Elisabeth Jiang

Control of lung vascular permeability and endotoxin-induced pulmonary oedema by changes in extracellular matrix mechanics

Increased vascular permeability contributes to many diseases, including acute respiratory distress syndrome, cancer and inflammation. Most past work on vascular barrier function has focused on soluble regulators, such as tumour-necrosis factor-α. Here we show that lung vascular permeability is controlled mechanically by changes in extracellular matrix structure. Our studies reveal that pulmonary vascular leakage can be increased by altering extracellular matrix compliance in vitro and by manipulating lysyl oxidase-mediated collagen crosslinking in vivo. Either decreasing or increasing extracellular matrix stiffness relative to normal levels disrupts junctional integrity and increases vascular leakage. Importantly, endotoxin-induced increases of vascular permeability are accompanied by concomitant increases in extracellular matrix rigidity and lysyl oxidase activity, which can be prevented by inhibiting lysyl oxidase activity. The identification of lysyl oxidase and the extracellular matrix as critical regulators of lung vascular leakage might lead to the development of new therapeutic approaches for the treatment of pulmonary oedema and other diseases caused by abnormal vascular permeability.

Inhibition of mammary tumor growth using lysyl oxidase-targeting nanoparticles to modify extracellular matrix.

A cancer nanotherapeutic has been developed that targets the extracellular matrix (ECM)-modifying enzyme lysyl oxidase (LOX) and alters the ECM structure. Poly(d,l-lactide-co-glycolide) nanoparticles (∼220 nm) coated with a LOX inhibitory antibody bind to ECM and suppress mammary cancer cell growth and invasion in vitro as well as tumor expansion in vivo, with greater efficiency than soluble anti-LOX antibody. This nanomaterials approach opens a new path for treating cancer with higher efficacy and decreased side effects.

Role of Collagen Matrix in Tumor Angiogenesis and Glioblastoma Multiforme Progression

Glioblastoma is a highly vascularized brain tumor, and antiangiogenic therapy improves its progression-free survival. However, current antiangiogenic therapy induces serious adverse effects including neuronal cytotoxicity and tumor invasiveness and resistance to therapy. Although it has been suggested that the physical microenvironment has a key role in tumor angiogenesis and progression, the mechanism by which physical properties of extracellular matrix control tumor angiogenesis and glioblastoma progression is not completely understood. Herein we show that physical compaction (the process in which cells gather and pack together and cause associated changes in cell shape and size) of human glioblastoma cell lines U87MG, U251, and LN229 induces expression of collagen types IV and VI and the collagen crosslinking enzyme lysyl oxidase and up-regulates in vitro expression of the angiogenic factor vascular endothelial growth factor. The lysyl oxidase inhibitor β-aminopropionitrile disrupts collagen structure in the tumor and inhibits tumor angiogenesis and glioblastoma multiforme growth in a mouse orthotopic brain tumor model. Similarly, d-penicillamine, which inhibits lysyl oxidase enzymatic activity by depleting intracerebral copper, also exhibits antiangiogenic effects on brain tumor growth in mice. These findings suggest that tumor microenvironment controlled by collagen structure is important in tumor angiogenesis and brain tumor progression.

Platelet rich plasma extract promotes angiogenesis through the angiopoietin1-Tie2 pathway

Development and regeneration of tissues and organs require precise coordination among endothelial, epithelial and mesenchymal morphogenesis. Angiogenesis plays key roles in normal development, wound healing, recovery from ischemic disease, and organ regeneration. It has been recognized that the combination of various angiogenic factors in an appropriate physiological ratio is critical for long-term functional blood vessel formation. Here we show that mouse soluble platelet-rich-plasma (PRP) extract, which includes abundant angiopoetin-1 (Ang1) and other angiogenic factors, stimulates endothelial cell growth, migration and differentiation in cultured human dermal microvascular endothelial cells in vitro and neonatal mouse retinal angiogenesis in vivo. Mouse platelet rich fibrin (PRF) matrix, the three-dimensional fibrin matrix that releases angiogenic factors with similar concentrations and proportions to the PRP extract, also recapitulates robust angiogenesis inside the matrix when implanted subcutaneously on the living mouse. Inhibition of Ang1-Tie2 signaling suppresses PRP extract-induced angiogenesis in vitro and angiogenic ability of the PRF matrix in vivo. Since human PRP extract and PRF matrix can be prepared from autologous peripheral blood, our findings may lead to the development of novel therapeutic interventions for various angiogenesis-related diseases as well as to the improvement of strategies for tissue engineering and organ regeneration.

Extracellular Matrix Structure and Tissue Stiffness Control Postnatal Lung Development through the Lipoprotein Receptor–Related Protein 5/Tie2 Signaling System

Physical properties of the tissues and remodeling of extracellular matrix (ECM) play an important role in organ development. Recently, we have reported that low-density lipoprotein receptor-related protein (LRP) 5/Tie2 signaling controls postnatal lung development by modulating angiogenesis. Here we show that tissue stiffness modulated by the ECM cross-linking enzyme, lysyl oxidase (LOX), regulates postnatal lung development through LRP5-Tie2 signaling. The expression of LRP5 and Tie2 is up-regulated twofold in lung microvascular endothelial cells when cultured on stiff matrix compared to those cultured on soft matrix in vitro. LOX inhibitor, β-aminopropionitrile, disrupts lung ECM (collagen I, III, and VI, and elastin) structures, softens neonatal mouse lung tissue by 20%, and down-regulates the expression of LRP5 and Tie2 by 20 and 60%, respectively, which leads to the inhibition of postnatal lung development (30% increase in mean linear intercept, 1.5-fold increase in air space area). Importantly, hyperoxia treatment (Postnatal Days 1-10) disrupts ECM structure and stiffens mouse lung tissue by up-regulating LOX activity, thereby increasing LRP5 and Tie2 expression and deregulating alveolar morphogenesis in neonatal mice, which is attenuated by inhibiting LOX activity. These findings suggest that appropriate physical properties of lung tissue are necessary for physiological postnatal lung development, and deregulation of this mechanism contributes to postnatal lung developmental disorders, such as bronchopulmonary dysplasia.

LRP5 Regulates Development of Lung Microvessels and Alveoli through the Angiopoietin-Tie2 Pathway

Angiogenesis is crucial for lung development. Although there has been considerable exploration, the mechanism by which lung vascular and alveolar formation is controlled is still not completely understood. Here we show that low-density lipoprotein receptor-related protein 5 (LRP5), a component of the Wnt ligand-receptor complex, regulates angiogenesis and alveolar formation in the lung by modulating expression of the angiopoietin (Ang) receptor, Tie2, in vascular endothelial cells (ECs). Vascular development in whole mouse lungs and in cultured ECs is controlled by LRP5 signaling, which is, in turn, governed by a balance between the activities of the antagonistic Tie2 ligands, Ang1 and Ang2. Under physiological conditions when Ang1 is dominant, LRP5 knockdown decreases Tie2 expression and thereby, inhibits vascular and alveolar development in the lung. Conversely, when Ang2 dominates under hyperoxia treatment in neonatal mice, high LRP5 and Tie2 expression suppress angiogenesis and lung development. These findings suggest that the LRP5-Tie2-Ang signaling axis plays a central role in control of both angiogenesis and alveolarization during postnatal lung development, and that deregulation of this signaling mechanism might lead to developmental abnormalities of the lung, such as are observed in bronchopulmonary dysplasia (BPD).

Twist1 Controls Lung Vascular Permeability and Endotoxin-Induced Pulmonary Edema by Altering Tie2 Expression

Tight regulation of vascular permeability is necessary for normal development and deregulated vascular barrier function contributes to the pathogenesis of various diseases, including acute respiratory distress syndrome, cancer and inflammation. The angiopoietin (Ang)-Tie2 pathway is known to control vascular permeability. However, the mechanism by which the expression of Tie2 is regulated to control vascular permeability has not been fully elucidated. Here we show that transcription factor Twist1 modulates pulmonary vascular leakage by altering the expression of Tie2 in a context-dependent way. Twist1 knockdown in cultured human lung microvascular endothelial cells decreases Tie2 expression and phosphorylation and increases RhoA activity, which disrupts cell-cell junctional integrity and increases vascular permeability in vitro. In physiological conditions, where Ang1 is dominant, pulmonary vascular permeability is elevated in the Tie2-specific Twist1 knockout mice. However, depletion of Twist1 and resultant suppression of Tie2 expression prevent increase in vascular permeability in an endotoxin-induced lung injury model, where the balance of Angs shifts toward Ang2. These results suggest that Twist1-Tie2-Angs signaling is important for controlling vascular permeability and modulation of this mechanism may lead to the development of new therapeutic approaches for pulmonary edema and other diseases caused by abnormal vascular permeability.

Paxillin controls endothelial cell migration and tumor angiogenesis by altering neuropilin 2 expression.

ABSTRACT Although a number of growth factors and receptors are known to control tumor angiogenesis, relatively little is known about the mechanism by which these factors influence the directional endothelial cell migration required for cancer microvessel formation. Recently, it has been shown that the focal adhesion protein paxillin is required for directional migration of fibroblasts in vitro. Here, we show that paxillin knockdown enhances endothelial cell migration in vitro and stimulates angiogenesis during normal development and in response to tumor angiogenic factors in vivo. Paxillin produces these effects by decreasing expression of neuropilin 2 (NRP2). Moreover, soluble factors secreted by tumors that stimulate vascular ingrowth, including vascular endothelial growth factor (VEGF), also decrease endothelial cell expression of paxillin and NRP2, and overexpression of NRP2 reverses these effects. These results suggest that the VEGF–paxillin–NRP2 pathway could represent a new therapeutic target for cancer and other angiogenesis-related diseases.

Platelet-rich plasma extract prevents pulmonary edema through angiopoietin-Tie2 signaling.

Increased vascular permeability contributes to life-threatening pathological conditions, such as acute respiratory distress syndrome. Current treatments for sepsis-induced pulmonary edema rely on low-tidal volume mechanical ventilation, fluid management, and pharmacological use of a single angiogenic or chemical factor with antipermeability activity. However, it is becoming clear that a combination of multiple angiogenic/chemical factors rather than a single factor is required for maintaining stable and functional blood vessels. We have demonstrated that mouse platelet-rich plasma (PRP) extract contains abundant angiopoietin (Ang) 1 and multiple other factors (e.g., platelet-derived growth factor), which potentially stabilize vascular integrity. Here, we show that PRP extract increases tyrosine phosphorylation levels of Tunica internal endothelial cell kinase (Tie2) and attenuates disruption of cell-cell junctional integrity induced by inflammatory cytokine in cultured human microvascular endothelial cells. Systemic injection of PRP extract also increases Tie2 phosphorylation in mouse lung and prevents endotoxin-induced pulmonary edema and the consequent decreases in lung compliance and exercise intolerance resulting from endotoxin challenge. Soluble Tie2 receptor, which inhibits Ang-Tie2 signaling, suppresses the ability of PRP extract to inhibit pulmonary edema in mouse lung. These results suggest that PRP extract prevents endotoxin-induced pulmonary edema mainly through Ang-Tie2 signaling, and PRP extract could be a potential therapeutic strategy for sepsis-induced pulmonary edema and various lung diseases caused by abnormal vascular permeability.

Modeling Hematopoiesis and Responses to Radiation Countermeasures in a Bone Marrow-on-a-Chip.

Studies on hematopoiesis currently rely on animal models because in vitro culture methods do not accurately recapitulate complex bone marrow physiology. We recently described a bone marrow-on-a-chip microfluidic device that enables the culture of living hematopoietic bone marrow and mimics radiation toxicity in vitro. In the present study, we used this microdevice to demonstrate continuous blood cell production in vitro and model bone marrow responses to potential radiation countermeasure drugs. The device maintained mouse hematopoietic stem and progenitor cells in normal proportions for at least 2 weeks in culture. Increases in the number of leukocytes and red blood cells into the microfluidic circulation also could be detected over time, and addition of erythropoietin induced a significant increase in erythrocyte production. Exposure of the bone marrow chip to gamma radiation resulted in reduction of leukocyte production, and treatment of the chips with two potential therapeutics, granulocyte-colony stimulating factor or bactericidal/permeability-increasing protein (BPI), induced significant increases in the number of hematopoietic stem cells and myeloid cells in the fluidic outflow. In contrast, BPI was not found to have any effect when analyzed using static marrow cultures, even though it has been previously shown to accelerate recovery from radiation-induced toxicity in vivo. These findings demonstrate the potential value of the bone marrow-on-a-chip for modeling blood cell production, monitoring responses to hematopoiesis-modulating drugs, and testing radiation countermeasures in vitro.