Magnetic Resonance Elastography and Computational Modeling Identify Heterogeneous Lung Biomechanical Properties during Cystic Fibrosis.

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.

A Micro-scale Humanized Ventilator-on-a-Chip to Examine the Injurious Effects of Mechanical Ventilation.

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.

Macrophage-Targeted Lipid Nanoparticle Delivery of microRNA-146a to Mitigate Hemorrhagic Shock-Induced Acute Respiratory Distress Syndrome.

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.

Macrophage-targeted lipid nanoparticle delivery of microRNA-146a to mitigate hemorrhagic shock-induced acute respiratory distress syndrome.

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.

Alveolar macrophages drive lung fibroblast function in cocultures of IPF and normal patient samples.

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.

Pulmonary drug delivery for acute respiratory distress syndrome.

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.

Biomechanical Force and Cellular Stiffness in Lung Fibrosis.

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.

mTORC1 is a mechanosensor that regulates surfactant function and lung compliance during ventilator-induced lung injury.

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.

Pten regulates collagen fibrillogenesis by fibroblasts through SPARC.

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.

Nanoparticle delivery of microRNA-146a regulates mechanotransduction in lung macrophages and mitigates injury during mechanical ventilation.

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.

A Novel Negative Pressure-Flow Waveform to Ventilate Lungs for Normothermic Ex Vivo Lung Perfusion.

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.

Reciprocal Signaling between Myeloid Derived Suppressor and Tumor Cells Enhances Cellular Motility and is Mediated by Structural Cues in the Microenvironment.

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.

Multi-scale modeling of an upper respiratory airway: Effect of mucosal adhesion on Eustachian tube function in young children.

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.

Stromal PTEN Regulates Extracellular Matrix Organization in the Mammary Gland.

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.

Myoferlin regulates epithelial cancer cell plasticity and migration through autocrine TGF-ß1 signaling.

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.

Lab-on-a-Chip Platforms for Biophysical Studies of Cancer with Single-Cell Resolution.

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.

Cellular Mechanics of Primary Human Cervical Fibroblasts: Influence of Progesterone and a Pro-inflammatory Cytokine.

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.

Quantification of nasal airflow resistance in English bulldogs using computed tomography and computational fluid dynamics.

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.

Stromal PDGFR-α Activation Enhances Matrix Stiffness, Impedes Mammary Ductal Development, and Accelerates Tumor Growth.

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.

Panel 2: Anatomy (Eustachian Tube, Middle Ear, and Mastoid-Anatomy, Physiology, Pathophysiology, and Pathogenesis).

Objective In this report, we review the recent literature (ie, past 4 years) to identify advances in our understanding of the middle ear-mastoid-eustachian tube system. We use this review to determine whether the short-term goals elaborated in the last report were achieved, and we propose updated goals to guide future otitis media research. Data Sources PubMed, Web of Science, Medline. Review Methods The panel topic was subdivided, and each contributor performed a literature search within the given time frame. The keywords searched included middle ear, eustachian tube, and mastoid for their intersection with anatomy, physiology, pathophysiology, and pathology. Preliminary reports from each panel member were consolidated and discussed when the panel met on June 11, 2015. At that meeting, the progress was evaluated and new short-term goals proposed. Conclusions Progress was made on 13 of the 20 short-term goals proposed in 2011. Significant advances were made in the characterization of middle ear gas exchange pathways, modeling eustachian tube function, and preliminary testing of treatments for eustachian tube dysfunction. Implications for Practice In the future, imaging technologies should be developed to noninvasively assess middle ear/eustachian tube structure and physiology with respect to their role in otitis media pathogenesis. The new data derived from these structure/function experiments should be integrated into computational models that can then be used to develop specific hypotheses concerning otitis media pathogenesis and persistence. Finally, rigorous studies on medical or surgical treatments for eustachian tube dysfunction should be undertaken.