Abstract
Background
Extracellular vesicles are vital mediators of cell-to-cell communications. We previously demonstrated that in the adult heart, exosomes released in the pericardial fluid (PF) have the functional capability to induce cell survival and angiogenesis in recipient endothelial cells. It has previously been shown that PF exosomes from patients with an existing myocardial infarction (MI) contain clusterin (CLU). Remarkably, secreted CLU is cardio-protective as evidenced by enhanced arteriogenesis and reduced apoptosis in animal models of MI. We hypothesize that type 2 Diabetes Mellitus (T2DM) along with ischemic heart disease (IHD) alters PF EV content impairing its reparative angiogenic potential.
Aim
To investigate the role of T2DM in modulating human PF exosomal content and its vascular action.
Methods and results
PF samples were collected from 3 different groups of patients (N=3): 1) Ischemic heart disease (IHD) with or 2) without T2DM, and 3) non-ischaemic, non-diabetic controls operated for mitral valve repair.
The impact of PF exosomes on human coronary microvascular ECs (HCMECs) was evaluated by cell based functional assays for measuring apoptosis, proliferation and angiogenesis. Interestingly, unlike PF exosomes from control patients, PF exosomes from DM patients with IHD promoted EC apoptosis and impaired angiogenesis.
To investigate the effect of T2DM on PF content, we performed high throughput proteomic and metabolomic analysis of whole PF and PF EVs. We identified proteins and metabolites that were differentially expressed under T2DM condition by using the R package Limma. Employing a network approach, protein and metabolic data were integrated by using our newly developed inhouse R package Metabosignal. This networks approach revealed an interesting interaction circuit involving protein CLU. The T2DM-associated reduction of CLU level in the PF, identified from the “omics” analyses was confirmed by ELISA (n=8 patients per group).
To examine the effects of DM and ischemia on CLU intracellular expression and secretion, HCMECs were exposed to high D-glucose (HG, 25 mM) and hypoxia (1%O2). In line with our PF data, HCMECs exposed to HG and hypoxia showed lower levels of both CLU mRNA and secreted CLU (ELISA). Importantly, recombinant CLU (rCLU) treatment on HCMECs rescued their angiogenic potential, induced cell proliferation and protected them from apoptosis under both HG and hypoxia conditions. In addition, silencing of endogenous levels of CLU in HCMECs impaired angiogenesis, suggesting that CLU might be an inherent component of the angiogenesis machinery in ECs.
Conclusions
This study suggests that DM reduces the level of secreted CLU in the PF, depriving myocardial micro-vessels of this protective and regenerative mediator. Restoring diminished CLU levels in the diabetic heart could be a possible therapeutic approach for contrasting diabetic micro-angiopathy.
Acknowledgement/Funding
British Heart Foundation Program Grant
Endothelin-1 Concentrations in Pericardial Fluid Are More Elevated in Patients with Ischemic Heart Disease Than in Patients With Nonischemic Heart Disease
