Nectin-2 (CD112) Is Expressed on Outgrowth Endothelial Cells and Regulates Cell Proliferation and Angiogenic Function

Abstract

Outgrowth endothelial cells (OECs) are a subpopulation of endothelial progenitor cells (EPCs) that have the capacity for proliferation and the ability to promote angiogenesis. In this study, we identified Nectin-2 as a surface protein of OECs through unbiased quantitative proteomics analysis. Using immunocytochemistry and flow cytometry, we confirmed that Nectin-2 is highly expressed on OECs. Nectin-2 (CD112) expression was limited or lower on mononuclear cells (MNCs) and mature tube-forming endothelial cells (ECs). Blocking Nectin-2 with a neutralizing monoclonal antibody significantly increased the trans-well migration and tube forming capacity of OECs. Similarly, Nectin-2 knockdown resulted in enhanced tube formation, cell migration and proliferation with p-Erk activation. Moreover, Nectin-2 deficiency resulted in compensatory increase of other Nectin family genes including Nectin-3 and Necl-4 which promote VEGFR signaling. These results indicate that Nectin-2 is a surface marker and an important regulator of OECs, with significant implications for the isolation of OECs and blocking Nectin-2 on OECs by an antibody for angiogenic applications.

Fig 1. Characterization of outgrowth endothelial progenitor cells (OECs).

(A) Quantitative real-time RT-PCR analysis of mRNA expression in MNCs, OECs and HUVECs. OECs and HUVECs expressed the endothelial cell markers, CD105, CD117 (c-Kit), CD144 (VE-cadherin)and CD146 (MCAM) but do not express the hematopoietic cell markers CD14 and CD45 (P < 0.01). (B) Immunofluorescence reveals that OECs were positive for anti-human CD31-FITC and anti-human CD144-FITC antibodies. Nuclei are stained blue with DAPI. (C) FACS analysis of OECs cell-surface-stained with the common endothelial markers CD105, CD144 (VE-cadherin) and CD146.

Fig 2. Nectin-2 is strongly expressed in OECs.

(A) Mass-spectrometric identification of glycoproteins expressed on OECs and HUVECs. Venn diagram showing cell-surface proteins detected only in OECs via analyses of glycoproteins from both total cell lysates and membrane fractions. (B) Nectin-2 mRNA levels expressed as median percentages relative to a housekeeping marker (P < 0.01). (C) Nectin-2 protein expression in MNCs. OECs and HUVECs were analyzed by western blotting using an anti-human Nectin-2 antibody. (D) Immunostaining without permeabilization. OECs were stained with anti-Nectin-2-FITC conjugated mAb. Arrowheads show the signal for Nectin-2 on the surface of OECs. (E) FACS analysis of cell-surface Nectin-2 and endothelial cell markers. Representative FACS analysis of OECs double stained with each of the following antibodies: Nectin-2 (CD112), common endothelial cell markers (CD105, CD144 (VE-Cadherin), CD146, CD31) and an endothelial progenitor marker (CD34).

Fig 3. Enhanced migration and tube formation induced by inhibitory anti-Nectin-2 monoclonal antibody and Nectin-2 knockdown.

(A) Increased tube formation by anti-Nectin-2 mAb treatment. Anti-Nectin-2 mAb-treated OECs and control OECs were cultured in Matrigel with 20 ng/ml VEGF, and the number of tubes was counted (P < 0.01). (B) Increased tube formation by Nectin-2 knockdown. OECs were infected with scrambled shRNA or Nectin-2 shRNA. After a 12-h incubation with shRNA, Puromycin selection was performed with 0.5 μg/ml Puromycin and continued for 3 days (p < 0.01). (C) Boyden-chamber assays were used to assess the migratory potential of OECs treated with anti Nectin-2 mAb. Nectin-2 mAb treatment increased OEC migration. (D) Scramble shRNA- or Nectin-2 shRNA-treated OECs were assessed by Boyden-chamber assays. Nectin-2 shRNA increased OEC and HUVEC migration compared with the scramble shRNA control. (E) Representative images of in vitro OEC scratch-wound healing assays. Nectin-2 knockdown or control OECs were seeded on gelatin-coated (1% gelatin) plates. Upon reaching confluency, cell layers were scratched with a pipette tip. Photos were taken immediately after wounding at 0 hr and 24 hr and the number of tubes was counted (P < 0.01).

Fig 4. Nectin-2 knockdown increases OEC proliferation.

OECs were infected with scramble shRNA or Nectin-2 shRNA lentivirus. (A) Nectin-2 mRNA expression levels were analyzed by quantitative RT-PCR (P < 0.01). (B) Nectin-2 protein expression was analyzed by western blotting. (C) Representative FACS analysis of Nectin-2-knockdown in OECs. Cell-surface expression of VE-Cadherin (CD144) and VEGFR-2 was reduced by Nectin-2 knockdown. Other endothelial cell-surface markers were not changed in OECs. Cell proliferation was evaluated by BrdU and MTT incorporation. (D) The percentage of BrdU incorporation (mean±SD) was analyzed for scramble and Nectin-2 shRNA in both OECs and HUVECs (P < 0.01). (E) MTT assays performed on OECs and HUVECs (P < 0.01). (F) Nectin-2 knockdown increased phospho-ERK activity. Nectin-2 was knocked down in OECs and HUVECs with shRNA, and the knockdown efficiency was monitored by anti-CD112 (Nectin-2) MAb in Western Blot.

Fig 5. Compensation of Nectin-2 function by other Nectin family members and related genes.

(A) Expression of the Nectin family genes Nectin-1, 2 and 3 and the related genes Necl-1 and Necl-4 was measured by real-time RT-PCR in MNCs, OECs and HUVECs. (B) Nectin-family and related gene expression levels were tested in Nectin-2-knockdown OECs. (C) Nectin-2 mRNA levels were assessed 24 hrs after CdCl₂ treatment in OECs and (D) in HUVECs. (E) OECs were cultured in Matrigel for 24 h, and the resulting tube-forming OECs were harvested and evaluated for Nectin and Nectin-like molecule expression (P< 0.01).