Journal of Huazhong University of Science and Technology [Med Sci] ~' ~1- ~ ~ �9 �9 ~ [ I~ ~ ( ~ ~ ~ ) I~ ] 23 (2): 121-123, 2003 121

Comparison between the Effects of Intraperitoneal Injection of LDL and Intravenous Injection of LDL on Arterial Endothelial Cells Apoptosis"

WANG Li ( ~ ~ ) , QIN Jin(JJlg ~ ) , LIU Zhengxiang (~l~$1) '

Department o f Cardiology, Tongji Hospital, Tongji Medical College, Huazhong University o f Science and Technology, Wuhan 430030

Summary: To observe the effect of oxidized low density lipoprotein (OxLDL) on arterial endothelial cells apoptosis in vivo, we established a model in which Sprague-Dawley rats were given intraperi- toneal and intravenous injection of unmodified LDL (8 mg/kg every day) via the tail vein. Seven days after the injection, the aortic endothelial cells specimens were prepared by an en face preparation of rat aorta. The apoptotic cells were identified and counted by in situ nick and labelling (TUNEL) method and light microcopy. The numbers of the apoptotic cells were 12. 52-t-4.71/field in the in- traperitoneal injection control group, 11.41-4-2. 94/field in the intravenous injection control group, 22.98-4-8.01/field in the intraperitoneal injection LDL group and 103. 8 5:11.5/field in the intra- venous injection LDL group, respectively. The difference was significant between injection LDL group and control ( P < 0 . 01), and the difference was also significant between two LDL injection groups (P<0. 01). These findings suggest that injection of LDL can induce apoptosis in arterial en- dothelial cells and the effect is especially significant with intravenous injection LDL. After injection, oxidative modification of LDL may occur in local arteries and cauls injury to the endothelial cells. Key words: arterial endothelial cells; apoptosis; LDL

OxLDL is believed to play an important role in the formation of atherosclerotic lesions !1,tt exhibits a wide spectrum of biological properties and is able to induce events involved in atherogenesis, such as monocyte chemotaxis, foam cell formation, smooth muscle proliferation and endothelial dysfunction. Many studies demonstrated that OxLDI. stimulated the cellar suicide pathway, leading to apoptosis of en- dothelial ceils and the effect could accelerate the for- mation of atherosclerosis r3-s3. However , most of these findings are based on in vitro studies in which LDL was oxidized by different methods, such as ex- posure to UV radiation, transition metals and lipoxy- genases, and then used in cukured ceils. Despite ex- tensive in vitro data, the in vivo relevance of LDL and apoptosis in endothelial cells is not clearly under- stood. Besides, there is no correlative study up to now. In this study, we developed an animal model to observe the effect of LDL on endothelial cells apopto- sis in vivo.

1 MATERIALS AND METHODS

1.1 LDL Isolation Plasma was obtained from healthy volunteers

under the age of 45 years after 12 h of fasting. LDL was isolated by two rapid consecutive steps of ultra- centrifugation in a NaBr gradient (1. 0 6 3 - - 1 . 2 0 g /

WANG Li, female, born in 1971, Postgraduate Student �9 The project was supported by a grant from the foundation of Scientific and Technological Key Project of Hubei Province (No. 2001AA307B03).

ml ) , as previously described Es'7]. The LDL was ster- ilised by passing it through a 0 .45 t~m filter and was kept on 4 ~ and protein content was determined by Lowery 's technique. I. 2 Animal Grouping and Establishment of Ani- mal Model

Twenty-four male Sprague-Dawley rats, of ap- proximately 250 g in weight, were provided by the Experimental Animal Centre of Traditional Chinese Medical Science College of Hubei Province and were randomly divided into four groups: intraperitoneal in- jection control, intravenous injection control, in- traperitoneal injection LDL and intravenous injection LDL, with 6 rats in each group. Two control groups were injected with PBS solution, and two LDL groups were injected intraperitoneally or intravenous- ly via the tail vein with LDL (8 mg/kg every day) for 7 days. I. 3 Sample Preparation

Seven days after the injection, all rats were anesthetized. Rats were fixed by perfusion with a so- lution containing 4 ~ paraformaldehyde in PBS (pH = 7 . 2 ) by inserting a catheter into the left cardiac ventricle. Fixation was continued for 5 min, and tho- racic aorta was excised and kept in the previous fixa- tive for 3 h. En face stretched preparation of en- dothelial cells was carried out based on literature Es]. Briefly, 3 aortic vessels sections from each animal were pressed against the collodin-coated slides so that the endothelium was in firm contact with the layer of dry coUodin. The vessel wall was removed from the collodin and the endothelium was left unremoved, then the endothelial sheets were stuck to the gelatine-

122 Journal of Huazhong University of Science and Technology ['Med SCi] 23 (2): 121-123, 2003

coated slides. En face specimens of endothelium were made. 1 . 4 Apoptosis Detect ion

Apoptosis in endothelial cells was assessed by T U N E L . Three en face specimens of each rat were stained by T U N E L kit (obtained from R ~ D System Inc, USA) and counterstained with methyl green. Apoptotic cells were counted per high-power field ( H R P , ) < 2 0 0 ) in en face specimens. For each speci- men 5 fields were counted. A positive control and a negative control for the T U N E L assay were set. 1 . 5 Statistical Analys i s

Three specimens per animal were analyzed to give a mean value. The data was presented as ~-=k s. t-test was used for difference evaluation. A value of P < 0 . 05 was considered to be statistically signifi- cant.

2 RESULTS

Under a light microscope, most nuclei of en- dothelial cells in two control groups were normal and displayed loose blue color (fig 1 ). Many apoptotic nuclei were observed in two LDL injection groups and displayed condensed brown color (fig. 2, 3). Table 1 showed the apoptotic nuclei numbers of aortic en- dothelial cells in the 4 groups (table 1). The induc- tion effect of LDL on apoptosis in the intravenous in- jection group was more apparent than in the in- traperitoneal injection LDL group ( P < 0 . 0 1 ) .

Table 1 Apoptotic nuclei numbers of aortic endothelial cells in the 4 groups

Apoptosis Groups n/field

Intraperitoneat injection control Intravenous injection control Intraperitoneal injection LDL Intravenous injection LDL

12. 52=t=4. 71 11.41-4-2.94 22.98-+8.01"

103.80• "zx " P~0 . 01, LDL groups vs control; a P ~ 0 . 01, intra-

venous injection LDL group vs intraperitoneal injection LDL group

3 DISCUSSION

Endothelial cells are the barrier of arterial wall, the integrity of endothelium depending on the balance of proliferation and apoptosis in endothelial cells. Many studies have demonstrated that OxLDL could induce excessive apoptosis of endothelial cells through stimulating apoptotic signal transduction pathways and the effect plays a key role in the formation of atherosclerosis E31. However, most of these findings were based on cell culture studies using LDL in a wide range of concentrations and oxidization by dif- ferent methods in vitro. The in vivo relevance of these findings is not clearly known and the mechanisms be- hind LDL oxidation in vivo have been difficult to elu- cidate. Brown et al proved that the cells of artery wall secrete oxidative products from multiple path-

Fig. 1 In control group, nuclei of endothelial Fig. 2 In the intraperitoneal injection LDL group,

cells displayed loosely blue color. Scarce apoptotic nuclei showed condensed brown col- or

Fig. 3 In the intravenous injection LDL group, densely covered apoptotic nuclei was of brown color

ways that can seed the LDL trapped in the suben- dothelial space and initiate lipid oxidation, mean- while, the matrix microenvironments provided by these artery wall cells could exclude the water-soluble antioxidants of plasma Eg]. It is suggested that the oxi- dation only occurs locally in vivo in vascular wall E~]. First, the modification results in the oxidation of lipids in LDL with little change in apoB before mono- cytes are recruited. The second stage begins when monocytes are recruited to the lesion, converted into macrophages and contribute their enormous oxidative capacity. In this stage, the LDL lipids are further oxidized, but the protein portion of LDL is also mod- ified, leading to a loss of recognition by the LDL re-

WANG Li et al. Effects of LDL and LDL on Apoptosis 123

ceptors and a shift to recognition by the scavenger re- ceptors or the oxidized LDL receptors. This shift in receptor recognition leads to cellular uptake of the LDL that are not regulated by the cholesterol content of the cell [~3. The result is massive accumulation of cholesterol and the subsequent formation of foam cells which are the hallmark of the arterial fatty streak.

Fedrico et ol observed the different changes be- tween LDL and OxLDL in vivo using rat model. By using antibodies against epitopes present on OxI.DL ( N A 5 9 ) , OxLDL could be detected in the arterial endothelia and media 6 h after one time injection of native LDL, and the accumulation of OxLDL peaked at the 24th h. Meanwhile, the expression of adhe- sion molecule ICAM-1 and the activation of NF-~:B could be detected in endothelial ceils 12 and 24 h after LDL injection. But, after injection of equal amounts of OxLDL, neither apoB nor OxLDL could be de- tected in the media or the endothelia at any time up to 48 h and the expression of ICAM-1 and NF-,:B could not be found [~~ Though OxLDL can induce an inflammation response and expression of many cy- tokines in the arterial wall, the absence of accumula- tion of OxLDL and of an inflammation response after injection of OxLDL is not surprising. Previous work has shown that injection of OxLDL in rats results in rapid clearance through scavenger receptors concen- trating on Kupffer ceils and endothelial liver cells, with a t]/2 Of a few minutes. Native LDL, on the other hand, disappears from serum at a low rate after injection, and it is trapped and oxidized in the en- dothelia, then exerts toxic effect on these arterial cells. The study also demonstrated that intravenous of LDI. resulted in an earlier and stronger modifica- tion and accumulation than intraperitoneal injec- tion ~~ The result is relevant with the different clearance between the two injection pathways.

The mechanisms involved in the effect of LDL on endothelial cells apoptosis in vivo are only poorly understood. In order to investigate the in vivo effect and further analyze the relevance with atheroscle-

rosis, we, for the first time, developed an animal model in which a successive injection of LDL resulted in an oxidation of LDL that induced apparent apopto- sis in endothelial cells. Thus , the model offers the opportunity to study the molecular mechanisms of the apoptosis of arterial endothelial ceils elicited by LDL and to analyze the relevance between endothelial apoptosis and atherosclerosis. And this model could also be used to evaluate the inhibitory effects of some drugs on cell apoptosis and therapeutic intervention of atherogenesis.

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(Received March 6, 2003)