Regulatory roles of interferon-inducible protein 204 on differentiation and vasculogenic activity of endothelial progenitor cells

Abstract

Background: Endothelial progenitor cells (EPCs) have shown great potential in angiogenesis either by their differentiation into endothelial cells or by secretion of angiogenic factors. Interferon-inducible protein 204 (Ifi204) has been reported to participate in the regulation of cell growth and differentiation. However, its role in differentiation of EPCs remains unknown. We proposed that Ifi204 could modulate the differentiation and regenerative abilities of EPCs.

Methods: Ifi204-expressing lentivirus and Ifi204 siRNA were introduced into EPCs to overexpress and suppress the expression of Ifi204. Using fluorescence-activated cell sorting, immunocytochemistry, and quantitative PCR, endothelial markers including CD31, VE-cadherin, and vWF were detected in the modified EPCs. An in-vitro incorporation assay and a colony-forming assay were also performed.

Results: Evidence showed that Ifi204 inhibition decreased the endothelial differentiation and vasculogenic activities of EPCs in vitro. In mice with hindlimb ischemia, downregulation of Ifi204 in EPCs, which was tracked by our newly synthesized nanofluorogen, impaired neovascularization, with a corresponding reduction in hindlimb blood reperfusion by postoperative day 14.

Conclusions: Ifi204 is required for EPC differentiation and neovascularization in vitro and in vivo. The regulatory roles of Ifi204 in EPC differentiation may benefit the clinical therapy of ischemic vascular diseases.

Keywords: Endothelial differentiation; Endothelial progenitor cells; Hindlimb ischemia; Ifi204; Vasculogenesis.

Fig. 1

Culture of EPCs and modulation of Ifi204 in EPCs. a Morphology of mouse bone marrow-derived EPCs cultured at day 3 and day 7. Bar, 100 μm and 50 μm. b Uptake of DiI-Ac-LDL by EPCs. Bar, 25 μm. c Infection of Ifi204 lentivirus in EPCs. Bar, 50 μm. d Expression of Ifi204 in EPCs after transduction of Ifi204 expressing lentivirus (n = 3). P < 0.01, LV5-Ifi204 EPCs vs LV-NC EPCs and normal EPCs. e Transfection of Ifi204 siRNA in EPCs. Bar, 100 μm. f Expression of Ifi204 in EPCs after transfection of Ifi204 siRNA (n = 3). P < 0.001, EPCs^siRNA vs normal EPCs and EPCs^scramble. EPC endothelial progenitor cell

Fig. 2

qPCR analysis of endothelial markers and growth factors in different EPC groups. mRNA expression levels of CD31 a, VE-cadherin b, vWF c, and VEGF d in 14-day cultured cells were assessed by quantitative real-time RT-PCR. The mRNA expressions were normalized to GAPDH (n = 3). All assays were performed in triplicate and demonstrated similar results. EPC endothelial progenitor cell

Fig. 3

Assessment of endothelial markers in cultured EPCs by immunocytostaining and FACS. Cells were cultured and induced with Ifi204 lentivirus or Ifi204 siRNA. Adherent cells were stained with endothelial marker CD31 a and VE-cadherin b. Bar, 50 μm. c Cell surface markers investigated by FACS were as follows: endothelial cell (CD31 and VE-cadherin) and stem cell (CD133). Cellular surface markers were analyzed on normal EPCs (upper panel), Ifi204 lentivirus-transduced EPCs (middle panel), and Ifi204 siRNA-transfected EPCs (lower panel). Percentages of each marker indicated in the histograms (n = 3). EPC endothelial progenitor cell

Fig. 4

Tube formation assay by HUVECs incorporated with modified EPCs. DiI-labeled cells and HUVECs were seeded onto Matrigel-coated 96-well plates in 10 % FBS/EBM2-MV without growth factors. After 24 hours in culture, incorporation of each cell population into tube-like structures formed with HUVECs was evaluated under fluorescence microscopy. a Morphology of cultured HUVECs forming a network on Matrigel with the incorporation of DiI-positive cells. Bar, 200 μm. b Number of incorporated DiI-positive cells into tubular structures was counted and averaged. All assays demonstrated similar results (n = 4). EPC endothelial progenitor cell

Fig. 5

Colony-forming assay by different EPC groups. a Representative staining for fluorescein isolectin B4 (ILB4) and DiI-Ac-LDL in EPC colonies. Bar, 250 μm. b Large cell colonies were counted and analyzed in each group. All assays demonstrated similar results (n = 4). EPC endothelial progenitor cell

Fig. 6

Blood flow patterns in ischemic hindlimbs. a LDPI was used to analyze blood flow 14 days after ischemia. Top panel: representative features just after surgery. Colors displayed correspond to intervals of perfusion value from 0 (dark) to 300 (red). Red, highest velocity; green, intermediate; blue, low; and dark, lowest velocity. b Blood-flow ratio of ischemic-to-contralateral hindlimb after 14 days (n = 6). P < 0.05, normal EPCs vs Ifi204 siRNA-EPCs; P < 0.01, Ifi204 LV-EPCs vs Ifi204 siRNA EPCs. EPC endothelial progenitor cell (Color figure online)

Fig. 7

Histological analysis for capillary density in ischemic hindlimb. Cells were intramuscularly injected immediately after hindlimb ischemia. Muscle samples were harvested following BSL I systemic perfusion 14 days after surgery. a Representative images of vessel morphology from each treatment group. Bar, 50 μm. b Numbers of capillaries were counted in the ischemic area and averaged in each group (n = 4), respectively. EPC endothelial progenitor cell

Fig. 8

In-vitro and in-vivo staining of EPCs by the nanofluorogen. a EPCs were incubated with TPE-11 for 12 hours. Fluorescent images of the cells observed by a confocal microscope at the 405 nm excitations. Bar, 20 μm and 8 μm. b Sections of the ischemic gastrocnemius muscle were observed by a confocal microscope at the 405 nm excitations. Bar, 8 μm. EPC endothelial progenitor cell

Fig. 9

Detection of cell incorporation into neovessels in ischemic hindlimb. Nonmodified EPCs and Ifi204 siRNA-transfected EPCs were first labeled with TPE-11, and then were intramuscularly injected together with Ifi204 lentivirus-transduced EPCs immediately after hindlimb ischemia. Muscle samples were harvested following BSL I systemic perfusion 14 days after surgery. a Colabeling with BSL I (red) and TPE-11 (green) appears (orange, marked by white arrows) in the section of ischemic calf muscle. Bar, 20 μm. b Numbers of incorporated cells into vessels were counted and averaged (n = 4). P < 0.05, normal EPCs vs Ifi204 siRNA-EPCs; P < 0.001, Ifi204 LV-EPCs vs Ifi204 siRNA EPCs. EPC endothelial progenitor cell