Citrate treatment reduces endothelial death and inflammation under hyperglycaemic conditions

Anna Bryland, Anders Wieslander, Ola Carlsson, Thomas Hellmark, Gabriela Godaly, Anna Bryland, Anders Wieslander, Ola Carlsson, Thomas Hellmark, Gabriela Godaly

Abstract

Hyperglycaemia and glucose degradation products (GDPs) are closely associated with oxidative stress and inflammation in diabetic patients, a condition that leads to endothelial dysfunction and cardiovascular problems. We evaluated the effect of citrate and gluconate on glucose- and GDP-induced endothelial inflammation by measuring changes in viability, inflammation and function in primary human umbilical vein endothelial cells (HUVECs). The extent of apoptosis/necrosis was measured by flow cytometry and visualised with confocal microscopy by staining with annexin V or propidium iodide, respectively. Protein kinase C-βII (PKC-βII) activation was evaluated with Western blotting. Incubation with glucose (30 mM) and GDP (50 µM) significantly increased PKC-βII expression, endothelial cell death and inflammation. The addition of citrate decreased hyperglycaemia-induced apoptosis (p = 0.021), necrosis (p = 0.04) and reduced PKC-βII expression (p = 0.021) down to background levels. Citrate improved endothelial function by reducing the inflammatory markers (p = 0.01) and by decreasing neutrophil diapedesis (p = 0.012). These results suggest that citrate may have therapeutic potential by reducing hyperglycaemia-induced endothelial inflammation and abolishing endothelial dysfunction.

Conflict of interest statement

None declared.

Figures

Figure 1.
Figure 1.
Citrate and citrate/gluconate decrease cell death. Glucose (a) and 3,4-dideoxyglucosone-3-ene (3,4-DGE) (b) treatment of endothelial cells increased total cell death. The figure shows the percentage of total cell death as well as the parts that are apoptotic (light grey) and necrotic (dark grey). Citrate and the 0.8 mM citrate and 1 mM gluconate (CAG) combination reduced apoptosis and necrosis after 48 h of incubation together with glucose (a) or 3,4-DGE (b). Glucose or 3,4-DGE treatment was compared to medium control, and citrate and CAG combination treatment of glucose and 3,4-DGE-damaged cells was compared to the respective additive. The results are the mean (± SEM) of four different experiments (a and b) (***p < 0.001, **p < 0.01).
Figure 2.
Figure 2.
Citrate and citrate/gluconate decrease apoptosis and PKC-βII expression. (a) Confocal microscopy revealed a significant increase of nuclear fragmentation in glucose- or 3,4-DGE-treated epithelial cells compared to control (indicated by the arrows). The cellular membrane was stained green for chemokine (C-X-C motif) receptor 2 (CXCR2) with primary mouse antibodies followed by labelling with secondary antibodies conjugated with fluorescein isothiocyanate (FITC) (R&D Systems, Copenhagen, Denmark). Cell nuclei were stained with propidium iodide (red). Original magnification × 300. (b and c) Western blot analysis revealed a continuous basal PKC-βII expression in endothelial cells. Treatment with glucose or 3,4-DGE increased the amount of PKC-βII proteins. Citrate or 0.8 mM citrate and 1 mM gluconate (CAG) treatment of hyperglycaemia-damaged cells decreased PKC-βII expression down to background level. Quantification of band intensity was performed using the ImageJ version 1.24 software (National Institutes of Health, Bethesda, Maryland, USA) and normalised to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein level. The results are the mean of three different experiments (± SEM) (**p < 0.01).
Figure 3.
Figure 3.
Citrate and the 0.8 mM citrate and 1 mM gluconate (CAG) combination decrease intercellular adhesion molecule-1 (ICAM-1) expression. ICAM-1 expression was analysed by flow cytometry on glucose or 3,4-DGE-treated endothelial cells and in the damaged cells in the presence of citrate or the CAG combination. Endothelial ICAM-1 expression after treatment with glucose and citrate (a), glucose and CAG combination (b), 3,4-DGE and citrate (c) and 3,4-DGE and CAG combination (d). Immunoglobulin G (IgG) negative control was used as the control. ICAM-1 expression was significantly upregulated by glucose or 3,4-DGE treatment (shift to the right). Addition of the CAG combination significantly decreased glucose or 3,4-DGE-induced ICAM-1 expression (shift to the left), while the addition of citrate only decreased glucose-induced ICAM-1 expression. The results are from one representative experiment out of a total of four experiments.
Figure 4.
Figure 4.
Citrate and the 0.8 mM citrate and 1 mM gluconate (CAG) combination decrease neutrophil diapedesis across infected cells. Endothelial cells were grown in glucose (grey) (panel a) or 3,4-DGE (grey) (panel b) alone or in the presence of citrate (dashed) and CAG combination (dotted) for 2 days before the addition of human neutrophils to the upper compartment of activated monolayers. Migrated neutrophils were calculated in the lower compartment after 3 h. (a) The addition of citrate and CAG combination decreased neutrophil migration across infected endothelial cells, but glucose treatment did not have any influence. (b) The 3,4-DGE treatment of endothelial cells had a small impact on neutrophil migration that was further reduced by citrate or CAG combination treatment. Cells grown in medium alone were used as a negative control (white) and untreated infected cells were used as positive control (black). The results are the mean (± SEM) of three different experiments (***p < 0.001, **p < 0.01, *p < 0.05, ns = not significant).
Figure 5.
Figure 5.
Citrate and 0.8 mM citrate and 1 mM gluconate (CAG) combination decrease inflammation. Secretion of interleukin-6 (IL-6) (a and b) and chemokine (C-X-C motif) ligand 8 (CXCL8) (c and d) from endothelial cells treated with glucose (grey) (panel c) or 3,4-DGE (grey) (panel d) alone or in the presence of citrate (dashed) or CAG combination (dotted). The presence of citrate and CAG combination decreased glucose- and 3,4-DGE-induced IL-6 secretion (a and b). CXCL8 secretion was not affected by glucose or 3,4-DGE treatment of endothelial cells, but the presence of citrate decreased endothelial CXCL8 secretion (c and d). Cells grown in medium alone were used as a negative control (white) and untreated infected cells were used as positive control (black). The results are the mean (±SEM) of three different experiments (**p < 0.01, *p < 0.05, ns = not significant).

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