RESUMO
The term 'fibroblast' often serves as a catch-all for a diverse array of mesenchymal cells, including perivascular cells, stromal progenitor cells and bona fide fibroblasts. Although phenotypically similar, these subpopulations are functionally distinct, maintaining tissue integrity and serving as local progenitor reservoirs. In response to tissue injury, these cells undergo a dynamic fibroblast-myofibroblast transition, marked by extracellular matrix secretion and contraction of actomyosin-based stress fibres. Importantly, whereas transient activation into myofibroblasts aids in tissue repair, persistent activation triggers pathological fibrosis. In this Review, we discuss the roles of mechanical cues, such as tissue stiffness and strain, alongside cell signalling pathways and extracellular matrix ligands in modulating myofibroblast activation and survival. We also highlight the role of epigenetic modifications and myofibroblast memory in physiological and pathological processes. Finally, we discuss potential strategies for therapeutically interfering with these factors and the associated signal transduction pathways to improve the outcome of dysregulated healing.
Assuntos
Fibrose , Miofibroblastos , Cicatrização , Humanos , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Animais , Fibrose/metabolismo , Cicatrização/fisiologia , Transdução de Sinais , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Epigênese GenéticaRESUMO
In regenerative medicine, natural protein-based polymers offer enhanced endogenous bioactivity and potential for seamless integration with tissue, yet form weak hydrogels that lack the physical robustness required for surgical manipulation, making them difficult to apply in practice. The use of higher concentrations of protein, exogenous cross-linkers, and blending synthetic polymers has all been applied to form more mechanically robust networks. Each relies on generating a smaller network mesh size, which increases the elastic modulus and robustness, but critically inhibits cell spreading and migration, hampering tissue regeneration. Here we report two unique observations; first, that colloidal suspensions, at sufficiently high volume fraction (Ï), dynamically assemble into a fully percolated 3D network within high-concentration protein polymers. Second, cells appear capable of leveraging these unique domains for highly efficient cell migration throughout the composite construct. In contrast to porogens, the particles in our system remain embedded within the bulk polymer, creating a network of particle-filled tunnels. Whereas this would normally physically restrict cell motility, when the particulate network is created using ultralow cross-linked microgels, the colloidal suspension displays viscous behavior on the same timescale as cell spreading and migration and thus enables efficient cell infiltration of the construct through the colloidal-filled tunnels.
Assuntos
Movimento Celular , Coloides/química , Animais , Materiais Biocompatíveis/química , Fibrina/química , Hidrogéis/química , Camundongos , Células NIH 3T3 , Polímeros/química , Medicina Regenerativa , Trombina/químicaRESUMO
Transforming growth factor-ß (TGFß) signaling through SMAD2/3 is an important driver of pathological fibrosis in multiple organ systems. TGFß signaling and extracellular matrix (ECM) stiffness form an unvirtuous pathological circuit in which matrix stiffness drives activation of latent TGFß, and TGFß signaling then drives cellular stress and ECM synthesis. Moreover, ECM stiffness also appears to sensitize cells to exogenously activated TGFß through unknown mechanisms. Here, using human fibroblasts, we explored the effect of ECM stiffness on a putative inner nuclear membrane protein, LEM domain-containing protein 3 (LEMD3), which is physically connected to the cell's actin cytoskeleton and inhibits TGFß signaling. We showed that LEMD3-SMAD2/3 interactions are inversely correlated with ECM stiffness and TGFß-driven luciferase activity and that LEMD3 expression is correlated with the mechanical response of the TGFß-driven luciferase reporter. We found that actin polymerization but not cellular stress or LEMD3-nuclear-cytoplasmic couplings were necessary for LEMD3-SMAD2/3 interactions. Intriguingly, LEMD3 and SMAD2/3 frequently interacted in the cytosol, and we discovered LEMD3 was proteolytically cleaved into protein fragments. We confirmed that a consensus C-terminal LEMD3 fragment binds SMAD2/3 in a stiffness-dependent manner throughout the cell and is sufficient for antagonizing SMAD2/3 signaling. Using human lung biopsies, we observed that these nuclear and cytosolic interactions are also present in tissue and found that fibrotic tissues exhibit locally diminished and cytoplasmically shifted LEMD3-SMAD2/3 interactions, as noted in vitro Our work reveals novel LEMD3 biology and stiffness-dependent regulation of TGFß by LEMD3, providing a novel target to antagonize pathological TGFß signaling.
Assuntos
Mecanotransdução Celular/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Actinas/metabolismo , Citosol/metabolismo , Proteínas de Ligação a DNA , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/química , Lâmina Nuclear/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosforilação , Proteína Fosfatase 2C/metabolismo , Proteína Smad2/antagonistas & inibidores , Proteína Smad2/química , Proteína Smad3/antagonistas & inibidores , Proteína Smad3/química , Fator de Crescimento Transformador beta/antagonistas & inibidoresRESUMO
Despite standardized postoperative care, some lung transplant patients suffer multiple episodes of acute and chronic rejection while others avoid graft problems for reasons that are poorly understood. Using an established model of C57BL/10 to C57BL/6 minor antigen mismatched single lung transplantation, we now demonstrate that the recipient microbiota contributes to variability in the alloimmune response. Specifically, mice from the Envigo facility in Frederick, Maryland contain nearly double the number of CD4+ Foxp3+ regulatory T cells (Tregs ) than mice from the Jackson facility in Bar Harbor, Maine or the Envigo facility in Indianapolis, Indiana (18 vs 9 vs 7%). Lung graft recipients from the Maryland facility thus do not develop acute or chronic rejection. Treatment with broad-spectrum antibiotics decreases Tregs and increases both acute and chronic graft rejection in otherwise tolerant strains of mice. Constitutive depletion of regulatory T cells, using Foxp3-driven expression of diphtheria toxin receptor, leads to the development of chronic rejection and supports the role of Tregs in both acute and chronic alloimmunity. Taken together, our data demonstrate that the microbiota of certain individuals may contribute to tolerance through Treg -dependent mechanisms and challenges the practice of indiscriminate broad-spectrum antibiotic use in the perioperative period.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Comércio/normas , Fatores de Transcrição Forkhead/fisiologia , Rejeição de Enxerto/prevenção & controle , Pneumopatias/imunologia , Transplante de Pulmão/efeitos adversos , Microbiota , Linfócitos T Reguladores/imunologia , Aloenxertos , Animais , Linfócitos T CD4-Positivos/microbiologia , Rejeição de Enxerto/etiologia , Rejeição de Enxerto/metabolismo , Sobrevivência de Enxerto/imunologia , Pneumopatias/microbiologia , Pneumopatias/cirurgia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Reguladores/microbiologia , TransplantadosRESUMO
BACKGROUND: Cancer-associated fibroblasts (CAFs) support tumour progression and invasion, and they secrete abundant extracellular matrix (ECM) that may shield tumour cells from immune checkpoint or kinase inhibitors. Targeting CAFs using drugs that revert their differentiation, or inhibit their tumour-supportive functions, has been considered as an anti-cancer strategy. METHODS: We have used human and murine cell culture models, atomic force microscopy (AFM), microarray analyses, CAF/tumour cell spheroid co-cultures and transgenic fibroblast reporter mice to study how targeting HDACs using small molecule inhibitors or siRNAs re-directs CAF differentiation and function in vitro and in vivo. RESULTS: From a small molecule screen, we identified Scriptaid, a selective inhibitor of HDACs 1/3/8, as a repressor of TGFß-mediated CAF differentiation. Scriptaid inhibits ECM secretion, reduces cellular contraction and stiffness, and impairs collective cell invasion in CAF/tumour cell spheroid co-cultures. Scriptaid also reduces CAF abundance and delays tumour growth in vivo. CONCLUSIONS: Scriptaid is a well-tolerated and effective HDACi that reverses many of the functional and phenotypic properties of CAFs. Impeding or reversing CAF activation/function by altering the cellular epigenetic regulatory machinery could control tumour growth and invasion, and be beneficial in combination with additional therapies that target cancer cells or immune cells directly.
Assuntos
Fibroblastos Associados a Câncer/efeitos dos fármacos , Inibidores de Histona Desacetilases/administração & dosagem , Hidroxilaminas/administração & dosagem , Neoplasias/tratamento farmacológico , Quinolinas/administração & dosagem , Fator de Crescimento Transformador beta/genética , Animais , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/ultraestrutura , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Técnicas de Cocultura , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/ultraestrutura , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/ultraestrutura , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Camundongos , Análise em Microsséries , Microscopia de Força Atômica , Neoplasias/patologia , Neoplasias/ultraestrutura , Fator de Crescimento Transformador beta/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Integrin binding to bioengineered hydrogel scaffolds is essential for tissue regrowth and regeneration, yet not all integrin binding can lead to tissue repair. Here, we show that through engineering hydrogel materials to promote α3/α5ß1 integrin binding, we can promote the formation of a space-filling and mature vasculature compared with hydrogel materials that promote αvß3 integrin binding. In vitro, α3/α5ß1 scaffolds promoted endothelial cells to sprout and branch, forming organized extensive networks that eventually reached and anastomosed with neighbouring branches. In vivo, α3/α5ß1 scaffolds delivering vascular endothelial growth factor (VEGF) promoted non-tortuous blood vessel formation and non-leaky blood vessels by 10 days post-stroke. In contrast, materials that promote αvß3 integrin binding promoted endothelial sprout clumping in vitro and leaky vessels in vivo. This work shows that precisely controlled integrin activation from a biomaterial can be harnessed to direct therapeutic vessel regeneration and reduce VEGF-induced vascular permeability in vivo.
Assuntos
Prótese Vascular , Permeabilidade Capilar , Fibronectinas/química , Células Endoteliais da Veia Umbilical Humana/metabolismo , Hidrogéis/química , Integrina alfa3/metabolismo , Integrina alfa5beta1/metabolismo , Bioprótese , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Engenharia Tecidual/métodosRESUMO
As platelets aggregate and activate at the site of vascular injury to stem bleeding, they are subjected to a myriad of biochemical and biophysical signals and cues. As clot formation ensues, platelets interact with polymerizing fibrin scaffolds, exposing platelets to a large range of mechanical microenvironments. Here, we show for the first time (to our knowledge) that platelets, which are anucleate cellular fragments, sense microenvironmental mechanical properties, such as substrate stiffness, and transduce those cues into differential biological signals. Specifically, as platelets mechanosense the stiffness of the underlying fibrin/fibrinogen substrate, increasing substrate stiffness leads to increased platelet adhesion and spreading. Importantly, adhesion on stiffer substrates also leads to higher levels of platelet activation, as measured by integrin αIIbß3 activation, α-granule secretion, and procoagulant activity. Mechanistically, we determined that Rac1 and actomyosin activity mediate substrate stiffness-dependent platelet adhesion, spreading, and activation to different degrees. This capability of platelets to mechanosense microenvironmental cues in a growing thrombus or hemostatic plug and then mechanotransduce those cues into differential levels of platelet adhesion, spreading, and activation provides biophysical insight into the underlying mechanisms of platelet aggregation and platelet activation heterogeneity during thrombus formation.
Assuntos
Coagulação Sanguínea/fisiologia , Plaquetas/citologia , Movimento Celular/fisiologia , Mecanotransdução Celular/fisiologia , Ativação Plaquetária/fisiologia , Adesividade Plaquetária/fisiologia , Resinas Acrílicas/metabolismo , Plaquetas/metabolismo , Microambiente Celular/fisiologia , Fibrina/metabolismo , Fibrinogênio/metabolismo , Humanos , Proteínas Imobilizadas/metabolismo , Microscopia Confocal , Selectina-P/metabolismo , Fosfatidilserinas/metabolismo , Agregação Plaquetária/fisiologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Estresse Mecânico , Trombose/fisiopatologia , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Fibronectin (Fn) is a promiscuous ligand for numerous cell adhesion receptors or integrins. The vast majority of Fn-integrin interactions are mediated through the Fn Arg-Gly-Asp (RGD) motif located within the tenth type III repeat. In the case of integrins αIIbß3 and α5ß1, the integrin binds RGD and the synergy site (PHSRN) located within the adjacent ninth type III repeat. Prior work has shown that these synergy-dependent integrins are exquisitely sensitive to perturbations in the Fn integrin binding domain conformation. Our own prior studies of epithelial cell responses to recombinant fragments of the Fn integrin binding domain led us to hypothesize that integrin α3ß1 binding may also be modulated by the synergy site. To explore this hypothesis, we created a variety of recombinant variants of the Fn integrin binding domain: (i) a previously reported (Leu â Pro) stabilizing mutant (FnIII9'10), (ii) an Arg to Ala synergy site mutation (FnIII9(R)â(A)10), (iii) a two-Gly (FnIII9(2G)10) insertion, and (iv) a four-Gly (FNIII9(4G)10) insertion in the interdomain linker region and used surface plasmon resonance to determine binding kinetics of integrin α3ß1 to the Fn fragments. Integrin α3ß1 had the highest affinity for FnIII9'10 and FnIII9(2G)10. Mutation within the synergy site decreased integrin α3ß1 binding 17-fold, and the four-Gly insertion decreased binding 39-fold compared with FnIII9'10. Cell attachment studies demonstrate that α3ß1-mediated epithelial cell binding is greater on FnIII9'10 compared with the other fragments. These studies suggest that the presence and spacing of the RGD and synergy sites modulate integrin α3ß1 binding to Fn.
Assuntos
Fibronectinas/metabolismo , Integrina alfa3beta1/metabolismo , Sequência de Aminoácidos , Adesão Celular , Linhagem Celular , Fibronectinas/química , Fibronectinas/genética , Humanos , Dados de Sequência Molecular , Mutação , Ligação ProteicaRESUMO
BACKGROUND: Quantitative and qualitative differences in the hemostatic systems exist between neonates and adults, including the presence of "fetal" fibrinogen, a qualitatively dysfunctional form of fibrinogen that exists until 1 yr of age. The consequences of "fetal" fibrinogen on clot structure in neonates, particularly in the context of surgery-associated bleeding, have not been well characterized. Here, the authors examine the sequential changes in clotting components and resultant clot structure in a small sample of neonates undergoing cardiac surgery and cardiopulmonary bypass (CPB). METHODS: Blood samples were collected from neonates (n = 10) before surgery, immediately after CPB, and after the transfusion of cryoprecipitate (i.e., adult fibrinogen component). Clots were formed from patient samples or purified neonatal and adult fibrinogen. Clot structure was analyzed using confocal microscopy. RESULTS: Clots formed from plasma obtained after CPB and after transfusion were more porous than baseline clots. Analysis of clots formed from purified neonatal and adult fibrinogen demonstrated that at equivalent fibrinogen concentrations, neonatal clots lack three-dimensional structure, whereas adult clots were denser with significant three-dimensional structure. Clots formed from a combination of purified neonatal and adult fibrinogen were less homogenous than those formed from either purified adult or neonatal fibrinogen. CONCLUSIONS: The results of this study confirm that significant differences exist in clot structure between neonates and adults and that neonatal and adult fibrinogen may not integrate well. These findings suggest that differential treatment strategies for neonates should be pursued to reduce the demonstrated morbidity of blood product transfusion.
Assuntos
Ponte Cardiopulmonar/efeitos adversos , Fibrina , Adulto , Coagulação Sanguínea , Perda Sanguínea Cirúrgica , Transfusão de Sangue , Fator XIII/análise , Feminino , Fibrinogênio/metabolismo , Humanos , Lactente , Recém-Nascido , Masculino , Microscopia Confocal , Protrombina/análiseRESUMO
Efforts to create platelet-like structures for the augmentation of haemostasis have focused solely on recapitulating aspects of platelet adhesion; more complex platelet behaviours such as clot contraction are assumed to be inaccessible to synthetic systems. Here, we report the creation of fully synthetic platelet-like particles (PLPs) that augment clotting in vitro under physiological flow conditions and achieve wound-triggered haemostasis and decreased bleeding times in vivo in a traumatic injury model. PLPs were synthesized by combining highly deformable microgel particles with molecular-recognition motifs identified through directed evolution. In vitro and in silico analyses demonstrate that PLPs actively collapse fibrin networks, an emergent behaviour that mimics in vivo clot contraction. Mechanistically, clot collapse is intimately linked to the unique deformability and affinity of PLPs for fibrin fibres, as evidenced by dissipative particle dynamics simulations. Our findings should inform the future design of a broader class of dynamic, biosynthetic composite materials.
Assuntos
Materiais Biocompatíveis/química , Coagulação Sanguínea/fisiologia , Plaquetas/fisiologia , Fibrina/química , Géis/química , Técnicas Hemostáticas , Modelos Biológicos , Plaquetas/citologia , Endotélio Vascular/citologia , Fibrina/metabolismo , Microscopia Confocal , Domínios e Motivos de Interação entre Proteínas , Propriedades de SuperfícieRESUMO
Neural stem cells (NSCs) possess great potential for neural tissue repair after traumatic injuries to the central nervous system (CNS). However, poor survival and self-renewal of NSCs after injury severely limits its therapeutic potential. Sulfated chondroitin sulfate glycosaminoglycans (CS-GAGs) linked to CS proteoglycans (CSPGs) in the brain extracellular matrix (ECM) have the ability to bind and potentiate trophic factor efficacy, and promote NSC self-renewal in vivo. In this study, we investigated the potential of CS-GAG hydrogels composed of monosulfated CS-4 (CS-A), CS-6 (CS-C), and disulfated CS-4,6 (CS-E) CS-GAGs as NSC carriers, and their ability to create endogenous niches by enriching specific trophic factors to support NSC self-renewal. We demonstrate that CS-GAG hydrogel scaffolds showed minimal swelling and degradation over a period of 15 days in vitro, absorbing only 6.5 ± 0.019% of their initial weight, and showing no significant loss of mass during this period. Trophic factors FGF-2, BDNF, and IL10 bound with high affinity to CS-GAGs, and were significantly (p < 0.05) enriched in CS-GAG hydrogels when compared to unsulfated hyaluronic acid (HA) hydrogels. Dissociated rat subventricular zone (SVZ) NSCs when encapsulated in CS-GAG hydrogels demonstrated â¼88.5 ± 6.1% cell viability in vitro. Finally, rat neurospheres in CS-GAG hydrogels conditioned with the mitogen FGF-2 demonstrated significantly (p < 0.05) higher self-renewal when compared to neurospheres cultured in unconditioned hydrogels. Taken together, these findings demonstrate the ability of CS-GAG based hydrogels to regulate NSC self-renewal, and facilitate growth factor enrichment locally.
Assuntos
Sulfatos de Condroitina/química , Hidrogéis/química , Células-Tronco Neurais/citologia , Alicerces Teciduais/química , Animais , Fator Neurotrófico Derivado do Encéfalo/administração & dosagem , Proliferação de Células , Células Cultivadas , Fator 2 de Crescimento de Fibroblastos/administração & dosagem , RatosRESUMO
Applied forces and the biophysical nature of the cellular microenvironment play a central role in determining cellular behavior. Specifically, forces due to cell contraction are transmitted into structural ECM proteins and these forces are presumed to activate integrin "switches." The mechanism of such switches is thought to be the partial unfolding of integrin-binding domains within fibronectin (Fn). However, integrin switches remain largely hypothetical due to a dearth of evidence for their existence, and relevance, in vivo. By using phage display in combination with the controlled deposition and extension of Fn fibers, we report the discovery of peptide-based molecular probes capable of selectively discriminating Fn fibers under different strain states. Importantly, we show that the probes are functional in both in vitro and ex vivo tissue contexts. The development of such tools represents a critical step in establishing the relevance of theoretical mechanotransduction events within the cellular microenvironment.
Assuntos
Microambiente Celular , Fibronectinas/metabolismo , Sondas Moleculares/metabolismo , Biblioteca de Peptídeos , Sequência de Aminoácidos , Animais , Bacteriófagos/genética , Bacteriófagos/metabolismo , Células Cultivadas , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibronectinas/química , Integrinas/metabolismo , Pulmão/citologia , Mecanotransdução Celular , Camundongos , Microscopia Confocal , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , Desdobramento de ProteínaRESUMO
Increased tissue stiffness and epithelial-to-mesenchymal transitions (EMTs) are two seemingly discrete hallmarks of fibrotic diseases. Despite recent findings highlighting the influence of tissue mechanical properties on cell phenotype, it remains unclear what role increased tissue stiffness has in the regulation of previously reported fibronectin-mediated EMTs associated with pulmonary fibrosis. Nano-indentation testing of lung interstitial spaces showed that in vivo cell-level Young's moduli increase with the onset of fibrosis from â¼2 to â¼17 kPa. In vitro, we found that stiff, but not soft, fibronectin substrates induce EMT, a response dependent on cell contraction-mediated integrin activation of TGFß. Activation or suppression of cell contractility with exogenous factors was sufficient to overcome the effect of substrate stiffness. Pulse-chase experiments indicate that the effect of cell contractility is dose- and time-dependent. In response to low levels of TGFß on soft surfaces, either added exogenously or produced through thrombin-induced contraction, cells will initiate the EMT programme, but upon removal revert to an epithelial phenotype. These results identify matrix stiffness and/or cell contractility as critical targets for novel therapeutics for fibrotic diseases.
Assuntos
Células Epiteliais Alveolares/patologia , Microambiente Celular , Transição Epitelial-Mesenquimal , Pulmão/patologia , Mecanotransdução Celular , Fibrose Pulmonar/patologia , Células Epiteliais Alveolares/metabolismo , Animais , Fenômenos Biomecânicos , Bleomicina , Células Cultivadas , Técnicas de Cocultura , Modelos Animais de Doenças , Módulo de Elasticidade , Fibronectinas/metabolismo , Integrinas/metabolismo , Laminina/metabolismo , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Força Atômica , Vison , Nanotecnologia , Fenótipo , Fibrose Pulmonar/metabolismo , Ratos , Ratos Sprague-Dawley , Trombina/metabolismo , Fatores de Tempo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Dysfunctional pulmonary homeostasis and repair, including diseases such as pulmonary fibrosis, chronic obstructive pulmonary disease (COPD*), and tumorigenesis, have been increasing steadily over the past decade, a fact that heavily implicates environmental influences. Several investigations have suggested that the lung "precursor cell"--the alveolar type II (ATII) epithelial cell--is central in the initiation and progression of pulmonary fibrosis. Specifically, ATII cells have been shown (Iwano et al. 2002) to be capable of undergoing an epithelial-to-mesenchymal transition (EMT). EMT, the de-differentiation of an epithelial cell into a mesenchymal cell, has been theorized to increase the number of extracellular matrix (ECM)-secreting mesenchymal cells, perpetuating fibrotic conditions and resulting in increased lung tissue stiffness. In addition, increased exposure to pollution and inhalation of particulate matter (PM) have been shown to be highly correlated with an increased incidence of pulmonary fibrosis. Although both of these events are involved in the progression of pulmonary fibrosis, the relationship between tissue stiffness, exposure to PM, and the initiation and course of EMT remains unclear. The hypothesis of this study was twofold: 1. That alveolar epithelial cells cultured on increasingly stiff substrates become increasingly contractile, leading to enhanced transforming growth factor beta (TGF-ß) activation and EMT; and 2. That exposure of alveolar epithelial cells to PM with an aerodynamic diameter ≤ 2.5 µm (PM2.5; also known as fine PM) results in enhanced cell contractility and EMT. Our study focused on the relationship between the micromechanical environment and external environmental stimuli on the phenotype of alveolar epithelial cells. This relationship was explored by first determining how increased tissue stiffness affects the regulation of fibronectin (Fn)-mediated EMT in ATII cells in vitro. We cultured ATII cells on substrates of increasing stiffness and evaluated changes in cell contractility and EMT. We found that stiff, but not soft, Fn substrates were able to induce EMT and that this event depended on a contractile phenotype of the cell and the subsequent activation of TGF-ß. In addition, we were able to show that activation or suppression of cell contractility by way of exogenous factors was sufficient to overcome the effect of substrate stiffness. Pulse-chase experiments indicated that the effect on cell contractility is dose- and time-dependent. In response to low levels of TGF-ß on soft surfaces, either added exogenously or produced through contraction induced by the stiffness agonist thrombin, cells initiate EMT; on removal of the TGF-ß, they revert to an epithelial phenotype. Overall, the results from this first part of our study identified matrix stiffness or cell contractility as critical targets for the control of EMT in fibrotic diseases. For the second part of our study, we wanted to investigate whether exposure to PM2.5, which might have higher toxicity than coarser PM because of its small size and large surface-to-mass ratio, altered the observed stiffness-mediated EMT. Again, we cultured ATII cells on increasingly stiff substrates with or without the addition of three concentrations of PM2.5. We found that exposure to PM2.5 was involved in increased stiffness-mediated EMT, as shown by increases in mesenchymal markers, cell contractility, and TGF-ß activation. Most notably, on substrates with an elastic modulus (E) of 8 kilopascals (kPa), a physiologically relevant range for pulmonary fibrosis, the addition of PM2.5 resulted in increased mesenchymal cells and EMT; these were not seen in the absence of the PM2.5. Overall, this study showed that there is a delicate balance between substrate stiffness, TGF-ß, and EMT. Furthermore, we showed that exposure to PM2.5 is able to further mediate this interaction. The higher levels of EMT seen with exposure to PM2.5 might have been a result of a positive feedback loop, in which enhanced exposure to PM2.5 through the loss of cell-cell junctions during the initial stages of EMT led to the cells being more susceptible to the effects of surrounding immune cells and inflammatory signals that can further activate TGF-ß and drive additional EMT progression. Overall, our work--showing increased cell contractility, TGF-ß activation, and EMT in response to substrate stiffness and PM2.5 exposure--highlights the importance of both the micromechanical and biochemical environments in lung disease. These findings suggest that already-fibrotic tissue might be more susceptible to further damage than healthy tissue when exposed to PM2.5.
Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Material Particulado , Alvéolos Pulmonares/citologia , Fibrose Pulmonar/fisiopatologia , Animais , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Progressão da Doença , Módulo de Elasticidade/fisiologia , Células Epiteliais , Matriz Extracelular/metabolismo , Immunoblotting , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Fibrose Pulmonar/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Fibrosis-associated fibroblasts have been identified across various fibrotic disorders, but not in the context of biomaterials, fibrotic encapsulation, and the foreign body response. In other fibrotic disorders, a fibroblast subpopulation defined by Thy-1 loss is strongly correlated with fibrosis yet we do not know what promotes Thy-1 loss. We have previously shown that Thy-1 is an integrin regulator enabling normal fibroblast mechanosensing, and here, leveraging nonfibrotic microporous annealed particle (MAP) hydrogels versus classical fibrotic bulk hydrogels, we demonstrate that Thy1-/- mice mount a fibrotic response to MAP gels that includes inflammatory signaling. We found that a distinct and cryptic α-smooth muscle actin-positive Thy-1- fibroblast population emerges in response to interleuklin-1ß (IL-1ß) and tumor necrosis factor-α (TNFα). Furthermore, IL-1ß/TNFα-induced Thy-1- fibroblasts consist of two distinct subpopulations that are strongly proinflammatory. These findings illustrate the emergence of a unique proinflammatory, profibrotic fibroblast subpopulation that is central to fibrotic encapsulation of biomaterials.
Assuntos
Materiais Biocompatíveis , Fibroblastos , Fibrose , Hidrogéis , Antígenos Thy-1 , Animais , Camundongos , Materiais Biocompatíveis/efeitos adversos , Materiais Biocompatíveis/toxicidade , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Hidrogéis/química , Interleucina-1beta/metabolismo , Camundongos Knockout , Antígenos Thy-1/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: The epididymis is important for sperm maturation and without its proper development, male infertility will result. Biomechanical properties of tissues/organs play key roles during their morphogenesis, including the Wolffian duct. It is hypothesized that structural/bulk stiffness of the capsule and mesenchyme/extracellular matrix that surround the duct is a major biomechanical property that regulates Wolffian duct morphogenesis. These data will provide key information as to the mechanisms that regulate the development of this important organ. OBJECTIVES: To measure the structural/bulk stiffness in Pascals (force/area) of the capsule and the capsule and mesenchyme together that surrounds the Wolffian duct during the development. To examine the relative membrane tension of mesenchymal cells during the Wolffian duct development. Since Ptk7 was previously shown to regulate ECM integrity and Wolffian duct elongation and coiling, the hypothesis that Ptk7 regulates structural/bulk stiffness and mesenchymal cell membrane tension was tested. MATERIALS AND METHODS: Atomic force microscopy and a microsquisher compression apparatus were used to measure the structural stiffness. Biomechanical properties within the membranes of cells within the capsule and mesenchyme were examined using a membrane-tension fluorescent probe. RESULTS AND DISCUSSION: The structural stiffness (Pascals) of the capsule and underlying mesenchyme was relatively constant during development, with a significant increase in the capsule at the later stages. However, this increase may reflect the ECM and associated mesenchyme being close to the capsule because the coiling of the duct pushed or compressed them into that space. Keeping the capsule and mesenchyme/ECM at constant stiffness would ensure that the duct will continue to coil under similar biomechanical forces throughout the development. Cells within the capsule and mesenchyme at different Wolffian duct regions during the development had varying degrees of membrane lipid tension. It is hypothesized that the dynamic changes ensure the duct is kept at a constant stiffness regardless of any external forces. Loss of Ptk7 resulted in an increase in stiffness at E18.5, which was presumable due to the loss of integrity of the ECM within the mesenchyme. CONCLUSION: Biomechanical properties of the capsule and the mesenchyme/extracellular matrix that surround the Wolffian duct play an important role toward Wolffian duct morphogenesis, thereby allowing for the proper development of the epididymis and subsequent male fertility.
RESUMO
Uncontrolled bleeding after trauma represents a substantial clinical problem. The current standard of care to treat bleeding after trauma is transfusion of blood products including platelets; however, donated platelets have a short shelf life, are in limited supply, and carry immunogenicity and contamination risks. Consequently, there is a critical need to develop hemostatic platelet alternatives. To this end, we developed synthetic platelet-like particles (PLPs), formulated by functionalizing highly deformable microgel particles composed of ultralow cross-linked poly (N-isopropylacrylamide) with fibrin-binding ligands. The fibrin-binding ligand was designed to target to wound sites, and the cross-linking of fibrin polymers was designed to enhance clot formation. The ultralow cross-linking of the microgels allows the particles to undergo large shape changes that mimic platelet shape change after activation; when coupled to fibrin-binding ligands, this shape change facilitates clot retraction, which in turn can enhance clot stability and contribute to healing. Given these features, we hypothesized that synthetic PLPs could enhance clotting in trauma models and promote healing after clotting. We first assessed PLP activity in vitro and found that PLPs selectively bound fibrin and enhanced clot formation. In murine and porcine models of traumatic injury, PLPs reduced bleeding and facilitated healing of injured tissue in both prophylactic and immediate treatment settings. We determined through biodistribution experiments that PLPs were renally cleared, possibly enabled by ultrasoft particle properties. The performance of synthetic PLPs in the preclinical studies shown here supports future translational investigation of these hemostatic therapeutics in a trauma setting.
Assuntos
Hemostáticos , Roedores , Animais , Camundongos , Suínos , Roedores/metabolismo , Distribuição Tecidual , Plaquetas/metabolismo , Hemorragia , Fibrina/química , Fibrina/metabolismoRESUMO
Investigating the molecular, cellular, and tissue-level changes caused by disease, and the effects of pharmacological treatments across these biological scales, necessitates the use of multiscale computational modeling in combination with experimentation. Many diseases dynamically alter the tissue microenvironment in ways that trigger microvascular network remodeling, which leads to the expansion or regression of microvessel networks. When microvessels undergo remodeling in idiopathic pulmonary fibrosis (IPF), functional gas exchange is impaired due to loss of alveolar structures and lung function declines. Here, we integrated a multiscale computational model with independent experiments to investigate how combinations of biomechanical and biochemical cues in IPF alter cell fate decisions leading to microvascular remodeling. Our computational model predicted that extracellular matrix (ECM) stiffening reduced microvessel area, which was accompanied by physical uncoupling of endothelial cell (ECs) and pericytes, the cells that comprise microvessels. Nintedanib, an FDA-approved drug for treating IPF, was predicted to further potentiate microvessel regression by decreasing the percentage of quiescent pericytes while increasing the percentage of pericytes undergoing pericyte-myofibroblast transition (PMT) in high ECM stiffnesses. Importantly, the model suggested that YAP/TAZ inhibition may overcome the deleterious effects of nintedanib by promoting EC-pericyte coupling and maintaining microvessel homeostasis. Overall, our combination of computational and experimental modeling can explain how cell decisions affect tissue changes during disease and in response to treatments.
RESUMO
Idiopathic pulmonary fibrosis (IPF) is a morbid fibrotic lung disease with limited treatment options. The pathophysiology of IPF remains poorly understood, and elucidation of the cellular and molecular mechanisms of IPF pathogenesis is key to the development of new therapeutics. B-1 cells are an innate B cell population which play an important role linking innate and adaptive immunity. B-1 cells spontaneously secrete natural IgM and prevent inflammation in several disease states. One class of these IgM recognize oxidation-specific epitopes (OSE), which have been shown to be generated in lung injury and to promote fibrosis. A main B-1 cell reservoir is the pleural space, adjacent to the typical distribution of fibrosis in IPF. In this study, we demonstrate that B-1 cells are recruited to the lung during injury where they secrete IgM to OSE (IgM OSE ). We also show that the pleural B-1 cell reservoir responds to lung injury through regulation of the chemokine receptor CXCR4. Mechanistically we show that the transcription factor Id3 is a novel negative regulator of CXCR4 expression. Using mice with B-cell specific Id3 deficiency, a model of increased B-1b cells, we demonstrate decreased bleomycin-induced fibrosis compared to littermate controls. Furthermore, we show that mice deficient in secretory IgM ( sIgM -/- ) have higher mortality in response to bleomycin-induced lung injury, which is partially mitigated through airway delivery of the IgM OSE E06. Additionally, we provide insight into potential mechanisms of IgM in attenuation of fibrosis through RNA sequencing and pathway analysis, highlighting complement activation and extracellular matrix deposition as key differentially regulated pathways.