Overexpression of hepatic plasminogen activator inhibitor type 1 mRNA in rabbits with fatty liver

INTRODUCTIONPlasminogen activator inhibitor type 1 ( PAI-I ), an approximately Mr 50000 glycoprotein, is the major physiological inhibitor of plasminogen activators. It is not only the priming factor for atherosclerosis and coronary thrombosis[1-3] , but also participates in the genesis of chronic hepatitis and liver fibrosis[4-11] . However, there has been no available report yet about the research of hepatic PAl-1 gene expression in hyperlipidemia and fatty liver. The present study aimed to explore the change of hepatic PAl-1 mRNA and its plasma activity by means of animal model.

INVESTIGATION OF THROMBOMODULIN AND PLASMINOGEN ACTIVATOR INHIBITOR TYPE-I IN PREGNANCY INDUCED HYPERTENSION AND ITS CLINICAL SIGNIFICANCE

Objective. To measure the circulating levels of thrombomodulin (TM) and plasminogen activator inhibitor type- I (PAI- I) in women with pregnancy induced hypertension (PIH). Methods. Blood samples were drawn from 97 pregnant women in their third trimester, grouped as 25 mild PIH,26 moderate PIH,22 severe PIH and 24 normotensive healthy pregnant women for determining levels of TM by ELISA,PAI- I by colorimetric assay methods, and creatinine (Cr) in serum by biochemical method. Results. Circulating levels of TM, PAI- I and TM/Cr ratio increased with increasing severity of PIH. There were no significant differences between mild and normotensive pregnant women. The parameters were significantly changed in the moderate and severe PIH groups. Conclusion. TM and PAI- I may serve as meaningful clinical markers for the assessment of the endothelial damage in PIH, which is very important in evaluating and following the development of PIH.

THE INCREASE IN PLASMINOGEN ACTIVATOR INHIBITOR TYPE-1 EXPRESSION BY STIMULATION OF ACTIVATORS FOR PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS IN HUMAN ENDOTHELIAL CELLS

Objective.To investigate the effect of peroxis ome proliferator-activated recept ors(PPARs )activators on plasminogen activator inhibitor ty pe-1(PAI-1)expression in human umbilical vein e ndothelial cells and the possi-ble mechanism.Methods.Human umbilical vein endothelial ce lls(HUVECs )were obtained from normal fetus,and cul-tured conventionally.Then the HUVECs were exposed to test agents(linolenic acid,linoleic acid,oleic acid,stearic acid and prostaglandin J 2 respectively)in varying concentrations with fresh media.RT -PCR and ELISA were applied to determine the expression of PPARs and PAI-1in HUVECs.Results.PPARα,PPARδand PPARγmRNA were detected by using RT-PCR in HUVECs.Treatment of HUVECs with PPARαand PPARγactivators---linolenic acid,linoleic acid,oleic acid and prostaglandin J 2 respectively,but not with stearic a cid could augment PAI-I mRNA expression and protein secretion in a concentration-dependent manner.However,the mRNA expressions of 3subclasses of PPAR with their activators in HUVECs were not changed compared w ith controls.Conclusion.HUVECs express PPARs.PPARs activators may increase PAI-1expression in ECs,but the underlying mechanism remains uncle ar.Although PPARs expression was not enhanced after stimulated by their activators in ECs,the role of functionally active PPARs in regulating PA I-1expression in ECs needs to be further investigated by using transient gen e transfection assay.

Expression and prognostic value of plasminogen activator inhibitor type 1 in node-negative breast cancer

Objective： To investigate the expressions of plasminogen activator inhibitor type 1 （PAl-1）, C-erbB-2, VEGF and Ki-67 by immunohistostaining and then to evaluate the prognostic value of PAl-1 in node-negative breast cancer. Methods： The study included a retrospective series of 62 female patients with axillary lymph node-negative breast cancer. Expressions of PAl-1, C-erbB-2, VEGF and Ki-67 were determined by immunohistostaining on formalin-fixed paraffin-embedded tissue sections from these patients after a median follow-up of 69 months （range 22-117 months）. Correlations with well known clinicopathologic factors were assessed and multivariate survival analyses were performed. Results： High PAl-1 level was positively associated with high histologic grade of the tumors. Disease-free survival （DFS） was significantly shorter for the patients with moderate to intensive expression of PAl-1 than for those with negative （χ^2 = 25.46, P 〈 0.001; χ^2 = 23.07, P 〈 0.001） to mild expression （χ^2 = 19.75, P 〈 0.001; χ^2 = 17.40, P 〈 0.001）. Although on univariate analysis of the prognostic factors, tumor size, location of primary tumor and age as well as expressions of PAl-1, VEGF and Ki-67 were all significantly prognostic factors for DFS （P 〈 0.05）, PAl-1 was the only independent prognostic factor on multivariate analysis （P 〈 0.0001; hazard ratio [HR], 4.041; 95% confidence interval [CI], 1.928-8.468）. Conclusion： These results of the current study indicate that intermediate or high expression of PAl-1 represents a strong and independent unfavorable prognostic factor for the development of recurrence or metastases in axillary node-negative breast cancer.

EFFECTS OF TGF-β_1 ON THE EXPRESSION OF PLASMINOGEN ACTIVATOR INHIBITOR TYPE 1 IN CULTURED HUMAN RENAL INTERSTITIAL FIBROBLASTS

Objective To investigate the effects of transforming growth factor-β1 (TGF-β1 ) on the expression of plasminogen activator inhibitor type 1 (PAI-1 ) mRNA in renal interstitial fibrosis in vitro. Methods Human renal interstitial fibroblasts were isolated and cultured in vitro. The cells wers stimulated by TGF-β1 with different concentration (0 to 10ng/ml ) at different time (0 to 48h). The expression of PAI-1 mRNA was assayed by RT-PCR. Results TGF-β1, had dose-dependent and time-dependent effects on the expression of PAI-1 mRNA in renal interstitial fibroblasts. Conclusion TGF-β1 may partic- ipate in renal fibrosis with inducing the expression of PAI-1 mRNA in renal fibroblasts and affecting the synthesis and degradation of extracellular matrix (ECM).

Tissue-type plasminogen activator and plasminogen activator inhibitor type-1 mRNA and their protein expression levels in human decidua after early pregnancy termination by mifepristone plus misoprostol

Objective To investigate the mechanism of prolonged uterine hemorrhage after terminating early pregnancy by mifepristone plus misoprostol.Methods Forty-five decidua specimens were obtained from 45 pregnant women with amenorrhea of 6-7 week duration. Fifteen women were treated with mifepristone and 15 were treated with mifepristone plus misoprostol. The remaining 15 served as controls. The tPA and PAI-1 mRNA levels were estimated by reverse transcription-polymerase chain reaction. Chromogenic assay and enzyme-linked immunosorbent assay were used to detect tPA activity and PAI-1 protein level in decidua. Results The activities of tPA in the mifepristone plus misoprostol group and in the mifepristone group were 46.91±20.74?IU/mg*protein and 64.25±35.81?IU/mg*protein respectively, lower than those in the normal decidua group (99.76±58.61?IU/mg*protein, P<0.05). tPA mRNA levels in the mifepristone plus misoprostol group were the highest (1.43±0.39) among the groups. In the mifepristone group, tPA mRNA level (0.90±0.16) was not significantly different from that in the normal decidua group (0.94±0.17). The protein and mRNA expression levels of PAI-1 were not significantly different among the three groups (P>0.05).Conclusions Mifepristone plus misoprostol decreased tPA activity in human early decidua by post-transcription pathways, which may influence decidua shedding, endometrial angiogenesis, endometrial remodeling, and cause prolonged uterine hemorrhage after drug abortion.

Perilla oil and exercise decrease expressions of tumor necrosis factor-α,plasminogen activator inhibitor-1 and highly sensitive C-reactive protein in patients with hyperlipidemia

OBJECTIVE:To verify the effects of perilla oil on the regulation of blood lipid levels in patients with hyperlipidemia.METHODS:Blood was taken from patients prior to and 8 weeks following treatment with perilla oil.Different ways to test for indexes which correlate to hyperlipidemia were performed.Some indexes,which correlate with inflammation and injury to endothelial cells,were tested using enzyme linked immunosorbent assays.RESULTS:Serum lipid levels [triglyceride(TG),total cholesterol(TC),and low-density lipoprotein-cholesterol(LDL-C)] changed significantly after 56 days of treatment.Differences were noted as early as 28 days after treatment began(P<0.05).Treatment with perilla oil showed statistically significant recovery levels of high-density lipoprotein-cholesterol(HDL-C) after 28 and 56 days of treatment.Plasma lipids levels were significantly lower after 56 days of treatment(P<0.05).Perilla oil reduced blood lipid levels in patients,and the regulation of cell signaling factor levels had no adverse effects on patients' liver or kidney function,or blood routine examinations.CONCLUSION:Perilla oil treatment is safe in clinical use,can regulate blood lipid levels and protects the function of endothelial cells.

Idiopathic pulmonary fibrosis in relation to gene polymorphisms of transforming growth factor-β1 and plasminogen activator inhibitor 1

Background Idiopathic pulmonary fibrosis （IPF） is a progressive and lethal fibrotic lung disease of unknown etiology. Host susceptibility or genetic factors may be important for the predisposition to it. Transforming growth factor-β1 （TGF-β1 a potent profibrotic cytokine） and plasminogen activator inhibitor 1 （PAl-1） play important roles in the development of pulmonary fibrosis. The objective of the study was to investigate the association between the gene polymorphisms of TGF-β1 869 T〉C and PAl-1 4G/5G and the susceptibility to IPF in Han ethnicity. Methods Polymerase chain reaction （PCR） and restriction fragment length polymorphism were performed to analyse the gene polymorphisms of TGF-β1 in 869T〉C and PAl-1 4G/5G in 85 IPF patients and 85 healthy controls matched in age, gender, race and smoker status. Results There was a significant difference in 869T〉C genotype distribution of TGF-β1 between IPF cases and controls, a significant negative association between TC genotype and the development of IPF （OR=0.508, 95% CI： 0.275-0.941） and a positive association between CC genotype and the development of IPF （OR=1.967, 95% CI： 1.063-3.641）. There was a significant positive association between PAl-1 5G/5G genotype and the development of IPF （OR=0.418, 95% CI： 0.193-0.904）. Conclusions Gene polymorphisms of TGF-β1 in 869T〉Cand PAl-1 4G/5G may affect the susceptibility to IPF in Han ethnicity. Further investigations are needed to confirm these findings and assess their biological significance in the development of the disease in this ethnic population.

Activation of peroxisome proliferator-activated receptor α in human endothelial cells increases plasminogen activator inhibitor type-1 expression

Objective To investigate the effect of peroxisome proliferator-activated receptors (PPARs) activators on plasminogen activator inhibitor 1 (PAI-1) expression in human umbilical vein endothelial cells and elucidate a possible mechanism. Methods Human umbilical vein endothelial cells (HUVECs) were obtained from normal fetus, and cultured conventionally. Then the HUVEC were exposed to fatty acids and prostaglandin J 2 in varying concentrations with fresh media. RT-PCR and ELISA were used to determine the expression of PPAR and PAI-1 in HUVECs. Transient co-transfection of PAI-1 promoter and PPARα gene or PPARγ gene to ECV304 was performed.Results PPARα, PPARδ and PPARγ mRNA in HUVECs were detected by RT-PCR. Treatment of HUVECs with PPARα and PPARγ activators-linolenic acid, linoleic acid, oleic acid and prostaglandin J 2, but not with stearic acid could augment PAI-I mRNA expression and protein secretion in a concentration-dependent manner. Proportional induction of PAI-1 promoter activity was observed through increasing amounts of PPARα DNA in HUVECs through a transient gene transfection assay, although the mRNA expression of the 3 subtypes of PPAR with their activators were not changed compared with controls.Conclusions HUVECs express PPARs. PPARs activators may increase PAI-1 expression in endothelial cells (EC). Although PPARs expression was not enhanced after being stimulated by their activators in EC, the functionally active PPARα is probably involved in regulating PAI-1 expression in EC.

Relationship of plasminogen activator inhibitor 1 gene 4G/5G polymorphisms to hypertension in Korean women

Background Hypertension （HTN） is a major determinant of various cardiovascular events. Plasma levels of plasminogen activator inhibitor 1 （PAl-l） modulate this risk. A deletion/insertion polymorphism within the PAl-1 loci （4G/4G, 4G/5G, 5G/5G） affects the expression of this gene. The present study investigated the association between PAl-1 loci polymorphisms and HTN in Korean women. Methods Korean women （n=1312） were enrolled in this study to evaluate the association between PAl-1 4G/5G gene polymorphisms and HTN as well as other metabolic risk factors. PAl-1 loci polymorphisms were investigated using polymerase chain reaction amplification and single-strand conformation polymorphism analysis. Results The three genotype groups differed with respect to systolic blood pressure （P=0.043）, and diastolic blood pressure （P=0.009） but not with respect to age, body mass index, total cholesterol, low or high density lipoprotein cholesterol, triglycerides, or fasting blood glucose. Carriers of the PAl-1 4G allele had more hypertension significantly （PAl-1 4G/5G vs. PAl-1 5G/5G, P=0.032; PAl-1 4G/4G vs. PAl-1 5G/5G, P=0.034）. When stratified according to PAl-1 4G/5G polymorphism, there was no significant difference in all metabolic parameters among PAl-1 genotype groups in patients with HTN as well as subjects with normal blood pressure. The estimated odds ratio of the 4G/4G genotype and 4G/5G for HTN was 1.7 （P=0.005）, and 1.6 （P=0.015）, respectively. Conclusion These findings might indicate that PAl-1 loci polymorphisms independently contribute to HTN and that gene-environmental interaction may be not associated in Korean women.

Roles of tissue plasminogen activator and its inhibitor in proliferative diabetic retinopathy

AIMTo investigate the role of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI) in proliferative diabetic retinopathy (PDR) and to discuss the correlations among t-PA, PAI and vascular endothelial growth factor (VEGF) expressions.

Plasminogen activator inhibitor-1 4G/5G gene polymorphism in patients with myocardial or cerebrovascular infarction in Tianjin, China

Objective To investigate the association between the plasminogen activator inhibitor-1 (PAI-1) 4G/ 5G gene polymorphism and the occurrence of myocardial and cerebrovascular infarctions in individuals from Tianjin, China.Methods The PAI-1 genotype was determined using allele-specific polymerase chain reaction (AS-PCR) in 56 myocardial infarction (Ml) patients, 54 cerebrovascular infarction (Cl) patients and 83 unrelated healthy controls. All subjects' clinical features and plasma PAI-1 activity levels were determined.Results The PAI-1 genotype distribution frequency of the single guanine deletion/insertion 4G/5G polymorphism (located -675 bp upstream from the start of transcription) significantly differed between the patients and healthy controls. In the Ml group, the4G/4G-genotype frequency was increased, but the 4G/5G-genotype is decreased when compared to the control group. In the Cl group, both the 4G/ 4G- and 4G/5G -genotypes occured at a lower frequency than those in the control group (P<0. 001) . The plasma PAI-1 activity level in the Ml group was lowered as the presence of the 4G allele decreases. In the Cl group, the frequency of 5G/5G was much higher than that of the control group (P<0. 001). The plasma PAI-1 activity level in the Cl group was elevated as the presence of the 5G allele increased. Furthermore, positive correlation between triglyceride, glucose levels and PAI-1 activity were found in all three groups (P<0. 001).Conclusions The PAI-1 4G/5G gene polymorphism is associated with a higher risk of Ml and Cl in individuals in Tianjin, China. The deletion/insertion polymorphism is probably an important hereditary risk factor for heart diseases. Moreover, triglyceride and glucose levels of plasma have functional importance in regulating PAI-1 activity.

Gene expression of fibrinolytic factors urokinase plasminogen activator and plasminogen activator inhibitor-1 in rabbit temporo-mandibular joint cartilage with disc displacement

Background The urokinase plasminogen activator system is believed to play an important role in degradation of the extracellular matrix associated with cartilage and bone destruction; however its precise roles in temporomandibular disorders have not yet been clarified. The aims of this study were to investigate the gene expression of fibrinolytic factors urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) in the articular cartilage of rabbit temporomandibular joint (TMJ) with disc displacement (DD) and to probe the relationship between fibrinolytic activity and cartilage remodeling. Methods Disc displacement of right joints was performed in 36 of 78 rabbits under investigation. The animals were sacrificed at 4 days and 1, 2, 4, 8 and 12 weeks after surgery, respectively. The right joints of these animals were harvested and processed for the examination of mRNA expression of uPA and PAI-1 in articular cartilage using in situ hybridization techniques. Results The expression of uPA and PAI-1 was co-expressed weakly in the chondrocytes from transitive zone to hypertrophic zone and mineralized zone, while no hybridizing signals were shown in proliferative zone and superficial zone in control rabbits. The most striking was the up-regulation of uPA and PAI-1 mRNA in 4-day rabbits postoperatively at the onset of cartilage degeneration. The strongest hybridizing signals for uPA and PAI-1 were seen in 2-week rabbits postoperatively. After 2 weeks, the expression of uPA and PAI-1 began to decrease and reached nearly normal level at 12 weeks. Conclusions The expression of the uPA/PAI-1 system coincides with the pathological changes in condylar cartilage after DD. The uPA/PAI-1 system may be one of the essential mediators in articular cartilage remodeling.

Comment on roles of tissue plasminogen activator and its inhibitor in proliferative diabetic retinopathy

Dear Sir,We congratulate Wu et al for their study entitled＂Roles of tissue plasminogen activator and its inhibitor in proliferative diabetic retinopathy＂.The authors investigated the effects of tissue plasminogen activator（t-PA）and plasminogen activator inhibitor（PAI）in the pathogenesis of proliferative diabetic retinopathy（PDR）.The authors reported that t-PA and PAI are involved in the pathogenesis of PDR.

tPA Involvement in Ovulation──Studies on Mechanism of Ovulation：Role of Tissue Type Plasminogen Activator

This review summarized our recent studies on involvement of tissue type plasminogen activator(tPA)and plasminogen activator inhibitor type 1(PAI-1) in process of ovulation.We have demonstrated that 1)hCG induces ovulation and coordinated tPA and PAI-1 gene expression in both rat and monkey ovaries;(2) GnRH and FSH are also capable of inducing ovulation by increasing ovarian tPA and PAI-1 gene expression in the same manner as hCG does;(3)Compounds which increase tPA production can induce oviation while compounds which decrease tPA and/or increase PAI-1 expression inhibit ovulation. Based on the data provided,a working model on the involvement of tPA in ovulation is presented.

An Experimental Study of Pathogenesis of Steroid-induced Avascular Necrosis of Femoral Head

Objective: To explore the pathogenesis of avascular necrosis of femoral head(ANFH) and search an effective method for clinical practice. Methods: Twenty-four Japanese rabbitswere divided into 2 groups of models and controls. ANFH models were produced byintramuscular-injection of large dosage of steroid to rabbits for 8 weeks. From the 4th, 8th and12th week after production of models, 2 rabbits of each group were sacrificed to observe thestructure of femoral head through light microscope and scanning electron microscope. The contents ofNitric Oxide (NO), tissue-type plasminogen activator (t-PA) and -plasminogen activator inhibitor(PAI) in plasma of the 4 rabbits in each group were estimated at the same time. Results: Comparedwith control group, the rabbits of model group exhibited many differences: such as osteoporosis offemoral head, the presence of more bone lacuna and fat cell through light microscope observing; thebroken and sunk bone trabecula, the loosen and broken collagen fibers on the surface of bone matrixthrough scanning electron microscope observing. Compared with control group, the Concentration ofNO and t-PA in plasma of the model rabbits decreased obviously, but the Concentration of the PAIincreased obviously. Conclusion: The steroid-induced ANFH might be related to the lower level of NOand the descent of fibrinolytic activity.

The involvement of p38 MAPK in transforming growth factor β1-induced apoptosis in murine hepatocytes

We reported in this manuscript that TGF-beta1 induces apoptosis in AML12 murine hepatocytes, which is associated with the activation of p38 MAPK signaling pathway. SB202190, a specific inhibitor of p38 MAPK, strongly inhibited the TGF-beta1-induced apoptosis and PAI-1 promoter activity. Treatment of cells with TGF-beta1 activates p38. Furthermore, over-expression of dominant negative mutant p38 also reduced the TGF-beta1-induced apoptosis. The data indicate that the activation of p38 is involved in TGF-beta1-mediated gene expression and apoptosis.

Analysis of Plasma Fibrinolysis in the Patients with Acute Cerebral Infarction

In the present study, the plasma fibrinolytic activity in 30 cases of cortical artery territory cerebral infarction (CACI) and 32 cases of perforating artery territory cerebral infarction (PACI) within 3 days after ictus, and 30 sexand age-matched controls without cardio-cerebrovascular diseases were evaluated by a comprehensive panel of assay, including the plasma tPA activity, PAI activity, endothelial capacity of tPA release and PAI/tPA ratio. The results showed that the plasma fibrinolysis and the endothelial potential to release tPA responding to stimulation in boch subtypes of the patients were significantly lower than those in the controls, which provide. the theoretical basis for carrying out the thrombolytic therapy in the ischemic stroke. And the study also suggested that increased Plasma PAI activity could increase the risk of AS chrombotic events with increased serum triglyceride level.

Evaluate the Expression of uPA,PAI-1 in Human Gastric Cancer and its Correlation with the Angiogenesis by the Application of Tissue Microarray

OBJECTIVE To investigate the expression of urokinase-type plasminogen （uPA）, its inhibitor-1 （PAI-1） mRNA and its protein in human gastric cancer and to find out the relationship among the tumor differentiation, angiogenesis, and other clinical pathologic factors. METHODS In situ hybridization （ISH） was used to get the uPA, PAI-lmRNA in 110 cases with human gastric cancer in 2-tissue microarray （TMA）. Immunohistochemical staining （S-P method） for uPA, PAI-1 protein and CD34 were performed in the 110 cases in 2 TMA. RESULTS The expression of the uPA, PAI-lmRNA and their protein happened in the cytoplasm of gastric cancer cells were induced by the poor differentiation of the GC, and the expression of uPA had an increasing trend while the expression of the PAI-1 had a decreasing trend. The microvessel density （MVD） had a positive correlation with the clinical stages and the significant relationship with the lymph node metastasis （P 〈 0.05）. The MVD in uPA positive group was significantly higher than those in uPA negative group （P 〈 0.05）. The expression of PAI-1 has no correlation neither with the clinical stages nor the lymph node metastasis. CONCLUSION The uPA play an important role in invasion and metastasis of GC through promoting angiogenesis. Interdicting the secretion and function of the uPA may allow the target therapy against the tumor invasion. As a new high-throughput technology, the tissue microarray is a valuable way to be used in clinical treatment.

The Physiopathological Crossroads of Aging

Stress, inflammation and Plasminogen activator inhibitor 1 (PAI-1) are key mechanisms throughout the development of aging, constituting a crossroad in the most frequent pathologies that accompany it. Among metabolic processes, obesity, metabolic syndrome and type 2 diabetes mellitus are included and Alzheimer’s disease among the neurodegenerative processes. Stress is a mechanism of defense of the organism against exogenous and endogenous actions called stressors. In the case of low intensity stimuli, the organism responds with actions aimed at a physiological adaptation (Homeostasis). On the other hand, when a high intensity (experimental level) or chronic stimulus (oxidative stress) is repeated, structural and functional changes are observed in different organs with activation of the hypothalamus-pituitary-adrenal axis, the renin angiotensin system and the sympathetic nervous system, stimulating the production of hormones that release cytokines with proin-flammatory/antiinflammatory properties that play an important role in the previously mentioned pathologies, as well as a marked increase in PAI-1, a gene regulated by stress and by cytokines, with manifest action at the origin of thromboembolic disease, so frequent in aging. The objective of this review is to highlight the importance of the binomial stress and PAI-1 in aging and in the pathologies that accompany it. Because PAI-1 is part of the pathology and complications in aging, some authors suggest the study of PAI-1 inhibitors to achieve its physiological levels, as part of the treatment of these diseases.