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Background We have shown previously that low‐density lipoprotein (LDL) can be oxidized in the lysosomes of macrophages, that this oxidation can be inhibited by cysteamine, an antioxidant that accumulates in lysosomes, and that this drug decreases atherosclerosis in LDL receptor–deficient mice fed a high‐fat diet. We have now performed a regression study with cysteamine, which is of more relevance to the treatment of human disease. Methods and Results LDL receptor–deficient mice were fed a high‐fat diet to induce atherosclerotic lesions. They were then reared on chow diet and drinking water containing cysteamine or plain drinking water. Aortic atherosclerosis was assessed, and samples of liver and skeletal muscle were analyzed. There was no regression of atherosclerosis in the control mice, but cysteamine caused regression of between 32% and 56% compared with the control group, depending on the site of the lesions. Cysteamine substantially increased markers of lesion stability, decreased ceroid, and greatly decreased oxidized phospholipids in the lesions. The liver lipid levels and expression of cluster of differentiation 68, acetyl–coenzyme A acetyltransferase 2, cytochromes P450 (CYP)27, and proinflammatory cytokines and chemokines were decreased by cysteamine. Skeletal muscle function and oxidative fibers were increased by cysteamine. There were no changes in the plasma total cholesterol, LDL cholesterol, high‐density lipoprotein cholesterol, or triacylglycerol concentrations attributable to cysteamine. Conclusions Inhibiting the lysosomal oxidation of LDL in atherosclerotic lesions by antioxidants targeted at lysosomes causes the regression of atherosclerosis and improves liver and muscle characteristics in mice and might be a promising novel therapy for atherosclerosis in patients.

Lifestyle- and genetically induced disorders related to disturbances in cholesterol metabolism have shown the detrimental impact of excessive cholesterol levels on a plethora of pathological processes such as inflammation. In this context, two-hydroxypropyl-β-cyclodextrin (CD) is increasingly considered as a novel pharmacological compound to decrease cellular cholesterol levels due to its ability to increase cholesterol solubility. However, recent findings have reported contra-indicating events after the use of CD questioning the clinical applicability of this compound. Given its potential as a therapeutic compound in metabolic inflammatory diseases, in this study, we evaluated the inflammatory effects of CD administration in the context of cholesterol-induced metabolic inflammation in vivo and in vitro. The inflammatory and cholesterol-depleting effects of CD were first investigated in low-density lipoprotein receptor knockout (Ldlr-/) mice that were transplanted with Npc1nih or Npc1wt bone marrow and were fed either regular chow or a high-fat, high-cholesterol (HFC) diet for 12 weeks, thereby creating an extreme model of lysosomal cholesterol-induced metabolic inflammation. In the final three weeks, these mice received daily injections of either control (saline) or CD subcutaneously. Subsequently, the inflammatory properties of CD were investigated in vitro in two macrophage cell lines and in murine bone marrow-derived macrophages (BMDMs). While CD administration improved cholesterol mobilization outside lysosomes in BMDMs, an overall pro-inflammatory profile was observed after CD treatment, evidenced by increased hepatic inflammation in vivo and a strong increase in cytokine release and inflammatory gene expression in vitro in murine BMDMs and macrophages cell lines. Nevertheless, this CD-induced pro-inflammatory profile was time-dependent, as short term exposure to CD did not result in a pro-inflammatory response in BMDM. While CD exerts desired cholesterol-depleting effects, its inflammatory effect is dependent on the exposure time. As such, using CD in the clinic, especially in a metabolic inflammatory context, should be closely monitored as it may lead to undesired, pro-inflammatory side effects.

Background & AimsThe lysosomal enzyme, cathepsin D (CTSD) has been implicated in the pathogenesis of non-alcoholic steatohepatitis (NASH), a disease characterised by hepatic steatosis and inflammation. We have previously demonstrated that specific inhibition of the extracellular CTSD leads to improved metabolic features in Sprague-Dawley rats with steatosis. However, the individual roles of extracellular and intracellular CTSD in NASH are not yet known. In the current study, we evaluated the underlying mechanisms of extracellular and intracellular CTSD fractions in NASH-related metabolic inflammation using specific small-molecule inhibitors.MethodsLow-density lipoprotein receptor knock out (Ldlr-/-) mice were fed a high-fat, high cholesterol (HFC) diet for ten weeks to induce NASH. Further, to investigate the effects of CTSD inhibition, mice were injected either with an intracellular (GA-12) or extracellular (CTD-002) CTSD inhibitor or vehicle control at doses of 50 mg/kg body weight subcutaneously once in two days for ten weeks.ResultsLdlr-/- mice treated with extracellular CTSD inhibitor showed reduced hepatic lipid accumulation and an associated increase in faecal bile acid levels as compared to intracellular CTSD inhibitor-treated mice. Furthermore, in contrast to intracellular CTSD inhibition, extracellular CTSD inhibition switched the systemic immune status of the mice to an anti-inflammatory profile. In line, label-free mass spectrometry-based proteomics revealed that extra- and intracellular CTSD fractions modulate proteins belonging to distinct metabolic pathways.ConclusionWe have provided clinically translatable evidence that extracellular CTSD inhibition shows some beneficial metabolic and systemic inflammatory effects which are distinct from intracellular CTSD inhibition. Considering that intracellular CTSD inhibition is involved in essential physiological processes, specific inhibitors capable of blocking extracellular CTSD activity, can be promising and safe NASH drugs.

Cathepsins are the most abundant lysosomal proteases that are mainly found in acidicendo/lysosomal compartments where they play a vital role in intracellular protein degradation,energy metabolism, and immune responses among a host of other functions. The discovery thatcathepsins are secreted and remain functionally active outside of the lysosome has caused a paradigmshift. Contemporary research has unraveled many versatile functions of cathepsins in extralysosomallocations including cytosol and extracellular space. Nevertheless, extracellular cathepsins are majorlyupregulated in pathological states and are implicated in a wide range of diseases including cancerand cardiovascular diseases. Taking advantage of the dierential expression of the cathepsinsduring pathological conditions, much research is focused on using cathepsins as diagnostic markersand therapeutic targets. A tailored therapeutic approach using selective cathepsin inhibitors isconstantly emerging to be safe and ecient. Moreover, recent development of proteomic-basedapproaches for the identification of novel physiological substrates oers a major opportunity tounderstand the mechanism of cathepsin action. In this review, we summarize the available evidenceregarding the role of cathepsins in health and disease, discuss their potential as biomarkers ofdisease progression, and shed light on the potential of extracellular cathepsin inhibitors as safetherapeutic tools.

Inflammatory bowel disease (IBD) is a chronic and relapsing intestinal inflammatory condition, hallmarked by a disturbance in the bidirectional interaction between gut and brain. In general, the gut/brain axis involves direct and/or indirect communication via the central and enteric nervous system, host innate immune system, and particularly the gut microbiota. This complex interaction implies that IBD is a complex multifactorial disease. There is increasing evidence that stress adversely affects the gut/microbiota/brain axis by altering intestinal mucosa permeability and cytokine secretion, thereby influencing the relapse risk and disease severity of IBD. Given the recurrent nature, therapeutic strategies particularly aim at achieving and maintaining remission of the disease. Alternatively, these strategies focus on preventing permanent bowel damage and concomitant long-term complications. In this review, we discuss the gut/microbiota/brain interplay with respect to chronic inflammation of the gastrointestinal tract and particularly shed light on the role of stress. Hence, we evaluated the therapeutic impact of stress management in IBD.

Dietary and lifestyle changes are leading to an increased occurrence of non-alcoholic fatty liver disease (NAFLD). Using a hyperlipidemic murine model for non-alcoholic steatohepatitis (NASH), we have previously demonstrated that the lysosomal protease cathepsin D (CTSD) is involved with lipid dysregulation and inflammation. However, despite identifying CTSD as a major player in NAFLD pathogenesis, the specific role of extracellular CTSD in NAFLD has not yet been investigated. Given that inhibition of intracellular CTSD is highly unfavorable due to its fundamental physiological function, we here investigated the impact of a highly specific and potent small-molecule inhibitor of extracellular CTSD (CTD-002) in the context of NAFLD. Treatment of bone marrow-derived macrophages with CTD-002, and incubation of hepatic HepG2 cells with a conditioned medium derived from CTD-002-treated macrophages, resulted in reduced levels of inflammation and improved cholesterol metabolism. Treatment with CTD-002 improved hepatic steatosis in high fat diet-fed rats. Additionally, plasma levels of insulin and hepatic transaminases were significantly reduced upon CTD-002 administration. Collectively, our findings demonstrate for the first time that modulation of extracellular CTSD can serve as a novel therapeutic modality for NAFLD.