Cell-Specific Protective Signaling Induced by the Novel AT2R-Agonist NP-6A4 on Human Endothelial and Smooth Muscle Cells

Figures

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  FIGURE 1. NP-6A4 increased Agtr2 mRNA and AT2R protein expression in hCAVSMC. (A) 1 μM NP-6A4 for 12-h and 7-day increased Agtr2 mRNA expression. (B) No change in Agtr1 mRNA was detected after 1 μM NP-6A4 for 7-days. n = at least three biological replicates and PCR replicates, respectively, for these experiment. (C) Representative images of hCAVSMCs treated with 1 μM NP-6A4 for 12-h. Blue indicates DAPI stained nuclei and green AT2R. Merged images are shown for all experimental conditions. (D) Quantification of relative fluorescent units (RFU) shows increased expression of AT2R protein after 1 μM NP-6A4 treatment for 12-h and 20 μM PD123319 prevented this increase. Immunofluorescent experiments consisted of quantification of >50 cells from triplicates per experiment. ∗Indicates p < 0.05 using One-way ANOVA. hCAVSMC, human coronary artery vascular smooth muscle cell; FC, fold change.

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  FIGURE 2. NP-6A4 increased Agtr2 mRNA and AT2R protein expression in hCAECs. (A) 1 μM NP-6A4 for 12-h showed no difference in Agtr2 mRNA, while 7-day treatment increased Agtr2 mRNA expression twofold. (B) No change in Atgr1 was detected after 1 μM NP-6A4 for 7-days. n = at least three biological replicates and PCR replicates, respectively, for these experiment. (C) Representative images of hCAECs treated with 1 μM NP-6A4 for 12-h. Blue indicates DAPI stained nuclei and green AT2R. Merged images are shown for all experimental conditions. (D) Quantification of relative fluorescent units (RFU) shows increased expression of AT2R protein after 1 μM NP-6A4 treatment for 12-h. AT2R-siRNA prevented NP-6A4-induced increase in AT2R expression. Immunofluorescent experiments consisted of quantification of >50 cells from triplicates per experiment. ∗Indicates p < 0.05 using One-way ANOVA. hCAEC, human coronary artery endothelial cell; FC, fold change.

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  FIGURE 3. NP-6A4 increased Agtr2 mRNA and AT2R protein expression in hUVEC. (A) 1 μM NP-6A4 for 12-h showed no difference in Agtr2 mRNA, while 7-day treatment increased Agtr2 mRNA expression eightfold. (B) No change in Atgr1 was detected after 1 μM NP-6A4 for 7-days. n = at least three biological replicates and PCR replicates, respectively, for these experiment. (C) Representative images of hUVECs treated with 1 μM NP-6A4 for 12-h. Blue indicates DAPI stained nuclei and green AT2R. Merged images are shown for all experimental conditions. (D) Quantification of relative fluorescent units (RFU) shows increased expression of AT2R protein after 1 μM NP-6A4 treatment for 12-h and 20 μM PD123319 prevented this increase. Immunofluorescent experiments consisted of quantification of >50 cells from triplicates per experiment. ∗Indicates p < 0.05 using One-way ANOVA. hUVEC, human umbilical vein endothelial cell; FC, fold change.

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  FIGURE 4. NP-6A4 increased hCAVSMC growth properties and mitochondrial energetics. (A) Cell Index in cells pretreated with NP-6A4 for 7-days and then treated with increasing doses of acute NP-6A4 from 0 to 20 μM. All doses increased CI compared to untreated (CTRL) cells. Data were analyzed using a two-way ANOVA with dose and time serving as independent variables. ∗indicates p < 0.05 vs. CTRL cells at baseline. #Indicates p < 0.05 vs. 0 μM at same time point. †Indicates p < 0.05 vs. 5 μM at the same time point. n = 4 per group. (B) OCR increased after 7-days of 1 μM NP-6A4 treatment measured using the Seahorse Extracellular Flux Assay. (C) ATP production was increased after NP-6A4 treatment. (D) Maximal respiration was increased after NP-6A4 treatment. (E) Spare respiratory capacity was increased after NP-6A4 treatment. n = 3 per group. ∗Indicates p < 0.05 using One-way ANOVA. hCAVSMC, human coronary artery vascular smooth muscle cell.

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  FIGURE 5. NP-6A4 reduces reactive oxygen species while not altering mitochondrial number in hCAVSMCs. (A) Cellular ROS was increased with 1 μM Doxorubicin, while 1 μM NP-6A4 reduced ROS generation in both normal media and 1 μM Doxorubicin. n = 4. (B) Mitochondrial DNA was non-significantly increased after 7 days of NP-6A4 treatment. n = 4. (C) Oxidative phosphorylation complexes quantification of Western Blot images. n = 3. (D) Representative image of the Western blot for oxidative phosphorylation complexes after 7-days of NP-6A4 treatment. ∗Indicates p < 0.05 vs. CTRL using two-tailed Student’s t-test. §Indicates p < 0.05 vs. CTRL cells (normal media). ‡Indicates p < 0.05 vs. NP-6A4 preTx cells (normal media). OCR, oxygen consumption rate; hCAVSMC, human coronary artery vascular smooth muscle cell.

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  FIGURE 6. Growth promoting effects of NP-6A4 are similar to Angiotensin II in hCAECs. (A) NP-6A4 did not increase CI in hCAEC, while AT2R-agonist PD123319 decreased CI initially but stabilized within 24-h. (B) Likewise, Ang II did not change CI compared to normal medium, while PD123319 attenuated CI at 6 h post treatment but stabilized within 24-h. n = 4. Control data is the same in A and B. CI, cell index; Ang II, angiotensin II; hCAECs, human coronary artery endothelial cells.

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  FIGURE 7. AT2R siRNA inhibited growth properties (CI) of hCAEC both in the absence and presence of NP-6A4. (A) Cell Index curve depicting change in cell index (CI). Arrow indicates addition of NP-6A4. (B) Differences in CI after 12 h of treatment with anti-AT2R siRNA (labeled siRNA) or siRNA-Scrambled (scRNA). At this point none of the cells had been treated with NP-6A4. n = 4 per group. (C) Differences in CI approximately 4-h after the addition of NP-6A4 (20 μM). n = 4 per group. ∗Indicates p < 0.05 vs. CTRL using one-way ANOVA at the indicated time points.

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  FIGURE 8. NP-6A4 promotes cellular protective signaling in hCAECs. (A) Representative images of DAF-FM diacetate staining in hCAEC. Blue DAPI images show nuclei, Red wheat germ agglutinin (WGA) images show endothelial cell membrane, Green DAF-FM images show presence of detectable nitric oxide, and lastly, merged images show al three colors on one image. n = 3. (B) Quantification of detectable NO showed that 5 μM NP-6A4 for 24 h increased NO, while PD123319 pretreatment prevented the increase. Fluorescent staining experiments consisted of quantification of >50 cells from triplicates. (C) Representative western blot images of eNOS and phospho-eNOS protein. (D) Quantification of eNOS and phosphos-eNOS show increased expression of eNOS after 12-h of 1 μM NP-6A4 treatment. The total to phospho-eNOS ratio was not affected. n = 4. (E) 5 μM NP-6A4 of hCAEC for 24-h decreased phosphorylated-JNK as determined by the Milliplex MAP Multi-Pathway Total and Phospho- Magnetic Bead 9-Plex Assay. ∗∗Indicates difference from CTRL and ∗Indicates difference from NP-6A4-treated at p < 0.05 using One-way ANOVA. Yellow scale bar = 100 microns. hCAEC, human coronary artery endothelial cell; DAF-FM =…