Functional Evaluation of the PTPase DEP-1 as a Novel Regulator of Monocytes and Macrophages in Diabetes and Inflammation

Abstract

Diabetes mellitus (DM) is one of the most emerging diseases regarding prevalence, mortality and health care expenses. The International Diabetes Federation (IDF) estimates the world-age adjusted prevalence of diabetes in Europe in adults between 20 and 79 years 6.3 % with an 16 % increase to 7.3 % until 2030. The IDF suggests a proportion of 32.9 % of all deaths in Europe before the age of 60 years to be due to diabetes (99). This data is concordant with 2020 data from the Centers for Disease Control and Prevention (28). A recent prospective trial performed on five continents could show that just a high-glycemic-index diet is associated with an increased rate of major cardiovascular events, independent of pre-existing cardiovascular disease or diabetes (103).

Along the autoimmune type 1 diabetes mellitus (T1DM), gestational diabetes and other specific secondary subtypes, such as genetic forms, endocrinopathies, DM following total pancreatectomy or pharmaceutically induced diabetes, type 2 diabetes mellitus (T2DM) is the leading subtype with a prevalence of 87 to 91% (99). This metabolic disease evolves roughly from long term elevated levels of blood glucose leading to increased stimulation of β-cells in the pancreatic islands to produce insulin. This phenomenon is typical for obesity (15) and leads by time to peripheral insulin resistance, requiring absolutely more insulin to keep the blood glucose levels in a normal range (109). At some point, the βcells' capability to produce sufficient insulin decreases and β-cells become dysfunctional. This is when the blood glucose levels begin to rise and impaired glucose tolerance develops to a manifest T2DM (264). Today, the disease’s pathophysiology is much more elucidated, including knowledge about genetic predisposition, regulatory mechanisms in the nervous system, remodeling of adipose tissue and concepts of systemic and local inflammation, for example within the β-cell islands, which all contribute to the feedback loops between insulin resistance, β-cell dysfunction and rising blood glucose levels (108).

The burden of patients suffering from T2DM, in contrast to T1DM, is usually not the diabetes itself, because typical acute complications like non-controlled blood glucose with hypo- and hyperglycemic episodes and symptoms such as nycturia and exsiccosis, unwanted weight loss, urinary tract infections or lassitude are much rarer in this subtype. However, T2DM disease burden, mortality, decreased quality of life, increased hospital admissions and associated health care costs, etc. are caused by the development of chronic complications from the disease (247), classified as chronic macrovascular (e. g. coronary artery disease, peripheral artery disease) and microvascular (e. g. diabetic nephropathy, diabetic retinopathy) complications (35, 250).

Atherosclerosis is the main pathology causing the chronic vascular complications of DM which are ischemic stroke and coronary as well as peripheral artery disease (178). Atherosclerosis is the accumulation of aggregated lipoproteins together with fibrous material in the subintimal layer, as well as proliferation of vascular smooth muscle cells and calcification within the walls of the macro- and microvasculature which leads to a chronic obstruction of the vessel lumen and ischemia of the tissue served. Tissue ischemia has functional implications (e. g. neurological deficits, decreased cardiac function and output, etc.) and causes clinical symptoms such as aphasia, paresis or angina pectoris. Most known from coronary artery disease, an atherosclerotic plaque can ultimately rupture (18) which exposes the thrombotic core to the bloodstream, leading to acute thrombosis of the respective vessel (47). In the heart, this pathology called type 1 myocardial infarction causes severe ischemia up to necrosis of the served tissue and acute and chronic lifethreatening conditions.

Alternate abstract:

Einleitung Diabetes mellitus (DM) ist eine Erkrankung mit hoher Morbidität und Mortalität. Die Inzidenz ist steigend. Monozyten und Makrophagen spielen eine aktive Rolle in der Pathogenese der Atherosklerose, der relevantesten Komplikation von DM. Densityenhanced phosphatase 1 (DEP-1), für die bereits relevante Funktionen in der Regulation von Tumor- und Endothelzellen gezeigt wurden, ist insbesondere in Makrophagen vergleichsweise stark exprimiert, ohne dass die funktionelle Relevanz in diesem Zelltyp hinreichend untersucht wurde.

Methoden DEP-1 mRNA-Expression wurde via rt-qPCR in isolierten, primären CD14⁺⁺CD16^- Monozyten hospitalisierter Patienten gemessen und zwischen einer an DM erkrankten und einer nicht an DM erkrankten Gruppe verglichen. Der Einfluss inflammatorischer und metabolischer Stimuli auf die Expression wurde in-vitro untersucht. Ein in-vitro DM-Modell humaner Makrophagen mit DEP-1 Knock-down (KD) via RNA-Interferenz wurde etabliert um mittels MTT-Tests, Migrationsversuchen und Expressionstests die funktionelle Relevanz von DEP-1 in DM-assoziierter Atherosklerose in Monozyten und Makrophagen zu untersuchen.

Ergebnisse DEP-1 mRNA war in der T2DM-Gruppe heraufreguliert (40%, p<0.05). Im Vergleich zu nicht stimulierten Monozyten war DEP-1 in TNF-α (260%, p<0.001) und unter hyperglykämischen Bedingungen kultivierten Monozyten heraufreguliert (466%, ns). Die DEP-1-Aktivität zeigte sich im Vergleich zwischen nicht-, M1- und M2-aktivierten Makrophagen in M1-Makrophagen am höchsten. DEP-1 KD führte zu reduzierter Makrophagen-Migration im Wundheilungsversuch. p65-Expression und -Phosphorylierung, sowie TNF-α-Expression zeigten sich unter DEP-1 KD erhöht, die Zellvitalität, gemessen im MTT-Test, war unbeeinflusst.

Diskussion Diese Arbeit untersucht und diskutiert die funktionelle Relevanz von DEP-1 in CD14⁺⁺CD16^- Monozyten, u. a. in Monozyten an DM erkrankter Patienten und in einem in-vitro Inflammations- und Hyperglykämie-Modell, sowie in in-vitro differenzierten Makrophagen. Es wird gezeigt und diskutiert, dass inflammatorische Signalwege, die NF-κB involvieren, von DEP-1 reguliert werden und umgekehrt auch DEP-1 regulieren. Mögliche Signalwege, die die Makrophagen-Migration sowie den NF-κB-Signalweg DEP-1-abhängig regulieren könnten, werden diskutiert.