Systemically administered wound-homing peptide accelerates wound healing by modulating syndecan-4 function.

CAR (CARSKNKDC) is a wound-homing peptide that recognises angiogenic neovessels. Here we discover that systemically administered CAR peptide has inherent ability to promote wound healing: wounds close and re-epithelialise faster in CAR-treated male mice. CAR promotes keratinocyte migration in vitro. The heparan sulfate proteoglycan syndecan-4 regulates cell migration and is crucial for wound healing. We report that syndecan-4 expression is restricted to epidermis and blood vessels in mice skin wounds. Syndecan-4 regulates binding and internalisation of CAR peptide and CAR-mediated cytoskeletal remodelling. CAR induces syndecan-4-dependent activation of the small GTPase ARF6, via the guanine nucleotide exchange factor cytohesin-2, and promotes syndecan-4-, ARF6- and Cytohesin-2-mediated keratinocyte migration. Finally, we show that genetic ablation of syndecan-4 in male mice eliminates CAR-induced wound re-epithelialisation following systemic administration. We propose that CAR peptide activates syndecan-4 functions to selectively promote re-epithelialisation. Thus, CAR peptide provides a therapeutic approach to enhance wound healing in mice; systemic, yet target organ- and cell-specific.

Dimeric IgA specifically disables intracellular mutated oncodrivers.

A prevailing belief in the immunotherapy field has been that antibody therapy can effectively target only extracellular antigens. In this issue of Immunity, Biswas et al. demonstrate therapeutically effective targeting, neutralization, and removal of mutated oncodriver proteins from within epithelial cancer cells by treatment with pIgR-dependent, transcytosing dimeric-IgA antibodies.

Selection of biophysically favorable antibody variants using a modified Flp-In CHO mammalian display platform.

Mammalian display enables the selection of biophysically favorable antibodies from a large IgG antibody library displayed on the plasma membrane of mammalian cells. We constructed and validated a novel mammalian display platform utilizing the commercially available Flp-In CHO cell line as a starting point. We introduced a single copy of a landing pad for Bxb1 integrase-driven recombinase-mediated cassette exchange into the FRT site of the Flp-In CHO line to facilitate the efficient single-copy integration of an antibody display cassette into the genome of the cell line. We then proceeded to demonstrate the ability of our platform to select biophysically favorable antibodies from a library of 1 × 106 displayed antibodies designed to improve the biophysical properties of bococizumab via randomization of problematic hydrophobic surface residues of the antibody. Enrichment of bococizumab variants via fluorescence-activated cell sorting selections was followed by next generation sequencing and thorough characterization of biophysical properties of 10 bococizumab variants that subsequently allowed attribution of the mutations to the biophysical properties of the antibody variants. The mammalian displayed variants exhibited reduced aggregation propensity and polyreactivity, while critically retaining its target binding thereby demonstrating the utility of this valuable tool.

Dual drug delivery collagen vehicles for modulation of skin fibrosisin vitro.

Single molecule drug delivery systems have failed to yield functional therapeutic outcomes, triggering investigations into multi-molecular drug delivery vehicles. In the context of skin fibrosis, although multi-drug systems have been assessed, no system has assessed molecular combinations that directly and specifically reduce cell proliferation, collagen synthesis and transforming growth factorß1 (TGFß1) expression. Herein, a core-shell collagen type I hydrogel system was developed for the dual delivery of a TGFßtrap, a soluble recombinant protein that inhibits TGFßsignalling, and Trichostatin A (TSA), a small molecule inhibitor of histone deacetylases. The antifibrotic potential of the dual delivery system was assessed in anin vitroskin fibrosis model induced by macromolecular crowding (MMC) and TGFß1. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and high performance liquid chromatography analyses revealed that ∼50% of the TGFßtrap and ∼30% of the TSA were released from the core and shell compartments, respectively, of the hydrogel system after 10 d (longest time point assessed) in culture. As a direct consequence of this slow release, the core (TGFßtrap)/shell (TSA) hydrogel system induced significantly (p< 0.05) lower than the control group (MMC and TGFß1) collagen type I deposition (assessed via SDS-PAGE and immunocytochemistry),αsmooth muscle actin (αSMA) expression (assessed via immunocytochemistry) and cellular proliferation (assessed via DNA quantification) and viability (assessed via calcein AM and ethidium homodimer-I staining) after 10 d in culture. On the other hand, direct TSA-TGFßsupplementation induced the lowest (p< 0.05) collagen type I deposition,αSMA expression and cellular proliferation and viability after 10 d in culture. Our results illustrate the potential of core-shell collagen hydrogel systems for sustained delivery of antifibrotic molecules.

Adapting the Scar-in-a-Jar to Skin Fibrosis and Screening Traditional and Contemporary Anti-Fibrotic Therapies.

Skin fibrosis still constitutes an unmet clinical need. Although pharmacological strategies are at the forefront of scientific and technological research and innovation, their clinical translation is hindered by the poor predictive capacity of the currently available in vitro fibrosis models. Indeed, customarily utilised in vitro scarring models are conducted in a low extracellular matrix milieu, which constitutes an oxymoron for the in-hand pathophysiology. Herein, we coupled macromolecular crowding (enhances and accelerates extracellular matrix deposition) with transforming growth factor ß1 (TGFß1; induces trans-differentiation of fibroblasts to myofibroblasts) in human dermal fibroblast cultures to develop a skin fibrosis in vitro model and to screen a range of anti-fibrotic families (corticosteroids, inhibitors of histone deacetylases, inhibitors of collagen crosslinking, inhibitors of TGFß1 and pleiotropic inhibitors of fibrotic activation). Data obtained demonstrated that macromolecular crowding combined with TGFß1 significantly enhanced collagen deposition and myofibroblast transformation. Among the anti-fibrotic compounds assessed, trichostatin A (inhibitors of histone deacetylases); serelaxin and pirfenidone (pleiotropic inhibitors of fibrotic activation); and soluble TGFß receptor trap (inhibitor of TGFß signalling) resulted in the highest decrease of collagen type I deposition (even higher than triamcinolone acetonide, the gold standard in clinical practice). This study further advocates the potential of macromolecular crowding in the development of in vitro pathophysiology models.

Systemically Administered Homing Peptide Targets Dystrophic Lesions and Delivers Transforming Growth Factor-ß (TGFß) Inhibitor to Attenuate Murine Muscular Dystrophy Pathology.

Muscular dystrophy is a progressively worsening and lethal disease, where accumulation of functionality-impairing fibrosis plays a key pathogenic role. Transforming growth factor-ß1 (TGFß1) is a central signaling molecule in the development of fibrosis in muscular dystrophic humans and mice. Inhibition of TGFß1 has proven beneficial in mouse models of muscular dystrophy, but the global strategies of TGFß1 inhibition produce significant detrimental side effects. Here, we investigated whether murine muscular dystrophy lesion-specific inhibition of TGFß1 signaling by the targeted delivery of therapeutic decorin (a natural TGFß inhibitor) by a vascular homing peptide CAR (CARSKNKDC) would reduce skeletal muscle fibrosis and pathology and increase functional characteristics of skeletal muscle. We demonstrate that CAR peptide homes to dystrophic lesions with specificity in two muscular dystrophy models. Recombinant fusion protein consisting of CAR peptide and decorin homes selectively to sites of skeletal muscle damage in mdxDBA2/J and gamma-sarcoglycan deficient DBA2/J mice. This targeted delivery reduced TGFß1 signaling as demonstrated by reduced nuclear pSMAD staining. Three weeks of targeted decorin treatment decreased both membrane permeability and fibrosis and improved skeletal muscle function in comparison to control treatments in the mdxD2 mice. These results show that selective delivery of decorin to the sites of skeletal muscle damage attenuates the progression of murine muscular dystrophy.

Exposed CendR Domain in Homing Peptide Yields Skin-Targeted Therapeutic in Epidermolysis Bullosa.

Systemic skin-selective therapeutics would be a major advancement in the treatment of diseases affecting the entire skin, such as recessive dystrophic epidermolysis bullosa (RDEB), which is caused by mutations in the COL7A1 gene and manifests in transforming growth factor-ß (TGF-ß)-driven fibrosis and malignant transformation. Homing peptides containing a C-terminal R/KXXR/K motif (C-end rule [CendR] sequence) activate an extravasation and tissue penetration pathway for tumor-specific drug delivery. We have previously described a homing peptide CRKDKC (CRK) that contains a cryptic CendR motif and homes to angiogenic blood vessels in wounds and tumors, but it cannot penetrate cells or tissues. In this study, we demonstrate that removal of the cysteine from CRK to expose the CendR sequence confers the peptide novel ability to home to normal skin. Fusion of the truncated CRK (tCRK) peptide to the C terminus of an extracellular matrix protein decorin (DCN), a natural TGF-ß inhibitor, resulted in a skin-homing therapeutic molecule (DCN-tCRK). Systemic DCN-tCRK administration in RDEB mice led to inhibition of TGF-ß signaling in the skin and significant improvement in the survival of RDEB mice. These results suggest that DCN-tCRK has the potential to be utilized as a novel therapeutic compound for the treatment of dermatological diseases such as RDEB.

The practicalities of establishing a porcine isolated heart model.

BACKGROUND: The isolated heart apparatus is over 100 years old, but remains a useful research tool today. While designs of many large animal systems have been described in the literature, trouble-shooting and refining such a model to yield a stable, workable system has not been previously described. This paper outlines the issues, in tabular form, that our group encountered in developing our own porcine isolated heart rig with the aim of assisting other workers in the field planning similar work. The paper also highlights some of the modern applications of the isolated heart apparatus. Methods Landrace pigs (50-80 kg) were used in a pilot project to develop the model. The model was then used in a study examining the effects of various cardioplegic solutions on function after reanimation of porcine hearts. During the two projects, non-protocol issues were documented as well as their solutions. These were aggregated in this paper. RESULTS: Issues faced by the group without explicit literature solutions included pig size selection, animal acclimatisation, porcine transoesophageal echocardiography, cannulation and phlebotomy for cross-clamping, cardioplegia delivery, heart suspension and rig tuning. CONCLUSION: Prior recognition of issues and possible solutions faced by workers establishing a porcine isolated heart system will speed progress towards a useable system for research. The isolated heart apparatus remains applicable in transplant, ischaemia reperfusion, heart failure and organ preservation research.

Role of carbonic anhydrases in skin wound healing.

Skin wound closure occurs when keratinocytes migrate from the edge of the wound and re-epithelialize the epidermis. Their migration takes place primarily before any vascularization is established, that is, under hypoxia, but relatively little is known regarding the factors that stimulate this migration. Hypoxia and an acidic environment are well-established stimuli for cancer cell migration. The carbonic anhydrases (CAs) contribute to tumor cell migration by generating an acidic environment through the conversion of carbon dioxide to bicarbonate and a proton. On this basis, we explored the possible role of CAs in tissue regeneration using mouse skin wound models. We show that the expression of mRNAs encoding CA isoforms IV and IX are increased (~25 × and 4 ×, respectively) during the wound hypoxic period (days 2-5) and that cells expressing CAs form a band-like structure beneath the migrating epidermis. RNA-Seq analysis suggested that the CA IV-specific signal in the wound is mainly derived from neutrophils. Due to the high level of induction of CA IV in the wound, we treated skin wounds locally with recombinant human CA IV enzyme. Recombinant CA IV significantly accelerated wound re-epithelialization. Thus, CA IV could contribute to wound healing by providing an acidic environment in which the migrating epidermis and neutrophils can survive and may offer novel opportunities to accelerate wound healing under compromised conditions.

R-Ras deficiency does not affect papain-induced IgE production in mice.

INTRODUCTION: R-Ras GTPase has recently been implicated in the regulation of immune functions, particularly in dendritic cell (DC) maturation, immune synapse formation, and subsequent T cell responses. METHODS: Here, we investigated the role of R-Ras in allergen-induced immune response (type 2 immune response) in Rras deficient (R-Ras KO) and wild type (WT) mice. RESULTS: Initially, we found that the number of conventional DC's in the lymph nodes (LNs) was reduced in R-Ras KO mice. The expression of co-stimulatory CD80 and CD86 molecules on these cells was also reduced on DC's from the R-Ras KO mice. However, there was no difference in papain-induced immune response between the R-Ras WT and KO as measured by serum IgE levels after the immunization. Interestingly, neither the DC number nor co-stimulatory molecule expression was different between WT and R-Ras KO animals after the immunization. CONCLUSIONS: Taken together, despite having reduced number of conventional DC's in the R-Ras KO mice and low expression of CD80 on DC's, the R-Ras KO mice are capable of mounting papain-induced IgE responses comparable to that of the WT mice. To our knowledge, this is the first report addressing potential differences in in vivo allergen responses regulated by the R-Ras GTPase.

Lack of R-Ras Leads to Increased Vascular Permeability in Ischemic Retinopathy.

PURPOSE: The role of R-Ras in retinal angiogenesis and vascular permeability was evaluated in an oxygen-induced retinopathy (OIR) model using R-Ras knockout (KO) mice and in human diabetic neovascular membranes. METHODS: Mice deficient for R-Ras and their wild-type (WT) littermates were subjected to 75% oxygen from postnatal day 7 (P7) to P12 and then returned to room air. At P17 retinal vascularization was examined from whole mounts, and retinal vascular permeability was studied using Miles assay. Real-time RT-PCR, Western blotting, and immunohistochemistry were used to assess the expression of R-Ras in retina during development or in the OIR model. The degree of pericyte coverage and vascular endothelial (VE)-cadherin expression on WT and R-Ras KO retinal blood vessels was quantified using confocal microscopy. The correlation of R-Ras with vascular endothelial growth factor receptor 2 (VEGFR2) and human serum albumin on human proliferative diabetic retinopathy membranes was assessed using immunohistochemistry. RESULTS: In retina, R-Ras expression was mostly restricted to the vasculature. Retinal vessels in the R-Ras KO mice were significantly more permeable than WT controls in the OIR model. A significant reduction in the direct physical contact between pericytes and blood vessel endothelium as well as reduced VE-cadherin immunostaining was found in R-Ras-deficient mice. In human proliferative diabetic retinopathy neovascular membranes, R-Ras expression negatively correlated with increased vascular leakage and expression of VEGFR2, a marker of blood vessel immaturity. CONCLUSIONS: Our results suggest that R-Ras has a role in controlling retinal vessel maturation and stabilization in ischemic retinopathy and provides a potential target for pharmacologic manipulation to treat diabetic retinopathy.

Functional evolution of IGF2:IGF2R domain 11 binding generates novel structural interactions and a specific IGF2 antagonist.

Among the 15 extracellular domains of the mannose 6-phosphate/insulin-like growth factor-2 receptor (M6P/IGF2R), domain 11 has evolved a binding site for IGF2 to negatively regulate ligand bioavailability and mammalian growth. Despite the highly evolved structural loops of the IGF2:domain 11 binding site, affinity-enhancing AB loop mutations suggest that binding is modifiable. Here we examine the extent to which IGF2:domain 11 affinity, and its specificity over IGF1, can be enhanced, and we examine the structural basis of the mechanistic and functional consequences. Domain 11 binding loop mutants were selected by yeast surface display combined with high-resolution structure-based predictions, and validated by surface plasmon resonance. We discovered previously unidentified mutations in the ligand-interacting surface binding loops (AB, CD, FG, and HI). Five combined mutations increased rigidity of the AB loop, as confirmed by NMR. When added to three independently identified CD and FG loop mutations that reduced the koff value by twofold, these mutations resulted in an overall selective 100-fold improvement in affinity. The structural basis of the evolved affinity was improved shape complementarity established by interloop (AB-CD) and intraloop (FG-FG) side chain interactions. The high affinity of the combinatorial domain 11 Fc fusion proteins functioned as ligand-soluble antagonists or traps that depleted pathological IGF2 isoforms from serum and abrogated IGF2-dependent signaling in vivo. An evolved and reengineered high-specificity M6P/IGF2R domain 11 binding site for IGF2 may improve therapeutic targeting of the frequent IGF2 gain of function observed in human cancer.

T-cell-expressed proprotein convertase FURIN inhibits DMBA/TPA-induced skin cancer development.

Proprotein convertases (PCSK) have a critical role in the body homeostasis as enzymes responsible for processing precursor proteins into their mature forms. FURIN, the first characterized member of the mammalian PCSK family, is overexpressed in multiple malignancies and the inhibition of its activity has been considered potential cancer treatment. FURIN has also an important function in the adaptive immunity, since its deficiency in T cells causes an impaired peripheral immune tolerance and accelerates immune responses. We addressed whether deleting FURIN from the immune cells would strengthen anticancer responses by subjecting mouse strains lacking FURIN from either T cells or macrophages and granulocytes to the DMBA/TPA two-stage skin carcinogenesis protocol. Unexpectedly, deficiency of FURIN in T cells resulted in enhanced and accelerated development of tumors, whereas FURIN deletion in macrophages and granulocytes had no effect. The epidermises of T-cell-specific FURIN deficient mice were significantly thicker with more proliferating Ki67+ cells. In contrast, there were no differences in the numbers of the T cells. The flow cytometric analyses of T-cell populations in skin draining lymph nodes showed that FURIN T-cell KO mice have an inherent upregulation of early activation marker CD69 as well as more CD4+CD25+Foxp3+ positive T regulatory cells. In the early phase of tumor promotion, T cells from the T-cell-specific FURIN knockout animals produced more interferon gamma, whereas at later stage the production of Th2- and Th17-type cytokines was more prominent than in wild-type controls. In conclusion, while PCSK inhibitors are promising therapeutics in cancer treatment, our results show that inhibiting FURIN specifically in T cells may promote squamous skin cancer development.

Decorin: A Growth Factor Antagonist for Tumor Growth Inhibition.

Decorin (DCN) is the best characterized member of the extracellular small leucine-rich proteoglycan family present in connective tissues, typically in association with or "decorating" collagen fibrils. It has substantial interest to clinical medicine owing to its antifibrotic, anti-inflammatory, and anticancer effects. Studies on DCN knockout mice have established that a lack of DCN is permissive for tumor development and it is regarded as a tumor suppressor gene. A reduced expression or a total disappearance of DCN has been reported to take place in various forms of human cancers during tumor progression. Furthermore, when used as a therapeutic molecule, DCN has been shown to inhibit tumor progression and metastases in experimental cancer models. DCN affects the biology of various types of cancer by targeting a number of crucial signaling molecules involved in cell growth, survival, metastasis, and angiogenesis. The active sites for the neutralization of different growth factors all reside in different parts of the DCN molecule. An emerging concept that multiple proteases, especially those produced by inflammatory cells, are capable of cleaving DCN suggests that native DCN could be inactivated in a number of pathological inflammatory conditions. In this paper, we review the role of DCN in cancer.

Systemically Administered, Target Organ-Specific Therapies for Regenerative Medicine.

Growth factors and other agents that could potentially enhance tissue regeneration have been identified, but their therapeutic value in clinical medicine has been limited for reasons such as difficulty to maintain bioactivity of locally applied therapeutics in the protease-rich environment of regenerating tissues. Although human diseases are treated with systemically administered drugs in general, all current efforts aimed at enhancing tissue repair with biological drugs have been based on their local application. The systemic administration of growth factors has been ruled out due to concerns about their safety. These concerns are warranted. In addition, only a small proportion of systemically administered drugs reach their intended target. Selective delivery of the drug to the target tissue and use of functional protein domains capable of penetrating cells and tissues could alleviate these problems in certain circumstances. We will present in this review a novel approach utilizing unique molecular fingerprints ("Zip/postal codes") in the vasculature of regenerating tissues that allows target organ-specific delivery of systemically administered therapeutic molecules by affinity-based physical targeting (using peptides or antibodies as an "address tag") to injured tissues undergoing repair. The desired outcome of targeted therapies is increased local accumulation and lower systemic concentration of the therapeutic payload. We believe that the physical targeting of systemically administered therapeutic molecules could be rapidly adapted in the field of regenerative medicine.

Resistance of R-Ras knockout mice to skin tumour induction.

The R-ras gene encodes a small GTPase that is a member of the Ras family. Despite close sequence similarities, R-Ras is functionally distinct from the prototypic Ras proteins; no transformative activity and no activating mutations of R-Ras in human malignancies have been reported for it. R-Ras activity appears inhibitory towards tumour proliferation and invasion, and to promote cellular quiescence. Contrary to this, using mice with a deletion of the R-ras gene, we found that R-Ras facilitates DMBA/TPA-induced skin tumour induction. The tumours appeared in wild-type (WT) mice on average 6 weeks earlier than in R-Ras knockout (R-Ras KO) mice. WT mice developed almost 6 times more tumours than R-Ras KO mice. Despite strong R-Ras protein expression in the dermal blood vessels, no R-Ras could be detected in the epidermis from where the tumours arose. The DMBA/TPA skin tumourigenesis-model is highly dependent upon inflammation, and we found a greatly attenuated skin inflammatory response to DMBA/TPA-treatment in the R-Ras KO mice in the context of leukocyte infiltration and proinflammatory cytokine expression. Thus, these data suggest that despite its characterised role in promoting cellular quiescence, R-Ras is pro-tumourigenic in the DMBA/TPA tumour model and important for the inflammatory response to DMBA/TPA treatment.

An exon splice enhancer primes IGF2:IGF2R binding site structure and function evolution.

Placental development and genomic imprinting coevolved with parental conflict over resource distribution to mammalian offspring. The imprinted genes IGF2 and IGF2R code for the growth promoter insulin-like growth factor 2 (IGF2) and its inhibitor, mannose 6-phosphate (M6P)/IGF2 receptor (IGF2R), respectively. M6P/IGF2R of birds and fish do not recognize IGF2. In monotremes, which lack imprinting, IGF2 specifically bound M6P/IGF2R via a hydrophobic CD loop. We show that the DNA coding the CD loop in monotremes functions as an exon splice enhancer (ESE) and that structural evolution of binding site loops (AB, HI, FG) improved therian IGF2 affinity. We propose that ESE evolution led to the fortuitous acquisition of IGF2 binding by M6P/IGF2R that drew IGF2R into parental conflict; subsequent imprinting may then have accelerated affinity maturation.

Structure and function of the human Gly1619Arg polymorphism of M6P/IGF2R domain 11 implicated in IGF2 dependent growth.

The mannose 6-phosphate/IGF 2 receptor (IGF2R) is comprised of 15 extra-cellular domains that bind IGF2 and mannose 6-phosphate ligands. IGF2R transports ligands from the Golgi to the pre-lysosomal compartment and thereafter to and from the cell surface. IGF2R regulates growth, placental development, tumour suppression and signalling. The ligand IGF2 is implicated in the growth phenotype, where IGF2R normally limits bioavailability, such that loss and gain of IGF2R results in increased and reduced growth respectively. The IGF2R exon 34 (5002A>G) polymorphism (rs629849) of the IGF2 specific binding domain has been correlated with impaired childhood growth (A/A homozygotes). We evaluated the function of the Gly1619Arg non-synonymous amino acid modification of domain 11. NMR and X-ray crystallography structures located 1619 remote from the ligand binding region of domain 11. Arg1619 was located close to the fibronectin type II (FnII) domain of domain 13, previously implicated as a modifier of IGF2 ligand binding through indirect interaction with the AB loop of the binding cleft. However, comparison of binding kinetics of IGF2R, Gly1619 and Arg1619 to either IGF2 or mannose 6-phosphate revealed no differences in 'on' and 'off' rates. Quantitative PCR, (35)S pulse chase and flow cytometry failed to demonstrate altered gene expression, protein half-life and cell membrane distribution, suggesting the polymorphism had no direct effect on receptor function. Intronic polymorphisms were identified which may be in linkage disequilibrium with rs629849 in certain populations. Other potential IGF2R polymorphisms may account for the correlation with childhood growth, warranting further functional evaluation.

Structural insights into the interaction of insulin-like growth factor 2 with IGF2R domain 11.

The insulin-like growth factor II/mannose-6-phosphate receptor (IGF2R) mediates trafficking of mannose-6-phosphate (M6P)-containing proteins and the mitogenic hormone IGF2. IGF2R also plays an important role as a tumor suppressor, as mutation is frequently associated with human carcinogenesis. IGF2 binds to domain 11, one of 15 extracellular domains on IGF2R. The crystal structure of domain 11 and the solution structure of IGF2 have been reported, but, to date, there has been limited success when using crystallography to study the interaction of IGFs with their binding partners. As an approach to investigate the interaction between IGF2 and IGF2R, we have used heteronuclear NMR in combination with existing mutagenesis data to derive models of the domain 11-IGF2 complex by using the program HADDOCK. The models reveal that the molecular interaction is driven by critical hydrophobic residues on IGF2 and IGF2R, while a ring of flexible, charged residues on IGF2R may modulate binding.