ABSTRACT
OBJECTIVE: Intrahepatic cholangiocarcinoma (ICC)-a rare liver malignancy with limited therapeutic options-is characterised by aggressive progression, desmoplasia and vascular abnormalities. The aim of this study was to determine the role of placental growth factor (PlGF) in ICC progression. DESIGN: We evaluated the expression of PlGF in specimens from ICC patients and assessed the therapeutic effect of genetic or pharmacologic inhibition of PlGF in orthotopically grafted ICC mouse models. We evaluated the impact of PlGF stimulation or blockade in ICC cells and cancer-associated fibroblasts (CAFs) using in vitro 3-D coculture systems. RESULTS: PlGF levels were elevated in human ICC stromal cells and circulating blood plasma and were associated with disease progression. Single-cell RNA sequencing showed that the major impact of PlGF blockade in mice was enrichment of quiescent CAFs, characterised by high gene transcription levels related to the Akt pathway, glycolysis and hypoxia signalling. PlGF blockade suppressed Akt phosphorylation and myofibroblast activation in ICC-derived CAFs. PlGF blockade also reduced desmoplasia and tissue stiffness, which resulted in reopening of collapsed tumour vessels and improved blood perfusion, while reducing ICC cell invasion. Moreover, PlGF blockade enhanced the efficacy of standard chemotherapy in mice-bearing ICC. Conclusion PlGF blockade leads to a reduction in intratumorous hypoxia and metastatic dissemination, enhanced chemotherapy sensitivity and increased survival in mice-bearing aggressive ICC.
Subject(s)
Bile Duct Neoplasms/pathology , Cholangiocarcinoma/pathology , Placenta Growth Factor/metabolism , Animals , Antibodies, Monoclonal/pharmacology , Bile Duct Neoplasms/metabolism , Cancer-Associated Fibroblasts/metabolism , Cell Line, Tumor , Cholangiocarcinoma/metabolism , Disease Progression , Drug Resistance, Neoplasm , Humans , Hypoxia/metabolism , Mice , Placenta Growth Factor/antagonists & inhibitorsABSTRACT
Preeclampsia (PE) and intrauterine growth retardation (IUGR) are the results of defective placentation associated with the downregulation of different genes in the human trophoblast including the Placental Growth Factor (PGF). TrophEctoderm (TE) biopsy is increasingly performed for Pre-implantation Genetic Testing of Aneuploidies and it involves the traumatical removal of an unpredictable number of mural TE cells from the human blastocyst. We observed strikingly similar obstetrical and neonatal complications in pregnancies where the placenta bears PGF downmodulation or a TE biopsy has been done. In both groups, the risk of PE, IUGR, congenital cardiac ventricular septal defects, caesarean section, sex ratio in favour of males and preterm birth is significantly increased compared to controls. Given the high degree of correlation, the observation may not be a casual one. We postulate herein that the TE biopsy may induce persistent dysregulation of different genes in the placenta including PGF. The mechanism proposed is the disruption of tight junctions caused by the TE biopsy.
Subject(s)
Ectoderm/surgery , Gene Expression Regulation , Genetic Testing/methods , Placenta Growth Factor/antagonists & inhibitors , Placenta/surgery , Surgical Procedures, Operative/adverse effects , Trophoblasts/chemistry , Female , Humans , PregnancyABSTRACT
During the past two decades, tumor therapy based on monoclonal antibody has been found as a confident therapeutic approach in solid tumors and hematologic malignancies. Nanobodies are the smallest fragment of an antigen-binding domain in heavy chain-only antibody originated from the Camelidae family. Accordingly, they are being recently developed rapidly as diagnostic and therapeutic agents. In this regard, targeting of angiogenic factors like Placenta growth factor (PLGF) via nanobodies show a high effectiveness. In the current study, we developed a recombinant anti-PLGF bivalent nanobody based on the affinity enhancement mutant form of anti-PLGF nanobody to suppress the angiogenesis progression. Thereafter, the bivalent nanobody (bi-Nb) was cloned and then expressed into a bacterial system. Afterward, the purity was authorized using western blot assay and the affinity was assessed using ELISA. In this regard, proliferation, 3D capillary tube formation, and migration assays were employed as functional assays. The obtained data were analyzed using t-test and P < 0.05 was considered as statistically significant. The results indicate that the bivalent nanobody could inhibit proliferation, mobility, and formation of endothelial cell capillary-like structure. Moreover, the EC50 was estimated for endothelial cell's proliferation and capillary tube's formation to be about 100 ng/ml and 65 ng/ml, respectively. Migration of MCF-7 was inhibited as about 69%, rather than the control. Accumulation of data have shown that targeting of angiogenic factors like VEGF via monoclonal antibodies or nanobodies can be useful in the suppression of tumor progression. Also, the inhibition of PLGF with monoclonal antibody indicated that it is significant in angiogenesis suppression. However, due to intrinsic properties of nanobodies, they are suggested to be used. Since the small size is rapidly removed through liver or kidney system, so it is important to use bivalent or polymeric forms for extending the half-life. Our findings indicated that the inhibition of PLGF can prevent growth and proliferation of endothelial cells and tumor cells through the bivalent nanobody. So, it is suggested as a novel therapeutic agent for angiogenesis suppression.
Subject(s)
Antibodies, Bispecific/genetics , Placenta Growth Factor/antagonists & inhibitors , Recombinant Proteins/pharmacology , Single-Domain Antibodies/genetics , Angiogenesis Inhibitors/pharmacology , Antibodies, Bispecific/metabolism , Antibodies, Bispecific/pharmacology , Antibody Affinity , Cell Movement/drug effects , Cell Proliferation/drug effects , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/growth & development , Human Umbilical Vein Endothelial Cells , Humans , MCF-7 Cells , Mutation , Placenta Growth Factor/genetics , Single-Domain Antibodies/metabolism , Single-Domain Antibodies/pharmacologyABSTRACT
Introduction: In colorectal cancer, anti-VEGF agents have demonstrated a survival benefit when combined with chemotherapy. However, development of resistance is very common. One of the mechanisms is due not to a failure in the VEGFR blockade, but rather to development of compensatory mechanisms of resistance, such as hypoxia-triggered upregulation of other proangiogenic factors, like placental growth factor (PlGF).Areas covered: This article summarizes the fundamental role of PlGF in the development of resistance to antiangiogenic treatment as well as the efficacy of aflibercept, ramucirumab, and regorafenib.Expert opinion: Aflibercept functions as a soluble decoy receptor precluding VEGFs and PlGF from binding to native VEGFR, and therefore preventing the emergence of resistance. Bevacizumab limits its function to preventing the interaction between VEGF-A and VEGFR. In combination with FOLFIRI (VELOUR trial), aflibercept improves survival in patients with metastatic CRC who are resistant or have progressed to oxaliplatin-based chemotherapy. Ramucirumab, a fully humanized immunoglobulin G1 (IgG-1) monoclonal antibody and regorafenib, a multikinase inhibitor, have significant improvement for overall survival as well as for progression-free survival in chemotherapy refractory settings.
Subject(s)
Colorectal Neoplasms/drug therapy , Placenta Growth Factor/antagonists & inhibitors , Angiogenesis Inhibitors/therapeutic use , Animals , Colorectal Neoplasms/metabolism , Drug Resistance, Neoplasm , Humans , Placenta Growth Factor/metabolismABSTRACT
BACKGROUND: Age-related macular degeneration (AMD) is a leading cause of visual impairment in the elderly. The neovascular (wet) form of AMD can be treated with intravitreal injections of different anti-vascular endothelial growth factor (VEGF) agents. Placental growth factor (PGF) is another member of the VEGF family of cytokines with pro-angiogenic and pro-inflammatory effects. Here, we aimed to compare single and combined inhibition of VEGF-A and PGF in the laser-induced mouse model of choroidal neovascularization (CNV) with a focus on the effects on retinal mononuclear phagocytes. METHODS: CNV was induced in C57BL/6J mice using a YAG-Laser. Immediately after laser damage antibodies against VEGF-A (aVEGF), anti-PGF (aPGF), aVEGF combined with aPGF, aflibercept, or IgG control were injected intravitreally in both eyes. Three and 7 days after laser damage, the vascular leakage was determined by fluorescence angiography. Lectin staining of retinal and RPE/choroidal flat mounts was used to monitor CNV. In situ mRNA co-expression of Iba1, VEGF and PGF were quantified using in situ hybridization. Retinal and RPE/choroidal protein levels of VEGF and PGF as well as the pro-inflammatory cytokines IL-6, IL1-beta, and TNF were determined by ELISA. RESULTS: Early (day 3) and intermediate (day 7) vascular leakage and CNV were significantly inhibited by PGF and VEGF-A co-inhibition, most effectively with the trap molecule aflibercept. While VEGF-A blockage alone had no effects, trapping PGF especially with aflibercept prevented the accumulation of reactive microglia and macrophages in laser lesions. The lesion-related mRNA expression and secretion of VEGF-A and PGF by mononuclear phagocytes were potently suppressed by PGF and partially by VEGF-A inhibition. Protein levels of IL-6 and IL1-beta were strongly reduced in all treatment groups. CONCLUSIONS: Retinal inhibition of PGF in combination with VEGF-A prevents vascular leakage and CNV possibly via modulating their own expression in mononuclear phagocytes. PGF-related, optimized strategies to target inflammation-mediated angiogenesis may help to increase efficacy and reduce non-responders in the treatment of wet AMD patients.
Subject(s)
Monocytes/metabolism , Neovascularization, Pathologic/prevention & control , Placenta Growth Factor/antagonists & inhibitors , Retinal Diseases/prevention & control , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Animals , Choroid Plexus/pathology , Cytokines/metabolism , Female , Interleukin-1/antagonists & inhibitors , Interleukin-1/biosynthesis , Interleukin-6/antagonists & inhibitors , Interleukin-6/biosynthesis , Male , Mice , Mice, Inbred C57BL , Monocytes/drug effects , Neovascularization, Pathologic/pathology , Placenta Growth Factor/biosynthesis , RNA, Messenger/biosynthesis , Retina/pathology , Retinal Diseases/pathology , Vascular Endothelial Growth Factor A/biosynthesisABSTRACT
Angiogenesis is essential in the early stage of solid tumor recurrence, but how a suspensive tumor is reactivated before angiogenesis is mostly unknown. Herein, we stumble across an interesting phenomenon that s.c. xenografting human lung cancer tissues can awaken the s.c. suspensive tumor in nude mice. We further found that a high level of insulin-like growth factor 1 (IGF1) was mainly responsible for triggering the transition from suspensive tumor to progressive tumor in this model. The s.c. suspensive tumor is characterized with growth arrest, avascularity, and a steady-state level of proliferating and apoptotic cells. Intriguingly, CD133+ lung cancer stem cells (LCSCs) are highly enriched in suspensive tumor compared with progressive tumor. Mechanistically, high IGF1 initiates LCSCs self-renewal from asymmetry to symmetry via the activation of a PI3K/Akt/ß-catenin axis. Next, the expansion of LCSC pool promotes angiogenesis by increasing the production of CXCL1 and PlGF in CD133+ LCSCs, which results in lung cancer recurrence. Clinically, a high level of serum IGF1 in lung cancer patients after orthotopic lung cancer resection as an unfavorable factor is strongly correlated with the high rate of recurrence and indicates an adverse progression-free survival. Vice versa, blocking IGF1 or CXCL1/PlGF with neutralizing antibodies can prevent the reactivation of a suspensive tumor induced by IGF1 stimulation in the mouse model. Collectively, the expansion of LCSC pool before angiogenesis induced by IGF1 is a key checkpoint during the initiation of cancer relapse, and targeting serum IGF1 may be a promising treatment for preventing recurrence in lung cancer patients.
Subject(s)
Carcinoma, Non-Small-Cell Lung/pathology , Insulin-Like Growth Factor I/metabolism , Lung Neoplasms/pathology , Neoplasm Recurrence, Local/pathology , Neoplastic Stem Cells/pathology , Neovascularization, Pathologic/pathology , AC133 Antigen/metabolism , Animals , Carcinoma, Non-Small-Cell Lung/blood , Cell Line, Tumor , Cell Proliferation , Chemokine CXCL1/antagonists & inhibitors , Chemokine CXCL1/metabolism , Female , Humans , Insulin-Like Growth Factor I/analysis , Insulin-Like Growth Factor I/antagonists & inhibitors , Lung Neoplasms/blood , Mice , Mice, Nude , Neoplasm Recurrence, Local/blood , Neovascularization, Pathologic/blood , Phosphatidylinositol 3-Kinases/metabolism , Placenta Growth Factor/antagonists & inhibitors , Placenta Growth Factor/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Xenograft Model Antitumor Assays , beta Catenin/metabolismABSTRACT
Our previous studies found that an anti-placental growth factor (PlGF) antibody protected the retina in light-induced retinal damage model, a model of non-exudative age-related macular degeneration (AMD). Aflibercept is an inhibitor of vascular endothelial growth factor (VEGF) and PlGF. In present study, we revealed that the intravitreal injection of aflibercept lessens light-induced retinal damage, while anti-VEGF antibody has no effect on the light-exposed retina. Moreover, PlGF disrupted the tight junctions between the human retinal pigment epithelial cells in vitro, and aflibercept blocked the disruption. These data suggest that the aflibercept may be an effective treatment of non-exudative AMD.
Subject(s)
Light/adverse effects , Macular Degeneration/etiology , Macular Degeneration/prevention & control , Photoreceptor Cells, Vertebrate/radiation effects , Receptors, Vascular Endothelial Growth Factor/administration & dosage , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/pharmacology , Animals , Cells, Cultured , Disease Models, Animal , Humans , Intravitreal Injections , Male , Mice, Inbred Strains , Photoreceptor Cells, Vertebrate/pathology , Placenta Growth Factor/adverse effects , Placenta Growth Factor/antagonists & inhibitors , Retinal Pigment Epithelium/cytology , Tight Junctions/drug effects , Tight Junctions/pathology , Vascular Endothelial Growth Factor A/antagonists & inhibitorsABSTRACT
PURPOSE: Several clinical trials have combined antiangiogenic agents and radiation therapy (RT), but evidence of its clinical benefit is insufficient. In this study, we rationalized and investigated the combination of vascular endothelial growth factor-Grab (VEGF-Grab), an antiangiogenic drug that inhibits VEGF-A and placental growth factor (PlGF) and radiotherapy for anti-cancer therapy. METHODS AND MATERIALS: To observe for changes in PlGF after radiation, HCT116, HCT15, SW480, BxPC3, and RAW264.7 cells and Lewis lung carcinoma (LLC) and BxPC3 tumors were given 10 Gy of radiation, and changes in the expression of PlGF were analyzed. Patients scheduled for RT for solid tumor mass were recruited, and their plasma VEGF-A and PlGF were analyzed at baseline and 2 and 4 weeks after the start of radiotherapy. To assess the effects of combining VEGF-Grab and radiotherapy, mice bearing LLC tumors were given 10 Gy of radiation once and 25 mg/kg of VEGF-Grab every 2 days for 5 rounds. To show that VEGF-Grab is effective in human tumors, mice bearing BxPC3 xenografts were given 2 doses of 15 mg/kg of VEGF-Grab or VEGF-Trap. To assess the efficacy of combination therapy in BxPC3 xenografts, the same experiment used in the LLC model was performed. RESULTS: We demonstrated that PlGF is increased as a direct consequence of irradiation in vitro and in vivo and in the plasma of patients being treated with radiation. Using a syngeneic tumor model, we showed that the combination of VEGF-Grab and RT most effectively inhibited tumor growth through antiangiogenesis, tumor vessel normalization, and tumor-associated macrophage polarization from protumorigenic M2-type to antitumorigenic M1-type. Finally, we demonstrated similar enhanced antitumor effects using a human xenograft model. CONCLUSIONS: This study shows that PlGF is a potential target in patients being treated with RT and suggests VEGF-Grab as a viable therapeutic option in the context of inhibiting secondarily activated pathways responsible for tumor recurrence.
Subject(s)
Neoplasms/drug therapy , Neoplasms/radiotherapy , Placenta Growth Factor/antagonists & inhibitors , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor A/pharmacology , Adult , Aged , Angiogenesis Inhibitors/pharmacology , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Combined Modality Therapy , Female , Flow Cytometry , HCT116 Cells , Humans , Macrophages/metabolism , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Nude , Middle Aged , Neoplasm Transplantation , Neoplasms/blood , Neovascularization, Pathologic/drug therapy , Placenta Growth Factor/metabolism , Protein Binding , RAW 264.7 Cells , Recombinant Proteins/metabolismABSTRACT
Hepatopulmonary syndrome (HPS) is a severe complication of cirrhosis with increased risk of mortality. Pulmonary microvascular alterations are key features of HPS; but underlying mechanisms are incompletely understood, and studies on HPS are limited to rats. Placental growth factor (PlGF), a proangiogenic molecule that is selectively involved in pathological angiogenesis, may play an important role in HPS development; however, its role has never been investigated. In this study, we validated an HPS model by common bile duct ligation (CBDL) in mice, investigated the kinetic changes in pulmonary angiogenesis and inflammation during HPS development, and provide evidence for a novel therapeutic strategy by targeting pathological angiogenesis. Mice with CBDL developed hypoxemia and intrapulmonary shunting on a background of liver fibrosis. Pulmonary alterations included increased levels of proangiogenic and inflammatory markers, which was confirmed in serum of human HPS patients. Increased PlGF production in HPS mice originated from alveolar type II cells and lung macrophages, as demonstrated by immunofluorescent staining. Dysfunctional vessel formation in CBDL mice was visualized by microscopy on vascular corrosion casts. Both prophylactic and therapeutic anti-PlGF (αPlGF) antibody treatment impeded HPS development, as demonstrated by significantly less intrapulmonary shunting and improved gas exchange. αPlGF treatment decreased endothelial cell dysfunction in vivo and in vitro and was accompanied by reduced pulmonary inflammation. Importantly, αPlGF therapy did not affect liver alterations, supporting αPlGF's ability to directly target the pulmonary compartment. CONCLUSION: CBDL in mice induces HPS, which is mediated by PlGF production; αPlGF treatment improves experimental HPS by counteracting pulmonary angiogenesis and might be an attractive therapeutic strategy for human HPS. (Hepatology 2017).
Subject(s)
Hepatopulmonary Syndrome/metabolism , Lung/pathology , Neovascularization, Pathologic/metabolism , Placenta Growth Factor/metabolism , Animals , Antibodies, Monoclonal/pharmacology , Biomarkers/metabolism , Common Bile Duct/surgery , Disease Models, Animal , Endoglin/blood , Hepatopulmonary Syndrome/physiopathology , Humans , Ligation/methods , Liver/pathology , Liver Cirrhosis/pathology , Male , Mice , Placenta Growth Factor/antagonists & inhibitorsABSTRACT
Purpose: The cellular immune response driven by mononuclear phagocytes (MPs) is crucial for choroidal neovascularization (CNV) progression. Case reports show that a switch from pure anti-vascular endothelial growth factor-A (VEGF-A) intravitreal treatment to aflibercept, a drug with combined anti-VEGF-A and anti-placenta growth factor (PlGF) activity, can be beneficial for patients who do not respond to anti-VEGF-A alone. Since MPs harbor VEGFR1, we hypothesize that the interplay of P1GF/vascular endothelial growth factor receptor 1 (VEGFR1) in immune cells plays a pivotal role for CNV. Methods: CNV was induced with laser, and immune cells and neovascularization were analyzed in vivo and ex vivo. Immunohistochemistry was employed for protein detection. Differential expression of angiogenic factors and macrophage polarization markers were assessed by quantitative PCR (qPCR). One day after laser, intravitreal injection of aflibercept or anti-PlGF was performed. Results: In the early inflammatory phase after laser, Plgf but not Vegfa was significantly upregulated. VEGF-A upregulation is limited to the scar, whereas PlGF shows a wider distribution. M1 (proinflammatory) macrophage markers were upregulated in the early phase of CNV. However, M2 (proangiogenic) markers showed more inconsistent dynamics. We demonstrated that both aflibercept and anti-PlGF treatments decrease the overall amount of activated subretinal MPs, and especially of those expressing PlGF. These data correlated with a reduction in leakage associated to CNV. Aflibercept showed a stronger reduction in both parameters. Conclusions: The results hint at an interplay between PlGF/VEGFR1 and MPs that is important in the early phase of CNV. A combined inhibition of VEGF-A and PlGF is superior to a specific anti-PlGF treatment in terms of subretinal MP recruitment.
Subject(s)
Choroidal Neovascularization/immunology , Phagocytes/immunology , Placenta Growth Factor/antagonists & inhibitors , Retina/immunology , Angiogenesis Inhibitors/pharmacology , Animals , Antibodies, Blocking/pharmacology , Calcium-Binding Proteins , Choroidal Neovascularization/drug therapy , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Gene Expression Regulation , Immunologic Factors/pharmacology , Intravitreal Injections , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microfilament Proteins , Microscopy, Fluorescence , Ophthalmoscopy , Placenta Growth Factor/genetics , Real-Time Polymerase Chain Reaction , Receptors, Vascular Endothelial Growth Factor , Recombinant Fusion Proteins/pharmacology , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor Receptor-1/genetics , Wet Macular Degeneration/drug therapy , Wet Macular Degeneration/immunologyABSTRACT
The current standard of care in clinical practice for diabetic retinopathy (DR), anti-vascular endothelial growth factor (VEGF) therapy, has shown a significant improvement in visual acuity. However, treatment response can be variable and might be associated with potential side effects. This study was designed to investigate inhibition of placental growth factor (PlGF) as a possible alternative therapy for DR. The effect of the anti-PlGF antibody (PL5D11D4) was preclinically evaluated in various animal models by investigating different DR hallmarks, including inflammation, neurodegeneration, vascular leakage and fibrosis. The in vivo efficacy was tested in diabetic streptozotocin (STZ) and Akimba models and in the laser induced choroidal neovascularization (CNV) mouse model. Intravitreal (IVT) administration of the anti-PlGF antibody was compared to anti-VEGFR-2 antibody (DC101), anti-VEGF antibody (B20), VEGF-Trap (aflibercept) and triamcinolone acetonide (TAAC). Vascular leakage was investigated in the mouse STZ model by fluorescein isothiocyanate labeled bovine serum albumin (FITC-BSA) perfusion and in the Akimba model by fluorescein angiography (FA). Repeated IVT administration of the anti-PlGF antibody reduced vascular leakage, which was comparable to a single administration of VEGFR-2 inhibition in the mouse STZ model. PL5D11D4 treatment did not alter retinal ganglion cell (RGC) density, as demonstrated by Brn3a staining, whereas DC101 significantly reduced RGC number with 20%. Immunohistological stainings were performed to investigate inflammation (CD45, F4/80) and fibrosis (collagen type 1a). In the CNV model, IVT injection(s) of PL5D11D4 dose-dependently reduced inflammation and fibrosis, as compared to PBS treatment. Equimolar single administration of the anti-PlGF antibody and aflibercept (21 nM) and TAAC decreased leukocyte and macrophage infiltration with 50%, whereas DC101 and B20 (21 nM) had no effect on the inflammatory response. Similar results were observed in the mouse STZ model on the number of microglia and macrophages in the retina. Repeated administration of PL5D11D4 (21 nM) and TAAC similarly reduced fibrosis, while no effect was observed after equimolar DC101, B20 nor aflibercept administration (21 nM). In summary, the anti-PlGF antibody showed comparable efficacy as well-characterized VEGF-inhibitor on the process of vascular leakage, but differentiates itself by also reducing inflammation and fibrosis, without triggering a neurodegenerative response.
Subject(s)
Antibodies, Blocking/therapeutic use , Antibodies, Monoclonal/therapeutic use , Diabetic Retinopathy/drug therapy , Placenta Growth Factor/antagonists & inhibitors , Angiogenesis Inhibitors/therapeutic use , Animals , Disease Models, Animal , Intravitreal Injections , Mice , Mice, Inbred C57BLABSTRACT
Fetal growth restriction (FGR) threatens perinatal health and is correlated with increased incidence of fetal original adult diseases. Most cases of FGR were idiopathic, which were supposed to be associated with placental abnormality. Decreased circulating placental growth factor (PGF) was recognized as an indication of placental deficiency in FGR. In this study, the epigenetic regulation of PGF in FGR placentas and the involvement of PGF in modulation of trophoblast activity were investigated. The expression level of PGF in placental tissues was determined by RT-qPCR, immunohistochemistry and ELISA. DNA methylation profile of PGF gene was analyzed by bisulfite sequencing. Trophoblastic cell lines were treated with ZM-306416, an inhibitor of PGF receptor FLT1, to observe the effect of PGF/FLT1 signaling on cell proliferation and migration. We demonstrated that PGF was downregulated in placentas from FGR pregnancies compared with normal controls. The villous expression of PGF was positively correlated with placental and fetal weight. The CpG island inside PGF promoter was hypomethylated without obvious difference in both normal and FGR placentas. However, the higher DNA methylation at another CpG island downstream exon 7 of PGF was demonstrated in FGR placentas. Additionally, we found FLT1 was expressed in trophoblast cells. Inhibition of PGF/FLT1 signaling by a selective inhibitor impaired trophoblast proliferation and migration. In conclusion, our data suggested that the PGF expression was dysregulated, and disrupted PGF/FLT1 signaling in trophoblast might contribute to placenta dysfunction in FGR. Thus, our results support the significant role of PGF in the pathogenesis of FGR.
Subject(s)
DNA Methylation , Fetal Growth Retardation/etiology , Gene Expression Regulation, Developmental , Placenta Diseases/physiopathology , Placenta Growth Factor/metabolism , Trophoblasts/metabolism , Adult , Birth Weight , Cell Line , Cell Movement/drug effects , Cell Proliferation/drug effects , CpG Islands/drug effects , DNA Methylation/drug effects , Epigenesis, Genetic/drug effects , Exons/drug effects , Female , Gene Expression Regulation, Developmental/drug effects , Humans , Placenta Diseases/blood , Placenta Diseases/metabolism , Placenta Diseases/pathology , Placenta Growth Factor/antagonists & inhibitors , Placenta Growth Factor/genetics , Placentation , Pregnancy , Promoter Regions, Genetic/drug effects , Quinazolines/pharmacology , RNA Interference , Trophoblasts/drug effects , Trophoblasts/pathology , Vascular Endothelial Growth Factor Receptor-1/antagonists & inhibitors , Vascular Endothelial Growth Factor Receptor-1/metabolismABSTRACT
Diabetic retinopathy (DR) is one of the common and specific microvascular complications of diabetes. This study aimed to investigate the anti-angiogenic effect of kaempferol and explore its underlying molecular mechanisms. The mRNA expression level of vascular endothelial growth factor (VEGF) and placenta growth factor (PGF) and the concentrations of secreted VEGF and PGF were measured by qTR-PCR and ELISA assay, respectively. Human retinal endothelial cells (HRECs) proliferation, migration, and sprouting were measured by CCK-8 and transwell, scratching wound, and tube formation assays, respectively. Protein levels were determined by western blot. High glucose (25 mM) increased the mRNA expression levels of VEGF and PGF as well as the concentrations of secreted VEGF and PGF in HRECs, which can be antagonized by kaempferol (25 µM). Kaempferol (5-25 µM) significantly suppressed cell proliferation, migration, migration distance and sprouting of HRECs under high glucose condition. The anti-angiogenic effect of kaempferol was mediated via downregulating the expression of PI3K and inhibiting the activation of Erk1/2, Src, and Akt1. This study indicates that kaempferol suppressed angiogenesis of HRECs via targeting VEGF and PGF to inhibit the activation of Src-Akt1-Erk1/2 signaling pathway. The results suggest that kaempferol may be a potential drug for better management of DR.
Subject(s)
Diabetic Retinopathy/metabolism , Endothelial Cells/drug effects , Kaempferols/pharmacology , Placenta Growth Factor/antagonists & inhibitors , Retina/pathology , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Cell Movement , Cell Proliferation , Diabetic Retinopathy/pathology , Enzyme-Linked Immunosorbent Assay , Humans , ImmunohistochemistryABSTRACT
Diabetic retinopathy (DR) is one of the common and specific microvascular complications of diabetes. This study aimed to investigate the anti-angiogenic effect of kaempferol and explore its underlying molecular mechanisms. The mRNA expression level of vascular endothelial growth factor (VEGF) and placenta growth factor (PGF) and the concentrations of secreted VEGF and PGF were measured by qTR-PCR and ELISA assay, respectively. Human retinal endothelial cells (HRECs) proliferation, migration, and sprouting were measured by CCK-8 and transwell, scratching wound, and tube formation assays, respectively. Protein levels were determined by western blot. High glucose (25 mM) increased the mRNA expression levels of VEGF and PGF as well as the concentrations of secreted VEGF and PGF in HRECs, which can be antagonized by kaempferol (25 µM). Kaempferol (5-25 µM) significantly suppressed cell proliferation, migration, migration distance and sprouting of HRECs under high glucose condition. The anti-angiogenic effect of kaempferol was mediated via downregulating the expression of PI3K and inhibiting the activation of Erk1/2, Src, and Akt1. This study indicates that kaempferol suppressed angiogenesis of HRECs via targeting VEGF and PGF to inhibit the activation of Src-Akt1-Erk1/2 signaling pathway. The results suggest that kaempferol may be a potential drug for better management of DR.
Subject(s)
Humans , Diabetic Retinopathy/metabolism , Endothelial Cells/drug effects , Kaempferols/pharmacology , Placenta Growth Factor/antagonists & inhibitors , Retina/pathology , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Cell Movement , Cell Proliferation , Diabetic Retinopathy/pathology , Enzyme-Linked Immunosorbent Assay , ImmunohistochemistryABSTRACT
Neuroblastoma (NB) is the most common extracranial solid tumor of childhood and is a rapidly growing, highly-vascularized cancer. NBs frequently express angiogenic factors and high tumor angiogenesis has been associated with poor outcomes. Placental growth factor (PlGF) is an angiogenic protein belonging to the vascular endothelial growth factor (VEGF) family and is up-regulated mainly in pathologic conditions. Recently, PlGF was identified as a member of a gene expression signature characterizing highly malignant NB stem cells drawing attention as a potential therapeutic target in NB. In the present study, we sought to investigate the expression of PlGF in NB patients and the effect of PlGF inhibition on high-risk MYCN-non-amplified SK-N-AS NB xenografts. Human SK-N-AS cells, which are poorly differentiated and express PlGF and VEGF-A, were implanted subcutaneously in athymic nude mice. Treatment was done by intratumoral injection of replication-incompetent adenoviruses (Ad) expressing PlGF- or VEGF-specific short hairpin (sh)RNA, or soluble (s)VEGF receptor 2 (VEGFR2). The effect on tumor growth and angiogenesis was analyzed. High PlGF expression levels were observed in human advanced-stage NBs. Down-regulating PlGF significantly reduced NB growth in established NB xenografts by reducing cancer cell proliferation but did not suppress angiogenesis. In contrast, blocking VEGF by administration of Ad(sh)VEGF and Ad(s)VEGFR2 reduced tumor growth associated with decreased tumor vasculature. These findings suggest that PlGF and VEGF-A modulate MYCN-non-amplified NB tumors by different mechanisms and support a role for PlGF in NB biology.
Subject(s)
N-Myc Proto-Oncogene Protein/genetics , Neuroblastoma/pathology , Placenta Growth Factor/metabolism , Vascular Endothelial Growth Factor A/metabolism , Adenoviridae/genetics , Animals , Cell Line, Tumor , Cell Proliferation , Child, Preschool , Female , Genetic Vectors/genetics , Genetic Vectors/metabolism , Humans , Infant , Male , Mice , Mice, Nude , Neovascularization, Pathologic , Neuroblastoma/metabolism , Neuroblastoma/prevention & control , Placenta Growth Factor/antagonists & inhibitors , Placenta Growth Factor/genetics , RNA Interference , RNA, Small Interfering/metabolism , Transplantation, Heterologous , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor Receptor-2/genetics , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
Placental growth factor (PlGF), a member of vascular endothelial growth factors (VEGF) family, is considered as an important antigen associated with pathological conditions such as cancer cell growth, and metastasis. PlGF-targeting via nanobody (Nb) therefore could be beneficial to modulate these pathologies. In this work, phage-display and computational approach was employed to develop a high affinity PlGF-specific Nb. An Nb library was constructed against human recombinant PlGF (rPlGF). After panning on immobilized rPlGF the periplasmic-extract (PE) of individual colonies were screened by ELISA (PE-ELISA). The 3D structures of selected Nbs were then homology modeled and energy minimized using the AMBER force field. Binding score calculations were also assessed to reveal possible Nb-PlGF interactions. Via ELISA-based affinity/specificity determinations, the best-qualified Nb was further evaluated by proliferation, migration, 3D capillary formation, invasion assays and on Chick chorioallantoic membrane (CAM) model. An immune library of 1.5×107 individual Nb clones was constructed. By PE-ELISA 12 clones with strong signals were selected. Three out of 12 sequenced Nbs (Nb-C13, Nb-C18 and Nb-C62) showed high binding scores ranging between -378.7 and -461kcal/mol. Compared to a control Nb, Nb-C18 significantly inhibited proliferation, migration and the 3D-capillary formation of HUVEC cells (p<0.05) with an EC50 of 35nM, 42nM and 24nM and invasion of MDA-MB231was significantly suppressed (p<0.05) with an EC50 of57nM. The result of the CAM assay shows that Nb-C18 could inhibit the vascular formation in the chicken chorioallantoic membrane. This Nb can be used as anti-angiogenesis agent in future.