Positive Allosteric Modulation of A2AR Alters Immune Cell Responses and Ameliorates Psoriasis-Like Dermatitis in Mice.

Psoriasis is an immune cell‒mediated inflammatory disease of the skin with a mixed T helper type 1/T helper type17 cytokine environment combined with an innate immune response engaging toll-like receptors. Inflammatory diseases are characterized by dysregulated immune cell responses and elevated levels of adenosine at disease sites. Adenosine, acting through the A2AR, regulates inflammation, immune response, T-cell homeostasis, and tissue repair. We have identified a unique means to enhance A2AR function using a positive allosteric modulator. We show that oral administration of the A2AR-positive allosteric modulator AEA061 reduced ear swelling, skin thickness, erythema, scale formation, and inflammatory cytokine expression in A2Ar+/+ but not in A2Ar-/- mice with imiquimod-induced psoriasis-like dermatitis. Similar clinical and mRNA improvements were observed with topical administration. AEA061 also reduced clinical scores and cytokine expression in a mouse model of IL-23‒induced psoriasis-like dermatitis. In addition, AEA061 attenuated imiquimod-induced expression of IFN-α in plasmacytoid dendritic cells in vivo and IL-23 and IL-36α in conventional dendritic cells. TCR-mediated IL-17 expression in γδT cells in vivo and IL-17 production by CD4+ T cells enriched for γδT cells in vitro were also inhibited. Thus, the enhancement of A2AR responsiveness to the endogenous agonist adenosine through positive allosteric modulation is sufficient to enhance intrinsic homeostatic mechanisms attenuating disease progression.

Positive effects of ferric iron on the systemic efficacy of nephrilin peptide in burn trauma.

INTRODUCTION: Nephrilin peptide is a designed inhibitor of Rictor complex (also known as mTORC2), an evolutionarily conserved assembly believed to modulate responses to cellular stress. We previously demonstrated the ability of nephrilin peptide to suppress neuroinflammation, loss of body mass, glycaemic control and kidney function in a rat scald model, as well as sepsis mortality in a mouse model. The present study explores the effect of nephrilin plus iron formulations on clinically relevant outcomes in the rat scald model. METHODS: Animals were treated with nephrilin by subcutaneous bolus injection on post-burn days 1-7. Equimolar ferric iron in the formulation improved the positive systemic effects of nephrilin on kidney function, glycaemic control, oxidative stress, early hyperinflammation, late inflammasome activation, hyperangiogenesis and body mass, all variables previously shown to bear upon clinically relevant burn injury outcomes. The sparing effects of nephrilin-iron were demonstrated in both sexes. DISCUSSION: Surprisingly, optimum daily treatment doses were in the range of 2-4 mg/kg, while 8 mg/kg was less effective, suggesting the possibility of marginal pro-oxidant effects from the 'free' iron fraction. Thus, although ferric iron in the nephrilin formulation is clearly helpful, care must be exercised to select an optimum treatment dose. CONCLUSION: Iron increases the efficacy of nephrilin peptide in burns.

Sox10+ Cells Contribute to Vascular Development in Multiple Organs-Brief Report.

OBJECTIVE: Previous genetic lineage tracing studies showed that Sox10+ cells differentiate into vascular mural cells, limited to neural crest-derived blood vessels in craniofacial tissues, aortic arch, pulmonary arch arteries, brachiocephalic, carotid arteries, and thymus. The purpose of this study was to investigate the contribution of Sox10+ cells to the vascular development in other tissues and organs and their relationship with neural crest. APPROACH AND RESULTS: Using genetic lineage tracing technique based on Cre/LoxP system, we examined blood vessels in the adult organs of the mice expressing Sox10-Cre/Rosa-LoxP-red fluorescent protein or Wnt1-Cre/Rosa-LoxP-red fluorescent protein by immunohistological analysis. In addition to previously reported tissues and organs derived from neural crest, we showed that Sox10+ cells also contributed to vascular mural cells in the lung, spleen, and kidney, which are derived from non-neural crest origin as evidenced by red fluorescent protein-negative blood vessels in these 3 organs of Wnt1-Cre/Rosa-LoxP-red fluorescent protein mice. CONCLUSIONS: This study demonstrates that Sox10+ cells contribute to pericytes and smooth muscle cells in most parts of the body, including those from neural crest and non-neural crest, which has significant implications in vascular remodeling under physiological and pathological conditions.

Angiopoietin-2 Blockade Promotes Survival of Corneal Transplants.

Purpose: Corneal transplantation remains the last hope for vision restoration, and lymphangiogenesis (LG) is a primary mediator of transplant rejection. This study was to investigate the specific role of angiopoietin-2 (Ang-2) in transplantation-associated LG and graft rejection. Methods: Orthotopic corneal transplantation was performed between fully mismatched C57BL/6 (donor) and BALB/c (recipient) mice to assess the effects of Ang-2 blockade via neutralizing antibody. Grafts were evaluated in vivo by ophthalmic slit-lamp biomicroscopy and anterior segment optical coherence tomography (OCT) up to 8 weeks after surgery. Additionally, whole-mount corneas were analyzed for lymphatic and blood vessels and macrophages by immunofluorescent microscopy, and draining lymph nodes were assessed for donor-derived cells by flow cytometry. Results: Anti-Ang-2 treatment significantly suppressed LG and graft rejection. In this study, we achieved 75% suppression of LG and 80% graft survival. Our approach also inhibited donor-derived cell trafficking to draining lymph nodes and affected macrophage morphologic phenotypes in the grafted corneas. Additionally, Ang-2 blockade also reduced central corneal thickening, a parameter strongly associated with graft rejection. Conclusions: Ang-2 is critically involved in corneal transplant rejection and anti-Ang-2 treatment significantly improves the outcomes of corneal grafts. Moreover, we have shown that anterior segment OCT offers a new tool to monitor murine corneal grafts in vivo. This study not only reveals new mechanisms for transplant rejection, but also offers a novel strategy to treat it.

Leukotriene B4 induces EMT and vimentin expression in PANC-1 pancreatic cancer cells: Involvement of BLT2 via ERK2 activation.

Leukotriene B4 (LTB4) is a leukocyte chemoattractant and plays a major role controlling inflammatory responses including pancreatitis. LTB4 is known to be correlated with cancer progression. LTB4 induces keratin phosphorylation and reorganization by activating extracellular regulated kinase (ERK) in PANC-1 pancreatic cancer cell lines. However, the role of LTB4 in epithelial mesenchymal transition (EMT) and vimentin expression in pancreatic cancer cells is unknown. We examined whether LTB4 induces EMT and vimentin expression by Western blot, si-RNA, and RT-PCR. LTB4 induced morphological change, decreased E-cadherin expression and increased N-cadherin and vimentin expression. LTB4 increased migration and invasion of PANC-1 cancer cells. LTB4 dose-dependently upregulated expression of vimentin in PANC-1 cancer cells. LTB4-induced vimentin expression was suppressed by LY255283 (BLT2 antagonist). Comp A, a BLT2 agonist, further increased vimentin expression. Gene silencing of BLT2 suppressed LTB4-or Comp A-induced vimentin expression in PANC-1 cells. The MEK inhibitor, PD98059 suppressed Comp A-induced vimentin expression. Comp A or transfection of plasmid containing BLT2 cDNA (pCBLT2) activated ERK, and BLT2 gene silencing suppressed Comp A-induced ERK activation. ERK2 siRNA abrogated Comp A-induced vimentin expression and ERK2 overexpression enhanced vimentin expression. One of well-known cause of ras mutation, cigarette smoke extracts increased BLT2 expression in PANC-1 cancer cells. Taken together, these results suggest that BLT2 is involved in LTB4-induced vimentin expression through ERK2 in PANC-1 cells.

Integrin Alpha 9 Blockade Suppresses Lymphatic Valve Formation and Promotes Transplant Survival.

PURPOSE: The lymphatic pathway mediates transplant rejection. We recently reported that lymphatic vessels develop luminal valves in the cornea during lymphangiogenesis, and these valves express integrin alpha 9 (Itga-9) and play a critical role in directing lymph flow. In this study, we used an allogeneic corneal transplantation model to investigate whether Itga-9 blockade could suppress valvulogenesis after transplantation, and how this effect would influence the outcomes of the transplants. METHODS: Orthotopic corneal transplantation was performed between fully mismatched C57BL/6 (donor) and BALB/c (recipient) mice. The recipients were randomized to receive subconjunctival injections of either Itga-9 blocking antibody or isotype control twice a week for 8 weeks. Corneal grafts were assessed in vivo by ophthalmic slit-lamp biomicroscopy and analyzed using Kaplan-Meier survival curves. Additionally, whole-mount full-thickness corneas were evaluated ex vivo by immunofluorescent microscopy on both lymphatic vessels and valves. RESULTS: Anti-Itga-9 treatment suppressed lymphatic valvulogenesis after transplantation. Our treatment did not affect lymphatic vessel formation or their nasal polarized distribution in the cornea. More importantly, Itga-9 blockade led to a significant promotion of graft survival. CONCLUSIONS: Lymphatic valvulogenesis is critically involved in transplant rejection. Itga-9 targeting may offer a new and effective strategy to interfere with the immune responses and promote graft survival.

Intravital Imaging Reveals Dynamics of Lymphangiogenesis and Valvulogenesis.

Lymphatic research signifies a field of rapid progression in recent years. Though lymphatic dysfunction has been found in a myriad of disorders, to date, few effective treatments are available for lymphatic diseases. It is therefore urgent to develop new experimental approaches and therapeutic protocols. The cornea offers an ideal site for lymphatic research due to its transparent nature, accessible location, and lymphatic-free but -inducible features. Moreover, we have recently discovered that corneal lymphatic vessels develop luminal valves as lymphangiogenesis proceeds. This tissue thus provides an optimal tool to study both lymphangiogenesis and valvulogenesis upon a pathological insult. In this paper, we show that the modified Prox-1-GFP mice carrying wildtype C57BL/6 background provide a valuable tool for intravital imaging of corneal lymphatic vessels and valves and can be used to study pathological lymphangiogenesis induced by various insults. Further, we demonstrate the multifaceted dynamics of lymphangiogenesis and valvulogenesis associated with transplantation, from the initiation to regression phases, and report several novel and critical phenomena and mechanisms that cannot be detected by conventional ex vivo approaches. Further investigation holds the great potential for divulging new mechanisms and therapeutic strategies for lymphangiogenesis and lymphangiogenesis-related diseases at various stages and inside or outside the eye.

Phenotype-based high-content chemical library screening identifies statins as inhibitors of in vivo lymphangiogenesis.

Lymphangiogenesis plays an important role in promoting cancer metastasis to sentinel lymph nodes and beyond and also promotes organ transplant rejection. We used human lymphatic endothelial cells to establish a reliable three-dimensional lymphangiogenic sprouting assay with automated image acquisition and analysis for inhibitor screening. This high-content phenotype-based assay quantifies sprouts by automated fluorescence microscopy and newly developed analysis software. We identified signaling pathways involved in lymphangiogenic sprouting by screening the Library of Pharmacologically Active Compounds (LOPAC)(1280) collection of pharmacologically relevant compounds. Hit characterization revealed that mitogen-activated protein kinase kinase (MEK) 1/2 inhibitors substantially block lymphangiogenesis in vitro and in vivo. Importantly, the drug class of statins, for the first time, emerged as potent inhibitors of lymphangiogenic sprouting in vitro and of corneal and cutaneous lymphangiogenesis in vivo. This effect was mediated by inhibition of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and subsequently the isoprenylation of Rac1. Supplementation with the enzymatic products of HMG-CoA reductase functionally rescued lymphangiogenic sprouting and the recruitment of Rac1 to the plasma membrane.