Pazopanib elicits remarkable response in metastatic porocarcinoma: a functional precision medicine approach.

Metastatic porocarcinomas (PCs) are vanishingly rare, highly aggressive skin adnexal tumors with mortality rates exceeding 70%. Their rarity has precluded the understanding of their disease pathogenesis, let alone the conduct of clinical trials to evaluate treatment strategies. There are no effective agents for unresectable PCs. Here, we successfully demonstrate how functional precision medicine was implemented in the clinic for a metastatic PC with no known systemic treatment options. Comprehensive genomic profiling of the tumor specimen did not yield any actionable genomic aberrations. However, ex vivo drug testing predicted pazopanib efficacy, and indeed, administration of pazopanib elicited remarkable clinicoradiological response. Pazopanib and its class of drugs should be evaluated for efficacy in other cases of PC, and the rationale for efficacy should be determined when PC tumor models become available. A functional precision medicine approach could be useful to derive effective treatment options for rare cancers.

Mapping Drug-Induced Neuropathy through In-Situ Motor Protein Tracking and Machine Learning.

Chemotherapy can induce toxicity in the central and peripheral nervous systems and result in chronic adverse reactions that impede continuous treatment and reduce patient quality of life. There is a current lack of research to predict, identify, and offset drug-induced neurotoxicity. Rapid and accurate assessment of potential neuropathy is crucial for cost-effective diagnosis and treatment. Here we report dynamic near-infrared upconversion imaging that allows intraneuronal transport to be traced in real time with millisecond resolution, but without photobleaching or blinking. Drug-induced neurotoxicity can be screened prior to phenotyping, on the basis of subtle abnormalities of kinetic characteristics in intraneuronal transport. Moreover, we demonstrate that combining the upconverting nanoplatform with machine learning offers a powerful tool for mapping chemotherapy-induced peripheral neuropathy and assessing drug-induced neurotoxicity.

Development of an Axon-Guiding Aligned Nanofiber-Integrated Compartmentalized Microfluidic Neuron Culture System.

Microfluidic-based neuron cell culture systems have recently gained a lot of attention due to their efficiency in supporting the spatial and temporal control of cellular microenvironments. However, the lack of axon guidance is the key limitation in current culture systems. To combat this, we have developed electrospun aligned nanofiber-integrated compartmentalized microfluidic neuron culture systems (NIMSs), where the nanofibers have enabled axonal guidance and stability. The resulting platform significantly improved axon alignment, length, and stability for both rat primary embryonic motor neurons (MNs) and dorsal root ganglia (DRG) neurons compared to the conventional glass-based microfluidic systems (GMSs). The results showed that axonal growth covered more than two times the area on the axonal chamber of NIMSs compared to the area covered for GMSs. Overall, this platform can be used as a valuable tool for fundamental neuroscience research, drug screening, and biomaterial testing.

Nanotunnels within Poly(3,4-ethylenedioxythiophene)-Carbon Nanotube Composite for Highly Sensitive Neural Interfacing.

Neural electrodes are developed for direct communication with neural tissues for theranostics. Although various strategies have been employed to improve performance, creating an intimate electrode-tissue interface with high electrical fidelity remains a great challenge. Here, we report the rational design of a tunnel-like electrode coating comprising poly(3,4-ethylenedioxythiophene) (PEDOT) and carbon nanotubes (CNTs) for highly sensitive neural recording. The coated electrode shows a 50-fold reduction in electrochemical impedance at the biologically relevant frequency of 1 kHz, compared to the bare gold electrode. The incorporation of CNT significantly reinforces the nanotunnel structure and improves coating adhesion by ∼1.5 fold. In vitro primary neuron culture confirms an intimate contact between neurons and the PEDOT-CNT nanotunnel. During acute in vivo nerve recording, the coated electrode enables the capture of high-fidelity neural signals with low susceptibility to electrical noise and reveals the potential for precisely decoding sensory information through mechanical and thermal stimulation. These findings indicate that the PEDOT-CNT nanotunnel composite serves as an active interfacing material for neural electrodes, contributing to neural prosthesis and brain-machine interface.

Visualization of Intra-neuronal Motor Protein Transport through Upconversion Microscopy.

Cargo transport along axons, a physiological process mediated by motor proteins, is essential for neuronal function and survival. A current limitation in the study of axonal transport is the lack of a robust imaging technique with a high spatiotemporal resolution to visualize and quantify the movement of motor proteins in real-time and in different depth planes. Herein, we present a dynamic imaging technique that fully exploits the characteristics of upconversion nanoparticles. This technique can be used as a microscopic probe for the quantitative in situ tracking of retrograde transport neurons with single-particle resolution in multilayered cultures. This study may provide a powerful tool to reveal dynamic neuronal activity and intra-axonal transport function as well as any associated neurodegenerative diseases resulting from mutation or impairment in the axonal transport machinery.

Electrospun nanofibers facilitate better alignment, differentiation, and long-term culture in an in vitro model of the neuromuscular junction (NMJ).

The neuromuscular junction (NMJ) is a specialized synapse between motor neurons and the muscle fibers they innervate. Due to the complexity of various signalling molecules and pathways, in vivo NMJs are difficult to study. Therefore, in vitro motor neuron-muscle co-culture plays a pivotal role in studying the mechanisms of NMJ formation associated with neurodegenerative diseases. There is a growing need to develop novel methodologies that can be used to develop long-term cultures of NMJs. To date, there have been few studies on NMJ development and long-term maintenance of the system, which is also the main challenge for the current in vitro models of NMJs. In this study, we demonstrate a long-term co-culture system of primary embryonic motor neurons from Sprague-Dawley rats and C2C12 cells on both random and aligned electrospun polylactic acid (PLA) nanofibrous scaffolds. This is the first study to explore the role of electrospun nanofibers in the long-term maintenance of NMJs. PLA nanofibrous scaffolds provide better contact guidance for C2C12 cells aligning along the fibers, thus guiding myotube formation. We can only maintain the co-culture system on a conventional glass substrate for 2 weeks, whilst 55% and 70% of the cells still survived on random and aligned PLA substrates after 7 weeks. Our nanofiber-based long-term co-culture system is used as an important tool for the fundamental research of NMJs.

Engineering PCL/lignin nanofibers as an antioxidant scaffold for the growth of neuron and Schwann cell.

Antioxidant is critical for the successful of nerve tissue regeneration, and biomaterials with antioxidant activity might be favorable for peripheral nerve repair. Lignin, a biopolymer from wood with excellent antioxidant properties, is still "unexplored" as biomaterials. To design an antioxidative bioscaffold for nerve regeneration, here we synthesized lignin-polycaprolactone (PCL) copolymers via solvent free ring-opening polymerization (ROP). Then such lignin-PCL copolymers were incorporated with PCL and engineered into nanofibrous scaffolds for supporting the growth of neuron and Schwann cell. Our results showed that the addition of lignin-PCL enhanced the mechanical properties of PCL nanofibers and endowed them with good antioxidant properties (up to 98.3 ±â€¯1.9% free radical inhibition within 4 h). Cell proliferation assay showed that PCL/lignin-PCL nanofibers increased cell viability compared to PCL fibers, especially after an oxidative challenge. Moreover, Schwann cells and dorsal root ganglion (DRG) neurons cultured on the nanofibers to assess their potential for nerve regeneration. These results suggested that nanofibers with lignin copolymers promoted cell proliferation of both BMSCs and Schwann cells, enhanced myelin basic protein expressions of Schwann cells and stimulated neurite outgrowth of DRG neurons. In all, these sustainable, intrinsically antioxidant nanofibers may be a potential candidate for nerve TE applications.

Supercritical carbon dioxide extracted extracellular matrix material from adipose tissue.

Adipose tissue is a rich source of extracellular matrix (ECM) material that can be isolated by delipidating and decellularizing the tissue. However, the current delipidation and decellularization methods either involve tedious and lengthy processes or require toxic chemicals, which may result in the elimination of vital proteins and growth factors found in the ECM. Hence, an alternative delipidation and decellularization method for adipose tissue was developed using supercritical carbon dioxide (SC-CO2) that eliminates the need of any harsh chemicals and also reduces the amount of processing time required. The resultant SC-CO2-treated ECM material showed an absence of nuclear content but the preservation of key proteins such as collagen Type I, collagen Type III, collagen Type IV, elastin, fibronectin and laminin. In addition, other biological factors such as glycosaminoglycans (GAGs) and growth factors such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) were also retained. Subsequently, the resulting SC-CO2-treated ECM material was used as a bioactive coating on tissue culture plastic (TCP). Four different cell types including adipose tissue-derived mesenchymal stem cells (ASCs), human umbilical vein endothelial cells (HUVECs), immortalized human keratinocyte (HaCaT) cells and human monocytic leukemia cells (THP-1) were used in this study to show that the SC-CO2-treated ECM coating can be potentially used for various biomedical applications. The SC-CO2-treated ECM material showed improved cell-material interactions for all cell types tested. In addition, in vitro scratch wound assay using HaCaT cells showed that the presence of SC-CO2-treated ECM material enhanced keratinocyte migration whilst the in vitro cellular studies using THP-1-derived macrophages showed that the SC-CO2-treated ECM material did not evoke pro-inflammatory responses from the THP-1-derived macrophages. Overall, this study shows the efficacy of SC-CO2 method for delipidation and decellularization of adipose tissue whilst retaining its ECM and its subsequent utilization as a bioactive surface coating material for soft tissue engineering, angiogenesis and wound healing applications.

Surface modification of PVDF using non-mammalian sources of collagen for enhancement of endothelial cell functionality.

Although polyvinylidene fluoride (PVDF) is non-toxic and stable in vivo, its hydrophobic surface has limited its bio-applications due to poor cell-material interaction and thrombus formation when used in blood contacting devices. In this study, surface modification of PVDF using naturally derived non-mammalian collagen was accomplished via direct surface-initiated atom transfer radical polymerisation (SI-ATRP) to enhance its cytocompatibility and hemocompatibility. Results showed that Type I collagen was successfully extracted from fish scales and bullfrog skin. The covalent immobilisation of fish scale-derived collagen (FSCOL) and bullfrog skin-derived collagen (BFCOL) onto the PVDF surface improves the attachment and proliferation of human umbilical vein endothelial cells (HUVECs). Furthermore, both FSCOL and BFCOL had comparable anti-thrombogenic profiles to that of commercially available bovine collagen (BVCOL). Also, cell surface expression of the leukocyte adhesion molecule was lower on HUVECs cultured on non-mammalian collagen surfaces than on BVCOL, which is an indication of lower pro-inflammatory response. Overall, results from this study demonstrated that non-mammalian sources of collagen could be used to confer bioactivity to PVDF, with comparable cell-material interactions and hemocompatibility to BVCOL. Additionally, higher expression levels of Type IV collagen in HUVECs cultured on FSCOL and BFCOL were observed as compared to BVCOL, which is an indication that the non-mammalian sources of collagen led to a better pro-angiogenic properties, thus making them suitable for blood contacting applications.

Early controlled release of peroxisome proliferator-activated receptor ß/δ agonist GW501516 improves diabetic wound healing through redox modulation of wound microenvironment.

Diabetic wounds are imbued with an early excessive and protracted reactive oxygen species production. Despite the studies supporting PPARß/δ as a valuable pharmacologic wound-healing target, the therapeutic potential of PPARß/δ agonist GW501516 (GW) as a wound healing drug was never investigated. Using topical application of polymer-encapsulated GW, we revealed that different drug release profiles can significantly influence the therapeutic efficacy of GW and consequently diabetic wound closure. We showed that double-layer encapsulated GW microparticles (PLLA:PLGA:GW) provided an earlier and sustained dose of GW to the wound and reduced the oxidative wound microenvironment to accelerate healing, in contrast to single-layered PLLA:GW microparticles. The underlying mechanism involved an early GW-mediated activation of PPARß/δ that stimulated GPx1 and catalase expression in fibroblasts. GPx1 and catalase scavenged excessive H2O2 accumulation in diabetic wound beds, prevented H2O2-induced ECM modification and facilitated keratinocyte migration. The microparticles with early and sustained rate of GW release had better therapeutic wound healing activity. The present study underscores the importance of drug release kinetics on the therapeutic efficacy of the drug and warrants investigations to better appreciate the full potential of controlled drug release.

From flab to fab: transforming surgical waste into an effective bioactive coating material.

Cellular events are regulated by the interaction between integrin receptors in the cell membrane and the extracellular matrix (ECM). Hence, ECM, as a material, can potentially play an instructive role in cell-material interactions. Currently, adipose tissue in the form of lipoaspirate is often discarded. Here, it is demonstrated how our chemical-free decellularization method could be used to obtain ECM from human lipoaspirate waste material. These investigations show that the main biological components are retained in the lipoaspirate-derived ECM (LpECM) material and that this LpECM material could subsequently be used as a coating material to confer bioactivity to an otherwise inert biodegradable material (i.e., polycaprolactone). Overall, lipoaspirate material, a complex blend of endogenous proteins, is effectively used a bioactive coating material. This work is an important stepping-stone towards the development of biohybrid scaffolds that contain cellular benefits without requiring the use of additional biologics based on commonly discarded lipoaspirate material.

Porous ovalbumin scaffolds with tunable properties: a resource-efficient biodegradable material for tissue engineering applications.

Natural materials are promising alternatives to synthetic materials used in tissue engineering applications as they have superior biocompatibility and promote better cell attachment and proliferation. Ovalbumin, a natural polymer found in avian egg white, is an example of a nature-derived material. Despite the availability and reported biocompatibility of ovalbumin, limited research has been carried out to investigate the efficacy of ovalbumin-based scaffolds for adipose tissue engineering applications. Hence, the current study was carried out to investigate the effect of different crosslinkers on ovalbumin scaffold properties as first step towards the development of ovalbumin-based scaffolds for adipose tissue engineering applications. In this study, highly porous three-dimensional scaffolds were fabricated by using three different crosslinkers: glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 1,4-butanediol diglycidyl ether. Results showed that the overall scaffold properties such as morphology, pore size and mechanical properties could be modulated based on the type and concentration of crosslinkers used during the fabrication process. Subsequently, the efficacy of the different scaffolds for supporting cell proliferation was investigated. In vitro degradation was also carried on for the best scaffold based on the mechanical and cellular results. Overall, this study is a demonstration of the viability of ovalbumin-based scaffolds as cell carriers for soft tissue engineering applications.

Angiopoietin-like 4 stimulates STAT3-mediated iNOS expression and enhances angiogenesis to accelerate wound healing in diabetic mice.

Impaired wound healing is a major source of morbidity in diabetic patients. Poor outcome has, in part, been related to increased inflammation, poor angiogenesis, and deficiencies in extracellular matrix components. Despite the enormous impact of these chronic wounds, effective therapies are lacking. Here, we showed that the topical application of recombinant matricellular protein angiopoietin-like 4 (ANGPTL4) accelerated wound reepithelialization in diabetic mice, in part, by improving angiogenesis. ANGPTL4 expression is markedly elevated upon normal wound injury. In contrast, ANGPTL4 expression remains low throughout the healing period in diabetic wounds. Exogenous ANGPTL4 modulated several regulatory networks involved in cell migration, angiogenesis, and inflammation, as evidenced by an altered gene expression signature. ANGPTL4 influenced the expression profile of endothelial-specific CD31 in diabetic wounds, returning its profile to that observed in wild-type wounds. We showed ANGPTL4-induced nitric oxide production through an integrin/JAK/STAT3-mediated upregulation of inducible nitric oxide synthase (iNOS) expression in wound epithelia, thus revealing a hitherto unknown mechanism by which ANGPTL4 regulated angiogenesis via keratinocyte-to-endothelial-cell communication. These data show that the replacement of ANGPTL4 may be an effective adjunctive or new therapeutic avenue for treating poor healing wounds. The present finding also confirms that therapeutic angiogenesis remains an attractive treatment modality for diabetic wound healing.

Bioactivated protein-based porous microcarriers for tissue engineering applications.

Microcarriers are commonly used in tissue engineering applications as they provide a large surface area for cell attachment. However, limited research has been done on ovalbumin (OVA), which is a relatively cheap protein found in avian egg white. Hence, in our current study OVA is fabricated into porous microcarriers and the effect of different OVA to alginate ratios on the properties of OVA microcarriers was investigated. Subsequently, in order to further improve cell-material interactions, the extracellular matrix (ECM) material isolated from the human lipoaspirate material was conjugated with the porous OVA microcarriers using carbodiimide chemistry. A waste-to-resource strategy was employed to obtain this ECM material from the human lipoaspirate material, which typically is discarded after surgery. This study illustrates the possibility of obtaining ECM material using a physical decellularization method as well as the novel application of ECM material as a coating to confer bioactivity to protein-based microcarriers such as OVA. The incorporation of lipoaspirate-derived ECM (LpECM) into the OVA microstructure has been shown to improve mechanical strength and promote cellular growth on the microcarriers. The resulting porous OVA-LpECM hybrid microcarriers with tunable mechanical properties are examples of bioactivated porous protein-based microcarriers that can be applied in the field of tissue engineering.

SMAD3 deficiency promotes inflammatory aortic aneurysms in angiotensin II-infused mice via activation of iNOS.

BACKGROUND: Ninety percent of the patients carrying distinct SMAD3 mutations develop aortic aneurysms and dissections, called aneurysms-osteoarthritis syndrome (AOS). However, the etiology and molecular events downstream of SMAD3 leading to the pathogenesis of aortic aneurysms in these patients still remain elusive. Therefore, we aimed to investigate the vascular phenotypes of SMAD3-knockout mice. METHODS AND RESULTS: We have shown that angiotensin II-induced vascular inflammation, but not hypertension, leads to aortic aneurysms and dissections, ultimately causing aortic rupture and death in mice. Lipopolysaccharide-triggered inflammation confirmed that enhanced aortic macrophage recruitment was essential for aneurysm formation in angiotensin II-infused SMAD3-knockout mice. In contrast, phenylephrine-triggered hypertension alone was insufficient to induce aortic aneurysms in mice. Using uniaxial tensile and contractility tests, we showed that SMAD3 deficiency resulted in defective aortic biomechanics and physiological functions, which caused weakening of the aortic wall and predisposed the mice to aortic aneurysms. Chromatin immunoprecipitation (ChIP) and re-ChIP assays revealed that the underlying mechanism involved aberrant upregulation of inducible nitric oxide synthase (iNOS)-derived nitric oxide production and activation of elastolytic matrix metalloproteinases 2 and 9. Administration of clodronate-liposomes and iNOS inhibitor completely abrogated these aortic conditions, thereby identifying iNOS-mediated nitric oxide secretion from macrophages as the downstream event of SMAD3 that drives this severe pathology. CONCLUSIONS: Macrophage depletion and iNOS antagonism represent 2 promising approaches for preventing aortic aneurysms related to SMAD3 mutations and merit further investigation as adjunctive strategies for the life-threatening manifestations of AOS.