[Efficacy and mechanism of local delivery of rapamycin and rapamycin-loaded poly(lactic-co-glycolic) acid nanoparticles on coronary restenosis of injury-stenosis model of minipigs].

OBJECTIVE: To determine whether intramural administration of rapamycin (RPM)-loaded polylactic-polyglycolic acid (PLGA) nanoparticles (NPs) can reduce intimal thickening and affect the mRNA expressions of matrix metalloproteinase (MMP)-2, tissue inhibitor of metalloproteinase (TIMP)-2 and p27(kipl) in a coronary injury-stenosis model of minipigs. METHODS: Twenty eight minipigs were randomly separated into four groups: saline group (n=7), blank PLGA NPs group (5.0 mg/ml)(n=7), RPM group (1.0 mg/ml)(n=7), and RPM-PLGA NPs(5.0 mg/ml)group (n=7), respectively. Different treatments were intracoronary locally delivered via a Dispatch™ catheter for 10 minutes. Serial angiography was performed pre-and post-modeling 30 days and the percent stenosis degree was assessed. Hematoxylin-Eosin (HE) staining, Weigert's resorcin fuchsin staining and picric acid-sirius red staining were used for morphometric analysis. Immunohistochemistry was performed to assess the levels of proliferating cell nuclear antigen (PCNA), MMP-2, and TIMP-2 at early and late time points, respectively. The expression of p27(kip1) mRNA was detected by in situ hybridization staining. RESULTS: Data from 21 minipigs had been collected at the end of the experiment with 6, 4, 5, and 6 from the former mentioned 4 groups, respectively. For the instant injury index, there was no significant difference among the four groups. The percent stenosis degree of RPM-PLGA NPs group was significantly lower than that of the other three groups respectively (all P< 0.05). The neointima area, net external elastic lamina area to external elastic laminal area ratio, and proliferative index of RPM-PLGA NPs group were significantly less than those of the other three groups, with all the P values less than 0.05. The mean value of integral optical density of p27(kip1)mRNA expression of RPM-PLGA group was 0.35 ± 0.06, higher than that of blank PLGA NPs group (0.12 ± 0.05, P< 0.01), saline group (0.16 ± 0.03, P< 0.05), and RPM group (0.15 ± 0.03, P< 0.05), respectively. The MMP-2/TIMP-2 ratio and the positive expression index of PCNA in RPM-PLGA group were lower than that of the other groups (P< 0.05). CONCLUSIONS: Locally delivered rapamycin-loaded PLGA NPs significantly reduces MMP-2/TIMP-2 ratio and PCNA expression, increases p27(kip1) mRNA expression and significantly relieves percent stenosis degree and shows excellent acute procedural results in the minipig interventional coronary artery oversized balloon injury model. The results from minipig model further support that this approach could be a potential clinical procedure for vascular proliferative disease.

[Effect of mm-LDL on NF-kB activation in endothelial cell].

OBJECTIVE: To investigate the signal transduction pathway of NF-kB activated by minimally modified low density lipoprotein (mm-LDL) in endothelial cells and the effect of NF-kB on platelet derived growth factor b (PDGFb) mRNA expression. METHODS: mm-LDL was prepared through iron oxidation by dialyzing the native LDL against FeSO4 in PBS. Endothelial cells were incubated in a medium containing mm-LDL, TNF, and IL-1 respectively and electrophoretic mobility shift assay (EMSA) was displayed to check on the activation of NF-kB. Luciferase reporter gene was analysed to investigate the effect of nuclear factor inducing kinase (NIK), inhibitor of NF-kB kinase alpha (IKK alpha) and inhibitor of NF-kB kinase beta (IKK beta) on NF-kB activation. In addition, endothelial cells were transfected using PDGFb promoter-luciferase for reporter gene analysis or transfected with mut-NIK for slot blot analysis to study the effect of NF-kB on PDGFb mRNA expression. RESULTS: mm-LDL was able to activate NF-kB in endothelial cells. mut-NIK and mut-IKK beta inhibited luciferase activity induced by mm-LDL. mm-LDL could also enhance luciferase activity controlled by upstream sequence of PDGFb promoter which contains element interacting with NF-kB. Result of slot blot showed inhibition of PDGFb mRNA expression by mut-NIK in the endothelial cells stimulated by mm-LDL. CONCLUSION: mm-LDL may activate NF-kB through NIK-IKK beta pathway and promote PDGFb mRNA expression in endothelial cells.

[Effect of oxLDL on the uptake and clearance rate of cholesterol in vascular smooth muscle cells originated from human apoAI transgenic mice].

OBJECTIVE: Study on (1) inhibition of oxidized low density lipoprotein (oxLDL) effect on the uptake and clearance of intra-cellular 3H-cholesterol in vascular smooth muscle cells (v-SMC) originated from the human-apoAI transgenic mice (C57BL/6); (2) change of human-apolipoprotein AI (h-apoAI) mRNA expression in v-SMC after oxLDL stimulation and the protective effect of expressed h-apoAI on v-SMC against oxLDL intoxication. METHODS: (1) v-SMC isolated from human apoAI transgenic mice possessing a recombined gene connected beforehand with a mouse metallothionein-I (MT-I) as the promoter; (2) study of h-apoAI mRNA expression from v-SMC of the transgenic mice by RT-PCR and Northern blot. RESULTS: oxLDL (30 micrograms/ml) strongly promoted v-SMC proliferation. No difference found on 3H-cholesterol uptake between smooth muscle from normal mouse aorta (n-SMC) and smooth muscle cells from transgenic mouse aorta (tr-SMC) of the control groups, the uptake rates of both kinds of SMC rose 100% after oxLDL stimulation. The efflux rates of 3H-cholesterol in tr-SMC were much higher than those of n-SMC (40%-50%). After oxLDL stimulation, the clearance rates fell by 28% and 10% respectively for n-SMC and tr-SMC. The result of RT-PCR and Northern blot showed a marked increase of h-apoAI gene expression after oxLDL stimulation. CONCLUSIONS: Expression of h-apoAI gene in C57BL/6 mice enables to decrease the accumulation of cholesterol in v-SMC. tr-SMC are capable to alleviate the harmful effect of oxLDL on v-SMC due to the increase of h-apoAI expression.

HDL and apolipoprotein A1 (apo A1). Their effects on retardation of lipid deposition in aortic intima.

The aim of this study is to clarify whether apo A1 displays a similar role as that of HDL in preventing the development of experimental atherosclerosis. Results were obtained from 4 groups of experiments. (1) Result from 4 successive experiments of tree shrews indicated that high serum HDL level is the main factor in preventing the development of experimental atherosclerosis; (2) Results from 4 successive experiments in rabbits showed that both HDL and apo A1 were able to decrease the extent of lipid deposition and atheromatous lesion developed in the aortic intima; (3) Apo A1 also inhibited the number of monocytes/macrophages infiltrated in aortic intima at the initial stage of fatty streak formation; (4) Similar as HDL, apo A1 phospholipid liposomes promoted markedly the clearance ability of smooth muscle cells on intracellular cholesterol. Conclusively, apo A1 is effective in preventing the development of experimental atherosclerosis. Further study, however, is required to detect an ideal combination of apo A1 with other component, e.g., phospholipid.

High density lipoproteins and prevention of experimental atherosclerosis with special reference to tree shrews.

According to data obtained from epidemiological and experimental survey, serum HDL level is known to be correlated conversely with the incidence of atherosclerosis. Experimental data collected in this article explained part of its mechanism, which is described in four parts as follows: 1. The result of 3 successive experiments on experimental atherosclerosis in tree shrews (total of 96 animals available including 40 as the controls) showed that the serum HDL level had been kept persistantly to 69-88% of the total serum lipoproteins even after a high cholesterol intake for 32 weeks. The incidence of atheromatous lesions developed was only 0-9%, but the incidence of gall stone was very high, 48-84% by gross examination by the end of these experiments. 2. HDL are also capable of (1) promotion of monocyte migration activity; (2) enhancement of cholesterol clearance rate of aortic smooth muscle cells originally isolated from either rabbits or tree shrews; (3) inhibition of 20% of LDL degradation but with no inhibitory effect obtained on Ac-LDL degradation in the endothelial cells; (4) presence of specific binding sites for apo E free HDL on the surface of aortic smooth muscle cells from either rabbits or tree shrews which recognizes apo A1 as a ligand. 3. Data from 2 successive experiments in rabbits showed that HDL lipoproteins (mainly apo A1) possess an inhibitory effect on the development of atheromatous plaques, but not a very strong one. 4. The colesterol clearance effect of smooth muscle cells was markedly enhanced by apo A1/phospholipid liposomes (the apo A1 used was isolated from either rabbit's or tree shrew's serum) in vitro.

Characterization of HDL binding sites and its ligand in cultured smooth muscle cells of rabbit's aorta.

Cultured smooth muscle cells of rabbit aorta were studied by 125I labelled rabbit HDL3. Saturation curves, measured at 4 C, showed the presence of two different components: the low-affinity non-saturable binding portion and the high-affinity binding portion (Kd about 5.6 x 10(-8) mol/L and Bmax about 0.321 micrograms/mg cell protein). Scatchard analyses of the high-affinity binding portion suggest the presence of single class binding sites. Binding of rabbit HDL3 to cultured smooth muscle cells was relatively resistant to trypsin or pronase, and independent to Ca2+. The binding rate of 125I-HDL3 to the smooth muscle cells was highest at 4 C and the optimal pH was 2. Additionally, presence of high concentration apoAI reduced 50% of the binding rate of 125I-HDL3, and 125I-HDL3, being pretreated (blocked) with rat anti-rabbit apoAI IgG of different concentrations lost 70% of its original binding rate with smooth muscle cells. The results suggest that rabbit aorta smooth muscle cells possess a specific binding sites for apoE-free HDL which recognizes apoAI as a ligand.