Stimulation of cell‐surface urokinase‐type plasminogen activator activity and cell migration in vascular endothelial cells by a novel hexapeptide analogue of neurotensin
Shin UshiroKazushige MizoguchiShigeo YoshidaSei‐ichiro JimiT. FujiwaraMasaya YoshidaEdward T. WeiPatrick KitabgiSakae AmagayaMayumi OnoMichihiko Kuwano
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To investigate if neurotensin (NT) could induce activation of urokinase‐type plasminogen activator (uPA) in vascular endothelial cells, we utilized the acetyl‐NT (8–13) analogue, TJN‐950, in which the C‐terminal leucine is reduced to leucinol. TJN‐950 inhibited the binding of 125 I‐NT to membranes of newborn rat brains and of COS‐7 cells transfected with rat NT receptor cDNA, but at 10 4 higher doses than NT (8–13). However, TJN‐950 was as effective as NT in inducing the fibrinolytic activity in bovine vascular aortic and human umbilical vein endothelial cells, and enhanced the migration of vascular endothelial cells. Moreover, administration of TJN‐950 induced neovascularization in the rat cornea in vivo. TJN‐950 had no effect on expression of uPA, plasminogen activator inhibitor‐1 or uPA receptor mRNA. The binding of 125 I‐TJN‐950 to cell membranes was blocked by unlabeled uPA and TJN‐950, but not the amino‐terminal or 12–32 fragment of uPA. TJN‐950 may enhance uPA activity in vascular endothelial cells by interacting with the uPA receptor, resulting in induction of angiogenesis.Neurotensin and several sequence analogues have been synthesized using solid‐phase technology. The purity of the following derivatives: neurotensin, neurotensin‐(10–13), neurotensin‐(9–13). neurotensin‐(8–13), neurotensin‐(6–13), neurotensin‐(4–13), [Cit 8 ]neurotensin‐(8–13), [Lys 8 ]neurotensin‐(8–13), [Cit 9 ]neurotensin‐(8–13),[Lys 9 ]neurotensin‐(8–13), [Phe 11 ]neurotensin‐(8–13), [Ala 12 ]neurotensin‐(8–13) and [Ala 13 ]‐ neurotensin‐(8–13) was verified by amino acid analyses after acid and enzymatic hydrolyses. reverse‐phase high‐ performance liquid chromatography in two systems and Edman degradation. The above analogues, those obtained after N‐acetylation of neurotensin‐(6–13), neurotensin‐(8–13), [Cit 8 ]neurotensin‐(8–13), [Cit 9 ]‐ neurotensin‐(8–13), [Lys 8 ]neurotensin‐(8–13), [Lys 9 ]neurotensin‐(8–13) and [Phe 11 ]neurotensin‐(8–13), as well as native xenopsin, were all tested for binding competition with [ 3 H]neurotensin on the specific fixation sites of rat brain synaptosomal membranes and on those of HT 29 cells. In addition to these radioreceptor assays on neural and extraneural targets, a pharmacological test (contraction of guinea pig ileum in the presence of neostigmine) was used to compare the behavior of the synthetic analogues. The use of these three biological systems enabled us to obtain consistent results. A good parallel was observed between the degree of fixation and pharmacological effects for entire neurotensin and for C‐terminal region analogues up to the size of neurotensin‐ (8–13). The two peptides neurotensin‐(6‐ 13) and neurotensin‐(4–13) had an abnormally high affinity for rat brain synaptic membrane binding sites compared to a relatively low contracting activity. The C‐terminal peptide ‐Arg‐Arg‐Pro‐Tyr‐Ile‐Leu fulfills all the structural requirements for mimicking the entire sequence, provided its α‐amino end is protected by acetylation. The guanidinium structure of residues 8 and 9 are not of vital importance, since they could be efficiently replaced by amino groups of lysyl side chains. Xenopsin, which can be considered as a natural analogue of neurotensin‐(8–13), acts similarly to acetyl‐neurotensin‐(8–13). Removal of the phenolic function of residue 11 induces a decrease in neurotensin effects. The C‐terminal isoleucyl and leucyl residues could not be replaced by alanine without complete loss of the three activities tested.
Neurotensin receptor
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Objective To investigate the relationship between plasma level of urokinase-type plasminogen activator(uPA),urokinase-type plasminogen activator receptor(uPAR)and plasminogen activator inhibitor type 1(PAI-1)and ovarian cancer.Method The concentration of uPA,uPAR and PAI-1 in 52 patients with ovarian cancer and 30 healthy subjects were simultaneously determined by ELISA.Results There were significant differences for uPA and uPAR in different grades of the patients with ovarian cancer(P0.01).However,there was no significant difference for PAI-1 in ovarian cancer patients with different grades.There were significant differences in PAI-1,uPA,uPAR between the patients with ovarian cancer and healthy subjects(P0.01).Conclusion uPA,uPAR and PAI-1 may play important roles and be used as the parameters for progression and reimplantation of ovarian malignant cancer cells.
SuPAR
Plasminogen activator inhibitor-1
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Abstract: The binding of [ 3 H]neurotensin(8–13) to membranes from human frontal cortex at 0°C was time dependent, specific, saturable, and reversible. Saturation isotherms provided an equilibrium dissociation constant ( K D ) of 0.52 n M , and the maximal number of binding sites ( B max) was 3.5 pmol/g original wet weight of tissue. Scat‐chard analysis yielded a straight line, and the Hill coefficient was equal to 1, a result indicating that [ 3 H]‐neurotensin(8–13) bound to single, noncooperative sites. The K D values of several analogs of neurotensin determined in competition with [ 3 H]neurotensin(8–13) were similar to those previously determined in competition with [ 3 H]‐neurotensin. The regional distribution of binding sites for [ 3 H]neurotensin(8–13) was also similar to that for [ 3 H]‐neurotensin. These results suggest that [ 3 H]neurotensin(8–13) binds to the same sites as [ 3 H]neurotensin and that [ 3 H]neurotensin(8–13) has a higher affinity than [ 3 H]‐neurotensin for these sites in human brain.
Dissociation constant
Neurotensin receptor
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Cigarette smoking has an influence on both arterial-type and venous-type thrombosis. However, little is known about the direct effect of cigarette smoke extract (CSE) on fibrinolytic activity of human umbilical vein endothelial cells (HUVECs). Most recently, simvastatin has been marked in its effect on endothelial cells protection and anticoagulation. In this study, the effect of CSE on the expression of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) in HUVECs was addressed. The role of simvastatin in CSE-induced fibrinolytic activity changes was investigated as well.The fourth to fifth generation of HUVECs were incubated respectively with 0, 5%, 10% and 20% CSE for 6 hours or exposed to 5% CSE for 0, 4, 6, 8, 12, 24 hours to determine the expression changes of t-PA and PAI-1 protein. Meanwhile, cells were also accordingly exposed either to 5% CSE alone or simvastatin pre-treated and 5% CSE for 24 hours to assess the role of simvastatin in CSE-induced t-PA and PAI-1 protein and mRNA expression in HUVECs. RT-PCR and ELISA techniques were used for detecting the t-PA or PAI-1 mRNA and protein.After 6-hour exposure to CSE, the expression levels of t-PA protein in 10% and 20% CSE-treated groups reduced significantly ((0.0365 +/- 0.0083) ng/ml, (0.0255 +/- 0.0087) ng/ml) when compared with that of control group ((0.0660 +/- 0.0120) ng/ml) (P < 0.05). In contrast, the levels of PAI-1 protein in 5%, 10% and 20% CSE-treated groups increased remarkably ((13.3225 +/- 0.5680) ng/ml, (14.2675 +/- 1.5380) ng/ml, (14.4292 +/- 1.6230) ng/ml) when compared with that of control group ((8.5193 +/- 0.7537) ng/ml) (P < 0.05). After stimulation with 5% CSE for 0, 4, 6, 8, 12, 24 hours, the levels of PAI-1 protein increased over time and reached the peak at 24 hours ((14.6400 +/- 1.0651) ng/ml), which was significantly higher than that of control group ((12.0656 +/- 0.6148) ng/ml) (P < 0.05). Additionally, CSE could up-regulate PAI-1 expression at both the mRNA and the protein levels. The levels of PAI-1 mRNA and protein increased significantly in 5% CSE-treated group ((8.8030 +/- 0.4745) ng/ml, (1.8155 +/- 0.0412) ng/ml) compared with those of control groups ((5.0588 +/- 0.2315) ng/ml, (1.3030 +/- 0.0647) ng/ml) (P < 0.01), and decreased after 2-hour simvastatin pre-treatment ((5.4875 +/- 0.3166) ng/ml, (1.3975 +/- 0.0297) ng/ml) (P < 0.01). No significant difference was found at the levels of t-PA protein and mRNA (P > 0.05).CSE inhibits the fibrinolytic activity of HUVECs in vitro. Simvastatin plays a protective role in CSE-induced fibrinolytic malfunction.
Plasminogen activator inhibitor-1
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Zymography
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Objectives To study the expressions of urokinase-type plasminogen activator (uPA) and its receptor (uPAR) in the gliomas, and to analysis the relationship between uPA and uPAR expressions and the grade of human gliomas. Methods The levels of uPA and uPAR protein expressions were determined by immunohistochemical technique in 58 cases of the gliomas and 10 samples of the normal brain tissue. Results The expressions of uPA and uPAR were not detected in normal brain tissures, and positively correlated with the pathological grade of the gliomas (P0.01). Conclusions The high levels of uPA and uPAR expressions may reflect a malignant biological behavior of the glioma cells.
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To investigate the pathogenesis of unknown nosebleed patients.The ELISA test were used to detected plasma Urokinase-type plasminogen activator (uPA) and Urokinase-type plasminogen activator receptor (uPAR) level in 19 cases unknown factor nosebleed patients and 36 health persons.The results showed uPAR and uPA level in nosebleed group (before treatment) uPAR (0.14 +/- 0.04) microg/L, uPA (0.24 +/- 0.09) microg/L; (after treatment) uPAR (0.08 +/- 0.02) microg/L, uPA (0.18 +/- 0.07) microg/L. And normal group uPAR (0.07 +/- 0.03) microg/L, uPA (0.17 +/- 0.05) microg/L. The uPAR and uPA level in nosebleed group before treatment is higher than that in normal group (P <0.05). There is no significant difference between nosebleed group after treatment and normal group (P>0.05).The reasons of uPAR and uPA level high in unknown factor nosebleed patients were not clear, maybe relation to vascular endothelial cell, smooth muscle cell and neutrophil-monocytic release more uPAR and uPA. So uPAR and uPA density of nostril accumulation is more high in its microenvironment, that fibrinolytic system activated increase and result in its hyperactivity, and happened nosebleed when blood be in hypocoagulable state.
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Abstract We investigated whether the expression levels of urokinase‐type plasminogen activator (uPA), uPA receptor (uPAR), and plasminogen activator inhibitor‐1 (PAI‐1) correlate with clinicopathological features of oral squamous cell carcinoma (SCC). We immunohistochemically examined the expression levels of uPA, uPAR, and PAI‐1 in 160 biopsy specimens of oral SCC. Positive stainings for uPA, uPAR, and PAI‐1 were observed mainly in SCC cells, and their intensity and number of positive cells were related to lymph node involvement ( p < 0.001, p < 0.001, and p < 0.001, respectively). The expression levels of uPA and uPAR were also related to the pattern of invasion ( p < 0.05 and p < 0.001, respectively), while both were associated with tumor size ( p < 0.05). Moreover, a poor survival rate was related to the expression of uPAR ( p < 0.01) and PAI‐1 ( p < 0.05). These findings suggest that the uPA system may regulate the invasion and metastasis of oral SCC cells.
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Abstract Eighteen patients with renal‐cell carcinoma have been investigated in an attempt to elucidate the ratio of active urokinase enzyme to urokinase antigen in the tumor and adjacent normal kidney. The tumor itself exhibited a significantly increased total fibrinolytic activity, an increase in the relative contribution of anti‐urokinase IgG‐inhibitable plasminogen activator activity and increased levels of urokinase antigen when compared to normal renal tissue. In tumor‐adjacent tissue total fibrinolytic activity was also, but not significantly, increased, this increase being completely due to tissue‐type plasminogen activator. Correlation of active urokinase‐type plasminogen activator with urokinase antigen revealed that in tumor tissue the enzyme was present to more than 70% in its active form whereas in tumor‐adjacent tissue and normal renal tissue only half of the enzyme appeared to be active. No correlation was obtained between urokinase antigen present in one of the 3 tissues investigated and plasma urokinase antigen.
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