Chemerin promotes angiogenesis in vivo

Figure 1. Chemerin promotes angiogenesis in vivo. (A) Matrigel plugs containing chemerin (10 nmol/L, n = 6), VEGF (5 nmol/L, n = 6), or phosphate‐buffered saline (control, n = 6) were injected subcutaneously into mice. Plugs were stained with the endothelial cell marker CD31 and vWF (arrow indicated). Bar = 100 μm. (B) Endothelial cell infiltration of the plugs was assessed by immunohistochemical analysis of CD31 and vWF positive cells. The frequency of DAPI (+) CD31 (+) vWF (+) cells in five low power fields was determined for each Matrigel plug. The data were presented as the number of DAPI (+) CD31 (+) vWF (+) cells per microscopic area. The mean ± SD was derived from six Matrigel plugs for each condition. *P < 0.05 compared with control. (C) Mouse corneal angiogenesis assay; Pellets containing chemerin (200 ng, n = 6), VEGF (100 ng, n = 6) were implanted in mouse cornea. Photographs of mouse eyes are shown (Control, chemerin 200 ng; VEGF, 100 ng). (D) The angiogenic activity was evaluated based on the number of newly formed capillaries per cornea. The results are shown as the mean ± SE **P < 0.01 versus control. (D) Rat aortic ring assay; The descending thoracic aorta of single male…

Figure 2. (A) Expression of chemerin receptors, CMKLR1 and CCRL2, in HUVECs. Chemerin receptors were identified by Western blot analyses using anti‐human CMKLR1 and CCRL2 antibodies. (B) The phosphorylation of Akt and ERK by chemerin. Chemerin stimulation (10 nmol/L) was conducted for the indicated time. The results of one of three experiments with similar results are shown. *P < 0.01 versus control. (C) Confocal immunofluorescence analysis of HUVECs treated with chemerin or VEGF using rabbit mAb against phospho‐Akt (Ser473) and phospho‐ERK (Thr202/Tyr204) (green). Actin filaments have been labeled with Alexa Fluor 568‐conjugated phalloidin (red). Blue pseudo color = DAPI.

Figure 3. Migration of HUVECs induced by chemerin. (A) Modified Boyden chamber assay was performed with chemerin (10 nmol/L) or VEGF (5 nmol/L) as chemoattractant. Chemerin stimulation (10 nmol/L) was conducted for the indicated time. Seven random microscopic fields per each well were quantified. All assays were performed in triplicate. The results are shown as the mean ± SE. *P < 0.05, **P < 0.01 versus control. (B) Representative images of the migrated HUVEC in the modified Boyden assay. Cells were stained with CD31 and DAPI (arrow indicated). Bar = 100 μm. (C) Chemerin‐induced enhancement of migration. After preincubation with LY294002 (1 μmol/L) or PD98059 (5 μmol/L), cells were incubated with chemerin (10 nmol/L). The migratory capacity was quantified by counting the migrated HUVECs on the lower surface of the filter. Seven random microscopic fields per each well were quantified. All assays were performed in triplicate. The results are shown as the mean ± SE. *P < 0.01 versus control.

Figure 4. Migration of HUVECs stimulated by chemerin assessed by cell scratch assay. (A) Modified wound healing assay was performed with chemerin (10 nmol/L) or VEGF (5 nmol/L) as chemoattractant. Chemerin stimulation (10 nmol/L) was conducted for the indicated time. After preincubation with LY294002 (1 μmol/L) or PD98059 (5 μmol/L), cells were incubated with chemerin (10 nmol/L). (B) The change rate of width between the leading edge covered by cells before and after 8 h of incubation was quantified. Seven random wounds per each well were quantified. All assays were performed in triplicate. *P < 0.05 versus serum‐free control.

Figure 5. Chemerin promotes proliferation of HUVECs. Cell growth was arrested using serum‐free EBM‐2 medium for 12 h. After preincubation with LY294002 (1 μmol/L) or PD98059 (5 μmol/L), cells were incubated with chemerin (10 nmol/L) or VEGF (5 nmol/L). (A) Cell proliferation was assessed by counting cells with a hemocytometer. For cell counting, HUVECs were detached from the each seven plates and counted using a hemocytometer. All assays were performed in triplicate. (B) Cell proliferation was assessed by the MTT assay. MTT reduction was read by each 10 wells at 550 nm using a spectrophotometer. All assays were performed in triplicate. The results are shown as the mean ± SE. *P < 0.05 versus control.

Figure 6. Chemerin promotes the differentiation of HUVECs into tube‐like structures. Tube formation assays were performed. HUVECs were seeded on Matrigel‐coated culture dishes in the presence of chemerin (10 nmol/L), VEGF (5 nmol/L), or BSA (10 g/mL: control). After preincubation with LY294002 (1 μmol/L) or PD98059 (5 μmol/L), cells were incubated with chemerin (10 nmol/L). (A) Representative cultures are shown. (B) Quantitative analysis of tube formation. Total tube length per each 10 microscopic area was calculated using image analyzer software. All assays were performed in triplicate. The results are shown as the mean ± SE. *P < 0.05 versus control.

Figure 7. (A) The siRNA‐mediated knockdown of CMKLR1 and CCRL2. (B) Chemerin‐induced phosphorylation of Akt and ERK was inhibited by siRNA against CMKLR1. (C) Modified Boyden chamber assay was performed with chemerin (10 nmol/L) as chemoattractant. Chemerin‐induced enhancement of migration was inhibited by siRNA against CMKLR1. Seven random microscopic fields per each well were quantified. All assays were performed in triplicate. The results are shown as the mean ± SE. *P < 0.05 versus control.

Figure 8. Chemerin promotes the differentiation of HUVECs into tube‐like structures via CMKLR1. HUVECs were seeded on Matrigel‐coated culture dishes in the presence of chemerin (10 nmol/L) Chemerin‐induced enhancement of angiogenesis was inhibited by siRNA against CMKLR1. (A) Representative cultures are shown. (B) Quantitative analysis of tube formation. Total tube length per each 10 microscopic area was calculated using image analyzer software. All assays were performed in triplicate. The results are shown as the mean ± SE. *P < 0.05 versus control.

Figure 9. Proposed scheme for chemerin‐stimulated signaling of proliferation and migration. Chemerin promotes angiogenesis and proliferation activity of HUVECs via MEK/ERK and PI3‐K/Akt, whereas chemerin promotes the migration activity only via PI3‐K/Akt.