BACKGROUND: The early stages of tumor bone metastasis are closely associated with changes in the vascular niche of the bone microenvironment, and abnormal angiogenesis accelerates tumor metastasis and progression. However, the effects of lung adenocarcinoma (LUAD) cells reprogrammed by the bone microenvironment on the vascular niche within the bone microenvironment and the underlying mechanisms remain unclear. This study investigates the effects and mechanisms of LUAD cells reprogrammed by the bone microenvironment on endothelial cells and angiogenesis, providing insights into the influence of tumor cells on the vascular niche within the bone microenvironment. METHODS: The culture media from bone-metastatic LUAD cell A549-GFP-LUC-BM3 (BM3-CM) and A549-GFP-LUC (A549-CM) were separately applied to human umbilical vein endothelial cell (HUVEC). A colony formation assay, scratch assay, and tube formation assay were conducted to evaluate the proliferation, migration, and angiogenesis of HUVEC. Gene set enrichment analysis (GSEA) was conducted to identify enriched pathways, while reverse transcription quantitative polymerase chain reaction (RT-qPCR) and enzyme linked immunosorbent assay (ELISA) were performed to quantify hepatocyte growth factor (HGF), a protein that plays a crucial role in angiogenesis. Furthermore, the pivotal function of HGF and its underlying molecular mechanisms have been substantiated through the utilisation of recombinant proteins, neutralising antibodies, pathway inhibitors, immunofluorescence staining, and Western blot. RESULTS: BM3-CM demonstrated a more pronounced impact on the proliferation, migration, and angiogenesis of HUVEC compared to A549-CM. Bioinformatics analysis, combined with in vitro experiment, demonstrated that the secretory protein HGF was significantly elevated in BM3 cells and BM3-CM (P<0.05). The addition of HGF neutralizing antibodies to BM3-CM inhibited the promoting effect of BM3-CM on HUVEC (P<0.05), while the addition of recombinant HGF to A549-CM reproduced that promoting effect of BM3-CM on HUVEC (P<0.05). HGF can enhance the activation of YAP (Yes-associated protein) in HUVEC, and this promotion effect may be achieved by activating Src and activating YAP into the nucleus (P<0.05), but this effect can be inhibited by HGF neutralizing antibodies (P<0.05). Furthermore, the addition of recombinant HGF to A549-CM can recapitulate the YAP activation effect of BM3-CM in HUVEC (P<0.05). CONCLUSIONS Bone microenvironment reprogrammed bone-metastatic LUAD cells BM3 promote the proliferation, migration, and angiogenesis of HUVEC through the HGF/YAP axis, potentially playing a significant role in the modifications of the vascular niche.
Humans ; Hepatocyte Growth Factor/genetics* ; Signal Transduction ; Neovascularization, Pathologic/genetics* ; Human Umbilical Vein Endothelial Cells ; Bone Neoplasms/blood supply* ; Adenocarcinoma of Lung/genetics* ; Adaptor Proteins, Signal Transducing/genetics* ; Lung Neoplasms/genetics* ; Cell Movement ; Cell Proliferation ; YAP-Signaling Proteins ; Transcription Factors/genetics* ; Cell Line, Tumor ; Tumor Microenvironment ; Angiogenesis

BACKGROUND: For an effective bone graft for reconstruction of the maxillofacial region, an adequate vascular network will be required to supply blood, osteoprogenitor cells, and growth factors. We previously reported that the secretomes of bone marrow-derived mesenchymal stem cells (MSC-CM) contain numerous growth factors such as insulin-like growth factor (IGF)-1, transforming growth factor (TGF)-β1, and vascular endothelial growth factor (VEGF), which can affect the cellular characteristics and behavior of regenerating bone cells. We hypothesized that angiogenesis is an important step for bone regeneration, and VEGF is one of the crucial factors in MSC-CM that would enhance its osteogenic potential. In the present study, we focused on VEGF in MSC-CM and evaluated the angiogenic and osteogenic potentials of MSC-CM for bone regeneration. METHODS: Cytokines in MSC-CM were measured by enzyme-linked immunosorbent assay (ELISA). Human umbilical vein endothelial cells (HUVECs) were cultured with MSC-CM or MSC-CM with anti-VEGF antibody (MSC-CM + anti-VEGF) for neutralization, and tube formation was evaluated. For the evaluation of bone and blood vessel formation with micro-computed tomography (micro-CT) and for the histological and immunohistochemical analyses, a rat calvarial bone defect model was used. RESULTS: The concentrations of IGF-1, VEGF, and TGF-β1 in MSC-CM were 1515.6 ± 211.8 pg/mL, 465.8 ± 108.8 pg/mL, and 339.8 ± 14.4 pg/mL, respectively. Tube formation of HUVECs, bone formation, and blood vessel formation were increased in the MSC-CM group but decreased in the MSC-CM + anti-VEGF group. Histological findings suggested that new bone formation in the entire defect was observed in the MSC-CM group although it was decreased in the MSC-CM + anti-VEGF group. Immunohistochemistry indicated that angiogenesis and migration of endogenous stem cells were much more abundant in the MSC-CM group than in the MSC-CM + anti-VEGF group. CONCLUSIONS: VEGF is considered a crucial factor in MSC-CM, and MSC-CM is proposed to be an adequate therapeutic agent for bone regeneration with angiogenesis.
Animals ; Blood Vessels ; Bone Regeneration ; Culture Media, Conditioned ; Cytokines ; Enzyme-Linked Immunosorbent Assay ; Human Umbilical Vein Endothelial Cells ; Humans* ; Immunohistochemistry ; Insulin-Like Growth Factor I ; Intercellular Signaling Peptides and Proteins ; Mesenchymal Stromal Cells* ; Osteogenesis ; Rats ; Stem Cells ; Transforming Growth Factors ; Transplants ; Vascular Endothelial Growth Factor A

This study was aimed to investigate the association of candidate gene polymorphisms and obesity or overweight in young Korean children. A total of 190 Korean preschool children (96 control, 48 overweight, and 46 obese children) were genotyped for the angiotensin converting enzyme (ACE) insertion (I)/deletion (D), angiotensin II type 2 receptor (AT2) C3123A, transforming growth factor (TGF)-β1 T869C, vascular endothelial growth factor (VEGF) T460C, and tumor necrosis factor (TNF)-α G308A polymorphisms. No differences were found among the groups with respect to age, sex, birth weight, blood pressure levels, and serum concentrations of glucose and total cholesterol. Obese children showed a higher incidence of ACE DD genotype and D allelic frequency compared to the controls (odds ratio [OR], 2.7, 95% confidence interval [CI], 1.01–7.21; OR, 2.5, 95% CI, 1.49–4.19; all P < 0.05). The frequency of TC genotype and C allele in the TGF-β1 T869C polymorphism (OR, 2.08, 95% CI, 1.01–4.27; OR, 1.93, 95% CI, 1.15–3.21) and that in the VEGF T460C polymorphism (OR, 2.5, 95% CI, 1.19–5.28; OR, 2.15, 95% CI, 1.26–3.68) was also higher in obese children than in control subjects (all P < 0.05). Overweight children exhibited a higher frequency of the A allele in the AT2 C3123A polymorphism compared to the controls (OR, 1.72, 95% CI, 1.03–2.88, P < 0.05). There were no differences in the TNF-α G308A polymorphism among the groups. The ACE I/D, AT2 C3123A, TGF-β1 T869C, and VEGF T460C polymorphisms can affect susceptibility to obesity or overweight in Korean children.
Alleles ; Angiogenic Proteins ; Birth Weight ; Blood Pressure ; Child ; Child, Preschool* ; Cholesterol ; Genetic Variation ; Genotype ; Glucose ; Humans ; Incidence ; Obesity ; Overweight ; Pediatric Obesity* ; Peptidyl-Dipeptidase A ; Receptor, Angiotensin, Type 2 ; Renin-Angiotensin System ; Transforming Growth Factors ; Tumor Necrosis Factor-alpha ; Vascular Endothelial Growth Factor A

PURPOSE: The aim of this study was to determine the relationship of hypoxia-inducible factor-2 (HIF-2α) and vascular endothelial growth factor (VEGF) with radiographic severity in primary osteoarthritis (OA) of the knee. Expression of these two factors in cartilage samples from OA knee joints was examined at mRNA and protein levels. MATERIALS AND METHODS: Knee joints were examined using plain radiographs, and OA severity was assessed using the Kellgren and Lawrence (KL) grading system. Specimens were collected from 29 patients (31 knees) who underwent total knee replacement because of severe medial OA of the knee (KL grades 3 and 4), 16 patients who underwent knee arthroscopy (KL grade 2), and 5 patients with traumatic knees (KL grade 0). HIF-2α and VEGF expression was quantified by real-time polymerase chain reaction and western blotting. RESULTS: Cartilage degeneration correlated with the radiographic severity grade. OA severity, determined using the Mankin scale, correlated positively with the KL grade (r=0.8790, p<0.01), and HIF-2α and VEGF levels with the radiographic severity of knee OA (r=0.7001, p<0.05; r=0.6647, p<0.05). CONCLUSION: In OA cartilage, HIF-2α and VEGF mRNA and protein levels were significantly and positively correlated. The expression of both factors correlated positively with the KL grade. HIF-2α and VEGF, therefore, may serve as biochemical markers as well as potential therapeutic targets in knee OA.
Adult ; Aged ; Arthroplasty, Replacement, Knee ; Arthroscopy ; Basic Helix-Loop-Helix Transcription Factors/*metabolism ; Biomarkers/*blood ; Cartilage/*metabolism ; Female ; Humans ; Knee Joint/*diagnostic imaging ; Male ; Middle Aged ; Osteoarthritis, Knee/*blood/diagnostic imaging/physiopathology ; RNA, Messenger ; Radiography ; Real-Time Polymerase Chain Reaction ; Severity of Illness Index ; Vascular Endothelial Growth Factor A/*metabolism

OBJECTIVETo explore the formation of pre-metastatic niche in the mouse lung and to study the underlying molecular mechanisms whereby primary breast carcinoma-derived factors mediate recruitment of bone marrow-derived cells (BMDCs) and affect the formation of pre-metastatic lung environment before the arrival of tumor cells.

METHODSMammary carcinoma 4T1 cells were inoculated into the mammary gland to construct mouse model of breast cancer. Confocal microscopy was used to detect the recruitment of BMDCs in the pre-metastatic lungs. The expression of factors in the mouse sera and 4T1 cell culture media was assayed using RayBio Custom mouse cytokine antibody array kit. The mice were injected daily with recombinant VEGF for 7 consecutive days to observe the effect of VEGF on BMDCs recruitment in the mouse lung.

RESULTSNo BMDCs were observed in the lungs of control and 4T1-tumor-bearing mice on day 0. On day 7 and 14, clusters of BMDCs observed in the lungs of 4T1-tumor-bearing mice were 8.7±2.2/objective field and 48.8±3.2/objective field, respectively, significantly higher than those in the control mice (1.1±0.8/objective field and 3.1±1.7/objective field) (P<0.05 for both). Confocal microscopic observation found that metastatic breast cancer cells preferentially facilitate BMDCs recruitment sites in the pre-metastatic mouse lungs. The levels of VEGF, GM-CSF, and IL-6 in the serum of 4T1-tumor-bearing mice were significantly increased compared with those in the control group (P<0.05 for all). However, VEGF was detected only in the culture media of 4T1 cells. The amount of BMDCs in the mouse lung tissue was (22.8±3.6)/objective field in the VEGF group and (3.1±0.4)/objective field in the control group (P<0.05). There were 36.8±5.4 metastatic foci in the lung tissue of VEGF group and 12.6±2.2 in the control group (P<0.05).

CONCLUSIONSThe results of this study demonstrate that primary breast cancer cells can alter the lung microenvironment during the pre-metastatic phase and induce the formation of pre-metastatic niche. Primary tumor cell-derived VEGF may be a crucial factor responsible for the formation of pre-metastatic niche.

Animals ; Bone Marrow Cells ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Disease Models, Animal ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; blood ; Humans ; Interleukin-6 ; blood ; Lung ; pathology ; Lung Neoplasms ; secondary ; Mice ; Recombinant Proteins ; administration & dosage ; Time Factors ; Tumor Microenvironment ; Vascular Endothelial Growth Factor A ; administration & dosage ; physiology ; secretion

The effects of the balance changes of pigment epithelium growth factor (PEDF) and vascular endothelial growth factor (VEGF) in whole-body and retinal tissue on rats with oxygen-induced retinopathy were investigated. Forty-eight neonatal SD rats at the age of 7 days were randomly divided into 4 groups. The neonatal rats in experimental groups were exposed to 75% to 80% oxygen for 5 days and then to normal air, and those in control groups were kept feeding in normal air. At the age of 17 and 22 days, all the neonatal rats received retina angiography with FITC-dextran and the pathological changes of retinal vessels and perfusion were observed. HE staining of the tissue section and the number counting of endothelial cells extending beyond the inner limiting membrane were performed to evaluate the endothelial proliferation. Immunohistochemistry was applied to detect the expression of PEDF and VEGF in retinal tissue, and ELISA to detect their expression in serum. A hypoxic-ischemic proliferation of retina and more endothelial cells extending beyond the inner limiting membrane were found in the neonatal rats in both experimental groups of 17-day old and 22-day old as compared with those in control group with the difference being statistically significant (P<0.01). VEGF staining of the rats in the 17-day old experimental group was significantly stronger, with an increasing positive rate, than that of the rats in the 17-day old control group (P<0.01). PEDF staining of the rats of 22 days old was weaker than that of the rats of 17 days old in the experimental groups (P<0.01). There was no significant difference in serum VEGF concentration among all groups (P>0.05). The serum PEDF concentration in the rats of 17 days old in experimental group was decreased significantly as compared with that in the rats of 17 days old in control group (P<0.01), and in experimental groups, the serum PEDF concentration of the rats of 22 days old was increased as compared with that of the rats of 17 days old (P<0.01). In conclusion, the obviously decreased serum PEDF concentration and the abnormal enhanced expression of VEGF density in local retinal tissue broke down the balance of PEDF/VEGF in whole-body or local tissues, which might play an important role in retinal vascular proliferation.
Animals ; Eye Proteins ; blood ; metabolism ; Nerve Growth Factors ; blood ; metabolism ; Oxygen ; adverse effects ; Rats ; Rats, Sprague-Dawley ; Retina ; metabolism ; Retinal Diseases ; etiology ; metabolism ; Serpins ; blood ; metabolism ; Time and Motion Studies ; Vascular Endothelial Growth Factor A ; blood ; metabolism

OBJECTIVETo investigate the effect of topical propranolol gel on the levels of plasma vascular endothelial growth factor (VEGF), basic fibroblastic growth factor (bFGF) and matrix metalloproteinases-9 (MMP-9) in proliferating infantile hemangiomas (IHs) of superficial type.

METHODS33 consecutive children with superficial IHs were observed pre-treatment, 1 and 3 months after application of topical propranolol gel for the levels of plasma VEGF, MMP-9 and bFGF by enzyme-linked immunosorbent assay (ELISA) in Department of General Surgery of Dongfang Hospital from February 2013 to February 2014. The plasma results of IHs were compared with those of 30 healthy infants. The clinical efficacy in IHs was evaluated by Achauer system. Differences of plasma results between the healthy group and the IHs group pre-treatment were analyzed using Mann-Whitney U-test. Paired sample comparisons of any two time points of pre-treatment, 1 month and 3 months after treatment in IHs were evaluated by Wilcoxon signed-rank test.

RESULTSThe clinical efficiency of topical propranolol gel at 1, 3 months after application were 45.45%, 81.82% respectively. The levels of plasma VEGF and MMP-9 in patients pre- treatment were higher than those in healthy infants [(362.16 ± 27.29) pg/ml vs (85.63 ± 8.14) pg/ml, (1376.41 ± 42.15) pg/ml vs (687.27 ± 44.1) pg/ml, P < 0.05], but the level of bFGF did not show significant difference [(176.03 ± 13.60 ) pg/ml vs (235.94 ± 35.43 ) pg/ml, P > 0. 05 ]. The concentrations of VEGF and bFGF at 1, 3 months after treatment decreased obviously [(271.51 ± 18.59) pg/ml vs (362.16 ± 27.29 ) pg/ml, (135.85 ± 12.66) pg/ml vs (176.03 ± 13.60) pg/ml], 1 month after treatment vs pre-treatment, P < 0.05; (240.80 ± 19.89) pg/ml vs (362.16 ± 27.29) pg/ml, (107.31 ± 5.82) pg/ml vs (176.03 ± 13.60) pg/ml, 3 month after treatment vs pre-treatment, P < 0.05, whereas the levels of plasma MMP-9 declined slightly [(1321.18 ± 48.74) pg/ml vs (1376.41 ± 42.15 ) pg/ml, (1468.68 ± 32.78) pg/ml vs (1376.41 ± 42 2.15 ) pg/ml, P > 0.05 ].

CONCLUSIONSPropranolol gel may suppress the proliferation of superficial infantile bemangiomas by reducing VEGF and bFGF.

Administration, Topical ; Case-Control Studies ; Child ; Enzyme-Linked Immunosorbent Assay ; Fibroblast Growth Factor 2 ; blood ; Gels ; Hemangioma ; blood ; drug therapy ; Humans ; Infant ; Matrix Metalloproteinase 9 ; blood ; Propranolol ; pharmacology ; Time Factors ; Vascular Endothelial Growth Factor A ; blood

OBJECTIVETo investigate the effect of storage time on accumulation of platelet-related growth factors in the supernatant of leukoreduced packed red blood cells (LR-pRBC) and on tumor cell proliferation in vitro.

METHODSLR-pRBC were quartered and stored at 2 °C-6 °C. The supernatant of pRBC was obtained by centrifugation with 1 006 × g for 10 min at day 0, 14, 21 and 35 d. The enzyme-linked immunosorbent assay (ELISA) was used to detect the expression of platelet-derived growth factor(PDGF) and vascular endothelial growth factor (VEGF). After HepG2 cells was cultured with the supernatant of LR pRBC at day 0 and day 35 together for 48 hours, methylthiazoliltetracolium (MTT) method was used to measure the proliferation of tumor cells in vitro.

RESULTSThe concentrations of 2 cytokines were still increased with the storage time prolonging. As compared to LR-pRBC at day 0 [611.84 (95%CI, 356.45-867.23) pg/ml], the level of VEGF reached 900.16 (95% CI, 552.26-1248.07) pg/ml (P<0.05). There was a similar tendency in PDGF level with less increment in the supernatant of LR pRBC at day 35 [2.23 (95% CI, 0-5.37) ng/L vs 5.66 (95% CI, 0-12.48), P=0.073], but there was no significant statistical difference. Likewise, in vitro study of HepG2 cell proliferation showed that the LR-pRBC at day 35 promoted more proliferation of tumor cells with OD value 0.40 (95% CI, 0.38-0.42) (P<0.05).

CONCLUSIONThe residual platelets in LR-pRBC were activated, disintegrated and released the dense granules and α-granules which induce the accumulation of VEGF and PDGF. It seemed that the supernatant of LR-pRBC promoted the proliferation of tumor cells in vitro.

Blood Platelets ; Blood Preservation ; Cell Proliferation ; Cytokines ; Enzyme-Linked Immunosorbent Assay ; Humans ; In Vitro Techniques ; Platelet-Derived Growth Factor ; Time Factors ; Vascular Endothelial Growth Factor A

Diabetic retinopathy(DR) is the leading cause of new onset blindness among working-aged groups in industrialized countries, and its incidence is expected to increase along with the rising incidence of diabetes mellitus. Primary interventions such as strict glycemic control, tight blood pressure regulation, and lipid-lowering therapy can significantly reduce the risk of DR occurrence and progression. Currently, laser photocoagulation is the mainstay of treatment of proliferative DR and some cases of diabetic macular edema (DME). However, a considerable number of DR patients still suffer from severe visual impairment in spite of the application of laser photocoagulation and even of pars plana vitrectomy. Considering the limitations of current DR treatments, ongoing efforts have been devoted to the development of new therapeutic strategies, and it has become necessary to focus on pharmacologic treatment. Since inflammation has been identified as playing a substantial role in the pathogenesis of DR, corticosteroids with an anti-inflammatory effect can be included in the treatment of DR, though this may cause cataract and intraocular pressure elevation. The recent discovery of inhibitors of vascular endothelial growth factor is a revolutionary event in the management of DR, specifically DME. Some new agents aiming at the process of angiogenesis and increased vascular permeability are still under investigation, offering hope for a more effective future treatment of this sight-threatening disease. This paper reviews the current state of knowledge of the clinical presentation, preventive management, and clinical therapeutic strategies of DR and DME.
Adrenal Cortex Hormones ; Blindness ; Blood Pressure ; Capillary Permeability ; Cataract ; Developed Countries ; Diabetes Mellitus ; Diabetic Retinopathy* ; Hope ; Humans ; Incidence ; Inflammation ; Intraocular Pressure ; Laser Coagulation ; Light Coagulation ; Macular Edema ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors ; Vision Disorders ; Vitrectomy

OBJECTIVE: The purpose of this study was to evaluate the expression of epidermal growth factor-like domain 7 (EGFL7) in epithelial ovarian cancer, and to assess its relevance to clinicopathological characteristics and patients' survival. METHODS: A total of 177 patients with epithelial ovarian cancer were enrolled in the current study. For each patient, a retrospective review of medical records was conducted. Immunohistochemical staining for EGFL7 was performed using tissue microarrays made with paraffin-embedded tissue block. EGFL7 expression levels were graded on a grade of 0 to 3 based on the percentage of positive cancer cells. We analyzed the correlations between the expression of EGFL7 and various clinical parameters, and also analyzed the survival outcome according to the EGFL7 expression. RESULTS: The expression of EGFL7 in ovarian cancer tissues was observed in 98 patients (55.4%). High expression of EGFL7 (grade 2 or 3) was significantly correlated with pathologic type, differentiation, stage, residual tumor after debulking surgery, lymphovascular space involvement, lymph node metastasis, high cancer antigen 125, peritoneal cytology, and ascites. Among these clinicopathologic factors, differentiation was significantly correlated with EGFL7 expression in multivariate analysis (p<0.05). Survival analysis showed that the patients with high EGFL7 expression had a poorer disease free survival than those with low EGFL7 expression (p=0.002). CONCLUSION: Our data suggest that EGFL7 expression is a novel predictive factor for the clinical progression of epithelial ovarian cancer, and may constitute a therapeutic target for antiangiogenesis therapy in patients with epithelial ovarian cancer.
Adult ; CA-125 Antigen/blood ; Cell Differentiation/physiology ; Endothelial Growth Factors/*metabolism ; Female ; Humans ; Lymphatic Metastasis ; Middle Aged ; Neoplasm Proteins/metabolism ; Neoplasm Staging ; Neoplasm, Residual ; Neoplasms, Glandular and Epithelial/*diagnosis/pathology/surgery ; Ovarian Neoplasms/*diagnosis/pathology/surgery ; Prognosis ; Retrospective Studies ; Survival Analysis ; Tumor Markers, Biological/*metabolism