**4. Cholesterol withdrawal increases plaque stability**

Numerous studies indicate that dietary modification is an important strategy for the prevention of cardiovascular disease [2, 25]. However, studies examining the effects of food restriction on atherosclerosis are scarce. Although short-term cholesterol withdrawal (4 weeks) does not alter plaque size (**Table 2**), a normal unrestricted diet results in a more stable plaque phenotype. Indeed, collagen content of the atherosclerotic plaques is increased, mainly due to an increase in type I collagen (**Figure 2**), which is essential for plaque stability. Moreover, VCAM-1 expression in endothelial cells declines (**Figure 3**). VCAM-1 is important for leucocyte recruitment and thereby contributes to plaque inflammation and macrophage accumulation. However, despite a decrease in VCAM-1 expression, the total amount of macrophages in the plaque does not alter within 4 weeks of cholesterol withdrawal (4 weeks). Indeed, only prolonged cholesterol withdrawal (12–24 weeks) results in a dramatic loss of plaque macrophages [26–28].


**5. Effect of food restriction on plaque development is controversial**

kines and chemotactic factors, thereby further aggravating plaque inflammation [30].

nutrient deprivation may be insufficient to counteract apoptosis.

In contrast with a normal unrestricted diet, severe food restriction does not promote beneficial effects such as increased collagen synthesis and decreased VCAM-1 expression. On the contrary, plaques of rabbits undergoing food restriction reveal an increase in apoptosis (**Figure 3**). Depending on the cell type and stage of the plaque, apoptosis could be detrimental for plaque stability [29]. Moreover, apoptosis can stimulate the release of inflammatory cyto-

**Figure 2.** Collagen content of atherosclerotic plaques in rabbits that were fed 0.3% cholesterol for 20 weeks (baseline), followed by cholesterol withdrawal for 4 weeks either via a normal diet or a restricted diet (20% of normal diet). (A) Sections of the proximal ascending aorta were stained with Sirius red for total collagen determination. Scale bar = 500 µm. (B) Analysis of Sirius red staining via polarized light microscopy. Collagen type I is displayed in red, type III in green. Scale bar = 500 µm. (C) Quantification of total collagen, type I and type III collagen as well as the type I/III collagen ratio in Sirius red stained sections. \*P < 0.05, \*\*P < 0.01, \*\*\*P < 0.001 (One-way ANOVA with post-hoc LSD, n = 8–10 in each group).

Food Restriction and Atherosclerotic Plaque Stabilization

http://dx.doi.org/10.5772/intechopen.76560

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Given that food restriction stimulates autophagy, a well-known cellular survival mechanism, increased apoptosis may seem surprising. However, autophagy induction after intensive

**Table 2.** Plaque area and cellular composition in the proximal ascending in cholesterol-fed rabbits (baseline, 20 weeks of cholesterol) followed by dietary lipid lowering for 4 weeks (normal diet) or a restricted diet for 4 weeks (restricted diet).

in serum lipids. SQSTM1/p62 is a scaffold protein that binds directly to the autophagosomal marker Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates via autophagy. Nutrient deprivation is a powerful autophagy-inducing condition [20]. Rabbits that undergo cholesterol withdrawal via a normal diet show high LC3-II levels but unaltered amounts of SQSTM1/p62 (**Figure 1A**), indicating moderate induction of autophagy. In contrast, rabbits undergoing severe food restriction show low levels of LC3-II and a clear reduction of SQSTM1/p62 (**Figure 1B**), which points to strong autophagy stimulation. It has been described that autophagy is strongly involved in managing intracellular lipids [21, 22]. Lipid droplets are taken up by lysosomes, where lysosomal acid lipases hydrolyze cholesteryl esters to generate free cholesterol for ATP-binding cassette transporter 1 (ABCA1)-mediated cholesterol efflux [21]. Impairment of autophagy in macrophages reduces reverse cholesterol transport [21], a condition that refers to net cholesterol flux from the peripheral tissues to the liver (for excretion via the bile). Conversely, pharmacological activation of the autophagy pathway attenuates lipid accumulation [23] and in some conditions (e.g., after treatment with mTOR

Numerous studies indicate that dietary modification is an important strategy for the prevention of cardiovascular disease [2, 25]. However, studies examining the effects of food restriction on atherosclerosis are scarce. Although short-term cholesterol withdrawal (4 weeks) does not alter plaque size (**Table 2**), a normal unrestricted diet results in a more stable plaque phenotype. Indeed, collagen content of the atherosclerotic plaques is increased, mainly due to an increase in type I collagen (**Figure 2**), which is essential for plaque stability. Moreover, VCAM-1 expression in endothelial cells declines (**Figure 3**). VCAM-1 is important for leucocyte recruitment and thereby contributes to plaque inflammation and macrophage accumulation. However, despite a decrease in VCAM-1 expression, the total amount of macrophages in the plaque does not alter within 4 weeks of cholesterol withdrawal (4 weeks). Indeed, only prolonged cholesterol withdrawal (12–24 weeks) results in a dramatic loss of plaque

) 3.3 ± 0.8 3.6 ± 0.6 3.6 ± 1.0

**Table 2.** Plaque area and cellular composition in the proximal ascending in cholesterol-fed rabbits (baseline, 20 weeks of cholesterol) followed by dietary lipid lowering for 4 weeks (normal diet) or a restricted diet for 4 weeks (restricted diet).

Macrophages (%) 22 ± 3 24 ± 4 34 ± 6 Smooth muscle cells (%) 26 ± 4 23 ± 2 26 ± 4 Fibrous cap thickness 0.4 ± 0.1 0.4 ± 0.1 0.5 ± 0.1

**Baseline Normal diet Restricted diet**

inhibitors) triggers hypercholesterolemia [24].

64 Atherosclerosis - Yesterday, Today and Tomorrow

macrophages [26–28].

Data are expressed as mean ± SEM.

Plaque area (mm2

**4. Cholesterol withdrawal increases plaque stability**

**Figure 2.** Collagen content of atherosclerotic plaques in rabbits that were fed 0.3% cholesterol for 20 weeks (baseline), followed by cholesterol withdrawal for 4 weeks either via a normal diet or a restricted diet (20% of normal diet). (A) Sections of the proximal ascending aorta were stained with Sirius red for total collagen determination. Scale bar = 500 µm. (B) Analysis of Sirius red staining via polarized light microscopy. Collagen type I is displayed in red, type III in green. Scale bar = 500 µm. (C) Quantification of total collagen, type I and type III collagen as well as the type I/III collagen ratio in Sirius red stained sections. \*P < 0.05, \*\*P < 0.01, \*\*\*P < 0.001 (One-way ANOVA with post-hoc LSD, n = 8–10 in each group).
