Endothelial Dysfunction in Youth-Onset Type 2 Diabetes: A Clinical Translational Study

Abstract

Background: Youth-onset type 2 diabetes (Y-T2D) is associated with increased risk for coronary atherosclerotic disease, but the timing of the earliest pathological features and evidence of cardiac endothelial dysfunction have not been evaluated in this population. Endothelial function magnetic resonance imaging may detect early and direct endothelial dysfunction in the absence of classical risk factors (severe hyperglycemia, hypertension, and hyperlipidemia). Using endothelial function magnetic resonance imaging, we evaluated peripheral and coronary artery structure and endothelial function in young adults with Y-T2D diagnosed ≤5 years compared with age-matched healthy peers. We isolated and characterized plasma-derived small extracellular vesicles and evaluated their effects on inflammatory and signaling biomarkers in healthy human coronary artery endothelial cells to validate the imaging findings.

Methods: Right coronary wall thickness, coronary artery flow-mediated dilation, and brachial artery flow-mediated dilation were measured at baseline and during isometric handgrip exercise using a 3.0T magnetic resonance imaging. Human coronary artery endothelial cells were treated with Y-T2D plasma-derived small extracellular vesicles. Protein expression was measured by Western blot analysis, oxidative stress was measured using the redox-sensitive probe dihydroethidium, and nitric oxide levels were measured by 4-amino-5-methylamino-2',7'-difluororescein diacetate.

Results: Y-T2D (n=20) had higher hemoglobin A1c and high-sensitivity C-reactive protein, but similar total and LDL (low-density lipoprotein)-cholesterol compared with healthy peers (n=16). Y-T2D had greater coronary wall thickness (1.33±0.13 versus 1.22±0.13 mm; P=0.04) and impaired endothelial function: lower coronary artery flow-mediated dilation (-3.1±15.5 versus 15.9±17.3%; P<0.01) and brachial artery flow-mediated dilation (6.7±14.7 versus 26.4±15.2%; P=0.001). Y-T2D plasma-derived small extracellular vesicles reduced phosphorylated endothelial nitric oxide synthase expression and nitric oxide levels, increased reactive oxygen species production, and elevated ICAM (intercellular adhesion molecule)-mediated inflammatory pathways in human coronary artery endothelial cells.

Conclusions: Coronary and brachial endothelial dysfunction was evident in Y-T2D who were within 5 years of diagnosis and did not have severe hyperglycemia or dyslipidemia. Plasma-derived small extracellular vesicles induced markers of endothelial dysfunction, which corroborated accelerated subclinical coronary atherosclerosis as an early feature in Y-T2D.

Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02830308 and NCT01399385.

Keywords: atherosclerosis; brachial artery; cardiovascular diseases; diabetes, type 2; pediatric obesity.

Figure 1.. Coronary arterial wall structure and function in youth-onset type 2 diabetes (Y-T2D) and lean healthy age-matched peers (Control).

(A) Right coronary wall thickness and (B) percent change from baseline in the right coronary and brachial arteries flow mediated dilation during the isometric handgrip exercise in Control (green) compared to Y-T2D (blue). Data are presented as violin plots (A) and bar graphs mean (B) with individual data points (black circles). Groups were compared with Student’s t-test. P< 0.05 was considered significant and shown inline.

Figure 2:. Characterization of plasma-derived small extracellular vesicles.

Plasma derived small extracellular vesicles (small ECVs) were isolated from healthy donor volunteer (HV, n=1) and youth-onset type 2 diabetes (Y-T2D, n=2) then stained with uranyl acetate (A,B), labeled with IgG control, illustrating the baseline distribution of particles without specific targeting (C), or 10-nm immunogold particles using antibodies against the exosomal membrane markers CD9 (D–F), and CD63 (G–I), and stained with uranyl acetate. Small extracellular vesicles were imaged by electron microscopy. Scale bar: 100 nm. (J) Western blot analysis was conducted on lysates sourced from the small ECVs fraction and cell pellet, utilizing antibodies against CD63, TSG101, ApoE, CD9, and Calnexin. The expression of TSG101, CD9, and CD63 were predominantly identified in plasma-derived small ECVs extracts, Calnexin was not detected, confirming the exclusion of endoplasmic reticulum components. ApoE and Calnexin were primarily observed in cell lysates and traces of observed in small ECVs extracts. (K-L) Nanoparticle tracking analysis (NTA) quantification of small ECVs size. The x-axis represents the small ECVs size in nanometers (nm). The y-axis represents the frequency (count) of particles found at each size interval.

Figure 3:. Visualization of the uptake of small extracellular vesicles by Human Coronary Artery Endothelial Cells (HCAEC).

(A) HCAECs were stained with DAPI (Blue) to visualize nuclei and (B) phallodin (red) for actin filaments. (C) HCAECs were incubated with RNASelect dye only, (D) HCAECs were incubated with SYTO RNASelect dye-labelled small extracellular vesicles (green) and imaged at 48h, Scale bar: 50 μm. (E) Flow cytogram showing the shift in fluorescence intensity in HCAECs after incubation with PKH26- labeled plasma derived Y-T2D small extracellular vesicles (red) and control (blue).

Figure 4:. Protein expression analysis in HCAECs post-small extracellular vesicles (small ECVs) treatment.

Small extracellular vesicles were isolated from the plasma of healthy donor volunteers (HV, n=3),youth-onset type 2 diabetes (Y-T2D, n=10), and adult-onset type 2 diabetes (A-T2D, n=1) and subsequently employed to treat HCAECs over a period of 48 hours. Post-treatment, the expression of target proteins phosphorylated eNOS (peNOS) and ICAM was assessed. (A-B): Western blot analysis showing the fold change in phosphorylated eNOS relative to total eNOS after treatment with plasma-derived small extracellular vesicles. (C-D): Western blot analysis depicting the fold change in ICAM-1 relative to β-actin after small extracellular vesicles treatment. Individual data (black circles) are presented with bar graphs for mean,with three replicates for panels (A) and (C), and four replicates for panels (B) and (D). Mann Whitney U tests were used to compare vs. Control (untreated cells, CTR) and SN (HCAECs treated with supernatant-depleted small extracellular vesicles). P< 0.05 was considered significant and shown inline.

Figure 5:. Analysis of reactive oxygen species and nitric oxide levels in HCAECs post-small extracellular vesicles (small ECVs) treatment.

Small extracellular vesicles were isolated from the plasma of healthy donor volunteers (HV, n=3),youth-onset type 2 diabetes (Y-T2D, n=10), and adult-onset type 2 diabetes (A-T2D, n=1) and subsequently employed to treat HCAECs over a period of 48 hours. Post-treatment (A-B): Flow cytometric analysis of nitric oxide (NO) levels using DAF-FM techniques after treatment with plasma-derived small extracellular vesicles. (C-D): Assessment of reactive oxygen species (ROS) production using dihydroethidium (DHE) after small extracellular vesicles treatment. Individual data (black circles) are presented with bar graphs for mean, with three replicates for panels (A-D). Mann Whitney U tests were used to compare vs. Control (untreated cells, CTR) and SN (HCAECs treated with supernatant-depleted small extracellular vesicles). P< 0.05 was considered significant and shown inline.