T he formation and progression of atherosclerotic plaques is characterized by accumulation of oxidized lipids and inflammatory cells covered by a fibrous cap of smooth muscle cells and collagen-rich matrix.1 Most severe clinical events result from plaque rupture, which exposes prothrombotic material in the plaque to the blood and causes intraluminal thrombus formation. Plaques vulnerable to rupture are characterized by a thin fibrous cap, a large lipid/necrotic core, positive vascular remodeling, and substantial infiltration of inflammatory cells.2 In particular, macrophages have emerged as a key cellular element of atherosclerotic plaque pathogenesis and pose a significant risk for causing plaque rupture.3 Recently, studies have observed heterogeneous subsets of macrophages, such as M1 and M2, in human atherosclerotic lesions. 4 Progressive necrosis of foam cells causes secretion of several factors that sustain the inflammatory processes and produce tissue-degrading proteases, such as matrix metalloproteinases and elastases.5 Plaque destabilization is attributed mostly to extracellular matrix (ECM) degradation (collagenolysis elastinolysis) by multiple ECM degrading enzymes, such as matrix metalloproteinases, serine hydrolyses, and cysteine proteases. Cysteine proteases, including cathepsins B, L, and S, display profound ECM catabolic activity in vitro and were found to be overexpressed in activated macrophages and advanced atherosclerotic lesions. 6,7 Cathepsin B, L, and S play key roles in macrophage function, with their mRNA expression and activity increasing significantly in the M2 phenotype.8 Additionally, M1 macrophages show elevated cathepsin S and L mRNA levels. Moreover, both mRNA and protein levels of cathepsin B, L, and S are overexpressed in murine atherosclerotic plaques. Similarly, in advanced human atherosclerotic plaques, cathepsin B and S were found overexpressed within macrophages, Background and Purpose-Atherosclerosis is a leading cause of mortality worldwide, contributing to both strokes and heart attacks. Macrophages are key players in atherogenesis, promoting vascular inflammation and arterial remodeling through cysteine cathepsin proteases. We used a cathepsin-targeted activity-based probe in human carotid plaque to assess its diagnostic potential and evaluate macrophage subtypes ex vivo. Methods-Carotid plaque specimens surgically removed during endarterectomy from 62 patients (age range, 38% female, 28% symptomatic) were graded pathologically as either stable (Grade 1) or unstable (Grade 2 or 3). A cathepsin activitybased probe was used to quantify individual cathepsins in plaque tissue and macrophage subtypes. Results-Cathepsin B and S activities were increased in unstable carotid plaques. They were quantified using the probe to biochemically investigate individual cathepsins (Cathepsin B and S: 0.97

Stroma-infiltrating immune cells, such as tumor-associated macrophages (TAM), play an important role in regulating tumor progression and chemoresistance. These effects are mostly conveyed by secreted mediators, among them several cathepsin proteases. In addition, increasing evidence suggests that stroma-infiltrating immune cells are able to induce profound metabolic changes within the tumor microenvironment. In this study, we aimed to characterize the impact of cathepsins in maintaining the TAM phenotype in more detail. For this purpose, we investigated the molecular effects of pharmacological cathepsin inhibition on the viability and polarization of human primary macrophages as well as its metabolic consequences. Pharmacological inhibition of cathepsins B, L, and S using a novel inhibitor, GB111-NH2, led to changes in cellular recycling processes characterized by an increased expression of autophagy- and lysosome-associated marker genes and reduced adenosine triphosphate (ATP) content. Decreased cathepsin activity in primary macrophages further led to distinct changes in fatty acid metabolites associated with increased expression of key modulators of fatty acid metabolism, such as fatty acid synthase (FASN) and acid ceramidase (ASAH1). The altered fatty acid profile was associated with an increased synthesis of the pro-inflammatory prostaglandin PGE2, which correlated with the upregulation of numerous NFkB-dependent pro-inflammatory mediators, including interleukin-1 (IL-1), interleukin-6 (IL-6), C-C motif chemokine ligand 2 (CCL2), and tumor necrosis factor-alpha (TNFα). Our data indicate a novel link between cathepsin activity and metabolic reprogramming in macrophages, demonstrated by a profound impact on autophagy and fatty acid metabolism, which facilitates a pro-inflammatory micromilieu generally associated with enhanced tumor elimination. These results provide a strong rationale for therapeutic cathepsin inhibition to overcome the tumor-promoting effects of the immune-evasive tumor micromilieu.

IntroductionMesenchymal stem cells (MSCs) are multipotent and have been derived from various tissues. Although MSCs share many basic features, they often display subtle tissue specific differences. We previously demonstrated that bone marrow (BM) MSCs frequently become polyploid in culture. This tendency was mediated by a reduction in the expression of H19 long non-coding RNA during the transition from a diploid to a polyploid state.MethodsMSCs were derived from both BM and adipose tissue of mice and expanded under normoxic and hypoxic culture conditions. Cells were stained by propidium iodide and their ploidy was evaluated by FACS. Gene expression of independent MSC preparations was compared by quantitative real time PCR and protein expression levels by Western blot analysis. p53 silencing in MSCs was performed by a specific small hairpin RNA (shRNA).ResultsWe set to examine whether genomic instability is common to MSCs originating from different tissues. It is demonstrated that adipose derived MSCs (ASCs) tend to remain diploid during culture while a vast majority of BM MSCs become polyploid. The diploid phenotype of ASCs is correlated with reduced H19 expression compared to BM MSCs. Under hypoxic conditions (3% oxygen) both ASCs and BM MSCs demonstrate increased RNA expression of H19 and Vascular endothelial growth factor A. Importantly, ASC gene expression is significantly less variable than BM MSCs under both oxygen conditions, indicating to their superior homogeneity. Gene expression analysis revealed that p53 target genes, often induced by DNA damage, are up-regulated in ASCs under basal conditions. However, p53 activation following treatment with DNA damaging agents was strongly elevated in BM MSCs compared to ASCs. We found that p53 is involved in maintaining the stable diploid state of ASCs as p53 shRNA induced ploidy changes in ASCs but not in BM MSCs.ConclusionsThe increased genomic stability of murine ASCs together with their lower H19 expression and relative homogeneity suggest a tissue specific higher stability of ASCs compared to BM MSCs, possibly due to higher activity of p53. The tissue specific differences between MSCs from a different tissue source may have important consequences on the use of various MSCs both in vitro and in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/scrt529) contains supplementary material, which is available to authorized users.