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.

Epitope-specific antibody fragments block aggregation of AGelD187N, an aberrant peptide in gelsolin amyloidosis.

Aggregation of aberrant fragment of plasma gelsolin, AGelD187N, is a crucial event underlying the pathophysiology of Finnish gelsolin amyloidosis, an inherited form of systemic amyloidosis. The amyloidogenic gelsolin fragment AGelD187N does not play any physiological role in the body, unlike most aggregating proteins related to other protein misfolding diseases. However, no therapeutic agents that specifically and effectively target and neutralize AGelD187N exist. We used phage display technology to identify novel single-chain variable fragments that bind to different epitopes in the monomeric AGelD187N that were further maturated by variable domain shuffling and converted to antigen-binding fragment (Fab) antibodies. The generated antibody fragments had nanomolar binding affinity for full-length AGelD187N, as evaluated by biolayer interferometry. Importantly, all four Fabs selected for functional studies efficiently inhibited the amyloid formation of full-length AGelD187N as examined by thioflavin fluorescence assay and transmission electron microscopy. Two Fabs, neither of which bound to the previously proposed fibril-forming region of AGelD187N, completely blocked the amyloid formation of AGelD187N. Moreover, no small soluble aggregates, which are considered pathogenic species in protein misfolding diseases, were formed after successful inhibition of amyloid formation by the most promising aggregation inhibitor, as investigated by size-exclusion chromatography combined with multiangle light scattering. We conclude that all regions of the full-length AGelD187N are important in modulating its assembly into fibrils and that the discovered epitope-specific anti-AGelD187N antibody fragments provide a promising starting point for a disease-modifying therapy for gelsolin amyloidosis, which is currently lacking.

Increased stable integration efficiency in CHO cells through enhanced nuclear localization of Bxb1 serine integrase.

BACKGROUND: Mammalian display is an appealing technology for therapeutic antibody development. Despite the advantages of mammalian display, such as full-length IgG display with mammalian glycosylation and its inherent ability to select antibodies with good biophysical properties, the restricted library size and large culture volumes remain challenges. Bxb1 serine integrase is commonly used for the stable genomic integration of antibody genes into mammalian cells, but presently lacks the efficiency required for the display of large mammalian display libraries. To increase the Bxb1 integrase-mediated stable integration efficiency, our study investigates factors that potentially affect the nuclear localization of Bxb1 integrase. METHODS: In an attempt to enhance Bxb1 serine integrase-mediated integration efficiency, we fused various nuclear localization signals (NLS) to the N- and C-termini of the integrase. Concurrently, we co-expressed multiple proteins associated with nuclear transport to assess their impact on the stable integration efficiency of green fluorescent protein (GFP)-encoding DNA and an antibody display cassette into the genome of Chinese hamster ovary (CHO) cells containing a landing pad for Bxb1 integrase-mediated integration. RESULTS: The nucleoplasmin NLS from Xenopus laevis, when fused to the C-terminus of Bxb1 integrase, demonstrated the highest enhancement in stable integration efficiency among the tested NLS fusions, exhibiting over a 6-fold improvement compared to Bxb1 integrase lacking an NLS fusion. Subsequent additions of extra NLS fusions to the Bxb1 integrase revealed an additional 131% enhancement in stable integration efficiency with the inclusion of two copies of C-terminal nucleoplasmin NLS fusions. Further improvement was achieved by co-expressing the Ran GTPase-activating protein (RanGAP). Finally, to validate the applicability of these findings to more complex proteins, the DNA encoding the membrane-bound clinical antibody abrilumab was stably integrated into the genome of CHO cells using Bxb1 integrase with two copies of C-terminal nucleoplasmin NLS fusions and co-expression of RanGAP. This approach demonstrated over 14-fold increase in integration efficiency compared to Bxb1 integrase lacking an NLS fusion. CONCLUSIONS: This study demonstrates that optimizing the NLS sequence fusion for Bxb1 integrase significantly enhances the stable genomic integration efficiency. These findings provide a practical approach for constructing larger libraries in mammalian cells through the stable integration of genes into a genomic landing pad.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Functional evaluation of novel soluble insulin-like growth factor (IGF)-II-specific ligand traps based on modified domain 11 of the human IGF2 receptor.

Ligands transported by the mannose 6-phosphate/insulin-like growth factor (IGF)-II receptor (IGF2R) include IGF-II- and mannose 6-phosphate-modified proteins. Increased extracellular supply of IGF-II, either secondary to loss of the clearance function of IGF2R, loss of IGF binding protein function, or increased IGF2 gene expression, can lead to embryonic overgrowth and cancer promotion. Reduced supply of IGF-II is detrimental to tumor growth, and this suggests that gain of function of IGF-II is a molecular target for human cancer therapy. Domain 11 of IGF2R binds IGF-II with high specificity and affinity. Mutagenesis studies have shown that substitution of glutamic acid for lysine at residue 1554 results in a 6-fold higher affinity for IGF-II (20.5 nmol/L) than native domain 11 (119 nmol/L). Here, we generate a novel high-affinity IGF-II ligand trap by fusion of mutated human 11(E1554K) to a COOH-terminal human IgG1 Fc domain (11(E1554K)-Fc). The resulting homodimer has a significantly increased affinity for IGF-II (1.79 nmol/L) when measured by surface plasmon resonance. IGF-II signaling via the IGF-I receptor and the proliferative effect of IGF-II were specifically inhibited by 11(E1554K)-Fc in both HaCaT and Igf2(-/-) mouse embryonic fibroblast cells. These data confirm that a novel engineered and soluble IGF2R-11(E1554K)-Fc protein functions as an IGF-II-specific and high-affinity ligand trap in vitro and that this protein has potential application as an IGF-II antagonist for cancer therapy following in vivo experimental evaluation.

Kinetics of insulin-like growth factor II (IGF-II) interaction with domain 11 of the human IGF-II/mannose 6-phosphate receptor: function of CD and AB loop solvent-exposed residues.

Ligands of the IGF-II/mannose 6-phosphate receptor (IGF2R) include IGF-II and mannose 6-phosphate modified proteins. Disruption of the negative regulatory effects of IGF2R on IGF-II-induced growth can lead to embryonic lethality and cancer promotion. Of the 15 IGF2R extracellular domains, domains 1-3 and 11 are known to have a conserved beta-barrel structure similar to that of avidin and the cation-dependent mannose 6-phosphate receptor, yet only domain 11 binds IGF-II with high specificity and affinity. In order to define the functional basis of this critical biological interaction, we performed alanine mutagenesis of structurally determined solvent-exposed loop residues of the IGF-II-binding site of human domain 11, expressed these mutant forms in Pichia pastoris, and determined binding kinetics with human IGF-II using isothermal calorimetry and surface plasmon resonance with transition state thermodynamics. Two hydrophobic residues in the CD loop (F1567 and I1572) were essential for binding, with a further non-hydrophobic residue (T1570) that slows the dissociation rate. Aside from alanine mutations of AB loop residues that decrease affinity by modifying dissociation rates (e.g. Y1542), a novel mutation (E1544A) of the AB loop enhanced affinity by threefold compared to wild-type. Conversion from an acidic to a basic residue at this site (E1544K) results in a sixfold enhancement of affinity via modification principally of the association rate, with enhanced salt-dependence, decreased entropic barrier and retained specificity. These data suggest that a functional hydrophobic binding site core is formed by I1572 and F1567 located in the CD loop, which initially anchors IGF-II. Within the AB loop, residues normally act to either stabilise or function as negative regulators of the interaction. These findings have implications for the molecular architecture and evolution of the domain 11 IGF-II-binding site, and the potential interactions with other domains of IGF2R.

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.

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.

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.

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.