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1.
Artigo em Inglês | MEDLINE | ID: mdl-38860845

RESUMO

COVID-19 syndrome is characterized by acute lung injury, hypoxemic respiratory failure, and high mortality. Alveolar Type 2 (AT2) cells are essential for gas exchange, repair, and regeneration of distal lung epithelium. We have shown that the causative agent, SARS-CoV-2 and other ß-coronavirus genus members induce an ER stress response in vitro, however the consequences for host AT2 function in vivo are less understood. To study this, two murine models of coronavirus infection were employed- mouse hepatitis virus-1 (MHV-1) in A/J mice and a mouse adapted SARS-CoV-2 strain. MHV-1 infected mice exhibited dose-dependent weight loss with histological evidence of distal lung injury accompanied by elevated bronchoalveolar lavage fluid (BALF) cell counts and total protein. AT2 cells showed evidence of both viral infection and increased BIP/GRP78 expression, consistent with activation of the unfolded protein response (UPR). The AT2 UPR included increased IRE1α signaling and a biphasic response in PERK signaling accompanied marked reductions in AT2 and BALF surfactant protein (SP-B, SP-C) content, increases in surfactant surface tension, and emergence of a re-programmed epithelial cell population (Krt8+, Cldn4+). The loss of a homeostatic AT2 endophenotype was attenuated by treatment with the IRE1α inhibitor OPK711. As proof-of-concept, C57BL6 mice infected with mouse-adapted SARS-CoV-2 demonstrated similar lung injury and evidence of disrupted surfactant homeostasis. We conclude that lung injury from ß-coronavirus infection results from an aberrant host response activating multiple AT2 UPR pathways, altering surfactant metabolism/function, and changing AT2 endophenotypes offering a mechanistic link between SARS-CoV-2 infection, AT2 cell biology, and acute respiratory failure.

2.
Res Sq ; 2024 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-38562870

RESUMO

The lung is a dynamic mechanical organ and several pulmonary disorders are characterized by heterogeneous changes in the lung's local mechanical properties (i.e. stiffness). These alterations lead to abnormal lung tissue deformation (i.e. strain) which have been shown to promote disease progression. Although heterogenous mechanical properties may be important biomarkers of disease, there is currently no non-invasive way to measure these properties for clinical diagnostic purposes. In this study, we use a magnetic resonance elastography technique to measure heterogenous distributions of the lung's shear stiffness in healthy adults and in people with Cystic Fibrosis. Additionally, computational finite element models which directly incorporate the measured heterogenous mechanical properties were developed to assess the effects on lung tissue deformation. Results indicate that consolidated lung regions in people with Cystic Fibrosis exhibited increased shear stiffness and reduced spatial heterogeneity compared to surrounding non-consolidated regions. Accounting for heterogenous lung stiffness in healthy adults did not change the globally averaged strain magnitude obtained in computational models. However, computational models that used heterogenous stiffness measurements predicted significantly more variability in local strain and higher spatial strain gradients. Finally, computational models predicted lower strain variability and spatial strain gradients in consolidated lung regions compared to non-consolidated regions. These results indicate that spatial variability in shear stiffness alters local strain and strain gradient magnitudes in people with Cystic Fibrosis. This imaged-based modeling technique therefore represents a clinically viable way to non-invasively assess lung mechanics during both health and disease.

3.
bioRxiv ; 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38464068

RESUMO

Patients with compromised respiratory function frequently require mechanical ventilation to survive. Unfortunately, non-uniform ventilation of injured lungs generates complex mechanical forces that lead to ventilator induced lung injury (VILI). Although investigators have developed lung-on-a-chip systems to simulate normal respiration, modeling the complex mechanics of VILI as well as the subsequent recovery phase is a challenge. Here we present a novel humanized in vitro ventilator-on-a-chip (VOC) model of the lung microenvironment that simulates the different types of injurious forces generated in the lung during mechanical ventilation. We used transepithelial/endothelial electrical resistance (TEER) measurements to investigate how individual and simultaneous application of the different mechanical forces alters real-time changes in barrier integrity during and after injury. We find that compressive stress (i.e. barotrauma) does not significantly alter barrier integrity while over-distention (20% cyclic radial strain, volutrauma) results in decreased barrier integrity that quickly recovers upon removal of mechanical stress. Conversely, surface tension forces generated during airway reopening (atelectrauma), result in a rapid loss of barrier integrity with a delayed recovery relative to volutrauma. Simultaneous application of cyclic stretching (volutrauma) and airway reopening (atelectrauma), indicate that the surface tension forces associated with reopening fluid-occluded lung regions is the primary driver of barrier disruption. Thus, our novel VOC system can monitor the effects of different types of injurious forces on barrier disruption and recovery in real-time and can be used to identify the biomechanical mechanisms of VILI.

4.
ACS Nano ; 17(17): 16539-16552, 2023 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-37595605

RESUMO

The pro-inflammatory response of alveolar macrophages to injurious physical forces during mechanical ventilation is regulated by the anti-inflammatory microRNA, miR-146a. Increasing miR-146a expression to supraphysiologic levels using untargeted lipid nanoparticles reduces ventilator-induced lung injury but requires a high initial dose of miR-146a making it less clinically applicable. In this study, we developed mannosylated lipid nanoparticles that can effectively mitigate lung injury at the initiation of mechanical ventilation with lower doses of miR-146a. We used a physiologically relevant humanized in vitro coculture system to evaluate the cell-specific targeting efficiency of the mannosylated lipid nanoparticle. We discovered that mannosylated lipid nanoparticles preferentially deliver miR-146a to alveolar macrophages and reduce force-induced inflammation in vitro. Our in vivo study using a clinically relevant mouse model of hemorrhagic shock-induced acute respiratory distress syndrome demonstrated that delivery of a low dose of miR-146a (0.1 nmol) using mannosylated lipid nanoparticles dramatically increases miR-146a levels in mouse alveolar macrophages and decreases lung inflammation. These data suggest that mannosylated lipid nanoparticles may have the therapeutic potential to mitigate lung injury during mechanical ventilation.


Assuntos
Lesão Pulmonar , MicroRNAs , Síndrome do Desconforto Respiratório , Choque Hemorrágico , Animais , Camundongos , Macrófagos , Síndrome do Desconforto Respiratório/tratamento farmacológico
5.
bioRxiv ; 2023 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-36824913

RESUMO

The pro-inflammatory response of alveolar macrophages to injurious physical forces during mechanical ventilation is regulated by the anti-inflammatory microRNA, miR-146a. Increasing miR-146a expression to supraphysiologic levels using untargeted lipid nanoparticles reduces ventilator-induced lung injury, but requires a high initial dose of miR-146a making it less clinically applicable. In this study, we developed mannosylated lipid nanoparticles that can effectively mitigate lung injury at the initiation of mechanical ventilation with lower doses of miR-146a. We used a physiologically relevant humanized in vitro co-culture system to evaluate the cell-specific targeting efficiency of the mannosylated lipid nanoparticle. We discovered that mannosylated lipid nanoparticles preferentially deliver miR-146a to alveolar macrophages and reduce force-induced inflammation in vitro . Our in vivo study using a clinically relevant mouse model of hemorrhagic shock-induced acute respiratory distress syndrome demonstrated that delivery of a low dose miR-146a (0.1 nmol) using mannosylated lipid nanoparticles dramatically increases miR-146a in mouse alveolar macrophages and decreases lung inflammation. These data suggest that mannosylated lipid nanoparticles may have therapeutic potential to mitigate lung injury during mechanical ventilation.

6.
Am J Physiol Lung Cell Mol Physiol ; 324(4): L507-L520, 2023 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-36791050

RESUMO

Idiopathic pulmonary fibrosis (IPF) is characterized by increased collagen accumulation that is progressive and nonresolving. Although fibrosis progression may be regulated by fibroblasts and alveolar macrophage (AM) interactions, this cellular interplay has not been fully elucidated. To study AM-fibroblast interactions, cells were isolated from IPF and normal human lung tissue and cultured independently or together in direct 2-D coculture, direct 3-D coculture, indirect transwell, and in 3-D hydrogels. AM influence on fibroblast function was assessed by gene expression, cytokine/chemokine secretion, and hydrogel contractility. Normal AMs cultured in direct contact with fibroblasts downregulated extracellular matrix (ECM) gene expression whereas IPF AMs had little to no effect. Fibroblast contractility was assessed by encapsulating cocultures in 3-D collagen hydrogels and monitoring gel diameter over time. Both normal and IPF AMs reduced baseline contractility of normal fibroblasts but had little to no effect on IPF fibroblasts. When stimulated with Toll-like receptor (TLR) agonists, IPF AMs increased production of pro-inflammatory cytokines TNFα and IL-1ß, compared with normal AMs. TLR ligand stimulation did not alter fibroblast contraction, but stimulation with exogenous TNFα and TGFß did alter contraction. To determine if the observed changes required cell-to-cell contact, AM-conditioned media and transwell systems were utilized. Transwell culture showed decreased ECM gene expression changes compared with direct coculture and conditioned media from AMs did not alter fibroblast contraction regardless of disease state. Taken together, these data indicate that normal fibroblasts are more responsive to AM crosstalk, and that AM influence on fibroblast behavior depends on cell proximity.


Assuntos
Fibrose Pulmonar Idiopática , Macrófagos Alveolares , Humanos , Macrófagos Alveolares/metabolismo , Técnicas de Cocultura , Fator de Necrose Tumoral alfa/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Meios de Cultivo Condicionados/farmacologia , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/metabolismo , Citocinas/metabolismo , Colágeno/metabolismo , Fibroblastos/metabolismo , Células Cultivadas
7.
Pulm Pharmacol Ther ; 79: 102196, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36682407

RESUMO

The acute respiratory distress syndrome (ARDS) is a life-threatening condition that causes respiratory failure. Despite numerous clinical trials, there are no molecularly targeted pharmacologic therapies to prevent or treat ARDS. Drug delivery during ARDS is challenging due to the heterogenous nature of lung injury and occlusion of lung units by edema fluid and inflammation. Pulmonary drug delivery during ARDS offers several potential advantages including limiting the off-target and off-organ effects and directly targeting the damaged and inflamed lung regions. In this review we summarize recent ARDS clinical trials using both systemic and pulmonary drug delivery. We then discuss the advantages of pulmonary drug delivery and potential challenges to its implementation. Finally, we discuss the use of nanoparticle drug delivery and surfactant-based drug carriers as potential strategies for delivering therapeutics to the injured lung in ARDS.


Assuntos
Surfactantes Pulmonares , Síndrome do Desconforto Respiratório , Humanos , Pulmão , Síndrome do Desconforto Respiratório/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Surfactantes Pulmonares/uso terapêutico , Portadores de Fármacos
8.
Am J Pathol ; 192(5): 750-761, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35183510

RESUMO

Lung fibrosis is characterized by the continuous accumulation of extracellular matrix (ECM) proteins produced by apoptosis-resistant (myo)fibroblasts. Lung epithelial injury promotes the recruitment and activation of fibroblasts, which are necessary for tissue repair and restoration of homeostasis. However, under pathologic conditions, a vicious cycle generated by profibrotic growth factors/cytokines, multicellular interactions, and matrix-associated signaling propagates the wound repair response and promotes lung fibrosis characterized not only by increased quantities of ECM proteins but also by changes in the biomechanical properties of the matrix. Importantly, changes in the biochemical and biomechanical properties of the matrix itself can serve to perpetuate fibroblast activity and propagate fibrosis, even in the absence of the initial stimulus of injury. The development of novel experimental models and methods increasingly facilitates our ability to interrogate fibrotic processes at the cellular and molecular levels. The goal of this review is to discuss the impact of ECM conditions in the development of lung fibrosis and to introduce new approaches to more accurately model the in vivo fibrotic microenvironment. This article highlights the pathologic roles of ECM in terms of mechanical force and the cellular interactions while reviewing in vitro and ex vivo models of lung fibrosis. The improved understanding of the fundamental mechanisms that contribute to lung fibrosis holds promise for identification of new therapeutic targets and improved outcomes.


Assuntos
Fibrose Pulmonar , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibrose , Humanos , Pulmão/patologia , Fibrose Pulmonar/patologia , Transdução de Sinais
9.
JCI Insight ; 6(14)2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34138757

RESUMO

The acute respiratory distress syndrome (ARDS) is a highly lethal condition that impairs lung function and causes respiratory failure. Mechanical ventilation (MV) maintains gas exchange in patients with ARDS but exposes lung cells to physical forces that exacerbate injury. Our data demonstrate that mTOR complex 1 (mTORC1) is a mechanosensor in lung epithelial cells and that activation of this pathway during MV impairs lung function. We found that mTORC1 is activated in lung epithelial cells following volutrauma and atelectrauma in mice and humanized in vitro models of the lung microenvironment. mTORC1 is also activated in lung tissue of mechanically ventilated patients with ARDS. Deletion of Tsc2, a negative regulator of mTORC1, in epithelial cells impairs lung compliance during MV. Conversely, treatment with rapamycin at the time MV is initiated improves lung compliance without altering lung inflammation or barrier permeability. mTORC1 inhibition mitigates physiologic lung injury by preventing surfactant dysfunction during MV. Our data demonstrate that, in contrast to canonical mTORC1 activation under favorable growth conditions, activation of mTORC1 during MV exacerbates lung injury and inhibition of this pathway may be a novel therapeutic target to mitigate ventilator-induced lung injury during ARDS.


Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Surfactantes Pulmonares/metabolismo , Respiração Artificial/efeitos adversos , Síndrome do Desconforto Respiratório/patologia , Lesão Pulmonar Induzida por Ventilação Mecânica/patologia , Animais , Modelos Animais de Doenças , Humanos , Pulmão/metabolismo , Pulmão/patologia , Complacência Pulmonar/fisiologia , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Camundongos , Síndrome do Desconforto Respiratório/tratamento farmacológico , Síndrome do Desconforto Respiratório/etiologia , Síndrome do Desconforto Respiratório/fisiopatologia , Sirolimo/farmacologia , Sirolimo/uso terapêutico , Lesão Pulmonar Induzida por Ventilação Mecânica/tratamento farmacológico , Lesão Pulmonar Induzida por Ventilação Mecânica/etiologia , Lesão Pulmonar Induzida por Ventilação Mecânica/fisiopatologia
10.
PLoS One ; 16(2): e0245653, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33534863

RESUMO

Collagen deposition contributes to both high mammographic density and breast cancer progression. Low stromal PTEN expression has been observed in as many as half of breast tumors and is associated with increases in collagen deposition, however the mechanism connecting PTEN loss to increased collagen deposition remains unclear. Here, we demonstrate that Pten knockout in fibroblasts using an Fsp-Cre;PtenloxP/loxP mouse model increases collagen fiber number and fiber size within the mammary gland. Pten knockout additionally upregulated Sparc transcription in fibroblasts and promoted collagen shuttling out of the cell. Interestingly, SPARC mRNA expression was observed to be significantly elevated in the tumor stroma as compared to the normal breast in several patient cohorts. While SPARC knockdown via shRNA did not affect collagen shuttling, it notably decreased assembly of exogenous collagen. In addition, SPARC knockdown decreased fibronectin assembly and alignment of the extracellular matrix in an in vitro fibroblast-derived matrix model. Overall, these data indicate upregulation of SPARC is a mechanism by which PTEN regulates collagen deposition in the mammary gland stroma.


Assuntos
Colágeno/metabolismo , Glândulas Mamárias Humanas/metabolismo , Osteonectina/metabolismo , PTEN Fosfo-Hidrolase/fisiologia , Animais , Linhagem Celular , Matriz Extracelular/metabolismo , Fibroblastos , Humanos , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/patologia , Camundongos , Camundongos Knockout
11.
Nat Commun ; 12(1): 289, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436554

RESUMO

Mechanical ventilation generates injurious forces that exacerbate lung injury. These forces disrupt lung barrier integrity, trigger proinflammatory mediator release, and differentially regulate genes and non-coding oligonucleotides including microRNAs. In this study, we identify miR-146a as a mechanosensitive microRNA in alveolar macrophages that has therapeutic potential to mitigate lung injury during mechanical ventilation. We use humanized in-vitro systems, mouse models, and biospecimens from patients to elucidate the expression dynamics of miR-146a needed to decrease lung injury during mechanical ventilation. We find that the endogenous increase in miR-146a following injurious ventilation is not sufficient to prevent lung injury. However, when miR-146a is highly overexpressed using a nanoparticle delivery platform it is sufficient to prevent injury. These data indicate that the endogenous increase in microRNA-146a during mechanical ventilation is a compensatory response that partially limits injury and that nanoparticle delivery of miR-146a is an effective strategy for mitigating lung injury during mechanical ventilation.


Assuntos
Técnicas de Transferência de Genes , Lesão Pulmonar/genética , Macrófagos Alveolares/metabolismo , Mecanotransdução Celular , Nanopartículas/química , Respiração Artificial/efeitos adversos , Transferência Adotiva , Animais , Lavagem Broncoalveolar , Feminino , Humanos , Inflamação/genética , Inflamação/patologia , Interleucina-8/metabolismo , Masculino , Camundongos Knockout , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Células THP-1 , Regulação para Cima/genética
12.
ASAIO J ; 67(1): 96-103, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-32404613

RESUMO

Ex vivo lung perfusion (EVLP) is increasingly used to treat and assess lungs before transplant. Minimizing ventilator induced lung injury (VILI) during EVLP is an important clinical need, and negative pressure ventilation (NPV) may reduce VILI compared with conventional positive pressure ventilation (PPV). However, it is not clear if NPV is intrinsically lung protective or if differences in respiratory pressure-flow waveforms are responsible for reduced VILI during NPV. In this study, we quantified lung injury using novel pressure-flow waveforms during normothermic EVLP. Rat lungs were ventilated-perfused ex vivo for 2 hours using tidal volume, positive end-expiratory pressure (PEEP), and respiratory rate matched PPV or NPV protocols. Airway pressures and flow rates were measured in real time and lungs were assessed for changes in compliance, pulmonary vascular resistance, oxygenation, edema, and cytokine secretion. Negative pressure ventilation lungs demonstrated reduced proinflammatory cytokine secretion, reduced weight gain, and reduced pulmonary vascular resistance (p < 0.05). Compliance was higher in NPV lungs (p < 0.05), and there was no difference in oxygenation between the two groups. Respiratory pressure-flow waveforms during NPV and PPV were significantly different (p < 0.05), especially during the inspiratory phase, where the NPV group exhibited rapid time-dependent changes in pressure and airflow whereas the PPV group exhibited slower changes in airflow/pressures. Lungs ventilated with PPV also had a greater transpulmonary pressure (p < 0.05). Greater improvement in lung function during NPV EVLP may be caused by favorable airflow patterns and/or pressure dynamics, which may better mimic human respiratory patterns.


Assuntos
Transplante de Pulmão , Perfusão/métodos , Transplantes , Animais , Circulação Extracorpórea/métodos , Pulmão/fisiopatologia , Transplante de Pulmão/métodos , Respiração com Pressão Positiva , Ratos , Ratos Sprague-Dawley , Respiradores de Pressão Negativa
13.
Adv Biosyst ; 4(6): e2000049, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32419350

RESUMO

Myeloid derived suppressor cells (MDSCs) have gained significant attention for their immunosuppressive role in cancer and their ability to contribute to tumor progression and metastasis. Understanding the role of MDSCs in driving cancer cell migration, a process fundamental to metastasis, is essential to fully comprehend and target MDSC-tumor cell interactions. This study employs microfabricated platforms, which simulate the structural cues present in the tumor microenvironment (TME) to elucidate the effects of MDSCs on the migratory phenotype of cancer cells at the single cell level. The results indicate that the presence of MDSCs enhances the motility of cancer-epithelial cells when directional cues (either topographical or spatial) are present. This behavior appears to be independent of cell-cell contact and driven by soluble byproducts from heterotypic interactions between MDSCs and cancer cells. Moreover, MDSC cell-motility is also impacted by the presence of cancer cells and the cancer cell secretome in the presence of directional cues. Epithelial dedifferentiation is the likely mechanism for changes in cancer cell motility in response to MDSCs. These results highlight the biochemical and biostructural conditions under which MDSCs can support cancer cell migration, and could therefore provide new avenues of research and therapy aimed at stemming cancer progression.


Assuntos
Comunicação Celular , Movimento Celular , Células Supressoras Mieloides/metabolismo , Neoplasias/metabolismo , Microambiente Tumoral , Animais , Linhagem Celular Tumoral , Feminino , Camundongos , Células Supressoras Mieloides/patologia , Metástase Neoplásica , Neoplasias/patologia
14.
Clin Biomech (Bristol, Avon) ; 66: 11-19, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-29395489

RESUMO

BACKGROUND: The Eustachian tube is a collapsible upper respiratory airway that is periodically opened to maintain a healthy middle ear. Young children, <10 years old, exhibit reduced Eustachian tube opening efficiency and are at risk for developing middle ear infections. Although these infections increase mucosal adhesion, it is not known how adhesion forces alters the biomechanics of Eustachian tube opening in young children. This study uses computational techniques to investigate how increased mucosal adhesion alters Eustachian tube function in young children. METHODS: Multi-scale finite element models were used to simulate the muscle-assisted opening of the Eustachian tube in healthy adults and young children. Airflow during opening was quantified as a function of adhesion strength, muscle forces and tissue mechanics. FINDINGS: Although Eustachian tube function was sensitive to increased mucosal adhesion in both adults and children, young children developed Eustachian tube dysfunction at significantly lower values of mucosal adhesion. Specifically, the critical adhesion value was 2 orders of magnitude lower in young children as compared to healthy adults. Although increased adhesion did not alter the sensitivity of Eustachian tube function to tensor and levator veli palatini muscles forces, increased adhesion in young children did reduced the sensitivity of Eustachian tube function to changes in cartilage and mucosal tissue stiffness. INTERPRETATIONS: These results indicate that increased mucosal adhesion can significantly alter the biomechanical mechanisms of Eustachian tube function in young children and that clinical assessment of adhesion levels may be important in therapy selection.


Assuntos
Tuba Auditiva/fisiopatologia , Aderências Teciduais/fisiopatologia , Adulto , Idoso , Fenômenos Biomecânicos , Cartilagem/fisiopatologia , Criança , Pré-Escolar , Feminino , Análise de Elementos Finitos , Humanos , Hidrodinâmica , Imageamento Tridimensional , Masculino , Pessoa de Meia-Idade , Mucosa/fisiopatologia , Músculo Esquelético , Músculos/fisiopatologia , Otite Média/fisiopatologia , Adulto Jovem
15.
Neoplasia ; 21(1): 132-145, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30550871

RESUMO

The organization of the extracellular matrix has a profound impact on cancer development and progression. The matrix becomes aligned throughout tumor progression, providing "highways" for tumor cell invasion. Aligned matrix is associated with breast density and is a negative prognostic factor in several cancers; however, the underlying mechanisms regulating this reorganization remain poorly understood. Deletion of the tumor suppressor Pten in the stroma was previously shown to promote extracellular matrix expansion and tumor progression. However, it was unknown if PTEN also regulated matrix organization. To address this question, a murine model with fibroblast-specific Pten deletion was used to examine how PTEN regulates matrix remodeling. Using second harmonic generation microscopy, Pten deletion was found to promote collagen alignment parallel to the mammary duct in the normal gland and further remodeling perpendicular to the tumor edge in tumor-bearing mice. Increased alignment was observed with Pten deletion in vitro using fibroblast-derived matrices. PTEN loss was associated with fibroblast activation and increased cellular contractility, as determined by traction force microscopy. Inhibition of contractility abrogated the increased matrix alignment observed with PTEN loss. Murine mammary adenocarcinoma cells cultured on aligned matrices derived from Pten-/- fibroblasts migrated faster than on matrices from wild-type fibroblasts. Combined, these data demonstrate that PTEN loss in fibroblasts promotes extracellular matrix deposition and alignment independently from cancer cell presence, and this reorganization regulates cancer cell behavior. Importantly, stromal PTEN negatively correlated with collagen alignment and high mammographic density in human breast tissue, suggesting parallel function for PTEN in patients.


Assuntos
Matriz Extracelular/metabolismo , Glândulas Mamárias Animais/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Células Estromais/metabolismo , Animais , Densidade da Mama , Linhagem Celular Tumoral , Movimento Celular , Colágeno/metabolismo , Feminino , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Humanos , Glândulas Mamárias Animais/patologia , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , Camundongos , Camundongos Transgênicos , PTEN Fosfo-Hidrolase/genética
16.
Oncotarget ; 9(27): 19209-19222, 2018 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-29721195

RESUMO

Epithelial cancer cells can undergo an epithelial-mesenchymal transition (EMT), a complex genetic program that enables cells to break free from the primary tumor, breach the basement membrane, invade through the stroma and metastasize to distant organs. Myoferlin (MYOF), a protein involved in plasma membrane function and repair, is overexpressed in several invasive cancer cell lines. Depletion of myoferlin in the human breast cancer cell line MDA-MB-231 (MDA-231MYOFKD) reduced migration and invasion and caused the cells to revert to an epithelial phenotype. To test if this mesenchymal-epithelial transition was durable, MDA-231MYOFKD cells were treated with TGF-ß1, a potent stimulus of EMT. After 48 hr with TGF-ß1, MDA-231MYOFKD cells underwent an EMT. TGF-ß1 treatment also decreased directional cell motility toward more random migration, similar to the highly invasive control cells. To probe the potential mechanism of MYOF function, we examined TGF-ß1 receptor signaling. MDA-MB-231 growth and survival has been previously shown to be regulated by autocrine TGF-ß1. We hypothesized that MYOF depletion may result in the dysregulation of TGF-ß1 signaling, thwarting EMT. To investigate this hypothesis, we examined production of endogenous TGF-ß1 and observed a decrease in TGF-ß1 protein secretion and mRNA transcription. To determine if TGF-ß1 was required to maintain the mesenchymal phenotype, TGF-ß receptor signaling was inhibited with a small molecule inhibitor, resulting in decreased expression of several mesenchymal markers. These results identify a novel pathway in the regulation of autocrine TGF-ß signaling and a mechanism by which MYOF regulates cellular phenotype and invasive capacity of human breast cancer cells.

17.
Trends Biotechnol ; 36(5): 549-561, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29559164

RESUMO

Recent cancer research has more strongly emphasized the biophysical aspects of tumor development, progression, and microenvironment. In addition to genetic modifications and mutations in cancer cells, it is now well accepted that the physical properties of cancer cells such as stiffness, electrical impedance, and refractive index vary with tumor progression and can identify a malignant phenotype. Moreover, cancer heterogeneity renders population-based characterization techniques inadequate, as individual cellular features are lost in the average. Hence, platforms for fast and accurate characterization of biophysical properties of cancer cells at the single-cell level are required. Here, we highlight some of the recent advances in the field of cancer biophysics and the development of lab-on-a-chip platforms for single-cell biophysical analyses of cancer cells.


Assuntos
Fenômenos Biofísicos , Dispositivos Lab-On-A-Chip , Neoplasias/patologia , Neoplasias/fisiopatologia , Análise de Célula Única/métodos , Humanos , Análise de Célula Única/instrumentação
18.
Ann Biomed Eng ; 46(1): 197-207, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28939933

RESUMO

The leading cause of neonatal mortality, pre-term birth, is often caused by pre-mature ripening/opening of the uterine cervix. Although cervical fibroblasts play an important role in modulating the cervix's extracellular matrix (ECM) and mechanical properties, it is not known how hormones, i.e., progesterone, and pro-inflammatory insults alter fibroblast mechanics, fibroblast-ECM interactions and the resulting changes in tissue mechanics. Here we investigate how progesterone and a pro-inflammatory cytokine, IL-1ß, alter the biomechanical properties of human cervical fibroblasts and the fibroblast-ECM interactions that govern tissue-scale mechanics. Primary human fibroblasts were isolated from non-pregnant cervix and treated with estrogen/progesterone, IL-1ß or both. The resulting changes in ECM gene expression, matrix remodeling, traction force generation, cell-ECM adhesion and tissue contractility were monitored. Results indicate that IL-1ß induces a significant reduction in traction force and ECM adhesion independent of pre-treatment with progesterone. These cell level effects altered tissue-scale mechanics where IL-1ß inhibited the contraction of a collagen gel over 6 days. Interestingly, progesterone treatment alone did not modulate traction forces or gel contraction but did result in a dramatic increase in cell-ECM adhesion. Therefore, the protective effect of progesterone may be due to altered adhesion dynamics as opposed to altered ECM remodeling.


Assuntos
Colo do Útero/citologia , Fibroblastos/efeitos dos fármacos , Interleucina-1beta/farmacologia , Progesterona/farmacologia , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Colágeno/metabolismo , Estradiol/farmacologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/fisiologia , Feminino , Fibroblastos/fisiologia , Humanos , Metaloproteinases da Matriz/metabolismo
19.
Vet Radiol Ultrasound ; 58(5): 542-551, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28718208

RESUMO

Stenotic nares, edematous intranasal turbinates, mucosal swelling, and an elongated, thickened soft palate are common sources of airflow resistance for dogs with brachycephalic airway syndrome. Surgery has focused on enlarging the nasal apertures and reducing tissue of the soft palate. However, objective measures of surgical efficacy are lacking. Twenty-one English bulldogs without previous surgery were recruited for this prospective, pilot study. Computed tomography was performed using conscious sedation and without endotracheal intubation using a 128 multidetector computed tomography scanner. Raw multidetector computed tomography data were rendered to create a three-dimensional surface mesh model by automatic segmentation of the air-filled nasal passage from the nares to the caudal soft palate. Three-dimensional surface models were used to construct computational fluid dynamics models of nasal airflow resistance from the nares to the caudal aspect of the soft palate. The computational fluid dynamics models were used to simulate airflow in each dog and airway resistance varied widely with a median 36.46 (Pa/mm)/(l/s) and an interquartile range of 19.84 to 90.74 (Pa/mm)/(/s). In 19/21 dogs, the rostral third of the nasal passage exhibited a larger airflow resistance than the caudal and middle regions of the nasal passage. In addition, computational fluid dynamics data indicated that overall measures of airflow resistance may significantly underestimate the maximum local resistance. We conclude that computational fluid dynamics models derived from nasal multidetector computed tomography can quantify airway resistance in brachycephalic dogs. This methodology represents a novel approach to noninvasively quantify airflow resistance and may have utility for objectively studying effects of surgical interventions in canine brachycephalic airway syndrome.


Assuntos
Resistência das Vias Respiratórias , Cães/anormalidades , Hidrodinâmica , Cavidade Nasal/diagnóstico por imagem , Tomografia Computadorizada por Raios X/veterinária , Animais , Biologia Computacional , Feminino , Masculino , Cavidade Nasal/anormalidades , Cavidade Nasal/patologia , Projetos Piloto , Estudos Prospectivos
20.
Neoplasia ; 19(6): 496-508, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28501760

RESUMO

The extracellular matrix (ECM) is critical for mammary ductal development and differentiation, but how mammary fibroblasts regulate ECM remodeling remains to be elucidated. Herein, we used a mouse genetic model to activate platelet derived growth factor receptor-alpha (PDGFRα) specifically in the stroma. Hyperactivation of PDGFRα in the mammary stroma severely hindered pubertal mammary ductal morphogenesis, but did not interrupt the lobuloalveolar differentiation program. Increased stromal PDGFRα signaling induced mammary fat pad fibrosis with a corresponding increase in interstitial hyaluronic acid (HA) and collagen deposition. Mammary fibroblasts with PDGFRα hyperactivation also decreased hydraulic permeability of a collagen substrate in an in vitro microfluidic device assay, which was mitigated by inhibition of either PDGFRα or HA. Fibrosis seen in this model significantly increased the overall stiffness of the mammary gland as measured by atomic force microscopy. Further, mammary tumor cells injected orthotopically in the fat pads of mice with stromal activation of PDGFRα grew larger tumors compared to controls. Taken together, our data establish that aberrant stromal PDGFRα signaling disrupts ECM homeostasis during mammary gland development, resulting in increased mammary stiffness and increased potential for tumor growth.


Assuntos
Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Humanas/crescimento & desenvolvimento , Neoplasias Mamárias Animais/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Animais , Diferenciação Celular/genética , Matriz Extracelular/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Ácido Hialurônico/administração & dosagem , Glândulas Mamárias Animais/patologia , Glândulas Mamárias Humanas/patologia , Neoplasias Mamárias Animais/patologia , Camundongos , Morfogênese/genética , Transdução de Sinais , Células Estromais/patologia
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