Atherosclerosis remains the leading cause of death in Western countries. Dipeptidyl peptidase (DPP) 4 has emerged as a novel target for the prevention and treatment of atherosclerosis. Family members DPP8 and 9 are abundantly present in macrophage-rich regions of atherosclerotic plaques, and DPP9 inhibition attenuates activation of human M1 macrophages in vitro. Studying this family in a mouse model for atherosclerosis would greatly advance our knowledge regarding their potential as therapeutic targets. We found that DPP4 is downregulated during mouse monocyte-to-macrophage differentiation. DPP8 and 9 expression seems relatively low in mouse monocytes and macrophages. Viability of primary mouse macrophages is unaffected by DPP4 or DPP8/9 inhibition. Importantly, DPP8/9 inhibition attenuates macrophage activation as IL-6 secretion is significantly decreased. Mouse macrophages respond similarly to DPP inhibition, compared to human macrophages. This shows that the mouse could become a valid model species for the study of DPPs as therapeutic targets in atherosclerosis.
Title: The Dipeptidyl Peptidase Family, Prolyl Oligopeptidase, and Prolyl Carboxypeptidase in the Immune System and Inflammatory Disease, Including Atherosclerosis Waumans Y, Baerts L, Kehoe K, Lambeir AM, De Meester I Ref: Front Immunol, 6:387, 2015 : PubMed
Research from over the past 20 years has implicated dipeptidyl peptidase (DPP) IV and its family members in many processes and different pathologies of the immune system. Most research has been focused on either DPPIV or just a few of its family members. It is, however, essential to consider the entire DPP family when discussing any one of its members. There is a substantial overlap between family members in their substrate specificity, inhibitors, and functions. In this review, we provide a comprehensive discussion on the role of prolyl-specific peptidases DPPIV, FAP, DPP8, DPP9, dipeptidyl peptidase II, prolyl carboxypeptidase, and prolyl oligopeptidase in the immune system and its diseases. We highlight possible therapeutic targets for the prevention and treatment of atherosclerosis, a condition that lies at the frontier between inflammation and cardiovascular disease.
Prolylcarboxypeptidase (PRCP, EC 3.4.16.2), a lysosomal carboxypeptidase, was discovered 45 years ago. However, research has been hampered by a lack of well-validated assays that are needed to measure low activities in biological samples. Two reversed-phase high-performance liquid chromatography (RP-HPLC) methods for quantifying PRCP activity in crude homogenates and plasma samples were optimized and validated. PRCP activity was determined by measuring the hydrolysis of N-benzyloxycarbonyl-l-proline (Z-Pro)-Phe. The enzymatically formed Z-Pro and Phe were measured independently under different HPLC conditions. The in-house methods showed good precision, linearity, accuracy, and specificity. Based on Michaelis-Menten constants, Z-Pro-Phe was chosen over Z-Pro-Ala as the substrate of preference. Cross-reactivity studies with dipeptidyl peptidases (DPPs) 2, 4, and 9 and prolyl oligopeptidase (PREP) confirmed the specificity of the PRCP activity assay. The average PRCP activity in plasma and serum of 32 healthy individuals was found to be 0.65 +/- 0.02 and 0.72 +/- 0.03 U/L, respectively. Both methods can be used to measure PRCP activity specifically in different biological samples and are well suited to evaluate PRCP inhibitors. These well-validated methods are valuable tools for studying PRCP's role in cardiovascular diseases, stroke, inflammation, and metabolic syndrome.
Atherosclerosis is a chronic inflammatory disorder of the arterial wall leading to coronary artery disease, stroke, and peripheral arterial disease. Along with the discovery of dipeptidyl peptidase 4 (DPP4) as a therapeutic target in type 2 diabetes, a role for DPP4 in atherosclerosis is emerging. However, until now the expression and role of other DPPs such as DPP8 and DPP9 in atherosclerosis is completely unknown. In the present study, we first investigated DPP expression in human atherosclerotic plaques. DPP4 could only be observed in endothelial cells of plaque neovessels in half of the specimens. In contrast, DPP8 and DPP9 were abundantly present in macrophage-rich regions of plaques. We then focused on DPP expression and function in macrophage differentiation, activation and apoptosis. DPP8/9 was responsible for most of the DPP activity in macrophages. During monocyte to macrophage differentiation, DPP9 was upregulated both in pro-inflammatory M1 (3.7 +/- 0.3-fold increase) and anti-inflammatory M2 macrophages (3.7 +/- 0.4-fold increase) whereas DPP8 expression remained unchanged. Inhibition of DPP8/9 activity with compound 1G244 reduced activation of M1 macrophages (IL-6 88 +/- 16 vs. 146 +/- 19 pg/ml; TNFalpha 3.8 +/- 1.0 vs. 6.6 +/- 1.9 ng/ml in treated vs. untreated cells), but not of M2 macrophages. Likewise, DPP9 silencing reduced TNFalpha and IL-6 secretion, pointing to a DPP9-mediated effect of the inhibitor. DPP8/9 inhibition also enhanced macrophage apoptosis (15 +/- 4 vs. 7 +/- 3 % in untreated cells). Because pro-inflammatory macrophages play a key role in atherogenesis, plaque rupture and subsequent infarction, DPP9 inhibition might provide interesting therapeutic prospects in reducing atherosclerosis and/or in the prevention of plaque rupture.
