Role of heparanase on hepatic uptake of intestinal derived lipoprotein and fatty streak formation in mice

Abstract

Background: Heparanase modulates the level of heparan sulfate proteoglycans (HSPGs) which have an important role in multiple cellular processes. Recent studies indicate that HSPGs have an important function in hepatic lipoprotein handling and processes involving removal of lipoprotein particles.

Principal findings: To determine the effects of decreased HSPGs chain length on lipoprotein metabolism and atherosclerosis, transgenic mice over-expressing the human heparanase gene were studied. Hepatic lipid uptake in hpa-Tg mice were evaluated by giving transgenic mice oral fat loads and labeled retinol. Sections of aorta from mice over-expressing heparanase (hpa-Tg) and controls (C57/BL6) fed an atherogenic diet were examined for evidence of atherosclerosis. Heparanase over-expression results in reduced hepatic clearance of postprandial lipoproteins and higher levels of fasting and postprandial serum triglycerides. Heparanase over-expression also induces formation of fatty streaks in the aorta. The mean lesion cross-sectional area in heparanase over-expressing mice was almost 6 times higher when compared to control mice (23,984 µm(2)±5,922 vs. 4,189 µm(2)±1,130, p<0.001).

Conclusions: Over-expression of heparanase demonstrates the importance of HSPGs for the uptake of intestinal derived lipoproteins and its role in the formation of fatty streaks.

Figure 1. Distribution of triglyceride (A) and cholesterol (B) in the plasma of hpa-Tg (open triangles) and control C67BL/6 mice (closed triangles).

Pooled plasma samples of 4 fasting mice from each group were applied to FPLC analysis. As indicated in the figure, triglyceride levels are increased in the non-HDL fractions (A), and cholesterol level is slightly increased in the VLDL+IDL fractions (B) of the hpa-Tg mice compared to controls. AUC (µg/ml x fraction) of C57BL/6 triglyceride was 281 compared to 393 of hpa-Tg whereas the AUC of C57BL/6 total cholesterol was 662 compared to 705 of hpa-Tg mice. The AUC of VLDL-cholesterol peak was 20 compared to 74 of hpa-Tg mice.

Figure 2. TG accumulation in plasma following oral administration.

Mice were fasted overnight and then administered a bolus of 100 ul corn oil via a stomach tube. TG levels in serum of hpa-Tg mice (n = 6) were significantly higher compared to TG levels in C57BL/6 mice (n = 6) during the first 3 hrs (p<0.005 for all measured time points).

Figure 3. ³H-retinol accumulation (A) and distribution (B) in plasma following oral administration.

(A) Mice were fasted overnight and then administered a bolus of 100 µl corn oil containing ³H-retinol via a stomach tube. Radioactivity in plasma of hpa-Tg mice (n = 3) was significantly higher compared to C57BL/6 (n = 3) during the 6 h following retinol administration (p<0.01). (B) 200 µl of plasma from hpa-Tg and C57BL/6 mice, taken 3 h following retinol load were analyzed by FPLC. As indicated in the figure, retinol radioactivity was increased in the VLDL and IDL fractions of hpa-Tg compared to control mice. AUC (dpm x fraction) of hpa-Tg was 16703 compared to 4640 in C57BL/6 mice.

Figure 4. Post-prandial lipid handling in hpa-Tg and C57BL/6 mice.

(A) Absorption of radio-labeled vitamin A (³H-retinol). Intestinal absorption was 12% of the total administered dose of ³H-retinol in both hpa-Tg (n = 4) and C57BL/6 mice (n = 4). (B) Distribution of absorbed vitamin A (³H-retinol) in blood, liver and carcass. 41% of the radioactive retinol were detected in serum of hpa-Tg mice compared to 9% detected in serum of C57BL/6 mice, p<0.0001. No difference was detected in radioactivity extracted from carcasses of the two experimental groups. The decrease in liver uptake of retinol was paralleled by an equivalent increase in the level of labeled retinol in the blood of hpa-Tg mice.

Figure 5. Fatty streaks in hpa-Tg and C57BL/6 mice.

(A) Frozen sections of aortic root from hpa-Tg and C57BL control mice (n = 15 for each group) were stained with Oil-Red-O for lipid content. (B) Mean lesion area in heparanase over-expressing mice was significantly higher compared to control mice, p<0.001.