RESUMO
All drugs recently developed in rodent models to treat inflammatory disease have failed in clinical trials. We therefore used our novel biomimetic microfluidic assay (bMFA) to determine whether the response of murine cells to inflammatory activation or anti-inflammatory treatment is predictive of the response in human cells. Under physiologically relevant flow conditions, permeability and transendothelial electrical resistance (TEER) of human or mouse lung microvascular endothelial cells (HLMVEC or MLMVEC), and neutrophil-endothelial cell interaction was measured. The differential impact of a protein kinase C-delta TAT peptide inhibitor (PKCδ-i) was also quantified. Permeability of HLMVEC and MLMVEC was similar under control conditions but tumor necrosis factor α (TNF-α) and PKCδ-i had a significantly higher impact on permeability of HLMVEC. TEER across HLMVEC was significantly higher than MLMVEC, but PKCδ-i returned TEER to background levels only in human cells. The kinetics of N-formylmethionyl-leucyl-phenylalanine (fMLP)-mediated neutrophil migration was significantly different between the two species and PKCδ-i was significantly more effective in attenuating human neutrophil migration. However, human and mouse neutrophil adhesion patterns to microvascular endothelium were not significantly different. Surprisingly, while intercellular adhesion molecule 1 (ICAM-1) was significantly upregulated on activated HLMVEC, it was not significantly upregulated on activated MLMVEC. Responses to activation and anti-inflammatory treatment in mice may not always be predictive of their response in humans.
Assuntos
Adesão Celular/fisiologia , Comunicação Celular/fisiologia , Endotélio Vascular/metabolismo , Neutrófilos/metabolismo , Animais , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/metabolismo , Humanos , Sepse/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
In the event of a radiologic catastrophe, endothelial cell and neutrophil dysfunction play important roles in tissue injury. Clinically available therapeutics for radiation-induced vascular injury are largely supportive. PKCδ was identified as a critical regulator of the inflammatory response, and its inhibition was shown to protect critical organs during sepsis. We used a novel biomimetic microfluidic assay (bMFA) to interrogate the role of PKCδ in radiation-induced neutrophil-endothelial cell interaction and endothelial cell function. HUVECs formed a complete lumen in bMFA and were treated with 0.5, 2, or 5 Gy ionizing radiation (IR). At 24 h post-IR, the cells were treated with a PKCδ inhibitor for an additional 24 h. Under physiologic shear flow, the role of PKCδ on endothelium function and neutrophil adherence/migration was determined. PKCδ inhibition dramatically attenuated IR-induced endothelium permeability increase and significantly decreased neutrophil migration across IR-treated endothelial cells. Moreover, neutrophil adhesion to irradiated endothelial cells was significantly decreased after PKCδ inhibition in a flow-dependent manner. PKCδ inhibition downregulated IR-induced P-selectin, intercellular adhesion molecule 1, and VCAM-1 but not E-selectin overexpression. PKCδ is an important regulator of neutrophil-endothelial cell interaction post-IR, and its inhibition can serve as a potential radiation medical countermeasure.-Soroush, F., Tang, Y., Zaidi, H. M., Sheffield, J. B., Kilpatrick, L. E., Kiani, M. F. PKCδ inhibition as a novel medical countermeasure for radiation-induced vascular damage.
RESUMO
BACKGROUND: Neuroinflammation often develops in sepsis leading to activation of cerebral endothelium, increased permeability of the blood-brain barrier (BBB), and neutrophil infiltration. We have identified protein kinase C-delta (PKCδ) as a critical regulator of the inflammatory response and demonstrated that pharmacologic inhibition of PKCδ by a peptide inhibitor (PKCδ-i) protected endothelial cells, decreased sepsis-mediated neutrophil influx into the lung, and prevented tissue damage. The objective of this study was to elucidate the regulation and relative contribution of PKCδ in the control of individual steps in neuroinflammation during sepsis. METHODS: The role of PKCδ in mediating human brain microvascular endothelial (HBMVEC) permeability, junctional protein expression, and leukocyte adhesion and migration was investigated in vitro using our novel BBB on-a-chip (B3C) microfluidic assay and in vivo in a rat model of sepsis induced by cecal ligation and puncture (CLP). HBMVEC were cultured under flow in the vascular channels of B3C. Confocal imaging and staining were used to confirm tight junction and lumen formation. Confluent HBMVEC were pretreated with TNF-α (10 U/ml) for 4 h in the absence or presence of PKCδ-i (5 µM) to quantify neutrophil adhesion and migration in the B3C. Permeability was measured using a 40-kDa fluorescent dextran in vitro and Evans blue dye in vivo. RESULTS: During sepsis, PKCδ is activated in the rat brain resulting in membrane translocation, a step that is attenuated by treatment with PKCδ-i. Similarly, TNF-α-mediated activation of PKCδ and its translocation in HBMVEC are attenuated by PKCδ-i in vitro. PKCδ inhibition significantly reduced TNF-α-mediated hyperpermeability and TEER decrease in vitro in activated HBMVEC and rat brain in vivo 24 h after CLP induced sepsis. TNF-α-treated HBMVEC showed interrupted tight junction expression, whereas continuous expression of tight junction protein was observed in non-treated or PKCδ-i-treated cells. PKCδ inhibition also reduced TNF-α-mediated neutrophil adhesion and migration across HBMVEC in B3C. Interestingly, while PKCδ inhibition decreased the number of adherent neutrophils to baseline (no-treatment group), it significantly reduced the number of migrated neutrophils below the baseline, suggesting a critical role of PKCδ in regulating neutrophil transmigration. CONCLUSIONS: The BBB on-a-chip (B3C) in vitro assay is suitable for the study of BBB function as well as screening of novel therapeutics in real-time. PKCδ activation is a key signaling event that alters the structural and functional integrity of BBB leading to vascular damage and inflammation-induced tissue damage. PKCδ-TAT peptide inhibitor has therapeutic potential for the prevention or reduction of cerebrovascular injury in sepsis-induced vascular damage.
Assuntos
Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/fisiopatologia , Proteína Quinase C-delta/metabolismo , Sepse/patologia , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Permeabilidade Capilar/efeitos dos fármacos , Permeabilidade Capilar/fisiologia , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Humanos , Masculino , Infiltração de Neutrófilos/efeitos dos fármacos , Infiltração de Neutrófilos/fisiologia , Peptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Proteína Quinase C-delta/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
Particle adhesion in vivo is highly dependent on the microvascular environment comprising of unique anatomical, geometrical, physiological fluid flow conditions and cell-particle and cell-cell interactions. Hence, proper design of vascular-targeted drug carriers that efficiently deliver therapeutics to the targeted cells or tissue at effective concentrations must account for these complex conditions observed in vivo. In this study, we build upon our previous results with the goal of characterizing the effects of bifurcations and their corresponding angle on adhesion of functionalized particles and neutrophils to activated endothelium. Our hypothesis is that adhesion is significantly affected by the type of biochemical interactions between particles and vessel wall as well as the presence of bifurcations and their corresponding angle. Here, we investigate adhesion of functionalized particles (2 µm and 7 µm microparticles) to protein coated channels as well as adhesion of human neutrophils to human endothelial cells under various physiological flow conditions in microfluidic bifurcating channels comprising of different contained angles (30°, 60°, 90°, or 120°). Our findings indicate that both functionalized particle and neutrophil adhesion propensity increase with a larger bifurcation angle. Moreover, the difference in the adhesion patterns of neutrophils and rigid, similar sized (7 µm) particles is more apparent in the junction regions with a larger contained angle. By selecting the right particle size range, enhanced targeted binding of vascular drug carriers can be achieved along with a higher efficacy at optimal drug dosage. Hence, vascular drug particle design needs to be tailored to account for higher binding propensity at larger bifurcation angles.
Assuntos
Vasos Sanguíneos/patologia , Adesão Celular , Microcirculação , Microvasos/fisiologia , Biotina/química , Portadores de Fármacos , Células Endoteliais/citologia , Humanos , Dispositivos Lab-On-A-Chip , Microesferas , Modelos Anatômicos , Neutrófilos/citologia , Tamanho da Partícula , Poliestirenos/química , Resistência ao Cisalhamento , Estresse MecânicoRESUMO
BACKGROUND: Neutrophil dysfunction plays an important role in inflammation-induced tissue injury. Previously, we identified protein kinase C-δ (PKCδ) as a critical controller of neutrophil activation and trafficking but how PKCδ is regulated in inflammation has not been delineated. PKCδ activity is regulated by tyrosine phosphorylation on multiple sites. Tyrosine155 is a key regulator of apoptosis and gene expression, but its role in proinflammatory signaling is not known. METHODS: In-vitro studies - superoxide anion (O2) and neutrophil extracellular traps (NETs) were measured in bone marrow neutrophils (BMN) isolated from wild type (WT) and PKCδY155F knock-in mice (PKCδ tyrosine 155 â phenylalanine). Our novel 3D biomimetic microfluidic assay (bMFA) was used to delineate PKCδ-mediated regulation of individual steps in neutrophil adhesion and migration using WT and PKCδY155F BMN and mouse lung microvascular endothelial cells (MLMVEC). In-vivo studies - WT and PKCδY155F knock-in mice underwent sham or cecal ligation and puncture surgery and the lungs harvested 24âh post-surgery. RESULTS: In vitro - PKCδY155F BMN had significantly reduced O2 and NETs release compared with WT. WT BMN, but not PKCδY155F BMN, demonstrated significant adhesion and migration across tumor necrosis factor-activated MLMVEC in bMFA. PKCδ inhibition significantly reduced WT BMN adhesion and migration under low shear and near bifurcations, but had no effect on PKCδY155F BMN. In vivo - mutation of PKCδ tyrosine 155 significantly decreased neutrophil migration into the lungs of septic mice. CONCLUSIONS: PKCδ tyrosine 155 is a key phosphorylation site controlling proinflammatory signaling and neutrophil-endothelial cell interactions. These studies provide mechanistic insights into PKCδ regulation during inflammation.
Assuntos
Células Endoteliais/citologia , Inflamação/metabolismo , Neutrófilos/citologia , Proteína Quinase C-delta/metabolismo , Animais , Apoptose , Células da Medula Óssea/citologia , Adesão Celular , Endotélio Vascular/metabolismo , Feminino , Fibronectinas/metabolismo , Técnicas de Introdução de Genes , Masculino , Camundongos , Camundongos Transgênicos , Microcirculação , Microfluídica , Ativação de Neutrófilo , Oxigênio/metabolismo , Permeabilidade , Peroxidase/metabolismo , Fenilalanina/química , Fosforilação , Proteína Quinase C-delta/genética , Sepse/fisiopatologia , Superóxidos/metabolismo , Tirosina/químicaRESUMO
It is crucial to dynamically analyze the movement of neutrophils (a type of white cell) during the process of inflammation. However, manually tracking and analyzing the cells is a time-consuming task due to the large volume of cells and similar appearance. To facilitate neutrophils analysis and address the issues mentioned above, we propose to leverage high-order temporal information as a cue to track neutrophils. A tensor-based approach is introduced to encode the high-order motion pattern and appearance variation for multi-frame multicell association. To evaluate the proposed method, we collected 354 sequences of cells from 200 frames of microscopic images. We conduct a systematic study on the collected data and show significant performance improvement over other solutions.
Assuntos
Neutrófilos/citologia , Algoritmos , Movimento CelularRESUMO
Chemotherapy has been widely used in breast cancer patients to reduce tumor size. However, most anticancer agents cannot differentiate between cancerous and normal cells, resulting in severe systemic toxicity. In addition, acquired drug resistance during the chemotherapy treatment further decreases treatment efficacy. With the proper treatment strategy, nanodrug carriers, such as liposomes/immunoliposomes, may be able to reduce undesired side effects of chemotherapy, to overcome the acquired multidrug resistance, and to further improve the treatment efficacy. In this study, a novel combinational targeted drug delivery system was developed by encapsulating antiangiogenesis drug bevacizumab into liposomes and encapsulating chemotherapy drug doxorubicin (DOX) into immunoliposomes where the human epidermal growth factor receptor 2 (HER2) antibody was used as a targeting ligand. This novel combinational system was tested in vitro using a HER2 positive and multidrug resistant breast cancer cell line (BT-474/MDR), and in vivo using a xenograft mouse tumor model. In vitro cell culture experiments show that immunoliposome delivery led to a high cell nucleus accumulation of DOX, whereas free DOX was observed mostly near the cell membrane and in cytoplasm due to the action of P-gp. Combining liposomal bevacizumab with immunoliposomal DOX achieved the best tumor growth inhibition and the lowest toxicity. Tumor size decreased steadily within a 60-day observation period indicating a potential synergistic effect between DOX and bevacizumab through the targeted delivery. Our findings clearly indicate that tumor growth was significantly delayed in the combinational liposomal drug delivery group. This novel combinational therapy has great potential for the treatment of patients with HER2/MDR double positive breast cancer.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Resistencia a Medicamentos Antineoplásicos , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Bevacizumab/farmacologia , Bevacizumab/uso terapêutico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Liberação Controlada de Fármacos , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Fluorescência , Humanos , Lipossomos , Camundongos Endogâmicos BALB C , Tamanho da Partícula , Distribuição Tecidual/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacosRESUMO
Real-time monitoring of tumor drug delivery in vivo is a daunting challenge due to the heterogeneity and complexity of the tumor microenvironment. In this study, we developed a biomimetic microfluidic tumor microenvironment (bMTM) comprising co-culture of tumor and endothelial cells in a 3D environment. The platform consists of a vascular compartment featuring a network of vessels cultured with endothelial cells forming a complete lumen under shear flow in communication with 3D solid tumors cultured in a tumor compartment. Endothelial cell permeability to both small dye molecules and large liposomal drug carriers were quantified using fluorescence microscopy. Endothelial cell intercellular junction formation was characterized by immunostaining. Endothelial cell permeability significantly increased in the presence of either tumor cell conditioned media (TCM) or tumor cells. The magnitude of this increase in permeability was significantly higher in the presence of metastatic breast tumor cells as compared to non-metastatic ones. Immunostaining revealed impaired endothelial cell-cell junctions in the presence of either metastatic TCM or metastatic tumor cells. Our findings indicate that the bMTM platform mimics the tumor microenvironment including the EPR effect. This platform has a significant potential in applications such as cell-cell/cell-drug carrier interaction studies and rapid screening of cancer drug therapeutics/carriers.
Assuntos
Biomimética , Sistemas de Liberação de Medicamentos , Microfluídica , Microambiente Tumoral , Biomimética/métodos , Comunicação Celular , Linhagem Celular Tumoral , Movimento Celular , Técnicas de Cocultura , Portadores de Fármacos , Células Endoteliais , Imunofluorescência , Humanos , Junções Intercelulares/metabolismo , Lipossomos , Microfluídica/métodos , Metástase Neoplásica , Neoplasias/metabolismo , Neoplasias/patologia , Permeabilidade , Microambiente Tumoral/efeitos dos fármacosRESUMO
Diabetic nephropathy (DN) is the leading cause of kidney disease; however, there are no early biomarkers and no cure. Thus, there is a large unmet need to predict which individuals will develop nephropathy and to understand the molecular mechanisms that govern this susceptibility. We compared the glomerular transcriptome from mice with distinct susceptibilities to DN at four weeks after induction of diabetes, but before histologic injury, and identified differential regulation of genes that modulate inflammation. From these genes, we identified endothelial cell specific molecule-1 (Esm-1), as a glomerular-enriched determinant of resistance to DN. Glomerular Esm-1 mRNA and protein were lower in DN-susceptible, DBA/2, compared to DN-resistant, C57BL/6, mice. We demonstrated higher Esm-1 secretion from primary glomerular cultures of diabetic mice, and high glucose was sufficient to increase Esm-1 mRNA and protein secretion in both strains of mice. However, induction was significantly attenuated in DN-susceptible mice. Urine Esm-1 was also significantly higher only in DN-resistant mice. Moreover, using intravital microscopy and a biomimetic microfluidic assay, we showed that Esm-1 inhibited rolling and transmigration in a dose-dependent manner. For the first time we have uncovered glomerular-derived Esm-1 as a potential non-invasive biomarker of DN. Esm-1 inversely correlates with disease susceptibility and inhibits leukocyte infiltration, a critical factor in protecting the kidney from DN.
Assuntos
Nefropatias Diabéticas/genética , Perfilação da Expressão Gênica , Predisposição Genética para Doença , Glomérulos Renais/metabolismo , Proteoglicanas/deficiência , Proteoglicanas/genética , Animais , Movimento Celular/efeitos dos fármacos , Nefropatias Diabéticas/metabolismo , Relação Dose-Resposta a Droga , Glucose/farmacologia , Humanos , Glomérulos Renais/efeitos dos fármacos , Leucócitos/citologia , Leucócitos/efeitos dos fármacos , Masculino , Camundongos , Proteínas de Neoplasias/farmacologia , Proteoglicanas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Especificidade da EspécieRESUMO
A key step in neutrophil-mediated tissue damage is the migration of activated neutrophils across the vascular endothelium. Previously, we identified protein kinase C δ as a critical regulator of neutrophil migration in sepsis but did not identify specific steps in migration. In this study, we used our novel biomimetic microfluidic assay to delineate systematically the mechanism by which protein kinase C δ regulates individual steps in human neutrophil-endothelial interaction during inflammation. The biomimetic microfluidic assay includes a network of vascular channels, produced from in vivo images connected to a tissue compartment through a porous barrier. HUVECs cultured in vascular channels formed a complete lumen under physiologic shear flow. HUVECs were pretreated with TNF-α ± a protein kinase C δ inhibitor, and the tissue compartment was filled with a chemoattractant (fMLP or IL-8). Under physiologic shear flow, the role of protein kinase C δ on spatial and temporal neutrophil adherence/migration was quantified. Protein kinase C δ inhibition significantly reduced neutrophil adhesion in response to fMLP and IL-8 only under low shear rate and near bifurcations. Protein kinase C δ inhibition also decreased adherence to nonactivated HUVECs in response to fMLP or IL-8. Protein kinase C δ inhibition reduced neutrophil migration into the tissue compartment in response to fMLP and to a lesser degree, to IL-8. Antibody-coated microparticles demonstrated that protein kinase C δ inhibition down-regulated E-selectin and ICAM-1 but not VCAM-1 expression. With the use of a physiologically relevant in vitro model system, we demonstrate that protein kinase C δ plays an important role in the regulation of neutrophil adherence/migration during inflammation and identifies key steps regulated by protein kinase C δ in neutrophil-endothelial interactions.
Assuntos
Endotélio Vascular/citologia , Neutrófilos/enzimologia , Proteína Quinase C-delta/fisiologia , Animais , Adesão Celular , Quimiotaxia de Leucócito/efeitos dos fármacos , Quimiotaxia de Leucócito/fisiologia , Modelos Animais de Doenças , Selectina E/biossíntese , Células Endoteliais da Veia Umbilical Humana , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Interleucina-8/farmacologia , Pulmão/imunologia , Pulmão/microbiologia , Masculino , Técnicas Analíticas Microfluídicas , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/citologia , Fragmentos de Peptídeos/farmacologia , Proteína Quinase C-delta/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/farmacologia , Reologia , Sepse/enzimologia , Sepse/imunologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
BACKGROUND: Patients with P-glycoprotein and HER2/neu (HER2) receptor-overexpressing breast cancer usually have poor clinical outcomes. However, there exist no commercially available breast cancer cell lines that are HER2/P-glycoprotein double-positive, which limits research in this field. MATERIALS AND METHODS: We report on the development and characterization of a drug-resistant sub-line from an HER2-positive breast cancer cell line by stable transfection of the ATP-binding cassette (ABC) subfamily B member 1 (ABCB1) gene which encodes P-glycoprotein. RESULTS: ABCB1 gene expression levels were higher after transfection, which led to a 40-fold increase in P-glycoprotein expression. Interestingly, the transfection of ABCB1 also led to a slight increase in HER2 gene and protein expression levels. The transfection of ABCB1 increased the P-glycoprotein expression levels significantly. CONCLUSION: The method used herein for developing this cell line is appropriate for fast, stable induction of P-glycoprotein-mediated drug resistance compared to traditional methods. The in vitro cytotoxicity test suggests this cell line has cross-resistance to a wide range of chemotherapeutic agents.