Predicting Angiogenesis by Endothelial Progenitor Cells Relying on In-Vitro Function Assays and VEGFR-2 Expression Levels

Clinical trials have demonstrated the safety and efficacy of autologous endothelial progenitor cell (EPC) therapy in various diseases. Since EPCs’ functions are influenced by genetic, systemic and environmental factors, the therapeutic potential of each individual EPCs is unknown and may affect treatment outcome. Therefore, our aim was to compare EPCs function among healthy donors in order to predict blood vessel formation (angiogenesis) before autologous EPC transplantation. Human EPCs were isolated from the blood of ten volunteers. EPCs proliferation rate, chemoattractant ability, and CXCR4 mRNA levels were different among donors (p < 0.0001, p < 0.01, p < 0.001, respectively). A positive correlation was found between SDF-1, CXCR4, and EPCs proliferation (R = 0.736, p < 0.05 and R = 0.8, p < 0.01, respectively). In-vivo, blood vessels were counted ten days after EPCs transplantation in a subcutaneous mouse model. Mean vessel density was different among donors (p = 0.0001); nevertheless, donors with the lowest vessel densities were higher compared to control (p < 0.05). Finally, using a linear regression model, a mathematical equation was generated to predict blood vessel density relying on: (i) EPCs chemoattractivity, and (ii) VEGFR-2 mRNA levels. Results reveal differences in EPCs functions among healthy individuals, emphasizing the need for a potency assay to pave the way for standardized research and clinical use of human EPCs.


Converting optical density (O.D) to cell number in XTT assay
Cells from donor #9 were seeded at gradual concentrations and incubated overnight; O.D values were measured using cell proliferation kit XTT in triplicate according to blank. The average results of four repeats are summarized (Table S1). XTT Kit was used to analyze the proliferation of human EPCs. During 72 hours, absorbance was examined every 24 hours in quadruplicate. After calculating mean values of relative O.D., results were converted to number of cells using a standardized calibration curve, and according to the mathematical equation: O.D = 10 -5 *(number of cells) ( Figure S1).  Table S1, a straight line equation is represented.

2.A primer listreal-time PCR
Expression levels of angiogenic and chemotactic genes were detected using Real-Time PCR for key genes critical to wound healing and bone repair: SDF1, VEGF-A, CCL2, PDGFβ, KDR, and CXCR4. As an internal control, levels of HPRT were quantified in parallel with target genes (Table  S2). Table S2. Humane primers for Real-Time PCR.

Function and Genotype is NOT Affected by Age
To examine whether age of donors influenced the cells' performance, a Pearson correlation test was carried out. The average result of each in-vitro assay for each individual was correlated with the age of the donor. Age-dependent dot plot diagrams were generated for each tested variable: proliferation, migration, and gene expression.
Unexpectedly, the proliferation potential of EPCs that was assessed by the number of cells after 48 and 72 hours did not correlate with the donors' age ( Figure S3.A: p = 0.7, R = 0.14 and S3.B: p = 0.09, R = 0.56). High proliferation capacity for more than 80,000 cells after 72 hours was detected in donors over 30 years old, while a lower proliferation was found in donors 23-37 years old ( Figure  S3.B).

Figure S3. Correlation between donor's age and the number of cells after 48 (A) and 72 (B) hours.
To verify whether the chemoatractive ability of EPCs' secretome might be affected by human age, the average number of migrated MSC toward EPCs condition media from a specific donor were correlated to the age of that donor. According to Pearson correlation test, no correlations were found between the age of healthy donors and their chemotaxis effect on MSCs (p = 0.4, R = 0.3) ( Figure S4).  In summary, we were surprised to find no significant correlation between donor's age and EPCs proliferation, chemotactic activity, or gene expression levels.

EPCs' Function and Genotype is NOT Affected by Gender
In order to evaluate the effect of patient's gender on cell function and genotype, the results of invitro assays were dichotomized to two groups according to gender. Wilcoxon-Mann-Whitney test was used to compare the groups.
. Comparing females and males for cell growth rate.
The chemotactic activity of EPCs secretome was also not affected by gender of the donor. In both groups, MSCs migration toward EPCs concentrated condition media was intensified compared to migration toward EGM-2 (medium without EPC supernatant). As shown in figure S7, both females and males show similar chemotaxis effect on MSCs, as their minimum, median, and maximum values fall into the same values. Correspondingly, no significant difference was noted between the two subpopulations, p = 0.8. Figure S7. Comparing females and males for chemoatractive activity.

A B
Similar to proliferation and chemoattractant ability, the expression levels of the investigated genes were not affected by the gender of the donor ( Figure S8).