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*Diabetes
The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 1 286-291
Copyright © 2002 by The Endocrine Society

Other Original Articles

Impact of Diabetes-Associated Lipoproteins on Generation of Fibrinolytic Regulators from Vascular Endothelial Cells

Song Ren, Harvey Lee, Limei Hu, Lin Lu and Garry X. Shen

Diabetes Research Group, University of Manitoba (S.R., L.H., L.L., G.X.S.), and Rossmere Medical Clinic (H.L.), Winnipeg, Manitoba, Canada R3E 3P4

Address all correspondence and requests for reprints to: Garry X. Shen, M.D., Ph.D., Diabetes Research Group, University of Manitoba, 835-715 McDermot Avenue, Winnipeg, Manitoba, Canada R3E 3P4. E-mail: gshen{at}ms.umanitoba.ca


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Increased thrombotic tendency and decreased fibrinolytic activity have been frequently found in patients with diabetes mellitus (DM). Previous studies by our group indicated that nonenzymatically glycated low density lipoprotein (LDL) increased plasminogen activator inhibitor-1 (PAI-1) production and decreased the generation of tissue plasminogen activator (tPA) from cultured human umbilical vein endothelial cells (HUVEC). The present study demonstrates that plasma levels of PAI-1 or PAI-1/tPA were significantly increased in patients with type 1 (n = 10) and type 2 DM (n = 14) compared with those in healthy controls (n = 10; P < 0.05 or 0.01). LDL from patients with type 1 or type 2 DM, and very low density lipoprotein (VLDL) from patients with type 2 DM induced significantly greater increases in the release of PAI-1 and more profound reduction in tPA generation from HUVEC compared with corresponding lipoproteins from healthy controls (P < 0.05 or 0.01). HDL from diabetic patients did not significantly alter the generation of PAI-1 or tPA from endothelial cells (EC) compared with HDL from controls. Comparable effects of lipoproteins from DM patients on the generation of PAI-1 and tPA were found in human coronary artery EC. LDL and VLDL from patients with type 2 DM enhanced the activation of PAI-1 promoter (-1528/+55)/luciferase reporter gene transiently transfected in HUVEC (P < 0.01). The results of the present study suggest that LDL and VLDL from patients with DM reduce the generation of tPA and increase PAI-1 production through the activation of the PAI-1 promoter in vascular EC.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
DIABETES MELLITUS (DM) has been diagnosed in 4–5% of the North American population with an increasing tendency (1). Cardiovascular complications are the predominant cause of death for patients with DM. Several lines of evidence suggest that endothelial dysfunction plays a crucial role in the development of cardiovascular complications in diabetes. Risk factors for cardiovascular complications, such as smoking, hypertension, hyperlipidemia, hyperglycemia, or insulin resistance, interfere the normal functions of endothelial cells (EC) (2). Endothelial dysfunction has been linked to multiple biological processes implicated in the development of cardiovascular diseases, including vascular tone regulation, thrombosis, fibrinolysis, atherosclerosis, inflammation, tissue remodeling, and smooth muscle cell proliferation (3).

Attenuated fibrinolytic activity has been frequently found in patients with DM (4, 5). The formation of plasmin, which is the biologically active product of the fibrinolytic system, is mainly modulated by tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) in the blood circulation. Elevated levels of PAI-1 or reduced tPA activity were detected in DM patients (6, 7). Hyperglycemia and dyslipidemia are two major biochemical markers of diabetes. Dyslipidemia in DM is characterized by increased levels of chylomicrons, triglycerides, very low density lipoproteins (VLDL), or low density lipoproteins (LDL) and decreased levels of high density lipoprotein (HDL) cholesterol. Conventional antihyperglycemic treatment frequently normalizes the dyslipidemia in type 1 DM, but often does not completely correct that in type 2 DM. Increased levels of glycated lipoproteins were detected in plasma of diabetic patients with and without glucose control (8). Recent studies in our laboratory demonstrated that nonenzymatic glycation amplified the effects of LDL on the generation of PAI-1 and tPA from human umbilical vein EC (HUVEC) (9). The impact of lipoproteins from patients with DM on the generation of fibrinolytic regulators has not been documented. VLDL activates a cis element in the PAI-1 promoter (10). The effect of LDL or its modified forms on the PAI-1 promoter remains unclear.

The present study examined the effects of LDL, VLDL, and HDL isolated from diabetic patients on the generation of fibrinolytic regulators from cultured human venous and arterial EC. The effects of LDL and VLDL from healthy and diabetic individuals on the activation of the PAI-1 promoter transiently transfected in EC were investigated.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

Twenty-four Caucasians with DM were recruited at the Rossmere Medical Center (Winnipeg, Canada). Ten of them were diagnosed as type 1 DM (7 males and 3 females), and 14 patients were diagnosed as type 2 DM (10 males and 4 females) according to 1998 Clinical Practice Guidelines for the Management of Diabetes in Canada (11). Ten healthy Caucasians (7 males and 3 females) were recruited from staffs of Health Sciences Center (Winnipeg, Canada) as controls. The study was approved by the research ethics board of University of Manitoba. Informed consent was signed by every participant. The ages of the control group were not significantly different from those of the study groups of patients. The ages of the type 2 DM group were significantly greater than those of the type 1 DM group. The patients with type 2 DM had greater body mass index and shorter duration of diabetes than the patients with type 1 DM, as expected (Table 1Go). Hypertension was detected in 30% of patients with type 1 DM and in 40% of patients with type 2 DM. Retinopathy was confirmed in 40% of patients with type 1 DM and in 33% of patients with type 2 DM. Coronary artery disease, stroke, and peripheral vascular diseases were diagnosed in 7%, 7%, and 13% of the patients with type 2 DM, but were not found in the patients with type 1 DM. Abnormal elevation of serum creatinine or blood urea nitrogen were detected in 20% of patients in both groups of DM. Antihyperglycemic treatments were not ceased during the study. All of the patients with type 1 DM and 5 of the patients with type 2 DM were treated with insulin during the period of blood withdrawal. Their insulin injections at that morning were postponed until after the blood withdrawal. Two of the type 2 DM patients had not received any treatment before the blood withdrawal. Seven of patients with type 2 DM were receiving oral hypoglycemic agents. Among the 7 patients, 5 received glyburide (5–10 mg, twice daily), metformin (500 mg, three times daily), and acarbose (25–50 mg, three times daily); one of them received glyburide alone; and the other was given acarbose and glyburide. None of participants was taking lipid-lowering medications within 30 d before blood sample collection. The levels of fasting blood glucose and hemoglobin A1c (HbA1c) were elevated in type 1 and type 2 DM patients to a similar extent. The total cholesterol/HDL cholesterol ratios were elevated in both types of DM after overnight fasting. Increased levels of triglycerides and LDL cholesterol were found in patients with type 2 DM, but not type 1 DM, compared with controls (Table 1Go).


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  		Table 1. Characterization of subjects

 
Blood sample collection and plasma lipoprotein preparation

Blood was withdrawn at 0800–0900 h after 12–14 h of fasting. Fasting blood glucose, HbA1c serum lipid profile, creatinine, and blood urea nitrogen were analyzed at Central Medical Laboratory (Winnipeg, Canada). Plasma was separated by centrifugation at 1000 x g at 4 C for 15 min. Aliquots of plasma from each individual were frozen for the analysis of fibrinolytic regulators. VLDL (density, 0.96–1.006), LDL (1.019–1.063), and HDL (1.063–1.210) were isolated from fresh plasma from each participant by serial floatation ultracentrifugation as previously described (12). Lipoproteins from each individual were stored individually in sealed tubes overlaid with nitrogen at 4 C in the dark to prevent oxidation. The lipoproteins were used within 1 month after separation.

Cell culture and experimental stimulation

HUVEC were obtained by collagenase digestion as previously described (13), and the nature of the cells was verified by morphology and the presence of factor VIII antigen. Cells were grown to confluence in medium 199 supplemented with 10% heat-inactivated FBS, 30 µg/ml EC growth supplements (Sigma), 100 µg/ml heparin, 0.1 mM nonessential amino acids, 200 U/ml penicillin, and 200 µg/ml streptomycin in a humidified incubator under 95% air/5% CO2 at 37 C. Confluent cells were treated with lipoproteins supplemented in heparin-free medium 199. Seed cells of human coronary artery EC (HCAEC) were obtained from Clonetics (San Diego, CA). HCAECs were cultured in endothelial growth medium-MV and required supplements as instructed by the provider (Clonetics) and used within passage 8 (14). Both types of EC were seeded at 10 4/cm2 in culture dishes. Cytotoxicity of lipoproteins was determined by incubating cells with 5 x 106 dpm/well [3H]leucine (54 Ci/mM; ICN Radiochemical, Irvine, CA) in leucine-free medium for 2 h after treatment with lipoproteins. No detectable reduction in the incorporation of radioactive leucine was found in EC treated with lipoproteins under the tested conditions.

Measurement of PAI-1 and tPA antigens

Conditioned media of HUVEC were collected at the end of the incubations. Cells were harvested in PBS (pH 7.4) containing 0.1% SDS and 0.5% Triton X-100. Total PAI-1 and tPA antigens (free and complex forms) in the media were measured using IMUBIND PAI-1 or tPA ELISA kits (American Diagnostica, Inc., Greenwich, CT), and expressed in micrograms of antigen per mg total cellular proteins (9). Plasma PAI-1 and tPA antigens were analyzed by the same method but expressed in micrograms per ml.

Preparation of PAI-1 promoter/luciferase reporter gene constructs

A fragment of PAI-1 promoter between -1557 bp and +55 bp (-1557/+55) was generated from human blood cell genomic DNA with the assistance of a pair of PCR primers (sense, 5'-CAGTTTCCACCCTCTACAGCA; antisense with flanking HindIII site, 5'-GTAAGCTTGCGTGTGGGTCTTCTTGAC) designed according to a reported sequence (GenBank J03764). The PCR product (1620 bp) was inserted into pGEM-T Easy vector (Promega Corp., Madison, WI) to generate pGEM-PAI-1(-1557/+55). A PAI-1 promoter luciferase (Luc) reporter construct, pPAI-1(-1528/+55)/Luc, was prepared by inserting a KpnI/SalI fragment from pGEM-PAI-1(-1557/+55) into the pXP1/Luc vector.

Transfection assay

One day before transfection, HUVECs were subcultured in six-well culture dishes in a density of 1 x 106/well. PAI-1 promoter/reporter constructs were precipitated in HEPES buffer (pH 7.05) containing 125 mM CaCl2 for 20 min. Cells were incubated with calcium phosphate-precipitated DNA for 4 h. pCDNA3-chloramphenicol acetyltransferase (CAT) expression vector was cotransfected with pPAI-1/Luc vector as an internal control. After transfection, cells were treated with medium containing 10% serum with or without addition of lipoproteins. Activities of luciferase in cell lysate were measured using the Luciferase Assay System (Promega Corp.) on Lumat LB9507 luminometer (Berthold, Nashua, NH) (15). CAT activity in cell lysate was determined by two-phase fluor diffusion assay as previously described (16).

Statistics

A t test was used for comparing the probability of values between two groups. Comparisons among multiple groups were achieved using one-way ANOVA, followed by Duncan’s test. The level of significance was defined as P < 0.05.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Plasma levels of PAI-1 and tPA in patients with DM

The levels of PAI-1 antigen were significantly increased in plasma of patients with type 2 DM compared with healthy individuals or patients with type 1 DM (P < 0.05). The plasma levels of PAI-1 in patients with type 1 DM were not significantly higher than control values. The levels of tPA antigen in plasma of both types of DM patients were significantly lower than control levels (P < 0.05 or 0.01). The ratios of PAI-1/tPA in patients with type 1 or type 2 DM were significantly greater than control values (P < 0.05 or 0.01; Fig. 1Go).



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  		Figure 1. Plasma PAI-1 and tPA in patients with DM. Blood was withdrawn from 10 healthy subjects (control), 10 patients with type 1 DM, and 14 patients with type 2 DM after 12–14 h of fasting. Plasma PAI-1 and tPA antigens were analyzed by ELISA. Values were expressed as micrograms per ml or as the ratio of PAI-1/tPA (mean ± SE). *, P < 0.05; P < 0.01 (vs. control group). +, P < 0.05 (vs. type 1 DM).

 
Effects of lipoproteins from diabetic patients on the generation of PAI-1 and tPA from HUVEC

Previous studies by our group indicated that incubation with 50–100 µg protein/ml native or glycated LDL for 48 h induced the maximal extent of changes in PAI-1 and tPA from HUVEC (9). Dose responses of lipoproteins from patients with type 2 DM on the generation of PAI-1 and tPA from HUVEC were determined in the present study. Lipoprotein levels above 200 µg/ml, but not 150 µg/ml or less, from healthy or diabetic patients noticeably affected the morphology and reduced cell numbers of EC (data not shown). One of possible interpretations for the finding is that ECs cultured in vitro may be less resistant to certain components of lipoproteins than in vivo, where ECs are supported by tissue and cells in the vessel wall. LDL from patients with type 2 DM (n = 4) at levels beyond 50 µg/ml significantly increased PAI-1 release from EC after 48 h of treatment compared with that in control cultures without LDL addition (P < 0.05). Increases in PAI-1 generation reached a plateau in EC treated with 100 µg/ml LDL or more. Addition of VLDL from diabetic patients at concentrations greater than 100 µg/ml increased the release of PAI-1 from EC (P < 0.05 or 0.01). Treatments with 100 µg/ml LDL or more from diabetic patients induced significantly greater increases in PAI-1 generation compared with equal concentrations of VLDL (P < 0.05 or 0.01). HDL from diabetic patients at concentrations up to 150 µg/ml did not significantly alter PAI-1 generation from HUVEC (Fig. 2Go, upper panel). The levels of tPA antigen in conditioned medium of EC treated with 50 µg/ml LDL or VLDL or more from patients with type 2 DM for 48 h were significantly lower than those in control cultures (P < 0.001). LDL treatment (>=100 µg/ml) induced a more profound decrease in tPA generation than equal amounts of VLDL (P < 0.05 or 0.001). Treatment with HDL (50–150 µg/ml) from patients with DM did not significantly reduce tPA generation from EC (Fig. 2Go, lower panel).



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  		Figure 2. Dose response of lipoproteins from patients with DM on PAI-1 and tPA release from HUVEC. VLDL, LDL, and HDL were isolated from patients with type 2 DM after 12–14 h of fasting. HUVECs were treated with 0–150 µg protein/ml lipoproteins from patients with DM for 48 h. The levels of PAI-1 and tPA antigens in postculture media were determined by ELISA. Values were expressed as micrograms of antigen per mg total cellular proteins (mean ± SD; n = 4 patients). Upper panel, PAI-1; lower panel, tPA. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (vs. cultures without addition of lipoprotein). +, P < 0.05; ++, P < 0.01; +++, P < 0.001 (vs. cultures treated with equal amounts of VLDL). x, P < 0.05; xx, P < 0.01; xxx, P < 0.001 (vs. cultures treated with equal amounts of HDL).

 
VLDL, LDL, and HDL (100 µg/ml) from patients with type 1 DM (n = 10), type 2 DM (n = 14), and healthy donors (n = 10) were incubated with HUVEC for 48 h. LDL, but not VLDL, from patients with type 1 DM increased PAI-1 release from EC compared with LDL from controls (P < 0.05). Both LDL and VLDL from patients with type 2 DM increased PAI-1 generation from EC (P < 0.01; Fig. 3Go, upper panel). LDL from patients with type 1 DM reduced the release of tPA from EC compared with LDL from controls (P < 0.05). Both VLDL and LDL from patients with type 2 DM reduced tPA generation from EC compared with corresponding lipoproteins from controls (P < 0.01) and type 1 DM (P < 0.05; Fig. 3Go, lower panel). HDL from patients with both types of DM did not significantly alter the generation of PAI-1 or tPA from EC compared with that in controls.



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  		Figure 3. Effects of various lipoproteins from patients with type 1 and type 2 DM on the generation of PAI-1 and tPA from HUVEC. VLDL, LDL, and HDL were isolated from 10 healthy subjects (control), 10 patients with type 1 DM, and 14 patients with type 2 DM after 12–14 h of fasting. HUVECs were treated with 100 µg protein/ml lipoproteins from diabetic patients for 48 h. The levels of PAI-1 and tPA antigens in postculture media were determined by ELISA. Values were expressed as the percentage of cultures without addition (mean ± SD). Upper panel, PAI-1; lower panel, tPA. *, P < 0.05; P < 0.01 (vs. corresponding lipoproteins from control group). +, P < 0.05; ++, P < 0.01 (vs. corresponding lipoproteins from type 1 DM group).

 
Effects of lipoproteins from DM patients on the generation of fibrinolytic regulators from HCAEC

The influence of lipoproteins from diabetes on the generation of PAI-1 and tPA was further investigated in HCAEC using VLDL, LDL, and HDL from type 2 DM and healthy subjects (n = 4). VLDL and LDL from patients with type 2 DM significantly increased PAI-1 generation and reduced the release of tPA from HCAEC (P < 0.05 or 0.01). HDL from the patients with type 2 DM did not significantly alter the generation of PAI-1 or tPA from arterial EC (Fig. 4Go). The levels of PAI-1 and tPA released from HCAEC (Fig. 4Go) were 2–4 times higher than those from HUVEC basally and under lipoprotein-stimulated conditions (Fig. 2Go).



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  		Figure 4. Effects of lipoproteins from patients with DM on the generation of fibrinolytic regulators from HCAEC. VLDL, LDL, and HDL (100 µg/ml) from healthy subjects (control) and type 2 DM patients after 12–14 of fasting were incubated with HCAEC for 48 h. The levels of PAI-1 and tPA antigens in postculture media were determined by ELISA. Values were expressed as micrograms per mg (mean ± SD; n = 4). Upper panel, PAI-1; lower panel, tPA. *, P < 0.05; **, P < 0.01 (vs. corresponding lipoproteins from control group).

 
Effects of lipoproteins from patients with DM on the activation of the PAI-1 promoter

The impact of LDL and VLDL from diabetic patients on activation of the PAI-1 promoter transiently transfected in HUVEC was determined. The PAI-1 promoter/reporter gene vector, pPAI-1(-1528/+55)/Luc, contains the VLDL response element (-672/-657) (10). LDL and VLDL from healthy individuals (n = 5) induced moderate increases in the activity of the PAI-1 promoter compared with cells transfected with same vectors, but without an addition of lipoproteins. VLDL and LDL from patients with type 2 DM (n = 5) significantly increased the activation of the PAI-1 promoter compared with EC treated with same concentrations of corresponding lipoproteins from healthy subjects (Fig. 5Go).



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  		Figure 5. Effects of lipoproteins from patients with DM on the activation of the PAI-1 promoter in HUVEC. Cells were transiently transfected with the PAI-1 promoter/luciferase reporter gene vector, pPAI-1(-1528/+55)/Luc. pCDNA3-CAT expression vector was cotransfected as an internal control. After transfection, cells were treated with medium with or without addition of 100 µg/ml LDL or VLDL from healthy controls or type 2 DM patients for 24 h. Activities of luciferase in cell lysates were measured using the Luciferase Assay System, and CAT activity was determined by a two-phase fluor diffusion assay. Values were expressed as fold increases after adjustment for CAT activity (mean ± SD; n = 5). *, P < 0.01 vs. lipoproteins from controls.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Previous studies indicated that VLDL from hypertriglyceridemic individuals induced greater increases in PAI-1 generation than VLDL of normolipidemics from HUVEC (17). Our group and others demonstrated that native and oxidized LDL stimulated PAI-1 generation from HUVEC (18, 19, 20, 21). Our recent studies demonstrated that nonenzymatic glycation enhances the effect of LDL on the generation of PAI-1 and reduces the release of tPA from HUVEC (9). The present study for the first time demonstrated that LDL and VLDL from patients with DM increased the generation of PAI-1 and decreased the release of tPA from venous or arterial EC compared with corresponding lipoproteins from healthy individuals. Imbalance between vascular EC-derived PAI-1 and tPA induced by diabetes-associated apoB-rich lipoproteins may lead to the attenuation of fibrinolytic activity in local blood circulation, which may lead to thrombotic tendency.

Dyslipoproteinemia, characterized by an increased ratio of total cholesterol/HDL cholesterol, is frequently detected in diabetic patients lacking optimal glucose control. Elevated levels of triglyceride-rich lipoproteins, mainly VLDL during the fasting state, have also been considered as a risk for coronary artery disease, although the results from various studies are not consistent (22). LDL particles are the major cholesterol carrier in plasma. Oxidatively modified LDL has been implicated in the pathogenesis of atherosclerosis in many aspects (23). The levels of glycated LDL were increased in diabetic patients (24). Glycated LDLs are susceptible to oxidation (25). The results of the present study demonstrated that LDL from both type 1 and type 2 DM patients with suboptimal glucose control (characterized by moderately elevated HbA1c) increased PAI-1 generation and decreased tPA release from vascular EC. VLDL from patients with type 2, but not type 1, DM stimulated the generation of PAI-1 and reduced the release of tPA from vascular EC. Increased levels of triglycerides were found in patients with type 2, but not those with type 1, DM in the present study. Our results support the previous findings that VLDL from hypertriglyceridemic individuals is one of lipoprotein stimulants for PAI-1 generation from EC (17). The results of the present study suggest that VLDL may also attenuate fibrinolytic activity through the reduction of tPA generation from EC. Both LDL and VLDL from diabetic patients may enhance the activation of the PAI-1 promoter transiently transfected in HUVEC compared with LDL from healthy subjects. This suggests that LDL as well as VLDL regulate PAI-1 production in EC at the transcriptional level. Previous studies in our laboratory demonstrated that native and glycated LDL did not significantly affect the mRNA levels of tPA (9), but decreased the de novo synthesis of tPA in HUVEC (unpublished observations). These findings suggest that LDL and its glycated form do not regulate tPA generation at the transcriptional level.

HDL has been considered a protective lipoprotein factor for atherosclerotic cardiovascular diseases (26). The results of the present study indicated that HDL from diabetic or healthy subjects did not significantly alter PAI-1 or tPA generation from EC. Recent studies by this laboratory indicate that cotreatment of HDL or its glycated form may normalize native or glycated LDL-induced changes in PAI-1 and tPA generation from HUVEC (14). The combination of increased levels of LDL or VLDL and decreased levels of HDL may substantially reduce EC-derived fibrinolytic activity in blood circulation of patients with diabetes.

Decreased levels of tPA were found in plasma of type 1 and type 2 DM patients. Elevated levels of PAI-1 was detected in plasma of type 2, but not type 1, DM patients. Similar changes in PAI-1 and tPA levels were reported by some, but not all, groups (27, 28, 29). Previous studies demonstrated that proinsulin increased the generation of PAI-1 from vascular EC (30). Proinsulin is deficient in type 1 DM, and the levels of proinsulin are increased in type 2 DM patients (31, 32). This may be one of reasons for the relatively higher levels of PAI-1 detected in the plasma of patients with type 2 DM compared with type 1 DM. Hyperglycemia is another biochemical marker in diabetic patients. The effect of glucose on the generation of PAI-1 was examined in previous studies, but the findings were not consistent (33, 34). Lipoproteins isolated from diabetic patients were thoroughly dialyzed. Free glucose was removed from the lipoprotein preparations through dialysis before they were added to cultured EC. Previous studies indicated that metformin moderately reduced the levels of PAI-1 antigen in plasma of patients with type 2 DM (35). Due to the consideration of ethics, hypoglycemic therapy was not interrupted in the diabetic patients during the study. Five of the patients with type 2 DM in the present study were receiving metformin. Plasma PAI-1 levels in the type 2 DM patients receiving metformin (60.3 ± 18.0 µg/ml; n = 5; mean ± SE) were not significantly different from that in the rest of patients with type 2 DM (55.0 ± 10.9 µg/ml; n = 9; P > 0.05). The possibility that the levels of PAI-1 in plasma of the type 2 DM patients receiving metformin in the present study were reduced by the treatment has not been excluded.

In summary, VLDL and/or LDL from patients with DM may reduce tPA generation and/or increase PAI-1 production from venous and arterial EC. HDL from DM patients did not significantly alter the generation of the fibrinolytic regulators from EC. The overproduction of PAI-1 induced by LDL and VLDL from diabetic patients may be mediated through the activation of the PAI-1 promoter. The alterations in the generation of fibrinolytic regulators from EC induced by lipoproteins from diabetic patients may attenuate fibrinolytic activity in vasculature.


   Acknowledgments
 
We thank Dr. Liam J Murphy (Departments of Internal Medicine and Physiology, University of Manitoba) for help with the analysis of luciferase activity, Drs. Peter Cattini and Mary-Lynn Duckworth (Department of Physiology, University of Manitoba) for providing important reagents, Fuqing Zhu for technical assistance, and Mrs. Kathy Demski (Rossmere Medical Center) for assistance with data collection.


   Footnotes
 
This work was supported by operating grants from the Canadian Diabetes Association in the memory of late Archibald Mitchell, the Canadian Institute of Health Research, the Health Sciences Centre Foundation, and the St. James Kiwanis Club (to G.X.S.).

Abbreviations: CAT, Chloramphenicol acetyltransferase; DM, diabetes mellitus; EC, endothelial cells; HbA1c, hemoglobin A1c; HCAEC, human coronary artery EC; HUVEC, human umbilical vein endothelial cells; LDL, low density lipoprotein; PAI-1, plasminogen activator inhibitor-1; tPA, tissue plasminogen activator; VLDL, very low density lipoprotein.

Received June 4, 2001.

Accepted October 9, 2001.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Ekoe JM 1999 Epidemiology of type 2 diabetes mellitus in North America. Can J Diabetes Care 23:43–46
  2. Quyyumi AA 1998 Endothelial function in health and disease: new insights into the genesis of cardiovascular disease. Am J Med 105:32S–39S
  3. Laight DW, Carrier MJ, Anggard EE 1999 Endothelial cell dysfunction and the pathogenesis of diabetic macroangiopathy. Diabetes Metab Res Rev 15:274–282[CrossRef][Medline]
  4. Feanley GR, Chakrabarti R, Avis PRD 1963 Blood fibrinolytic activity in diabetes mellitus and its bearing on ischaemic heart disease and obesity. Br Med J 1:921–923
  5. Sharma SC 1981 Platelet adhesiveness, plasma fibrinogen, and fibrinolytic activity in juvenile-onset and maturity-onset diabetes mellitus. J Clin Pathol 34:501–503[Abstract/Free Full Text]
  6. Gough SC, Rice PJ, McCormack L, Chapman C, Grant PJ 1993 The relationship between plasminogen activator inhibitor-1 and insulin resistence in newly diagnosed type 2 diabetes mellitus. Diabet Med 10:638–642[Medline]
  7. Matsuo T, Kadowaki S, Okada K, Matsuo O 1990 Activity of tissue plasminogen activator and plasminogen activator inhibitor in noninsulin-dependent diabetes mellitus. Hals-, Nasen-, and Ohrenheikunde 4:119–121
  8. Lyons TJ, Baynes JW, Patrick JS, Colwell JA, Lopes-Virella MF 1986 Glycosylation of low density lipoprotein in patients with type I (insulin-dependent) diabetes: correlation with other parameters of glycaemic control. Diabetologia 29:685–689[CrossRef][Medline]
  9. Zhang J, Ren S, Sun D, Shen GX 1998 Influence of glycation on LDL-induced generation of fibrinolytic regulators in vascular endothelial cells. Arterioscler Thromb Vasc Biol 18:1040–1048
  10. Eriksson P, Nilsson L, Karpe F, Hamsten A 1998 Very low density lipoprotein response element in the promoter region of the human plasminogen activator inhibitor-1 gene implicated in the impaired fibrinolysis of hypertriglyceridemia. Arterioscler Thromb Vasc Biol 18:20–26[Abstract/Free Full Text]
  11. Meltzer S, Leiter L, Daneman D, Gerstein HC, Lau D, Luswig S, Yale JF, Zinman B, Lillie D 1998 Clinical Practice Guidelines for the Management of Diabetes in Canada. Canadian Diabetes Association. Can Med Assoc J 159(Suppl):S1–S29
  12. Havel RJ, Eder HA, Bragdon JH 1955 The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest 34:1345–1353
  13. Shen XY, Figard PH, Kaduce TL, Spector AA 1988 Conversion of 15-hydroxyeicosatetraenoic acid to 11-hydroxyhexadecatrienoic acid by endothelial cells. Biochemistry 27:996–1004[CrossRef][Medline]
  14. Ren S, Shen GX 2000 Impact of antioxidants and HDL on glycated LDL-induced generation of fibrinolytic regulators from vascular endothelial cells. Arterioscler Thromb Vasc Biol 20:688–693
  15. Hu M, Roberston G, Murphy LJ 1996 Growth hormone modulates insulin regulation of hepatic insulin-like growth factor binding protein-1 transcription. Endocrinology 137:3702–3709[Abstract]
  16. Nickel BE, Kadami E, Cattini PA 1990 Differential expression of human placental growth hormone variant and chorionic somatomammotropin in cultures. Biochem J 267:653–658[Medline]
  17. Latron Y, Chautan M, Anfosso F, Alessi MC, Nalnone G, Lafont H, Juhan-Vague I 1991 Stimulating effect of oxidized low density lipoprotein on plasminogen activator inhibitor-1 synthesis by endothelial cells. Arterioscler Thromb 11:1821–1829[Abstract/Free Full Text]
  18. Kugiyama K, Sakamoto T, Misumi I, Sugiysma S, Ohgushi M, Ogawa H, Horiguchi M, Yasue H 1993 Transferable lipids in oxidized low-density lipoprotein stimulate plasminogen activator inhibitor-1 and inhibit tissue-type plasminogen activator from endothelial cells. Circ Res 73:335–343[Abstract/Free Full Text]
  19. Tremoli E, Camera M, Maderna P, Sironi L, Prati L, Colli S, Piovella F, Bernini F, Corsini A, Mussoni L 1993 Increased synthesis of plasminogen activator inhibitor-1 by cultured human endothelial cells exposed to native and modified LDLs. An LDL receptor-independent phenomenon. Arterioscler Thromb 113:338–346
  20. Ren S, Man RYK, Angel A Shen GX 1997 Oxidative modification enhances lipoprotein(a)-induced overproduction of plasminogen activator inhibitor-1 in cultured vascular endothelial cells. Atherosclerosis 128:1–10[CrossRef][Medline]
  21. Allison BA, Nilsson L, Karpe F, Hamsten A, Eriksson P 1999 Effects of native, triglyceride-enriched, and oxidatively modified LDL on plasminogen activator inhibitor-1 expression in human endothelial cells. Arterioscler Thromb Vasc Biol 19:1354–1360[Abstract/Free Full Text]
  22. Austin MA 1991 Plasma triglyceride and coronary artery disease. Arterioscler Thromb 11:2–14[Abstract/Free Full Text]
  23. Young SG, Parthasarathy S 1994 Why are low-density lipoproteins atherogenic? West J Med 160:183–184[Medline]
  24. Lyons TJ, Baynes JW, Patrick JS, Colwell JA, Lopes-Virella MF 1986 Glycosylation of low density lipoprotein in patients with type I (insulin-dependent) diabetes: correlation with other parameters of glycaemic control. Diabetologia 29:685–689
  25. Kobayashi K, Watanabe J, Umeda F, Nawata H 1995 Glycation accelerates the oxidation of low density by copper ions. Endocrinol J 42:461–465
  26. Gorden T, Casterlli WP, Hajortland MC, Kennel WB, Dawber TR 1977 High-density lipoprotein as a protective factor against coronary heart disease: the Framingham study. Am J Med 62:707–730[CrossRef][Medline]
  27. Schalkwijk CG, Smulders RA, Lambert J, Donker AJ, Sthouwer CD 2000 ACE-inhibition modulates some endothelial functions in health subjects and in normotensive type 1 diabetic patients. Eur J Clin Invest 30:853–860[CrossRef][Medline]
  28. Gasic S, Wagner OF, Fasching P, Ludwig C, Veitl M, Kapiotis S, Jilma B 1999 Fosinopril decreases levels of soluble vascular cell adhesion molecule-1 in borderline hypertensive type II diabetic patients with microalbuminuria. Am J Hypertens 12:217–222[Medline]
  29. Gough SC, Rice PJ, McCormack L, Chapman C, Grant PJ 1993 The relationship between plasminogen activator inhibitor-1 and insulin resistance in newly diagnosed type 2 diabetes mellitus. Diabet Med 10:638–642
  30. Schneider DJ, Nordt TK, Sobel BE 1992 Stimulation by proinsulin of expression of plasminogen activator inhibitor 1 in endothelial cells. Diabetes 41:890–895[Abstract]
  31. Bergnestal RM, Cohen RM, Lever E, Polonsky K, Jaspan J, Blix PM, Revers R, Olefsky JM, Kolterman O, Steiner K, Cherrington A, Frank B, Rubenstein AH 1984 The metablic effects of biosynthetic human proinsulin in individuals with type 1 diabetes. J Clin Endocrinol Metab 58:973–979[Abstract]
  32. Schneider DJ, Nordt TK, Sobel BE 1992 Stimulation by proinsulin of expression of plasminogen activator type-1 in endothelial cells. Diabetes 41:890–895
  33. Nordt TK, Klassen KJ, Schneider DJ, Sobel BE 1993 Augmentation of synthesis of plasminogen activator inhibitor type-1 in arterial endothelial cells by glucose and its implication for local fibrinolysis. Arterioscler Thromb 13:1822–1828[Abstract/Free Full Text]
  34. Kollros PR, Konkle BA, Ambarian AP, Henrikson P 1994 Plasminogen activator inhibitor-1 expression by brain microvascular endothelial cells is inhibited by elevated glucose. J Neurochem 63:903–909[Medline]
  35. Grant PJ 1996 The effects of high- and medium-dose metformin therapy on cardiovascular risk factors in patients with type II diabetes. Diabetes Care 19:64–66[Abstract]



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