Impact of antioxidants and HDL on glycated LDL-induced generation of fibrinolytic regulators from vascular endothelial cells

Hyperglycemia and dyslipoproteinemia are biochemical markers of diabetes mellitus (DM). Elevated levels of plasminogen activator inhibitor-1 (PAI-1) with and without reduction of tissue plasminogen activator (tPA) in plasma have been frequently found in patients with DM. Our previous studies indicated that glycation enhances low density lipoprotein (LDL)-induced production of PAI-1 and further decreases tPA generation in vascular endothelial cells (ECs). The present study demonstrated that treatment with antioxidants, butylated hydroxytoluene or vitamin E, blocked native LDL- and glycated LDL-induced changes in PAI-1 and tPA generation in ECs. Native or glycated high density lipoprotein (HDL) did not significantly alter tPA generation in ECs. Glycated but not native HDL (>/=100 microg/mL) moderately increased PAI-1 release from ECs. Cotreatment with native or glycated HDL inhibited LDL-induced or glycated LDL-induced changes in PAI-1 and tPA generation in ECs. The abundance of conjugated dienes was increased in glycated or EC-modified LDL. Treatment with butylated hydroxytoluene, vitamin E, or HDL reduced the abundance of conjugated dienes in glycated or EC-modified LDL. The effects of antioxidants and HDL on LDL-induced or its glycated LDL-induced changes in the generation of PAI-1 and tPA were also found in cultured human coronary artery ECs. The findings of the present study suggest that antioxidants and HDL may attenuate native LDL- or glycated LDL-induced changes in the generation of fibrinolytic regulators from vascular ECs, which possibly results from their inhibition on the lipid peroxidation of LDL particles. Treatment with antioxidants or hypolipidemic agents potentially improves fibrinolytic activity and reduces thrombotic tendencies in patients with DM.     

Glycation amplifies lipoprotein(a)-induced alterations in the generation of fibrinolytic regulators from human vascular endothelial cells

Increased lipoprotein(a) [Lp(a)] in plasma is an independent risk factor for premature cardiovascular diseases. The levels of glycated Lp(a) are elevated in diabetic patients. The present study demonstrated that glycation enhanced Lp(a)-induced production of plasminogen activator inhibitor-1 (PAI-1), and further decreased the generation of tissue-type plasminogen activator (t-PA) from human umbilical vein endothelial cells (HUVEC) and human coronary artery EC. The levels of PAI-1 mRNA and its antigen in the media of HUVEC were significantly increased following treatments with 5 μg/ml of glycated Lp(a) compared to equal amounts of native Lp(a). The secretion and de novo synthesis of t-PA, but not its mRNA level, in EC were reduced by glycated Lp(a) compared to native Lp(a). Treatment with aminoguanidine, an inhibitor for the formation of advanced glycation end products (AGEs), during glycation normalized the generation of PAI-1 and t-PA induced by glycated Lp(a). Butylated hydroxytoluene, a potent antioxidant, inhibited native and glycated Lp(a)-induced changes in PAI-1 and t-PA generation in EC. The results indicate that glycation amplifies Lp(a)-induced changes in the generation of PAI-1 and t-PA from venous and arterial EC. This may attenuate fibrinolytic activity in blood circulation and potentially contributes to the increased incidence of cardiovascular complications in diabetic patients with hyperlipoprotein(a). EC-mediated oxidative modification and the formation of AGEs may be implicated in glycated Lp(a)-induced alterations in the generation of fibrinolytic regulators from vascular EC.     

Abnormalities in platelets and vascular endothelial cells induced by glycated lipoproteins.

We studied the effects of glycated lipoproteins of low- and high-density (LDL and HDL) on platelets and vascular endothelial cells. After pretreatment for 5 minutes at 37 degrees C, the thrombin-induced synthesis of thromboxane B2 in washed platelets was significantly increased by glycated LDL as compared with native LDL (198.9 +/- 16.2 vs 90.3 +/- 29.4 ng/10(9) platelets, n = 8, p less than 0.01). Platelet aggregation was also increased by glycated LDL as compared with native LDL. After treatment with platelet-rich plasma for 5 hours at 37 degrees C, these values were suppressed by native HDL vs the control (buffer), but not by glycated HDL. Abnormalities in the release of 6-keto prostaglandin F1 alpha and lactate dehydrogenase from vascular endothelial cells were also induced by glycated LDL and/or HDL. These observations suggest that abnormalities induced in platelets and vascular endothelial cells by glycated lipoproteins may play an important role in the development of atherosclerosis in patients with diabetes mellitus.     

Influence of endothelin on human platelet aggregation and prostacyclin generation from human vascular endothelial cells in culture.

The effects of endothelin (ET) on the function of cultured human umbilical vein endothelial cells (HUVEC) and that of human platelets were investigated with reference to endothelium-derived relaxing factor (EDRF) and PGI2. Considering the platelets, ET had no effect on platelet-rich plasma (PRP) aggregation, the generation of thromboxane A2 ([TXA2]) from platelets, and cytosolic free calcium ion concentration ([Ca++]i), cAMP content ([cAMP]i) or cGMP content ([cGMP]i) in platelets. In contrast, the addition of the solution in which HUVEC had been incubated with ET to PRP produced a decrease in PRP aggregation, [TXA2], and [Ca++]i, and an increase not only in [cAMP]i but also in [cGMP]i in platelets. In the HUVEC pretreated with acetylsalicylic acid (aspirin), this increase of [cGMP]i was not affected, but the HUVEC-mediated decrease in PRP aggregation, [TXA2], and [Ca++]i induced by ET were not completely abolished. However, the pretreatment of HUVEC with a combination of aspirin and L-NG-monomethyl arginine (LNMMA) as an inhibitor of EDRF completely abolished the HUVEC-mediated decrease in PRP aggregation, [TXA2] and [Ca++]i induced by ET, and also abolished the enhancement of [cGMP]i and [cAMP]i in platelets. The PGI2 of HUVEC was enhanced by ET with no changes in [Ca++]i, [cAMP]i and [cGMP]i. The ET-induced enhancement was remarkably attenuated by pretreating the HUVEC with aspirin, but not with LNMMA. We conclude that ET attenuates the aggregation of platelets through a decrease in [TXA2] by an increase in [cAMP]i via the increase in PGI2 of HUVEC, and by an increase in [cGMP]i via EDRF.     

Effect of inflammatory cytokines on the metabolism of low-density lipoproteins by human vascular endothelial cells

Cytokines have been shown to activate multiple, varied metabolic pathways in endothelial cells. Little information is available concerning the effects of inflammatory cytokines on lipoprotein metabolism by vascular endothelial cells. Human umbilical vein endothelial cells (HuECs) and bovine aortic endothelial cells (BAECs) were incubated with the inflammatory cytokines recombinant human interleukin-1beta (IL-1), tumor necrosis factor alpha (TNF), interferon gamma (gamma-IF), and interferon beta (beta-IF) at increasing concentrations (0.1 to 1,000 U/mL), for increasing periods (6 to 72 hours). After the incubation period, the media were removed and replaced with serum-free media containing radiolabeled native or acetylated low-density lipoprotein (Ac-LDL) and the rates of degradation and accumulation of radiolabeled LDL were determined. The degradation and accumulation of 125I-LDL were significantly increased (P < .02) in HuECs preincubated with IL-1 (100 U/mL) compared with control incubations without the cytokine or incubations containing gamma-IF, beta-IF, or TNF. This resulted from a 38% increase in LDL receptor protein in cells incubated with IL-1. The increased rate of LDL catabolism by HuECs incubated with IL-1 was accompanied by a significant increase (P < .05) in the rate of cholesteryl ester synthesis in the cells. Cholesteryl ester synthesis rates in HuECs preincubated with gamma-IF, beta-IF, or TNF did not differ significantly from the rates in control incubations. The effect of preincubation with cytokine on the activity of the scavenger receptor was also determined. There were no significant differences in the rate of degradation or accumulation of radiolabeled Ac-LDL in control incubations compared with cultures preincubated with IL-1, gamma-IF, beta-IF, or TNF. There also were no significant differences in the rate of catabolism of native LDL or Ac-LDL in BAECs preincubated with cytokines. Although cytokines have been shown previously to alter the binding of monocytes to endothelial cells, there was no significant increase in the binding of monocytes to cultures incubated with IL-1 plus LDL compared with IL-1 alone. In summary, we now demonstrate that cytokines, specifically IL-1, may alter LDL metabolism by human vascular endothelial cells and alter endothelial cell cholesterol metabolism. These changes in endothelial cell metabolism provide additional evidence supporting the critical role of cytokines in atherogenesis.     

Diabetes mellitus and diabetes-associated vascular disease.

Diabetes-related cardiovascular disease remains the leading cause of death in patients with type 2 diabetes. Hypertension is common among diabetics and has the same pathogenetic mechanisms as insulin resistance, in which the activated renin-angiotensin system contributes to the emerging high blood pressure and hyperglycemia. Hyperglycemia is one of the triggering factors for vascular dysfunction and clotting abnormalities and, therefore, for accelerated atherosclerosis in diabetes. Glycated hemoglobin levels, as a reflection of the degree of glycemia, are strongly associated with the risk of cardiovascular disease in diabetics and in the general population. Tight glycemic control, the treatment of dyslipidemia and raised blood pressure, in addition to the use of antiplatelet therapy, all powerfully reduce the risks associated with diabetes. Furthermore, angiotensin-converting enzyme inhibitors might offer additional cardioprotection to diabetics above that provided by blood pressure reduction.     

Effect of cyclic stretch on endothelial cells from different vascular beds.

Endothelial cells (EC) mediate many of the organ responses to shock. Much of our knowledge of EC are obtained from cell culture studies. However, compared to the dynamic milieu in vivo, the stationary environment for large-vessel EC may be artificial and inappropriate. In this study, the morphology, growth rate, and production of prostacyclin (PGI2) by EC obtained from different vascular beds under stationary and dynamic conditions were examined. EC were harvested from the thoracic aorta (Ao), pulmonary artery (PA), and vena cava (VC) of the same calves and exposed to 0.5 sec 24% deformation alternating with 0.5 sec relaxation (i.e., 60 cycles/min). Our results show that in response to the cyclic regimen, VCEC were elongated perpendicular to the force vector and their actin filaments aligned in the same direction, while AoEC and PAEC did not exhibit any morphological changes. The growth rate of AoEC (but not PAEC or VCEC) was significantly enhanced when stimulated by cyclic stretch. In addition, AoEC demonstrated an increased PGI2 synthetic activity with cyclic stretch, while PAEC and VCEC were unaltered. We conclude that the maintenance of EC phenotype and function is dependent on the hemodynamic milieu in vivo and may be influenced by the vascular origin of the cultured EC.     

Vasostatin基因对胰腺癌细胞和血管内皮细胞增殖的影响

目的研究Vasostatin转基因对胰腺癌细胞、血管内皮细胞的作用,探讨其对胰腺癌生长抑制的作用机制。方法应用腺病毒载体将Vasostatin基因转入人胰腺癌细胞SW1990、人脐静脉血管内皮细胞ECV304,应用MTT方法检测转基因后细胞的生长活力。同时,应用小管形成实验研究Vasostatin对血管内皮细胞ECV304体外血管生成的影响。结果Vasostatin转基因对人胰腺癌SW1990细胞生长无显著影响。Vasostatin转基因(MOI分别为25和50)对人脐静脉血管内皮细胞ECV304作用72h后,Ad-Vasostatin组的ECV304细胞数目显著少于PBS组和Ad-lacZ组(P<0.05)。小管形成实验结果显示,Ad-Vasostatin组内皮细胞数目较少,细胞排列连续性差,可见条索状的细胞链,但中空闭合的管状结构缺如。结论腺病毒介导的Vasostatin基因可显著抑制人脐静脉血管内皮细胞ECV304的体外细胞增殖,抑制其体外血管生成,而对人胰腺癌肿瘤细胞SW1990的体外细胞增殖无明显影响。     

Phosphatidylethanol in high density lipoproteins increases the vascular endothelial growth factor in smooth muscle cells

To study whether qualitative changes in high density lipoprotein (HDL) phospholipids mediate part of the advantageous effects of ethanol on atherosclerosis, we investigated whether HDL associated phosphatidylethanol (PEth) affects the secretion of vascular endothelial growth factor (VEGF) from cultured human smooth muscle cells. Serum-starved human umbilical vein HUVS-112D smooth muscle cells were incubated in the presence of PEth–HDL, HDL, or buffer. The phosphorylation of protein kinase C (PKC) and mitogen activated protein kinase (p44/42 MAPK) was determined by specific antibodies against phosphorylated and total proteins. VEGF concentrations were measured from cell culture medium of the cells. PEth increased the secretion of VEGF into the culture medium of HUVS cells. PEth–HDL increased the PKC phosphorylation by 2.1-fold and p44/42 MAPK phosphorylation by 3.3-fold compared with HDL, indicating that PEth-containing HDL particles influence vascular smooth muscle cells by PKC and p44/42 MAPK signalling. This may mediate the effects of ethanol on vascular wall by increasing the VEGF secretion from smooth muscle cells. The secreted VEGF may inhibit the formation of neointima and in doing so helps prevent atherosclerosis.     

Synthesis of fibrinolytic activator and inhibitor by endothelial cells

Vascular endothelial cells derived from rabbit vena cava and maintained in continuous culture exhibited properties characteristic of the intact endothelium. These cells were used as a model for characterizing the fibrinolytic components specified by the endothelium. Endothelial cells in culture digested radiolabeled fibrinogen. Digestion resulted from the synthesis and secretion of a plasminogen activator. Fibrinolysis was not detected when cells were grown in medium lacking plasminogen, indicating the absence of plasminogen-independent fibrinolytic enzymes. Phorbol-myristate-acetate increased extracellular plasminogen activator activity dramatically. This increase was prevented when actinomycin D or cycloheximide was included in the growth medium, indicating that new gene expression was required for it. Intracellular plasminogen activator could not be detected unless the cell extracts were exposed briefly to mildly acidic conditions. Mixing experiments between acid-treated and untreated extracts suggested that the cells contained a potent, acid-labile inhibitor of fibrinolysis. As little as 10 mug of protein from whole cell extracts inhibited both cell and urokinase-mediated fibrinolysis by more than 70%. Cell fractionation studies localized the inhibitor to the cytosol whereas plasminogen activator activity was restricted to the membrane-rich fraction. This membrane fraction did not require acidification for activity, suggesting that the inhibitor had been removed and that acidification did not activate a plasminogen proactivator. These observations demonstrate that regulation of endothelial fibrinolytic activity is far more complex than had been anticipated and raise several uncertainties in regard to detecting the presence of plasminogen activators in cells and tissues.     

Characterization and fibrinolytic properties of human omental tissue mesothelial cells. Comparison with endothelial cells

It has been reported that omental fat tissue is a good source of human microvascular endothelial cells. By characterization we demonstrate that the epitheloid cells isolated from omental tissue are not endothelial cells, but mesothelial cells. They contain abundant cytokeratins 8 and 18, which are absent in endothelial cells, and vimentin. No staining with the endothelial-specific antibodies EN-4 and PAL-E is observed. A faint and diffuse staining of von Willebrand factor (vWF) is seen in mesothelial cells, whereas microvascular endothelial cells from subcutaneous fat display vWF in distinct granular structures. Human peritoneal mesothelium produces plasminogen activator-dependent fibrinolytic activity, which is essential in the resolution of fibrous exudates and may therefore be important in preventing the formation of fibrous peritoneal adhesions. This fibrinolytic activity is plasminogen activator-dependent, but has not been fully characterized. We report here that human omental tissue mesothelial cells in vitro produce large amounts of tissue-type plasminogen activator (t-PA), together with type 1 and 2 plasminogen activator inhibitor (PAI-1 and PAI-2). PAI-1 is predominantly secreted into the culture medium, whereas the major part of PAI-2 is found in the cells. No urokinase-type plasminogen activator is detected. On stimulation with the inflammatory mediator tumor necrosis factor (TNF), at least a threefold decrease in t-PA antigen is observed, together with an increase in both PAI-1 and PAI-2. TNF also induces a marked change in cell shape. Whereas TNF and bacterial lipopolysaccharide (LPS) have similar effects on the production of PA inhibitor by human endothelial cells, LPS has no or only a relatively small effect on the fibrinolytic properties of mesothelial cells. The decreased fibrinolytic activity induced by the cytokine TNF may impair the natural dissolution of fibrin deposits at the peritoneum in the presence of an inflammatory reaction.     

The extracellular matrix on atherogenesis and diabetes-associated vascular disease

Atherosclerosis is remarkably increased in type 2 diabetes suggesting that mechanisms causing arterial lesion are enhanced by the metabolic disturbances of insulin resistance (IR) and diabetes. Several lines of research suggest that processes taking place in the arterial intima extracellular matrix may be part of a shared pathogenic mechanism. The intima extracellular matrix is where atherogenesis takes place. This layer contains fibrilar macromolecules like collagens, proteoglycans (PGs), hyaluronate, and extracellular multi-domain proteins. Specific interaction of lysine, arginine-rich segments of the apoB-100 lipoproteins, LDL, IDL and Lp (a), with the negatively charged glycosaminoglycans (GAGs) of PGs cause retention of the lipoproteins, one of the initiation process of atherogenesis. Such interactions cause structural modifications of the lipid and protein moieties of the lipoproteins that appear to increase their susceptibility to proteases, phospholipases and free radical-mediated processes. The association of apoB-lipoproteins, specially small and dense LDL, with intima PGs increases their uptake by macrophages and human arterial smooth muscle cells (HASMC) leading to 'foam cell' formation. In vitro, elevated levels of non-esterified fatty acids (NEFA) alter the matrix of endothelial cells basement membrane making them more permeable to macromolecules. NEFA cause changes in the expression of genes controlling the PGs composition of the PGs secreted by HASMC causing formation of a matrix with high affinity for LDL. These results lead us to speculate that an important component of the dyslipidemia of IR and type 2 diabetes, chronic high NEFA, may contribute to cellular alterations that cause changes of the arterial intima extracellular matrix. Such changes may increase the atherogenicity of the retention of apoB lipoproteins in the intima and contribute to the systemic alteration of the arterial wall frequently observed in IR and type 2 diabetes.     

Detection of an unusually stable fibrinolytic inhibitor produced by bovine endothelial cells.

Fibrin/agar films were prepared and used to detect plasminogen activators produced by cultured bovine aortic endothelial cells (fibrin autography). One preparation of fibrin underwent spontaneous lysis upon incubation at 37 degrees C. This lysis was prevented by antibodies to tissue-type plasminogen activator but not by antibodies to urokinase. Conditioned medium from the confluent endothelial cells was fractionated by polyacrylamide gel electrophoresis in the presence of NaDodSO4. The gels were analyzed on indicator films prepared with the spontaneously lysing fibrin (reverse fibrin autography). Unexpectedly, as the opaque fibrin film cleared, a distinct lysis-resistant zone appeared in the indicator gel at a region corresponding to Mr 55,000. Experiments were devised to determine whether the lysis-resistant zone in the indicator film reflected the presence of a cellular inhibitor in the polyacrylamide gel. The corresponding region was excised from a polyacrylamide gel, extracted with buffer, and tested directly for antifibrinolytic activity by the 125I-labeled fibrin plate method. Urokinase-mediated fibrinolytic activity was inhibited by the gel extract in a dose-dependent manner indicating the presence of such an inhibitor. Inhibitor activity was detected in Triton X-100 extracts of washed monolayers and in conditioned medium, where it accumulated with time. The endothelial cell inhibitor not only survived exposure to NaDodSO4 but also was active after incubation at pH 12 or treatment with 5% (vol/vol) 2-mercaptoethanol, 6 M urea, 4 M guanidine hydrochloride, or 1 M acetic acid. Considerable activity also remained after heating at 100 degrees C for 30 min. These results indicate that cultured bovine aortic endothelial cells synthesize and secrete a previously undetected, unusually stable fibrinolytic inhibitor of Mr 55,000. Reverse fibrin autography offers a convenient approach for studying such molecules.     

Antithrombotic regulation in human endothelial cells by fibrinolytic factors

Vascular endothelial cells (ECs) modulate the blood fibrinolytic system by secreting tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), and their inhibitor, type-1 plasminogen activator inhibitor (PAI-1). ECs also express t-PA receptors (t-PAR) and u-PA receptors (u-PAR) on their cell surfaces, assembling both enzymes to regulate the cellular fibrinolytic activity. In addition, ECs modulate these factors in response to several stimuli. Fibrin clots on ECs induce the up- and downregulation of t-PA and PAI-1 production, respectively, thus causing an effective lysis of the fibrin clot. Heat shock (43 degrees C) increases the expression of u-PA, t-PA, PAI-1, and u-PAR by which ECs become more fibrinolytic around the cells. Furthermore, because ECs possess t-PAR and u-PAR on their cell surfaces, the binding of t-PA and u-PA is a critical event, which affords ECs the localized and condensed fibrinolytic potential. Therefore, ECs play a central role in antithrombotic activity by regulating the levels of these fibrinolytic factors.     

Effect of shear stress on asymmetric dimethylarginine release from vascular endothelial cells

We demonstrated recently that plasma concentrations of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthase, are increased by high salt intake concomitantly with a decrease in plasma levels of NO in human hypertension. We investigated the effect of shear stress on ADMA release in 2 types of cells: transformed human umbilical vein endothelial cells (HUVECs; cell line ECV-304) and HUVECs. Exposure of ECV-304 cells and HUVECs to shear stress with the use of a cone-plate viscometer enhanced gene expression of protein arginine methyltransferase (PRMT-1), ADMA synthase. In HUVECs, the ratio of PRMT-1 to glyceraldehyde 3-phosphate dehydrogenase mRNA was increased by 2-fold by a shear stress of > or =15 dyne/cm2. A dominant-negative mutant of IkappaB kinase alpha and troglitazone at 8 micromol/L, an activator of peroxisome proliferator-activated receptor gamma, abolished the shear stress-induced increase in PRMT-1 gene expression in parallel with the blockade of nuclear factor (NF)-kappaB translocation into the nucleus. The activity of dimethylarginine dimethylaminohydrolase, the degradation enzyme of ADMA, was unchanged after shear stress < or =15 dyne/cm2 and was enhanced by 1.48+/-0.06-fold (P<0.05) by shear stress at 25 dyne/cm2. The release of ADMA was increased by 1.64+/-0.10-fold (P<0.05) by shear stress at 15 dyne/cm2 but was not affected by shear stress at 25 dyne/cm2. These results indicate that shear stress enhances gene expression of PRMT-1 and ADMA release via activation of the NF-kappaB pathway. Shear stress at higher magnitudes facilitates the degradation of ADMA, thus returning ADMA release levels to baseline.