Figure 1.
NE increased EPCs in peripheral blood in mice with limb ischemia.
Cells from peripheral blood were lysed and analyzed by flow cytometry. Cells were sequentially gated based on CD45 (A), CD34 and KDR expression (C). Circulating EPCs were defined as CD45-/CD34+/KDR+ cells. A gate was used to select the total CD45- cell population (A). Corresponding flow cytometric analysis was used to detect CD34+/KDR+ cells in the gated CD45- cell population. Proportion of EPCs in peripheral blood was increased from 0.15±0.04% to 0.31±0.05% after intraperitoneal injection of NE in limb ischemia model (B, * P<0.05 compared with the model group). Representative flow cytometric analysis of EPCs (CD34/Flk-1 cells) were showed in part C.
Figure 2.
NE increased EPCs in BM in mice with limb ischemia.
Cells from peripheral blood were lysed and analyzed with flow cytometry. BM derived EPCs were defined as CD34+/KDR+ cells (B). Proportion of EPCs in BM was increased from 1.47±0.29% to 4.23±1.01% after intraperitoneal injection of NE in mice with limb ischemia (A, * P<0.05 compared with model group). Representative flow cytometric analysis of EPCs (CD34+/Flk-1+cells) were showed in part B.
Figure 3.
NE increased EPCs in spleen in mice bearing limb ischemia.
Cells from splenic tissue homogenates were lysed and analyzed with flow cytometry. EPCs in spleen were defined as CD34+/KDR+ cells (B). Proportion of EPCs in spleen was increased from 1.94±0.39% to 4.89±0.36% after intraperitoneal injection of NE in mice with limb ischemia (A, * P<0.05 compared with model group). Representative flow cytometric analysis of EPCs (CD34+/Flk-1+cells) were showed in part B.
Figure 4.
The angiogenesis in the caudal gastrocnemius was augmented by NE and blocked by Phentolamine and 127 in vivo.
Cells positive for CD31 and muscle fiber number were both counted. The ratio of capillary number to muscle fiber number was determined. NE significantly increased hind-limb vascularization as assessed by capillary densitometry (2.35-fold increase relative to PBS-treated mice; P<0.01, Figure 4). The increase of neovessel formation in ischaemic muscles was inhibited by co-treatment of Phentolamine (1.56-fold increase relative to PBS-treated mice; P<0.01) and selective β 2 adrenoceptor blocker I127 (0.81-fold increase relative to PBS-treated mice; P<0.01), but not Metoprolol (2.71-fold increase relative to PBS-treated mice; P<0.01).
Figure 5.
Effects of NE on VEGF production.
The blood samples, BM homogenates, skeletal muscle and splenic tissue homogenates were collected from limb ischemic mice and measured by ELISA. NE significantly increased VEGF concentrations in both skeletal muscle and blood sumples, but did not affect it in bone marrow and spleen. Co-treatment of Phentolamine, I127attenuated the increase of VEGF in bone marrow and skeletal muscle, but did not affect it in blood samples and spleen.
Figure 6.
Characterization of EPCs derived from human peripheral circulation.
EPCs exhibitedspindle-shaped or cobblestone-like morphology and were stained by DAPI and double labeled by Dil-Ac-LDL and FITC-UEA-I.
Figure 7.
Proliferation potential of EPCs was increased by NE and blocked by Phentolamine, I127, LY294002 and L-NAME in vitro.
Growth capacity of PBMC-derived EPCs after 8 days of culture was evaluated (n = 6). As shown by multiwell spectrophotometer, the treatment of NE induced a significant increase of the number of EPCs in the S-phase in a dose-dependent manner, as well as proliferation of EPC (* P<0.05 compared with model group). Pretreatment of phentolamine, I127, LY294002 and L-NAME significantly blocked the effect of NE on EPCs proliferation (Figure 5), but Metoprolol could not.
Figure 8.
Migrative activity of EPCs was increased by NE and blocked by Phentolamine and 127 in vitro.
The migrative activity of EPCs was detected using transwell chamber assay. EPCs stayed on the membrane in the upper chamber were wiped off with a cotton swab. EPCs stayed on the lower membrane of transwell filter were stained with 1% crystal violet solution and counted in six random high-power (100×) microscope fields. NE increased migration of EPCs in a dose dependent manner (* P<0.05 compared with control group). This effect could be blocked by either pretreatment of phentolamine or I127, but not Metoprolol. Representative figure of EPCs passed through the holes into the lower chamber of each group are showed here.
Figure 9.
Inhibition of α adrenoceptor and β 2 adrenoceptor attenuated the elevation of the proportion of EPCs in peripheral circulation.
Cells from peripheral blood were lysed and analyzed with flow cytometry. Cells were sequentially gated based on CD45 (A), CD34 and KDR expression (C). Circulating EPCs were defined as CD45-/CD34+/KDR+ cells. A gate was used to select the total CD45- cell population (A). Corresponding flow cytometric analysis was used to detect CD34+/KDR+ cells in the gated CD45- cell population. Co-treatment of either phentolamine or I127 with NE significantly attenuated the increases of EPCs in peripheral circulation, but Metoprolol didn't (B, *P<0.05 compared with the model group.). Representative flow cytometric analysis of EPCs (CD34/Flk-1 cells) were showed in part C.
Figure 10.
Inhibition of α adrenoceptor and β 2 adrenoceptor attenuated the elevation of EPCs in BM.
Cells from BM homogenates were lysed and analyzed with flow cytometry. BM derived EPCs were defined as CD34+/KDR+ cells (B). Co-treatment of either Phentolamine or I127 with NE significantly attenuated the increases of EPCs in peripheral circulation, but Metoprolol didn't (B, *P<0.05 compared with model group). Representative flow cytometric analysis of EPCs (CD34+/Flk-1+cells) were showed in part B.
Figure 11.
Inhibition of α adrenoceptor and β 2 adrenoceptor attenuated the elevation of EPCs in spleen. Cells from splenic tissue homogenates were lysed and analyzed with flow cytometry.
EPCs in spleen were defined as CD34+/KDR+ cells (B). Co-treatment of either Phentolamine or I127 with NE significantly attenuated the increases of EPCs in spleen, but Metoprolol didn't (B, *P<0.05 compared with model group.). Representative flow cytometric analyses of EPCs (CD34+/Flk-1+cells) were showed in part B.
Figure 12.
NE activated EPCs via Akt/eNOS pathways.
Treatment of NE significantly increased phosphorylation of Akt, eNOS. Addition of 10 µM Phentolamine and 10 µM I127 attenuated the activation of Akt/eNOS pathway. However, addition of 10 µM Metorolol could not. Representative immunoblots (top) and densitometric quantification (bottom) demonstrated phosphorylation of Akt and eNOS in the following groups: control, 0.1 µM NE, 1 µM NE, 10 µM NE, 100 µM NE, NE plus Phentolamine, NE plus Metorolol and NE plus I127. *P<0.05 vs control.