The purpose of this study was to devise an expanded ischemic flap model and to investigate the role of AMD-3100 (Plerixafor, chemokine receptor 4 inhibitor) in this model by confirming its effect on mobilization of stem cells from the bone marrow. Male Sprague-Dawley rats were used as an animal research model. The mobilization of stem cells from the bone marrow was confirmed in the AMD-3100-treated group. The fractions of endothelial progenitor cells (EPC) and the vascular endothelial growth factor receptor (VEGFR) 2+ cells in the peripheral blood were increased in groups treated with AMD-3100. The expression of vascular endothelial growth factor (VEGF) was increased in response to expansion or AMD injection. The expression of stromal cell derived factor (SDF)-1 and VEGFR2 were increased only in unexpanded flap treated with AMD-3100. Treatment with AMD-3100 increased both the number and area of blood vessels. However, there were no statistically significant differences in the survival area or physiologic microcirculation in rats from the other groups. This endogenous neovascularization induced by AMD-3100 may be a result of the increase in both the area and number of vessels, as well as paracrine augmentation of the expression of VEGF and EPCs. However, the presence of a tissue expander under the flap could block the neovascularization between the flap and the recipient regardless of AMD-3100 treatment and expansion.
Animals ; Anti-HIV Agents/pharmacology ; Bone Marrow Cells/cytology ; Chemokine CXCL12/biosynthesis ; Endothelial Progenitor Cells/*cytology ; Hematopoietic Stem Cells/*cytology ; Heterocyclic Compounds/*pharmacology ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism ; Male ; Neovascularization, Physiologic ; Nitric Oxide Synthase Type III/metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, CXCR4/antagonists & inhibitors ; Surgical Flaps/*blood supply/surgery ; Tissue Expansion/*methods ; Vascular Endothelial Growth Factor A/biosynthesis ; Vascular Endothelial Growth Factor Receptor-2/biosynthesis/metabolism

This study sought to construct recombinant eukaryotic plasmid pcDNA3. 1-sFlt-1 and observe its effect on proliferation of vascular endothelial cells. Total RNA was extracted from human umbilical vein endothelial cells (HUVECs) firstly. The 1st-3rd Ig-like domains of Flt were amplified by polymerase chain reaction (PCR) from the full-length cDNA. Subsequently, the PCR product was cloned into the eukaryotic plasmid pcDNA3.l(+)/myc-His. The constructed recombinant plasmid pcDNA3. 1-sFlt-1 was sequenced. Then recombinant plasmid was transfected into Lewis lung cancer cells. RT-PCR and SDS-PAGE were used to detect the expression of soluble vascular endothelial growth factor (VEGF) receptor mRNA and protein, respectively. MTT method was used to evaluate the effect of sFlt-1 protein on proliferation of HUVECs induced by VEGF. The results showed: (1) The sequence of inserted fragment was correct. (2) Lewis lung cancer cells with recombinant plasmid transfection could express the soluble VEGF receptor mRNA and protein stably. (3) Culture supernatant of Lewis lung cancer cells with sFlt-1 could significantly inhibit the proliferation of HUVECs induced by VEGF. These data suggested that recombinant eukaryotic plasmids pcDNA3. 1-sFlt-1 was constructed successfully, sFlt-1 mRNA and protein were expressed in eukaryotic system stably and sFlt-1 protein could significantly inhibit the proliferaton of endothelial cells induced by VEGF.
Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cells, Cultured ; Genetic Vectors ; genetics ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; Transfection ; Vascular Endothelial Growth Factor A ; pharmacology ; Vascular Endothelial Growth Factor Receptor-1 ; biosynthesis ; genetics ; pharmacology

OBJECTIVETo study the expressions of the vascular endothelial growth factor (VEGF) and its receptor fms-like tyrosine kinase-1 (Flt-1) in the testis, epididymis and epididymal sperm of adolescent rats and explore the functions of both the proteins in the male reproductive system.

METHODSThe expressions of VEGF and Flt-1 were detected in 20 adolescent SD rats, immunohistochemical staining used for both the testis and the epididymis and immunofluorescent staining for sperm.

RESULTSVEGF and Flt-1 proteins were specifically present in the testis, epididymis and sperm. In the testis, VEGF immunoreactive particles were localized in the cytoplasm of spermatogenic cells, the developing acrosome of spermatids, Sertoli cells and Leydig cells, while Flt-1 expressed mainly in the developing acrosome of spermatids and Leydig cells. In the epididymis, the cell-specific and region-specific expressions of VEGF and Flt-1 proteins were observed in the principal cells of epididymal epithelia, VEGF in the whole epididymis, while Flt-1 only in the caput and cauda segments. Both VEGF and Flt-1 were localized in the acrosome of the sperm head as well as in the neck, middle and principal segments of the sperm tail.

CONCLUSIONThe specific expression patterns of VEGF and Flt-1 in the rat testis, epididymis and sperm indicate that they may independently or collectively affect spermatogenesis and spermiotelcosis in either an autocrinological or a

Animals ; Epididymis ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Sexual Maturation ; Spermatozoa ; metabolism ; Testis ; metabolism ; Vascular Endothelial Growth Factor A ; biosynthesis ; Vascular Endothelial Growth Factor Receptor-1 ; biosynthesis

OBJECTIVETo investigate the expressions of VEGF in prostate cancer (PCa) and benign prostatic hyperplasia (BPH), their clinical significance and their relationship with that of ET-1.

METHODSA total of 44 specimens of PCa and 36 of BPH tissues were examined by the immunohistochemical Elivision plus method for the expressions of VEGF and ET-1. The intensity of staining for VEGF and ET-1 was assessed by light microscopy on a scale from "-" to "+ + +".

RESULTSThe rates of positive expression of VEGF were 69.4% in BPH and 80.9% in PCa, positive staining mostly in the cytoplasm of glandular epithelia and cancer cells, and strongly positive in all the stroma vascular endothelial cells. The staining intensity of VEGF was significantly higher in the PCa than in the BPH group (P < 0.05) , in the bone metastasis (BM) than in the non-BM group (P < 0.01), and in the lowly than in the highly and moderately differentiated PCa tissues (P < 0.01). The expression of VEGF was positively correlated with that of ET-1 ( r(s) = 0.780, P < 0.01).

CONCLUSIONVEGF is involved in the development, progression and metastasis of PCa. VEGF and ET-1 may play a joint role in its development and progression.

Aged ; Aged, 80 and over ; Endothelin-1 ; biosynthesis ; Humans ; Male ; Middle Aged ; Neoplasm Metastasis ; Neoplasm Staging ; Prostatic Hyperplasia ; metabolism ; pathology ; Prostatic Neoplasms ; metabolism ; pathology ; Vascular Endothelial Growth Factor Receptor-1 ; biosynthesis

The current study was purposed to investigate the inhibitory effect of human soluble vascular endothelial growth factor-1 (sFLT-1) on the proliferation of leukemic cells in vitro. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the VEGF mRNA and VEGF-R1 (FLT-1) mRNA in K562, HL60, U937 leukemic cell lines and bone marrow LTC-IC. Flow cytometry was used to detect the VEGF and VEGF-R1 (FLT-1) in all above-mentioned cells. VEGF concentrations in the cell culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). Cell proliferation was determined by MTT after adding sFLT-1 to K562, HL60 and LTC-IC culture system. The result showed that expression of VEGF could be detected in K562, HL60, U937 leukemic cell lines and LTC-IC, especially K562, K562 and HL60 cell lines also expressed FLT-1, but a little expression was found in U937 and LTC-IC. sFLT-1 could effectively inhibit the growth of K562 and HL60 cell lines in dose-dependent manner. The highest inhibition rate was found at 48 hours after adding sFLT-1. It is concluded that sFLT-1 can inhibit the growth of some leukemic cell lines, and the inhibition effect enhances as the concentration of the sFLT-1 increase, but sFLT -1 not influence the proliferation of normal marrow cells.
Cell Proliferation ; drug effects ; HL-60 Cells ; Humans ; K562 Cells ; RNA, Messenger ; biosynthesis ; genetics ; U937 Cells ; Vascular Endothelial Growth Factor A ; antagonists & inhibitors ; biosynthesis ; genetics ; Vascular Endothelial Growth Factor Receptor-1 ; biosynthesis ; genetics ; physiology

OBJECTIVETo investigate the expression of Angiotensin II type 1 receptor (AT1R) in tissue and cell lines of squamous cervical carcinomas and its clinical significance, and to explore the molecular mechamisms of angiotensin II and AT1R activity in the process of cervical carcinogenesis.

METHODS(1) The levels of AT1R mRNA were examined by quantitative reverse transcriptase-polymerase chain reaction( RT-PCR) in paraffin-embedded tissues from 35 cases of cervical squamous cell carcinoma, 15 cases of cervical intraepithelial neoplasia (CIN), and 15 cases of normal cervix, and in Siha and C33a cells. The expression of AT1R protein in 65 specimens of cervix tissue sections was evaluated by immunohistochemistry. The corelation between the expressions of AT1R and its clinicopathologic features was analyzed accordingly. (2) After the Siha and C33a cells were treated at different concentrations of Angiotensin II (0, 10(-10) mol/L, 10(-9) mol/L, 10(-8) mol/L, 10(-7) mol/L, 10(-6) mol/L, 10(-5) mol/L) for different time in culture, the cell proliferation was determined by methylthiazolyl tetrazolium (MTT) assay. The vascular endothelial growth factor (VEGF) expression was examined by enzyme-linked immuno-absordent assay (ELISA).

RESULTS(1) AT1R mRNA expression was detected in the two cervix cancer cell lines. The positive rate of ATIR mRNA was 77.1%, 40.0% and 0, respectively, in squamous cell carcinomas, cervical intraepithelial neoplasia and normal cervical tissues, while their mRNA quantities were 0.3863 +/- 0.041, 0.0768 +/- 0.035 and 0, respectively. There was statistically a significant difference between them (P < 0.01). The average staining intensity of AT1R protein was stronger in invasive carcinoma cells than that in dysplasia tissues and normal ones (P < 0.01). Among 65 cases of squamous cell carcinomas, the expressions of AT1R mRNA and protein increased with pathological grading (P < 0.05), while it was neither correlated with clinical stage nor pelvic lymph node metastasis (P > 0.05). The level of AT1R protein expression corresponded to that of its mRNA. (2) Angiotensin II promoted the cell growth of cervical cancer cell lines Siha and C33a and induced secretion of VEGF from cells in a dose-dependent manner (P < 0.01), and the expression of VEGF was reversed by the addition of valsatan (an antagonist of angiotensin II type 1 receptor) (P < 0.01).

CONCLUSIONAngiotensin II is involved in the progression of cervical carcinoma, since it may increase the proliferation activity of cancer cells, induce secretion of VEGF through AT1R synchronously, and results in an increase of angiogenesis in tumors. It suggests that use of AT1R antagonists may be an useful therapeutic strategy for cervical carcinoma.

Angiotensin II ; pharmacology ; Angiotensin II Type 1 Receptor Blockers ; pharmacology ; Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cervical Intraepithelial Neoplasia ; genetics ; metabolism ; pathology ; Cervix Uteri ; metabolism ; pathology ; Dose-Response Relationship, Drug ; Enzyme-Linked Immunosorbent Assay ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Immunohistochemistry ; Lymphatic Metastasis ; Neoplasm Staging ; RNA, Messenger ; biosynthesis ; genetics ; Receptor, Angiotensin, Type 1 ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Tetrazoles ; pharmacology ; Uterine Cervical Neoplasms ; genetics ; metabolism ; pathology ; Valine ; analogs & derivatives ; pharmacology ; Valsartan ; Vascular Endothelial Growth Factor A ; secretion

The purpose of this study was to determine whether the levels of soluble fms-like tyrosine kinase-1 (sFlt-1) and placenta growth factor (PlGF) are altered during the second trimester in the plasma of women who subsequently develop preeclampsia. We performed a case-control study to compare the levels of sFlt-1 and PlGF in the preeclamptic (n=46) and normal pregnant women (n=100). The maternal plasma levels of sFlt-1 and PlGF were measured by enzyme-linked immunosorbent assay. The sFlt-1 levels were significantly higher in the preeclamptic women than in normal controls (p<0.001), while the PlGF levels were significantly lower (p<0.001). In normal controls, sFlt-1 levels were positively correlated (r=0.27, p=0.008), whereas, in the preeclamptic women, those were negatively correlated with the PlGF levels (r=-0.423, p=0.005). Furthermore, the log[sFlt-1/PlGF] ratio was significantly higher in the preeclamptic women than in normal controls (p<0.001). The receiver operating characteristic curve revealed a specificity of 78% with a diagnostic sensitivity of 80.4%; the optimal cut-off value of the log[sFlt-1/PlGF] ratio was 1.4 (95% CI 0.756-0.910, p<0.001). Preeclampsia showed a strong association with increased levels of sFlt-1 and decreased levels of PlGF in the second trimester maternal plasma. Accordingly, the sFlt-1/PlGF ratio may provide early prediction of subsequent development of preeclampsia.
Adult ; Biological Markers/metabolism ; Case-Control Studies ; Female ; Humans ; Immunoassay ; Middle Aged ; Placenta/metabolism ; Pre-Eclampsia/*diagnosis/*metabolism ; Pregnancy ; Pregnancy Proteins/*biosynthesis ; Pregnancy Trimester, Second ; ROC Curve ; Sensitivity and Specificity ; Vascular Endothelial Growth Factor Receptor-1/*biosynthesis

Angiogenesis is required for solid tumor growth and facilitates tumor progression and metastasis. The inhibition effects of O-(chloroacetyl-carbamoyl) fumagillol (TNP-470), an angiogenesis inhibitor, and gemcitabine, a chemotherapeutic agent, on expression of growth factors were investigated using human pulmonary adenocarcinoma cell line, A549. The A549 cells were divided into four groups: control group, 10(-6) mg/ml gemcitabine treated group, 10(-4) mg/ml TNP-470 treated group and gemcitabine+TNP-470 treated group. The mRNA and protein expression of vascular endothelial growth factor (VEGF) and its receptors, FMS-like tyrosine kinase-1 (FLT-1) and kinase insert domain-containing receptor (KDR), in different groups were measured. The growth of A549 cell cultured with gemcitabine or TNP-470 was inhibited in an almost dose-dependent manner. Although gemcitabine (10(-6) mg/ml) alone and TNP-470 (10(-4) mg/ml) alone had no effect on the mRNA and protein expression of VEGF and its receptors (FLT-1, KDR) in A549 cells compared to the control (P>0.05), 10(-6) mg/ml gemcitabine in combination with 10(-4) mg/ml TNP-470 had significant effect (P<0.01). Moreover, combination of the two drugs significantly inhibited the mRNA expression of VEGF, FLT-1 and KDR compared to either drug alone (P<0.05). This study suggests that combined treatment with TNP-470 plus gemcitabine may augment the antiangiogenic and antineoplastic effects in lung cancer cells in vitro.
Adenocarcinoma ; drug therapy ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Cell Line, Tumor ; Cell Proliferation ; Cyclohexanes ; administration & dosage ; Deoxycytidine ; administration & dosage ; analogs & derivatives ; Disease Progression ; Gene Expression Regulation, Neoplastic ; Humans ; Lung Neoplasms ; drug therapy ; Neoplasm Metastasis ; Protein Structure, Tertiary ; Sesquiterpenes ; administration & dosage ; Vascular Endothelial Growth Factor A ; biosynthesis ; Vascular Endothelial Growth Factor Receptor-1 ; biosynthesis ; Vascular Endothelial Growth Factor Receptor-2 ; biosynthesis

OBJECTIVE: To investigate the effect of probucol and losartan on the prevention of restenosis after balloon angioplasty in hypercholesterolaemic rabbits, and to examine the expression of growth factors. METHODS: Forty male New Zealand white rabbits were randomly divided into high cholesterol diet group, probucol group, losartan group and combined drugs group. After one week of diet, all rabbits were injured on iliac arteries with balloon. Four weeks after the injury, the morphology of the iliac arteries of the rabbits were observed, and the insulin-like growth factor-I receptor (IGF-IR) and vascular endothelial growth factor (VEGF) were examined by immunohistochemical methods. RESULTS: Compared with the high cholesterol diet group, the lumen areas of the probucol group, losartan group and combined drugs group were larger (P < 0.01), the intimal areas were smaller (P < 0.05), and the expression of IGF-IR and VEGF significantly decreased (P < 0.05). There was no statistically significant difference among the three groups. CONCLUSION Probucol and losartan can prevent the restenosis of rabbits' iliac artery from balloon injury, and inhibit the expression of IGF-IR and VEGF. There is no statistical difference between combined drugs and single drug administration.
Angioplasty, Balloon ; adverse effects ; Animals ; Anticholesteremic Agents ; pharmacology ; therapeutic use ; Coronary Restenosis ; prevention & control ; Drug Therapy, Combination ; Hypercholesterolemia ; therapy ; Losartan ; pharmacology ; therapeutic use ; Male ; Probucol ; pharmacology ; therapeutic use ; Rabbits ; Random Allocation ; Receptor, IGF Type 1 ; biosynthesis ; genetics ; Vascular Endothelial Growth Factor A ; biosynthesis ; genetics

OBJECTIVETo investigate the effect of rhodiola on expression of vascular endothelial growth factors receptors (VEGFR) in myocardium of rats after myocardial infarction.

METHODSOn the basis of successful establishment of myocardial infarction rat model, the experimental animals were divided into the model group, the rhodiola group, the positive control group and the sham-operated group, they were sacrificed after 6 weeks feeding. Their hearts were resected and embedded in paraffin to make sections with standard immunohistochemistry stain. Then the stained slices were analyzed in the IMS cell imagine analysis system using immunohistochemical quantitative analysis software. The field of vision of left ventricular myocardial tissue in three sites selected from the marginal area of infarction in each slice were determined, the mean value was then converted to positive area. Meanwhile, the mean optical density (OD) was calculated and the various expressions of VEGFR, i.e. Flt-1, KDR and angiopoietin receptor (Tie-2) were measured.

RESULTSThe expressions of Flt-1 and Tie-2 in myocardial tissue were significantly increased in the rhodiola treated group after treatment, showing significant difference as compared with those in the positive control group and the model group (P < 0.05). The expression of KDR in myocardium after rhodiola intervention was higher than that in the sham-operated and nonintervened group (P < 0.05), but insignificantly different to that in the positive control group and model group.

CONCLUSIONRhodiola could improve angiogenesis to ameliorate myocardial ischemia by regulating the expression of Flt-1 and Tie-2 in ischemic myocardium.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Male ; Myocardial Infarction ; metabolism ; Myocardium ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptor, TIE-2 ; biosynthesis ; genetics ; Receptors, Vascular Endothelial Growth Factor ; biosynthesis ; genetics ; Rhodiola ; Vascular Endothelial Growth Factor Receptor-1 ; biosynthesis ; genetics ; Vascular Endothelial Growth Factor Receptor-2 ; biosynthesis ; genetics