UDP-6-glucose dehydrogenase in hormonally responsive breast cancers.

Survival for metastatic breast cancer is low and thus, continued efforts to treat and prevent metastatic progression are critical. Estrogen is shown to promote aggressive phenotypes in multiple cancer models irrespective of estrogen receptor (ER) status. Similarly, UDP-Glucose 6-dehydrogenase (UGDH) a ubiquitously expressed enzyme involved in extracellular matrix precursors, as well as hormone processing increases migratory and invasive properties in cancer models. While the role of UGDH in cellular migration is defined, how it intersects with and impacts hormone signaling pathways associated with tumor progression in metastatic breast cancer has not been explored. Here we demonstrate that UGDH knockdown blunts estrogen-induced tumorigenic phenotypes (migration and colony formation) in ER+ and ER- breast cancer in vitro. Knockdown of UGDH also inhibits extravasation of ER- breast cancer ex vivo, primary tumor growth and animal survival in vivo in both ER+ and ER- breast cancer. We also use single cell RNA-sequencing to demonstrate that our findings translate to a human breast cancer clinical specimen. Our findings support the role of estrogen and UGDH in breast cancer progression provide a foundation for future studies to evaluate the role of UGDH in therapeutic resistance to improve outcomes and survival for breast cancer patients.

Gene Therapy and Spinal Fusion: Systematic Review and Meta-analysis of the Available Data.

OBJECTIVE: To analyze the extant literature describing the application of gene therapy to spinal fusion. METHODS: A systematic review of the English-language literature was performed. The search query was designed to include all published studies examining gene therapy approaches to promote spinal fusion. Approaches were classified as ex vivo (delivery of genetically modified cells) or in vivo (delivery of growth factors via vectors). The primary endpoint was fusion rate. Random effects meta-analyses were performed to calculate the overall odds ratio (OR) of fusion using a gene therapy approach and overall fusion rate. Sub-group analyses of fusion rate were also performed for each gene therapy approach. RESULTS: Of 1179 results, 35 articles met criteria for inclusion (all preclinical), of which 26 utilized ex vivo approaches and 9 utilized in vivo approaches. Twenty-seven articles (431 animals) were included in the meta-analysis. Gene therapy use was associated with significantly higher fusion rates (OR 77; 95% CI: [31, 192]; p<0.001); ex vivo strategies had a greater effect (OR 136) relative to in vivo strategies (OR 18) (p=0.017). The overall fusion rate using a gene therapy approach was 80% (95% CI: [62%, 93%]; p<0.001); overall fusion rates were significantly higher in subjects treated with ex vivo compared to in vivo strategies (90% vs 42%; p=0.011). For both ex vivo and in vivo approaches, the effect of gene therapy on fusion was independent of animal model. CONCLUSIONS: Gene therapy may augment spinal fusion; however, future investigation in clinical populations is necessary.

Active immunotherapy for C5a-mediated inflammation using adjuvant-free self-assembled peptide nanofibers.

The terminal protein in the complement cascade C5a is a potent inflammatory molecule and chemoattractant that is involved in the pathology of multiple inflammatory diseases including sepsis and arthritis, making it a promising protein to target with immunotherapies. Active immunotherapies, in which patients are immunized against problematic self-molecules and generate therapeutic antibodies as a result, have received increasing interest as an alternative to traditional monoclonal antibody treatments. In previous work, we have designed supramolecular self-assembling peptide nanofibers as active immunotherapies with defined combinations of B- and T-cell epitopes. Herein, the self-assembling peptide Q11 platform was employed to generate a C5a-targeting active immunotherapy. Two of three predicted B-cell epitope peptides from C5a were found to be immunogenic when displayed within Q11 nanofibers, and the nanofibers were capable of reducing C5a serum concentrations following immunization. Contrastingly, C5a's precursor protein C5 maintained its original concentration, promising to minimize side effects heretofore associated with C5-targeted therapies. Immunization protected mice against an LPS-challenge model of sepsis, and it reduced clinical severity in a model of collagen-antibody induced arthritis. Together, this work indicates the potential for targeting terminal complement proteins with active immunotherapies by leveraging the immunogenicity of self-assembled peptide nanomaterials. STATEMENT OF SIGNIFICANCE: Chronic inflammatory diseases such as rheumatoid arthritis, psoriasis, and inflammatory bowel disease are currently treated primarily with monoclonal antibodies against key inflammatory mediators. While helpful for many patients, they have high non-response rates, are costly, and commonly fail as anti-drug antibodies are raised by the patient. The approach we describe here explores a fundamentally different treatment paradigm: raising therapeutic antibody responses with an active immunotherapy. We employ innovative supramolecular peptide nanomaterials to elicit neutralizing antibody responses against complement component C5a and demonstrate therapeutic efficacy in preclinical mouse models of sepsis and rheumatoid arthritis. The strategy reported may represent a potential alternative to monoclonal antibody therapies.

Differential roles of normal and lung cancer-associated fibroblasts in microvascular network formation.

Perfusable microvascular networks offer promising three-dimensional in vitro models to study normal and compromised vascular tissues as well as phenomena such as cancer cell metastasis. Engineering of these microvascular networks generally involves the use of endothelial cells stabilized by fibroblasts to generate robust and stable vasculature. However, fibroblasts are highly heterogenous and may contribute variably to the microvascular structure. Here, we study the effect of normal and cancer-associated lung fibroblasts on the formation and function of perfusable microvascular networks. We examine the influence of cancer-associated fibroblasts on microvascular networks when cultured in direct (juxtacrine) and indirect (paracrine) contacts with endothelial cells, discovering a generative inhibition of microvasculature in juxtacrine co-cultures and a functional inhibition in paracrine co-cultures. Furthermore, we probed the secreted factors differential between cancer-associated fibroblasts and normal human lung fibroblasts, identifying several cytokines putatively influencing the resulting microvasculature morphology and functionality. These findings suggest the potential contribution of cancer-associated fibroblasts in aberrant microvasculature associated with tumors and the plausible application of such in vitro platforms in identifying new therapeutic targets and/or agents that can prevent formation of aberrant vascular structures.

Grand Challenges at the Interface of Engineering and Medicine.

Over the past two decades Biomedical Engineering has emerged as a major discipline that bridges societal needs of human health care with the development of novel technologies. Every medical institution is now equipped at varying degrees of sophistication with the ability to monitor human health in both non-invasive and invasive modes. The multiple scales at which human physiology can be interrogated provide a profound perspective on health and disease. We are at the nexus of creating "avatars" (herein defined as an extension of "digital twins") of human patho/physiology to serve as paradigms for interrogation and potential intervention. Motivated by the emergence of these new capabilities, the IEEE Engineering in Medicine and Biology Society, the Departments of Biomedical Engineering at Johns Hopkins University and Bioengineering at University of California at San Diego sponsored an interdisciplinary workshop to define the grand challenges that face biomedical engineering and the mechanisms to address these challenges. The Workshop identified five grand challenges with cross-cutting themes and provided a roadmap for new technologies, identified new training needs, and defined the types of interdisciplinary teams needed for addressing these challenges. The themes presented in this paper include: 1) accumedicine through creation of avatars of cells, tissues, organs and whole human; 2) development of smart and responsive devices for human function augmentation; 3) exocortical technologies to understand brain function and treat neuropathologies; 4) the development of approaches to harness the human immune system for health and wellness; and 5) new strategies to engineer genomes and cells.

Multi-Functional Small Molecule Alleviates Fracture Pain and Promotes Bone Healing.

Bone injuries such as fractures are one major cause of morbidities worldwide. A considerable number of fractures suffer from delayed healing, and the unresolved acute pain may transition to chronic and maladaptive pain. Current management of pain involves treatment with NSAIDs and opioids with substantial adverse effects. Herein, we tested the hypothesis that the purine molecule, adenosine, can simultaneously alleviate pain and promote healing in a mouse model of tibial fracture by targeting distinctive adenosine receptor subtypes in different cell populations. To achieve this, a biomaterial-assisted delivery of adenosine is utilized to localize and prolong its therapeutic effect at the injury site. The results demonstrate that local delivery of adenosine inhibited the nociceptive activity of peripheral neurons through activation of adenosine A1 receptor (ADORA1) and mitigated pain as demonstrated by weight bearing and open field movement tests. Concurrently, local delivery of adenosine at the fracture site promoted osteogenic differentiation of mesenchymal stromal cells through adenosine A2B receptor (ADORA2B) resulting in improved bone healing as shown by histological analyses and microCT imaging. This study demonstrates the dual role of adenosine and its material-assisted local delivery as a feasible therapeutic approach to treat bone trauma and associated pain.

Radiation-induced bone loss in mice is ameliorated by inhibition of HIF-2α in skeletal progenitor cells.

Radiotherapy remains a common treatment modality for cancer despite skeletal complications. However, there are currently no effective treatments for radiation-induced bone loss, and the consequences of radiotherapy on skeletal progenitor cell (SPC) survival and function remain unclear. After radiation, leptin receptor-expressing cells, which include a population of SPCs, become localized to hypoxic regions of the bone and stabilize the transcription factor hypoxia-inducible factor-2α (HIF-2α), thus suggesting a role for HIF-2α in the skeletal response to radiation. Here, we conditionally knocked out HIF-2α in leptin receptor-expressing cells and their descendants in mice. Radiation therapy in littermate control mice reduced bone mass; however, HIF-2α conditional knockout mice maintained bone mass comparable to nonirradiated control animals. HIF-2α negatively regulated the number of SPCs, bone formation, and bone mineralization. To test whether blocking HIF-2α pharmacologically could reduce bone loss during radiation, we administered a selective HIF-2α inhibitor called PT2399 (a structural analog of which was recently FDA-approved) to wild-type mice before radiation exposure. Pharmacological inhibition of HIF-2α was sufficient to prevent radiation-induced bone loss in a single-limb irradiation mouse model. Given that ~90% of patients who receive a HIF-2α inhibitor develop anemia because of off-target effects, we developed a bone-targeting nanocarrier formulation to deliver the HIF-2α inhibitor to mouse bone, to increase on-target efficacy and reduce off-target toxicities. Nanocarrier-loaded PT2399 prevented radiation-induced bone loss in mice while reducing drug accumulation in the kidney. Targeted inhibition of HIF-2α may represent a therapeutic approach for protecting bone during radiation therapy.

Spinal cord repair is modulated by the neurogenic factor Hb-egf under direction of a regeneration-associated enhancer.

Unlike adult mammals, zebrafish regenerate spinal cord tissue and recover locomotor ability after a paralyzing injury. Here, we find that ependymal cells in zebrafish spinal cords produce the neurogenic factor Hb-egfa upon transection injury. Animals with hb-egfa mutations display defective swim capacity, axon crossing, and tissue bridging after spinal cord transection, associated with disrupted indicators of neuron production. Local recombinant human HB-EGF delivery alters ependymal cell cycling and tissue bridging, enhancing functional regeneration. Epigenetic profiling reveals a tissue regeneration enhancer element (TREE) linked to hb-egfa that directs gene expression in spinal cord injuries. Systemically delivered recombinant AAVs containing this zebrafish TREE target gene expression to crush injuries of neonatal, but not adult, murine spinal cords. Moreover, enhancer-based HB-EGF delivery by AAV administration improves axon densities after crush injury in neonatal cords. Our results identify Hb-egf as a neurogenic factor necessary for innate spinal cord regeneration and suggest strategies to improve spinal cord repair in mammals.

Extracellular adenosine signaling in bone health and disease.

Purinergic signaling is a key molecular pathway in the maintenance of bone health and regeneration. P1 receptor signaling, which is activated by extracellular adenosine, has emerged as a key metabolic pathway that regulates bone tissue formation, function, and homeostasis. Extracellular adenosine is mainly produced by ectonucleotidases, and alterations in the function of these enzymes or compromised adenosine generation can result in bone disorders, such as osteoporosis and impaired fracture healing. This mini review discusses the key role played by adenosine in bone health and how its alterations contribute to bone diseases, as well as potential therapeutic applications of exogenous adenosine to combat bone diseases like osteoporosis and injury.

Self-assembled innervated vasculature-on-a-chip to study nociception.

Nociceptor sensory neurons play a key role in eliciting pain. An active crosstalk between nociceptor neurons and the vascular system at the molecular and cellular level is required to sense and respond to noxious stimuli. Besides nociception, interaction between nociceptor neurons and vasculature also contributes to neurogenesis and angiogenesis.In vitromodels of innervated vasculature can greatly help delineate these roles while facilitating disease modeling and drug screening. Herein, we report the development of a microfluidic-assisted tissue model of nociception in the presence of microvasculature. The self-assembled innervated microvasculature was engineered using endothelial cells and primary dorsal root ganglion (DRG) neurons. The sensory neurons and the endothelial cells displayed distinct morphologies in presence of each other. The neurons exhibited an elevated response to capsaicin in the presence of vasculature. Concomitantly, increased transient receptor potential cation channel subfamily V member 1 (TRPV1) receptor expression was observed in the DRG neurons in presence of vascularization. Finally, we demonstrated the applicability of this platform for modeling nociception associated with tissue acidosis. While not demonstrated here, this platform could also serve as a tool to study pain resulting from vascular disorders while also paving the way towards the development of innervated microphysiological models.

Protective effects of omega-3 fatty acids in a blood-brain barrier-on-chip model and on postoperative delirium-like behaviour in mice.

BACKGROUND: Peripheral surgical trauma can trigger neuroinflammation and ensuing neurological complications, such as delirium. The mechanisms whereby surgery contributes to postoperative neuroinflammation remain unclear and without effective therapies. Here, we developed a microfluidic-assisted blood-brain barrier (BBB) device and tested the effects of omega-3 fatty acids on neuroimmune interactions after orthopaedic surgery. METHODS: A microfluidic-assisted BBB device was established using primary human cells. Tight junction proteins, vascular cell adhesion molecule 1 (VCAM-1), BBB permeability, and astrocytic networks were assessed after stimulation with interleukin (IL)-1ß and in the presence or absence of a clinically available omega-3 fatty acid emulsion (Omegaven®; Fresenius Kabi, Bad Homburg, Germany). Mice were treated 1 h before orthopaedic surgery with 10 µl g-1 body weight of omega-3 fatty acid emulsion i.v. or equal volumes of saline. Changes in pericytes, perivascular macrophages, BBB opening, microglial activation, and inattention were evaluated. RESULTS: Omega-3 fatty acids protected barrier permeability, endothelial tight junctions, and VCAM-1 after exposure to IL-1ß in the BBB model. In vivo studies confirmed that omega-3 fatty acid treatment inhibited surgery-induced BBB impairment, microglial activation, and delirium-like behaviour. We identified a novel role for pericyte loss and perivascular macrophage activation in mice after surgery, which were rescued by prophylaxis with i.v. omega-3 fatty acids. CONCLUSIONS: We present a new approach to study neuroimmune interactions relevant to perioperative recovery using a microphysiological BBB platform. Changes in barrier function, including dysregulation of pericytes and perivascular macrophages, provide new targets to reduce postoperative delirium.

Mindin (SPON2) Is Essential for Cutaneous Fibrogenesis in a Mouse Model of Systemic Sclerosis.

Systemic sclerosis is a fibrotic disease that initiates in the skin and progresses to internal organs, leading to a poor prognosis. Unraveling the etiology of a chronic, multifactorial disease such as systemic sclerosis has been aided by various animal models that recapitulate certain aspects of the human pathology. We found that the transcription factor SNAI1 is overexpressed in the epidermis of patients with systemic sclerosis, and a transgenic mouse recapitulating this expression pattern is sufficient to induce many clinical features of the human disease. Using this mouse model as a discovery platform, we have uncovered a critical role for the matricellular protein Mindin (SPON2) in fibrogenesis. Mindin is produced by SNAI1 transgenic skin keratinocytes and aids fibrogenesis by inducing early inflammatory cytokine production and collagen secretion in resident dermal fibroblasts. Given the dispensability of Mindin in normal tissue physiology, targeting this protein holds promise as an effective therapy for fibrosis.

Deciphering the Mechanics of Cancer Spheroid Growth in 3D Environments through Microfluidics Driven Mechanical Actuation.

Uncontrolled growth of tumor cells is a key contributor to cancer-associated mortalities. Tumor growth is a biomechanical process whereby the cancer cells displace the surrounding matrix that provides mechanical resistance to the growing cells. The process of tumor growth and remodeling is regulated by material properties of both the cancer cells and their surrounding matrix, yet the mechanical interdependency between the two entities is not well understood. Herein, this work develops a microfluidic platform that precisely positions tumor spheroids within a hydrogel and mechanically probes the growing spheroids and surrounding matrix simultaneously. By using hydrostatic pressure to deform the spheroid-laden hydrogel along with confocal imaging and finite element (FE) analysis, this work deduces the material properties of the spheroid and the matrix in situ. For spheroids embedded within soft hydrogels, decreases in the Young's modulus of the matrix are detected at discrete locations accompanied by localized tumor growth. Contrastingly, spheroids within stiff hydrogels do not significantly decrease the Young's modulus of the surrounding matrix, despite exhibiting growth. Spheroids in stiff matrices leverage their high bulk modulus to grow and display a uniform volumetric expansion. Collectively, a quantitative platform is established and new insights into tumor growth within a stiff 3D environment are provided.

Characterization of bending balloon actuators.

The emerging field of soft robotics often relies on soft actuators powered by pressurized fluids to obtain a variety of movements. Strategic incorporation of soft actuators can greatly increase the degree of freedom of soft robots thereby bestowing them with a range of movements. Balloon actuators are extensively used to achieve various motions such as bending, twisting, and expanding. A detailed understanding of how material properties and architectural designs of balloon actuators influence their motions will greatly enable the application of these soft actuators. In this study, we developed a framework involving experimental and theoretical analyses, including computational analysis, delineating material and geometrical parameters of balloon actuators to their bending motions. Furthermore, we provide a simple analytical model to predict and control the degree of bending of these actuators. The described analytical tool could be used to predict the actuating function of balloon actuators and thereby help generate optimal actuators for functions which require control over the extent and direction of actuation.

Microgel-Assisted Delivery of Adenosine to Accelerate Fracture Healing.

Extracellular adenosine plays a key role in promoting bone tissue formation. Local delivery of adenosine could be an effective therapeutic strategy to harness the beneficial effect of extracellular adenosine on bone tissue formation following injury. Herein, we describe the development of an injectable in situ curing scaffold containing microgel-based adenosine delivery units. The two-component scaffold includes adenosine-loaded microgels and functionalized hyaluronic acid (HA) molecules. The microgels were generated upon copolymerization of 3-acrylamidophenylboronic acid (3-APBA)- and 2-aminoethylmethacrylamide (2-AEMA)-conjugated HA (HA-AEMA) in an emulsion suspension. The PBA functional groups were used to load the adenosine molecules. Mixing of the microgels with the HA polymers containing clickable groups, dibenzocyclooctyne (DBCO) and azide (HA-DBCO and HA-Azide), resulted in a 3D scaffold embedded with adenosine delivery units. Application of the in situ curing scaffolds containing adenosine-loaded microgels following tibial fracture injury showed improved bone tissue healing in a mouse model as demonstrated by the reduced callus size, higher bone volume, and increased tissue mineral density compared to those treated with the scaffold without adenosine. Overall, our results suggest that local delivery of adenosine could potentially be an effective strategy to promote bone tissue repair.

Loss of ATRX promotes aggressive features of osteosarcoma with increased NF-κB signaling and integrin binding.

Osteosarcoma (OS) is a lethal disease with few known targeted therapies. Here, we show that decreased ATRX expression is associated with more aggressive tumor cell phenotypes, including increased growth, migration, invasion, and metastasis. These phenotypic changes correspond with activation of NF-κB signaling, extracellular matrix remodeling, increased integrin αvß3 expression, and ETS family transcription factor binding. Here, we characterize these changes in vitro, in vivo, and in a data set of human OS patients. This increased aggression substantially sensitizes ATRX-deficient OS cells to integrin signaling inhibition. Thus, ATRX plays an important tumor-suppression role in OS, and loss of function of this gene may underlie new therapeutic vulnerabilities. The relationship between ATRX expression and integrin binding, NF-κB activation, and ETS family transcription factor binding has not been described in previous studies and may impact the pathophysiology of other diseases with ATRX loss, including other cancers and the ATR-X α thalassemia intellectual disability syndrome.

pH-Sensitive nanocarrier assisted delivery of adenosine to treat osteoporotic bone loss.

Bone tissue undergoes continuous remodeling via osteoclast-mediated bone resorption and osteoblast-mediated bone formation. An imbalance in this process with enhanced osteoclastic activity can lead to excessive bone resorption, resulting in bone thinning. Once activated, osteoclasts bind to the bone surface and acidify the local niche. This acidic environment could serve as a potential trigger for the delivery of therapeutic agents into the osteoporotic bone tissue. To this end, we developed a pH-responsive nanocarrier-based drug delivery system that binds to the bone tissue and delivers an osteoanabolic molecule, adenosine. Adenosine is incorporated into a hyaluronic acid (HA)-based nanocarrier through a pH-sensitive ketal group. The HA-nanocarrier is further functionalized with alendronate moieties to improve binding to the bone tissues. Systemic administration of the nanocarrier containing adenosine attenuated bone loss in ovariectomized mice and showed comparable bone qualities to that of healthy mice. Delivery of osteoanabolic small molecules that can contribute to bone formation and inhibit excessive osteoclast activity by leveraging the tissue-specific milieu could serve as viable therapeutics for osteoporosis.

An In Vitro Microfluidic Alveolus Model to Study Lung Biomechanics.

The gas exchange units of the lung, the alveoli, are mechanically active and undergo cyclic deformation during breathing. The epithelial cells that line the alveoli contribute to lung function by reducing surface tension via surfactant secretion, which is highly influenced by the breathing-associated mechanical cues. These spatially heterogeneous mechanical cues have been linked to several physiological and pathophysiological states. Here, we describe the development of a microfluidically assisted lung cell culture model that incorporates heterogeneous cyclic stretching to mimic alveolar respiratory motions. Employing this device, we have examined the effects of respiratory biomechanics (associated with breathing-like movements) and strain heterogeneity on alveolar epithelial cell functions. Furthermore, we have assessed the potential application of this platform to model altered matrix compliance associated with lung pathogenesis and ventilator-induced lung injury. Lung microphysiological platforms incorporating human cells and dynamic biomechanics could serve as an important tool to delineate the role of alveolar micromechanics in physiological and pathological outcomes in the lung.

Molecularly Tailored Interface for Long-Term Xenogeneic Cell Transplantation.

Encapsulation of therapeutic cells in a semipermeable device can mitigate the need for systemic immune suppression following cell transplantation by providing local immunoprotection while being permeable to nutrients, oxygen, and different cell-secreted biomolecules. However, fibrotic tissue deposition around the device has been shown to compromise the long-term function of the transplanted cells. Herein, a macroencapsulation device design that improves long-term survival and function of the transplanted cells is reported. The device is comprised of a semipermeable chitosan pouch with a tunable reservoir and molecularly engineered interface. The chitosan pouch interface decorated with 1,12-dodecanedioic acid (DDA), limits the cell adhesion and vigorous foreign body response while maintaining the barrier properties amenable to cell encapsulation. The device provides long-term protection to the encapsulated human primary hepatocytes in the subcutaneous space of immunocompetent mice. The device supports the encapsulated cells for up to 6 months as evident from cell viability and presence of human specific albumin in circulation. Solutions that integrate biomaterials and interfacial engineering such as the one described here may advance development of easy-to manufacture and retrievable devices for the transplantation of therapeutic cells in the absence of immunosuppression.

Self-Healing of Hyaluronic Acid to Improve In Vivo Retention and Function.

Convergent advances in the field of soft matter, macromolecular chemistry, and engineering have led to the development of biomaterials that possess autonomous, adaptive, and self-healing characteristics similar to living systems. These rationally designed biomaterials can surpass the capabilities of their parent material. Herein, the modification of hyaluronic acid (HA) to exhibit self-healing properties is described, and its physical and biological function both in vitro and in vivo is studied. The in vitro findings showed that self-healing HA designed to undergo self-repair improves lubrication, enhances free radical scavenging, and attenuates enzymatic degradation compared to unmodified HA. Longitudinal imaging following intraarticular injection of self-healing HA shows improved in vivo retention despite its low molecular weight. Concomitant with these functions, intraarticular injection of self-healing HA mitigates anterior cruciate ligament injury-mediated cartilage degeneration in rodents. This proof-of-concept study shows how incorporation of functional properties such as self-healing can be used to surpass the existing capabilities of biolubricants.