INTRODUCTION
Type 2 diabetes mellitus (T2DM) is a global public health problem characterized by chronic hyperglycemia contributing to atherosclerosis development and other vasculopathies.[1] Impaired fasting glucose (IFG) is a risk factor for T2DM development.[1,2] Moreover, IFG is associated with micro- and macrovascular pathologies.[2,3] Angiogenesis is essential for forming collateral circulation that may counteract vascular flow obstruction consequences accompanying diabetic complications.[4] For this reason, diverse researchers are aimed to discover the mechanisms that regulate new blood vessel formation in chronic hyperglycemia states.
Extracellular vesicles (EVs) are spherical or cup-shaped microfragments of cell membranes, with diameters from 0.01 to 1 μm. EVs are released by most cell types, such as endothelial cells, leukocytes, erythrocytes, platelets, adipocytes, and stem cells.[5] EVs include exosomes, microvesicles (MVs), and apoptotic bodies released by a calcium-dependent cellular mechanism involving the cytoskeleton; they contain diverse biomolecules such as lipids, structural and functional proteins, and nucleic acids that mediate inflammation, coagulation, vascular functions, apoptosis, cell proliferation, and differentiation.[5] EV number in peripheral blood of T2DM subjects is significantly higher than in healthy subjects.[6] The content of EVs isolated from patients with T2DM is mainly composed of miRNAs and proteins related to inflammation, thrombosis, endothelial activation or dysfunction, and angiogenesis.[7,8] The pathophysiological role that EVs released in different dysglycemia conditions may have been not fully understood;[7] however, some evidence suggests that miRNAs in EVs may signal progression from IFG or prediabetes to T2DM.[9]
Since cell migration and endothelial cell tube formation are critical steps in angiogenesis, research has focused on the capacity of EVs to promote these effects. Circulating EVs from patients with diabetic retinopathy and diabetic foot ulcers stimulate in vitro vascular tube formation, although tube networks are unstable and subsequently collapse.[8] Other studies demonstrated that EVs from fibrocytes and stem cells stimulate angiogenesis and endothelial and mesenchymal stem cell migration. Moreover, EVs from T2DM patients induce greater endothelial cell migration than EVs from normoglycemic controls.[10] Furthermore, in vivo treatment with fibrocyte-derived exosomes accelerates wound closure in diabetic mice.[11]
The effects of plasma-derived EVs from patients with IFG on vascular endothelium remain undescribed. Here, we studied the in vitro effect of EVs isolated from patients with IFG on angiogenic score and migration on a human vascular endothelial cell line.
METHODS
Materials
The insulin enzyme-linked immunosorbent assay kit (Cat. no IS130D) was from Calbiotech Inc. (Spring Valley, CA, USA). Mouse monoclonal anti-MHC Class I antibody (Ab) BRA23/9 (Cat. no sc-66205) and mouse anti-flotillin 2 Ab B-6 (Cat. no sc-28320) were from Santa Cruz Biotechnology, Inc. AF647-conjugated mouse anti-human endoglin (CD105) clone 266 (Cat. no sc-561439) and AF647-conjugated mouse anti-human vascular endothelial growth factor receptor (VEGFR-2) (CD309) clone 89,106 (Cat. no sc-560495) were from BD Pharmingen. Recombinant human vascular endothelial growth factor (VEGF) was from Sigma-Aldrich (St. Louis, MO, USA). Matrigel matrix basement membrane high concentrate growth factor reduced was from Corning (Bedford, MA, USA). All the other used reagents had the highest purity grade.
Patients
One hundred seventy-six subjects who underwent medical and biochemical assessment at the Laboratory of Clinical Biochemistry of the Department of Medicine and Health Sciences of the University of Sonora in Mexico were categorized according to the American Diabetes Association criteria.[1] Peripheral blood samples and anthropometric and clinical data were collected. Five patients with T2DM, five with IFG, and five subjects with normoglycemia were included. The study was executed following the ethical principles mentioned in the Declaration of Helsinki, with the approval of the Bioethics and Research Committee of the Department of Medicine and Health Sciences of the University of Sonora (DMCS/CBIDMCS/D-68). Written informed consent from each subject was obtained.
Isolation of extracellular vesicles from plasma samples
EVs were isolated using ultracentrifugation as previously described.[12] Briefly, all venous blood samples were collected from subjects that fasted overnight (8–12 h) in citrate-containing BD Vacutainer tubes. Plasma from blood samples was immediately separated by centrifugation at 1500 × g/15 min. The separated plasmas were subsequently centrifuged at 3500 × g/30 min at 4°C to remove platelets and cellular detritus and subjected to centrifugation at 15,000 × g/30 min at 4°C; the resulting supernatants plasma fractions were collected, aliquoted, and keep frozen − 80°C until analysis. Next, the aliquoted plasma was thawed on ice and ultracentrifuged at 100,000 × g/2 h at 4°C. The final pellet-containing EVs were reconstituted in 100 μL of phosphate-buffered saline (PBS)/citrate solution and used for assays on cells, flow cytometry [Supplementary Figure 1], and vesicle characterization techniques [Figure 1a] .
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Supplementary Figure 1: Flow cytometric classification strategy. Analysis of EVs (d) using submicron particles shows (a) and (e) gating of 0.1 μm beads according to FITC-A MIF (R1); (b) and (f) gating of 0.5 μm beads according to FITC-A mean fluorescence intensity (R2); (c) and (g) gating of 1.0 μm beads according to FITC-A mean fluorescence intensity (R3). (d) Representative MV regions according to R1, R2, and R3 limited as described previously in the materials and methods section (R4). EVs: Extracellular vesicles, FITC-A: Fluorescein isothiocyanate channel, MFI: Mean fluorescence intensity, MVs: Microvesicles, SSC-A: Side Scatter parameter, FSC-A: Forward Scatter parameter
Figure 1: EV’s characterization. (a) Protocol diagram (BioRender.com). (b) Representative micrographs, arrows point to plasma derived EVs. (c) The bars present average size (gray) and polydispersity index (white). Values correspond to mean ± standard deviation. (d) WB detection of EV markers. EVs: Extracellular vesicles, T2DM: Type 2 diabetes mellitus, IFG: Impaired fasting glucose, WB: Western blot, MHC-1: Major Histocompatibility Complex type 1
Western blotting
Western blot (WB) was carried out to analyze pooled EVs as previously described,[12] anti-flotillin 2 and anti-MHC-1 as primary Abs, and a horseradish peroxidase-conjugated donkey anti-rabbit Ab as secondary. The films were scanned, and the bands were quantified using ImageJ (NIH, USA).
Electron microscopy
EV structures were visualized using a scanning transmission electron microscopy (STEM) mode (STEM) (Model JSM 7800 F Field Emission Scanning Electron Microscope; JEOL Ltd., Akishima-shi, Japan). Ten microliters of EVs were deposited on a formvar/carbon-coated copper TEM grid (300 Mesh), and after 20 s, the excess was removed with a filter paper. Samples were stained using 10 μL of 2% phosphotungstic acid solution for 15 s. The grid was vacuum dried for 24 h before observations (20 kV).
Dynamic light scattering
EV sizes were measured by dynamic light scattering (DLS) of Zetasizer Nano ZS (Malvern Instruments, UK) with a resolution of 0.5 nm and sensitivity of 0.1 ppm to 40% w/v. The instrument determines the size by first measuring the Brownian motion of particles in the samples using DLS and interpreting a size using established theories. The relationship between particle size and its speed due to Brownian motion is defined in the Stokes–Einstein equation.
where D is the diffusion coefficient, KB is the Boltzmann constant, T is the temperature of the sample, h is the viscosity, and R is the hydrodynamic radio, representing particle size in nm. Each sample was measured at room temperature (25°C) in triplicate to verify reproducibility. EVs size data were analyzed and plotted with Origin 2016 software.
Cell culture
EA.hy926 (ATCC CRL-2922) were cultured until they reached confluence in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 3.7 g/L sodium bicarbonate,[13] 10% fetal bovine serum (FBS), and antibiotics in a humidified atmosphere containing 5% CO2 and 95% air at 37°C. All experiments were performed on EA.hy926 cultures with no more than 6 passages and starved in FBS-free DMEM for 12 h before treatment with pooled EVs, FBS, or VEGF.
Scratch-wound assay
EA.hy926 confluent cultures were serum starved >12 h and treated for 2 h with 12 μm mitomycin C. Cell cultures were scratch wounded using a sterile yellow pipette tip, washed twice with PBS, and incubated in DMEM containing pooled EVs (50 μg protein), and cell migration was measured after 24 h. The assays were carried out in quadruple. As a positive control, cells treated with 10% FBS were used. Cells were fixed in 4% paraformaldehyde and dyed with 0.1% crystal violet.
Angiogenesis assay
Growth Factor Reduced Matrigel (Corning, New York, USA) (8 mg/mL) was transferred to 24-well plates. Serum starved >12 h and EA.hy926 cells were added to wells (40 × 103 cells/well) containing DMEM with 0.1% FBS. Cells were exposed to EVs (20 μg) from each study group. EV-free DMEM and VEGF (50 ŋg) were used as negative and positive controls. After incubation (5 h), cells were dyed and photographed using a bright-field Nikon Eclipse Ci microscopy at ×4 magnification with a DS-Vi1 Nikon camera.
Angiogenesis score (AS) was calculated using a modified protocol.[14] ImageJ software counted sprouting cells, numbers of polygonal structures, and complex meshes comprising 2–3 cells thick that formed under each condition. AS was calculated according to Aranda and Owen, 2009,[14] as follows:
Data analysis
Data are presented as mean + standard deviation or median (interquartile range). The Kolmogorov–Smirnov test was applied to test the variable distribution. Differences between groups were analyzed using the Kruskal–Wallis test (Bonferroni posthoc test); P < 0.05 indicates a significant difference. Data analyses were performed using SPSS 23 Statistical Software (IBM SPSS Statistics for Windows, version 23.0. Armonk, NY, USA: IBM Corp.).
RESULTS
Studied groups’ characteristics
The selected characteristics of the subjects in the three study groups are shown in Table 1. In the normoglycemic group, 40% of the subjects were under oral treatment for high blood pressure; their systolic pressure was not significantly higher than in patients with IFG and T2DM (P = 0.654). In addition, the homeostatic model assessment of insulin resistance (HOMA-IR) was significantly higher in T2DM subjects than in IFG patients (P = 0.007) and more than in the control group (P = 0.028). However, body mass index and fasting plasma insulin showed no significant differences between groups (P = 0.113 and P = 0.208, respectively). High-density cholesterol was below the normal range values in all three subject groups (P = 0.295), while total cholesterol was significantly higher in IFG and T2DM patients compared to control subjects (P = 0.021). Low-density cholesterol (P = 0.145), triglycerides (P = 0.691), and uric acid (P = 0.236) did not show significant differences between groups.
Table 1: Clinical and biochemical characteristics of the donors of venous blood samples
Extracellular vesicle characterization
TSEM revealed well-integrated spherical-shaped and small vesicles in the three groups of plasma-derived EVs [Figure 1b], thus corroborating its integrity after plasma isolation. EV sizes measured by DLS indicate theoretical sizes of isolated EVs in a range of 450–750 nm with a polydispersity index comparable between samples [Figure 1c]. Flotillin 2 and MHC-1 proteins were detected by WB [Figure 1d], and TSG-101 and CD9 tetraspanin were detected by flow cytometry in isolated EVs [Supplementary Figures 1 and 2].
Supplementary Figure 2: CD9 and TSG-101 expression. (a) CD9 in PE-A channel and the attached histogram were performed on plasma EVs. (b) TSG-101 in PE-A channel and the attached histogram were performed on plasma EVs. Classification strategy described in
Supplementary Figure 1. Ten thousand events were acquired. EVs: Extracellular vesicles, PE-A: Phycoerythrin channel, FSC-A: Forward Scatter parameter
EVs from IFG patients induce human endothelial cell migration
The effect of plasma-derived EVs from normoglycemic subjects and patients with IFG or T2DM on EA.hy926 cell migration was evaluated using a scratch assay [Figure 2a]. Control cultures with 10% FBS added significantly induced cell migration compared to basal cultures without EVs (P < 0.001) [Figure 2a and b]. Similarly, EA.hy926 cells incubated with EVs from IFG and T2DM exhibited significant migration compared with basal cultures (P = 0.001 and P = 0.042, respectively). Furthermore, migration induced by EVs from IFG patients was significantly higher than that induced for EVs from normoglycemic subjects (P = 0.023).
Figure 2: Migration of EA.hy926 cells. (a) Representative migration micrograph. (b) The graph shows the fold-basal migration mean ± standard deviation of four independent experiments. * EVs versus basal cultures (*P < 0.05, **P < 0.001), and # EVs of patients versus controls (# P < 0.05). EVs: Extracellular vesicles, FBS: Fetal bovine serum, T2DM: Type 2 diabetes mellitus, IFG: Impaired fasting glucose
Extracellular vesicles from impaired fasting glucose or Type 2 diabetes mellitus patients induce angiogenesis
The angiogenic potential for plasma-derived EVs was analyzed in the three groups of subjects [Figure 3a]. AS of EVs from patients with IFG and normoglycemic subjects calculated by Aranda and Owen model[14] did not show a significant difference compared to basal cultures (without added plasma-derived EVs). In contrast, AS of EVs from patients with T2DM was significantly higher than basal culture [P = 0.042, Figure 3b]. Likewise, EVs from T2DM or IFG patients had a higher AS compared to EVs from normoglycemic subjects [P = 0.012 vs. P = 0.036, respectively, Figure 3b].
Figure 3: Angiogenic potential of EVs. (a) Arrows indicate protruding cells, and asterisks indicate polygonal cell morphology. (b) Angiogenic score, *denotes differences between EVs versus basal cultures (*P < 0.05, **P < 0.01), and #EVs of patients versus controls (# P < 0.05). EVs: Extracellular vesicles, IFG: Impaired fasting glucose, T2DM: Type 2 diabetes mellitus, VEGF: Vascular endothelial growth factor, IFG: Impaired fasting glucose
Extracellular vesicle endoglin and vascular endothelial growth factor receptor 2 detection
Vesicle numbers were different among plasma pools from the three groups, so it was necessary to adjust the volume of each pool before flow cytometry assays. Endoglin and VEGFR-2 levels in EVs from each group were not significantly different [Figure 4a and b].
Figure 4: Endoglin and VEGFR-2 detection. (a) Endoglin expression of the EVs. (b) VEGFR-2 expression histogram of the EVs. The right panel shows the MFI/20,000 events ± standard deviation. VEGFR-2: Vascular endothelial growth factor receptor 2, EVs: Extracellular vesicles, IFG: Impaired fasting glucose, T2DM: Type 2 diabetes mellitus, MFI: Mean fluorescence intensity, APC-A: Allophycocyanin channel A, CTL: control
DISCUSSION
Chronic abnormalities in glycemia are strongly associated with endothelial dysfunction development.[15] Inadequate glucose uptake in target tissues leads to glucose accumulation and its metabolites, inducing endothelial cell injury that contributes to atherosclerosis.[16] In those circumstances, collateral circulation to counteract the consequences of any vascular flow obstruction is necessary.[17] Endothelial damage is an early hallmark of the onset of macro and microvascular complications in patients with IFG and T2DM.[18,19] Angiogenesis occurs in response to an injury and aims to restore tissues from the blood supply and promote wound healing.[4] Sprouting angiogenesis comprises enzymatic degradation of the vessel’s basement membrane and proliferation, migration, sprouting, branching, and tube formation by endothelial cells.[20]
EVs have been considered markers of inflammation, thrombosis, endothelial activation or dysfunction, and angiogenesis.[7,8] However, chronic hyperglycemia effects on EVs in humans are still controversial. EV elevation in plasma from patients with T2DM and prediabetes suggests that chronic hyperglycemia is related to EVs release; furthermore, high glucose levels increase EVs released from HUVEC cultures.[21] HOMA-IR high value is a hallmark of impaired glucose metabolism in T2DM patients;[22] in this study, HOMA-IR is lower in IFG than in T2DM patients but is not different from the control subjects. A remarkable difference between the normoglycemic subjects versus IFG and T2DM patients was the highest total cholesterol level in the latter two groups.
The present study explores if plasma-derived EVs from patients with IFG would modulate endothelial cell behavior invitro, and migration and ASs of human endothelial cells exposed to EVs from IFG patients were analyzed. We found that plasma-derived EVs from patients with IFG and T2DM induced a greater EA.hy926 cell migration than EVs from subjects with normoglycemia, and it is the first study to show that plasma-derived EVs from patients with IFG guide to in vitro migration of an endothelial cell line.
Moreover, EVs derived from endothelial progenitor cells induce in vitro islet endothelial cell migration, proliferation, an organization in vessel-like structures, and resistance to apoptosis.[23] Likewise, Trinh etal., 2016, showed that EVs from stem cells and EVs from diabetic individuals stimulate cell migration and improve revascularization.[24]
Here, we have shown that the effect on cell migration with plasma-derived EVs from patients with T2DM was similar even to that produced by EVs from subjects with IFG. In contrast to our results, others found that circulating MVs derived from T2DM patients decreased the migration of endothelial progenitor cells from healthy people while circulating MVs derived from healthy individuals induce uncontrolled cell migration and decrease the apoptosis of endothelial progenitor cells isolated from T2DM patients.[25]
Moreover, our data show that plasma-derived EVs from T2DM or IFG patients had a more extensive AS than those obtained from normoglycemic subjects; a similar effect was present in a recent work in which plasma-derived EVs from diabetic individuals increased cell lamellipodia and cell migration compared to EVs from euglycemic individuals.[10] Tsimerman etal. in 2011 found that circulating EVs from diabetic retinopathy and diabetic foot ulcer patients contribute to forming vascular tube networks in vitro that were the longest and most stable and formed by endothelial cells incubated with EVs from healthy controls.[8]
Our results suggest that plasma EV cargo from T2DM and IFG patients could contribute to activating mechanisms to compensate for endothelial damage through activation of cell migration and angiogenesis, even if the insulin resistance evaluated by HOMA-IR in IFG patients is like normoglycemic subjects levels. However, extensive in vivo studies are necessary to corroborate this effect.
Endoglin and VEGFR-2 are involved in the dynamic vascular processes leading to angiogenesis.[26,27] Endoglin (also called CD105) is a transmembrane glycoprotein expressed primarily in vascular endothelium that plays a vital role in angiogenesis and vascular remodeling,[26] while VEGFR-2 is the most prominent VEGFR, which is the main angiogenic growth factor that modulates angiogenesis.[27] For these reasons, we analyzed endoglin and VEGFR-2 expression in plasma-derived MVs. We found that endoglin and VEGFR-2 levels in MVs from patients with IFG or T2DM were not significantly different from those with normoglycemic patients. Tramontano et al. found that plasma levels of endothelial EVs endoglin positive in diabetic patients are higher than in healthy subjects.[28]
Malik et al. suggests elevated levels of EVs endoglin positive are associated with developing and progressing diabetic retinopathy in human patients.[29] By another hand, Wieczór et al. showed that the angiogenic factor VEGF-A and angiogenic inhibitors sVEGFR-2 and sVEGFR-1 are increased in the serum of T2DM patients with peripheral arterial disease.[30] Nevertheless, we found no significant differences in VEGFR-2 and endoglin expression between the EVs obtained from the study groups; however, the small sample sizes of the groups studied here could influence this result.
CONCLUSION
This pilot study presents evidence that supports the possible proangiogenic role of plasma-derived EVs from IFG subjects with normal HOMA-IR values, at a similar level of the effect induced from T2DM EVs. Data were robust and significant independently of the relatively small sample of blood donors; however, extended studies are necessary to identify the underlying mechanisms involved in EV angiogenic effect and its role in IFG since EVs from patients here evaluated no difference in endoglin and VEGFR-2 expression. Therefore, plasma-derived EVs from IFG patients are excellent candidates for exploring early biomarkers of vascular complications in chronic dysglycemic states even before establishing an insulin resistance state.
Financial support and sponsorship
This research was funded by CONACyT, grant number 285059.
Conflicts of interest
There are no conflicts of interest.
Acknowledgments
We would like to thank R. C. Carrillo Torres and P. Cortez Reynosa for technical support, CONACyT for founding the project (Grant 285059) and for the doctoral scholarship to R-R AN, and staff of the Signal Transduction Laboratory of the Cell Biology Department of Cinvestav-IPN for providing the EA.hy926 cell line.
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