Hyperlipemia and oxidation of LDL induce vascular smooth muscle cell growth: an effect mediated by the HLH factor Id3

Abstract

Hyperlipemia and oxidized LDL (ox-LDL) are important independent cardiovascular risk factors. Ox-LDL has been shown to stimulate vascular smooth muscle cell (VSMC) proliferation. However, the effects of hyperlipemia and the molecular mechanisms mediating hyperlipemia and ox-LDL effects on VSMC growth are poorly understood. The helix-loop-helix (HLH) transcription factor, Id3, is a redox-sensitive gene expressed in VSMC in response to mitogen stimulation and vascular injury. Accordingly, we hypothesize that Id3 is an important mediator of ox-LDL and hyperlipemia-induced VSMC growth. Aortas harvested from hyperlipemic pigs demonstrated significantly more Id3 than normolipemic controls. Primary VSMC were stimulated with ox-LDL, native LDL, sera from hyperlipemic pigs, or normolipemic pigs. VSMC exposed to hyperlipemic sera demonstrated increased Id3 expression, VSMC growth and S-phase entry and decreased p21cip1 expression and transcription. Cells stimulated with ox-LDL demonstrated similar findings of increased growth and Id3 expression and decreased p21cip1 expression. Moreover, the effects of ox-LDL on growth were abolished in cells devoid of the Id3 gene. Results provide evidence that the HLH factor Id3 mediates the mitogenic effect of hyperlipemic sera and ox-LDL in VSMC via inhibition of p21cip1 expression, subsequently increasing DNA synthesis and proliferation.

Fig. 1

ox-LDL increases linearly with total cholesterol. Levels of LDL oxidation were determined using a commercially available antibody assay. As total cholesterol increases in the porcine model, ox-LDL increases.

Fig. 2

Hyperlipemia induces Id3 expression in the vessel wall. After 20 weeks of high-fat feeding, vessels were harvested and equal amounts of protein were analyzed for Id3. A representative Western blot is shown (a). Quantitative densitometry is shown from 4 animals from each group (b). Results are normalized to quantitative densitometry of the tubulin blot for each sample. Normolipemic animals had serum cholesterol levels of 65–105 mg/dl. Hyper-lipemic animals had cholesterol levels of 287–450 mg/dl. Hyperlipemic aortas demonstrated significantly more Id3 relative to normolipemic aortas (* p < 0.001).

Fig. 3

Hyperlipemia increases Id3 expression, VSMC growth, and S-phase entry. Porcine VSMC were plated in equal densities, quiesced for 48 h and stimulated with media containing either hyperlipemic or normolipemic sera. Normolipemic animals had serum cholesterol levels of 65–105 mg/ dl. Hyperlipemic animals had cholesterol levels of 287–450 mg/dl. a Forty-eight hours after stimulation, equal amounts of protein were analyzed for Id3 expression by Western blotting. The blot was stripped and reprobed with an anti-tubulin antibody to control for protein loading. b VSMC stimulated with hyperlipemic sera grew faster at all time points relative to normolipemic sera (* p = 0.008 at 24 h, ^† p = 0.01 at 48 h). c Eighteen hours after stimulation, cells were stained with PI and analyzed for cell cycle components. VSMC stimulated with hyperlipemic sera had significantly greater S-phase entry relative to normolipemic sera (* p = 0.016). Results are the average of three experiments performed in triplicate utilizing sera from 7 animals from each group.

Fig. 4

Hyperlipemia increases Id3 transcription and decreases p21^cip1 transcription. Cells were cotransfected with pK7GFP and either pId3Luc or p21Luc. Cells were quiesced for 48 h and stimulated with either hyperlipemic or normolipemic sera. Lucif-erase activity was measured (normalized to protein concentration and GFP fluorescence) was measured 24 and 48 h later. a Hyperlipemic sera increased Id3 promoter reporter activity relative to normolipemic sera (* p < 0.01 at 24 and 48 h). b Hyperlipemic sera decreased p21 promoter reporter activity relative to normolipemic sera (* p < 0.01 at 24 h, ^† p = 0.02 at 48 h). Results are average of three experiments performed in triplicate utilizing sera from 7 animals from each group.

Fig. 5

Id3 is essential for ox-LDL-induced growth. Control and Id3 knockout primary mouse aortic VSMC were plated at equal densities, quiesced for 48 h and stimulated with either 20 μg/ml of n-LDL or ox-LDL. a Control B6 cells stimulated with ox-LDL grew faster at all time points relative to n-LDL (* p < 0.05 at 24 h, ^† p < 0.001 at 48 h). Id3 KO cells grew significantly slower than wild-type cells at all time points (p < 0.05 at 24 h, p < 0.001 at 48 h) and demonstrated no increase in growth in response to ox-LDL. Results are averages of three experiments performed in triplicate. b At 48 h, equal amounts of protein from control cell lysates were immunoblotted using antibodies to Id3 or p21^cip1 . The blot was stripped and reprobed with an anti-tubulin antibody to control for protein loading.

Fig. 6

AdId3 infection rescues decreased proliferation of Id3–/– cells. Wild-type and Id3 knockout VSMC were infected with AdId3 or AdGFP control virus and assayed for cell number 24 h after infection. Control infected Id3 knockout cells demonstrated decreased proliferation relative to control infected wild type cells. Proliferation of Id3 knockout cells is rescued with AdId3 infection.