Immunomodulatory glc/man‐directed Dolichos lablab lectin (DLL) evokes anti‐tumour response in vivo by counteracting angiogenic gene expressions

Neovascularization and jeopardized immunity has been critically emphasized for the establishment of malignant progression. Lectins are the diverse class of carbohydrate interacting proteins, having great potential as immunopotentiating and anti‐cancer agents. The present investigation sought to demonstrate the anti‐proliferative activity of Dolichos lablab lectin (DLL) encompassing immunomodulatory attributes. DLL specific to glucose and mannose carbohydrate moieties has been purified to homogeneity from the common dietary legume D. lablab. Results elucidated that DLL agglutinated blood cells non‐specifically and displayed striking mitogenicity to human and murine lymphocytes in vitro with interleukin (IL)‐2 production. The DLL‐conditioned medium exerted cytotoxicity towards malignant cells and neoangiogenesis in vitro. Similarly, in‐vivo anti‐tumour investigation of DLL elucidated the regressed proliferation of ascitic and solid tumour cells, which was paralleled with blockade of tumour neovasculature. DLL‐treated mice showed an up‐regulated immunoregulatory cytokine IL‐2 in contrast to severely declined levels in control mice. Mechanistic validation revealed that DLL has abrogated the microvessel formation by weakening the proangiogenic signals, specifically nuclear factor kappa B (NF‐κB), hypoxia inducible factor 1α (HIF‐1 α), matrix metalloproteinase (MMP)‐2 and 9 and vascular endothelial growth factor (VEGF) in malignant cells leading to tumour regression. In summary, it is evident that the dietary lectin DLL potentially dampens the malignant establishment by mitigating neoangiogenesis and immune shutdown. For the first time, to our knowledge, this study illustrates the critical role of DLL as an immunostimulatory and anti‐angiogenic molecule in cancer therapeutics.     

The angiogenic pathway "vascular endothelial growth factor/flk-1(KDR)-receptor" in rheumatoid arthritis and osteoarthritis

Active angiogenesis, together with an up-regulation of angiogenic factors, is evident in the synovium of both rheumatoid arthritis (RA) and osteoarthritis (OA). The present study assessed, by immunohistochemistry, the microvessel density in the synovium of these arthritides and in normal controls, in relation to the expression of the angiogenic factors vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF) and the apoptosis-related proteins bcl-2 and p53. More importantly, using the novel 11B5 MAb, the activated "VEGF/flk-1(KDR)-receptor" microvessel density was assessed. VEGF expression in fibroblasts was diffuse in both RA and OA. Diffuse PD-ECGF expression of fibroblasts was noted in all cases of RA, while fibroblast reactivity was focal in the OA material. The standard microvessel density (sMVD), as assessed with the anti-CD31 monoclonal antibody (MAb), was higher in RA (64+/-12) and in OA (65+/-16) than in normal tissues (52+/-8; p=0.008 and 0.0004, respectively). The activated microvessel density (aMVD), assessed with the 11B5 MAb, was significantly higher in RA (29+/-10) than in OA (17+/-4; p<0.0001) and than in normal tissues (14+/-2; p<0.0001). The "activation ratio" (aMVD/sMVD) was statistically higher in RA (0.46+/-0.17) than in OA and normal synovial tissues, the latter two having a similar ratio (0.28+/-0.08 and 0.26+/-0.03, respectively). Cytoplasmic bcl-2 expression was frequent in the synovial cells of OA, but rare in RA. Nuclear p53 protein accumulation was never observed. It is suggested that the angiogenic pathway VEGF/flk-1(KDR) may play an important role in the pathogenesis of RA and OA. Thus, failure of VEGF/flk-1(KDR) activation, in the presence of increased VEGF expression, may indicate a synovium with an impaired capacity to establish a viable vasculature, consistent with the degenerative nature of OA. On the other hand, the activated angiogenesis in RA shows a functional, still pathologically up-regulated VEGF/flk-1(KDR) pathway. Whether restoration of an impaired VEGF/flk-1(KDR) pathway in OA, or inhibition of this in RA, would prove of therapeutic importance requires further investigation.     

Vascular endothelial growth factor and its type 2 receptor in tumors and serum of patients with renal cancer

It was found that renal cancer tissue is characterized by increased content of VEGF not depending on the parameters of normal tissue and expression of VEGFR2 receptor. At the same time, the content of VEGFR2 in the tumor is determined by the state of the surrounding tissue and increases in low number of patients with tumors invading the renal capsule. Serum concentrations of VEGF and VEGFR2 in patients with renal cancer did not significantly differ from the corresponding values in the control group. No significant correlations between the levels of VEGF and VEGFR2 in the blood and tumor tissue were found. __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 145, No. 6, pp. 691–694, June, 2008     

Regulation of embryonic lung vascular development by vascular endothelial growth factor receptors,Flk‐1 and Flt‐1

The biological effects of vascular endothelial growth factor A (VEGF‐A) are mediated by fetal liver kinase‐1 (Flk‐1) and fms‐like tyrosine kinase‐1 (Flt‐1). In lung tissue, VEGF‐A is diffusely expressed throughout the embryonic stages, whereas the development of vascular endothelial cells is not uniform. Noting the signaling properties of the two receptors, we hypothesized that Flk‐1 and Flt‐1 regulate the embryonic development of lung vasculature. We herein show the spatiotemporal expression and experimental inhibition of Flk‐1 and Flt‐1 of embryonic mouse lung tissue. When Flk‐1 was predominantly expressed (embryonic day [E] 9.5–E13.5), then vascular endothelial cells actively proliferated. When Flt‐1 was enhanced (E14.5–E16.5), these cells less actively proliferated, thereby constituting organized networks. The treatment of cultured lung buds (E11.5) with antisense oligonucleotides complementary to Flk‐1 inhibited branching of capillaries and proliferation of endothelial cells. In contrast, the inhibition of Flt‐1 promoted the branching of capillaries and enhanced proliferation of endothelial cells. Of interest, inhibition of Flt‐1 promoted Flk‐1 expression. These results suggest that the two VEGF‐A receptors regulate pulmonary vascular development by modulating the VEGF‐A signaling. Anat Rec, 290:958–973, 2007. © 2007 Wiley‐Liss, Inc.     

ANGPTL4–αvβ3 interaction counteracts hypoxia‐induced vascular permeability by modulating Src signalling downstream of vascular endothelial growth factor receptor 2

Dynamic control of endothelial cell junctions is essential for vascular homeostasis and angiogenesis. We recently provided genetic evidence that ANGPTL4 is a key regulator of vascular integrity both during developmental and in hypoxia‐induced pathological conditions. The purpose of the present study was to decipher the molecular mechanisms through which ANGPTL4 regulates vascular integrity. Using surface plasmon resonance and proximity ligation assays, we show that ANGPTL4 binds integrin αvβ3. In vitro and in vivo functional assays with Angptl4‐deficient mice demonstrate that ANGPTL4–αvβ3 interaction is necessary to mediate ANGPTL4 vasoprotective effects. Mechanistically, ANGPTL4–αvβ3 interaction enhances Src recruitment to integrin αvβ3 and inhibits Src signalling downstream of vascular endothelial growth factor receptor 2 (VEFGR2), thereby repressing hypoxia‐induced breakdown of VEGFR2–VE‐cadherin and VEGFR2–αvβ3 complexes. We further demonstrate that intravitreal injection of recombinant human ANGPTL4 limits vascular permeability and leads to increased adherens junction and tight junction integrity. These findings identify a novel mechanism by which ANGPTL4 counteracts hypoxia‐driven vascular permeability through integrin αvβ3 binding, modulation of VEGFR2–Src kinase signalling, and endothelial junction stabilization. We further demonstrate that Angptl4‐deficient mice show increased vascular leakage in vivo in a model of laser‐induced choroidal neovascularization, indicating that this newly identified ANGPTL4–αvβ3 axis might be a target for pharmaceutical intervention in pathological conditions. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

B7‐1/2, but not PD‐L1/2 molecules,are required on IL‐10‐treated tolerogenic DC and DC‐derived exosomes for in vivo function

Costimulatory molecules, such as B7‐1/2 and PD‐L1/2 play an important role in the function of APC. The regulation of the surface levels of costimulatory molecules is one mechanism by which APC maintain the balance between tolerance and immunity. We examined the contributions of B7‐1/2 and PD‐L1/2 to the function of IL‐10‐treated, immunosuppressive DC as well as therapeutic exosomes derived from these DC. IL‐10 treatment of DC significantly downregulated surface expression of MHC II, B7‐1, B7‐2, and decreased levels of MHC I and PD‐L2. IL‐10 treatment of DC resulted in a modified costimulatory profile of DC‐secreted exosomes with a reduction in B7‐1, PD‐L1 and PD‐L2. We further demonstrate that absence of B7‐1 or B7‐2 on donor DC results in a loss of ability of IL‐10‐treated DC and their exosomes to suppress the delayed‐type hypersensitivity response, whereas IL‐10‐treated DC deficient in PD‐L1/2 as well as their secreted exosomes retained the ability to suppress delayed‐type hypersensitivity responses. We conclude that B7‐1 and B7‐2, but not PD‐L1 and PD‐L2, on IL‐10‐treated DC and DC‐derived exosomes play a critical role in immunosuppressive functions of both DC and exosomes.     

Programmed death (PD)‐1 molecule and its ligand PD‐L1 distribution among memory CD4 and CD8 T cell subsets in human immunodeficiency virus‐1‐infected individuals

Human immunodeficiency virus (HIV)‐1 causes T cell anergy and affects T cell maturation. Various mechanisms are responsible for impaired anti‐HIV‐1‐specific responses: programmed death (PD)‐1 molecule and its ligand PD‐L1 are negative regulators of T cell activity and their expression is increased during HIV‐1 infection. This study examines correlations between T cell maturation, expression of PD‐1 and PD‐L1, and the effects of their blockade. Peripheral blood mononuclear cells (PBMC) from 24 HIV‐1⁺ and 17 uninfected individuals were phenotyped for PD‐1 and PD‐L1 expression on CD4⁺ and CD8⁺ T cell subsets. The effect of PD‐1 and PD‐L1 blockade on proliferation and interferon (IFN)‐γ production was tested on eight HIV‐1⁺ patients. Naive (CCR7⁺CD45RA⁺) CD8⁺ T cells were reduced in HIV‐1 aviraemic (P = 0·0065) and viraemic patients (P = 0·0130); CD8 T effector memory subsets [CCR7^‐CD45RA^–(T_EM)] were increased in HIV‐1⁺ aviraemic (P = 0·0122) and viraemic (P = 0·0023) individuals versus controls. PD‐1 expression was increased in CD4 naive (P = 0·0496), central memory [CCR7⁺CD45RA^– (T_CM); P = 0·0116], T_EM (P = 0·0037) and CD8 naive T cells (P = 0·0133) of aviraemic HIV‐1⁺versus controls. PD‐L1 was increased in CD4 T_EMRA (CCR7^‐CD45RA⁺, P = 0·0119), CD8 T_EM (P = 0·0494) and CD8 T_EMRA (P = 0·0282) of aviraemic HIV‐1⁺versus controls. PD‐1 blockade increased HIV‐1‐specific proliferative responses in one of eight patients, whereas PD‐L1 blockade restored responses in four of eight patients, but did not increase IFN‐γ‐production. Alteration of T cell subsets, accompanied by increased PD‐1 and PD‐L1 expression in HIV‐1 infection contributes to anergy and impaired anti‐HIV‐1‐specific responses which are not rescued when PD‐1 is blocked, in contrast to when PD‐L1 is blocked, due possibly to an ability to bind to receptors other than PD‐1.     

The in vivo effects of tumour necrosis factor blockade on the early cell mediated immune events and syndrome expression in rat adjuvant arthritis

Anti-TNF therapy is effective in rheumatoid arthritis (RA); however, its mechanisms of action are incompletely understood. T cell-driven mechanisms are thought to play an important role in RA and the effects of TNF blockade on these mechanisms are unclear. Adjuvant arthritis (AA) is a T cell dependent model of inflammatory arthritis. The aims of this study were to investigate the effects of TNF blockade on in vivo T cell cytokine expression and to clarify the role of TNF in the inguinal lymph nodes (ILN) in early arthritis. AA was induced in male DA rats. Rats received either 3 mg/kg or 10 mg/kg PEG sTNF-RI at days 0, 2 and 4 postinduction or 10 mg/kg anti-TNF antibody on day of arthritis induction. Control rats received either saline or normal sheep serum. Paw volume was assessed every 3-4 days. Rats were sacrificed on days 0, 6, 13 and 21 postinduction. Ankles were removed for quantitative radiology and histology. Synovium and ILN were removed for cell culture and to determine mRNA expression of cytokines using semiquantitative RT-PCR. TNF and IFN-gamma protein production was measured using a bioassay and an ELISA. TNF blockade did not suppress mRNA expression of T cell cytokines in the ILN of rats in the early phase of AA, suggesting ongoing T cell activity. TNF protein production by ILN cells in culture was reduced in PEG sTNF-RI treated rats, although mRNA expression was increased in the ILN prior to culture. Early administration of PEG sTNF-RI did not attenuate AA, in contrast to an anti-TNF antibody, which suppressed disease. A shorter half-life for the PEG sTNF-RI compared with the anti-TNF antibody or the development of anti-PEG sTNF-RI antibodies may account for these results.     

The role of high‐mobility group box protein 1 in collagen antibody‐induced arthritis is dependent on vascular endothelial growth factor

High‐mobility group box 1 (HMGB1) has been implicated in angiogenesis and rheumatoid arthritis (RA). The aim of this study was to define more clearly the role of HMGB1 in the synovial angiogenesis and pathogenesis of an immune model of arthritis. BALB/c mice were injected with monoclonal anti‐collagen antibody cocktail followed by lipopolysaccharide to induce arthritis. HMGB1 and vascular endothelial growth factor (VEGF) were over‐expressed in the areas of the synovium where more inflammation and neoangiogenesis were present. The selective blockade of HMGB1 or VEGF resulted alternatively in a lower severity of arthritis evaluated by the arthritis index. Furthermore, exogenous HMGB1 administration caused a worsening of arthritis, associated with VEGF up‐regulation and increased synovial angiogenesis. The selective inhibition of VEGF also resulted in no induction of arthritis in mice receiving exogenous HMGB1. Cytokine enzyme‐linked immunosorbent assay (ELISA) analyses performed on peripheral blood and synovial fluid demonstrated a significant reduction of interleukin (IL)?1β, IL‐6 and tumour necrosis factor (TNF)‐α in mice where HMGB1 and VEGF pathways were blocked. Interestingly, the selective blockade of HMGB1 and VEGF resulted in an increase of the peripheral IL‐17A concentration. The development of arthritis mediated by HMGB1 and the synovial angiogenesis can be blocked by inhibiting the VEGF activity. The proinflammatory and proangiogenic cytokine IL‐17A was increased when HMGB1 is inhibited, but the synovial angiogenesis was nevertheless reduced in this model of arthritis. Taken together, these findings shed new light on the role of this nuclear protein in the pathogenesis of arthritis in an RA‐like model.     

Vascular endothelial growth factor (VEGF) receptor neuropilin-1's distribution in astrocytic tumors

Neuropilin-1 is a VEGF165- and semaphorin receptor expressed by endothelial cells and tumor cells. The specific function of neuropilin-1 is not fully known, but in the developing nervous system neuropilin, as a semaphorin receptor, has been shown to influence neuronal guidance. The expression of neuropilin-1 was studied in low-grade and high-grade astrocytic tumors, the latter characterized by extensive angiogenesis. We examined 20 low-grade astrocytomas (WHO grade II) and 46 glioblastomas (WHO grade IV) immunohistochemically for neuropilin-1, p53 and EGFR. The glioblastomas were according to the p53 and EGFR expression classified as 35 primary--de novo--glioblastomas, 9 secondary glioblastomas, and 2 uncertain cases. Furthermore, the presence of mast cells was evaluated to search for any potential function in angiogenesis. The glioblastomas expressed neuropilin-1 in the endothelial cells of the proliferating vessels and the majority of the glioblastomas had immunoreactive neoplastic astrocytes, with no difference between the glioblastoma subgroups. Six out of twenty of the low-grade astrocytomas were negative in the endothelial cells and 8 out of 20 in the tumor cells for neuropilin-1. Mast cells were observed in the collagen matrix around larger vessels in the leptomeninges, but not adjacent to malignant tumor vessels or as part of the tumor process itself. Increased expression of neuropilin-1 is shown in endothelial cells and in neoplastic astrocytes of glioblastomas. Less neuropilin-1 expression is found in about half of the low-grade astrocytomas in both neoplastic astrocytes and endothelial cells. The results suggest a correlation between neuropilin-1 and vascularity in human astrocytic tumors and a possible role for neuropilin-1 as a receptor for VEGF-induced angiogenesis.