The Effect of High and Variable Glucose on the Viability of Endothelial Cells Co-Cultured with Smooth Muscle Cells

Abstract

Diabetes mellitus causes endothelial dysfunction. The aim of this study was to investigate the effect of normal (5 mmol/L), high (20 mmol/L), and fluctuating (5 and 20 mmol/L changed every day) glucose concentration in the culture medium on the viability of human umbilical vein endothelial cells (HUVECs) co-cultured with human umbilical artery smooth muscle cells (HUASMCs). The cultures were conducted on semi-permeable flat polysulfone (PSU) fibronectin-coated membranes immobilized in self-made inserts. The insert contained either HUVECs on a single membrane or HUASMCs and HUVECs on two membranes close to each other. Cultures were conducted for 7 or 14 days. Apoptosis, mitochondrial potential, and the production of reactive oxygen species and lactate by HUVECs were investigated. The results indicate that fluctuations in glucose concentration have a stronger negative effect on HUVECs viability than constant high glucose concentration. High and fluctuating glucose concentrations slow down cell proliferation compared to the culture carried out in the medium with normal glucose concentration. In conclusion, HUASMCs affect the viability of HUVECs when both types of cells are co-cultured in medium with normal or variable glucose concentration.

Keywords: DM; HUASMCs; HUVECs; co-culture; diabetes; diabetes complications; endothelial cells; high concentration of glucose; reactive oxygen species; smooth muscle cells; variable concentration of glucose; viability.

Figure 1

Percentage of early apoptotic HUVECs: after 7 days of culturing without HUASMCs or co-culturing with HUASMCs (a); after 14 days of culturing without HUASMCs or co-culturing with HUASMCs (b); after 7 and 14 days of culturing without HUASMCs (c); after 7 and 14 days of co-culturing with HUASMCs (d); in normal (N), high (H), or variable (H/N) glucose concentration on PSU flat membranes covered with fibronectin. The data are presented as scatter plot (yellow, green, red and blue dots) with corresponding bar graphs.

Figure 2

Total percentage of the early and late apoptotic HUVECs: after 7 days of culturing without HUASMCs or co-culturing with HUASMCs (a); after 14 days of culturing without HUASMCs or co-culturing with HUASMCs (b); after 7 and 14 days of culturing without HUASMCs (c); after 7 and 14 days of co-culturing with HUASMCs (d); in normal (N), high (H), or variable (H/N) glucose concentration on PSU flat membranes covered with fibronectin. The data are presented as scatter plot (yellow, green, red and blue dots) with corresponding bar graphs. The ANOVA results showed that all of the independent factors but the presence or absence of muscle cells, as well as all interactions of the independent factors in pairs and all three together, significantly influenced the percentage of HUVECs in early or late apoptosis.

Figure 3

Mitochondrial membrane potential of HUVECs: after 7 days of culturing without HUASMCs or co-culturing with HUASMCs (a); after 14 days of culturing without HUASMCs or co-culturing with HUASMCs (b); after 7 and 14 days of culturing without HUASMCs (c); after 7 and 14 days of co-culturing with HUASMCs (d); in normal (N), high (H), or variable (H/N) glucose concentration on PSU flat membranes covered with fibronectin. The data are presented as scatter plot (yellow, green, red and blue dots) with corresponding bar graphs.

Figure 4

ROS production (DCFDA geometrical mean of the fluorescence intensity in cytometric analysis) by HUVECs: after 7 days of culturing without HUASMCs or co-culturing with HUASMCs (a); after 14 days of culturing without HUASMCs or co-culturing with HUASMCs (b); after 7 and 14 days of culturing without HUASMCs (c); after 7 and 14 days of co-culturing with HUASMCs (d); in normal (N), high (H), or variable (H/N) glucose concentration on PSU flat membranes covered with fibronectin. The data are presented as scatter plot (yellow, green, red and blue dots) with corresponding bar graphs.

Figure 5

The daily (blue bars) and total (red line) lactate production during a 14-day culture of HUVECs in medium with N (a), H (b), and H/N (c) glucose concentration; L_tp—total lactate production.

Figure 6

Lactate production by HUVECs after 7 and 14 days of culturing in medium with normal (N), high (H), or variable (H/N) glucose concentration on PSU flat membranes covered with fibronectin. The data are presented as scatter plot (yellow, green, red and blue dots) with corresponding bar graphs.

Figure 7

Angiopoietin 1/Tie 2 pathway as a reaction to high- and fluctuating-glucose stimulation. VSMC—Vascular Smooth Muscle Cells, ICAM-1 - Intercellular Adhesion Molecule 1, VCAM-1—Vascular Cell Adhesion Molecule 1, Ang-1—Angiopoietin 1, Ang-2—Angiopoietin 2, NF-κB—Nuclear Factor-κB, PI3K/Akt—the phosphatidylinositol 3-kinase/protein kinase B pathway, IQGAP1—IQ motif of the GTPase-1 activating protein 1, Rac 1—GTPase Rac1, EC—Endothelial Cell, WPB—Weibel-Palade Body.

Figure 8

Culture of HUVECs seeded on the PSU membrane modified with fibronectin (a); co-culture of HUVECs and HUASMCs (HUVECs/HUASMCs) seeded on two flat membranes modified with fibronectin immobilized in the one insert (b); establishing a cell culture in a 12-well culture plate (c); 12-well culture plate with inserts with membranes and seeded cells (d).