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Inhibition of lysophosphatidic acid receptors 1 and 3 attenuates atherosclerosis development in LDL-receptor deficient mice

View Article: PubMed Central - PubMed

ABSTRACT

Lysophosphatidic acid (LPA) is a natural lysophospholipid present at high concentrations within lipid-rich atherosclerotic plaques. Upon local accumulation in the damaged vessels, LPA can act as a potent activator for various types of immune cells through its specific membrane receptors LPA1/3. LPA elicits chemotactic, pro-inflammatory and apoptotic effects that lead to atherosclerotic plaque progression. In this study we aimed to inhibit LPA signaling by means of LPA1/3 antagonism using the small molecule Ki16425. We show that LPA1/3 inhibition significantly impaired atherosclerosis progression. Treatment with Ki16425 also resulted in reduced CCL2 production and secretion, which led to less monocyte and neutrophil infiltration. Furthermore, we provide evidence that LPA1/3 blockade enhanced the percentage of non-inflammatory, Ly6Clow monocytes and CD4+ CD25+ FoxP3+ T-regulatory cells. Finally, we demonstrate that LPA1/3 antagonism mildly reduced plasma LDL cholesterol levels. Therefore, pharmacological inhibition of LPA1/3 receptors may prove a promising approach to diminish atherosclerosis development.

No MeSH data available.


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The LPA1/3 antagonist, Ki16425, reduces atherosclerosis development.(a) Atherosclerotic plaque size in the aortic root of the heart was determined by an Oil-Red-O staining on 10 μm sections; representative pictures are shown. Blockade of receptors LPA1/3 resulted in a 40% reduction in atherosclerosis. (b) The Ki16425 treated mice had significantly lower atherosclerotic plaque development throughout the entire three valve area of the aortic root. (c) Macrophage expression levels were measured using a MOMA-2 staining; LPA1/3 antagonism led to 45% less macrophage accumulation within the aortic root of the hearts. (d) The relative amount of macrophages in the atherosclerotic plaques was not significantly affected by the Ki16425 treatment. (e) Mast cell numbers (#) and (f) activation state, as well as (g) neutrophil numbers were manually quantified using a Naphthol AS-D chloro-acetate esterase staining; no difference was observed in the number or degranulation status of mast cells in the aortic root. Neutrophil numbers were found significantly reduced by 31% in the aortic root of the Ki16425 group as compared to the control. All values (n = 12/grp) are depicted as mean ± SEM (*P < 0.05, **P < 0.01).
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f1: The LPA1/3 antagonist, Ki16425, reduces atherosclerosis development.(a) Atherosclerotic plaque size in the aortic root of the heart was determined by an Oil-Red-O staining on 10 μm sections; representative pictures are shown. Blockade of receptors LPA1/3 resulted in a 40% reduction in atherosclerosis. (b) The Ki16425 treated mice had significantly lower atherosclerotic plaque development throughout the entire three valve area of the aortic root. (c) Macrophage expression levels were measured using a MOMA-2 staining; LPA1/3 antagonism led to 45% less macrophage accumulation within the aortic root of the hearts. (d) The relative amount of macrophages in the atherosclerotic plaques was not significantly affected by the Ki16425 treatment. (e) Mast cell numbers (#) and (f) activation state, as well as (g) neutrophil numbers were manually quantified using a Naphthol AS-D chloro-acetate esterase staining; no difference was observed in the number or degranulation status of mast cells in the aortic root. Neutrophil numbers were found significantly reduced by 31% in the aortic root of the Ki16425 group as compared to the control. All values (n = 12/grp) are depicted as mean ± SEM (*P < 0.05, **P < 0.01).

Mentions: To assess the effect of LPA1/3 inhibition on atherosclerosis, LDLr−/− mice were injected intraperitoneally with either Ki16425 (5 mg/kg) or a vehicle-control for 6 weeks, (3x/week). Plaque size quantification, using an Oil-Red-O staining showed that mice treated with the LPA1/3 inhibitor had significantly smaller plaque size (−40%) compared to control mice (Fig. 1a, Ki16425: 89*103 ± 9*103 μm2 vs control: 147*103 ± 21*103 μm2, P = 0.023). In fact, plaque size was significantly lower in the treated group at each distance measured from the start of the three-valve area up to its end (Fig. 1b). A MOMA-2 staining was performed to evaluate the intra-plaque macrophage levels. The absolute macrophage content of the Ki16425 treated group was significantly lower (−45%) compared to the control (Fig. 1c, Ki16425: 36*103 ± 8*103 μm2 vs control: 65*103 ± 6*103 μm2, P = 0.006), whereas the relative amount (% macrophage levels of the plaque) was not significantly different (Fig. 1d, P = 0.11). Furthermore, the aortic root area was analyzed for mast cell and neutrophil content, since both immune cell types express LPA1/3 and are involved in atherosclerosis progression. No differences in the number or activation status of mast cells were detected between the two groups (Fig. 1e, P = 0.38 for mast cell # and Fig. 1f, P = 0.88 for activated mast cell #). However, a substantial reduction in the number of infiltrated neutrophils (−31%) upon LPA1/3 blockade was observed (Fig. 1g, Ki16425:8.5 ± 0.7 neutrophils/μm2 tissue vs. control: 12.4 ± 0.9 neutrophils/μm2 tissue, P = 0.004).


Inhibition of lysophosphatidic acid receptors 1 and 3 attenuates atherosclerosis development in LDL-receptor deficient mice
The LPA1/3 antagonist, Ki16425, reduces atherosclerosis development.(a) Atherosclerotic plaque size in the aortic root of the heart was determined by an Oil-Red-O staining on 10 μm sections; representative pictures are shown. Blockade of receptors LPA1/3 resulted in a 40% reduction in atherosclerosis. (b) The Ki16425 treated mice had significantly lower atherosclerotic plaque development throughout the entire three valve area of the aortic root. (c) Macrophage expression levels were measured using a MOMA-2 staining; LPA1/3 antagonism led to 45% less macrophage accumulation within the aortic root of the hearts. (d) The relative amount of macrophages in the atherosclerotic plaques was not significantly affected by the Ki16425 treatment. (e) Mast cell numbers (#) and (f) activation state, as well as (g) neutrophil numbers were manually quantified using a Naphthol AS-D chloro-acetate esterase staining; no difference was observed in the number or degranulation status of mast cells in the aortic root. Neutrophil numbers were found significantly reduced by 31% in the aortic root of the Ki16425 group as compared to the control. All values (n = 12/grp) are depicted as mean ± SEM (*P < 0.05, **P < 0.01).
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f1: The LPA1/3 antagonist, Ki16425, reduces atherosclerosis development.(a) Atherosclerotic plaque size in the aortic root of the heart was determined by an Oil-Red-O staining on 10 μm sections; representative pictures are shown. Blockade of receptors LPA1/3 resulted in a 40% reduction in atherosclerosis. (b) The Ki16425 treated mice had significantly lower atherosclerotic plaque development throughout the entire three valve area of the aortic root. (c) Macrophage expression levels were measured using a MOMA-2 staining; LPA1/3 antagonism led to 45% less macrophage accumulation within the aortic root of the hearts. (d) The relative amount of macrophages in the atherosclerotic plaques was not significantly affected by the Ki16425 treatment. (e) Mast cell numbers (#) and (f) activation state, as well as (g) neutrophil numbers were manually quantified using a Naphthol AS-D chloro-acetate esterase staining; no difference was observed in the number or degranulation status of mast cells in the aortic root. Neutrophil numbers were found significantly reduced by 31% in the aortic root of the Ki16425 group as compared to the control. All values (n = 12/grp) are depicted as mean ± SEM (*P < 0.05, **P < 0.01).
Mentions: To assess the effect of LPA1/3 inhibition on atherosclerosis, LDLr−/− mice were injected intraperitoneally with either Ki16425 (5 mg/kg) or a vehicle-control for 6 weeks, (3x/week). Plaque size quantification, using an Oil-Red-O staining showed that mice treated with the LPA1/3 inhibitor had significantly smaller plaque size (−40%) compared to control mice (Fig. 1a, Ki16425: 89*103 ± 9*103 μm2 vs control: 147*103 ± 21*103 μm2, P = 0.023). In fact, plaque size was significantly lower in the treated group at each distance measured from the start of the three-valve area up to its end (Fig. 1b). A MOMA-2 staining was performed to evaluate the intra-plaque macrophage levels. The absolute macrophage content of the Ki16425 treated group was significantly lower (−45%) compared to the control (Fig. 1c, Ki16425: 36*103 ± 8*103 μm2 vs control: 65*103 ± 6*103 μm2, P = 0.006), whereas the relative amount (% macrophage levels of the plaque) was not significantly different (Fig. 1d, P = 0.11). Furthermore, the aortic root area was analyzed for mast cell and neutrophil content, since both immune cell types express LPA1/3 and are involved in atherosclerosis progression. No differences in the number or activation status of mast cells were detected between the two groups (Fig. 1e, P = 0.38 for mast cell # and Fig. 1f, P = 0.88 for activated mast cell #). However, a substantial reduction in the number of infiltrated neutrophils (−31%) upon LPA1/3 blockade was observed (Fig. 1g, Ki16425:8.5 ± 0.7 neutrophils/μm2 tissue vs. control: 12.4 ± 0.9 neutrophils/μm2 tissue, P = 0.004).

View Article: PubMed Central - PubMed

ABSTRACT

Lysophosphatidic acid (LPA) is a natural lysophospholipid present at high concentrations within lipid-rich atherosclerotic plaques. Upon local accumulation in the damaged vessels, LPA can act as a potent activator for various types of immune cells through its specific membrane receptors LPA1/3. LPA elicits chemotactic, pro-inflammatory and apoptotic effects that lead to atherosclerotic plaque progression. In this study we aimed to inhibit LPA signaling by means of LPA1/3 antagonism using the small molecule Ki16425. We show that LPA1/3 inhibition significantly impaired atherosclerosis progression. Treatment with Ki16425 also resulted in reduced CCL2 production and secretion, which led to less monocyte and neutrophil infiltration. Furthermore, we provide evidence that LPA1/3 blockade enhanced the percentage of non-inflammatory, Ly6Clow monocytes and CD4+ CD25+ FoxP3+ T-regulatory cells. Finally, we demonstrate that LPA1/3 antagonism mildly reduced plasma LDL cholesterol levels. Therefore, pharmacological inhibition of LPA1/3 receptors may prove a promising approach to diminish atherosclerosis development.

No MeSH data available.


Related in: MedlinePlus