New therapeutic approaches to modulate metabolic alterations in the cardiovascular system. In vivo studies

Abstract

[eng] BACKGROUND: Low-density lipoprotein receptor-related protein 1 (LRP1) plays a key role in fatty acid metabolism and glucose homeostasis. In the context of dyslipidemia, LRP1 is positively regulated in the heart. The LRP1 domain (CR9) and, in particular, the sequence Gly1127-Cys1140 (P3 peptide) is essential in the binding and internalization of aggregated LDL (agLDL). AgLDL internalization leads to intracellular cholesterol ester (CE) loading and foam cell formation in different cell types. The link between intracellular CE accumulation and insulin response is largely unknown. The first study aimed to evaluate the impact of cardiomyocyte Lrp1 deficiency on high-fat diet (HFD)-induced cardiac and metabolic alterations in a murine model and to explore the possible mechanisms involved. In the second and third studies, we aimed to evaluate whether immunization of rabbits with the P3 peptide reduces HFD-induced atherosclerosis and HFD-induced alterations in cardiac insulin response, respectively. METHODS STUDY I: We used TnT-iCre transgenic mice to generate an experimental mouse model with conditional Lrp1 deficiency in cardiomyocytes (TNT-iCre+-LRP1flox/flox). cm- Lrp1-/- (deficient) and cm-Lrp1+/+ (control) mice were fed with HFD and treated daily with Doxycycline Cyclate in drinking water for six weeks to inhibit Lrp1. After two weeks, mice were randomly assigned to untreated (PBS) or treated with natriuretic peptide receptor type A (NPRA) inhibitor until the end of the experiment. The indirect calorimetry study was performed at week 5 and glucose tolerance test at week 6. At the end of the experiment, target tissues were collected for molecular, lipidic, proteomic and immunohistochemical studies. METHODS STUDY II AND III: Rabbits immunized with irrelevant peptides (IrP) or P3 were randomized into HFD- or Chow-fed groups. Anti-P3 antibody (Abs) levels were determined by ELISA. Lipoprotein profile, circulating and tissue lipids, and vascular proinflammatory mediators were determined by standardized methods. Atherosclerosis was determined by confocal microscopy and noninvasive imaging studies. LRP1, Insulin receptor (InsR) and glucose transporter type 4 (GLUT4) levels were determined in rabbit heart membranes and total lysates. The interaction between InsR and LRP1 was analyzed by immunoprecipitation and confocal microscopy. Ex-vivo studies of cultured human macrophages (hMΦ), HL-1 cardiomyocytes, and vascular smooth muscle cells (VSMC) exposed to rabbit serum were conducted to explore the cell-specific effects of anti-P3 Abs. FINDINGS OF THE STUDIES: cm-Lrp1-/- mice have normal cardiac function combined with a favorable metabolic phenotype against HFD-induced glucose intolerance and obesity. Protection against glucose intolerance was associated with higher hepatic fatty acid oxidation (FAO), lower hepatic steatosis, and higher whole-body energy expenditure. Proteomic studies of the heart revealed reduced levels of cardiac pro-atrial natriuretic peptide (pro-ANP), which paralleled higher circulating levels and signaling of ANP.

In the rabbit model, anti-P3 Abs reduced HFD-induced vascular CE accumulation and intracellular CE loading of hMΦ and VSMC as well as HFD-induced pro-inflammatory effects in these cells. Microscopy studies revealed that anti-P3 abs reduced the percentage of lipids, macrophages, and SMCs in the arterial intima, as well as the atherosclerotic extent and injured area in the aorta of hypercholesterolemic rabbits. Noninvasive imaging studies showed that aortic standardized mean uptake value (SUVmean) and carotid arterial resistance index (ARI) were upregulated by HFD to a much higher extent in control groups than in the P3-immunized group. In addition, anti-P3 abs not only decreases the amount of HFD-induced generation of lipid droplets (LDs) and glycogen drops into cardiomyocytes but also modify LD size, content and electrodensity. In addition, anti-P3 Abs restored the downregulatory effect of HFD on membrane InsR and GLUT4 levels as well as on LRP1/InsR interactions in rabbit hearts, and normalized the insulin signaling cascade and glucose uptake in HL-1 cells exposed to rabbit HFD serums.