2009;122(21):2662-2665

Diabetes mellitus known as its macro- and micro- angiopathy has caused thousands of mortality per year. Recent researches showed that hyperglycemia, advanced glycation end products (AGEs) and some other factors acted on the process of atherogenesis. AGEs can combine with receptors of AGEs (RAGEs), which exist on the vascular endothelium, smooth muscle cells, macrophage, lymphocyte and so on. They can stimulate series of signal transduction systems including nuclear factor κB (NF-κB) pathway, finally promote the secretion of inflammatory factor such as interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1),^1,2 as well as increase the synthesis and secretion of coagulant modulatory factors such as vascular cell adhesion molecule-1 (VCAM-1) and von Willebrand factor (vWF).³       

 

The non-enzymatic glycation of low density lipoprotein (LDL) is a naturally occurring chemical modification of lysine residues of apolipoprotein (apo)-B of LDL by blood glucose. Evidences showed that glycated LDL contributed to the increased atherosclerotic risk of diabetes. It is well demonstrated that the in vitro glycated LDL stimulates the expression of the inflammatory factors such as heat shock factor-1 (HSF-1), plasminogen activator inhibitor-1 (PAI-1), ICAM-1, in cultured Human umbilical vein cells (HUVECs). Little is known about the effect of in vivo glycated LDL on the proliferation, the expression of inflammatory and coagulant modulatory factors of endothelial cells. In this study, we investigated the effects of glycated LDL on the proliferation, the expression of inflammatory ICAM-1 and procoagulant factors vWF of human umbilical endothelial cells.  

 

 

LDL (d=1.019 to 1.063) was separated from plasma of 9 normal and 8 diabetes volunteers by sequential density gradient ultracentrifuge in a 5819R ultracentrifuge.⁴ It was dialyzed extensively at 4°C in the dark against 10 mmol/L phosphate buffer solution (PBS) containing 0.15mmol/L NaCl, 0.3mmol/L EDTA with pH of 7.4. The glycation rate of LDL was determined by frutosamine method. The LDL preparations were passed through sterile filters and stored in the dark under nitrogen at 4°C. LDL derived from diabetic serum was nominated as LDL_D, and LDL extracted from normal serum was named as LDL_N.  In order to avoid the potential influence of high lipid, the serum lipid levels (LDL-cholesterol) of normal and diabetes volunteers were in the normal range.

 

HUVECs were obtained through the enzyme dissociation method. The cells were identified by the typical “cobblestone” morphology exhibited by confluent monolayer, and positive staining for vWF.⁵ Cells were subcultured in 7 days, and the second generation of the cells was used in this study.

 

The proliferation of HUVECs was observed by the method MTT as the reagent instruction described. The confluent monolayer cells were detached by trypsin, centrifuged at 1000 rounds per minute for 5 minutes, and diluted by serum-containing culture medium. Cells were seeded in 96-well culture plate (10⁵ per well), and incubated with LDL_D (0 mg/L, 50 mg/L, 100 mg/L, 150 mg/L, 200 mg/L and 500 mg/L) for 24 hours in the constant temperature box at 37°C in a humidified atmosphere of 95% air and 5% CO₂. Furthermore, cells were incubated with LDL_D and LDL_N (100 mg/L separately) for 12 hours, 24 hours and 36 hours, respectively. Then MTT solution was added, cells were incubated for 4 hours at 37°C, the supernatant fluid was drawn out, 150 µl DMSO (Sigma, USA) was added in the plates, vibrated for 10 minutes on the vibrator. The optical density (OD) value was measured by enzyme-labeled instrument.

 

The second generation of HUVECs were planted on 96-well plates (10⁵ per well), and incubated in the condition described previously. Cells were divided into three groups: 12-hour group, 24-h our group, 36-hour group. Diabetic LDL was added respectively (the final consistency is 100 mg/L) , and cells were incubated for 12 hours, 24 hours, 36 hours respectively in the same surrounding as described precedingly. Normal LDL was added in the control group. The sICAM-1 level of the supernatant fluid was determined by enzyme linked immunosorbent assay (ELISA), and experiment was done as the instruction of the human sICAM-1 quantity EIA kit (senxongbiotech, China) described. The level of supernatant vWF was evaluated in the same method.

 

The level of gene expression was measured by reverse transcriptional polymerase chain reaction (PCR). Total RNA was isolated from HUVECs using TRIZOL reagent (Invitrogen corporation, USA) according to the manufacturer′s instructions. A 1-µg aliquot of total RNA was reverse-transcribed using oligo (dT) primers and reverse transcritase (Invitrogen co, USA). The product was denatured and amplified in the PCR system. Aliquots of cDNA were amplified with Taq polymerase and the following primers: ICAM-1 sense, 5′-TATGGCAAC- GACTCCTCCT-3′ and antisense, 5′-CATTCAGCGT- CACCTTGG-3′; vWF sense, 5′-ATGGATCCTGTCACC- TTGAATCCCAGT-3′ and antisense, 5′-ATAAGCTT- CTTGGGCCCCAGGGT-3′; β-actin sense, 5′-CCCTGG- ACTTCGAGCAAGAGAT-3′ and antisense, 5′-GTTTT- CTGCGAAGTTAGG-3′. Thirty cycles were conducted in thermocycler (Biometra, Germany) under the following condition: initial denaturation at 94°C for 3 minutes followed by 94°C for 45 seconds, annealing at 55°C for 45 seconds, extension at 72°C for 60 seconds and a final extension at 72°C for 5 minutes. PCR products were electrophoresed on a 2% agarose gel containing ethidium bromide. The OD was measured by a BIO RAD 5000 image system and arbitrarily compared before treatment. PCRs resulted in the amplication of a single product of the predicted size for ICAM-1 (369 bp), vWF (854 bp), β-actin (531 bp). We chose β-actin as housekeeping gene and used their gene product to normanize RT-PCR data.        

 

Data were expressed as the mean ± standard error (SE). statistical analysis was performed by SPSS 12.0, and analyzed by t test of variance and Dunnett test. P <0.05 was considered statistically significant.

 

 

The consistency of glycated LDL from the diabetic serum was 610.12±50.92 µmol/L, while that from the normal volunteers was (235.47±40.53) µmol/L.

 

Experiments showed that different consistence of glycated LDL had different influence on the proliferation of HUVECs. In the vacant group (0 mg/L group), the OD value was the highest, along with the increase of LDL_D, the OD value decreased gradually. Statistically, when the consistence reached 100 mg/L or more, the OD value was significantly lower than that of the vacant group (P <0.01) (Figure 1A). 

 

Further study showed that LDL_D had a negative time-dependent relationship on the proliferation of HUVECs. Compared with 0-hour group, the OD values of 12-hour and 36-hour group were significantly lower (P <0.05). While in the control group, which added LDL_N, the difference of OD values of groups was not obvious statistically. What is more, in the same time point, the OD value of LDL_D group was significantly lower than that of the control group (P <0.01) (Figure 1B).

 

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Figure 1. A: Diabetic LDL exhibited a negative dose-dependent relationship on the proliferation of HUVECs — the higher consistence of LDLD the HUVECs were incubated with, the lower OD value was examined. B: Described as the red curve, LDLD had a negative time-dependent relationship on the proliferation of HUVECs. In the same time point, the OD value of LDLD group was lower than that of LDLN group.

 

 

Level of supernatant sICAM-1 displayed a time-dependent pattern. The level of sICAM-1 of 24-hour group was 19.3% higher than that of LDL_D 12-hour group, and 36-hour group was 38.7% higher than that of LDL_D 12-hour group (Figure 2A). At the same time point, the sICAM-1 level of the LDL_D group was significantly higher than that of control group (P <0.05), LDL_D has a similarly time dependent influence on the secretion of vWF (Figure 2B). What′s more, LDL_D showed a dose- dependent promotional pattern to the supernatant level of vWF (Figure 2C).

 

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Figure 2. A: The supernatant level of sICAM-1 increased in positive proportion to the incubating period. B: The supernatant level of vWF showed a similar pattern as sICAM-1. C: The vWF level displayed a dose-dependent pattern; the vWF level in the latter four groups was obviously higher than that in LDLN 100 group.

 

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Figure 3. A: RNA electrophoretogram, 18s and 28s rRNA stripe can be observed. B: Electrophoretogram of ICAM-1 mRNA RT-PCR products. 1:12-hour LDLD group. 2:24-hour LDLD group. 3:36-hour LDLD group. 4:12-hour LDLN group. C: Electrophoretogram of vWF mRNA RT-PCR products. 1:12-hour LDLD group; 2:24-hour LDLD group; 3:36-hour LDLD group; 4:36-hour LDLN group; 5:12-hour LDLN group. D: LDLD enhances ICAM-1 gene expression in a time-dependent pattern. E: LDLD also enhances vWF gene expression in a time-dependent pattern, and LDLD has a stronger potential on the vWF gene expression than LDLN in the same time point. M: maker. N: vacant.

 

Data of RT-PCR of groups of LDL_D of 12-hour, 24-hour and 36-hour were 22.7%, 44.7%, 84.9% higher than that of LDL_N 12-hour group. Also, LDL_D displayed a positive time dependent pattern on the vWF expression. Importantly, LDL_D promoted vWF mRNA expression at any time point compared with LDL_N, which characterized a low glycation degree.

 

 

The endothelium has many important biological functions additional to its role as a mechanical lining. The endothelium releases multiple mediators, not only regulators of vasomotor function but also important physiological and pathophysiological mediator. Endothelial dysfunction is caused by chronic exposure to various stressors such as oxidative and modified low density lipoprotein cholesterol, resulting in impaired nitric oxide (NO) production and chronic inflammation.⁶ Endothelial dysfunction is thought to be an early event in the atherosclerotic process and has been implicated in the pathogenesis of diabetic atherosclerotic vascular disease. 

 

Several clues indicated that glycated LDL can inhibit proliferation and promote apoptosis of the HUVECs.^7,8  A former research indicated that LDL_D possessed a more prominent inhibitory potential to the HUVECs proliferation than in vitro glycated LDL (LDL_iv), while LDL_D exhibits a 2.7 folds higher proapoptotic activity in endothelial cells than LDLiv in relation to its degree of glycation. We observed the OD value examined in the LDL_D group is obviously lower than that in the LDL_N group. In addition, OD value examined was in negative correlation to the LDL_D concentration. These data coincided with the existing conclusion. The difference between LDL_D and LDL_N on the proliferation of HUVECs may be due to their varied degree of glycation, or their different property. 

 

Inflammatory process is considered as a result of endothelial dysfunction, and plays a key role in the pathogenesis of atherosclerosis. Increased vascular inflammation, including enhanced expression of IL-6, ICAM-1, monocyte chemoattractant protein and PAI-1 was observed in type 2 diabetes. It is well demonstrated that glycated LDL stimulated the generation of PAI-1.⁹ In this study, we observed a higher level of ICAM-1 in the LDL_D group than that in the LDL_N group. Further study showed that LDL_D increased ICAM-1 secretion in a time-dependent pattern. What is more, LDL_D increased ICAM-1 gene expression. All these implied that LDL_D stimulate inflammatory process and thus resulting in atherogenesis in DM.

 

Plasma vWF is a coagulant modulatory factor, and the level is elevated in patients with DM.¹⁰ This was also found to be associated with markers of increased oxidative stress and therefore reflect the severity of biochemical abnormalities. Furthermore, vWF is considered as a strong predictive index for diabetic angiopathy¹¹ and its prognosis.^12-15 In this study, we observed a higher level of vWF in LDLD group than that in LDL_N group, also LDL_D stimulated vWF gene expression. 

 

In conclusion, LDL_D inhibits proliferation of HUVECs, stimulates the secretion of vWF and sICAM-1. Therefore, LDL_D may play an important role in the formation of diabetic angiopathy. What′s more, LDL_D promoted the gene expression of vWF and ICAM-1, which may mean that LDL_D have a long-term effect in the atherogenesis. Howevert, we now have little information about the signal transduction pathway how glycated LDL upgrade the ICAM-1 and vWF gene expression. In our future work, the signal transduction pathway may be studied.

 

 

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