RESUMO
BACKGROUND: Astrocytes are the most abundant cell type of the central nervous system and are fundamentally involved in homeostasis, neuroprotection, and synaptic plasticity. This regulatory function of astrocytes on their neighboring cells in the healthy brain is subject of current research. In the ischemic brain we assume disease specific differences in astrocytic acting. The renin-angiotensin-aldosterone system regulates arterial blood pressure through endothelial cells and perivascular musculature. Moreover, astrocytes express angiotensin II type 1 and 2 receptors. However, their role in astrocytic function has not yet been fully elucidated. We hypothesized that the angiotensin II receptors impact astrocyte function as revealed in an in vitro system mimicking cerebral ischemia. Astrocytes derived from neonatal wistar rats were exposed to telmisartan (angiotensin II type 1 receptor-blocker) or PD123319 (angiotensin II type 2 receptor-blocker) under normal conditions (control) or deprivation from oxygen and glucose. Conditioned medium (CM) of astrocytes was harvested to elucidate astrocyte-mediated indirect effects on microglia and cortical neurons. RESULT: The blockade of angiotensin II type 1 receptor by telmisartan increased the survival of astrocytes during ischemic conditions in vitro without affecting their proliferation rate or disturbing their expression of S100A10, a marker of activation. The inhibition of the angiotensin II type 2 receptor pathway by PD123319 resulted in both increased expression of S100A10 and proliferation rate. The CM of telmisartan-treated astrocytes reduced the expression of pro-inflammatory mediators with simultaneous increase of anti-inflammatory markers in microglia. Increased neuronal activity was observed after treatment of neurons with CM of telmisartan- as well as PD123319-stimulated astrocytes. CONCLUSION: Data show that angiotensin II receptors have functional relevance for astrocytes that differs in healthy and ischemic conditions and effects surrounding microglia and neuronal activity via secretory signals. Above that, this work emphasizes the strong interference of the different cells in the CNS and that targeting astrocytes might serve as a therapeutic strategy to influence the acting of glia-neuronal network in de- and regenerative context.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II , Bloqueadores do Receptor Tipo 2 de Angiotensina II , Astrócitos , AVC Isquêmico , Microglia , Neurônios , Ratos Wistar , Receptor Tipo 1 de Angiotensina , Receptor Tipo 2 de Angiotensina , Telmisartan , Animais , Ratos , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Bloqueadores do Receptor Tipo 2 de Angiotensina II/farmacologia , Animais Recém-Nascidos , Astrócitos/metabolismo , Astrócitos/efeitos dos fármacos , Benzimidazóis/farmacologia , Comunicação Celular/fisiologia , Comunicação Celular/efeitos dos fármacos , Células Cultivadas , Imidazóis/farmacologia , AVC Isquêmico/metabolismo , AVC Isquêmico/patologia , Microglia/metabolismo , Microglia/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Piridinas/farmacologia , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Telmisartan/farmacologiaRESUMO
Concussion, caused by a rotational acceleration/deceleration injury mild enough to avoid structural brain damage, is insufficiently captured in recent preclinical models, hampering the relation of pathophysiological findings on the cellular level to functional and behavioral deficits. We here describe a novel model of unrestrained, single vs. repetitive concussive brain injury (CBI) in male C56Bl/6j mice. Longitudinal behavioral assessments were conducted for up to seven days afterward, alongside the evaluation of structural cerebral integrity by in vivo magnetic resonance imaging (MRI, 9.4 T), and validated ex vivo by histology. Blood-brain barrier (BBB) integrity was analyzed by means of fluorescent dextran- as well as immunoglobulin G (IgG) extravasation, and neuroinflammatory processes were characterized both in vivo by positron emission tomography (PET) using [18F]DPA-714 and ex vivo using immunohistochemistry. While a single CBI resulted in a defined, subacute neuropsychiatric phenotype, longitudinal cognitive testing revealed a marked decrease in spatial cognition, most pronounced in mice subjected to CBI at high frequency (every 48 h). Functional deficits were correlated to a parallel disruption of the BBB, (R2 = 0.29, p < 0.01), even detectable by a significant increase in hippocampal uptake of [18F]DPA-714, which was not due to activation of microglia, as confirmed immunohistochemically. Featuring a mild but widespread disruption of the BBB without evidence of macroscopic damage, this model induces a characteristic neuro-psychiatric phenotype that correlates to the degree of BBB disruption. Based on these findings, the BBB may function as both a biomarker of CBI severity and as a potential treatment target to improve recovery from concussion.
Assuntos
Barreira Hematoencefálica , Concussão Encefálica , Camundongos , Barreira Hematoencefálica/diagnóstico por imagem , Barreira Hematoencefálica/patologia , Concussão Encefálica/diagnóstico por imagem , Concussão Encefálica/patologia , Animais , Tomografia por Emissão de Pósitrons , Masculino , Rotação , ComportamentoRESUMO
In the lung, pulmonary epithelial cells undergo mechanical stretching during ventilation. The associated cellular mechanoresponse is still poorly understood at the molecular level. Here, we demonstrate that activation of the mechanosensitive cation channel Piezo1 in a human epithelial cell line (H441) and in primary human lung epithelial cells induces the proteolytic activity of the metalloproteinases ADAM10 and ADAM17 at the plasma membrane. These ADAMs are known to convert cell surface expressed proteins into soluble and thereby play major roles in proliferation, barrier regulation and inflammation. We observed that chemical activation of Piezo1 promotes cleavage of substrates that are specific for either ADAM10 or ADAM17. Activation of Piezo1 also induced the synthesis and ADAM10/17-dependent release of the growth factor amphiregulin (AREG). In addition, junctional adhesion molecule A (JAM-A) was shed in an ADAM10/17-dependent manner resulting in a reduction of cell contacts. Stretching experiments combined with Piezo1 knockdown further demonstrated that mechanical activation promotes shedding via Piezo1. Most importantly, high pressure ventilation of murine lungs increased AREG and JAM-A release into the alveolar space, which was reduced by a Piezo1 inhibitor. Our study provides a novel link between stretch-induced Piezo1 activation and the activation of ADAM10 and ADAM17 in lung epithelium. This may help to understand acute respiratory distress syndrome (ARDS) which is induced by ventilation stress and goes along with perturbed epithelial permeability and release of growth factors.
Assuntos
Secretases da Proteína Precursora do Amiloide , Pulmão , Humanos , Camundongos , Animais , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Pulmão/metabolismo , Proteína ADAM10/genética , Proteína ADAM10/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Células Epiteliais/metabolismo , Canais Iônicos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Metaloproteases/metabolismo , Proteína ADAM17/genética , Proteína ADAM17/metabolismoRESUMO
Epithelia maintain a functional barrier during tissue turnover while facing varying mechanical stress. This maintenance requires both dynamic cell rearrangements driven by actomyosin-linked intercellular adherens junctions and ability to adapt to and resist extrinsic mechanical forces enabled by keratin filament-linked desmosomes. How these two systems crosstalk to coordinate cellular movement and mechanical resilience is not known. Here we show that in stratifying epithelia the polarity protein aPKCλ controls the reorganization from stress fibers to cortical actomyosin during differentiation and upward movement of cells. Without aPKC, stress fibers are retained resulting in increased contractile prestress. This aberrant stress is counterbalanced by reorganization and bundling of keratins, thereby increasing mechanical resilience. Inhibiting contractility in aPKCλ-/- cells restores normal cortical keratin networks but also normalizes resilience. Consistently, increasing contractile stress is sufficient to induce keratin bundling and enhance resilience, mimicking aPKC loss. In conclusion, our data indicate that keratins sense the contractile stress state of stratified epithelia and balance increased contractility by mounting a protective response to maintain tissue integrity.
Assuntos
Actomiosina , Transdução de Sinais , Actomiosina/metabolismo , Epitélio/metabolismo , Citoesqueleto/metabolismo , Queratinas/metabolismo , Células Epiteliais/metabolismoRESUMO
Cell contractility regulates epithelial tissue geometry development and homeostasis. The underlying mechanobiological regulation circuits are poorly understood and experimentally challenging. We developed an elastomeric pillar cage (EPC) array to quantify cell contractility as a mechanoresponse of epithelial microtissues to substrate stiffness and topography. The spatially confined EPC geometry consisted of 24 circularly arranged slender pillars (1.2 MPa, height: 50 µm; diameter: 10 µm, distance: 5 µm). These high-aspect-ratio pillars were confined at both ends by planar substrates with different stiffness (0.15-1.2 MPa). Analytical modeling and finite elements simulation retrieved cell forces from pillar displacements. For evaluation, highly contractile myofibroblasts and cardiomyocytes were assessed to demonstrate that the EPC device can resolve static and dynamic cellular force modes. Human breast (MCF10A) and skin (HaCaT) cells grew as adherence junction-stabilized 3D microtissues within the EPC geometry. Planar substrate areas triggered the spread of monolayered clusters with substrate stiffness-dependent actin stress fiber (SF)-formation and substantial single-cell actomyosin contractility (150-200 nN). Within the same continuous microtissues, the pillar-ring topography induced the growth of bilayered cell tubes. The low effective pillar stiffness overwrote cellular sensing of the high substrate stiffness and induced SF-lacking roundish cell shapes with extremely low cortical actin tension (11-15 nN). This work introduced a versatile biophysical tool to explore mechanobiological regulation circuits driving low- and high-tensional states during microtissue development and homeostasis. EPC arrays facilitate simultaneously analyzing the impact of planar substrate stiffness and topography on microtissue contractility, hence microtissue geometry and function.
Assuntos
Actinas , Actomiosina , Humanos , Citoesqueleto de Actina , Contração Muscular/fisiologiaRESUMO
The efficient and biocompatible transfer of nucleic acids into mammalian cells for research applications or medical purposes is a long-standing, challenging task. Viral transduction is the most efficient transfer system, but often entails high safety levels for research and potential health impairments for patients in medical applications. Lipo- or polyplexes are commonly used transfer systems but result in comparably low transfer efficiencies. Moreover, inflammatory responses caused by cytotoxic side effects were reported for these transfer methods. Often accountable for these effects are various recognition mechanisms for transferred nucleic acids. Using commercially available fusogenic liposomes (Fuse-It-mRNA), we established highly efficient and fully biocompatible transfer of RNA molecules for in vitro as well as in vivo applications. We demonstrated bypassing of endosomal uptake routes and, therefore, of pattern recognition receptors that recognize nucleic acids with high efficiency. This may underlie the observed almost complete abolishment of inflammatory cytokine responses. RNA transfer experiments into zebrafish embryos and adult animals fully confirmed the functional mechanism and the wide range of applications from single cells to organisms.
RESUMO
In their natural environment, most cells and tissues are continuously exposed to cyclic mechanical strain. Sensing these stimuli by mechanosensory proteins and subsequent conversion into a variety of biological responses (referred to as mechanotransduction) are key processes for tissue homeostasis, survival, and differentiation. Perturbations of underlying signaling pathways lead to severe diseases in vivo (Urciuoli E, Peruzzi B, Int J Mol Sci 21(24). https://doi.org/10.3390/ijms21249426, (2020)). In addition, cellular mechanoresponses to cyclic stretching of an isolated single cell differ from those of a cell monolayer, network, or even three-dimensional tissue. Since these processes depend on various physical and biological parameters, the development of a precise, well-characterized, and highly reproducible but also easily tunable stretcher assay is indispensable. Here, we describe the fabrication of defined elastic substrates and their application in cyclic stretching of cultured cells in a custom-made cell stretcher device. We focus on the detailed description of the system and provide a possibility for mechanoresponse characterization, using the analysis of actin stress fiber orientation as exemplary mechanoresponse to cyclic stretching of adherent cells.
Assuntos
Mecanotransdução Celular , Macas , Mecanotransdução Celular/fisiologia , Células Cultivadas , Transdução de Sinais , Actinas , Estresse MecânicoRESUMO
Fusogenic liposomes have been widely used for molecule delivery to cell membranes and cell interior. However, their physicochemical state is still little understood. We tested mechanical material behavior by micropipette aspiration of giant vesicles from fusogenic lipid mixtures and found that the membranes of these vesicles are fluid and under high mechanical tension even before aspiration. Based on this result, we developed a theoretical framework to determine the area expansion modulus and membrane tension of such pre-tensed vesicles from aspiration experiments. Surprisingly high membrane tension of 2.1 mN m-1 and very low area expansion modulus of 63 mN m-1 were found. We interpret these peculiar material properties as the result of a mechanically driven phase transition between the usual lamellar phase and an, as of now, not finally determined three dimensional phase of the lipid mixture. The free enthalpy of transition between these phases is very low, i.e. on the order of the thermal energy.
RESUMO
BACKGROUND & AIMS: Desmosomes are intercellular junctions connecting keratin intermediate filaments of neighboring cells. The cadherins desmoglein 2 (Dsg2) and desmocollin 2 mediate cell-cell adhesion, whereas desmoplakin (Dsp) provides the attachment of desmosomes to keratins. Although the importance of the desmosome-keratin network is well established in mechanically challenged tissues, we aimed to assess the currently understudied function of desmosomal proteins in intestinal epithelia. METHODS: We analyzed the intestine-specific villin-Cre DSP (DSPΔIEC) and the combined intestine-specific DSG2/DSPΔIEC (ΔDsg2/Dsp) knockout mice. Cross-breeding with keratin 8-yellow fluorescent protein knock-in mice and generation of organoids was performed to visualize the keratin network. A Dsp-deficient colorectal carcinoma HT29-derived cell line was generated and the role of Dsp in adhesion and mechanical stress was studied in dispase assays, after exposure to uniaxial cell stretching and during scratch assay. RESULTS: The intestine of DSPΔIEC mice was histopathologically inconspicuous. Intestinal epithelial cells, however, showed an accelerated migration along the crypt and an enhanced shedding into the lumen. Increased intestinal permeability and altered levels of desmosomal proteins were detected. An inconspicuous phenotype also was seen in ΔDsg2/Dsp mice. After dextran sodium sulfate treatment, DSPΔIEC mice developed more pronounced colitis. A retracted keratin network was seen in the intestinal epithelium of DSPΔIEC/keratin 8-yellow fluorescent protein mice and organoids derived from these mice presented a collapsed keratin network. The level, phosphorylation status, and solubility of keratins were not affected. Dsp-deficient HT29 cells had an impaired cell adhesion and suffered from increased cellular damage after stretch. CONCLUSIONS: Our results show that Dsp is required for proper keratin network architecture in intestinal epithelia, mechanical resilience, and adhesion, thereby protecting from injury.
Assuntos
Desmossomos , Queratinas , Animais , Adesão Celular , Desmoplaquinas/metabolismo , Desmossomos/metabolismo , Queratina-8/metabolismo , Queratinas/metabolismo , CamundongosRESUMO
Efficient and reliable transfer of nucleic acids for therapy applications is a major challenge. Stabilization of lipo- and polyplexes has already been successfully achieved by PEGylation. This modification reduces the interaction with serum proteins and thus prevents the lipoplexes from being cleared by the reticuloendothelial system. Problematically, this stabilization of lipoplexes simultaneously leads to reduced transfer efficiencies compared to non-PEGylated complexes. However, this reduction in transfer efficiency can be used to advantage since additional modification of PEGylated lipoplexes with functional groups enables improved selective transfer into target cells. Cancer cells overexpress folate receptors because of a significantly increased need of folate due to high cell proliferation rates. Thus, additional folate functionalization of PEGylated lipoplexes improves uptake into cancer cells. We demonstrate herein that NHS coupling chemistries can be used to modify two commercially available transfection reagents (Fuse-It-DNA and Lipofectamine® 3000) with NHS-PEG-folate for increased uptake of nucleic acids into cancer cells. Lipoplex characterization and functional analysis in cultures of cancer- and healthy cells clearly demonstrate that functionalization of PEGylated lipoplexes offers a promising method to generate efficient, stable and selective nucleic acid transfer systems.
RESUMO
The neural stem cell (NSC) niche is a highly vascularized microenvironment that supplies stem cells with relevant biological and chemical cues. However, the NSCs' proximity to the vasculature also means that the NSCs are subjected to permanent tissue deformation effected by the vessels' heartbeat-induced pulsatile movements. Cultivating NSCs under common culture conditions neglects the-yet unknown-influence of this cyclic mechanical strain on neural stem cells. Under the hypothesis that pulsatile strain should affect essential NSC functions, a cyclic uniaxial strain was applied under biomimetic conditions using an in-house developed stretching system based on cross-linked polydimethylsiloxane (PDMS) elastomer. While lineage commitment remained unaffected by cyclic deformation, strain affected NSC quiescence and cytoskeletal organization. Unexpectedly, cyclically stretched stem cells aligned in stretch direction, a phenomenon unknown for other types of cells in the mammalian organism. The same effect was observed for young astrocytes differentiating from NSCs. In contrast, young neurons differentiating from NSCs did not show mechanoresponsiveness. The exceptional orientation of NSCs and young astrocytes in the stretch direction was blocked upon RhoA activation and went along with a lack of stress fibers. Compared to postnatal astrocytes and mature neurons, NSCs and their young progeny displayed characteristic and distinct mechanoresponsiveness. Data suggest a protective role of young astrocytes in mixed cultures of differentiating neurons and astrocytes by mitigating the mechanical stress of pulsatile strain on developing neurons.
RESUMO
The cellular mechanisms of basement membrane (BM) invasion remain poorly understood. We investigated the invasion-promoting mechanisms of actin cytoskeleton reorganization in BM-covered MCF10A breast acini. High-resolution confocal microscopy has characterized actin cell protrusion formation and function in response to tumor-resembling ECM stiffness and soluble EGF stimulation. Traction force microscopy quantified the mechanical BM stresses that invasion-triggered acini exerted on the BM-ECM interface. We demonstrate that acini use non-proteolytic actin microspikes as functional precursors of elongated protrusions to initiate BM penetration and ECM probing. Further, these microspikes mechanically widened the collagen IV pores to anchor within the BM scaffold via force-transmitting focal adhesions. Pre-invasive basal cells located at the BM-ECM interface exhibited predominantly cortical actin networks and actin microspikes. In response to pro-invasive conditions, these microspikes accumulated and converted subsequently into highly contractile stress fibers. The phenotypical switch to stress fiber cells matched spatiotemporally with emerging high BM stresses that were driven by actomyosin II contractility. The activation of proteolytic invadopodia with MT1-MMP occurred at later BM invasion stages and only in cells already disseminating into the ECM. Our study demonstrates that BM pore-widening filopodia bridge mechanical ECM probing function and contractility-driven BM weakening. Finally, these EMT-related cytoskeletal adaptations are critical mechanisms inducing the invasive transition of benign breast acini.
Assuntos
Actinas/metabolismo , Membrana Basal/metabolismo , Miosina Tipo II/metabolismo , Fibras de Estresse/metabolismo , Células Acinares/citologia , Células Acinares/metabolismo , Mama/citologia , Mama/metabolismo , Adesão Celular , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Matriz Extracelular/metabolismo , Feminino , Humanos , Microscopia Confocal , Podossomos/metabolismo , Pseudópodes/metabolismo , Fibras de Estresse/químicaRESUMO
Formation of a barrier capable of protecting tissue from external damage, chemical factors, and pathogens is one of the main functions of the epidermis. Furthermore, upon development and during aging, mechanoprotective epidermal functions change dramatically. However, comparative studies between embryonic and adult skin in comparison to skin equivalents are still scarce which is especially due to the lack of appropriate measurement systems with sufficient accuracy and long-term tissue compatibility. Our studies fill this gap by developing a combined bioreactor and tensile testing machine for biomechanical analysis of living epithelia. Based on this tissue stretcher, our data clearly show that viscoelastic and plastic deformation behavior of embryonic and adult skin differ significantly. Tissue responses to static strain compared to cyclic strain also show a clear dependence on differentiation stage. Multilayered unkeratinized epidermis equivalents, on the other hand, respond very similar to mechanical stretch as adult tissue. This mechanical similarity is even more evident after a single cycle of mechanical preconditioning. Our studies therefore suggest that skin equivalents are well suited model systems to analyze cellular interactions of epidermal cells in natural tissues.
Assuntos
Envelhecimento/fisiologia , Epitélio/fisiologia , Queratinócitos/citologia , Mecanotransdução Celular/fisiologia , Pele Artificial , Pele/citologia , Animais , Fenômenos Biomecânicos , Materiais Biomiméticos/química , Reatores Biológicos , Comunicação Celular , Elasticidade , Embrião de Mamíferos , Epitélio/anatomia & histologia , Queratinócitos/fisiologia , Camundongos , Ratos , Resistência à Tração , ViscosidadeRESUMO
Anterior cruciate ligament (ACL) ruptures are usually treated with autograft implantation to prevent knee instability. Tissue engineered ACL reconstruction is becoming promising to circumvent autograft limitations. The aim was to evaluate the influence of cyclic stretch on lapine (L) ACL fibroblasts on embroidered scaffolds with respect to adhesion, DNA and sulphated glycosaminoglycan (sGAG) contents, gene expression of ligament-associated extracellular matrix genes, such as type I collagen, decorin, tenascin C, tenomodulin, gap junctional connexin 43 and the transcription factor Mohawk. Control scaffolds and those functionalized by gas phase fluorination and cross-linked collagen foam were either pre-cultured with a suspension or with spheroids of LACL cells before being subjected to cyclic stretch (4%, 0.11 Hz, 3 days). Stretch increased significantly the scaffold area colonized with cells but impaired sGAGs and decorin gene expression (functionalized scaffolds seeded with cell suspension). Stretching increased tenascin C, connexin 43 and Mohawk but decreased decorin gene expression (control scaffolds seeded with cell suspension). Pre-cultivation of functionalized scaffolds with spheroids might be the more suitable method for maintaining ligamentogenesis in 3D scaffolds compared to using a cell suspension due to a significantly higher sGAG content in response to stretching and type I collagen gene expression in functionalized scaffolds.
Assuntos
Ligamento Cruzado Anterior/fisiologia , Esferoides Celulares/citologia , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Ligamento Cruzado Anterior/citologia , Adesão Celular , Proliferação de Células , Células Cultivadas , Conexinas/genética , Conexinas/metabolismo , Decorina/genética , Decorina/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Feminino , Fibroblastos/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Homeostase , Masculino , Poliésteres/química , Coelhos , Regeneração , Esferoides Celulares/metabolismo , Estresse MecânicoRESUMO
Basically, all mammalian tissues are constantly exposed to a variety of environmental mechanical signals. Depending on the signal strength, mechanics intervenes in a multitude of cellular processes and is thus capable of inducing simple cellular adaptations but also complex differentiation processes and even apoptosis. The underlying recognition typically depends on mechanosensitive proteins, which most often sense the mechanical signal for the induction of a cellular signaling cascade by changing their protein conformation. However, the fate of mechanosensors after mechanical stress application is still poorly understood, and it remains unclear whether protein degradation pathways affect the mechanosensitivity of cells. Here, we show that cyclic stretch induces autophagosome formation in a time-dependent manner. Formation depends on the cochaperone BAG family molecular chaperone regulator 3 (BAG3) and thus likely involves BAG3-mediated chaperone-assisted selective autophagy. Furthermore, we demonstrate that strain-induced cell reorientation is clearly delayed upon inhibition of autophagy, suggesting a bidirectional cross-talk between mechanotransduction and autophagic degradation. The strength of the observed delay depends on stable adhesion structures and stress fiber formation in a Ras homologue family member A (RhoA)-dependent manner.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Mecanorreceptores/metabolismo , Animais , Apoptose/fisiologia , Autofagossomos/metabolismo , Autofagia/fisiologia , Fenômenos Biomecânicos , Linhagem Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Mecanorreceptores/citologia , Mecanotransdução Celular , Camundongos , Músculo Liso/citologia , Músculo Liso/metabolismo , Proteólise , Ratos , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismoRESUMO
Cell survival, tissue integrity and organismal health depend on the ability to maintain functional protein networks even under conditions that threaten protein integrity. Protection against such stress conditions involves the adaptation of folding and degradation machineries, which help to preserve the protein network by facilitating the refolding or disposal of damaged proteins. In multicellular organisms, cells are permanently exposed to stress resulting from mechanical forces. Yet, for long time mechanical stress was not recognized as a primary stressor that perturbs protein structure and threatens proteome integrity. The identification and characterization of protein folding and degradation systems, which handle force-unfolded proteins, marks a turning point in this regard. It has become apparent that mechanical stress protection operates during cell differentiation, adhesion and migration and is essential for maintaining tissues such as skeletal muscle, heart and kidney as well as the immune system. Here, we provide an overview of recent advances in our understanding of mechanical stress protection.
Assuntos
Dobramento de Proteína , Proteostase , Sobrevivência Celular , Proteoma/metabolismo , Estresse MecânicoRESUMO
HYPOTHESIS: Colloidal aggregation phenomena have been found responsible for the supersaturation of poorly water-soluble drugs, potentially leading to bioavailability enhancements. Unlike coarse precipitates, phase separation in the form of colloids, is expected to enhance drug supersaturation performance. Therefore, a high proportion of these colloids should correlate with the extent and the kinetics of supersaturation. The prime objective of the current study is to provide a mechanistic understanding on supersaturation for the model drug albendazole (ALB) in combination with twelve polymers. EXPERIMENTS: Species separated after a pH-shift were characterized by dynamic light scattering (DLS), freeze-fracture electron microscopy (FF-EM) and transmission X-ray diffraction (XRD). Laser diffraction (LD) in a liquid cell was introduced for a relative quantification of the colloidally separated species, described as colloid fraction. The pH-dependent supersaturation was assessed online using a miniaturized dissolution assay. FINDINGS: Here, a measure of the extent of amorphous colloidal phase separation was established, and its impact on supersaturation was evaluated. As a result, a correlation was found between the extent of supersaturation and the colloid fraction. This confirmed the dependence of polymer-mediated enabling and preservation of supersaturation on the ability of polymers to stabilize colloid fractions. Furthermore, a fixed ratio was suggested between the dissolved drug and colloidally separated drug as the kinetic profiles of both species showed similar trajectories. In conclusion, colloid fractions were identified to be responsible for dissolved and potentially bioavailable drug molecules.
Assuntos
Coloides , Polímeros , Disponibilidade Biológica , Liberação Controlada de Fármacos , Cinética , SolubilidadeRESUMO
Local basement membrane (BM) disruption marks the initial step of breast cancer invasion. The activation mechanisms of force-driven BM-weakening remain elusive. We studied the mechanical response of MCF10A-derived human breast cell acini with BMs of tuneable maturation to physical and soluble tumour-like extracellular matrix (ECM) cues. Traction force microscopy (TFM) and elastic resonator interference stress microscopy (ERISM) were used to quantify pro-invasive BM stress and protrusive forces. Substrate stiffening and mechanically impaired BM scaffolds induced the invasive transition of benign acini synergistically. Robust BM scaffolds attenuated this invasive response. Additional oncogenic EGFR activation compromised the BMs' barrier function, fuelling invasion speed and incidence. Mechanistically, EGFR-PI3-Kinase downstream signalling modulated both MMP- and force-driven BM-weakening processes. We show that breast acini form non-proteolytic and BM-piercing filopodia for continuous matrix mechanosensation, which significantly push and pull on the BM and ECM under pro-invasive conditions. Invasion-triggered acini further shear and compress their BM by contractility-based stresses that were significantly increased (3.7-fold) compared to non-invasive conditions. Overall, the highest amplitudes of protrusive and contractile forces accompanied the highest invasiveness. This work provides a mechanistic concept for tumour ECM-induced mechanically misbalanced breast glands fuelling force-driven BM disruption. Finally, this could facilitate early cell dissemination from pre-invasive lesions to metastasize eventually.
Assuntos
Mama/metabolismo , Fator de Crescimento Epidérmico/genética , Neoplasias/genética , Células Acinares/metabolismo , Células Acinares/patologia , Membrana Basal/metabolismo , Membrana Basal/patologia , Mama/patologia , Linhagem Celular Tumoral , Receptores ErbB/genética , Matriz Extracelular/genética , Matriz Extracelular/patologia , Feminino , Humanos , Glândulas Mamárias Humanas/patologia , Fenômenos Mecânicos , Invasividade Neoplásica/genética , Neoplasias/patologia , Pseudópodes/genética , Pseudópodes/patologiaRESUMO
Mechanical stress of ligaments varies; hence, ligament fibroblasts must adapt their expression profile to novel mechanomilieus to ensure tissue resilience. Activation of the mechanoreceptors leads to a specific signal transduction, the so-called mechanotransduction. However, with regard to their natural three-dimensional (3D) microenvironment cell reaction to mechanical stimuli during emigrating from a 3D spheroid culture is still unclear. This study aims to provide a deeper understanding of the reaction profile of anterior cruciate ligament (ACL)-derived fibroblasts exposed to cyclic uniaxial strain in two-dimensional (2D) monolayer culture and during emigration from 3D spheroids with respect to cell survival, cell and cytoskeletal orientation, distribution, and expression profile. Monolayers and spheroids were cultured in crosslinked polydimethyl siloxane (PDMS) elastomeric chambers and uniaxially stretched (14% at 0.3 Hz) for 48 h. Cell vitality, their distribution, nuclear shape, stress fiber orientation, focal adhesions, proliferation, expression of ECM components such as sulfated glycosaminoglycans, collagen type I, decorin, tenascin C and cell-cell communication-related gap junctional connexin (CXN) 43, tendon-related markers Mohawk and tenomodulin (myodulin) were analyzed. In contrast to unstretched cells, stretched fibroblasts showed elongation of stress fibers, cell and cytoskeletal alignment perpendicular to strain direction, less rounded cell nuclei, increased numbers of focal adhesions, proliferation, amplified CXN43, and main ECM component expression in both cultures. The applied cyclic stretch protocol evoked an anabolic response and enhanced tendon-related marker expression in ACL-derived fibroblasts emigrating from 3D spheroids and seems also promising to support in future tissue formation in ACL scaffolds seeded in vitro with spheroids.