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Inhibition of hepatic scavenger receptor-class B type I by RNA interference decreases atherosclerosis in rabbits.

Demetz E, Tancevski I, Duwensee K, Stanzl U, Huber E, Heim C, Handle F, Theurl M, Schroll A, Tailleux A, Dietrich H, Patsch JR, Eller P, Ritsch A - Atherosclerosis (2012)

Bottom Line: After 2 weeks of treatment hepatic SR-BI mRNA levels were reduced by 80% accompanied by reduced SR-BI protein levels and a modulation of the lipoprotein profile.In a long-term study, this gene therapeutic intervention led to a similar modulation of the lipoprotein profile, to lower total cholesterol levels, and most importantly to a 50% reduction of the relative atherosclerotic lesion area.Our results are another indication that the role of SR-BI in lipoprotein metabolism and atherogenesis in rabbits--a CETP-expressing animal model displaying a manlike lipoprotein profile may be different from the one found in rodents.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine I, Innsbruck Medical University, Innsbruck, Austria.

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Rabbits on cholesterol-rich diet were treated with SR-BI specific small hairpin RNA (grey circles) and respective scrambled controls (black circles) for 8 weeks. Total cholesterol levels were analyzed weekly (A). The insert shows the same curves in linear scale. Cholesterol measurements of lipoproteins fractions isolated from hyperlipidemic plasma samples by stepwise ultracentrifugation are shown in (B). Down-regulation of hepatic SR-BI expression on RNA level was shown by real-time PCR (C). Two representative aortas with Sudan IV stained lipid depositions in the intima (D), and the relative areas of atherosclerotic plaques in whole thoracic aortas and in corresponding aortic arches are presented (E). siRNA = small interfering RNA.
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fig0015: Rabbits on cholesterol-rich diet were treated with SR-BI specific small hairpin RNA (grey circles) and respective scrambled controls (black circles) for 8 weeks. Total cholesterol levels were analyzed weekly (A). The insert shows the same curves in linear scale. Cholesterol measurements of lipoproteins fractions isolated from hyperlipidemic plasma samples by stepwise ultracentrifugation are shown in (B). Down-regulation of hepatic SR-BI expression on RNA level was shown by real-time PCR (C). Two representative aortas with Sudan IV stained lipid depositions in the intima (D), and the relative areas of atherosclerotic plaques in whole thoracic aortas and in corresponding aortic arches are presented (E). siRNA = small interfering RNA.

Mentions: In order to study the effect of hepatic SR-BI inhibition on the development of atherosclerosis, we put the rabbits on a Western type diet supplemented with 2% cholesterol for 8 weeks. Two independent long-term experiments were performed. The animals were injected either with galactosylated poly-l-lysine-pENTR214 complexes (30 μg/kg body weight; n = 10), or its scrambled control construct (n = 9). To check for potential toxic effects of repeated treatment with poly-l complexes, we measured serum levels of aspartate-aminotransferase (AST), alanine-aminotransferase (ALT), γ-glutamyl transferase, alkaline phosphatase and C-reactive protein. The observed moderately increased serum levels of AST and ALT in both groups are in line with previous studies by us, where high cholesterol/high fat diet had the same effect in New Zealand White Rabbits [18]. However, all other parameters were not elevated upon treatment suggesting that that rabbits treated with PLL-Gal complexes did not develop severe cholestasis or systemic inflammation (Table 2). Additionally, we observed a steady increase in body weight in both groups of animals (Suppl. Fig. 4A). We monitored total cholesterol levels over time during the experiment (Fig. 3A). As observed in the short term experiment, there was again a shift in the lipoprotein profile from HDL-cholesterol to VLDL-cholesterol after two weeks of treatment (data not shown). Lower HDL-cholesterol levels were associated with decreased apoA-I levels (83% of control; p = 0.057) in the treatment group. However, HDL particle size was not altered upon siRNA treatment for two weeks (Suppl. Fig. 4B). These changes resulted in reduced total cholesterol levels. Animals treated with SR-BI-specific small hairpin RNA had total cholesterol levels of 38.4 ± 4.7 mg/dL, while controls displayed cholesterol levels of 51.2 ± 6.6 mg/dL (p = 0.014). This difference disappeared at the end of the experiment, when the total cholesterol levels rose to a plateau of nearly 5000 mg/dL in both trial arms (Fig. 3A; Table 2). No differences in levels of lipoprotein fractions isolated by stepwise ultracentrifugation were detected at this stage of the experiment (Fig. 3B). This was confirmed by FPLC analysis of chylomicron depleted plasma samples (Suppl Fig. 4C). The same was true for levels of free cholesterol as well as for initial and total CE transfer (Table 2). Biochemical characteristics of the animals at the end of the experiment are given in Table 2.


Inhibition of hepatic scavenger receptor-class B type I by RNA interference decreases atherosclerosis in rabbits.

Demetz E, Tancevski I, Duwensee K, Stanzl U, Huber E, Heim C, Handle F, Theurl M, Schroll A, Tailleux A, Dietrich H, Patsch JR, Eller P, Ritsch A - Atherosclerosis (2012)

Rabbits on cholesterol-rich diet were treated with SR-BI specific small hairpin RNA (grey circles) and respective scrambled controls (black circles) for 8 weeks. Total cholesterol levels were analyzed weekly (A). The insert shows the same curves in linear scale. Cholesterol measurements of lipoproteins fractions isolated from hyperlipidemic plasma samples by stepwise ultracentrifugation are shown in (B). Down-regulation of hepatic SR-BI expression on RNA level was shown by real-time PCR (C). Two representative aortas with Sudan IV stained lipid depositions in the intima (D), and the relative areas of atherosclerotic plaques in whole thoracic aortas and in corresponding aortic arches are presented (E). siRNA = small interfering RNA.
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fig0015: Rabbits on cholesterol-rich diet were treated with SR-BI specific small hairpin RNA (grey circles) and respective scrambled controls (black circles) for 8 weeks. Total cholesterol levels were analyzed weekly (A). The insert shows the same curves in linear scale. Cholesterol measurements of lipoproteins fractions isolated from hyperlipidemic plasma samples by stepwise ultracentrifugation are shown in (B). Down-regulation of hepatic SR-BI expression on RNA level was shown by real-time PCR (C). Two representative aortas with Sudan IV stained lipid depositions in the intima (D), and the relative areas of atherosclerotic plaques in whole thoracic aortas and in corresponding aortic arches are presented (E). siRNA = small interfering RNA.
Mentions: In order to study the effect of hepatic SR-BI inhibition on the development of atherosclerosis, we put the rabbits on a Western type diet supplemented with 2% cholesterol for 8 weeks. Two independent long-term experiments were performed. The animals were injected either with galactosylated poly-l-lysine-pENTR214 complexes (30 μg/kg body weight; n = 10), or its scrambled control construct (n = 9). To check for potential toxic effects of repeated treatment with poly-l complexes, we measured serum levels of aspartate-aminotransferase (AST), alanine-aminotransferase (ALT), γ-glutamyl transferase, alkaline phosphatase and C-reactive protein. The observed moderately increased serum levels of AST and ALT in both groups are in line with previous studies by us, where high cholesterol/high fat diet had the same effect in New Zealand White Rabbits [18]. However, all other parameters were not elevated upon treatment suggesting that that rabbits treated with PLL-Gal complexes did not develop severe cholestasis or systemic inflammation (Table 2). Additionally, we observed a steady increase in body weight in both groups of animals (Suppl. Fig. 4A). We monitored total cholesterol levels over time during the experiment (Fig. 3A). As observed in the short term experiment, there was again a shift in the lipoprotein profile from HDL-cholesterol to VLDL-cholesterol after two weeks of treatment (data not shown). Lower HDL-cholesterol levels were associated with decreased apoA-I levels (83% of control; p = 0.057) in the treatment group. However, HDL particle size was not altered upon siRNA treatment for two weeks (Suppl. Fig. 4B). These changes resulted in reduced total cholesterol levels. Animals treated with SR-BI-specific small hairpin RNA had total cholesterol levels of 38.4 ± 4.7 mg/dL, while controls displayed cholesterol levels of 51.2 ± 6.6 mg/dL (p = 0.014). This difference disappeared at the end of the experiment, when the total cholesterol levels rose to a plateau of nearly 5000 mg/dL in both trial arms (Fig. 3A; Table 2). No differences in levels of lipoprotein fractions isolated by stepwise ultracentrifugation were detected at this stage of the experiment (Fig. 3B). This was confirmed by FPLC analysis of chylomicron depleted plasma samples (Suppl Fig. 4C). The same was true for levels of free cholesterol as well as for initial and total CE transfer (Table 2). Biochemical characteristics of the animals at the end of the experiment are given in Table 2.

Bottom Line: After 2 weeks of treatment hepatic SR-BI mRNA levels were reduced by 80% accompanied by reduced SR-BI protein levels and a modulation of the lipoprotein profile.In a long-term study, this gene therapeutic intervention led to a similar modulation of the lipoprotein profile, to lower total cholesterol levels, and most importantly to a 50% reduction of the relative atherosclerotic lesion area.Our results are another indication that the role of SR-BI in lipoprotein metabolism and atherogenesis in rabbits--a CETP-expressing animal model displaying a manlike lipoprotein profile may be different from the one found in rodents.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine I, Innsbruck Medical University, Innsbruck, Austria.

Show MeSH
Related in: MedlinePlus