ABSTRACT
Immunological disorders are one of the main causes of recurrent spontaneous abortions (RSA). A rapidly expanding body of evidence indicates that excessive activation of the complement system is critically involved in the development of miscarriages. In the CBA/J × DBA/2 murine model of recurrent miscarriage, exaggerated and unrestrained complement activation is reported to be the underlying cause of angiogenic factor imbalance and persistent inflammation. We have previously shown that mesenchymal stem cell (MSC) therapy can significantly reduce the abortion rate in abortion-prone mice through regulating the feto-maternal immune response. In the present study, we hypothesized that MSCs might improve the balance of angiogenic factors at the feto-maternal unit of CBA/J × DBA/2 mice by restraining complement activation and deposition. To explore this hypothesis, autologous adipose tissue-derived mesenchymal stem cells (AD-MSCs) were administered intra-peritoneally to abortion-prone mice on the 4.5th day of gestation. Control mice received PBS as vehicle. On day 13.5 of pregnancy, deposition of the complement component C3 and expression levels of Crry, CFD (adipsin), VEGF, PlGF and FLT-1 were measured at the feto-maternal interface by immunohistochemistry and real-time PCR, respectively. Decidual cells were also cultured in RPMI 1640 medium for 48 h and VEGF and sFLT-1 protein levels were quantified in supernatants using enzyme-linked immunosorbent assay (ELISA). Our results indicated that MSC therapy significantly reduced C3 deposition and adipsin transcription in the fetal-maternal interface of abortion-prone mice. Furthermore, administration of MSCs robustly upregulated the mRNA expression levels of Crry, VEGF, PlGF and FLT-1 in the placenta and decidua of CBA/J × DBA/2 mice. Consistently, the in vitro results demonstrated that decidual cells obtained from MSC-treated dams produced increased concentrations of VEGF in culture supernatants, with concomitant decreased levels of sFLT-1 protein. Here, we show for the first time that adoptive transfer of MSCs rectifies the disturbed balance of angiogenic factors observed at the feto-maternal unit of CBA/J × DBA/2 mice, in part at least, through inhibiting excessive complement activation and promoting the production of angiogenic factors. Collectively, these alterations seem to play a pivotal role in reducing the abortion rate and improving the intrauterine condition for the benefit of the fetus.
Subject(s)
Abortion, Habitual/immunology , Angiogenic Proteins/immunology , Complement C3/immunology , Mesenchymal Stem Cells/immunology , Abortion, Induced/methods , Animals , Cytokines/immunology , Disease Models, Animal , Female , Male , Mesenchymal Stem Cell Transplantation/methods , Mice , Mice, Inbred BALB C , Mice, Inbred CBA , Mice, Inbred DBA , Placenta/immunology , PregnancyABSTRACT
The Myo/Nog cell lineage was discovered in the chick embryo and is also present in adult mammalian tissues. The cells are named for their expression of mRNA for the skeletal muscle specific transcription factor MyoD and bone morphogenetic protein inhibitor Noggin. A third marker for Myo/Nog cells is the cell surface molecule recognized by the G8 monoclonal antibody (mAb). G8 has been used to detect, track, isolate and kill Myo/Nog cells. In this study, we screened a membrane proteome array for the target of the G8 mAb. The array consisted of >5,000 molecules, each synthesized in their native confirmation with appropriate post-translational modifications in a single clone of HEK-293T cells. G8 mAb binding to the clone expressing brain-specific angiogenesis inhibitor 1 (BAI1) was detected by flow cytometry, re-verified by sequencing and validated by transfection with the plasmid construct for BAI1. Further validation of the G8 target was provided by enzyme-linked immunosorbent assay. The G8 epitope was identified by screening a high-throughput, site directed mutagenesis library designed to cover 95-100% of the 954 amino acids of the extracellular domain of the BAI1 protein. The G8 mAb binds within the third thrombospondin repeat of the extracellular domain of human BAI1. Immunofluorescence localization experiments revealed that G8 and a commercially available BAI1 mAb co-localize to the subpopulation of Myo/Nog cells in the skin, eyes and brain. Expression of the multi-functional BAI1 protein in Myo/Nog cells introduces new possibilities for the roles of Myo/Nog cells in normal and diseased tissues.
Subject(s)
Angiogenic Proteins/biosynthesis , Myofibroblasts/metabolism , Receptors, G-Protein-Coupled/biosynthesis , Amino Acid Substitution , Angiogenic Proteins/chemistry , Angiogenic Proteins/genetics , Angiogenic Proteins/immunology , Animals , Antibodies, Monoclonal/immunology , Antibody Specificity , Antigen-Antibody Reactions , Brain/cytology , Carrier Proteins/analysis , Cell Lineage , Epitopes/immunology , Eye Proteins/biosynthesis , Eye Proteins/chemistry , Eye Proteins/genetics , Eye Proteins/immunology , Humans , Male , Mice , Mice, Inbred C57BL , Models, Molecular , Muscle Development , MyoD Protein/analysis , Organ Specificity , Protein Conformation , Protein Domains , Rabbits , Rats, Sprague-Dawley , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/immunology , Repetitive Sequences, Amino Acid , Skin/cytology , Species Specificity , Tattooing , Young AdultABSTRACT
BACKGROUND: Chimeric antigen receptors (CARs) presented on T cell surfaces enable redirection of T cell specificity, which has enormous promise in antitumor therapy. However, excessive activity and poor control over such engineered T cells cause significant safety challenges, such as cytokine release syndrome and organ toxicities. To enhance the specificity and controllable activity of CAR-T cells, we report a novel switchable dual-receptor CAR-engineered T (sdCAR-T) cell and a new switch molecule of FITC-HM-3 bifunctional molecule (FHBM) in this study. METHODS: We designed a fusion molecule comprising FITC and HM-3. HM-3, an antitumor peptide including an Arg-Gly-Asp sequence, can specifically target integrin αvß3 that is presented on some tumor cells. Moreover, to improve the specificity of CAR-T cells, we also generated the sdCAR-T cell line against cognate tumor cells expressing human mesothelin (MSLN) and integrin αvß3. Finally, the activity of sdCAR-T cell and FHBM is verified via in vitro and in vivo experiments. RESULTS: In the presence of FHBM, the designed sdCAR-T cells exerted high activity including activation and proliferation and had specific cytotoxicity in a time- and dose-dependent manner in vitro. Furthermore, using a combination of FHBM in nude mice, sdCAR-T cells significantly inhibited the growth of MSLN+ K562 cells and released lower levels of the cytokines (e.g., interleukin-2, interferon γ, interleukin-6, and tumor necrosis factor α) relative to conventional CAR-T cells, obtaining specific, controllable, and enhanced cytotoxicity. CONCLUSIONS: Our data indicate that FHBM can accurately control timing and dose of injected CAR-T cells, and sdCAR-T cells exert significant antitumor activity while releasing lower levels of cytokines for the cognate tumor cells expressing both MSLN and integrin αvß3. Therefore, combination therapies using sdCAR-T cells and the switch molecule FHBM have significant potential to treat malignancies.
Subject(s)
Angiogenic Proteins/immunology , Receptors, Chimeric Antigen/immunology , T-Lymphocytes/immunology , Animals , Humans , Mesothelin , MiceABSTRACT
There are two members of the vasohibin (VASH) family, VASH1 and VASH2. VASH1 is expressed mainly in endothelial cells to inhibit angiogenesis, whereas VASH2 is expressed mainly in cancer cells to stimulate tumor growth. The aim of the present study was to establish neutralizing monoclonal antibody (mAb) against human VASH2 and apply it as an anti-cancer treatment. We previously raised mAb against several synthetic peptides of hVASH1, and found that one of them exhibited neutralizing activity against hVASH1. Because of the similarity in the amino acid sequences between VASH1 and VASH2, we hypothesized that they shared the bioactive center. When we mutated four amino acids within the region, the mutant VASH2 lost its pro-angiogenic activity. Therefore, we raised mAb against a synthetic peptide overlapping the mutated amino acids of hVASH2, and isolated one clone (1760) that almost completely inhibited the stimulatory effect of hVASH2 on the migration of and tube formation by endothelial cells. When we used this clone 1760 antibody for cancer treatment, the peritoneal injection of it inhibited both tumor growth and angiogenesis in a mouse xenograft model of human cancer cells. In terms of anti-tumor activity, 25 mg/kg of clone 1760 was equivalent to 5 mg/kg of bevacizmab. From these results, we propose the targeting of human VASH2 with neutralizing mAb as a new strategy for cancer treatment.
Subject(s)
Angiogenesis Inhibitors/therapeutic use , Angiogenic Proteins/antagonists & inhibitors , Antibodies, Monoclonal/therapeutic use , Antibodies, Neutralizing/therapeutic use , Neovascularization, Pathologic/prevention & control , Ovarian Neoplasms/drug therapy , Angiogenic Proteins/immunology , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/immunology , Bevacizumab/therapeutic use , Cell Line, Tumor , Cell Movement , Cell Proliferation , Endothelial Cells/metabolism , Female , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Neovascularization, Pathologic/immunology , Ovarian Neoplasms/blood supply , Ovarian Neoplasms/pathology , Xenograft Model Antitumor AssaysABSTRACT
Milk fat globule epidermal growth factor-factor 8 (MFG-E8) is a peripheral glycoprotein that acts as a bridging molecule between the macrophage and apoptotic cells, thus executing a pivotal role in the scavenging of apoptotic cells from affected tissue. We have previously reported that apoptotic cell clearance activity or efferocytosis is compromised in diabetic wound macrophages. In this work, we test the hypothesis that MFG-E8 helps resolve inflammation, supports angiogenesis, and accelerates wound closure. MFG-E8(-/-) mice displayed impaired efferocytosis associated with exaggerated inflammatory response, poor angiogenesis, and wound closure. Wound macrophage-derived MFG-E8 was recognized as a critical driver of wound angiogenesis. Transplantation of MFG-E8(-/-) bone marrow to MFG-E8(+/+) mice resulted in impaired wound closure and compromised wound vascularization. In contrast, MFG-E8(-/-) mice that received wild-type bone marrow showed improved wound closure and improved wound vascularization. Hyperglycemia and exposure to advanced glycated end products inactivated MFG-E8, recognizing a key mechanism that complicates diabetic wound healing. Diabetic db/db mice suffered from impaired efferocytosis accompanied with persistent inflammation and slow wound closure. Topical recombinant MFG-E8 induced resolution of wound inflammation, improvements in angiogenesis, and acceleration of closure, upholding the potential of MFG-E8-directed therapeutics in diabetic wound care.
Subject(s)
Antigens, Surface/immunology , Antigens, Surface/metabolism , Diabetes Mellitus/physiopathology , Inflammation/drug therapy , Milk Proteins/immunology , Milk Proteins/metabolism , Wound Healing , Angiogenic Proteins/immunology , Angiogenic Proteins/isolation & purification , Angiogenic Proteins/metabolism , Animals , Antigens, Surface/genetics , Antigens, Surface/pharmacology , Apoptosis , Diabetes Mellitus/immunology , Humans , Macrophages/immunology , Mice , Mice, Inbred C57BL , Milk Proteins/genetics , Milk Proteins/pharmacology , PhagocytosisABSTRACT
Adhesion GPCRs (aGPCRs) form a subfamily of the large GPCR super family. Most aGPCRs are characterised by a non-covalent bipartite structure that consists of a large extracellular domain and a membrane-spanning 7 transmembrane domain. Typically, aGPCRs can combine cell adhesion by the large extracellular domain with intracellular signalling by the 7 transmembrane domain. Immune responses rely on cellular communication and subsequent defence reactions. Indeed, aGPCR ADGRB1 and members of the ADGRE class have been linked to processes like phagocytosis, leucocyte activation and migration. Nevertheless, research is hampered by absence of endogenous ligands, unknown activity of generated antibodies and non-identified signalling pathways. Yet, based on their membrane localisation and important function, aGPCRs could be novel drug targets to modulate leucocyte function.
Subject(s)
Cell Adhesion Molecules/immunology , Cell Adhesion/immunology , Receptors, G-Protein-Coupled/immunology , Angiogenic Proteins/immunology , Angiogenic Proteins/metabolism , Animals , Cell Adhesion Molecules/metabolism , Gram-Negative Bacteria/immunology , Humans , Leukocytes/cytology , Leukocytes/immunology , Phagocytosis/immunology , Receptors, G-Protein-Coupled/metabolism , Signal Transduction/immunologyABSTRACT
Angiogenesis is a complex and balanced process in which new blood vessels form from preexisting ones by sprouting, splitting, growth and remodeling. This phenomenon plays a vital role in many physiological and pathological processes. However, the disturbance in physiological process can play a role in pathogenesis of some chronic inflammatory diseases, including multiple sclerosis (MS) in human and its animal model. Although the relation between abnormal blood vessels and MS lesions was established in previous studies, but the role of pathological angiogenesis remains unclear. In this study, the link between proangiogenic factors and multiple sclerosis pathogenesis was examined by conducting a systemic review. Thus we searched the English medical literature via PubMed, ISI web of knowledge, Medline and virtual health library (VHL) databases. In this review, we describe direct and indirect roles of some proangiogenic factors in MS pathogenesis and report the association of these factors with pathological and inflammatory angiogenesis.
Subject(s)
Angiogenic Proteins/metabolism , Central Nervous System/metabolism , Inflammation Mediators/metabolism , Multiple Sclerosis/metabolism , Neovascularization, Pathologic , Angiogenic Proteins/immunology , Animals , Blood-Brain Barrier/immunology , Blood-Brain Barrier/metabolism , Blood-Brain Barrier/pathology , Central Nervous System/immunology , Central Nervous System/pathology , Humans , Inflammation Mediators/immunology , Multiple Sclerosis/immunology , Multiple Sclerosis/pathology , Signal TransductionSubject(s)
Blood Platelets/immunology , Cell-Derived Microparticles/immunology , Dendritic Cells/immunology , Liver X Receptors/immunology , ATP Binding Cassette Transporter 1/genetics , ATP Binding Cassette Transporter 1/immunology , Angiogenic Proteins/genetics , Angiogenic Proteins/immunology , Benzoates/pharmacology , Benzylamines/pharmacology , Blood Platelets/cytology , Blood Platelets/drug effects , Cell-Derived Microparticles/chemistry , Dendritic Cells/cytology , Dendritic Cells/drug effects , Endothelial Cells/cytology , Endothelial Cells/drug effects , Endothelial Cells/immunology , Gene Expression Regulation , Humans , Hydrocarbons, Fluorinated/pharmacology , Hydroxycholesterols/pharmacology , Imidazoles/pharmacology , Immunity, Innate , Liver X Receptors/agonists , Liver X Receptors/antagonists & inhibitors , Liver X Receptors/genetics , NF-kappa B/genetics , NF-kappa B/immunology , Oligodeoxyribonucleotides/genetics , Oligodeoxyribonucleotides/immunology , Phenylenediamines/pharmacology , Primary Cell Culture , Receptors, G-Protein-Coupled , Signal Transduction , Sterol Regulatory Element Binding Protein 1/genetics , Sterol Regulatory Element Binding Protein 1/immunology , Sulfonamides/pharmacology , Toll-Like Receptor 7/antagonists & inhibitors , Toll-Like Receptor 7/genetics , Toll-Like Receptor 7/immunology , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunologyABSTRACT
Angiogenic factor with G patch and FHA domains 1 (AGGF1) is a newly identified proangiogenic protein, which plays an important role in vascular disease and angiogenesis. However, its role in myocardial ischemia/reperfusion (I/R) injury remains unknown. This study investigated whether AGGF1 is involved in the pathogenesis of mouse myocardial I/R injury and the underlying mechanisms. Wild-type (WT) C57BL/6 J mice were treated at 30 min prior to I/R injury with anti-AGGF1 neutralizing antibody (3 mg/kg) or recombinant human AGGF1 (rhAGGF1, 0.25 mg/kg). After I/R injury, the infarct size, the number of TUNEL-positive cardiomyocytes, Bax/Bcl2 ratio, inflammatory cytokine expression and angiogenesis were markedly increased as compared with sham control. Treatment of WT mice with anti-AGGF1 neutralizing antibody resulted in exaggeration of myocardial I/R injury but reducing angiogenesis. In contrast, administration of rhAGGF1 markedly reversed these effects. Furthermore, anti-AGGF1- or rhAGGF1-mediated effects on I/R-induced cardiac apoptosis, inflammation and angiogenesis were dose dependent. In addition, the protective effects of AGGF1 on cardiomyocyte apoptosis and inflammation were confirmed in cultured cardiomyocytes after I/R. Finally, these effects were associated with activation of ERK1/2, Stat3 and HIF-1α/VEGF pathways and inhibition of activation of NF-κB, p53 and JNK1/2 pathways. In conclusion, we report the first in vivo and in vitro evidence that AGGF1 reduces myocardial apoptosis and inflammation and enhances angiogenesis, leading to decreased infarct size after I/R injury. These results may provide a novel therapeutic approach for ischemic heart diseases.
Subject(s)
Angiogenic Proteins/metabolism , Apoptosis , Myocardial Reperfusion Injury/metabolism , Myocardium/pathology , Neovascularization, Physiologic/drug effects , Angiogenic Proteins/immunology , Angiogenic Proteins/pharmacology , Animals , Antibodies, Neutralizing/pharmacology , Cells, Cultured , Humans , Inflammation/metabolism , Male , Mice, Inbred C57BL , Myocardial Infarction/metabolism , Myocardial Infarction/pathology , Myocardial Reperfusion Injury/drug therapy , Myocardial Reperfusion Injury/pathology , Myocardium/metabolism , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Rats, Sprague-Dawley , Recombinant Proteins/pharmacology , Signal Transduction/drug effectsABSTRACT
Vasohibin-2 was recently identified as an important pro-angiogenesis factor in solid tumor and intracellular localization of its variants is important for elucidating the downstream mechanism(s) of its effects. Currently there are no reported antibodies affordable for intracellular localization. The aim of this study was to generate and characterize polyclonal antibodies against Vasohibin-2 and to determine the intracellular localization of Vasohibin-2. In this study, two polypeptides were synthesized and one prokaryotic Vasohibin-2 recombinant protein was custom-made. New Zealand rabbits were immunized with the polypeptide mixture and prokaryotic recombinant protein, respectively. The purified antibodies from the antiserum were validated by ELISA, western blotting (WB), immunofluorescence (IF), immunohistochemistry (IHC) and immunoprecipitation (IP). In order to determine intracellular localization, the cytoplasmic and nuclear proteins of the human liver cancer cell line HepG2 were isolated for the detection of Vasohibin-2 by western blotting. Vasohibin-2 cDNA, coding for 311 and 355 amino acid residues, fused with or without a DDK/V5 tag at the c-terminus, respectively, was cloned into the Lv-CMV-EGFP vector. Lentiviruses were successfully packaged. Vasohibin-2-overexpressing HepG2-VASH2 (355 amino acid residues) and HepG2-VASH2-V5 (311 amino acid residues fused with V5 tag at the c-terminus) human liver cancer cell lines were established. Approximately 1-2x106 HepG2, HepG2-VASH2 and HepG2-VASH2-V5 cells were injected subcutaneously into the flanks of BALB/c nude mice. Xenograft tumors were harvested for immunohistochemistry. HepG2 cells were transiently transfected with the Lv-CMV-EGFP vectors containing Vasohibin-2 cDNA (coding for 311/355 amino acid residues with a DDK tag at the c-terminal), followed by anti-DDK immunofluorescence. The antibodies obtained were able to detect human VASH2 successfully as applied in western blotting, IF, IHC and IP. Results from IF, IHC and WB (post cytoplasmic/nuclear protein isolation) showed a quite different intracellular localization of VASH2 protein. The VASH2 (with 355 amino acid residues) was located in the cytoplasm while VASH2 (with 311 amino acid residues) was located in the nucleus. The former was found to be a relatively low abundance protein. We successfully generated three rabbit anti-human Vasohibin-2 polyclonal antibodies which can be used for western blotting, IF, IP and IHC. These antibodies will provide a convenient tool for further studies on Vasohibin-2. This is the first study to report differences in the intracellular localization of the VASH2 protein and, hence, a new research direction on the study of VASH2.
Subject(s)
Angiogenic Proteins/immunology , Antibodies/immunology , Hepatoblastoma/pathology , Liver Neoplasms/pathology , Recombinant Proteins/immunology , Angiogenic Proteins/genetics , Animals , Hep G2 Cells/ultrastructure , Hepatoblastoma/ultrastructure , Humans , Immunohistochemistry , Liver Neoplasms/ultrastructure , Mice , Mice, Nude , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/immunology , Rabbits , Recombinant Proteins/geneticsABSTRACT
BACKGROUND: We have previously reported that serum levels of 70 kDa heat shock protein (Hsp70, HSPA1A) are increased and reflect systemic inflammation, oxidative stress and hepatocellular injury in preeclampsia. The purpose of this study was to determine whether increased serum Hsp70 concentrations in women with preeclampsia are related to circulating levels of cytokines, chemokines, adhesion molecules and angiogenic factors, the key players in the pathogenesis of the disease. METHODS: Sixty preeclamptic patients and 60 normotensive, healthy pregnant women were involved in this case-control study. Levels of Hsp70 (HSPA1A) and transforming growth factor (TGF)-beta1 in maternal sera were assessed by ELISA. Serum levels of interleukin (IL)-1 beta, IL-1 receptor antagonist, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, IL-12p70, IL-18, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interferon-gamma-inducible protein (IP)-10, monocyte chemotactic protein (MCP)-1, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 were determined by multiplex suspension array. Serum total soluble fms-like tyrosine kinase-1 (sFlt-1) and biologically active placental growth factor (PlGF) levels were measured by electrochemiluminescence immunoassay. For statistical analyses, the Mann-Whitney U-test, the Fisher exact and Pearson chi-square tests, the Spearman rank order correlation, multiple linear regression and logistic regression were applied. RESULTS: Serum levels of Hsp70 were significantly higher in preeclamptic patients than in healthy pregnant women. Additionally, most of the measured inflammatory variables differed significantly between the two study groups except for serum IL-1 beta and TGF-beta1 levels and IL-18/IL-12p70 and IL-12p70/IL-12p40 ratios, indicating a bias toward a pro-inflammatory status in preeclampsia. Preeclamptic patients had significantly higher sFlt-1 levels and sFlt-1/PlGF ratio and significantly lower PlGF concentrations as compared to healthy pregnant women. In the preeclamptic group, serum Hsp70 concentrations showed significant correlations with serum levels of IL-12p40 (R=0.59, p<0.001), MCP-1 (R=0.43, p<0.001), ICAM-1 (R=0.39, p=0.0020) and VCAM-1 (R=0.46, p<0.001). Furthermore, elevated serum Hsp70 level and sFlt-1/PlGF ratio had a synergistic (joint) effect in the risk of preeclampsia, as shown by the substantially higher odds ratios of their combination than of either alone. CONCLUSIONS: Increased serum Hsp70 concentrations in women with preeclampsia were associated with pro-inflammatory changes in circulating cytokine profile, suggesting that circulating Hsp70 might contribute to the development of the excessive systemic inflammatory response characteristic of the maternal syndrome of the disease.
Subject(s)
Angiogenic Proteins/blood , Cell Adhesion Molecules/blood , Cytokines/blood , HSP70 Heat-Shock Proteins/blood , Pre-Eclampsia/blood , Adult , Angiogenic Proteins/immunology , Biomarkers/blood , Case-Control Studies , Cell Adhesion Molecules/immunology , Cytokines/immunology , Female , HSP70 Heat-Shock Proteins/immunology , Humans , Pre-Eclampsia/immunology , PregnancyABSTRACT
The chronic inflammatory environment of tumors is a target for novel antitumor therapeutic strategies. Besides cholesterol lowering effects, statins have been studied for their anti-inflammatory and immunomodulatory properties. These pleiotropic effects result mainly from the altered post-translational modification of GTP-binding proteins which regulate many intracellular pathways involved in cell growth and survival. Although pre-clinical studies suggest that statins may be effective anticancer agents required doses that are 100 to 500 fold higher than those needed to lower cholesterol levels. Furthermore, in view of their wide-ranging effects on cellular metabolism, target site-specific delivery is preferred. In this study, we investigated tumor-specific delivery of pravastatin using small long-circulating liposomes. In vitro studies on the effects of (liposomal) pravastatin on viability and proliferation of tumor cells, endothelial cells and macrophages revealed that the latter were the most sensitive cell type towards (liposomal) pravastatin treatment. In vivo, liposome-encapsulated pravastatin (5mg/kg) inhibited murine B16F10-melanoma growth over 70% as compared to free pravastatin, which was ineffective. As expected, treatments did not influence serum cholesterol levels within the time frame of the study. At 48 h post-injection, 3 µg of pravastatin could still be recovered from the tumors of liposomal pravastatin treated mice, whereas pravastatin could not be detected in tumors of the free drug treated mice (i.e. < 20 ng). In contrast to the free drug, liposomal pravastatin treatment effectively inhibited the production of several pro-inflammatory/pro-angiogenic mediators involved in inflammation and angiogenesis, out of a range of a panel of 24 proteins studied. Furthermore, liposomal pravastatin treatment increased MHC class I protein expression in the tumor tissue whereas free drug showed no effect. Taken together, targeted delivery of statins can improve their tumor growth inhibiting activity by increasing local drug concentration and direct modulation of macrophage function. The antitumor activity seems to result primarily from a local inhibition of tumor inflammation and stimulation of antitumor immune response.
Subject(s)
Anti-Inflammatory Agents/administration & dosage , Anti-Inflammatory Agents/therapeutic use , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/therapeutic use , Inflammation/drug therapy , Neoplasms/drug therapy , Pravastatin/administration & dosage , Pravastatin/therapeutic use , Angiogenic Proteins/immunology , Animals , Anti-Inflammatory Agents/pharmacokinetics , Anti-Inflammatory Agents/pharmacology , Anticholesteremic Agents/administration & dosage , Anticholesteremic Agents/pharmacokinetics , Anticholesteremic Agents/pharmacology , Anticholesteremic Agents/therapeutic use , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cytokines/immunology , Genes, MHC Class I/drug effects , Humans , Inflammation/etiology , Lipid Metabolism/drug effects , Liposomes , Male , Mice , Mice, Inbred C57BL , Neoplasms/complications , Neoplasms/genetics , Neoplasms/immunology , Pravastatin/pharmacokinetics , Pravastatin/pharmacologyABSTRACT
Adhesion-GPCRs are unusual, owing to their unique structure, comprising a large and complex extracellular domain composed of various common protein modules. Adhesion-GPCR family members are expressed ubiquitously; however the expression of each receptor is highly regulated and often restricted to specific cell types. The EGF-TM7 adhesion-GPCR subfamily members are predominantly expressed by leukocytes and involved in coordinating both the innate and acquired immune responses. Here we highlight some immunological insights in relation to EGF-TM7 proteins and other members of the adhesion-GPCR family.
Subject(s)
Immune System/immunology , Receptors, G-Protein-Coupled/immunology , Angiogenic Proteins/chemistry , Angiogenic Proteins/immunology , Animals , Humans , Ligands , Receptors, G-Protein-Coupled/chemistryABSTRACT
Extracellular pH (pH(e)) is lower in many tumors than in the corresponding normal tissue. The significance of acidic pH(e) in the development of metastatic disease was investigated in the present work. Human melanoma cells (A-07, D-12, and T-22) were cultured in vitro at pH(e) 6.8 or 7.4 (control) before being inoculated into the tail vein of BALB/c nu/nu mice for formation of experimental pulmonary metastases. Cell invasiveness was studied in vitro by using Matrigel invasion chambers and angiogenesis was studied in vivo by using an intradermal assay. Protein secretion was measured by ELISA and immunocapture assays. Cells cultured at acidic pH(e) showed increased secretion of proteinases and proangiogenic factors, enhanced invasive and angiogenic potential, and enhanced potential to develop experimental metastases. Acidity-induced metastasis was inhibited by treatment with the general matrix metalloproteinase (MMP) inhibitor GM6001, the general cysteine proteinase inhibitor E-64, or blocking antibody against vascular endothelial growth factor-A (VEGF-A) or interleukin-8 (IL-8). Our study indicates that acidic pH(e) promotes experimental pulmonary metastasis in A-07, D-12, and T-22 human melanoma cells by a common mechanism involving acidity-induced up-regulation of the proteolytic enzymes MMP-2, MMP-9, cathepsin B, and cathepsin L and acidity-induced up-regulation of the proangiogenic factors VEGF-A and IL-8. One consequence of this observation is that treatment strategies involving deliberate tumor acidification to improve the efficacy of chemotherapy, photodynamic therapy, and hyperthermia should be avoided. Moreover, the possibility that the pH(e) of the primary tumor may be an important prognostic parameter for melanoma patients merits clinical investigation.
Subject(s)
Lung Neoplasms/metabolism , Lung Neoplasms/secondary , Melanoma/metabolism , Melanoma/secondary , Angiogenic Proteins/antagonists & inhibitors , Angiogenic Proteins/immunology , Animals , Antibodies/pharmacology , Extracellular Fluid/metabolism , Female , Humans , Hydrogen-Ion Concentration , Lung Neoplasms/blood supply , Melanoma/blood supply , Melanoma/pathology , Mice , Mice, Inbred BALB C , Mice, Nude , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Peptide Hydrolases/immunology , Peptide Hydrolases/metabolism , Protease Inhibitors/pharmacology , Transplantation, HeterologousABSTRACT
In vivo phage display has been used extensively to screen for novel targets of tumor therapy. Phage display peptide libraries can express random peptides or protein fragments and the aim of phage display is to identify peptide molecules that bind stably to a given target. Angiogenesis is essential to tumor development. Both blood and lymphatic vessels of tumors are different from those of normal tissues. Phage display has been used to analyze the structure and molecular diversity of tumor vasculature and to select tumor-specific antigens which have revealed stage- and type-specific markers of tumor blood vessels. Furthermore, peptides identified by in vivo phage display also work as vehicles to transport cargo therapeutic reagents to tumors. These peptides and their corresponding cellular proteins and ligands may provide molecular tools to selectively target the addresses of tumors and their pathological blood vessels and might increase the efficacy of therapy while decreasing side effects.
Subject(s)
Angiogenic Proteins/metabolism , Antigens, Neoplasm/metabolism , Neoplasms/metabolism , Peptide Library , Amino Acid Sequence , Angiogenic Proteins/immunology , Animals , Endothelium, Vascular/metabolism , Humans , Molecular Sequence Data , Neoplasms/blood supply , Neoplasms/immunology , Neovascularization, Pathologic/immunology , Neovascularization, Pathologic/metabolismABSTRACT
A characteristic feature of chronic inflammatory reactions is their persistence and predilection for certain sites. The molecular basis for such tissue tropism (as, for example, seen with metastatic spread) has until recently remained obscure, but recent studies have strongly implicated tissue-resident, stromal cells, such as macrophages, endothelial cells and fibroblasts. These cell types make attractive therapeutic targets as they help define the three-dimensional structure of tissues and are key orchestrators of the inflammatory infiltrate. Most current anti-inflammatory therapies target immune cells in an attempt to inhibit the production of pro-inflammatory mediators; however, an equally important target is the active induction of anti-inflammatory mediators involved in the resolution of inflammation. Recent work suggests that stromal cells are an important source of these mediators. Targeting of multiple signals may be required to inhibit tissue damage associated with inflammatory disease. Cells of the monocyte lineage are present as tissue-resident cells and interact closely with other stromal populations. These cells form an ideal target for modulation of the inflammatory environment as, in some cases, they appear to induce tissue repair. Therapeutic manipulation of the stromal microenvironment has been particularly effective in treating cancer and is likely to provide a novel method to achieve improved control of chronic inflammatory disease.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Inflammation/therapy , Stromal Cells/drug effects , Angiogenic Proteins/immunology , Angiogenic Proteins/metabolism , Animals , Anti-Inflammatory Agents/therapeutic use , Antibodies, Monoclonal/therapeutic use , Chronic Disease , Cytokines/immunology , Cytokines/metabolism , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Endothelial Cells/pathology , Fibroblasts/drug effects , Fibroblasts/metabolism , Fibroblasts/pathology , Humans , Inflammation/metabolism , Inflammation/pathology , Monocytes/drug effects , Monocytes/metabolism , Monocytes/pathology , Neoplasms/drug therapy , Neoplasms/metabolism , Neoplasms/pathology , Stromal Cells/metabolism , Stromal Cells/pathology , Transforming Growth Factor beta/immunologyABSTRACT
OBJECTIVE: The release of four diffusible angiogenic growth factors in human dental pulp following orthodontic force was investigated by using neutralising growth factor antibodies (NAs), individually and in four different combinations to block their effects. This study investigated if increasing the number of NAs (anti h vascular endothelial growth factor (VEGF), anti h fibroblast growth factor (FGF2), anti h platelet derived growth factor (PDGF) and anti Transforming growth factor beta (TGFbeta)) in combination resulted in a progressive reduction of the angiogenic response of the pulp. MATERIALS AND METHODS: The dental pulps from two groups of 40 premolar teeth, four teeth from each of 20 patients treated with fixed appliances for 2 weeks, were divided vertically, and sections from each half pulp co-cultured with sections of rat aorta in collagen. In one group, one of each of the four NAs, and in the other group, one of the four different NA combinations were added to the media of the co-cultures from one half of the pulp from each of the four teeth of each patient; the other half pulp co-cultures were controls. Cultures were examined daily by light microscopy for growth and number of microvessels. RESULTS: NAs significantly reduced microvessel numbers in the co-cultures when added individually (P<0.004), and in each of the four combinations (P<0.002), with a trend to progressively reduced microvessel numbers with increasing number of NAs in combination. CONCLUSIONS: Results indicated that all four angiogenic growth factors examined were released following orthodontic force application and play a role in the angiogenic response of the pulp, and that these factors may be more effective in combination.
Subject(s)
Angiogenic Proteins/metabolism , Dental Pulp/metabolism , Angiogenic Proteins/immunology , Animals , Antibodies, Monoclonal , Coculture Techniques , Dental Pulp/blood supply , Fibroblast Growth Factor 2/immunology , Fibroblast Growth Factor 2/metabolism , Humans , Neovascularization, Physiologic , Orthodontics , Platelet-Derived Growth Factor/immunology , Platelet-Derived Growth Factor/metabolism , Rats , Rats, Wistar , Stress, Mechanical , Transforming Growth Factor beta/immunology , Transforming Growth Factor beta/metabolism , Vascular Endothelial Growth Factor A/immunology , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Tumor growth and metastasis are angiogenesis-dependent. Anti-angiogenic therapy may be a useful approach to cancer therapy. This review discussed tumor angiogenesis and immunotherapy of targeting tumor angiogenesis from two main aspects: (1) active vaccination to induce effective anti-angiogenesis immunity; (2) passive immunotherapy with anti-pro-angiogenic molecules relevant antibody. Evidence from the recent years suggested that anti-angiogenic therapy should be one of the most promising approaches to cancer therapy.