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. 2015 Mar;24(1):55-70.
doi: 10.5607/en.2015.24.1.55. Epub 2015 Mar 13.

COMP-Ang1 Potentiates EPC Treatment of Ischemic Brain Injury by Enhancing Angiogenesis Through Activating AKT-mTOR Pathway and Promoting Vascular Migration Through Activating Tie2-FAK Pathway

Hyo Eun Moon  1 , Kyunghee Byun  2 , Hyung Woo Park  1 , Jin Hyun Kim  3 , Jin Hur  4 , Joong Shin Park  5 , Jong Kwan Jun  5 , Hyo-Soo Kim  4 , Seung Leal Paek  6 , In Keyoung Kim  7 , Jae Ha Hwang  7 , Jin Wook Kim  7 , Dong Gyu Kim  7 , Young Chul Sung  8 , Gou-Young Koh  9 , Chang W Song  10 , Bonghee Lee  2 , Sun Ha Paek  1
Affiliations

COMP-Ang1 Potentiates EPC Treatment of Ischemic Brain Injury by Enhancing Angiogenesis Through Activating AKT-mTOR Pathway and Promoting Vascular Migration Through Activating Tie2-FAK Pathway

Hyo Eun Moon et al. Exp Neurobiol. 2015 Mar.

Abstract

Successful recovery from brain ischemia is limited due to poor vascularization surrounding the ischemic zone. Cell therapy with strong angiogenic factors could be an effective strategy to rescue the ischemic brain. We investigated whether cartilage oligomeric matrix protein (COMP)-Ang1, a soluble, stable and potent Ang1 variant, enhances the angiogenesis of human cord blood derived endothelial progenitor cells (hCB-EPCs) for rescuing brain from ischemic injury. COMP-Ang1 markedly improved the tube formation of capillaries by EPCs and incorporation of EPCs into tube formation with human umbilical vein endothelial cells (HUVECs) upon incubation on matrigel in vitro. COMP-Ang1 stimulated the migration of EPCs more than HUVECs in a scratch wound migration assay. The transplanted EPCs and COMP-Ang1 were incorporated into the blood vessels and decreased the infarct volume in the rat ischemic brain. Molecular studies revealed that COMP-Ang1 induced an interaction between Tie2 and FAK, but AKT was separated from the Tie2-FAK-AKT complex in the EPC plasma membrane. Tie2-FAK increased pp38, pSAPK/JNK, and pERK-mediated MAPK activation and interacted with integrins ανβ3, α4, β1, finally leading to migration of EPCs. AKT recruited mTOR, SDF-1, and HIF-1α to induce angiogenesis. Taken together, it is concluded that COMP-Ang1 potentiates the angiogenesis of EPCs and enhances the vascular morphogenesis indicating that combination of EPCs with COMP-Ang1 may be a potentially effective regimen for ischemic brain injury salvage therapy.

Keywords: COMP-Ang1; Tie2-FAK-AKT pathway; angiogenesis; ischemia.

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Figures

Fig. 1
Fig. 1. COMP-Ang1 enhances tube formation and migration of hCB-EPCs. (A, C) COMP-Ang1 promoted tube formation, more in EPCs than in HUVECs. COMP-Ang1 (C.A1, 200 ng/mL) was incubated for 24 h (A) followed by densitometric analyses (C). Scale bar, 100 µm. All values are the means±SEM. ****p <0.0001, **p <0.01. (B) COMP-Ang1 promoted the number of EPC incorporated HUVECs. CFSE-labeled EPCs were incorporated on DiI-labeled HUVECs. COMP-Ang1 (200 ng/mL) was incubated for 24 h. Scale bar, 100 µm. (D, E) COMP-Ang1 promoted cell migration in EPCs better than that in HUVECs. COMP-Ang1 (200 ng/mL) was incubated for 24 h. Scale bar, 50 µm.
Fig. 2
Fig. 2. hCB-EPCs enhance angiogenesis and protect against degeneration in a rat ischemic model. (A, B) Measurement of infarct size of the control, EPC and EPC-C.A1 in the rat model. (A) Densitometric analyses (B), Scale bar, 1000 µm. All values are the means±SEM. ***p<0.001, *p<0.05. (C, D) The behavior test for the control, EPC and EPC-C.A1 in rat model. Limb placement test (C) and Rota-rod test (D). All values are the means±SEM. *p<0.05. (E, F) Confocal images of laminin staining (green) and HuNu staining (red) with the DAPI nuclear marker (blue) in ischemic rats with/without EPC treatment in the absence or presence of co-treatment with COMP-Ang1 (E) and densitometric analyses (F). Scale bar, 50 µm. All values are the means±SEM. ***p<0.001.
Fig. 3
Fig. 3. COMP-Ang1 increase the Tie2/FAK interaction after translocation in hCB-EPCs. (A) Confocal images of Tie2, FAK, and AKT (red) with one of two location markers (CY or PM, green) and the DAPI nuclear marker (blue) with or without treatment of COMP-Ang1 in EPC. Yellow or cyan color indicate overlap between protein and the corresponding location markers. Scale bar, 200 µm. (B, C) Interactions between Tie2: FAK (left), Tie2: AKT (middle), or AKT: FAK (right) with or without COMP-Ang1 treatment in EPCs. A proximity ligation assay (PLA) was used to measure the population of close physical interactions. Red spots indicate the physical proximity of the corresponding protein pair. Scale bar, 200 µm. (B). Number of blobs (or interactions) per cell between Tie2 and FAK with or without COMP-Ang1 treatment in EPCs (C). Average number of "blobs" (spots or interactions) per cell for Tie2: FAK, Tie2: AKT, or AKT: FAK under the COMP-Ang1 treatment condition showing that the number of interacting pairs between Tie2: FAK increased dramatically, but interaction between Tie2: AKT and AKT: FAK decreased after COMP-Ang1 treatment. All values are the means±SEM. ***p<0.001. (D, E) Confocal images of Tie2, FAK, and AKT (red) with one of two location markers (CY or PM, green) and the DAPI nuclear marker (blue) in a rat model. (D) Densitometric analyses (E). Scale bar, 20 µm. All values are the means±SEM. ***p<0.001, **p <0.01, *p<0.05.
Fig. 4
Fig. 4. COMP-Ang1 increases Tie2/FAK/ITG-mediated migration and tube formation. (A, C) Tube formation assay with COMP-Ang1 treated Tie2, FAK and AKT silencing (A). COMP-Ang1 (200 ng/mL) was incubated for 24 h followed by densitometric analyses (C). Scale bar, 100 µm. All values are the means±SEM. ****p <0.0001, *p<0.05. (B) HUVEC-DiI incorporation assays with COMP-Ang1 treated Tie2, FAK, and Akt silencing. COMP-Ang1 (200 ng/mL) was incubated for 24 h. Scale bar, 100 µm. (D) Scratch wound migration assay with COMP-Ang1 treated Tie2, FAK and AKT silencing. COMP-Ang1 (200 ng/mL) was incubated for 24 h. Scale bar, 50 µm. (E, F) The numbers of interacting pairs between ITGανβ3: ITGα4, ITGανβ3: ITGαV or ITGανβ3: ITGβ1 did not change after Tie2, FAK or AKT silencing. Interactions between ITGανβ3: ITGα4 (top), ITGανβ3: ITGαV (middle), or ITGανβ3: ITGβ1 (bottom) with/without COMP-Ang1 treatment in EPCs before or after Tie2 or FAK silencing. Red spots indicate the physical proximity of the corresponding protein pair. Scale bar, 200 µm. (E). Number of blobs (or interactions) per cell between ITGανβ3: ITGα4, ITGανβ3: ITGαV or ITGανβ3: ITGβ1 with/without COMP-Ang1 treatment in EPCs before or after Tie2 or FAK silencing (F). All values are the means±SEM. ***p<0.001.
Fig. 5
Fig. 5. COMP-Ang1 increases migration and tube formation through the MAPK pathway. (A, B) Relative protein expression levels of MAPK kinase with/without COMP-Ang1 treatment in EPCs before or after Tie2 or FAK silencing. Whole cell lysates (30 µg protein/lane) were subjected to Western blot analyses to determine the levels of ERK1/2, pERK1/2, p38, pp38, and SAPK/JNK, pSAPK/JNK. β-actin was used as an internal control for equal protein loading of each lane. (C, D) Confocal images of pp38, pERK1/2, and pSAPK/JNK (red) with the DAPI nuclear marker (blue) in a rat model (C) followed by densitometric analyses (D). Scale bar, 20 µm. All values are the means±SEM. ***p<0.001, **p<0.01.
Fig. 6
Fig. 6. COMP-Ang1 increase angiogenesis by AKT/mTOR interaction. (A,B) Interaction between AKT and mTOR with/without COMP-Ang1 treatment in EPCs before or after Tie2 silencing. Red spots indicate the physical proximity of the corresponding protein pair. Scale bar, 200 µm. (A). Blobs showing interaction between AKT and mTOR with/without COMP-Ang1 treatment in EPCs before or after Tie2 silencing. The numbers of interacting pairs between AKT: mTOR no changed after Tie2 silencing (B). All values are the means±SEM. ***p<0.001. (C) Confocal images of ERK1/2, SAPK/JNK and mTOR (red) with DAPI of nucleus marker (blue) in rat model. Scale bar, 50 µm. (D, E) The relative protein expression levels of MAPK kinase with/without COMP-Ang1 treatment in EPC before or after AKT silencing or mTOR inhibition. Whole cell lysates (30 µg protein/lane) were subjected to Western blot analyses to determine the levels of ERK1/2, pERK1/2, p38, pp38, and SAPK/JNK, pSAPK/JNK. β-actin was used as an internal control for equal protein loading for each lane.
Fig. 7
Fig. 7. COMP-Ang1 induces SDF-1/CXCR4/HIF-1α mediated angiogenesis. (A, B) Confocal images of CXCR4 (green) with DAPI for nucleus marker (blue)with/without COMP-Ang1 treatment in EPC before or after FAK or AKT silencing or mTOR inhibition (A) and densitometric analyses (B). Scale bar, 200 µm. All values are the means±SEM. ***p<0.001. (C, E) Confocal images of SDF-1 (red) and FLAG (green) with DAPI of nucleus marker (blue) in ischemia rat with/without EPC treatment in the absence or presence of co-treatment with COMP-Ang1 (C) followed by densitometric analysis (E). Scale bar, 20 µm. All values are the means±SEM. **p <0.01, *p<0.05. (D) The time-dependent changes of extracellular (supernatant) SDF-1 in EPC and HUVEC, treated with COMP-Ang1 for 1, 3, 6, 12, 24 h, were determined by ELISA. All values are the means±SEM. *p<0.05. (F) The relative mRNA expression level of HIF-1α before and after COMP-Ang1 treatment in EPC with/without Tie2, FAK, or AKT silencing or mTOR inhibition. All values are the means±SEM. ***p<0.001. (G) Western blot analyses of HIF-1α protein before or after COMP-Ang1 treatment in EPC with/without Tie2, FAK, or AKT silencing or mTOR inhibition. Whole cell lysates (30 µg protein/lane) were subjected to Western blot analysis to determine the levels of HIF-1α. β-actin was used as an internal control for equal protein loading for each lane. The mRNA and protein expression levels of HIF-1α were increased dramatically after COMP-Ang1 treatment, but that of HIF-1α no changed after Tie2 and AKT silencing.
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