In order to further investigate the effect of annexin II (Ann-II) on tissue plasminogen activator (t-PA)-dependent plasminogen (PLG) activation and its interactive mechanism, recombinant native Ann-II bound t-PA, PLG and plasmin with high affinity was examined. The flow cytometric assay showed that the ann-II expression rate was higher in the human umbilical vein endothelial cell (HUVEC) (87.65%) than in the HL-60 cells as controls (35.79%). Two irrelevant proteins, bovine serum albumin (BSA) and equine IgG (EIG) had no effect on the production of plasmin. Ann-II-mediated enhancement of t-PA-dependent PLG activation was inhibited by epsilon-aminocaproic acid or by pretreatment of Ann-II with carboxypeptidase B with the inhibitive rate being 77.8% and 77.0%, respectively. It was revealed that the effect of Ann-II on PLG activation was specific for t-PA. Urokinase didn't bind to Ann-II, demonstrating the role of receptor-related lysine residues on activation of PLG, showing that the Ann-II-PLG interaction was dependent upon carboxyl-terminal lysine residues. These findings suggest that annexin II-mediated co-assembly of t-PA and PLG may promote plasmin generation and play a key role in modulating fibrinolysis on the endothelial surface.
Annexin A2/*pharmacology ; Cells, Cultured ; Endothelium, Vascular/cytology ; Endothelium, Vascular/*metabolism ; Fibrinolysis ; Plasminogen/*metabolism ; Recombinant Proteins/pharmacology ; Tissue Plasminogen Activator/*metabolism ; Umbilical Veins/cytology

The effects of 3, 4-Dihydroxyacetophenone (3, 4-DHAP) on cytosolic free calcium [Ca2+]i in pulmonary artery endothelia (PAECs) and smooth muscle cells (PASMCs) during acute hypoxia were studied. Porcine pulmonary artery endothelial and smooth muscle cells (PASMCs) were cultured primarily, and they were divided into 4 groups: groups incubated under normoxia or hypoxia and those with or without treatment with 3,4-DHAP. The [Ca2+]i of both PAECs and PASMCs was measured by determining the fluorescence of fura 2 AM on spetrofluorometer. Our results showed that hypoxia caused significant elevation of [Ca2+]i, in both PAECs and PASMCs, 3,4-DHAP could attenuate the hypoxic elevation of [Ca2+]i only in PASMCs but not in PAECs. It is concluded that 3,4-DHAP decreases the hypoxic elevation of [Ca2+]i in PASMCs. This might contribute to its inhibitory effect on hypoxic pulmonary vasoconstriction.
Acetophenones/*pharmacology ; Calcium/*metabolism ; Cell Hypoxia ; Cells, Cultured ; Endothelium, Vascular/cytology ; Endothelium, Vascular/*metabolism ; Muscle, Smooth, Vascular/cytology ; Muscle, Smooth, Vascular/*metabolism ; Pulmonary Artery/cytology ; Pulmonary Artery/metabolism ; Swine

The effects of 3, 4-Dihydroxyacetophenone (3, 4-DHAP) on cytosolic free calcium [Ca2+]i in pulmonary artery endothelia (PAECs) and smooth muscle cells (PASMCs) during acute hypoxia were studied. Porcine pulmonary artery endothelial and smooth muscle cells (PASMCs) were cultured primarily, and they were divided into 4 groups: groups incubated under normoxia or hypoxia and those with or without treatment with 3,4-DHAP. The [Ca2+]i of both PAECs and PASMCs was measured by determining the fluorescence of fura 2 AM on spetrofluorometer. Our results showed that hypoxia caused significant elevation of [Ca2+]i, in both PAECs and PASMCs, 3,4-DHAP could attenuate the hypoxic elevation of [Ca2+]i only in PASMCs but not in PAECs. It is concluded that 3,4-DHAP decreases the hypoxic elevation of [Ca2+]i in PASMCs. This might contribute to its inhibitory effect on hypoxic pulmonary vasoconstriction.
Acetophenones ; pharmacology ; Animals ; Calcium ; metabolism ; Cell Hypoxia ; Cells, Cultured ; Endothelium, Vascular ; cytology ; metabolism ; Muscle, Smooth, Vascular ; cytology ; metabolism ; Pulmonary Artery ; cytology ; metabolism ; Swine

AIMTo investigate the role of ICAM-1 on the surface of vascular endothelial cell (VEC) in freezing/thawing injury of VEC, in order to elucidate the pathogenesis of freezing/thawing injury.

METHODSVEC separated and cultured from rat aorta and PMN separated from rat peripheral blood were selected as experiment materials. The frozen/thawed VEC model was founded by freezing VEC with the type WKL-V rate cooling instrument and then rewarming them in a water bath. ICAM-1 expression on the surface of frozen/thawed VEC was detected at 4, 12 and 24 h after freezing/thawing with immunohistochemical method. After coincubating frozen/thawed VEC with normal PMN, the adhesion of VEC to PMN was monitored with rose bengal staining assay and the injury level of VEC was indicated by measuring LDH activity in nutrient solution.

RESULTSThe ICAM-1 expression on the surface of VEC increased from 13.2% +/- 3.6% before freezing/thawing of VEC to 22.3% +/- 4.4% at 4 hour after freezing/thawing, and reached the peak (37.9% +/- 2.5%) at 12 hour after freezing/thawing of VEC. After coincubation of frozen/thawed VEC with normal PMN, the adherence of frozen/thawed VEC to PMN increased from group control 0.204 +/- 0.025 to 0.363 +/- 0.022 (P < 0.01), LDH activity in nutrient solution increased from group control 104.64 +/- 20.14 U/L to 162.33 +/- 27.88 U/L (P < 0.01), monoclonal antibody against ICAM-1 (ICAM-1 Mab) could partially block the adherence of frozen/thawed VEC to PMN (0.270 +/- 0.021, P < 0.01), and diminish LDH activity in nutrient solution (125.39 +/- 22.26 U/L, P < 0.05).

CONCLUSIONThe freezing/thawing of VEC can elicit an increase in ICAM-1 expression on the surface of VEC, and then proceed to VEC-PMN adherence and lead to VEC injury.

Animals ; Cells, Cultured ; Endothelial Cells ; metabolism ; Endothelium, Vascular ; cytology ; Freezing ; Intercellular Adhesion Molecule-1 ; metabolism ; Neutrophils ; cytology ; Rats

Cells, Cultured ; Endothelial Cells ; drug effects ; metabolism ; Endothelium, Vascular ; cytology ; metabolism ; Gene Expression Profiling ; Humans ; Ouabain ; pharmacology ; Umbilical Veins ; cytology

Endothelial injury, smooth muscle cells (SMCs) proliferation and migration are the same common pathophysiological processes of many cardiovascular diseases, such as atherosclerosis, hypertension, diabetes and restenosis. It is important to determine the functional interactions between endothelial cells (ECs) and SMCs under pathologic conditions. This work was to study the effects of ECs growth states on the proliferation and migration of vascular SMCs in cell coculture system. (3)H-TdR incorporation and flow cytometry were used to determine the effects of ECs growth states on the proliferation of SMCs. The number of migrating SMCs was counted. RT-PCR was used to analyze the expression of alpha-SM-actin mRNA. The results showed that (3)H-TdR incorporation decreased significantly from 14,900+/-1035 cpm/well in the control group to 8,575+/-749 cpm/well in the confluent ECs group (n=6, P<0.01), and increased to 27,268+/-2310 cpm/well in the proliferative ECs group ( n=6, P<0.01). The transition of SMCs from G(0)/G(1) phase to G(2)/M and S phases was blocked in the confluent ECs group but promoted in the proliferative ECs group. Compared with the control group, the number of migrating cells was about 4 times higher in the proliferative ECs group (n=6, P<0.01), while it in the confluent ECs group was only the half of the number of the control(n=6, P<0.05). The expression of alpha-SM-actin mRNA was increased significantly in the confluent ECs group(2.3+/-0.11 vs 1.4+/-0.12, P<0.05), but decreased significantly in the proliferative ECs group(0.92+/-0.08 vs 1.4+/-0.12, P<0.05). The results suggest that the biologic features of SMCs are influenced by ECs growth states. The proliferative ECs promote SMCs proliferation, migration and downregulate alpha-SM-actin mRNA expression significantly.
Actins ; metabolism ; Aorta ; cytology ; Cell Differentiation ; Cell Division ; Cell Movement ; Cells, Cultured ; Coculture Techniques ; Endothelium, Vascular ; cytology ; Epithelial Cells ; cytology ; Muscle, Smooth, Vascular ; cytology

OBJECTIVETo investigate relationship between expression of inducible nitric oxide synthase (iNOS) and proliferative potency of endothelium in hemangioma (HM).

METHODSImmunohistochemical staining was used to detect expression of iNOS and Ki-67 protein in 49 cases of HM and 29 cases of vascular malformation (VM).

RESULTSExpressive rate of iNOS and Ki-67 protein of 49 cases of HM was 38% and (10.98 +/- 7.93)%. Expressive rate of iNOS and Ki-67 protein of 29 cases of VM was respectively 3% and (0.03 +/- 0.19)%. The expressive rate of iNOS and Ki-67 protein of HM was significantly higher than that of VM (P = 0.001 and 0). The expressive rate of Ki-67 protein of HM of proliferative phase was (12.67 +/- 7.65)% , which was significantly higher than that of HM of extinctive phase, (7.27 +/- 7.49)% (P = 0.028).

CONCLUSIONSExpression of iNOS and Ki-67 protein of HM is significantly higher than that of VM, and the proliferative potency of HM is significantly higher than that of VM.

Child, Preschool ; Endothelium ; metabolism ; Endothelium, Vascular ; cytology ; Female ; Hemangioma ; metabolism ; pathology ; Humans ; Hyperplasia ; Infant ; Ki-67 Antigen ; metabolism ; Male ; Neovascularization, Pathologic ; metabolism ; Nitric Oxide Synthase Type II ; metabolism

OBJECTIVETo compare the differences in nitric oxide (NO) release and endothelium-derived hyperpolarizing factor (EDHF)-mediated hyperpolarization between human radial artery (RA) and saphenous vein (SV) through direct measurement of NO and membrane potential.

METHODSRA (n = 8), SV (n = 23), and surgical prepared SV (PV, n = 9, dilatation with normal saline solution at a pressure of 100 - 600 mmHg, 1 mmHg = 0.133 kPa) segments (5 mm long) taken from patients undergoing coronary artery bypass grafting were placed in an organ chamber. The NO-sensitive electrode and intracellular glass microelectrode was used to directly measure the NO release and the membrane potential changes in response to acetylcholine (ACh) and bradykinin (BK) before and after incubation with NG-nitro-L-arginine, indomethacin, and oxyhemoglobin.

RESULTSThe basal release of NO in RA [(11.9 ± 1.8) nmol/L] was significantly greater than that in SV [(9.9 ± 2.8) nmol/L, P = 0.041]. BK-induced NO release in RA was lower than that in SV [for BK 10(-7) mol/L: (25.8 ± 3.6) nmol/L vs. (43.7 ± 8.2) nmol/L, P = 0.006]. Both basal and ACh- or BK-induced NO release in PV were significantly reduced [basal release: PV (3.4 ± 1.4) nmol/L; P = 0.006 vs. RA; P = 0.002 vs. SV; stimulated release: for ACh 10(-5) mol/L: PV (4.8 ± 3.2) nmol/L; vs. RA (28.6 ± 7.9) nmol/L, P = 0.005; vs. SV (27.4 ± 3.7) nmol/L, P = 0.003; for BK 10(-7) mol/L: PV (7.0 ± 3.6) nmol/L; vs. RA (25.8 ± 3.6) nmol/L, P = 0.016; vs. SV (43.7 ± 8.2) nmol/L, P = 0.004]. EDHF-mediated hyperpolarization was greater in RA than that in SV [ACh 10(-5) mol/L: (-9.7 ± 1.9) mV vs. (-4.5 ± 1.1) mV, n = 17, P = 0.002].

CONCLUSIONSRA is superior to SV in terms of NO basal release and EDHF-mediated endothelial function. Surgical preparation and pressure dilatation may severely impair the NO-mediated endothelial function of SV, which may contribute to the poor long-term patency of SV coronary graft.

Biological Factors ; metabolism ; Endothelial Cells ; metabolism ; physiology ; Endothelium, Vascular ; cytology ; metabolism ; Female ; Humans ; Male ; Membrane Potentials ; physiology ; Middle Aged ; Nitric Oxide ; metabolism ; Radial Artery ; cytology ; Saphenous Vein ; cytology

To explore the biological effects of shear stress on intact artery and the change of growth factor during stress-induced vascular remodeling, we established an artery organ-cultured system under stress in vitro, and the common carotid arteries of pigs were cultured under shear stress of 20, 5 and 0 dyn/cm2. PDGF-A synthesis of vascular smooth muscle cells (VSMCs) cultured for 1, 4 and 7 days were studied by immunohistochemical and computer image processing methods, and PDGF-B secretion of endothelial cells (ECs) cultured within 12 h were studied by ELISA. Results showed that PDGF-B increased obviously under shear stress of 5 dyn/cm2, and reached the highest point at about 3 h; PDGF-A synthesis also obviously increased under low shear stress in 7 days. Increasing of PDGF synthesis promotes phenotype switch and proliferation of VSMC. It may have important influence on artery remodeling under low shear stress.
Animals ; Carotid Artery, Common ; chemistry ; metabolism ; Cells, Cultured ; Endothelium, Vascular ; cytology ; metabolism ; Muscle, Smooth, Vascular ; cytology ; metabolism ; Platelet-Derived Growth Factor ; biosynthesis ; Shear Strength ; Stress, Mechanical ; Swine

Fluid shear stress plays an important role in many physiological and pathological processes of the cardiovascular system. Being constantly exposed to mechanical shear stress, vascular endothelial cells can sense the changes of blood flow forces and regulate vascular structure and function. Previous studies demonstrated that IL-8 mRNA expression in endothelial cells was modulated by fluid shear stress. To identify the effect of fluid shear stress on IL-8 protein production of human umbilical vein endothelial cells (HUVECs), we employed quantitative sandwich enzyme-linked immunosorbent assay (ELISA) to measure the IL-8 protein. It was found that the HUVECs not treated with fluid shear stress secreted very little IL-8 in culture media. However, after 1 hour of exposure to shear stress, the secretion of IL-8 increased; at 5 hours of exposure, the seceretion reached the summit; at 8 hours of exposure, the secretion of IL-8 decreased and then remained at a constant level till the end (12 hours) of the experiment. The increase of IL-8 secretion induced by shear stress was time-dependent. The biphasic response of IL-8 protein production was found in experiments in which the shear stress applied was 2.09 dyne/cm2, 4.61 dyne/cm2, and 6.19 dyne/cm2. The IL-8 protein production in response to shear stress was very similar to the IL-8 gene expression in response to shear stress, and had the obvious delay. The induction of IL-8 protein production by fluid shear stress is probably due to the gene expression. This in vitro study demonstrates that the production of IL-8 can be regulated by fluid shear stress. Fluid shear stress induces a biphasic response of human HUVECs' production of IL-8 protein. These observations suggest that the process of the fluid shear stress induced HUVECs' production of IL-8 may play an important role in the genesis and development of both inflammation and atherosclerosis.
Cells, Cultured ; Endothelial Cells ; metabolism ; Endothelium, Vascular ; cytology ; Humans ; Interleukin-8 ; biosynthesis ; Stress, Mechanical ; Umbilical Veins ; cytology