The Proteome of Circulating Large Extracellular Vesicles in Diabetes and Hypertension.

Hypertension and diabetes induce vascular injury through processes that are not fully understood. Changes in extracellular vesicle (EV) composition could provide novel insights. Here, we examined the protein composition of circulating EVs from hypertensive, diabetic and healthy mice. EVs were isolated from transgenic mice overexpressing human renin in the liver (TtRhRen, hypertensive), OVE26 type 1 diabetic mice and wild-type (WT) mice. Protein content was analyzed using liquid chromatography-mass spectrometry. We identified 544 independent proteins, of which 408 were found in all groups, 34 were exclusive to WT, 16 were exclusive to OVE26 and 5 were exclusive to TTRhRen mice. Amongst the differentially expressed proteins, haptoglobin (HPT) was upregulated and ankyrin-1 (ANK1) was downregulated in OVE26 and TtRhRen mice compared with WT controls. Conversely, TSP4 and Co3A1 were upregulated and SAA4 was downregulated exclusively in diabetic mice; and PPN was upregulated and SPTB1 and SPTA1 were downregulated in hypertensive mice, compared to WT mice. Ingenuity pathway analysis identified enrichment in proteins associated with SNARE signaling, the complement system and NAD homeostasis in EVs from diabetic mice. Conversely, in EVs from hypertensive mice, there was enrichment in semaphroin and Rho signaling. Further analysis of these changes may improve understanding of vascular injury in hypertension and diabetes.

High glucose increases the formation and pro-oxidative activity of endothelial microparticles.

AIMS/HYPOTHESIS: Individuals with diabetes exhibit increases in circulating endothelial microparticles (eMPs, also referred to as endothelial microvesicles), which are associated with endothelial dysfunction and a heightened risk of cardiovascular complications. We have shown that eMPs are markers and mediators of vascular injury although their role in diabetes is unclear. We hypothesised that the composition and biological activity of eMPs are altered in response to high glucose exposure. We assessed the effects of high glucose on eMP formation, composition and signalling in cultured HUVECs. METHODS: eMPs were isolated from the media of HUVECs cultured under conditions of normal glucose (eMPNG), high glucose (eMPHG) or osmotic control of L-glucose (eMPLG). eMP size, concentration and surface charge were assessed by nanoparticle tracking analysis and flow cytometry. eMP protein composition was assessed by liquid chromatography-tandem mass spectrometry, and eMP-mediated effects on coagulation, reactive oxygen species (ROS) production and vessel function were assessed. RESULTS: Exposure of HUVECs to high glucose for 24 h caused a threefold increase in eMP formation, increased mean particle size (269 ± 18 nm vs 226 ± 11 nm) and decreased surface charge. Compared with eMPNG or eMPLG, eMPHG possessed approximately threefold greater pro-coagulant activity, stimulated HUVEC ROS production to a greater extent (~250% of eMPNG) and were more potent inhibitors of endothelial-dependent relaxation. Proteomic analysis of eMPs identified 1212 independent proteins of which 68 were exclusively found in eMPHG. Gene ontology analysis revealed that eMPHG-exclusive proteins were associated with signalling pathways related to blood coagulation, cell signalling and immune cell activation. CONCLUSIONS/INTERPRETATION: Our results indicate that elevated glucose is a potent stimulus for eMP formation that also alters their molecular composition leading to increased bioactivity. Such effects may contribute to progressive endothelial injury and subsequent cardiovascular complications in diabetes.

Podocyte-derived microparticles promote proximal tubule fibrotic signaling via p38 MAPK and CD36.

Tubulointerstitial fibrosis is a hallmark of advanced diabetic kidney disease that is linked to a decline in renal function, however the pathogenic mechanisms are poorly understood. Microparticles (MPs) are 100-1000 nm vesicles shed from injured cells that are implicated in intercellular signalling. Our lab recently observed the formation of MPs from podocytes and their release into urine of animal models of type 1 and 2 diabetes and in humans with type 1 diabetes. The purpose of the present study was to examine the role of podocyte MPs in tubular epithelial cell fibrotic responses. MPs were isolated from the media of differentiated, untreated human podocytes (hPODs) and administered to cultured human proximal tubule epithelial cells (PTECs). Treatment with podocyte MPs increased p38 and Smad3 phosphorylation and expression of the extracellular matrix (ECM) proteins fibronectin and collagen type IV. MP-induced responses were attenuated by co-treatment with the p38 inhibitor SB202190. A transforming growth factor beta (TGF-ß) receptor inhibitor (LY2109761) blocked MP-induced Smad3 phosphorylation and ECM protein expression but not p38 phosphorylation suggesting that these responses occurred downstream of p38. Finally, blockade of the class B scavenger receptor CD36 completely abrogated MP-mediated p38 phosphorylation, downstream Smad3 activation and fibronectin/collagen type IV induction. Taken together our results suggest that podocyte MPs interact with proximal tubule cells and induce pro-fibrotic responses. Such interactions may contribute to the development of tubular fibrosis in glomerular disease.

Survival Motor Neuron Protein is Released from Cells in Exosomes: A Potential Biomarker for Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is caused by homozygous mutation of the survival motor neuron 1 (SMN1) gene. Disease severity inversely correlates to the amount of SMN protein produced from the homologous SMN2 gene. We show that SMN protein is naturally released in exosomes from all cell types examined. Fibroblasts from patients or a mouse model of SMA released exosomes containing reduced levels of SMN protein relative to normal controls. Cells overexpressing SMN protein released exosomes with dramatically elevated levels of SMN protein. We observed enhanced quantities of exosomes in the medium from SMN-depleted cells, and in serum from a mouse model of SMA and a patient with Type 3 SMA, suggesting that SMN-depletion causes a deregulation of exosome release or uptake. The quantity of SMN protein contained in the serum-derived exosomes correlated with the genotype of the animal, with progressively less protein in carrier and affected animals compared to wildtype mice. SMN protein was easily detectable in exosomes isolated from human serum, with a reduction in the amount of SMN protein in exosomes from a patient with Type 3 SMA compared to a normal control. Our results suggest that exosome-derived SMN protein may serve as an effective biomarker for SMA.

Endothelial Microparticle-Derived Reactive Oxygen Species: Role in Endothelial Signaling and Vascular Function.

Endothelial microparticles are effectors of endothelial damage; however mechanisms involved are unclear. We examined the effects of eMPs on cultured endothelial cells (ECs) and isolated vessels and investigated the role of eMP-derived reactive oxygen species (ROS) and redox signaling in these processes. eMPs were isolated from EC media and their ability to directly produce ROS was assessed by lucigenin and liquid chromatography. Nicotinamide adenine dinucleotide phosphate oxidase (Nox) subunits were probed by Western blot. ECs were treated with eMPs and effects on kinase signaling, superoxide anion (O2 (∙-)) generation, and nitric oxide (NO) production were examined. Acetylcholine-mediated vasorelaxation was assessed by myography in eMP-treated mesenteric arteries. eMPs contained Nox1, Nox2, Nox4, p47(phox), p67(phox), and p22(phox) and they produced ROS which was inhibited by the Nox inhibitor, apocynin. eMPs increased phosphorylation of ERK1/2 and Src, increased O2 (∙-) production, and decreased A23187-induced NO production in ECs. Pretreatment of eMPs with apocynin diminished eMP-mediated effects on ROS and NO production but had no effect on eMP-mediated kinase activation or impairment in vasorelaxation. Our findings identify a novel mechanism whereby eMP-derived ROS contributes to MP bioactivity. These interactions may be important in conditions associated with vascular injury and increased eMP formation.