To determine the effect of andrographolide (Andro) on angiogenesis of human umbilical vein endothelial cells (HUVECs).
 Methods: HUVECs were treated with different concentrations of Andro and the cell viability was detected with Cell Counting Kit-8 (CCK-8). HUVECs were treated with half lethal dose (IC50) of Andro. Matrigel was used to make capillary formation of HUVECs and the effect of Andro on capillary formation was evaluated by calculating the percentage of capillary formation. Moreover, the effects of Andro and the supernatant from cultured A549 tumor cells on capillary formation were evaluated by calculating the percentage of capillary formation. The effect of Andro on the expression of matrix metalloproteinase-9 (MMP-9) was determined with Western blot.
 Results: The cell viability of HUVECs decreased with the increase of Andro concentrations. IC50 was 20 μmol/L. The capillary formation of HUVECs was inhibited when treated with 20 μmol/L Andro for 24 hours. Moreover, Andro was able to antagonize the promotion of the capillary formation induced by the supernatant from cultured tumor cells. Andro could suppress the expression of MMP-9 and antagonize the capillary formation.
 Conclusion: Andro inhibits the capillary formation of HUVECs and can antagonize the promotion of angiogenesis induced by the supernatant from cultured tumor cells.
Capillaries ; drug effects ; Cell Survival ; Collagen ; Culture Media ; Diterpenes ; pharmacology ; Drug Combinations ; Human Umbilical Vein Endothelial Cells ; drug effects ; Humans ; Laminin ; Matrix Metalloproteinase 9 ; metabolism ; Neovascularization, Pathologic ; enzymology ; etiology ; prevention & control ; Proteoglycans ; Tumor Cells, Cultured

OBJECTIVETo investigate the mechanisms of baicalin on anti-cerebral ischemic through observing the effect of baicalin on human brain microvascular endothelial cell under the glucose deprivation combined with hypoxia condition.

METHODSHuman brain microvascular endothelial cells (HBMVECs) cultured in vitro were divided into the following groups: normal group, model group, baicalin high dose group, baicalin middle dose group, baicalin low dose group, nimodipine group. The kits were used to detect the cell viability, leakage rate of lactate dehydrogenase (LDH), Na(+)-K(+)-ATPase, Ca(2+)-ATPase, the concentration of Ca2+ in each group, and apoptosis rates of each group were analyzed by flow cytometry (FCM).

RESULTSCompared with the normal group, the cell viability, Na(+)-K(+)-ATPase and Ca(2+)-ATPase in model group decreased significantly (P < 0.01, P < 0.05). But the leakage rate of LDH, Ca2+ in cells and apoptosis rates increased remarkably (P < 0.01). Compared with the model group, the cell viability, Na(+)-K(+)-ATPase and Ca(2+)-ATPase increased obviously (P < 0.01, P < 0.05) in baicalin high dose group. But the leakage rate of LDH and Ca2+ in cells in baicalin high dose group decreased significantly comparing with that of model group (P < 0.05, P < 0.01). And the reduction of intracellular Ca2+ was superior to that of nimodipine group. Meanwhile, the apoptosis rates decreased significantly in both baicalin high and middle dose groups (P < 0.01).

CONCLUSIONBaicalin could improve the cell viability of HBMVECs under the glucose deprivation combined with hypoxia condition. And the mechanisms were related with improving the energy metabolism, inhibiting intracellular calcium overload and decreasing the apoptosis rate of cells further.

Apoptosis ; Brain ; blood supply ; Calcium ; metabolism ; Capillaries ; cytology ; Cell Hypoxia ; Cell Survival ; Cells, Cultured ; Endothelial Cells ; cytology ; drug effects ; metabolism ; Flavonoids ; pharmacology ; Glucose ; chemistry ; Humans

Kallistatin (Kal) is a negative acute phase endogenous protein which can inhibit tumor angiogenesis, growth and metastasis effectively. To express and purify recombinant human kallistatin (rHKal), and characterize its biological activity, P. pastoris was transformed with pPIC9-Kal/GS115 (His4) to express rHKal. The fermentation was carried out in a 7.5 L bioreactor with high density cell culture. 1%-2% methanol was added to the medium to induce the expression of rHKal. The secretion was purified with phenyl sepharose, G-25 sepharose, heparin sepharose and Sephacryl S-100 chromatography. The biological activity of purified bulk rHKal on HUVEC was evaluated with MTT and tube formation assays. The final expression of rHKal in the supernatant reached 50 mg x L(-1), the purity of bulk rHKal after purification was above 98%. A dose-dependent inhibition of rHKal on HUVEC proliferation was observed, however, a U-shaped dose-response curve of rHKal on capillary formation of HUVEC was revealed. The described protocol provides an effective means for preparing rHKal that could be used for anti-angiogenesis therapy in the future.
Bioreactors ; Capillaries ; drug effects ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Electrophoresis, Polyacrylamide Gel ; Fermentation ; Human Umbilical Vein Endothelial Cells ; Humans ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Serpins ; biosynthesis ; genetics ; pharmacology

A major advance in the understanding of the regulation of food intake has been the discovery of the adipokine leptin a hormone secreted by the adipose tissue. After crossing the blood-brain barrier, leptin reaches its main site of action at the level of the hypothalamic cells where it plays fundamental roles in the control of appetite and in the regulation of energy expenditure. At first considered as a hormone specific to the white adipose tissue, it was rapidly found to be expressed by other tissues. Among these, the gastric mucosa has been demonstrated to secrete large amounts of leptin. Secretion of leptin by the gastric chief cells was found to be an exocrine secretion. Leptin is secreted towards the gastric lumen into the gastric juice. We found that while secretion of leptin by the white adipose tissue is constitutive, secretion by the gastric cells is a regulated one responding very rapidly to secretory stimuli such as food intake. Exocrine-secreted leptin survives the hydrolytic conditions of the gastric juice by forming a complex with its soluble receptor. This soluble receptor is synthesized by the gastric cells and the leptin-leptin receptor complex gets formed at the level of the gastric chief cell secretory granules before being released into the gastric lumen. The leptin-leptin receptor upon resisting the hydrolytic conditions of the gastric juice is channelled, to the duodenum. Transmembrane leptin receptors expressed at the luminal membrane of the duodenal enterocytes interact with the luminal leptin. Leptin is actively transcytosed by the duodenal enterocytes. From the apical membrane it is transferred to the Golgi apparatus where it binds again its soluble receptor. The newly formed leptin-leptin receptor complex is then secreted baso-laterally into the intestinal mucosa to reach the blood capillaries and circulation thus reaching the hypothalamus where its action regulates food intake. Exocrine-secreted gastric leptin participates in the short term regulation of food intake independently from that secreted by the adipose tissue. Adipose tissue leptin on the other hand, regulates in the long term energy storage. Both tissues work in tandem to ensure management of food intake and energy expenditure.
Adipokines ; Adipose Tissue ; Adipose Tissue, White ; Appetite ; Blood-Brain Barrier ; Capillaries ; Chief Cells, Gastric ; Dietary Sucrose ; Duodenum ; Eating ; Energy Metabolism ; Enterocytes ; Gastric Juice ; Gastric Mucosa ; Golgi Apparatus ; Hand ; Hypothalamus ; Intestinal Mucosa ; Leptin ; Membranes ; Phenobarbital ; Receptors, Leptin ; Secretory Vesicles

OBJECTIVE: To explore the degree, mechanism and clinical significance of three-dimensional tumor microvascular architecture phenotype heterogeneity (3D-TMAPH) in non-small cell carcinoma (NSCLC). METHODS: Twenty-one samples of solitary pulmonary nodules were collected integrally. To establish two-dimensional tumor microvascular architecture phenotype (2D-TMAP) and three-dimensional tumor microvascular architecture phenotype (3D-TMAP), five layers of each nodule were selected and embedded in paraffin. Test indices included the expressions of vascular endothelial growth factor (VEGF), proliferating cell nuclear antigen (PCNA), EphB4, ephfinB2 and microvascular density marked by anti-CD34 (CD34-MVD). The degrees of 3D-TMAPH were evaluated by the coefficient of variation and extend of heterogeneity. Spearman rank correlation analysis was used to investigate the relationships between 2D-TMAP, 3D-TMAP and clinicopathological features. RESULTS: 3D-TMAPH showed that 2D-TMAP heterogeneity was expressed in the tissues of NSCLC. The heterogeneities in the malignant nodules were significantly higher than those in the active inflammatory nodules and tubercular nodules. In addition, different degrees of heterogeneity of CD34-MVD and PCNA were found in NSCLC tissues. The coefficients of variation of CD34- MVD and PCNA were positively related to the degree of differentiation (all P<0.05), but not related to the P-TNM stages, histological type or lymphatic metastasis (all P>0.05). The level of heterogeneity of various expression indexes (ephrinB2, EphB4, VEGF) in NSCLC tissues were inconsistent, but there were no significant differences in heterogeneity in NSCLC tissues with different histological types (P>0.05). CONCLUSION 3D-TMAPH exists widely in the microenvironment during the genesis and development of NSCLC and has a significant impact on its biological complexity.
Adult ; Aged ; Capillaries ; ultrastructure ; Carcinoma, Non-Small-Cell Lung ; blood supply ; Ephrin-B2 ; metabolism ; Female ; Humans ; Lung Neoplasms ; blood supply ; Male ; Middle Aged ; Neovascularization, Pathologic ; pathology ; Phenotype ; Proliferating Cell Nuclear Antigen ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism

Blood Glucose ; analysis ; Capillaries ; metabolism ; Humans ; Hypoglycemia ; diagnosis ; Infant, Newborn ; Neonatal Screening ; methods ; Reference Standards

OBJECTIVETo study the effects of Compound Danshen Dripping Pill (CDDP) on the structure and functions of sternohyoid muscle in metabolic syndrome (MS) rats, and to study whether it has therapeutic effects on obstructive sleep apnea-hypopnea syndrome (OSAHS).

METHODSTwenty-one healthy male SD rats were randomly divided into three groups, i.e., the normal control group (n = 6), the MS group (n = 8), and the CDDP group (n = 7). Rats in the normal control group were routinely fed. High lipid forage was given to rats in the rest two groups. Nine weeks later, CDDP (at the dose of 375 mg/kg) was additionally given to rats in the CDDP group by gastrogavage, and then rats in the CDDP group and the MS group were fed with the same high lipid forage for 12 successive weeks. The content of malondialdehyde (MDA) and the activities of superoxide dismutase (SOD) in the sternohyoid muscle were detected in the three groups. The capillary density, capillary-to-fiber ratio (C/F), the section area of type I muscle fiber were detected using myosin-ATPase histochemical assay. The contractile changes of isometric stemohyoid muscles were determined under electric stimulation by different frequencies.

RESULTSThe contents of MDA were obviously lower in the CDDP group than in the MS group, while the activities of SOD, the capillary density, C/F, the section area of type I muscle fiber, the tension of stemohyoid muscle at 10 -60 Hz, and the 1-5 min tension percentages of the stemohyoid muscle were higher in the CDDP group than in the MS group (P < 0.05, P < 0.01).

CONCLUSIONCDDP could improve oxidative stress induced intramuscularly microcirculation disturbance and changes of muscular fiber structures of the upper airway muscles, and elevate their contractile functions, thus possibly contributing to favorable effects on OSAHS.

Animals ; Capillaries ; Drugs, Chinese Herbal ; pharmacology ; Male ; Metabolic Syndrome ; metabolism ; Muscle Contraction ; drug effects ; Neck Muscles ; drug effects ; metabolism ; Oxidative Stress ; drug effects ; Phenanthrolines ; pharmacology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effect of recombinant adenovirus-mediated triple mutant of hypoxia inducible factor-1alpha (Ad-HIF-1alpha-564/402/803) in modulating angiogenesis in vitro.

METHODSThe recombinant adenoviruses Ad-lacZ, Ad-Null, Ad-HIF-1alpha-nature, and Ad-HIF-1alpha-564/402/803 were amplified in HEK293A cells and purified by ultracentrifugation in CsCl step gradient solutions, and the adenoviral titer was determined by end-point dilution assay. The recombinant adenovirus was confirmed by PCR and DNA sequence analysis, and the infection efficiency was observed by X-gal staining. Human microvascular endothelial cells (HMVECs) were infected with Ad- HIF-1alpha-564/402/803, Ad- HIF-1alpha-nature, or Ad-Null to compare the number of capillary-like tube structures in vitro. The effect of Ad- HIF-1alpha-564/402/803, Ad-HIF-1alpha-nature, and Ad-Null on angiogenesis was evaluated using a chick embryo chorioallantoic membrane (CAM) model.

RESULTSPCR and gene sequencing suggested the correct construction of the recombinant adenovirus HIF-1alpha, and the adenoviral titer reached 1011-1012 PFU/ml. Infection of the hMVECs with Ad-HIF-1alpha-564/402/803 at the optimal multiplicity of infection of 100 pfu/cell resulted in a significantly greater number of capillary-like tube structures than infection by Ad-HIF-1alpha-nature and Ad-Null (P=0.000). Ad-HIF-1alpha-564/402/803 group showed significantly higher microvessel density than Ad-HIF-1alpha-nature, Ad-Null, and PBS groups, with also higher angiogenesis area to CAM area ratio (P=0.01, 0.000, and 0.000, respectively).

CONCLUSIONThe triple mutant Ad-HIF-1alpha-564/402/803 can obviously promote the formation of capillary-like tube structures in vitro and modulate angiogenesis in the CAM model, suggesting the capacity of Ad-HIF-1alpha-564/402/803 in promoting angiogenesis under normoxic condition.

Adenoviridae ; genetics ; metabolism ; Animals ; Capillaries ; Chick Embryo ; Chorioallantoic Membrane ; blood supply ; Endothelial Cells ; cytology ; metabolism ; HEK293 Cells ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; biosynthesis ; genetics ; Mutation ; Neovascularization, Physiologic ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVESTo explore the effects and significance of Huanshuai Recipe Oral Liquid (, HSR), a formula with supplementing qi, nourishing blood and activating blood on restructuring glomerular microvasculature and expression of vascular endothelial growth factor (VEGF) in subtotal nephrectomized (SNX) rats.

METHODSA total of 76 male Wistar rats were randomly divided into four groups: 16 in the sham-operated group and fed with tap water 10 mL/kg per day; 20 in the model group were operated with 5/6 SNX and fed with tap water 10 mL/ kg per day; 20 SNX rats in the HSR group were treated with HSR 10 mL/kg per day; 20 SNX rats in the losartan group were treated with losartan 40 mg/kg per day. Serum creatinine (SCr) and urinary protein excretion (Upro) were examined at the 2nd, 4th, 8th, and 12th weeks of the treatment, and the remnant kidneys were harvested. Changes in histological microstructure were evaluated using light microscopy, and the expression of VEGF was detected by using ELISA.

RESULTSUpro, microvasculature injury and glomerulosclerosis were found to be alleviated in HSR and Losartan groups, respectively. The change of VEGF expression showed positive correlation with glomerular capillary area and peritubular capillary number (r=0.448, r=0.422, P<0.01), but negative correlation with that of SCr and Upro (r=-0.592, r=-0.481, P<0.01).

CONCLUSIONSHSR could regulate the VEGF expression, reduce the loss of microvasculature, which demonstrated similar renal protective effects to losartan in SNX rats. Examination of Chinese herbal medicine influence on VEGF signaling and restructuring renal microvasculature may elucidate the molecular mechanism of renal protection to a certain degree.

Administration, Oral ; Animals ; Capillaries ; drug effects ; metabolism ; pathology ; Collagen Type IV ; metabolism ; Creatinine ; blood ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; Extracellular Matrix ; drug effects ; metabolism ; Fibronectins ; metabolism ; Immunohistochemistry ; Kidney Cortex ; drug effects ; metabolism ; pathology ; Kidney Function Tests ; Kidney Glomerulus ; blood supply ; drug effects ; pathology ; physiopathology ; Microvessels ; drug effects ; Nephrectomy ; Proteinuria ; blood ; drug therapy ; physiopathology ; Rats ; Rats, Wistar ; Time Factors ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo investigate the expression of vascular endothelial growth factor (VEGF) and microvessel density (MVD) in hippocampus of rats with aging.

METHODSParaffin sections of brain tissue of rats at the age of 3, 18, 24, 30 months were stained by immunohistochemistry, the expression of VEGF and MVD was quantitatively analyzed.

RESULTSInnunohistochemical staining showed that the VEGF-positive cells were mainly pyramidal neuron in hippocampus; the intensity of VEGF-positivity in neuron cells was decreased with the aging (P<0.05). The MVD in hippocampus was also decreased with the aging of rats (P<0.05).

CONCLUSIONIncreasing VEGF contents and improving blood circulation in brain tissue may prevent or treat vascular dementia and cerebrovascular diseases.

Aging ; Animals ; Capillaries ; pathology ; Hippocampus ; blood supply ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Vascular Endothelial Growth Factor A ; genetics ; metabolism