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

To establish vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) as secretary biomarkers for cell growth on topographic substrates, we have evaluated the secretion and expression of these 2 factors by SH-SY5Y human neuroblastoma cells on poly-L-lactide (PLLA) micropillar arrayed topographic substrates. We fabricated topographic substrates with UV lithography, silicon etching and polydimethylsiloxane-based replica molding, and interfaced SH-SY5Y human neuroblastoma cells with both the topographic substrates and PLLA flat substrates. Cell morphology and spreading were examined with scanning electron microscopy. The secretion and mRNA expression of VEGF and IL-8 were evaluated with enzyme linked immunosorbent assay (ELISA) and real time qPCR, respectively, 24 hours after cell plating. We successfully achieved 4 topographic substrates with a nominal pillar diameter of 2 microm and 4 microm, and a nominal pillar spacing of 2 microm and 7 microm. We found that the secretion and mRNA expression of VEGF and/or IL-8 by SH-SY5Y cells on 2-2 microm (pillar diameter-spacing), 4-2 microm and 4-7 microm topographic substrates were upregulated in comparison to those by cells on PLLA flat substrate, 24 hours after cell plating. Furthermore, both cytokines were even more substantially upregulated on the 2-7 microm substrate than on the other 3 topographic substrates. Compared to those on PLLA flat substrate, cells on topographic substrates showed significant changes in morphology (spreading area, perimeter and roundness), and the increase in the secretion and mRNA expression of VEGF and IL-8 was accompanied with a decrease in cell spreading areas. These results provided evidence that pillar arrayed topography was an important microenvironmental factor in affecting VEGF and IL-8 expression or secretion, and VEGF and IL-8 might serve as important secretary biomarkers for growth on topographic substrates by SH-SY5Y cells.
Biomarkers ; Cell Line ; Cell Proliferation ; Cellular Microenvironment ; Humans ; Interleukin-8 ; genetics ; secretion ; Neuroblastoma ; secretion ; Polyesters ; chemistry ; RNA, Messenger ; genetics ; Vascular Endothelial Growth Factor A ; genetics ; secretion

OBJECTIVETo discuss the mechanism of traditional Chinese medicines reducing phlegm and resolving masses in treatment of iodine deficiency-induced goiter by observing the expression of growth factors and the balance-regulating mechanism of proliferation and apoptosis.

METHOD180 four-week-old Wistar rats were selected to establish the iodine deficiency model. After the modeling, the rats were randomly divided into six groups: the normal control group, the model control group, the iodine group, the phlegm compound group, the L-T4 group and the phlegm compound and L-T4 group. At the 21st day and 77th day after administration, 15 rats in each group were killed to collect specimens. Doses were calculated and adjusted according to body surface area and body weight. TT3, TT4 radioimmunoassay, TSH, immunoradiometric method were adopted. Fas, FasL and PCNA protein expressions are detected using immunohistochemical methods.

RESULTCompared with the normal group and the model group, the expressions of fas and FasL in the phlegm Group significantly increased, the expressions of fas and FasL in the phlegm and L-T4 group were also increased significantly. The expression of fas in the L-T4 Group was significantly lower than that of the L-T4 group and the phlegm compound and L-T4 group. Compared with the normal group, the expression of PCNA of the phlegm group and the phlegm and L-T4 group was significantly lower. Compared with the model group, the expression of PCNA of the iodine group, the phlegm groups and the phlegm and L-T4 group were significantly lower. Compared with the normal group, the expression of VEGF in the iodine group significantly decreased after treatment. Compared with the iodine group, the expression of VEGF in the phlegm group and the L-T4 group significantly reduced. Compared with the normal group, the expression of TGF-beta1 in the model group and the phlegm group significantly increased. Compared with model group, the expression of TGF-beta1 in the iodine group significantly reduced. Compared with the phlegm group, the expression of TGF-beta1 in the phlegm compound and L-T4 group was significantly reduced.

CONCLUSIONTraditional Chinese medicines reducing phlegm and resolving masses can completely recover goiter by promoting apoptosis of thyroid cells, inhibiting their proliferation and the expression of growth factors and enhancing the expression of TGF-beta, without causing injury on thyroid cells.

Animals ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Gene Expression ; drug effects ; Goiter ; drug therapy ; genetics ; metabolism ; Humans ; Male ; Proliferating Cell Nuclear Antigen ; genetics ; metabolism ; Rats ; Rats, Wistar ; Thyroid Hormones ; secretion ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

OBJECTIVETo explore the role of RhoA in regulating vascular endothelial growth factor (VEGF) secretion level in breast cancer cells and in the proliferation, migration and tube formation of human umbilical vascular endothelial cells (HUVECs) under hypoxia.

METHODSEnzyme-linked immunosorbent assay was used to examine the effect of V14RhoA plasmid transfection-induced RhoA activation and RhoA knockdown on VEGF secretion level in breast cancer MCF-7 cells under hypoxic condition. A MCF-7/HUVEC co-culture model was established to assess the effect of the changes in RhoA expressions in MCF-7 cells on HUVEC proliferation, migration, and tube formation under hypoxia.

RESULTSUnder hypoxic condition, RhoA activation promoted VEGF secretion in MCF-7 cells, and RhoA knockdown inhibited VEGF secretion. In the co-culture model, RhoA activation in the MCF-7 cells enhanced HUVEC proliferation, migration, and tube formation, and RhoA knockdown inhibited these changes.

CONCLUSIONUnder hypoxic condition, RhoA indirectly influences HUVECs to affect tumor angiogenesis by regulating VEGF level in breast cancer cells.

Breast Neoplasms ; metabolism ; pathology ; Cell Hypoxia ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Female ; Gene Knockdown Techniques ; Human Umbilical Vein Endothelial Cells ; cytology ; secretion ; Humans ; Transfection ; Vascular Endothelial Growth Factor A ; secretion ; rhoA GTP-Binding Protein ; metabolism

To investigate the effects of oleanolic acid (OA) on the proliferation, migration and the formation of tube-like structure in human vascular endothelial cells (HUVECs). MTT assay, flat plate scarification, Transwell plates and matrigel-induced tube formation assay were performed to detect the effects of OA on proliferation, migration and tube formation. MTT assay showed that the inhibition rates of HUVECs treated with 60 and 100 microg x mL(-1) of OA for 24 h were 19% and 83% respectively. Treatment of HUVECs significantly inhibited the cell migration in a dose-dependent manner. The vascular indexes of HUVECs treated with 40 and 60 microg x mL(-1) OA were 33% and 20% respectively. Western blotting analysis showed that treatment of the cells with OA significantly attenuated the expression and secretion of VEGF. Additionally, VEGF could in part reverse the effects of OA on migration and tube formation of HUVECs. In conclusion, OA inhibits the proliferation, and VEGF plays an important role in OA induced decreased migration and tube formation of HUVECs.
Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Human Umbilical Vein Endothelial Cells ; cytology ; metabolism ; Humans ; Neovascularization, Physiologic ; drug effects ; Oleanolic Acid ; administration & dosage ; pharmacology ; Signal Transduction ; drug effects ; Vascular Endothelial Growth Factor A ; metabolism ; secretion

To investigate the potential ability of the nitrite to induce neuronal differentiation of PC12 cells, cultured PC12 cells planted on matrigel in the presence or absence of sodium nitrite were employed as model, nerve growth factor (NGF) served as a positive control. After 48 h, sodium nitrite enhanced cell viability and vascular endothelial growth factor (VEGF) secretion. Same as the effect of NGF, sodium nitrite (1.4 mmol x L(-1)) treated cultures contained a greater proportion of cells bearing neurites and neurites were much longer than those found in negative control cultures (P < 0.05). Compared with the negative control, sodium nitrite (1.4 mmol x L(-1)) also upregulated the expression of VEGF mRNA (P < 0.05) and hypoxia inducible factor 1 alpha (HIF-1 alpha) or VEGF protein expression (P < 0.05) in cultures of PC12 cells. On the other hand, these effects of the sodium nitrite were likely mediated by HIF-1alpha, since their effects were antagonized by addition of HIF-1alpha inhibitor YC-1. Taken together, these results suggest that low doses of sodium nitrite could induce neurite outgrowth in PC12 cells by activating the HIF-1alpha-VEGF pathway.
Animals ; Cell Differentiation ; drug effects ; Cell Survival ; drug effects ; Food Preservatives ; pharmacology ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Neurites ; drug effects ; PC12 Cells ; RNA, Messenger ; metabolism ; Rats ; Sodium Nitrite ; pharmacology ; Up-Regulation ; Vascular Endothelial Growth Factor A ; genetics ; secretion

Platelet-rich plasma (PRP) contains growth factors that promote tissue regeneration. Previously, we showed that heparin-conjugated fibrin (HCF) exerts the sustained release of growth factors with affinity for heparin. Here, we hypothesize that treatment of skin wound with a mixture of PRP and HCF exerts sustained release of several growth factors contained in PRP and promotes skin wound healing. The release of fibroblast growth factor 2, platelet-derived growth factor-BB, and vascular endothelial growth factor contained in PRP from HCF was sustained for a longer period than those from PRP, calcium-activated PRP (C-PRP), or a mixture of fibrin and PRP (F-PRP). Treatment of full-thickness skin wounds in mice with HCF-PRP resulted in much faster wound closure as well as dermal and epidermal regeneration at day 12 compared to treatment with either C-PRP or F-PRP. Enhanced skin regeneration observed in HCF-PRP group may have been at least partially due to enhanced angiogenesis in the wound beds. Therefore, this method could be useful for skin wound treatment.
Animals ; Blotting, Western ; *Cell Proliferation ; Dermis/cytology/metabolism ; Female ; Fibrin/*metabolism ; Fibroblast Growth Factor 2/genetics/metabolism ; Heparin/metabolism ; Immunoenzyme Techniques ; Intercellular Signaling Peptides and Proteins/*secretion ; Mice ; Mice, Inbred BALB C ; Platelet-Rich Plasma/*metabolism ; Proto-Oncogene Proteins c-sis/genetics/metabolism ; RNA, Messenger/genetics ; Real-Time Polymerase Chain Reaction ; Regeneration ; Skin/*cytology/*metabolism ; Vascular Endothelial Growth Factor A/genetics/metabolism ; Wound Healing/*physiology

OBJECTIVETo study the biological effects of the paracrine from ADSC after being stimulated by insulin on vascular endothelial cells.

METHODS(1) ADSC was isolated from human adipose tissue and cultured in vitro. The third generation cells were collected and divided into insulin group (I, cultured with serum-free DMEM containing 1 x 10(-7) mol/L insulin) and control group (C, cultured with serum-free DMEM) according to the random number table, with 6 slots in each group. Three days later, ADSC culture medium (ADSC-CM) was collected for determination of levels of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) by ELISA. (2) Human umbilical vein endothelial cells (HUVEC) were cultured to the third generation, and they were cultured with special nutrient solution and divided into ADSC-CM with insulin stimulation group (AI), ADSC-CM without insulin stimulation group (AC), insulin group (I, with same concentration as above), blank control group (BC) according to the random number table. Three days later, proliferation of HUVEC was determined with MTT method (with expression of absorbance value). Another two samples of HUVEC were respectively divided into 4 groups as above for determination of apoptosis rate with Annexin V/FITC double-staining 12 hours after culture, and HUVEC migration with scratch adhesion test at post scratch hour (PSH) 12, 24, 36, 48. Data were processed with t test.

RESULTS(1) Compared with those in C group [(287 +/- 47), (577 +/- 84) pg/mL, respectively], the secretion levels of VEGF and HGF in I group [(643 +/- 64), (930 +/- 68) pg/mL, respectively] were significantly increased (with t value respectively 18.869, 18.475, P values all below 0.05). (2) The absorbance value of HUVEC in AI and AC groups was 0.847 +/- 0.042, 0.798 +/- 0.022, respectively, which were higher than that in I and BC groups [0.665 +/- 0.028 (with t value respectively 4.579, 3.732), 0.674 +/- 0.031 (with t value respectively 3.761, 4.073), P values all below 0.01], and that in AI group was higher than that in AC group (t = 2.576, P < 0.05). The apoptosis rates of HUVEC in AI and AC groups [(5.8 +/- 1.9)%, (9.0 +/- 2.0)%, respectively] were obviously lower as compared with that in I and BC groups [(30.4 +/- 6.0)% (with t value respectively 12.891, 10.417), (31.4 +/- 7.4)% (with t value respectively 11.474, 9.783), P values all below 0.05 ], and that in AC group was higher than that in AI group (t = 8.548, P < 0.05). The distance of migration of HUVEC in AI and AC groups were greater than that in I and BC groups at PSH 36, 48, and that in AI group was greater as compared with that in AC group (with t value respectively 4.076, 4.573, P values all below 0.05).

CONCLUSIONSParacrine from ADSC after being stimulated by insulin can promote proliferation and migration of HUVEC, and suppress its apoptosis, and it is beneficial for tissue vascularization.

Adipocytes ; cytology ; secretion ; Adipose Tissue ; cytology ; Apoptosis ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; Endothelial Cells ; cytology ; metabolism ; Hepatocyte Growth Factor ; metabolism ; Human Umbilical Vein Endothelial Cells ; cytology ; metabolism ; Humans ; Insulin ; pharmacology ; Stem Cells ; cytology ; secretion ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo explore the expression of integrin-linked kinase (ILK) and its effect on VEGF expression in fibroblasts from human hypertrophic scar.

METHODSFibroblasts were isolated from hypertrophic scar of 8 patients and cultured in vitro. Then the cells were divided into three groups: (1) Cells were cultured only in DMEM containing 10% FCS in the control group; (2) Cells were transfected with empty plasmid in the empty plasmid group; (3) Cells were transfected with plasmid expressing ILKcDNA in the ILK cDNA plasmid transfection group. First, the expression of ILK and VEGF was observed by immunocytochemistry before and after ILK cDNA transfection. Second, ILK and VEGF mRNA expression was investigated by real-time PCR (RT-PCR). Third, the protein expression of ILK and VEGF was detected by Western blot. Finally, the protein level of VEGF in supernatant of fibroblasts was measured by ELISA.

RESULTSBefore ILK cDNA transfection, the expression of ILK was positive and the VEGF expression was weak in cytoplasm of fibroblasts . After ILK cDNA transfection, both the expression of ILK and VEGF was enhanced. The level of VEGF mRNA was significantly higher in ILK cDNA transfection group (0.338 +/- 0.060) than that in control group (0.022 +/- 0.001) and empty plasmid group (0.028 +/- 0.005, P < 0.05). The level of VEGF protein was significantly higher in ILK cDNA transfection group (0.819 +/- 0.019) than that in control group (0.607 +/- 0.033) and empty plasmid group (0. 591 +/- 0.024, P<0. 05). Secretion of VEGF increased remarkably in ILK cDNA transfection group comparing with the other two groups (P < 0.05).

CONCLUSIONSILK could up-regulate the VEGF mRNA and protein level in human scar fibroblasts. It may play an important role in the angiogenesis in hypertrophic scar.

Cells, Cultured ; Cicatrix, Hypertrophic ; genetics ; metabolism ; pathology ; Fibroblasts ; secretion ; Humans ; Plasmids ; Protein-Serine-Threonine Kinases ; genetics ; RNA, Messenger ; genetics ; Transfection ; Vascular Endothelial Growth Factor A ; metabolism

Angiogenesis is a multi-step process that involves the activation, proliferation, and migration of endothelial cells. We have recently shown that TGF-beta1 can induce mouse macrophages to produce VEGF, a potent angiogenic factor. In the present study, we explored whether TGF-beta1 has a similar effect on mouse dendritic cells. First, we show that under hypoxic conditions, TGF-beta1 induced the expression of VEGF transcripts in bone marrow-derived dendritic cells. Overexpression of Smad3/4 further augmented TGF-beta1-induced VEGF transcription, while overexpression of DN-Smad3 decreased VEGF transcription in DC2.4 cells, a mouse dendritic cell line. We also show that TGF-beta1 and Smads are involved in the induction of VEGF protein secretion. Interestingly, under the same conditions, the expression of VEGF receptor 1 (Flt-1) was also elevated at both the transcriptional and protein levels. Additionally, we found that the TGF-beta1-induced VEGF secretion in activated DC2.4 cells has wound-healing properties. Finally, Smad7 and Smurf1 negatively regulated the TGF-beta1-induced and Smad3/4-mediated VEGF expression. Taken together, these results indicate that TGF-beta1 can enhance the expression of VEGF and Flt-1 through the typical Smad pathway in mouse dendritic cells.
Angiogenesis Inhibitors/*metabolism ; Animals ; Cell Line ; Dendritic Cells/*metabolism ; Macrophages/metabolism ; Mice ; Mice, Inbred BALB C ; RNA, Messenger/metabolism ; Signal Transduction ; Smad2 Protein/metabolism ; Smad3 Protein/metabolism ; Smad4 Protein/metabolism ; Smad7 Protein/metabolism ; Transforming Growth Factor beta1/metabolism/*pharmacology ; Vascular Endothelial Growth Factor A/*secretion ; Vascular Endothelial Growth Factor Receptor-1/*metabolism