Dextran induces differentiation of circulating endothelial progenitor cells

Abstract

Abstract Endothelial progenitor cells (EPCs) have been demonstrated to be effective for the treatment of cardiovascular diseases. However, the differentiation process from circulation to adhesion has not been clarified because circulating EPCs rarely attached to dishes in EPC cultures previously. Here we investigated whether immature circulating EPCs differentiate into mature adhesive EPCs in response to dextran. When floating-circulating EPCs derived from ex vivo expanded human cord blood were cultured with 5% and 10% dextran, they attached to fibronectin-coated dishes and grew exponentially. The bioactivities of adhesion, proliferation, migration, tube formation, and differentiated type of EPC colony formation increased in EPCs exposed to dextran. The surface protein expression rate of the endothelial markers vascular endothelial growth factor (VEGF)-R1/2, VE-cadherin, Tie2, ICAM1, VCAM1, and integrin αv/β3 increased in EPCs exposed to dextran. The mRNA levels of VEGF-R1/2, VE-cadherin, Tie2, endothelial nitric oxide synthase, MMP9, and VEGF increased in EPCs treated with dextran. Those of endothelium-related transcription factors ID1/2, FOXM1, HEY1, SMAD1, FOSL1, NFkB1, NRF2, HIF1A, EPAS1 increased in dextran-treated EPCs; however, those of hematopoietic- and antiangiogenic-related transcription factors TAL1, RUNX1, c-MYB, GATA1/2, ERG, FOXH1, HHEX, SMAD2/3 decreased in dextran-exposed EPCs. Inhibitor analysis showed that PI3K/Akt, ERK1/2, JNK, and p38 signal transduction pathways are involved in the differentiation in response to dextran. In conclusion, dextran induces differentiation of circulating EPCs in terms of adhesion, migration, proliferation, and vasculogenesis. The differentiation mechanism in response to dextran is regulated by multiple signal transductions including PI3K/Akt, ERK1/2, JNK, and p38. These findings indicate that dextran is an effective treatment for EPCs in regenerative medicines.

Keywords: Culture; endothelial progenitor cell; signal transduction; transcription.

Figure 1

Effect of dextran on the culture, adhesion, and proliferation. 3 × 10⁴/cm² floating endothelial progenitor cells (EPCs) were cultured in medium with 5% dextran (A‐b and ‐e) and 10% dextran (A‐c and ‐f) or without dextran (A‐a and ‐d) on human fibronectin‐coated dishes. After 4 days (A‐a, ‐b, and ‐c) and 7 days (A‐d, ‐e, and ‐f) EPCs were observed by a phase contrast microscope (×10) (A). Dextran induced differentiation of circulating EPCs toward adhesive EPCs. Floating EPCs exposed to various densities of dextran for 24 h were cultured for 6 h and the adhesive cells were observed by a phase contrast microscope (×10) (B). EPCs exposed to dextran significantly increased adhesion. The number of adhesive cells per high‐power field (HPF) was counted (C). N = 3. Floating EPCs exposed to various density of dextran for 24 h were cultured for 24 h and the proliferation activity was measured (D). Dextran increased proliferation. N = 5. Data are means ± SD. **P < 0.01, *P < 0.05 versus dextran‐free control.

Figure 2

Effect of dextran on the migration, tube formation, and endothelial progenitor cell (EPC) colony formation. Floating EPCs were cultured with or without 10% dextran for 24 h and they were used for measuring following bioactivities. Nuclei of migrated EPCs were stained with DAPI (×10) (A). The number of migrated cells was counted (B). Dextran increased migration. N = 3. EPCs under exposure of dextran for 24 h were cultured in matrigel with HUVECs and were observed by a phase contrast microscope (×4) (C). Dextran apparently increased tube formation. The number of tubes per low power field (LPF) was measured (D). N = 5. EPCs were cultured in methylcellulose‐containing medium for 15 days, and EPC colonies were observed (E‐a and ‐b, x4; E‐c and ‐d, ×10). Representative pictures of a primitive EPC colony (E‐a and ‐c) and a definitive EPC colony (E‐b and ‐d). Dextran decreased the number of primitive EPC colonies and increased that of definitive EPC colonies (F). N = 3. Data are means ± SD. **P < 0.01, *P < 0.05 versus dextran‐free control.

Figure 3

Effect of dextran on the protein and mRNA expression levels of endothelial markers. The expression rates of surface protein in floating endothelial progenitor cells (EPCs) under exposure of 5% and 10% dextran for 24 h (24 h) or 48 h (48 h) were analyzed (A). In 24 h‐EPCs, 10% dextran increased the protein expression of VCAM1. In 48 h‐EPCs, 5% and/or 10% dextran increased vascular endothelial growth factor (VEGF)‐R1, VEGF‐R2, VE‐cadherin, Tie2, ICAM1, VCAM1, and integrin αv/β3. The mRNA expression levels of EPCs under exposure of 5% and 10% dextran for 48 h were analyzed (B). 5% and/or 10% dextran increased gene expression levels of VEGF‐R1, VEGF‐R2, VE‐cadherin, Tie2, endothelial nitric oxide synthase, MMP9, and VEGF. Values are means ± SD of five samples. **P < 0.01, *P < 0.05 versus dextran‐free control.

Figure 4

Effect of dextran on the transcription factors. The mRNA expression levels of transcription factors in floating EPCs under exposure of 10% dextran for 48 h were analyzed. Expression levels of 69 genes per 10,000 GAPDH copies are shown in A. The horizontal (x) axis indicates copy number in dextran‐free EPCs (control intensity). The vertical (y) axis indicates copy number in dextran EPC (dextran intensity). The lines display y = 1.5x, y = x, and y = 2/3 x, respectively. Relative expression levels of 10 selected genes are shown in B and C. Dextran increased gene expression levels of ID1/2, FOXM1, HEY1, SMAD1, FOSL1, NFkB1, NRF2, HIF1A, and EPAS1. While dextran decreased those of TAL1, RUNX1, c‐MYB, GATA1/2, ERG, FOXH1, HHEX, and SMAD2/3. N = 5. Data are means ± SD. **P < 0.01, *P < 0.05 versus dextran‐free control.

Figure 5

Inhibitor analysis of the adhesion, proliferation, tube formation, endothelial progenitor cell (EPC) colony formation, and differentiation. The abilities of proliferation (A), adhesion (B), tube formation (C), and EPC colony formation were analyzed (D) after floating EPCs were exposed to 10% dextran for 24 h with various inhibitors of signal transduction pathways,. All inhibitors decreased proliferation and adhesion. PD98059, JNK inhibitor II, and SB203580 decreased tube formation. Every inhibitor decreased definitive EPC colony formation, meanwhile, increased primitive EPC colony formation. EPCs were exposed to 10% dextran for 48 h with various inhibitors and the mRNA expression levels were analyzed (E). Inhibitors decreased almost all mRNA expression levels of vascular endothelial growth factor (VEGF)‐R1, VEGF‐R2, VE‐cadherin, Tie2, and endothelial nitric oxide synthase. However, SB203580 increased the mRNA expression level of Tie2. LY, LY294002; PD, PD98059; JNK, JNK inhibitor II; and SB, SB203580. Values are means ± SD of 3–5 samples. **P < 0.01, *P < 0.05 versus dextran control.

Figure 6

A schematic diagram which shows that dextran induces differentiation of circulating endothelial progenitor cells.