Figure 1.
Effect of AT2R Activation on Cardiomyocyte Protection of BMMNCs in vitro.
(A to F) BMMNCs were initially pre-incubated with DMEM, Ang II, CGP42112A, AngII+Valsartan, or AngII+Valsartan+PD123319 for 2 hours at 37°C, respectively. Then preconditioned BMMNCs were co-cultured with NRCMs under hypoxia in serum free medium for 48 hours. Apoptotic NRCMs were detected using TUNEL assay. Bar=100μm; Green represents TUNEL positive cells; Red represents Troponin T (TnT); Blue represents nuclei; and yellow arrowhead represents apoptotic NRCMs. (G) Quantification of apoptotic NRCMs. n=3 for each group; *P < 0.05 versus NRCMs single culture group and BMMNCs group, #P < 0.05 versus BMMNCs+AngII+Valsartan+PD123319 group.
Figure 2.
Activation of AT2R/p-ERK/eNOS/NO Pathway in BMMNCs upon AT2R Stimulation.
(A) Dynamic change of ERK phosphorylation after 10 nM CGP42112A treatment at different time points. n=3-4 for each group; *P < 0.05 versus baseline (0 min). (B) Dynamic change of ERK phophorylation after 10 nM CGP42112A and 1μM PD123319 co-treatment at different time points. n=5 for each group. (C) Expression of ERK phosphorylation in preconditioned BMMNCs. n=6 for each group; *P < 0.05 versus BMMNCs group, #P < 0.05 versus BMMNCs+AngII+Valsartan+PD123319 group. (D) eNOS expression of preconditioned BMMNCs was assessed by Western blot. n=6 for each group; *P < 0.05 versus all other groups. (E) NO production in cultured medium was determined with a commercial kit. BMMNCs+CGP42112A group n=3; and n=4 for other groups. *P < 0.05 versus all other groups.
Figure 3.
AT2R/p-ERK/eNOS/NO Pathway was Involved in Cardioprotection of BMMNC in vitro.
(A to I) BMMNCs were initially pre-incubated with DMEM, CGP42112A±U0126, CGP42112A±L-NAME, AngII+Valsartan±U0126, AngII+Valsartan±L-NAME, or AngII+Valsartan+PD123319 for 2 hours at 37 °C, respectively. Then preconditioned BMMNCs were co-cultured with NRCMs under hypoxia in serum free medium for 48 hours. Apoptotic NRCMs were detected using TUNEL assay. Bar=100μm; Green represents TUNEL positive cells; Red represents Troponin T (TnT); Blue represents nuclei; and yellow arrowhead represents apoptotic NRCMs. (J) Quantification of apoptotic NRCMs. Single culture NRCMs group n=6; and other groups n=4 for each group; *P < 0.05 versus all other groups.
Figure 4.
Transplantation of AT2R Stimulated BMMNCs Improved Global Heart Function and Reduced Infarct Size.
(A) Representative M-mode images showing cardiac function in each group. (B) Ejection Fraction; (C) Fraction Shortening; (D) LVIDd; and (E) LVIDs. Sham n=5; DMEM group n=8; BMMNCs group n=9; BMMNCs + AngII + Valsartan group n=10; BMMNCs +CGP42112A group n=10; and BMMNCs + AngII + Valsartan + PD123319 group n=9. *P <0.05 versus DMEM group and BMMNCs group, #P < 0.05 versus BMMNCs + AngII + Valsartan +PD123319 group. (F) Intramyocardial transplantation of AT2R stimulated BMMNCs reduced infarct size. DMEM group n=4; BMMNCs group n=5; BMMNCs+AngII+Valsartan group n=6; BMMNCs+CGP42112A group n=5; and BMMNCs+AngII+Valsartan+PD123319 group n=4. *P < 0.05 versus DMEM group and BMMNCs group, #P < 0.05 versus BMMNCs+AngII+Valsartan+PD123319 group.
Figure 5.
AT2R Stimulation Enhanced Survival of Transplanted BMMNCs in the Region of Ischemic Myocardium in vivo.
Donor BMMNCs derived from males rats were injected into female rats intramyocardially. 3 days after injection, the abundance of sry gene in ischemic (A) and non-ischemic region (B) of heart tissue was determined by real-time PCR. BMMNCs group n=3; and other groups n=4. *P < 0.05 versus BMMNCs group, and #P < 0.05 versus BMMNCs+AngII+Valsartan+PD123319 group.
Figure 6.
Effect of AT2R Stimulated BMMNCs on Protection of Adult Cardiac Cells 3 days After Myocardial Infarction.
(A to E) TUNEL assay of adult cardiac cells in peri-infarct region of heart tissue. (Bar=100μm; Green represents TUNEL positive cells; Blue represents nuclei; yellow arrowhead represents apoptotic cells). (F) Quantification of TUNEL positive cells. DMEM group n=6; BMMNCs group n=7; BMMNCs+AngII+Valsartan group n=5; BMMNCs+CGP42112A group n=6; and BMMNCs+AngII+Valsartan+PD123319 group n=4. *P < 0.05 versus DMEM group and BMMNCs group, #P < 0.05 versus BMMNCs+AngII+Valsartan+PD123319 group. (G) Western blot analysis of several apoptotic related proteins expression level (Bax, Bcl2, cleaved caspase3, cleaved caspase9, p-Akt, Akt) in border zone respectively. Rat GAPDH was used as control. Quantification of protein level using densitometry analysis (sham n=3; n=3-5 for other each group). *P < 0.05 versus DMEM group and BMMNCs group; #P < 0.05 versus BMMNCs+AngII+Valsartan+PD123319 group.
Figure 7.
Effect of AT2R Stimulated BMMNCs on Inflammatory Markers of Heart Tissue 3 days After Myocardial Infarction.
(A) Real-time PCR of mRNA (IL-1β, IL-6, and MCP-1) expression level in border zone of heart tissues (n=3-4 for each group). *P < 0.01 versus DMEM group and BMMNCs group; #P < 0.01 versus BMMNCs+AngII+Valsartan+PD123319 group. (B &C) Immunofluorescent staining of inflammatory cells (Bar=50μm; Red represents CD68+ cells; Blue represents nuclei) in border zone of heart tissues on 3 days post-MI. Bar graph shows quantitative analysis of infiltrating CD68 positive cells at 3 days post-MI (n=4 for each group). *P < 0.001 versus DMEM group and BMMNCs group, #P < 0.001 versus BMMNCs+AngII+Valsartan+PD123319 group.
Figure 8.
Transplantation of Preconditioned BMMNCs by AT2R Activation Enhanced Angiogenesis in Border Zone on 28 days After MI.
(A) Immunofluorescent staining of vWF and alpha-SMA in border zone of heart tissue on 28 days post-MI; (B) Bar graph showed quantitative analysis of vWF positive vessel density. Bar=100μm; DMEM group n=6; BMMNCs group n=4; BMMNCs+AngII+Valsartan group n=5; BMMNCs+CGP42112A group n=4; BMMNCs+AngII+Valsartan+PD123319 group n=5; *P < 0.05 versus DMEM group and BMMNCs group, #P < 0.05 versus BMMNCs+AngII+Valsartan+PD123319 group. (C) Bar graph showed quantitative analysis of alpha-SMA positive vessel density. Bar=100μm; DMEM group n=6; BMMNCs group n=4; BMMNCs+AngII+Valsartan group n=4; BMMNCs+CGP42112A group n=4; BMMNCs+AngII+Valsartan+PD123319 group n=5; *P < 0.05 versus DMEM group and BMMNCs group; #P < 0.05 versus BMMNCs+AngII+Valsartan+PD123319 group.