D-Carnosine octylester attenuates atherosclerosis and renal disease in ApoE null mice fed a Western diet through reduction of carbonyl stress and inflammation

Abstract

Background and purpose: Lipoxidation-derived reactive carbonyl species (RCS) such as 4-hydroxy-2-nonenal (HNE) react with proteins to form advanced lipoxidation end products (ALEs), which have been implicated in both atherosclerosis and renal disease. L-carnosine acts as an endogenous HNE scavenger, but it is rapidly inactivated by carnosinase. This study aimed at assessing the effect of the carnosinase-resistant, D-carnosine, on HNE-induced cellular injury and of its bioavailable prodrug D-carnosine octylester on experimental atherosclerosis and renal disease.

Experimental approach: Vascular smooth muscle cells (VSMCs) were exposed to HNE or H₂O₂ plus D-carnosine. ApoE null mice fed a Western, pro-atherogenic diet were treated with D-carnosine octylester for 12 weeks.

Key results: In vitro, D-carnosine attenuated the effect of HNE, but not of H₂O₂, on VSMCs. In vivo, D-carnosine octylester-treated mice showed reduced lesion area and a more stable plaque phenotype compared with untreated animals, with reduced foam cell accumulation, inflammation and apoptosis and increased clearance of apoptotic bodies and collagen deposition, resulting in decreased necrotic core formation. Likewise, renal lesions were attenuated in D-carnosine octylester-treated versus untreated mice, with lower inflammation, apoptosis and fibrosis. This was associated with increased urinary levels of HNE-carnosine adducts and reduced protein carbonylation, circulating and tissue ALEs, expression of receptors for these products, and systemic and tissue oxidative stress.

Conclusions and implications: These data indicate RCS quenching with a D-carnosine ester was highly effective in attenuating experimental atherosclerosis and renal disease by reducing carbonyl stress and inflammation and that this may represent a promising therapeutic strategy in humans.

Figure 1

In vitro studies. Quantification of cell viability (panel A), Annexin-V-FLUOS fluorescence from representative monolayers and quantification of apoptosis rate (panel B) and DCF fluorescence from representative monolayers and quantification of ROS levels (panel C) in VSMCs incubated with saline CTR, HNE (10 µM for 4 h) or H₂O₂ (300 µM for 1 h), in the presence or absence of D-carnosine 20 mM. *P < 0.001 versus CTR monolayers; †P < 0.001 or ‡P < 0.01 versus monolayers incubated with HNE without D-carnosine. CTR = control.

Figure 2

Carnosine levels. Plasma (panel A) and urinary (panel B) total carnosine, urinary D-carnosine octylester (panel C) and urine CAR-HNE (panel D) levels, in untreated and D-carnosine octylester-treated ApoE null mice fed a NFD or a HFD (mean ± SD; n= 4 per group). *P < 0.001 or †P < 0.05 versus NFD-fed mice; ‡P < 0.001 or §P < 0.01 versus untreated mice. DCO = D-carnosine octylester.

Figure 3

Aortic lesions. Oil Red O and Weigert-Van Gieson's staining of aortic sinus from representative untreated and D-carnosine octylester-treated ApoE null mice fed a HFD (panel A); and quantification of lesion area (panel B), necrotic core (panel C) and collagen content (panel D) in untreated and D-carnosine octylester-treated ApoE null mice fed a NFD (only for lesion area) or a HFD (mean ± SD; n= 10 per group); scale bar = 100 µm. *P < 0.001 versus NFD-fed mice; †P < 0.001 or ‡P < 0.01 versus untreated mice. L = lumen; M = macrophages; NC = necrotic core; C = collagen; DCO = D-carnosine octylester.

Figure 4

Aortic apoptosis, inflammation and VSMCs. Immunohistochemistry of aortic sinus from representative untreated and D-carnosine octylester-treated ApoE null mice fed a HFD and quantification of expression (mean ± SD; n= 5 per group) for active caspase-3 (panel A), F4/80 (panel B), CXCR3 (panel C) and α-SMA (panel D) in untreated and D-carnosine octylester-treated ApoE null mice fed a HFD; scale bar = 50 µm. *P < 0.001 or †P < 0.01 versus untreated mice. CXCR3 = chemokine receptor; α-SMA =α- smooth muscle actin; L = lumen; NC = necrotic core; TM = tunica media; FC = fibrous cap; DCO = D-carnosine octylester.

Figure 5

Aortic ALE levels and oxidative stress. Immunohistochemistry of aortic sinus from representative untreated and D-carnosine octylester-treated ApoE null mice fed a HFD and quantification of expression (mean ± SD; n= 5 per group) for HNE adducts (panel A), oxLDLs (panel B) and nitrotyrosine (panel C); scale bar = 100 µm. *P < 0.001 or †P < 0.01 versus the untreated mice. L = lumen; DCO = D-carnosine octylester.

Figure 6

Kidney lesions. PAS and Masson's trichrome staining of kidneys from representative untreated and D-carnosine octylester-treated ApoE null mice fed a HFD (panel A; *= microaneurysms containing foam cells); and quantification of mGA (panel B), mGV (panel C), glomerular matrix (panel D), foam cells (panel E), microaneurysms (panel F) and tubulo-interstitial fibrosis (panel G) in untreated and D-carnosine octylester-treated ApoE null mice fed a NFD or a HFD (mean ± SD; n= 10 per group); scale bar = 50 µm. *P < 0.001 versus NFD-fed mice; †P < 0.001 versus untreated mice. DCO = D-carnosine octylester.