RESUMO
Muscle stem cells (MuSCs) are specialized cells that reside in adult skeletal muscle poised to repair muscle tissue. The ability of MuSCs to regenerate damaged tissues declines markedly with aging and in diseases such as Duchenne muscular dystrophy, but the underlying causes of MuSC dysfunction remain poorly understood. Both aging and disease result in dramatic increases in the stiffness of the muscle tissue microenvironment from fibrosis. MuSCs are known to lose their regenerative potential if cultured on stiff plastic substrates. We sought to determine whether MuSCs harbor a memory of their past microenvironment and if it can be overcome. We tested MuSCs in situ using dynamic hydrogel biomaterials that soften or stiffen on demand in response to light and found that freshly isolated MuSCs develop a persistent memory of substrate stiffness characterized by loss of proliferative progenitors within the first three days of culture on stiff substrates. MuSCs cultured on soft hydrogels had altered cytoskeletal organization and activity of Rho and Rac guanosine triphosphate hydrolase (GTPase) and Yes-associated protein mechanotransduction pathways compared to those on stiff hydrogels. Pharmacologic inhibition identified RhoA activation as responsible for the mechanical memory phenotype, and single-cell RNA sequencing revealed a molecular signature of the mechanical memory. These studies highlight that microenvironmental stiffness regulates MuSC fate and leads to MuSC dysfunction that is not readily reversed by changing stiffness. Our results suggest that stiffness can be circumvented by targeting downstream signaling pathways to overcome stem cell dysfunction in aged and disease states with aberrant fibrotic tissue mechanics.
Assuntos
Materiais Biocompatíveis , Hidrogéis , Músculo Esquelético , Animais , Hidrogéis/química , Materiais Biocompatíveis/química , Músculo Esquelético/metabolismo , Camundongos , Mecanotransdução Celular , Células-Tronco/metabolismo , Células-Tronco/citologia , Proteína rhoA de Ligação ao GTP/metabolismo , Células CultivadasRESUMO
Vimentin intermediate filaments form part of the cytoskeleton of mesenchymal cells, but under pathological conditions often associated with inflammation, vimentin filaments depolymerize as the result of phosphorylation or citrullination, and vimentin oligomers are secreted or released into the extracellular environment. In the extracellular space, vimentin can bind surfaces of cells and the extracellular matrix, and the interaction between extracellular vimentin and cells can trigger changes in cellular functions, such as activation of fibroblasts to a fibrotic phenotype. The mechanism by which extracellular vimentin binds external cell membranes and whether vimentin alone can act as an adhesive anchor for cells is largely uncharacterized. Here, we show that various cell types (normal and vimentin null fibroblasts, mesenchymal stem cells, and A549 lung carcinoma cells) attach to and spread on polyacrylamide hydrogel substrates covalently linked to vimentin. Using traction force microscopy and spheroid expansion assays, we characterize how different cell types respond to extracellular vimentin. Cell attachment to and spreading on vimentin-coated surfaces is inhibited by hyaluronic acid degrading enzymes, hyaluronic acid synthase inhibitors, soluble heparin or N-acetyl glucosamine, all of which are treatments that have little or no effect on the same cell types binding to collagen-coated hydrogels. These studies highlight the effectiveness of substrate-bound vimentin as a ligand for cells and suggest that carbohydrate structures, including the glycocalyx and glycosylated cell surface proteins that contain N-acetyl glucosamine, form a novel class of adhesion receptors for extracellular vimentin that can either directly support cell adhesion to a substrate or fine-tune the glycocalyx adhesive properties.
Assuntos
Vimentina , Acetilglucosamina/química , Adesão Celular , Movimento Celular , Ácido Hialurônico/química , Filamentos Intermediários/metabolismo , Vimentina/metabolismo , Humanos , Linhagem Celular TumoralRESUMO
In this work, we investigate whether stiffening in compression is a feature of single cells and whether the intracellular polymer networks that comprise the cytoskeleton (all of which stiffen with increasing shear strain) stiffen or soften when subjected to compressive strains. We find that individual cells, such as fibroblasts, stiffen at physiologically relevant compressive strains, but genetic ablation of vimentin diminishes this effect. Further, we show that unlike networks of purified F-actin or microtubules, which soften in compression, vimentin intermediate filament networks stiffen in both compression and extension, and we present a theoretical model to explain this response based on the flexibility of vimentin filaments and their surface charge, which resists volume changes of the network under compression. These results provide a new framework by which to understand the mechanical responses of cells and point to a central role of intermediate filaments in response to compression.
Assuntos
Citoesqueleto , Filamentos Intermediários , Citoesqueleto de Actina , Actinas , VimentinaRESUMO
Cellular mechanics encompass both mechanical properties that resist forces applied by the external environment and internally generated forces applied at the location of cell-cell and cell-matrix junctions. Here, the authors demonstrate that microindentation of cellular domes formed by cell monolayers that locally lift off the substrate provides insight into both aspects of cellular mechanics in multicellular structures. Using a modified Hertz contact equation, the force-displacement curves generated by a micro-tensiometer are used to measure an effective dome stiffness. The results indicate the domes are consistent with the Laplace-Young relationship for elastic membranes, regardless of biochemical modulation of the RhoA-ROCK signaling axis. In contrast, activating RhoA, and inhibiting ROCK both alter the relaxation dynamics of the domes deformed by the micro-tensiometer, revealing an approach to interrogate the role of RhoA-ROCK signaling in multicellular mechanics. A finite element model incorporating a Mooney-Rivlin hyperelastic constitutive equation to describe monolayer mechanics predicts effective stiffness values that are consistent with the micro-tensiometer measurements, verifying previous measurements of the response of cell monolayers to tension. Overall, these studies establish microindentation of fluid-filled domes as an avenue to investigate the contribution of cell-generated forces to the mechanics of multicellular structures.
Assuntos
Transdução de SinaisRESUMO
Infection of human cells by pathogens, including SARS-CoV-2, typically proceeds by cell surface binding to a crucial receptor. The primary receptor for SARS-CoV-2 is the angiotensin-converting enzyme 2 (ACE2), yet new studies reveal the importance of additional extracellular co-receptors that mediate binding and host cell invasion by SARS-CoV-2. Vimentin is an intermediate filament protein that is increasingly recognized as being present on the extracellular surface of a subset of cell types, where it can bind to and facilitate pathogens' cellular uptake. Biophysical and cell infection studies are done to determine whether vimentin might bind SARS-CoV-2 and facilitate its uptake. Dynamic light scattering shows that vimentin binds to pseudovirus coated with the SARS-CoV-2 spike protein, and antibodies against vimentin block in vitro SARS-CoV-2 pseudovirus infection of ACE2-expressing cells. The results are consistent with a model in which extracellular vimentin acts as a co-receptor for SARS-CoV-2 spike protein with a binding affinity less than that of the spike protein with ACE2. Extracellular vimentin may thus serve as a critical component of the SARS-CoV-2 spike protein-ACE2 complex in mediating SARS-CoV-2 cell entry, and vimentin-targeting agents may yield new therapeutic strategies for preventing and slowing SARS-CoV-2 infection.
Assuntos
Ligação Proteica , SARS-CoV-2 , Vimentina , Anticorpos/farmacologia , COVID-19 , Humanos , Glicoproteína da Espícula de Coronavírus , Vimentina/antagonistas & inibidores , Vimentina/metabolismoRESUMO
Uropathogenic E. coli (UPEC) infection in vivo is characterized by invasion of bladder umbrella epithelial cells followed by endosomal escape and proliferation in the cytoplasm to form intracellular bacterial communities. By contrast, UPEC infection in tissue culture models results in bacteria being trapped within Lamp1-positive endosomes where proliferation is limited. Pharmacological disruption of the actin cytoskeleton has been shown to facilitate UPEC endosomal escape in vitro and extracellular matrix stiffness is a well-characterized physiological regulator of actin dynamics; therefore, we hypothesized that substrate stiffness may play a role in UPEC endosomal escape. Using functionalized polyacrylamide substrates, we found that at physiological stiffness, UPEC escaped the endosome and proliferated rapidly in the cytoplasm of bladder epithelial cells. Dissection of the cytoskeletal signaling pathway demonstrated that inhibition of the Rho GTPase RhoB or its effector PRK1 was sufficient to increase cytoplasmic bacterial growth and that RhoB protein level was significantly reduced at physiological stiffness. Our data suggest that tissue stiffness is a critical regulator of intracellular bacterial growth. Due to the ease of doing genetic and pharmacological manipulations in cell culture, this model system may provide a useful tool for performing mechanistic studies on the intracellular life cycle of uropathogens.
Assuntos
Endossomos/microbiologia , Endossomos/fisiologia , Escherichia coli Uropatogênica/fisiologia , Actinas/metabolismo , Animais , Técnicas de Cultura de Células , Proliferação de Células , Citoesqueleto/fisiologia , Células Epiteliais/microbiologia , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Feminino , Humanos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Bexiga Urinária , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
The migration of cells through constricting spaces or along fibrous tracks in tissues is important for many biological processes and depends on the mechanical properties of a cytoskeleton made up of three different filaments: F-actin, microtubules, and intermediate filaments. The signaling pathways and cytoskeletal structures that control cell motility on 2D are often very different from those that control motility in 3D. Previous studies have shown that intermediate filaments can promote actin-driven protrusions at the cell edge, but have little effect on overall motility of cells on flat surfaces. They are however important for cells to maintain resistance to repeated compressive stresses that are expected to occur in vivo. Using mouse embryonic fibroblasts derived from wild-type and vimentin-null mice, it is found that loss of vimentin increases motility in 3D microchannels even though on flat surfaces it has the opposite effect. Atomic force microscopy and traction force microscopy experiments reveal that vimentin enhances perinuclear cell stiffness while maintaining the same level of acto-myosin contractility in cells. A minimal model in which a perinuclear vimentin cage constricts along with the nucleus during motility through confining spaces, providing mechanical resistance against large strains that could damage the structural integrity of cells, is proposed.
Assuntos
Movimento Celular , Vimentina/deficiência , Animais , Fenômenos Biomecânicos , Capilares/efeitos dos fármacos , Colágeno/farmacologia , Citoesqueleto/metabolismo , Hidrogéis/farmacologia , Camundongos , Miosina Tipo II/metabolismo , Células NIH 3T3 , Vimentina/metabolismoRESUMO
Unlike many other cancer cells that grow in tumors characterized by an abnormally stiff collagen-enriched stroma, glioma cells proliferate and migrate in the much softer environment of the brain, which generally lacks the filamentous protein matrix characteristic of breast, liver, colorectal, and other types of cancer. Glial cell-derived tumors and the cells derived from them are highly heterogeneous and variable in their mechanical properties, their response to treatments, and their properties in vitro. Some glioma samples are stiffer than normal brain when measured ex vivo, but even those that are soft in vitro stiffen after deformation by pressure gradients that arise in the tumor environment in vivo. Such mechanical differences can strongly alter the phenotype of cultured glioma cells. Alternatively, chemical signaling might elicit the same phenotype as increased stiffness by activating intracellular messengers common to both initial stimuli. In this study the responses of three different human glioma cell lines to changes in substrate stiffness are compared with their responses on very soft substrates composed of a combination of hyaluronic acid and a specific integrin ligand, either laminin or collagen I. By quantifying cell morphology, stiffness, motility, proliferation, and secretion of the cytokine IL-8, glioma cell responses to increased stiffness are shown to be nearly identically elicited by substrates containing hyaluronic acid, even in the absence of increased stiffness. PI3-kinase activity was required for the response to hyaluronan but not to stiffness. This outcome suggests that hyaluronic acid can trigger the same cellular response, as can be obtained by mechanical force transduced from a stiff environment, and demonstrates that chemical and mechanical features of the tumor microenvironment can achieve equivalent reactions in cancer cells.
Assuntos
Movimento Celular , Proliferação de Células , Neuroglia/citologia , Alicerces Teciduais/química , Linhagem Celular Tumoral , Elasticidade , Glioma/metabolismo , Humanos , Ácido Hialurônico/química , Ácido Hialurônico/farmacologia , Interleucina-8/metabolismo , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Propriedades de Superfície , Alicerces Teciduais/efeitos adversosRESUMO
Filamentous hyphae of the human pathogen, Candida albicans, invade mucosal layers and medical silicones. In vitro, hyphal tips reorient thigmotropically on contact with small obstacles. It is not known how surface topography is sensed but hyphae lacking the cortical marker, Rsr1/Bud1, are unresponsive. We show that, on surfaces, the morphology of hyphal tips and the position of internal polarity protein complexes are asymmetrically skewed towards the substratum and biased towards the softer of two surfaces. In nano-fabricated chambers, the Spitzenkörper (Spk) responded to touch by translocating across the apex towards the point of contact, where its stable maintenance correlated with contour-following growth. In the rsr1Δ mutant, the position of the Spk meandered and these responses were attenuated. Perpendicular collision caused lateral Spk oscillation within the tip until after establishment of a new growth axis, suggesting Spk position does not predict the direction of growth in C. albicans. Acute tip reorientation occurred only in cells where forward growth was countered by hyphal friction sufficient to generate a tip force of â¼ 8.7 µN (1.2 MPa), more than that required to penetrate host cell membranes. These findings suggest mechanisms through which the organization of hyphal tip growth in C. albicans facilitates the probing, penetration and invasion of host tissue.
Assuntos
Candida albicans/crescimento & desenvolvimento , Hifas/crescimento & desenvolvimento , Candida albicans/citologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Hifas/citologia , Microscopia , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Ceragenins constitute a novel family of cationic antibiotics characterized by a broad spectrum of antimicrobial activities, which have mostly been assessed in vitro. Using a polarized human lung epithelial cell culture system, we evaluated the antibacterial activities of the ceragenin CSA-13 against two strains of Pseudomonas aeruginosa (PAO1 and Xen5). Additionally, the biodistribution and bactericidal activity of a CSA-13-IRDye 800CW derivate were assessed using an animal model of peritoneal infection after PAO1 challenge. In cell culture, CSA-13 bactericidal activities against PAO1 and Xen5 were higher than the activities of the human cathelicidin peptide LL-37. Increased CSA-13 activity was observed in polarized human lung epithelial cell cultures subjected to butyric acid treatment, which is known to increase endogenous LL-37 production. Eight hours after intravenous or intraperitoneal injection, the greatest CSA-13-IRDye 800CW accumulation was observed in mouse liver and kidneys. CSA-13-IRDye 800CW administration resulted in decreased bacterial outgrowth from abdominal fluid collected from animals subjected to intraperitoneal PAO1 infection. These observations indicate that CSA-13 may synergistically interact with antibacterial factors that are naturally present at mucosal surfaces and it maintains its antibacterial activity in the infected abdominal cavity. Cationic lipids such as CSA-13 represent excellent candidates for the development of new antibacterial compounds.
Assuntos
Antibacterianos/farmacologia , Peritonite/tratamento farmacológico , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Esteroides/farmacologia , Animais , Antibacterianos/farmacocinética , Peptídeos Catiônicos Antimicrobianos/farmacocinética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Disponibilidade Biológica , Ácido Butírico/farmacologia , Linhagem Celular , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Feminino , Corantes Fluorescentes/química , Humanos , Injeções Intraperitoneais , Rim/efeitos dos fármacos , Rim/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Camundongos Nus , Testes de Sensibilidade Microbiana , Peritonite/microbiologia , Peritonite/patologia , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/microbiologia , Mucosa Respiratória/patologia , Esteroides/farmacocinética , CatelicidinasRESUMO
BACKGROUND: Biofilm formation is associated with various aspects of bacterial and fungal infection. This study was designed to assess the impact of diverse natural polyelectrolytes, such as DNA, F-actin, neurofilaments (NFs), vimentin and purified Pf1 bacteriophage on biofilm formation and swarming motility of select pathogens including Pseudomonas aeruginosa associated with lung infections in CF patients. RESULTS: The bacteriophage Pf1 (1 mg/ml) significantly increased biofilm mass produced by Pseudomonas aeruginosa P14, Escherichia coli RS218 and Bacillus subtilis ATCC6051. DNA, F-actin, NFs and Pf1 also increased biofilm mass of the fungal C. albicans 1409 strain. Addition of F-actin, DNA or Pf1 bacteriophage to 0.5% agar plates increased swarming motility of Pseudomonas aeruginosa Xen5. CONCLUSIONS: The presence of polyelectrolytes at infection sites is likely to promote biofilm growth and bacterial swarming.
Assuntos
Fenômenos Fisiológicos Bacterianos/efeitos dos fármacos , Bacteriófago Pf1/fisiologia , Biofilmes/crescimento & desenvolvimento , Eletrólitos/farmacologia , Polímeros/farmacologia , Actinas/farmacologia , Linhagem Celular , DNA/farmacologia , Humanos , Filamentos Intermediários/metabolismo , Vimentina/farmacologiaRESUMO
The signals that initiate cell invasion are not well understood, but there is increasing evidence that extracellular physical signals play an important role. Here we show that epithelial cell invasion in the intestine of zebrafish meltdown (mlt) mutants arises in response to unregulated contractile tone in the surrounding smooth muscle cell layer. Physical signaling in mlt drives formation of membrane protrusions within the epithelium that resemble invadopodia, matrix-degrading protrusions present in invasive cancer cells. Knockdown of Tks5, a Src substrate that is required for invadopodia formation in mammalian cells blocked formation of the protrusions and rescued invasion in mlt. Activation of Src-signaling induced invadopodia-like protrusions in wild type epithelial cells, however the cells did not migrate into the tissue stroma, thus indicating that the protrusions were required but not sufficient for invasion in this in vivo model. Transcriptional profiling experiments showed that genes responsive to reactive oxygen species (ROS) were upregulated in mlt larvae. ROS generators induced invadopodia-like protrusions and invasion in heterozygous mlt larvae but had no effect in wild type larvae. Co-activation of oncogenic Ras and Wnt signaling enhanced the responsiveness of mlt heterozygotes to the ROS generators. These findings present the first direct evidence that invadopodia play a role in tissue cell invasion in vivo. In addition, they identify an inducible physical signaling pathway sensitive to redox and oncogenic signaling that can drive this process.
Assuntos
Movimento Celular , Intestinos/patologia , Intestinos/fisiopatologia , Tono Muscular/fisiologia , Músculo Liso/fisiologia , Peixe-Zebra/fisiologia , Actinas/metabolismo , Animais , Membrana Basal/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Matriz Extracelular/metabolismo , Heterozigoto , Homozigoto , Larva/metabolismo , Mecanotransdução Celular , Contração Muscular/fisiologia , Mutação/genética , Oncogenes/genética , Oxirredução , Estresse Oxidativo , Pseudópodes/metabolismo , Transdução de Sinais , Proteínas de Peixe-Zebra/metabolismo , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismoRESUMO
BACKGROUND: Tick-borne encephalitis (TBE) is a serious acute central nervous system infection that can result in death or long-term neurological dysfunctions. We hypothesize that changes in sphingosine-1-phosphate (S1P) concentration occur during TBE development. METHODS: S1P and interleukin-6 (IL-6) concentrations in blood plasma and cerebrospinal fluid (CSF) were measured using HPLC and ELISA, respectively. The effects of S1P on cytoskeletal structure and IL-6 production were assessed using rat astrocyte primary cultures with and without addition of plasma gelsolin and the S1P receptor antagonist fingolimod phosphate (FTY720P). RESULTS: We report that acute inflammation due to TBE virus infection is associated with elevated levels of S1P and IL-6 in the CSF of infected patients. This elevated concentration is observed even at the earliest neurologic stage of disease, and may be controlled by glucocorticosteroid anti-inflammatory treatment, administered to patients unresponsive to antipyretic drugs and who suffer from a fever above 39°C. In vitro, treatment of confluent rat astrocyte monolayers with a high concentration of S1P (5 µM) results in cytoskeletal actin remodeling that can be prevented by the addition of recombinant plasma gelsolin, FTY720P, or their combination. Additionally, gelsolin and FTY720P significantly decreased S1P-induced release of IL-6. CONCLUSIONS: TBE is associated with increased concentration of S1P and IL-6 in CSF, and this increase might promote development of inflammation. The consequences of increased extracellular S1P can be modulated by gelsolin and FTY720P. Therefore, blocking the inflammatory response at sites of infection by agents modulating S1P pathways might aid in developing new strategies for TBE treatment.
Assuntos
Encefalite Transmitida por Carrapatos/líquido cefalorraquidiano , Encefalite Transmitida por Carrapatos/imunologia , Lisofosfolipídeos/líquido cefalorraquidiano , Esfingosina/análogos & derivados , Adulto , Animais , Astrócitos/efeitos dos fármacos , Biomarcadores/líquido cefalorraquidiano , Cromatografia Líquida de Alta Pressão , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Interleucina-6/líquido cefalorraquidiano , Lisofosfolipídeos/farmacologia , Masculino , Pessoa de Meia-Idade , Ratos , Esfingosina/líquido cefalorraquidiano , Esfingosina/farmacologiaRESUMO
Many cell types, including neurons, astrocytes and other cells of the central nervous system respond to changes in extracellular matrix or substrate viscoelasticity, and increased tissue stiffness is a hallmark of several disease states including fibrosis and some types of cancers. Whether the malignant tissue in brain, an organ that lacks the protein-based filamentous extracellular matrix of other organs, exhibits the same macroscopic stiffening characteristic of breast, colon, pancreatic, and other tumors is not known. In this study we show that glioma cells like normal astrocytes, respond strongly in vitro to substrate stiffness in the range of 100 to 2000 Pa, but that macroscopic (mm to cm) tissue samples isolated from human glioma tumors have elastic moduli on the order of 200 Pa that are indistinguishable from those of normal brain. However, both normal brain and glioma tissues increase their shear elastic moduli under modest uniaxial compression, and glioma tissue stiffens more strongly under compression than does normal brain. These findings suggest that local tissue stiffness has the potential to alter glial cell function, and that stiffness changes in brain tumors might arise not from increased deposition or crosslinking of collagen-rich extracellular matrix but from pressure gradients that form within the tumors in vivo.
RESUMO
The structure and dynamics of the cell nucleus regulate nearly every facet of the cell. Changes in nuclear shape limit cell motility and gene expression. Although the nucleus is generally seen as the stiffest organelle in the cell, cells can nevertheless deform the nucleus to large strains by small mechanical stresses. Here, we show that the mechanical response of the cell nucleus exhibits active fluidization that is driven by the BRG 1 motor of the SWI/SNF/BAF chromatin-remodeling complex. Atomic force microscopy measurements show that the nucleus alters stiffness in response to the cell substrate stiffness, which is retained after the nucleus is isolated and that the work of nuclear compression is mostly dissipated rather than elastically stored. Inhibiting BRG 1 stiffens the nucleus and eliminates dissipation and nuclear remodeling both in isolated nuclei and in intact cells. These findings demonstrate a novel link between nuclear motor activity and global nuclear mechanics.
RESUMO
OBJECTIVES: We aim to develop antibacterial peptide mimics resistant to protease degradation, with broad-spectrum activity at sites of infection. METHODS: The bactericidal activities of LL-37, ceragenins CSA-13, CSA-90 and CSA-92 and the spermine-conjugated dexamethasone derivative D2S were evaluated using MIC and MBC measurements. Gingival fibroblast counting, interleukin-8 (IL-8) and lactate dehydrogenase (LDH) release from keratinocytes (HaCat) were used to determine effects on cell growth, pro-inflammatory response and toxicity. RESULTS: All tested cationic lipids showed stronger bactericidal activity than LL-37. Incubation of Staphylococcus aureus with half the MIC of LL-37 led to the appearance of bacteria resistant to its bactericidal effects, but identical incubations with CSA-13 or D2S did not produce resistant bacteria. Cathelicidin LL-37 significantly increased the total number of gingival fibroblasts, but ceragenins and D2S did not alter gingival fibroblast growth. Cationic lipids showed no toxicity to HaCat cells at concentrations resulting in bacterial killing. CONCLUSIONS: These data suggest that cationic lipids such as ceragenins warrant further testing as potential novel antibacterial agents.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Boca/microbiologia , Infecções Respiratórias/microbiologia , Adolescente , Bactérias/isolamento & purificação , Humanos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacosRESUMO
In addition to its antibacterial activity, the cathelicidin-derived LL-37 peptide induces multiple immunomodulatory effects on host cells. Atomic force microscopy, F-actin staining with phalloidin, passage of FITC-conjugated dextran through a monolayer of lung epithelial cells, and assessment of bacterial outgrowth from cells subjected to Pseudomonas aeruginosa infection were used to determine LL-37's effect on epithelial cell mechanical properties, permeability, and bacteria uptake. A concentration-dependent increase in stiffness and F-actin content in the cortical region of A549 cells and primary human lung epithelial cells was observed after treatment with LL-37 (0.5-5 µM), sphingosine 1-phosphate (1 µM), or LPS (1 µg/ml) or infection with PAO1 bacteria. Other cationic peptides, such as RK-31, KR-20, or WLBU2, and the antibacterial cationic steroid CSA-13 did not reproduce the effect of LL-37. A549 cell pretreatment with WRW4, an antagonist of the transmembrane formyl peptide receptor-like 1 protein attenuated LL-37's ability to increase cell stiffness. The LL-37-mediated increase in cell stiffness was accompanied by a decrease in permeability and P. aeruginosa uptake by a confluent monolayer of polarized normal human bronchial epithelial cells. These results suggested that the antibacterial effect of LL-37 involves an LL-37-dependent increase in cell stiffness that prevents epithelial invasion by bacteria.
Assuntos
Peptídeos Catiônicos Antimicrobianos/fisiologia , Catelicidinas/fisiologia , Permeabilidade da Membrana Celular/imunologia , Inibição de Migração Celular/imunologia , Pulmão/imunologia , Pseudomonas aeruginosa/patogenicidade , Mucosa Respiratória/imunologia , Mucosa Respiratória/microbiologia , Sequência de Aminoácidos , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Inibição de Migração Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Pulmão/citologia , Pulmão/efeitos dos fármacos , Dados de Sequência Molecular , Pseudomonas aeruginosa/efeitos dos fármacos , Mucosa Respiratória/efeitos dos fármacosRESUMO
Scaffolds delivered to injured spinal cords to stimulate axon connectivity often match the anisotropy of native tissue using guidance cues along the rostral-caudal axis, but current approaches do not mimic the heterogeneity of host tissue mechanics. Although white and gray matter have different mechanical properties, it remains unclear whether tissue mechanics also vary along the length of the cord. Mechanical testing performed in this study indicates that bulk spinal cord mechanics do differ along anatomical level and that these differences are caused by variations in the ratio of white and gray matter. These results suggest that scaffolds recreating the heterogeneity of spinal cord tissue mechanics must account for the disparity between gray and white matter. Digital light processing (DLP) provides a means to mimic spinal cord topology, but has previously been limited to printing homogeneous mechanical properties. We describe a means to modify DLP to print scaffolds that mimic spinal cord mechanical heterogeneity caused by variation in the ratio of white and gray matter, which improves axon infiltration compared to controls exhibiting homogeneous mechanical properties. These results demonstrate that scaffolds matching the mechanical heterogeneity of white and gray matter improve the effectiveness of biomaterials transplanted within the injured spinal cord.
Assuntos
Traumatismos da Medula Espinal , Humanos , Medula Espinal , Axônios , Materiais Biocompatíveis , Impressão Tridimensional , Alicerces TeciduaisRESUMO
LL-37 peptide is a multifunctional host defense molecule essential for normal immune responses to infection or tissue injury. In this study we assess the impact of LL-37 on endothelial stiffness and barrier permeability. Fluorescence microscopy reveals membrane localization of LL-37 after its incubation with human umbilical vein endothelial cells (HUVECs). A concentration-dependent increase in stiffness was observed in HUVECs, bovine aortic endothelial cells (BAECs), human pulmonary microvascular endothelial cells, and mouse aorta upon LL-37 (0.5-5 µM) addition. Stiffening of BAECs by LL-37 was blocked by P2X7 receptor antagonists and by the intracellular Ca²(+) chelator BAPTA-AM. Increased cellular stiffness correlated with a decrease in permeability of HUVEC cell monolayers after LL-37 addition compared with nontreated cells, which was similar to the effect observed upon treatment with sphingosine 1-phosphate, and both treatments increased F-actin content in the cortical region of the cells. These results suggest that the antiinflammatory effect of LL-37 at the site of infection or injury involves an LL-37-mediated increase in cell stiffening that prevents increased pericellular permeability. Such a mechanism may help to maintain tissue fluid homeostasis.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Actinas/fisiologia , Junções Aderentes/fisiologia , Animais , Aorta/efeitos dos fármacos , Bovinos , Membrana Celular , Células Cultivadas , Citoesqueleto/fisiologia , Humanos , Camundongos , Permeabilidade/efeitos dos fármacos , CatelicidinasRESUMO
The mechanical properties of tissues and cells including renal glomeruli are important determinants of their differentiated state, function, and responses to injury but are not well characterized or understood. Understanding glomerular mechanics is important for understanding renal diseases attributable to abnormal expression or assembly of structural proteins and abnormal hemodynamics. We use atomic force microscopy (AFM) and a new technique, capillary micromechanics, to measure the elastic properties of rat glomeruli. The Young's modulus of glomeruli was 2,500 Pa, and it was reduced to 1,100 Pa by cytochalasin and latunculin, and to 1,400 Pa by blebbistatin. Cytochalasin or latrunculin reduced the F/G actin ratios of glomeruli but did not disrupt their architecture. To assess glomerular biomechanics in disease, we measured the Young's moduli of glomeruli from two mouse models of primary glomerular disease, Col4a3(-/-) mice (Alport model) and Tg26(HIV/nl) mice (HIV-associated nephropathy model), at stages where glomerular injury was minimal by histopathology. Col4a3(-/-) mice express abnormal glomerular basement membrane proteins, and Tg26(HIV/nl) mouse podocytes have multiple abnormalities in morphology, adhesion, and cytoskeletal structure. In both models, the Young's modulus of the glomeruli was reduced by 30%. We find that glomeruli have specific and quantifiable biomechanical properties that are dependent on the state of the actin cytoskeleton and nonmuscle myosins. These properties may be altered early in disease and represent an important early component of disease. This increased deformability of glomeruli could directly contribute to disease by permitting increased distension with hemodynamic force or represent a mechanically inhospitable environment for glomerular cells.