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Comparative Study
. 2013 Oct 5:12:142.
doi: 10.1186/1475-2840-12-142.

Hyperglycemia induces differential change in oxidative stress at gene expression and functional levels in HUVEC and HMVEC

Hemang Patel  1 , Juan Chen, Kumuda C Das, Mahendra Kavdia
Affiliations
Comparative Study

Hyperglycemia induces differential change in oxidative stress at gene expression and functional levels in HUVEC and HMVEC

Hemang Patel et al. Cardiovasc Diabetol. .

Abstract

Background: Endothelial dysfunction precedes pathogenesis of vascular complications in diabetes. In recent years, the mechanisms of endothelial dysfunction were investigated to outline strategies for its treatment. However, the therapies for dysfunctional endothelium resulted in multiple clinical trial failures and remain elusive. There is a need for defining hyperglycemia-induced endothelial dysfunction with both generic and specific dysfunctional changes in endothelial cells (EC) using a systems approach. In this study, we investigated hyperglycemia-induced endothelial dysfunction in HUVEC and HMVEC. We investigated hyperglycemia-induced functional changes (superoxide (O₂⁻), and hydrogen peroxide (H₂O₂) production and mitochondrial membrane polarization) and gene expression fingerprints of related enzymes (nitric oxide synthase, NAD(P)H oxidase, and reactive oxygen species (ROS) neutralizing enzymes) in both ECs.

Method: Gene expression of NOS2, NOS3, NOX4, CYBA, UCP1, CAT, TXNRD1, TXNRD2, GPX1, NOX1, SOD1, SOD2, PRDX1, 18s, and RPLP0 were measured using real-time PCR. O₂⁻ production was measured with dihydroethidium (DHE) fluorescence measurement. H2O2 production was measured using Amplex Red assay. Mitochondrial membrane polarization was measured using JC-10 based fluorescence measurement.

Results: We showed that the O₂⁻ levels increased similarly in both ECs with hyperglycemia. However, these endothelial cells showed significantly different underlying gene expression profile, H₂O₂ production and mitochondrial membrane polarization. In HUVEC, hyperglycemia increased H₂O₂ production, and hyperpolarized mitochondrial membrane. ROS neutralizing enzymes SOD2 and CAT gene expression were downregulated. In contrast, there was an upregulation of nitric oxide synthase and NAD(P)H oxidase and a depolarization of mitochondrial membrane in HMVEC. In addition, ROS neutralizing enzymes SOD1, GPX1, TXNRD1 and TXNRD2 gene expression were significantly upregulated in high glucose treated HMVEC.

Conclusion: Our findings highlighted a unique framework for hyperglycemia-induced endothelial dysfunction. We showed that multiple pathways are differentially affected in these endothelial cells in hyperglycemia. High occurrences of gene expression changes in HMVEC in this study supports the hypothesis that microvasculature precedes macrovasculature in epigenetic regulation forming vascular metabolic memory. Identifying genomic phenotype and corresponding functional changes in hyperglycemic endothelial dysfunction will provide a suitable systems biology approach for understanding underlying mechanisms and possible effective therapeutic intervention.

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Figures

Figure 1
Figure 1
Expression levels of NOS enzymes in HUVEC, and HMVEC after 24 hours of high glucose exposure. Gene expression of NOS2 and NOS3 were measured following 24 hours of HG-M199 exposure to (a) HUVEC and (b) HMVEC. Fold changes were calculated by 2-∆∆CT method using 18s and RPLP0 as housekeeping genes and cells grown in M199 media with 5.6 mM D-glucose as control. **p ≤ 0.05 significantly higher than control for respective cells type.
Figure 2
Figure 2
Expression levels of NAD(P)H oxidase family enzymes in a) HUVEC, and) HMVEC cells after 24 hours of high glucose exposure. Gene expression of NOX1, NOX4, and CYBA were measured using real-time PCR in both (a) HUVEC and (b) HMVEC. Fold changes were calculated by 2-∆∆CT method using 18s and RPLP0 as housekeeping genes and cells grown in M199 media with 5.6 mM D-glucose as control. **p ≤ 0.05 significantly higher than control for HMVEC. *p ≤ 0.07 higher than control for HMVEC.
Figure 3
Figure 3
Expression levels of ROS clearance enzymes in HUVEC, and HMVEC cells after 24 hours of high glucose exposure. Gene expression of SOD1, SOD2, and CAT were measured in (a) HUVEC and (b) HMVEC following 24 hours of treatment with HG-M199. Fold changes were calculated by 2-∆∆CT method using 18s and RPLP0 as housekeeping genes and cells grown in M199 media with 5.6 mM D-glucose as control. **p ≤ 0.05 significantly different than control for respective cells type.
Figure 4
Figure 4
Expression levels of peroxide clearance enzymes in HUVEC, and HMVEC cells after 24 hours of high glucose exposure. Gene expression of GPX1, TXNRD1, TXNRD2, and PRDX1 were measured in (a) HUVEC and (b) HMVEC following 24 hours of exposure to HG-M199. Fold changes were calculated by 2-∆∆CT method using 18s and RPLP0 as housekeeping genes and cells grown in M199 media with 5.6 mM D-glucose as control. **p ≤ 0.05 significantly higher than control for HMVEC.
Figure 5
Figure 5
Expression levels of oxidative stress responding transcription factor and uncoupling protein in HUVEC, and HMVEC cells after 24 hours of high glucose exposure. Gene expression of NFE2L2 and UCP1 were measured in (a) HUVEC and (b) HMVEC following 24 hours of exposure to HG-M199. Fold changes were calculated by 2-∆∆CT method using 18s and RPLP0 as housekeeping genes and cells grown in M199 media with 5.6 mM D-glucose as control. *p ≤ 0.06 higher than control for HMVEC.
Figure 6
Figure 6
O2‾ production in HUVEC and HMVEC cells following high glucose exposure for 24 hours. Using DHE levels of O2‾ were measured in both HUVEC and HMVEC following 24 hours of high glucose treatment. Fluorescence imaging was used to capture DHE fluorescence in (a-b) HUVEC and (c-d) HMVEC. Fluorescence microplate reader based measurements were also performed to measure specific fluorescence intensity of 2-OH-E+ in (e) HUVEC and HMVEC treated with high glucose. For both types of DHE measurements, cells grown in 5.6 mM D-glucose were used as control. **p ≤ 0.01 significantly higher than control for respective cells type.
Figure 7
Figure 7
H2O2 production profile in high glucose treated HUVEC and HMVEC. Levels of H2O2 were measured in media supernatant of (a) HUVEC and (b) HMVEC treated with either 5.6 mM (control) or 25 mM D-glucose. Cells grown in media with 5.6 mM D-glucose were used as control. **p ≤ 0.006 significantly higher than control for respective cells type.
Figure 8
Figure 8
Mitochondrial membrane polarization in HUVEC and HMVEC following high glucose treatment for 24 hours. Using JC-10 mitochondrial membrane polarization was measured in both HUVEC and HMVEC after 24 hours of high glucose treatment. Mitochondrial membrane polarization of high glucose treated HUVEC was evaluated through measurement of JC-10 aggregates (Red) and monomers (Green) using fluorescence microscopy and microplate reader. a) Overlapped fluorescence images of red and green fluorescence intensities along with Hoescht-33342 based nuclear stain were used to show hyperpolarized mitochondrial membrane in high glucose treated HUVEC. b) Similarly, ratio of red and green fluorescence intensities were plotted to illustrate mitochondrial membrane hyperpolarization in high glucose treated HUVEC. **p ≤ 0.05 significantly higher than 5.6 mM D-glucose exposure treatment in HUVEC. c) Overlapped fluorescence images of red and green fluorescence intensities along with Hoescht-33342 based nuclear stain were used to show depolarized mitochondrial membrane in high glucose treated HMVEC. d) Ratio of red and green fluorescence intensities were plotted to show high glucose exposure-induced mitochondrial membrane depolarization in HMVEC. **p ≤ 0.05 significantly higher than 25 mM D-glucose exposure concentration in HMVEC.
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