Silk fibroin-based inks for in situ 3D printing using a double crosslinking process.

The shortage of tissues and organs for transplantation is an urgent clinical concern. In situ 3D printing is an advanced 3D printing technique aimed at printing the new tissue or organ directly in the patient. The ink for this process is central to the outcomes, and must meet specific requirements such as rapid gelation, shape integrity, stability over time, and adhesion to surrounding healthy tissues. Among natural materials, silk fibroin exhibits fascinating properties that have made it widely studied in tissue engineering and regenerative medicine. However, further improvements in silk fibroin inks are needed to match the requirements for in situ 3D printing. In the present study, silk fibroin-based inks were developed for in situ applications by exploiting covalent crosslinking process consisting of a pre-photo-crosslinking prior to printing and in situ enzymatic crosslinking. Two different silk fibroin molecular weights were characterized and the synergistic effect of the covalent bonds with shear forces enhanced the shift in silk secondary structure toward ß-sheets, thus, rapid stabilization. These hydrogels exhibited good mechanical properties, stability over time, and resistance to enzymatic degradation over 14 days, with no significant changes over time in their secondary structure and swelling behavior. Additionally, adhesion to tissues in vitro was demonstrated.

Silk fibroin, gelatin, and human placenta extracellular matrix-based composite hydrogels for 3D bioprinting and soft tissue engineering.

BACKGROUND: There is a great clinical need and it remains a challenge to develop artificial soft tissue constructs that can mimic the biomechanical properties and bioactivity of natural tissue. This is partly due to the lack of suitable biomaterials. Hydrogels made from human placenta offer high bioactivity and represent a potential solution to create animal-free 3D bioprinting systems that are both sustainable and acceptable, as placenta is widely considered medical waste. A combination with silk and gelatin polymers can bridge the biomechanical limitations of human placenta chorion extracellular matrix hydrogels (hpcECM) while maintaining their excellent bioactivity. METHOD: In this study, silk fibroin (SF) and tyramine-substituted gelatin (G-TA) were enzymatically crosslinked with human placental extracellular matrix (hpcECM) to produce silk-gelatin-ECM composite hydrogels (SGE) with tunable mechanical properties, preserved elasticity, and bioactive functions. The SGE composite hydrogels were characterized in terms of gelation kinetics, protein folding, and bioactivity. The cyto- and biocompatibility of the SGE composite was determined by in vitro cell culture and subcutaneous implantation in a rat model, respectively. The most cell-supportive SGE formulation was then used for 3-dimensional (3D) bioprinting that induced chemical crosslinking during extrusion. CONCLUSION: Addition of G-TA improved the mechanical properties of the SGE composite hydrogels and inhibited crystallization and subsequent stiffening of SF for up to one month. SGE hydrogels exhibit improved and tunable biomechanical properties and high bioactivity for encapsulated cells. In addition, its use as a bioink for 3D bioprinting with free reversible embedding of suspended hydrogels (FRESH) has been validated, opening the possibility to fabricate highly complex scaffolds for artificial soft tissue constructs with natural biomechanics in future.

Engineered porosity for tissue-integrating, bioresorbable lifetime-based phosphorescent oxygen sensors.

Versatile silk protein-based material formats were studied to demonstrate bioresorbable, implantable optical oxygen sensors that can integrate with the surrounding tissues. The ability to continuously monitor tissue oxygenation in vivo is desired for a range of medical applications. Silk was chosen as the matrix material due to its excellent biocompatibility, its unique chemistry that facilitates interactions with chromophores, and the potential to tune degradation time without altering chemical composition. A phosphorescent Pd (II) benzoporphyrin chromophore was incorporated to impart oxygen sensitivity. Organic solvent-based processing methods using 1,1,1,3,3,3-hexafluoro-2-propanol were used to fabricate: 1) silk-chromophore films with varied thickness and 2) silk-chromophore sponges with interconnected porosity. All compositions were biocompatible and exhibited photophysical properties with oxygen sensitivities (i.e., Stern-Volmer quenching rate constants of 2.7-3.2 × 104 M-1) useful for monitoring physiological tissue oxygen levels and for detecting deviations from normal behavior (e.g., hyperoxia). The potential to tune degradation time without significantly impacting photophysical properties was successfully demonstrated. Furthermore, the ability to consistently monitor tissue oxygenation in vivo was established via a multi-week rodent study. Histological assessments indicated successful tissue integration for the sponges, and this material format responded more quickly to various oxygen challenges than the film samples.

Defining the fine structure of promoter activity on a genome-wide scale with CISSECTOR.

Classic promoter mutagenesis strategies can be used to study how proximal promoter regions regulate the expression of particular genes of interest. This is a laborious process, in which the smallest sub-region of the promoter still capable of recapitulating expression in an ectopic setting is first identified, followed by targeted mutation of putative transcription factor binding sites. Massively parallel reporter assays such as survey of regulatory elements (SuRE) provide an alternative way to study millions of promoter fragments in parallel. Here we show how a generalized linear model (GLM) can be used to transform genome-scale SuRE data into a high-resolution genomic track that quantifies the contribution of local sequence to promoter activity. This coefficient track helps identify regulatory elements and can be used to predict promoter activity of any sub-region in the genome. It thus allows in silico dissection of any promoter in the human genome to be performed. We developed a web application, available at cissector.nki.nl, that lets researchers easily perform this analysis as a starting point for their research into any promoter of interest.

3D Printability of Silk/Hydroxyapatite Composites for Microprosthetic Applications.

Micro-prosthetics requires the fabrication of mechanically robust and personalized components with sub-millimetric feature accuracy. Three-dimensional (3D) printing technologies have had a major impact on manufacturing such miniaturized devices for biomedical applications; however, biocompatibility requirements greatly constrain the choice of usable materials. Hydroxyapatite (HA) and its composites have been widely employed to fabricate bone-like structures, especially at the macroscale. In this work, we investigate the rheology, printability, and prosthetic mechanical properties of HA and HA-silk protein composites, focusing on the roles of composition and water content. We correlate key linear and nonlinear shear rheological parameters to geometric outcomes of printing and explain how silk compensates for the inherent brittleness of printed HA components. By increasing ink ductility, the inclusion of silk improves the quality of printed items through two mechanisms: (1) reducing underextrusion by lowering the required elastic modulus and, (2) reducing slumping by increasing the ink yield stress proportional to the modulus. We demonstrate that the elastic modulus and compressive strength of parts fabricated from silk-HA inks are higher than those for rheologically comparable pure-HA inks. We construct a printing map to guide the manufacturing of HA-based inks with excellent final properties, especially for use in biomedical applications for which sub-millimetric features are required.

Development of a novel bioengineered 3D brain-like tissue for studying primary blast-induced traumatic brain injury.

Primary blast injury is caused by the direct impact of an overpressurization wave on the body. Due to limitations of current models, we have developed a novel approach to study primary blast-induced traumatic brain injury. Specifically, we employ a bioengineered 3D brain-like human tissue culture system composed of collagen-infused silk protein donut-like hydrogels embedded with human IPSC-derived neurons, human astrocytes, and a human microglial cell line. We have utilized this system within an advanced blast simulator (ABS) to expose the 3D brain cultures to a blast wave that can be precisely controlled. These 3D cultures are enclosed in a 3D-printed surrogate skull-like material containing media which are then placed in a holder apparatus inside the ABS. This allows for exposure to the blast wave alone without any secondary injury occurring. We show that blast induces an increase in lactate dehydrogenase activity and glutamate release from the cultures, indicating cellular injury. Additionally, we observe a significant increase in axonal varicosities after blast. These varicosities can be stained with antibodies recognizing amyloid precursor protein. The presence of amyloid precursor protein deposits may indicate a blast-induced axonal transport deficit. After blast injury, we find a transient release of the known TBI biomarkers, UCHL1 and NF-H at 6 h and a delayed increase in S100B at 24 and 48 h. This in vitro model will enable us to gain a better understanding of clinically relevant pathological changes that occur following primary blast and can also be utilized for discovery and characterization of biomarkers.

Molecular simulations of the interfacial properties in silk-hydroxyapatite composites.

Biomineralization is a common strategy used in Nature to improve the mechanical strength and toughness of biological materials. This strategy, applied in materials like bone or nacre, serves as inspiration for materials scientists and engineers to design new materials for applications in healthcare, soft robotics or the environment. In this regard, composites consisting of silk and hydroxyapatite have been extensively researched for bone regeneration applications, due to their reported cytocompatibility and osteoinduction capacity that supports bone formation in vivo. Thus, it becomes relevant to understand how silk and hydroxyapatite interact at their interface, and how this affects the overall mechanical properties of these composites. This theoretical-experimental work investigates the interfacial dynamic and structural properties of silk in contact with hydroxyapatite, combining molecular dynamics simulations with analytical characterization. Our data indicate that hydroxyapatite decreases the ß-sheets in silk, which are a key load-bearing element of silk. The ß-sheets content can usually be increased in silk biomaterials via post-processing methods, such as water vapor annealing. However, the presence of hydroxyapatite appears to reduce also for the formation of ß-sheets via water vapor annealing. This work sheds light into the interfacial properties of silk-hydroxyapatite composite and their relevance for the design of composite biomaterials for bone regeneration.

Photoacoustic Carbon Nanotubes Embedded Silk Scaffolds for Neural Stimulation and Regeneration.

Neural interfaces using biocompatible scaffolds provide crucial properties, such as cell adhesion, structural support, and mass transport, for the functional repair of nerve injuries and neurodegenerative diseases. Neural stimulation has also been found to be effective in promoting neural regeneration. This work provides a generalized strategy to integrate photoacoustic (PA) neural stimulation into hydrogel scaffolds using a nanocomposite hydrogel approach. Specifically, polyethylene glycol (PEG)-functionalized carbon nanotubes (CNT), highly efficient photoacoustic agents, are embedded into silk fibroin to form biocompatible and soft photoacoustic materials. We show that these photoacoustic functional scaffolds enable nongenetic activation of neurons with a spatial precision defined by the area of light illumination, promoting neuron regeneration. These CNT/silk scaffolds offered reliable and repeatable photoacoustic neural stimulation, and 94% of photoacoustic-stimulated neurons exhibit a fluorescence change larger than 10% in calcium imaging in the light-illuminated area. The on-demand photoacoustic stimulation increased neurite outgrowth by 1.74-fold in a rat dorsal root ganglion model, when compared to the unstimulated group. We also confirmed that promoted neurite outgrowth by photoacoustic stimulation is associated with an increased concentration of neurotrophic factor (BDNF). As a multifunctional neural scaffold, CNT/silk scaffolds demonstrated nongenetic PA neural stimulation functions and promoted neurite outgrowth, providing an additional method for nonpharmacological neural regeneration.

Functionalized 3D-printed silk-hydroxyapatite scaffolds for enhanced bone regeneration with innervation and vascularization.

Our goal was to generate functionalized 3D-printed scaffolds for bone regeneration using silk-hydroxyapatite bone cements and osteoinductive, proangiogenic and neurotrophic growth factors or morphogens for accelerated bone formation. 3D printing was utilized to generate macroporous scaffolds with controlled geometries and architectures that promote osseointegration. We build on the knowledge that the osteoinductive factor Bone Morphogenetic Protein-2 (BMP2) can also positively impact vascularization, Vascular Endothelial Growth Factor (VEGF) can impact osteoblastic differentiation, and that Neural Growth Factor (NGF)-mediated signaling can influence bone regeneration. We assessed functions on the 3D printed construct via the osteogenic differentiation of human mesenchymal stem cells; migration and proliferation of human umbilical vein endothelial cells; and proliferation of human induced neural stem cells. The scaffolds provided mechanical properties suitable for bone and the materials were cytocompatible, osteoconductive and maintained the activity of the morphogens and cytokines. Synergistic outcomes between BMP-2, VEGF and NGF in terms of osteoblastic differentiation in vitro were identified, based on the upregulation of genes associated with osteoblastic differentiation (Runt-related transcription factor-2, Osteopontin, Bone Sialoprotein). Additional studies will be required to assess these scaffold designs in vivo. These results are expected to have a strong impact in bone regeneration in dental, oral and maxillofacial surgery.

Silk Polymers and Nanoparticles: A Powerful Combination for the Design of Versatile Biomaterials.

Silk fibroin (SF) is a natural protein largely used in the textile industry but also in biomedicine, catalysis, and other materials applications. SF is biocompatible, biodegradable, and possesses high tensile strength. Moreover, it is a versatile compound that can be formed into different materials at the macro, micro- and nano-scales, such as nanofibers, nanoparticles, hydrogels, microspheres, and other formats. Silk can be further integrated into emerging and promising additive manufacturing techniques like bioprinting, stereolithography or digital light processing 3D printing. As such, the development of methodologies for the functionalization of silk materials provide added value. Inorganic nanoparticles (INPs) have interesting and unexpected properties differing from bulk materials. These properties include better catalysis efficiency (better surface/volume ratio and consequently decreased quantify of catalyst), antibacterial activity, fluorescence properties, and UV-radiation protection or superparamagnetic behavior depending on the metal used. Given the promising results and performance of INPs, their use in many different procedures has been growing. Therefore, combining the useful properties of silk fibroin materials with those from INPs is increasingly relevant in many applications. Two main methodologies have been used in the literature to form silk-based bionanocomposites: in situ synthesis of INPs in silk materials, or the addition of preformed INPs to silk materials. This work presents an overview of current silk nanocomposites developed by these two main methodologies. An evaluation of overall INP characteristics and their distribution within the material is presented for each approach. Finally, an outlook is provided about the potential applications of these resultant nanocomposite materials.

Self-Folding 3D Silk Biomaterial Rolls to Facilitate Axon and Bone Regeneration.

Biomaterial scaffold designs are needed for self-organizing features related to tissue formation while also simplifying the fabrication processes involved. Toward this goal, silk protein-based self-folding scaffolds to support 3D cell culture, while providing directional guidance and promotion of cell growth and differentiation, are reported. A simple and robust one-step self-folding approach is developed using bilayers consisting of a hydrogel and silk film in aqueous solution. The 3D silk rolls, with patterns transferred from the initially prepared 2D films, guide the directional outgrowth of neurites and also promote the osteogenic differentiation of human mesenchymal stem cells (hMSCs). The osteogenic outcomes are further supported by enhanced biomechanical performance. By utilizing this self-folding method, cocultures of neurons and hMSCs are achieved by patterning cells on silk films and then converting these materials into a 3D format with rolling, mimicking aspects of the structure of osteons and providing physiologically relevant structures to promote bone regeneration. These results demonstrate the utility of self-folded silk rolls as efficient scaffold systems for tissue regeneration, while exploiting relatively simple 2D designs programmed to form more complex 3D structures.

From Silk Spinning to 3D Printing: Polymer Manufacturing using Directed Hierarchical Molecular Assembly.

Silk spinning offers an evolution-based manufacturing strategy for industrial polymer manufacturing, yet remains largely inaccessible as the manufacturing mechanisms in biological and synthetic systems, especially at the molecular level, are fundamentally different. The appealing characteristics of silk spinning include the sustainable sourcing of the protein material, the all-aqueous processing into fibers, and the unique material properties of silks in various formats. Substantial progress has been made to mimic silk spinning in artificial manufacturing processes, despite the gap between natural and artificial systems. This report emphasizes the universal spinning conditions utilized by both spiders and silkworms to generate silk fibers in nature, as a scientific and technical framework for directing molecular assembly into high-performance structures. The preparation of regenerated silk feedstocks and mimicking native spinning conditions in artificial manufacturing are discussed, as is progress and challenges in fiber spinning and 3D printing of silk-composites. Silk spinning is a biomimetic model for advanced and sustainable artificial polymer manufacturing, offering benefits in biomedical applications for tissue scaffolds and implantable devices.

High-throughput identification of human SNPs affecting regulatory element activity.

Most of the millions of SNPs in the human genome are non-coding, and many overlap with putative regulatory elements. Genome-wide association studies (GWAS) have linked many of these SNPs to human traits or to gene expression levels, but rarely with sufficient resolution to identify the causal SNPs. Functional screens based on reporter assays have previously been of insufficient throughput to test the vast space of SNPs for possible effects on regulatory element activity. Here we leveraged the throughput and resolution of the survey of regulatory elements (SuRE) reporter technology to survey the effect of 5.9 million SNPs, including 57% of the known common SNPs, on enhancer and promoter activity. We identified more than 30,000 SNPs that alter the activity of putative regulatory elements, partially in a cell-type-specific manner. Integration of this dataset with GWAS results may help to pinpoint SNPs that underlie human traits.

Automated Buildup of Biomimetic Films in Cell Culture Microplates for High-Throughput Screening of Cellular Behaviors.

An automatic method is established for layer-by-layer (LbL) assembly of biomimetic coatings in cell culture microplates using a commercial liquid-handling robot. Highly homogeneous thin films are formed at the bottom of each microwell. The LbL film-coated microplates are compatible with common cellular assays, using microplate readers and automated microscopes. Cellular adhesion is screened on crosslinked and peptide-functionalized LbL films and stem cell differentiation in response to increasing doses of bone morphogenetic proteins (2, 4, 7, 9). This method paves the way for future applications of LbL films in cell-based assays for regenerative medicine and high-throughput drug screening.

SelexGLM differentiates androgen and glucocorticoid receptor DNA-binding preference over an extended binding site.

The DNA-binding interfaces of the androgen (AR) and glucocorticoid (GR) receptors are virtually identical, yet these transcription factors share only about a third of their genomic binding sites and regulate similarly distinct sets of target genes. To address this paradox, we determined the intrinsic specificities of the AR and GR DNA-binding domains using a refined version of SELEX-seq. We developed an algorithm, SelexGLM, that quantifies binding specificity over a large (31-bp) binding site by iteratively fitting a feature-based generalized linear model to SELEX probe counts. This analysis revealed that the DNA-binding preferences of AR and GR homodimers differ significantly, both within and outside the 15-bp core binding site. The relative preference between the two factors can be tuned over a wide range by changing the DNA sequence, with AR more sensitive to sequence changes than GR. The specificity of AR extends to the regions flanking the core 15-bp site, where isothermal calorimetry measurements reveal that affinity is augmented by enthalpy-driven readout of poly(A) sequences associated with narrowed minor groove width. We conclude that the increased specificity of AR is correlated with more enthalpy-driven binding than GR. The binding models help explain differences in AR and GR genomic binding and provide a biophysical rationale for how promiscuous binding by GR allows functional substitution for AR in some castration-resistant prostate cancers.

Signal mingle: Micropatterns of BMP-2 and fibronectin on soft biopolymeric films regulate myoblast shape and SMAD signaling.

In vivo, bone morphogenetic protein 2 (BMP-2) exists both in solution and bound to the extracellular matrix (ECM). While these two modes of presentation are known to influence cell behavior distinctly, their role in the niche microenvironment and their functional relevance in the genesis of a biological response has sparsely been investigated at a cellular level. Here we used the natural affinity of BMP-2 for fibronectin (FN) to engineer cell-sized micropatterns of BMP-2. This technique allowed the simultaneous control of the spatial presentation of fibronectin-bound BMP-2 and cell spreading. These micropatterns induced a specific actin and adhesion organization around the nucleus, and triggered the phosphorylation and nuclear translocation of SMAD1/5/8 in C2C12 myoblasts and mesenchymal stem cells, an early indicator of their osteoblastic trans-differentiation. We found that cell spreading itself potentiated a BMP-2-dependent phosphorylation of SMAD1/5/8. Finally, we demonstrated that FN/BMP-2-mediated early SMAD signaling depended on LIM kinase 2 and ROCK, rather than myosin II activation. Altogether, our results show that FN/BMP-2 micropatterns are a useful tool to study the mechanisms underlying BMP-2-mediated mechanotransduction. More broadly, our approach could be adapted to other combinations of ECM proteins and growth factors, opening an exciting avenue to recreate tissue-specific niches in vitro.

Genome-wide mapping of autonomous promoter activity in human cells.

Previous methods to systematically characterize sequence-intrinsic activity of promoters have been limited by relatively low throughput and the length of the sequences that could be tested. Here we present 'survey of regulatory elements' (SuRE), a method that assays more than 108 DNA fragments, each 0.2-2 kb in size, for their ability to drive transcription autonomously. In SuRE, a plasmid library of random genomic fragments upstream of a 20-bp barcode is constructed, and decoded by paired-end sequencing. This library is used to transfect cells, and barcodes in transcribed RNA are quantified by high-throughput sequencing. When applied to the human genome, we achieve 55-fold genome coverage, allowing us to map autonomous promoter activity genome-wide in K562 cells. By computational modeling we delineate subregions within promoters that are relevant for their activity. We show that antisense promoter transcription is generally dependent on the sense core promoter sequences, and that most enhancers and several families of repetitive elements act as autonomous transcription initiation sites.

Stiffness-dependent cellular internalization of matrix-bound BMP-2 and its relation to Smad and non-Smad signaling.

Surface coatings delivering BMP are a promising approach to render biomaterials osteoinductive. In contrast to soluble BMPs which can interact with their receptors at the dorsal side of the cell, BMPs presented as an insoluble cue physically bound to a biomimetic matrix, called here matrix-bound (bBMP-2), are presented to cells by their ventral side. To date, BMP-2 internalization and signaling studies in cell biology have always been performed by adding soluble (sBMP-2) to cells adhered on cell culture plates or glass slides, which will be considered here as a "reference" condition. However, whether and how matrix-bound BMP-2 can be internalized by cells and its relation to canonical (SMAD) and non-canonical signaling (ALP) remain open questions. In this study, we investigated the uptake and processing of BMP-2 by C2C12 myoblasts. This BMP-2 was presented either embedded in polyelectrolyte multilayer films (matrix-bound presentation) or as soluble form. Using fluorescently labeled BMP-2, we showed that the amount of matrix-bound BMP-2 internalized is dependent on the level of crosslinking of the polyelectrolyte films. Cav-1-mediated internalization is related to both SMAD and ALP signaling, while clathrin-mediated is only related to ALP signaling. BMP-2 internalization was independent of the presentation mode (sBMP-2 versus bBMP-2) for low crosslinked films (soft, EDC10) in striking contrast with high crosslinked (stiff, EDC70) films where internalization was much lower and slower for bBMP-2. As anticipated, internalization of sBMP-2 barely depended on the underlying matrix. Taken together, these results indicate that BMP-2 internalization can be tuned by the underlying matrix and activates downstream BMP-2 signaling, which is key for the effective formation of bone tissue. STATEMENT OF SIGNIFICANCE: The presentation of growth factors from material surfaces currently presents significant challenges in academic research, clinics and industry. Being able to deliver efficiently these growth factors by a biomaterial will open new perspectives for regenerative medicine. However, to date, very little is known about how matrix-bound growth factors are delivered to cells, especially whether they are internalized and how they are signaling to drive key differentiation events. These initial steps are crucial as they will guide the subsequent processes leading to tissue regeneration. In this work, we investigate the uptake and processing by cells of BMP-2 ligands embedded in polyelectrolyte multilayer films in comparison to soluble BMP-2. We show that BMP-2 responsive cells can internalize matrix-bound BMP-2 and that internalization is dependent on the cross-linking level of the polyelectrolyte films. In addition, we show that internalization is mediated by both clathrin- and caveolin-dependent pathways. While inhibiting clathrin-dependent endocytosis affects only non-canonical signaling, blocking caveolin-1-dependent endocytosis reduces both canonical and non-canonical BMP signaling. The signaling pathways found for matrix-bound BMP-2 are similar to those found for soluble BMP-2. These results highlight that BMP-2 presented by a biomaterial at the ventral side of the cell can trigger major endocytic and associated signaling pathways leading to bone regeneration.

Complementary effects of two growth factors in multifunctionalized silk nanofibers for nerve reconstruction.

With the aim of forming bioactive guides for peripheral nerve regeneration, silk fibroin was electrospun to obtain aligned nanofibers. These fibers were functionalized by incorporating Nerve Growth Factor (NGF) and Ciliary NeuroTrophic Factor (CNTF) during electrospinning. PC12 cells grown on the fibers confirmed the bioavailability and bioactivity of the NGF, which was not significantly released from the fibers. Primary neurons from rat dorsal root ganglia (DRGs) were grown on the nanofibers and anchored to the fibers and grew in a directional fashion based on the fiber orientation, and as confirmed by growth cone morphology. These biofunctionalized nanofibers led to a 3-fold increase in neurite length at their contact, which was likely due to the NGF. Glial cell growth, alignment and migration were stimulated by the CNTF in the functionalized nanofibers. Organotypic culture of rat fetal DRGs confirmed the complementary effect of both growth factors in multifunctionalized nanofibers, which allowed glial cell migration, alignment and parallel axonal growth in structures resembling the 'bands of Bungner' found in situ. Graftable multi-channel conduits based on biofunctionalized aligned silk nanofibers were developed as an organized 3D scaffold. Our bioactive silk tubes thus represent new options for a biological and biocompatible nerve guidance conduit.

Inhibition of WISE preserves renal allograft function.

Wnt-modulator in surface ectoderm (WISE) is a secreted modulator of Wnt signaling expressed in the adult kidney. Activation of Wnt signaling has been observed in renal transplants developing interstitial fibrosis and tubular atrophy; however, whether WISE contributes to chronic changes is not well understood. Here, we found moderate to high expression of WISE mRNA in a rat model of renal transplantation and in kidneys from normal rats. Treatment with a neutralizing antibody against WISE improved proteinuria and graft function, which correlated with higher levels of ß-catenin protein in kidney allografts. In addition, treatment with the anti-WISE antibody reduced infiltration of CD68(+) macrophages and CD8(+) T cells, attenuated glomerular and interstitial injury, and decreased biomarkers of renal injury. This treatment reduced expression of genes involved in immune responses and in fibrogenic pathways. In summary, WISE contributes to renal dysfunction by promoting tubular atrophy and interstitial fibrosis.