular matrix separating and encasing vascular cells-provides biological and mechanical cues to endothelial cells, pericytes, and migrating leukocytes. In contrast, experimental scaffolds typically used to replicate basement membranes are stiff and bio-inert. Here, we present thin, porated polyethylene glycol hydrogels to replicate human vascular basement membranes. Like commercial transwells, our hydrogels are approximately 10µm thick, but like basement membranes, the hydrogels presented here are elastic (E: 50-80kPa) and contain a dense network of small pores. Moreover, the inclusion of bioactive domains introduces receptor-mediated biochemical signaling. We compare elastic hydrogels to common culture substrates (E: >2GPa) for human endothelial cell and pericyte monolayers and bilayers to replicate postcapillary venules in vitro. Our data demonstrate that substrate elasticity facilitates differences in vascular phenotype, supporting expression of vascular markers that are increasingly replicative of venules. Endothelial cells differentially express vascular markers, like EphB4, and leukocyte adhesion molecules, such as ICAM-1, with decreased mechanical stiffness. With porated PEG hydrogels we demonstrate the ability to evaluate and observe leukocyte recruitment across endothelial cell and pericyte monolayers and bilayers, reporting that basement membrane scaffolds can significantly alter the rate of vascular migration in experimental systems. Overall, this study demonstrates the creation and utility of a new and accessible method to recapture the mechanical and biological complexity of human basement membranes in vitro.
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Membrana Basal/química , Células Endoteliales/citología , Neutrófilos/citología , Pericitos/citología , Ingeniería de Tejidos/métodos , Membrana Basal/metabolismo , Membrana Basal/ultraestructura , Biomarcadores/metabolismo , Materiales Biomiméticos/química , Materiales Biomiméticos/metabolismo , Movimiento Celular , Módulo de Elasticidad , Elasticidad , Células Endoteliales/metabolismo , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Expresión Génica , Humanos , Hidrogeles/química , Hidrogeles/metabolismo , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Neutrófilos/metabolismo , Pericitos/metabolismo , Polietilenglicoles/química , Porosidad , Cultivo Primario de Células , Receptor EphB4/genética , Receptor EphB4/metabolismo , Transducción de SeñalRESUMEN
The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.