RESUMEN
BACKGROUND AND AIMS: Inflammatory cells within atherosclerotic lesions secrete proteolytic enzymes that contribute to lesion progression and destabilization, increasing the risk for an acute cardiovascular event. Elastase is a serine protease, secreted by macrophages and neutrophils, that may contribute to the development of unstable plaque. We previously reported interaction of endogenous protease-inhibitor proteins with high-density lipoprotein (HDL), including alpha-1-antitrypsin, an inhibitor of elastase. These findings support a potential role for HDL as a modulator of protease activity. In this study, we test the hypothesis that enhancement of HDL-associated elastase inhibitor activity is protective against atherosclerotic lesion progression. METHODS: We designed an HDL-targeting protease inhibitor (HTPI) that binds to HDL and confers elastase inhibitor activity. Lipoprotein binding and the impact of HTPI on atherosclerosis were examined using mouse models. Histology and immunofluorescence staining of aortic root sections were used to examine the impact of HTPI on lesion morphology and inflammatory features. RESULTS: HTPI is a small (1.6 kDa) peptide with an elastase inhibitor domain, a soluble linker, and an HDL-targeting domain. When incubated with human plasma ex vivo, HTPI predominantly binds to HDL. Intravenous administration of HTPI to mice resulted in its binding to plasma HDL and increased elastase inhibitor activity on isolated HDL. Accumulation of HTPI within plaque was observed after administration to Apoe-/- mice. To examine the effect of HTPI treatment on atherosclerosis, prevention and progression studies were performed using Ldlr-/- mice fed Western diet. In both study designs, HTPI-treated mice had reduced lipid deposition in plaque. CONCLUSIONS: These data support the hypothesis that HDL-associated anti-elastase activity can improve the atheroprotective potential of HDL and highlight the potential utility of HDL enrichment with anti-protease activity as an approach for stabilization of atherosclerotic lesions.
Asunto(s)
Aterosclerosis , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Lipoproteínas HDL , Animales , Aterosclerosis/patología , Aterosclerosis/prevención & control , Aterosclerosis/enzimología , Aterosclerosis/metabolismo , Aterosclerosis/tratamiento farmacológico , Lipoproteínas HDL/metabolismo , Humanos , Ratones Endogámicos C57BL , Ratones , Ratones Noqueados para ApoE , Placa Aterosclerótica , Masculino , Elastasa Pancreática/metabolismo , Aorta/patología , Aorta/efectos de los fármacos , Aorta/enzimología , Aorta/metabolismo , Enfermedades de la Aorta/prevención & control , Enfermedades de la Aorta/patología , Enfermedades de la Aorta/enzimología , Enfermedades de la Aorta/metabolismo , Inhibidores de Proteasas/farmacología , alfa 1-Antitripsina/farmacología , alfa 1-Antitripsina/metabolismoRESUMEN
Background: Inflammatory cells within atherosclerotic lesions secrete various proteolytic enzymes that contribute to lesion progression and destabilization, increasing the risk for an acute cardiovascular event. The relative contributions of specific proteases to atherogenesis is not well understood. Elastase is a serine protease, secreted by macrophages and neutrophils, that may contribute to the development of unstable plaque. We have previously reported interaction of endogenous protease-inhibitor proteins with high-density lipoprotein (HDL), including alpha-1-antitrypsin, an inhibitor of elastase. These findings support a potential role for HDL as an endogenous modulator of protease activity. In this study, we test the hypothesis that enhancement of HDL-associated elastase inhibitor activity is protective against atherosclerotic lesion progression. Methods: We designed an HDL-targeting protease inhibitor (HTPI) that binds to HDL and confers elastase inhibitor activity. Lipoprotein binding and the impact of HTPI on atherosclerosis was examined using mouse models. Results: HTPI is a small (1.6 kDa) peptide with an elastase inhibitor domain, a soluble linker, and an HDL-targeting domain. When incubated with human plasma ex vivo , HTPI predominantly binds to HDL. Intravenous administration of HTPI to mice resulted in its binding to plasma HDL and increased elastase inhibitor activity on isolated HDL. Accumulation of HTPI within plaque was observed after systemic administration to Apoe -/- mice. To examine the effect of HTPI treatment on atherosclerosis, prevention and progression studies were performed using Ldlr -/- mice fed Western diet. In both study designs, HTPI-treated mice had reduced lipid deposition in plaque. Histology and immunofluorescence staining of aortic root sections were used to examine the impact of HTPI on lesion morphology and inflammatory features. Conclusions: These data support the hypothesis that HDL-associated anti-elastase activity can improve the atheroprotective potential of HDL and highlight the potential utility of HDL enrichment with anti-protease activity as an approach for stabilization of atherosclerotic lesions.
RESUMEN
Dennd5b plays a pivotal role in intestinal absorption of dietary lipids in mice and is associated with body mass index in humans. This study examined the impact of whole-body Dennd5b deletion on plasma lipid concentrations, atherosclerosis, and hepatic lipid metabolism in mice. Hypercholesterolemia was induced in Dennd5b-/- mice by infection with an adeno-associated virus expressing the proprotein convertase subtilisin/kexin type 9 serine protease (PCSK9) gain-of-function mutation (PCSK9D377Y) and feeding a Western diet for 12 weeks. Body weight and plasma lipid concentrations were monitored over 12 weeks, and then aortic atherosclerosis and hepatic lipid content were quantified. Compared to Dennd5b+/+ mice, Dennd5b-/- mice were resistant to diet-induced weight gain and PCSK9-induced hypercholesterolemia. Atherosclerosis quantified by en face analysis and in aortic root sections, revealed significantly smaller lesions in Dennd5b-/- compared to Dennd5b+/+ mice. Additionally, Dennd5b-/- mice had significantly less hepatic lipid content (triglyceride and cholesterol) compared to Dennd5b+/+ mice. To gain insight into the basis for reduced hepatic lipids, quantitative PCR was used to measure mRNA abundance of genes involved in hepatic lipid metabolism. Key genes involved in hepatic lipid metabolism and lipid storage were differentially expressed in Dennd5b-/- liver including Pparg, Cd36, and Pnpla3. These findings demonstrate a significant impact of Dennd5b on plasma and hepatic lipid concentrations and resistance to PCSK9-induced hypercholesterolemia in the absence of Dennd5b.