It has been demonstrated that the activated protein C (APC) plays an important role in the inhibition of inflammation. The activation of protein C can be significantly enhanced by the endothelial cell protein C receptor (EPCR). Previous studies proposed that the APC regulates the inflammatory response in endothelial cells by suppressing the expression of adhesion molecules and the secretion of chemokines and cytokines. However, the precise mechanism of the inhibitory effect of APC on inflammation is still poorly understood. In the present study, we evaluated the anti-inflammatory effect of recombinant human APC (rhAPC) and whether its inhibitory effect is conducted through the EPCR-dependent mechanism on human umbilical vein endothelial cells (HUVECs). By exposing HUVECs to: (1) TNF-alpha; (2) rhAPC plus TNF-alpha; (3) anti EPCR antibody that prevents rhAPC interaction with EPCR; (4) TNF-alpha plus anti EPCR antibody; (5) rhAPC plus TNF-alpha in the presence of anti EPCR antibody, we found that APC was able to significantly inhibit the TNF-alpha-induced secretion of cytokines such as IL-1beta and IL-8, as well as the expression of adhesion molecules such as ICAM-1, VCAM-1 and E-selction in HUVECs. These results reveal a novel pathway by which APC protects endothelial cells from inflammatory mediators through an EPCR-dependent mechanism.
Antigens, CD ; metabolism ; Cells, Cultured ; Cytokines ; metabolism ; Endothelial Protein C Receptor ; Human Umbilical Vein Endothelial Cells ; drug effects ; Humans ; Inflammation ; metabolism ; Protein C ; pharmacology ; Receptors, Cell Surface ; metabolism ; Tumor Necrosis Factor-alpha ; antagonists & inhibitors

The protein C anticoagulant pathway plays a fundamental role in the control of coagulation system and inflammatory response. It has been well established that physiological levels of shear stress induce endothelial structural change and modulate gene and protein expression. However, the role of shear stress in protein C pathway remains unknown. In the present study, we evaluated the effect of shear stress on the activation of protein C as well as on the expression of endothelial protein C receptor (EPCR) and thrombomodulin (TM) in human umbilical vein endothelial cells (HUVECs) which were exposed to TNF-alpha alone, shear stress alone, and TNF-alpha under shear stress. We found: (1) Either TNF-alpha or shear stress alone significantly reduced EPCR expression and protein C activation in HUVECs; and simultaneous exposure of HUVECs to TNF-alpha and shear stress resulted in a further decrease of EPCR expression and protein C activation (P<0.05); (2) Simultaneous exposure of HUVECs to TNF-alpha and shear stress resulted in the increase of soluble EPCR level more significantly than did the exposure of HUVECs to either TNF-alpha or shear stress alone (P<0.05); (3) Shear stress significantly increased TM expression on HUVECs, whereas TNF-alpha inhibited TM expression; shear stress could strongly neutralize TNF-alpha's inhibitive effect on TM expression. We therefore conclude that shear stress may play an important role in protein C pathway, which may be fulfilled by regulating EPCR expression and TM expression in endothelial cells.
Antigens, CD ; genetics ; metabolism ; Biomechanical Phenomena ; Cells, Cultured ; Endothelial Cells ; cytology ; metabolism ; Endothelial Protein C Receptor ; Humans ; Protein C ; metabolism ; Receptors, Cell Surface ; genetics ; metabolism ; Shear Strength ; Stress, Mechanical ; Thrombomodulin ; genetics ; metabolism ; Umbilical Veins ; cytology

endothelial growth factor (VEGF)-C and its tyrosine kinase receptor, VEGF receptor (VEGFR)-3 are recently known to have lymphangiogenic activities in various tumor types. In this study, we determined whether the expression of lymphangiogenic factors correlate with nodal metastasis or survival in a nude mouse model of oral squamous cell carcinoma (OSCC).METHODS: Three OSCC cells (KB, SCC4, SCC9) were xenografted into the right mandibular gland of athymic nude mice. The mice were followed for tumor development and growth, and the mice were sacrificed when they had lost more than 20% of their initial body weight, or the diameter of the induced tumor exceeds 20 mm. After necropsy, the murine tumors were examined histologically and radiologically (micro-positron emission tomography computed tomography) for regional or distant metastasis. We performed immunohistochemical assays with anti-VEGF-C, VEGFR-3, CD105, and D2-40 antibodies. Immunofluorescence double staining for LYVE-1/CD31 was also performed. To quantify the VEGF-C and VEGFR-3 level in the cancer tissue, Western blotting was performed. Finally, we determined the correlation between the degree of expression of VEGF-C/VEGFR-3 and the mean survival time.RESULTS: OSCC tumor cells into the mandibular gland of the nude mice successfully resulted in the formation of recapitulating orthotopic tumor. Tumor cells of the induced tumor did not express VEGF-C. VEGF-C/VEGFR-3 expression was mainly distributed in the endothelial cells of the stromal area. There were no correlation between the degree of expression of VEGF-C/VEGFR-3 and the mean survival time of mice injected with different OSCC cell lines.CONCLUSION: An recapitulating orthotopic model of OSCC in nude mice was established, which copies the cervical nodal metastasis of human OSCC. Overexpression of lymphangiogenic factors seems to have no effect on survival of hosts in this in vivo experiment.]]>
Animals ; Antibodies ; Blotting, Western ; Body Weight ; Carcinoma, Squamous Cell ; Coat Protein Complex I ; Endothelial Cells ; Fluorescent Antibody Technique ; Growth and Development ; Humans ; Mice ; Mice, Nude ; Neoplasm Metastasis ; Protein-Tyrosine Kinases ; Receptors, Vascular Endothelial Growth Factor ; Survival Rate ; Transplantation, Heterologous ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factor C ; Vascular Endothelial Growth Factor Receptor-3

BACKGROUNDTwist is a highly conserved epithelial-mesenchymal transcription factor that has been reported to be a key factor in tumor malignancy, including lymph node metastasis. It represents the major step of dissemination and serves as a chief prognostic indicator of disease progression. However, the mechanism by which Twist regulates lymph node metastasis remains incompletely understood. Studies on the mechanism of metastasis are thus required for determining appropriate therapeutic strategies.

METHODSImmunohistochemistry for lymphatic vessel endothelial receptor 1 (LYVE-1), Ki-67, Twist, vascular endothelial growth factor C (VEGF-C), and vascular endothelial growth factor receptor 3 (VEGFR-3) was performed to detect lymphatic vessel density (LVD), cell proliferation levels and the expressions of Twist, VEGF-C, and VEGFR-3 were determined from 66 primary supraglottic carcinoma tissue samples from 36 patients with lymph node metastasis (pathological N+, pN+) and 30 patients without metastasis (pathological N0, pN0). Western blotting analysis of the proteins in pN+ and pN0 primary tumors was used to characterize the expressions of Twist, VEGF-C, and VEGFR-3 further.

RESULTSThe LVD was 22.4 ± 10.3 in pN+ patients and 6.8 ± 4.1 in pN0 ones. For Ki-67, the number of proliferous cells in pN+ patients was greater than that in pN0 ones. Both, however, were associated with their clinical nodal stages. In pN+ patients, Twist, VEGF-C, and VEGFR-3 expressions were 86.11% (31/36), 80.56% (29/36), and 58.33% (21/36), respectively. These values were higher than those found for pN0 patients (i.e., 13/30, 11/30, and 7/30, respectively) (P < 0.05). Among the samples with Twist expression, 88.64% were VEGF-C-positive and 59.09% were VEGFR-3-positive. The pN0 counterparts were 4.55% and 9.09%, respectively (P < 0.05). The expressions of Twist, VEGF-C, and VEGFR-3 in pN+ patients obtained through Western blotting analysis were significantly higher than those in pN0 patients, and the levels of VEGF-C and VEGFR-3 were positively correlated with that of Twist.

CONCLUSIONSTwist expression correlates with lymph node metastasis. The mechanism involved in such a correlation may be related to lymphangiogenesis.

Adult ; Aged ; Blotting, Western ; Female ; Humans ; Immunohistochemistry ; Laryngeal Neoplasms ; complications ; metabolism ; Lymphangiogenesis ; genetics ; physiology ; Lymphatic Metastasis ; genetics ; pathology ; Male ; Middle Aged ; Twist-Related Protein 1 ; genetics ; metabolism ; Vascular Endothelial Growth Factor C ; metabolism ; Vascular Endothelial Growth Factor Receptor-3 ; metabolism

endothelial growth factor (VEGF)-C, VEGF-D and their tyrosine kinase receptor, VEGF receptor (VEGFR)-3 are recently known to have lymphangiogenic activities in various tumor types. Oral mucosal squamous cell carcinoma (OMSCC) easily metastasizes to cervical lymph nodes, so we determined the expression levels of VEGF-C, VEGF-D and VEGFR-3 in oral squamous cell carcinoma.MATERIALS AND METHODS: We performed Western blot analyses with 4 OMSCC cultured tumor cell lines (SCC9, KB, YD-10B, YD-38), and with 7 surgical specimens of OMSCC for the detection of VEGF-C, VEGF-D and VEGFR-3 proteins. Expression of VEGF-C mRNA as well as mRNA for VEGFR-3 in 4 OMSCC cell lines (KB, SCC-4, SCC-9, YD-10B) was investigated by RT-PCR. We also measured VEGFC/VEGF-D protein concentrations in the media and protein concentration of VEGFR-3 in cell lysates of 4 OMSCC cell lines (SCC9, KB, YD-10B, YD-38) using commerical ELISA kits. Finally, we performed immunoprecipitation for the detection of VEGF-C in cell lysates of 4 OMSCC cells (KB, SCC-4, SCC-9, YD-10B) and real-time RT-PCR for the quantification of VEGF-C mRNA.RESULTS: In the result of Western blotting with cell lysates of 4 OMSCC cells, we could not detect the protein expression of VEGF-C, VEGF-D, and VEGFR-3. But, all tumor tissues demonstrated VEGF-C and VEGFR-3. VEGF-C mRNA was detected at various levels in 4 OMSCC cell lines. Moreover, OMSCC cellssecreted VEGF-C, not VEGF-D and VEGFR-3 was also detected in cell lysates of OMSCC by ELISA.Immunoprecipitation and real-time RT-PCR revealed VEGF-C was also expressed in 4 OMSCC cell lines.CONCLUSION: Taken together, tumor cells of OMSCC secrete VEGF-C, not VEGF-D. And VEGFR-3 is expressed tumor cells as well as OMSCC tumor tissues, needs further study.]]>
Blotting, Western ; Carcinoma, Squamous Cell ; Cell Line ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Immunoprecipitation ; Lymph Nodes ; Protein-Tyrosine Kinases ; Proteins ; Receptors, Vascular Endothelial Growth Factor ; RNA, Messenger ; Tumor Cells, Cultured ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factor C ; Vascular Endothelial Growth Factor D ; Vascular Endothelial Growth Factor Receptor-3

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

OBJECTIVETo investigate the expressions of vascular endothelial growth factor-C (VEGF-C), C-erbB-2, p53, estrogen receptor (ER), and progesterone receptor (PR) in breast cancer tissue and their clinical significance.

METHODSThe expressions of C-erbB-2, p53, ER, and PR in 60 breast cancer tissues were detected using immunohistochemistry, and their clinical significance was analyzed.

RESULTSThe positivity rates of VEGF-C, C-erbB-2, p53, ER, and PR in the 60 breast cancer tissues were 56.7%, 38.3%, 46.7%, 48.3% and 53.3%, respectively. The expressions of VEGF-C and C-erbB-2 differed significantly in relation to the lymph node status (P<0.05), but not to the patient's age or tumor volume (P>0.05). The expression of VEGF-C was positively correlated to that of C-erbB-2 (P<0.05). The expression of p53 was positively correlated to the tumor volume (P<0.05). The expressions of ER and PR were not correlated to the patient's age, tumor volume and lymph node status (P>0.05), but were inversely correlated to C-erbB-2 expression (P<0.05) independent of VEGF-C and p53 expressions (P>0.05).

CONCLUSIONSThe high expressions of VEGF-C and C-erbB-2 are closely related to lymph node metastasis in breast cancer patients, and may cooperate in promoting lymph node metastasis of breast carcinoma. Combined detection of VEGF-C, C-erbB-2, p53, ER and PR may help clinical treatment and prognostic evaluation of breast cancer patients.

Adult ; Aged ; Breast Neoplasms ; metabolism ; pathology ; Female ; Humans ; Lymphatic Metastasis ; Middle Aged ; Progesterone ; metabolism ; Receptor, ErbB-2 ; metabolism ; Receptors, Estrogen ; metabolism ; Tumor Suppressor Protein p53 ; metabolism ; Vascular Endothelial Growth Factor C ; metabolism

The present study was aimed to investigate whether Bcl-2, Fas and Bax are involved in monocyte chemotacitic protein-1 (MCP-1)-induced apoptosis of human umbilical vein endothelial cells (hUVECs). hUVECs were cultured, and the purity was identified by immunofluorescence and immunohistochemistry with specific anti-von Willebrand factor (vWF) and anti-VEGF receptor-2 (KDR) antibodies. With 90% confluence hUVECs were serum-starved for 12 h, and then treated with different concentrations of MCP-1 (0.1, 1.0, 10, 100 ng/mL) for 24 and 48 h respectively. The expressions of apoptosis related proteins Fas, Bcl-2, Bax were detected by flow cytometry (FACS) and Western blot. As shown in our preliminary study, MCP-1 induced apoptosis of hUVECs in a dose-dependent manner at both 24 h and 48 h. FACS and Western blot analysis results in the present study indicated that MCP-1 promoted the expression of proapoptotic proteins Bax and Fas and inhibited the expression of antiapoptotic protein Bcl-2. These results suggest that MCP-1 may induce the apoptosis of hUVECs through evoking the imbalance between proapoptotic Fas/Bax and antiapoptotic Bcl-2 protein.
Apoptosis ; Atherosclerosis ; physiopathology ; Cells, Cultured ; Chemokine CCL2 ; metabolism ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; bcl-2-Associated X Protein ; metabolism ; fas Receptor ; metabolism

In order to explore the distribution frequency of endothelial protein C receptor (EPCR) gene A6936G variant and to study the correlation between this mutation and cerebral infarction in Chinese Han population of Hubei province district. The genotype and allele frequencies of EPCR A6936G were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 380 cerebral infarction patients and 380 healthy Chinese Han persons. The results indicated that the frequencies of A/A, A/G and G/G genotypes in cerebral infarction group were 77.1%, 22.1% and 0.8% respectively. While the frequencies of A/A and A/G genotypes in control group were 88.2% and 11.8%, and without G/G genotype. It is concluded that A6936G polymorphism of EPCR can be detected in Chinese Han population of Hubei province district, which may be correlated with the increasing risk of thrombosis in cerebral infarction patients.
Adult ; Aged ; Aged, 80 and over ; Antigens, CD ; genetics ; Asian Continental Ancestry Group ; genetics ; Case-Control Studies ; Cerebral Infarction ; genetics ; Endothelial Protein C Receptor ; Female ; Gene Frequency ; Genotype ; Humans ; Male ; Middle Aged ; Polymorphism, Genetic ; Receptors, Cell Surface ; genetics

OBJECTIVETo explore anti-angiogenesis effect of Mer, a member of tyrosine kinase receptor family, and its mechanism.

METHODSHuman Mer full length plasmid was transfected into HMEC-1 cells through liposome. G418 was used to select positive clone. Expression of Mer at mRNA and protein level was detected by real-time PCR and Western-blot, respectively. Transwell and Matrigel were used to evaluate the effect of overexpressed Mer on migration and angiogenesis of HMEC-1 cells. Primary angiogenesis associated factor VEGF-A, VEGF-B, VEGF-C, VEGF-D and VEGFR-1, VEGFR-2 were screened by real-time PCR.

RESULTSAfter G418 selection, the Mer expression in transfected HMEC-1 cells was increased 3.61- and 2.12 fold at mRNA and protein level, respectively. Compared with negative control, the migration of Mer-HMEC-1 was decreased (21 +/- 6 vs 36 +/- 11), and angiogenesis capability on Matrigel significantly decreased. By real-time PCR, the expression of VEGF-C and VEGFR-2 was down-regulated to 44.7% and 25.6% of the negative control.

CONCLUSIONOverexpressed Mer tyrosine kinase receptor can inhibit the migration and angiogenesis of HMEC-1 cells through VEGF-C/VEGFR-2 signal pathway.

Cell Line ; Cell Movement ; Cell Proliferation ; Endothelial Cells ; metabolism ; physiology ; Endothelium, Vascular ; cytology ; Humans ; Neovascularization, Physiologic ; Proto-Oncogene Proteins ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Receptor Protein-Tyrosine Kinases ; genetics ; metabolism ; Receptors, Vascular Endothelial Growth Factor ; metabolism ; Transfection ; Vascular Endothelial Growth Factors ; metabolism ; c-Mer Tyrosine Kinase