Release of miR-29 Target Laminin C2 Improves Skin Repair.

miRNAs are small noncoding RNAs that regulate mRNA targets in a cell-specific manner. miR-29 is expressed in murine and human skin, where it may regulate functions in skin repair. Cutaneous wound healing model in miR-29a/b1 gene knockout mice was used to identify miR-29 targets in the wound matrix, where angiogenesis and maturation of provisional granulation tissue was enhanced in response to genetic deletion of miR-29. Consistently, antisense-mediated inhibition of miR-29 promoted angiogenesis in vitro by autocrine and paracrine mechanisms. These processes are likely mediated by miR-29 target mRNAs released upon removal of miR-29 to improve cell-matrix adhesion. One of these, laminin (Lam)-c2 (also known as laminin γ2), was strongly up-regulated during skin repair in the wound matrix of knockout mice. Unexpectedly, Lamc2 was deposited in the basal membrane of endothelial cells in blood vessels forming in the granulation tissue of knockout mice. New blood vessels showed punctate interactions between Lamc2 and integrin α6 (Itga6) along the length of the proto-vessels, suggesting that greater levels of Lamc2 may contribute to the adhesion of endothelial cells, thus assisting angiogenesis within the wound. These findings may be of translational relevance, as LAMC2 was deposited at the leading edge in human wounds, where it formed a basal membrane for endothelial cells and assisted neovascularization. These results suggest a link between LAMC2, improved angiogenesis, and re-epithelialization.

Interaction of the NRF2 and p63 transcription factors promotes keratinocyte proliferation in the epidermis.

Epigenetic regulation of cell and tissue function requires the coordinated action of transcription factors. However, their combinatorial activities during regeneration remain largely unexplored. Here, we discover an unexpected interaction between the cytoprotective transcription factor NRF2 and p63- a key player in epithelial morphogenesis. Chromatin immunoprecipitation combined with sequencing and reporter assays identifies enhancers and promoters that are simultaneously activated by NRF2 and p63 in human keratinocytes. Modeling of p63 and NRF2 binding to nucleosomal DNA suggests their chromatin-assisted interaction. Pharmacological and genetic activation of NRF2 increases NRF2-p63 binding to enhancers and promotes keratinocyte proliferation, which involves the common NRF2-p63 target cyclin-dependent kinase 12. These results unravel a collaborative function of NRF2 and p63 in the control of epidermal renewal and suggest their combined activation as a strategy to promote repair of human skin and other stratified epithelia.

Genetically-programmed, mesenchymal stromal cell-laden & mechanically strong 3D bioprinted scaffolds for bone repair.

Additive manufacturing processes used to create regenerative bone tissue engineered implants are not biocompatible, thereby restricting direct use with stem cells and usually require cell seeding post-fabrication. Combined delivery of stem cells with the controlled release of osteogenic factors, within a mechanically-strong biomaterial combined during manufacturing would replace injectable defect fillers (cements) and allow personalized implants to be rapidly prototyped by 3D bioprinting. Through the use of direct genetic programming via the sustained release of an exogenously delivered transcription factor RUNX2 (delivered as recombinant GET-RUNX2 protein) encapsulated in PLGA microparticles (MPs), we demonstrate that human mesenchymal stromal (stem) cells (hMSCs) can be directly fabricated into a thermo-sintered 3D bioprintable material and achieve effective osteogenic differentiation. Importantly we observed osteogenic programming of gene expression by released GET-RUNX2 (8.2-, 3.3- and 3.9-fold increases in OSX, RUNX2 and OPN expression, respectively) and calcification (von Kossa staining) in our scaffolds. The developed biodegradable PLGA/PEG paste formulation augments high-density bone development in a defect model (~2.4-fold increase in high density bone volume) and can be used to rapidly prototype clinically-sized hMSC-laden implants within minutes using mild, cytocompatible extrusion bioprinting. The ability to create mechanically strong 'cancellous bone-like' printable implants for tissue repair that contain stem cells and controlled-release of programming factors is innovative, and will facilitate the development of novel localized delivery approaches to direct cellular behaviour for many regenerative medicine applications including those for personalized bone repair.

In vivo delivery of VEGF RNA and protein to increase osteogenesis and intraosseous angiogenesis.

Deficient bone vasculature is a key component in pathological conditions ranging from developmental skeletal abnormalities to impaired bone repair. Vascularisation is dependent upon vascular endothelial growth factor (VEGF), which drives both angiogenesis and osteogenesis. The aim of this study was to examine the efficacy of blood vessel and bone formation following transfection with VEGF RNA or delivery of recombinant human VEGF165 protein (rhVEGF165) across in vitro and in vivo model systems. To quantify blood vessels within bone, an innovative approach was developed using high-resolution X-ray computed tomography (XCT) to generate quantifiable three-dimensional reconstructions. Application of rhVEGF165 enhanced osteogenesis, as evidenced by increased human osteoblast-like MG-63 cell proliferation in vitro and calvarial bone thickness following in vivo administration. In contrast, transfection with VEGF RNA triggered angiogenic effects by promoting VEGF protein secretion from MG-63VEGF165 cells in vitro, which resulted in significantly increased angiogenesis in the chorioallantoic (CAM) assay in ovo. Furthermore, direct transfection of bone with VEGF RNA in vivo increased intraosseous vascular branching. This study demonstrates the importance of continuous supply as opposed to a single high dose of VEGF on angiogenesis and osteogenesis and, illustrates the potential of XCT in delineating in 3D, blood vessel connectivity in bone.

Post-Modified Polypeptides with UCST-Type Behavior for Control of Cell Attachment in Physiological Conditions.

Upper Critical Solution Temperature (UCST)-type thermally responsive polypeptides (TRPs) with phase transition temperatures around 37 °C in phosphate-buffered saline (PBS) buffer (pH 7.4, 100 mM) were prepared from poly(l-ornithine) hydrobromide and coated on non-tissue culture-treated plastic plates (nTCP). Cell adhesion was observed at temperatures above the phase transition temperature of the coating polymer (39 °C), while cell release was triggered when the culture temperature was switched to 37 °C. Approximately 65% of the attached cells were released from the surface within 6 h after changing the temperature, and more than 96% of the released cells were viable. Water contact angle measurements performed at 39 and 37 °C demonstrated that the surface hydrophobicity of the new TRP coatings changed in response to applied temperature. The cell attachment varied with the presence of serum in the media, suggesting that the TRP coatings mediated cell attachment and release as the underlying polymer surface changed conformation and consequently the display of adsorbed protein. These new TRP coatings provide an additional means to mediate cell attachment for application in cell-based tissue regeneration and therapies.

Controlled release of GAG-binding enhanced transduction (GET) peptides for sustained and highly efficient intracellular delivery.

Controlled release systems for therapeutic molecules are vital to allow the sustained local delivery of their activities which direct cell behaviour and enable novel regenerative strategies. Direct programming of cells using exogenously delivered transcription factors can by-pass growth factor signalling but there is still a requirement to deliver such activity spatio-temporally. We previously developed a technology termed GAG-binding enhanced transduction (GET) to efficiently deliver a variety of cargoes intracellularly, using GAG-binding domains which promote cell targeting, and cell penetrating peptides (CPPs) which allow cell entry. Herein we demonstrate that GET system can be used in controlled release systems to mediate sustained intracellular transduction over one week. We assessed the stability and activity of GET peptides in poly(dl-lactic acid-co-glycolic acid) (PLGA) microparticles (MPs) prepared using a S/O/W double emulsion method. Efficient encapsulation (∼65%) and tailored protein release profiles could be achieved, however intracellular transduction was significantly inhibited post-release. To retain GET peptide activity we optimized a strategy of co-encapsulation of l-Histidine, which may form a complex with the PLGA degradation products under acidic conditions. Simulations of the polymer microclimate showed that hydrolytic acidic PLGA degradation products directly inhibited GET peptide transduction activity, and use of l-Histidine significantly enhanced released protein delivery. The ability to control the intracellular transduction of functional proteins into cells will facilitate new localized delivery methods and allow approaches to direct cellular behaviour for many regenerative medicine applications. STATEMENT OF SIGNIFICANCE: The goal for regenerative medicine is to restore functional biological tissue by controlling and augmenting cellular behaviour. Either Transcription (TFs) or growth factors (GFs) can be presented to cells in spatio-temporal gradients for programming cell fate and gene expression. Here, we have created a sustained and controlled release system for GET (Glycosaminoglycan-enhanced transducing)-tagged proteins using S/O/W PLGA microparticle fabrication. We demonstrated that PLGA and its acidic degradants inhibit GET-mediated transduction, which can be overcome by using pH-activated l-Histidine. l-Histidine inhibits the electrostatic interaction of GET/PLGA and allows enhanced intracellular transduction. GET could provide a powerful tool to program cell behaviour either in gradients or with sustained delivery. We believe that our controlled release systems will allow application of GET for tissue regeneration directly by TF cellular programming.

Osteogenic Programming of Human Mesenchymal Stem Cells with Highly Efficient Intracellular Delivery of RUNX2.

Mesenchymal stem cells (MSCs) are being exploited in regenerative medicine due to their tri-lineage differentiation and immunomodulation activity. Currently, there are two major challenges when directing the differentiation of MSCs for therapeutic applications. First, chemical and growth factor strategies to direct osteogenesis in vivo lack specificity for targeted delivery with desired effects. Second, MSC differentiation by gene therapy is difficult as transfection with existing approaches is clinically impractical (viral transfection) or have low efficacy (lipid-mediated transfection). These challenges can be avoided by directly delivering nonvirally derived recombinant protein transcription factors with the glycosaminoglycan-binding enhanced transduction (GET) delivery system (P21 and 8R peptides). We used the osteogenic master regulator, RUNX2 as a programming factor due to its stage-specific role in osteochondral differentiation pathways. Herein, we engineered GET-fusion proteins and compared sequential osteogenic changes in MSCs, induced by exposure to GET fusion proteins or conventional stimulation methods (dexamethasone and Bone morphogenetic protein 2). By assessing loss of stem cell-surface markers, upregulation of osteogenic genes and matrix mineralization, we demonstrate that GET-RUNX2 efficiently transduces MSCs and triggers osteogenesis by enhancing target gene expression directly. The high transduction efficiency of GET system holds great promise for stem cell therapies by allowing reproducible transcriptional control in stem cells, potentially bypassing problems observed with high-concentration growth-factor or pleiotropic steroid therapies. Stem Cells Translational Medicine 2017;6:2146-2159.

Upper critical solution temperature thermo-responsive polymer brushes and a mechanism for controlled cell attachment.

We report the synthesis of thermo-responsive polymer brushes with Upper Critical Solution Temperature (UCST)-type behaviour on glass to provide a new means to control cell attachment. Thermoresponsive poly(N-acryloyl glycinamide)-stat-poly(N-phenylacrylamide) (PNAGAm-PNPhAm) brushes with three different monomer ratios were synthesized to give tunable phase transition temperatures (Tp) in solution. Surface energies of surface-grafted brushes of these polymers at 25, 32, 37 and 50 °C were calculated from contact angle measurements and atomic force microscopy (AFM) studies confirmed that these polymers were highly extended at temperatures close to Tp in physiologically-relevant media. Importantly, NIH-3T3 cells were attached on the collapsed PNAGAm-PNPhAm brush surface at 30 °C after 20 h incubation, while release of cells from the extended brushes was observed within 2 h after the culture temperature was switched to 37 °C. Furthermore, the changes in cell attachment followed changes in the Lewis base component of surface energy. The results indicate that, in contrast to the established paradigm of enhanced cell attachment to surfaces where polymers are above a Lower Critical Solution Temperature (LCST), these novel substrates enable detachment of cells from surfaces at temperatures above a UCST. In turn these responsive materials open new avenues for the use of polymer-modified surfaces to control cell attachment for applications in cell manufacture and regenerative medicine.

TBX2 represses CST6 resulting in uncontrolled legumain activity to sustain breast cancer proliferation: a novel cancer-selective target pathway with therapeutic opportunities.

TBX2 is an oncogenic transcription factor known to drive breast cancer proliferation. We have identified the cysteine protease inhibitor Cystatin 6 (CST6) as a consistently repressed TBX2 target gene, co-repressed through a mechanism involving Early Growth Response 1 (EGR1). Exogenous expression of CST6 in TBX2-expressing breast cancer cells resulted in significant apoptosis whilst non-tumorigenic breast cells remained unaffected. CST6 is an important tumor suppressor in multiple tissues, acting as a dual protease inhibitor of both papain-like cathepsins and asparaginyl endopeptidases (AEPs) such as Legumain (LGMN). Mutation of the CST6 LGMN-inhibitory domain completely abrogated its ability to induce apoptosis in TBX2-expressing breast cancer cells, whilst mutation of the cathepsin-inhibitory domain or treatment with a pan-cathepsin inhibitor had no effect, suggesting that LGMN is the key oncogenic driver enzyme. LGMN activity assays confirmed the observed growth inhibitory effects were consistent with CST6 inhibition of LGMN. Knockdown of LGMN and the only other known AEP enzyme (GPI8) by siRNA confirmed that LGMN was the enzyme responsible for maintaining breast cancer proliferation. CST6 did not require secretion or glycosylation to elicit its cell killing effects, suggesting an intracellular mode of action. Finally, we show that TBX2 and CST6 displayed reciprocal expression in a cohort of primary breast cancers with increased TBX2 expression associating with increased metastases. We have also noted that tumors with altered TBX2/CST6 expression show poor overall survival. This novel TBX2-CST6-LGMN signaling pathway, therefore, represents an exciting opportunity for the development of novel therapies to target TBX2 driven breast cancers.