Human mononuclear cells (MNCs) differentiate into Human Endothelial Progenitor cells (hEPC) in vitro.
The hEPCs from diabetic patients have their basic angiogenic functions such as migration, the ability to form blood vessels and adhesion capacity altered; however, it is not entirely clear how hyperglycemia impacts these functions. Therefore, in order to understand how hyperglycemia can induce failure of hEPC adhesion, we used hEPC cultures as our biological model. hEPC were isolated from MNC primary culture derived from non-diabetic patients (Table 1). As can be seen in figures 1A and 1B, after four days of culture the cells showed a rounded shape, without morphological signs characteristic of a mature endothelial cell (splinde shape). To further characterized our cultures, we determined surface markers expression using reverse transcription polymerase chain reaction analysis RT-PCR. Our results demonstrate the presence of mRNA for markers of cell immaturity such as Oct-4 (169 bp) and CD34 (380 bp) (figure 1C upper and middle panel respectively, lanes 8 and 9), as well as mRNA expression for endothelial marker CD31 (240 bp) (figure 1C lower panel lanes 8 and 9).
mRNA expression is not a guarantee of protein expression; therefore, we additionally analyzed expression of the endothelial marker CD34 and KDR at protein level by flow cytometry as can be seen in Figure 1D, our results indicate the presence of three distinct cell subpopulations, one with immaturity characteristics but without endothelial characteristics (KDR-/CD34+) corresponding to 8,7% of the cell population; a second subpopulation without immaturity characteristics but with endothelial characteristics (KDR+/CD34-) corresponding to 16,7% of the cell population and a third cell subpopulation with immature and endothelial characteristics (KDR+/CD34+), the one with the highest number of cells within the culture equivalent to 44% (p> 0.01). Therefore, under our culture conditions, the higher percentage of cells exhibits an immature immunophenotype with endothelial characteristics. Taken together, our results indicate that from our four-day primary culture methodology, we obtain mainly hEPCs identified as CD34+ Oct 4+ CD31+ KDR+.
High concentrations of D-glucose do not affect the identity of hEPCs or the integrity of their DNA.
In order to evaluate how hyperglycemia, affect the adhesion of hEPC, it was necessary to simulate the physiological conditions in which the hEPC of diabetic patients are found. To this end, we cultured hEPC in media supplemented with 5 -35 mM of D-Glucose.
Our results show that under this conditions cell viability is close to 100% (Figure 2A). Although a slight decrease can be observed at 35mM glucose of D-glucose and 35 mM mannitol (which was used as osmotic control), it is not statistically significant. Importantly in these growing conditions no DNA damage is observed (Figure 2B).
All following experiments were carried out using media supplemented with 5 mM (90 mg/dl) as a control since it is the concentration of D-glucose contained in the growth media and equivalent to normal glycemia, 10 mM (180 mg/dl) which is a clinically acceptable value for a diabetic patient, and 20 mM of D-glucose (360 mg/dl) corresponding to the value of hyperglycemia in a patient with poor glycemic control.
To characterize cells growing under high glucose conditions we evaluated expression of the characteristic surface markers OCT4, CD34 and CD31. As show figure 2C, under all growing conditions hEPC exhibit expression of these markers (lanes 8 to 13), however, this expression seems not to be the same in the different treatments (5, 10 and 20 mM of D-glucose), in the OCT-4 and CD34 genes. Moreover, as shown in Figure 2D, flow cytometry analysis of CD34 and KDR reveal the presence of the three previously described cell subpopulations KDR-/CD34+; KDR+/CD34-; KDR+/CD34+. All subpopulations exhibit similar distribution in media supplemented with 5mM, 10mM or 20mM D-glucose. Our population of interest, CD34+/KDR+ represent a 40,3 % (10mM D-glucose) and 40,5% (20mM D-glucose), compared with cell grown in media supplemented with 5mM of D-glucose 44%. Therefore, culturing hEPC cells in medium containing 5, 10 and 20 mM of D-glucose for four days does not affect cell viability, DNA integrity or cell populations distribution.
Human Endothelial Progenitor Cell adhesion is impaired by high D-Glucose concentrations.
hEPC from diabetic patients show a decreased adhesion capacity; therefore, in the following experiment we evaluate if this phenomenon is reproduced when cells are cultured in media supplemented with the selected D-glucose concentrations.
hEPC cultured at 5, 10 and 20mM of D-Glucose were grown in fibronectin matrix for 4 hours, adhered cells were subsequently counted by fluorescence microscopy (Figure 3A). Cell adhesion was quantified by counting the cells that adhered to fibronectin in different microscopic fields. As expected, there is a statistically significant decrease (p> 0.05) when cells were cultured in 20 mM of D-glucose, a concentration that represents the glycemia from a patient with poor glycemic control (Figure 3B). Interestingly, when cells were cultured with 10mM D-glucose, which is equivalent to clinically acceptable hyperglycemia, no changes in adhesion activity were observed in comparison with the control of 5 mM of D-glucose equivalent to a normal glycemia.
High D-glucose generates global hypomethylation in human endothelial progenitor cells.
Our results indicate that hEPC cultured in high concentrations of D-glucose (20mM) exhibit changes in its adhesion capacity. The most studied mechanisms by which the environment triggers changes at the gene expression level reflected in function are epigenetic modifications, such as miRNA-based mechanisms, histone modifications, and DNA methylation.
It is well documented that high glucose concentrations cause DNA damage which, when repaired by various enzymatic mechanisms, generate changes in the global methylation state of the genome. Therefore, we decided to evaluate changes that could be occurring in hEPC DNA methylation.
DNA methylation analysis was performed by slot blot using an anti-methylated DNA antibody. Ad seen in figure 4A, unmethylated DNA is not recognized by our antibody, thus demonstrating its specificity for the recognition of the 5-methylcytosine epitope.
As a positive control for the technique, we evaluated the effect of D-glucose on the methylation MNCs because previously published data show that the high D-glucose concentrations produces DNA hypermethylation in leucocytes (Wu et al., 2017). MNCs DNA was analyzed by a slot blot, using the total level of DNA (DNA stained on the membrane) to standardize the antibody signal. As can be seen in Figure 4B MNCs behave according to what was expected showing hypermethylation, with significantly statistically differences when cells cultured in media supplemented with 20 mM of glucose is compared to control (p> 0.05). Surprisingly, when hEPC where grown in 10mM and 20mM of D-Glucose we observed a decrease in the overall methylation of the genome compared to control, media containing of 5mM of D-Glucose, (p> 0.0001) (Figure 4C).
These results were unexpected considering that high glucose concentration generate hypermethylation in peripheral blood cells and hEPC are derived from them. Furthermore, we hypothesize that decrease in hEPC adhesion function could be result a silencing of the genes involved in this process, an effect triggered by DNA hypermethylation and not by DNA hypomethylation.
It is noteworthy that cells cultured at 10mM D-glucose present a statistically significant hypomethylation of the genome (p> 0.001) compared to the control of 5mM D-Glucose (figure 4C), even though, at this glucose concentration, no changes in cell adhesion activity were observed (Figure 3B).
Demethylation of the hPEC genome leads to decreased cell adhesion
Our results indicate that high concentrations of D-glucose in vitro induce a decrease in hEPC adhesion to fibronectin matrix and also cause hypomethylation of the genome. To evaluate whether there is a causal relationship between these two phenomena, D-glucose- independent DNA demethylation was induced in hEPC using 5-aza-2'-deoxycytidine (5 Aza-2dC).
Our results indicate that under these culture conditions cell viability is close to 100% (Figure 5A) and although slight decrease was observed in cultures treated with 5 Aza-2dC, this is not statistically significant; furthermore, no DNA damage was observed under these conditions (Figure 5B).
To characterize cells grown under demethylating conditions, we evaluated the expression of their characteristic surface markers OCT4, CD34 and CD31 and, as seen in Figure 5C, similar mRNA levels of markers OCT4, CD34 were observed for cells cultured with 5 Aza-2dC (lanes 10-11) and with DMSO (vehicle) (lanes 8-9) but apparently there is a decrease in the mRNA expression levels of the surface marker CD31 compared to control (lanes 6-7). As expected, 5 Aza-2dC induces a statistically significant hypomethylation hEPC genome compared to control (p> 0.001) and vehicle (p> 0.01) (Figure 5D).
Moreover, hEPC adhesion to fibronectin matrix of cell treated with 5 Aza-2dC was lower (Figure 5E, right panel) compared to vehicle and control (Figure 5E, central panel and left panel respectively). Cell adhesion was quantified by counting the cells in different microscopic fields that adhered to fibronectin, observing a statistically significant decrease in cells cultured with 5 Aza-2dC (p> 0.001) compared to the control and the vehicle (Figure 5F).
Therefore, our results indicate that global DNA hypomethylation of hEPC results in a
decreased cell adhesion capacity.
High D-glucose causes active demethylation of the hEPC genome
Our results indicate that culture hEPC in high D-glucose concentration results in DNA hypomethylation, to confirm that this effect was produced by the metabolism of D-glucose and not by an osmotic effect, cells were grown in media supplemented with 10mM and 20mM L-Glucose (not metabolizable). As observed in figure 6A, DNA methylation of hEPC cultured in 20mM L-Glucose does not exhibit statistically significant changes when compared to the control (5mM of D-Glucose). Therefore, these results demonstrate that Glucose must be metabolized (D-Glucose) to induce the hypomethylation of hEPC DNA.
It has been previously show that the increase in glucose metabolism generates DNA damage, which, when repaired, can lead to hypomethylation of the genome through the action of the enzymes TET1 and TET 2, which transform 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) (Lio and Rao, 2019; Yuan et al., 2019)
To evaluated if hEPC DNA hypomethylation is generated as result DNA repair activities, the levels of 5-hmC in cells cultured with different concentrations of D-Glucose were measured through slot blot. As seen in Figure 6B, a statistically significant increase (p <0.05) in the levels of 5-hmC was observed in cells cultured with 20mM of D-glucose compared to the control and an increasing trend when they were cultured with 10mM of D-Glucose. Interestingly, although the expression of the TET1 and TET2 genes is observed in the three culture conditions (5, 10 and 20 mM of D-glucose) (figure 6 and lines 8 to 16), no statistically significant changes in the expression levels of the messenger for TET1 and TET2 genes were observed when cells were cultured at the selected D-Glucose concentrations compared to the control (figure 6C and 6D).
Therefore, our results demonstrated that hEPC genome hypomethylation observed when cells were cultured in high concentrations D-Glucose is independent of TET1 and TET2 expression levels.