FURIN Inhibition Reduces Vascular Remodeling and Atherosclerotic Lesion Progression in Mice.

Objective- Atherosclerotic coronary artery disease is the leading cause of death worldwide, and current treatment options are insufficient. Using systems-level network cluster analyses on a large coronary artery disease case-control cohort, we previously identified PCSK3 (proprotein convertase subtilisin/kexin family member 3; FURIN) as a member of several coronary artery disease-associated pathways. Thus, our objective is to determine the role of FURIN in atherosclerosis. Approach and Results- In vitro, FURIN inhibitor treatment resulted in reduced monocyte migration and reduced macrophage and vascular endothelial cell inflammatory and cytokine gene expression. In vivo, administration of an irreversible inhibitor of FURIN, α-1-PDX (α1-antitrypsin Portland), to hyperlipidemic Ldlr-/- mice resulted in lower atherosclerotic lesion area and a specific reduction in severe lesions. Significantly lower lesional macrophage and collagen area, as well as systemic inflammatory markers, were observed. MMP2 (matrix metallopeptidase 2), an effector of endothelial function and atherosclerotic lesion progression, and a FURIN substrate was significantly reduced in the aorta of inhibitor-treated mice. To determine FURIN's role in vascular endothelial function, we administered α-1-PDX to Apoe-/- mice harboring a wire injury in the common carotid artery. We observed significantly decreased carotid intimal thickness and lower plaque cellularity, smooth muscle cell, macrophage, and inflammatory marker content, suggesting protection against vascular remodeling. Overexpression of FURIN in this model resulted in a significant 67% increase in intimal plaque thickness, confirming that FURIN levels directly correlate with atherosclerosis. Conclusions- We show that systemic inhibition of FURIN in mice decreases vascular remodeling and atherosclerosis. FURIN-mediated modulation of MMP2 activity may contribute to the atheroprotection observed in these mice.

Therapeutic modulation of the bile acid pool by Cyp8b1 knockdown protects against nonalcoholic fatty liver disease in mice.

Bile acids (BAs) are surfactant molecules that regulate the intestinal absorption of lipids. Thus, the modulation of BAs represents a potential therapy for nonalcoholic fatty liver disease (NAFLD), which is characterized by hepatic accumulation of fat and is a major cause of liver disease worldwide. Cyp8b1 is a critical modulator of the hydrophobicity index of the BA pool. As a therapeutic proof of concept, we aimed to determine the impact of Cyp8b1 inhibition in vivo on BA pool composition and as protection against NAFLD. Inhibition of Cyp8b1 expression in mice led to a remodeling of the BA pool, which altered its signaling properties and decreased intestinal fat absorption. In a model of cholesterol-induced NAFLD, Cyp8b1 knockdown significantly decreased steatosis and hepatic lipid content, which has been associated with an increase in fecal lipid and BA excretion. Moreover, inhibition of Cyp8b1 not only decreased hepatic lipid accumulation, but also resulted in the clearance of previously accumulated hepatic cholesterol, which led to a regression in hepatic steatosis. Taken together, our data demonstrate that Cyp8b1 inhibition is a viable therapeutic target of crucial interest for metabolic diseases, such as NAFLD.-Chevre, R., Trigueros-Motos, L., Castaño, D., Chua, T., Corlianò, M., Patankar, J. V., Sng, L., Sim, L., Juin, T. L., Carissimo, G., Ng, L. F. P., Yi, C. N. J., Eliathamby, C. C., Groen, A. K., Hayden, M. R., Singaraja, R. R. Therapeutic modulation of the bile acid pool by Cyp8b1 knockdown protects against nonalcoholic fatty liver disease in mice.

Integrin ανß5 in vitro inhibition limits pro-fibrotic response in cardiac fibroblasts of spontaneously hypertensive rats.

BACKGROUND: To date the TGF-ß1 activation mediated by integrin ανß5 during fibrosis is well-known. This process has been shown also in the heart, where cardiac fibroblasts (CF) differentiate into α-smooth muscle actin (α-SMA)-positive myofibroblasts (MyoFB). Here, we studied the effects on CF, isolated by spontaneously hypertensive rats (SHR), of integrin ανß5 inhibition in MyoFB differentiation. METHODS: Staining and immunohistochemistry were performed on rat cardiac tissue. CF were isolated by enzymatic digestion from SHR (SHR-CF) and normotensive WKY (WKY-CF) rat hearts and then treated for in vitro evaluation. RESULTS: SHR heart tissues revealed a higher TGF-ß1 expression vs. WKY samples. SHR-CF showed an enhanced SMAD2/3 activation and an up-regulated expression of α-SMA, a typical MyoFB marker, especially after TGF-ß1 treatment. Immunostaining on cardiac tissues revealed a higher expression of integrin ανß5 in SHR vs. WKY rat hearts. In vitro results confirmed the up-regulation of integrin ανß5 expression in SHR-CF at basal condition and after TGF-ß1 treatment, in comparison with WKY-CF. Inhibition of integrin ανß5 by cilengitide treatment led a decreased expression of ανß5, collagen I, and α-SMA in SHR-CF vs. WKY-CF, resulting in a diminished differentiation of CF into MyoFB. Taking together, results suggested that SHR-CF are more susceptible to TGF-ß1, showing an up-regulated activation of SMAD2/3 signaling, and an increased ανß5, α-SMA, and collagen I expression. Hypertension stimulus promoted an up-regulation of integrin ανß5 on SHR cardiac tissue and its in vitro inhibition reverted pro-fibrotic events of SHR-CF. CONCLUSION: Inhibition of integrin ανß5 exerted by cilengitide strongly diminished SHR-CF differentiation into detrimental MyoFB. So, integrin ανß5 might be considered a novel therapeutic target and cilengitide an effective pharmacological tool to limit the progression of hypertension-induced cardiac fibrosis.

Haploinsufficiency of CYP8B1 associates with increased insulin sensitivity in humans.

BACKGROUNDCytochrome P450 family 8 subfamily B member 1 (CYP8B1) generates 12α-hydroxylated bile acids (BAs) that are associated with insulin resistance in humans.METHODSTo determine whether reduced CYP8B1 activity improves insulin sensitivity, we sequenced CYP8B1 in individuals without diabetes and identified carriers of complete loss-of-function (CLOF) mutations utilizing functional assays.RESULTSMutation carriers had lower plasma 12α-hydroxylated/non-12α-hydroxylated BA and cholic acid (CA)/chenodeoxycholic acid (CDCA) ratios compared with age-, sex-, and BMI-matched controls. During insulin clamps, hepatic glucose production was suppressed to a similar magnitude by insulin, but glucose infusion rates to maintain euglycemia were higher in mutation carriers, indicating increased peripheral insulin sensitivity. Consistently, a polymorphic CLOF CYP8B1 mutation associated with lower fasting insulin in the AMP-T2D-GENES study. Exposure of primary human muscle cells to mutation-carrier CA/CDCA ratios demonstrated increased FOXO1 activity, and upregulation of both insulin signaling and glucose uptake, which were mediated by increased CDCA. Inhibition of FOXO1 attenuated the CDCA-mediated increase in muscle insulin signaling and glucose uptake. We found that reduced CYP8B1 activity associates with increased insulin sensitivity in humans.CONCLUSIONOur findings suggest that increased circulatory CDCA due to reduced CYP8B1 activity increases skeletal muscle insulin sensitivity, contributing to increased whole-body insulin sensitization.FUNDINGBiomedical Research Council/National Medical Research Council of Singapore.

Bile Acids: A Communication Channel in the Gut-Brain Axis.

Bile acids are signalling hormones involved in the regulation of several metabolic pathways. The ability of bile acids to bind and signal through their receptors is modulated by the gut microbiome, since the microbiome contributes to the regulation and synthesis of bile acids as well to their physiochemical properties. From the gut, bacteria have been shown to send signals to the central nervous system via their metabolites, thus affecting the behaviour and brain function of the host organism. In the last years it has become increasingly evident that bile acids affect brain function, during normal physiological and pathological conditions. Although bile acids may be synthesized locally in the brain, the majority of brain bile acids are taken up from the systemic circulation. Since the composition of the brain bile acid pool may be regulated by the action of intestinal bacteria, it is possible that bile acids function as a communication bridge between the gut microbiome and the brain. However, little is known about the molecular mechanisms and the physiological roles of bile acids in the central nervous system. The possibility that bile acids may be a direct link between the intestinal microbiome and the brain is also an understudied subject. Here we review the influence of gut bacteria on the bile acid pool composition and properties, as well as striking evidence showing the role of bile acids as neuroactive molecules.

Lipid efflux mechanisms, relation to disease and potential therapeutic aspects.

Lipids are hydrophobic and amphiphilic molecules involved in diverse functions such as membrane structure, energy metabolism, immunity, and signaling. However, altered intra-cellular lipid levels or composition can lead to metabolic and inflammatory dysfunction, as well as lipotoxicity. Thus, intra-cellular lipid homeostasis is tightly regulated by multiple mechanisms. Since most peripheral cells do not catabolize cholesterol, efflux (extra-cellular transport) of cholesterol is vital for lipid homeostasis. Defective efflux contributes to atherosclerotic plaque development, impaired ß-cell insulin secretion, and neuropathology. Of these, defective lipid efflux in macrophages in the arterial walls leading to foam cell and atherosclerotic plaque formation has been the most well studied, likely because a leading global cause of death is cardiovascular disease. Circulating high density lipoprotein particles play critical roles as acceptors of effluxed cellular lipids, suggesting their importance in disease etiology. We review here mechanisms and pathways that modulate lipid efflux, the role of lipid efflux in disease etiology, and therapeutic options aimed at modulating this critical process.

Cyclophilin A/EMMPRIN Axis Is Involved in Pro-Fibrotic Processes Associated with Thoracic Aortic Aneurysm of Marfan Syndrome Patients.

: Background: Marfan syndrome (MFS) is a genetic disease, characterized by thoracic aortic aneurysm (TAA), which treatment is to date purely surgical. Understanding of novel molecular targets is mandatory to unveil effective pharmacological approaches. Cyclophilin A (CyPA) and its receptor EMMPRIN are associated with several cardiovascular diseases, including abdominal aortic aneurysm. Here, we envisioned the contribution of CyPA/EMMPRIN axis in MFS-related TAA. METHODS: We obtained thoracic aortic samples from healthy controls (HC) and MFS patients' aortas and then isolated vascular smooth muscle cells (VSMC) from the aortic wall. RESULTS: our findings revealed that MFS aortic tissue samples isolated from the dilated zone of aorta showed higher expression levels of EMMPRIN vs. MFS non-dilated aorta and HC. Interestingly, angiotensin II significantly stimulated CyPA secretion in MFS-derived VSMC (MFS-VSMC). CyPA treatment on MFS-VSMC led to increased levels of EMMPRIN and other MFS-associated pro-fibrotic mediators, such as TGF-ß1 and collagen I. These molecules were downregulated by in vitro treatment with CyPA inhibitor MM284. Our results suggest that CyPA/EMMPRIN axis is involved in MFS-related TAA development, since EMMPRIN is upregulated in the dilated zone of MFS patients' TAA and the inhibition of its ligand, CyPA, downregulated EMMPRIN and MFS-related markers in MFS-VSMC. CONCLUSIONS: these insights suggest both a novel detrimental role for CyPA/EMMPRIN axis and its inhibition as a potential therapeutic strategy for MFS-related TAA treatment.

Cyclophilin A modulates bone marrow-derived CD117(+) cells and enhances ischemia-induced angiogenesis via the SDF-1/CXCR4 axis.

BACKGROUND: Critical limb ischemia (CLI) is a major health problem with no adequate treatment. Since CLI is characterized by insufficient tissue vascularization, efforts have focused on the discovery of novel angiogenic factors. Cyclophilin A (CyPA) is an immunophilin that has been shown to promote angiogenesis in vitro and to enhance bone marrow (BM) cell mobilization in vivo. However, its potential as an angiogenic factor in CLI is still unknown. Thus, this study aimed to evaluate whether CyPA might induce neo-angiogenesis in ischemic tissues. METHODS AND RESULTS: Wild-type C57Bl/6j mice underwent acute hind-limb ischemia (HLI) and received a single intramuscular administration of recombinant CyPA or saline. Limb perfusion, capillary density and arteriole number in adductor muscles were significantly increased after CyPA treatment. Interestingly, BM-derived CD117(+) cell recruitment was significantly higher in ischemic adductor tissue of mice treated with CyPA versus saline. Therefore, the effect of CyPA on isolated BM-derived CD117(+) cells in vitro was evaluated. Low concentrations of CyPA stimulated CD117(+) cell proliferation while high concentrations promoted cell death. Moreover, CyPA enhanced CD117(+) cell adhesion and migration in a dose-dependent manner. Mechanistic studies revealed that CyPA up-regulated CXCR4 in CD117(+) cells and in adductor muscles after ischemia. Additionally, SDF-1/CXCR4 axis inhibition by the CXCR4 antagonist AMD3100 decreased CyPA-mediated CD117(+) cell recruitment in the ischemic limb. CONCLUSION: CyPA induces neo-angiogenesis by recruiting BM-derived CD117(+) cell into ischemic tissues, at least in part, through SDF-1/CXCR4 axis.