PGC1α Regulates the Endothelial Response to Fluid Shear Stress via Telomerase Reverse Transcriptase Control of Heme Oxygenase-1

Abstract

Objective: Fluid shear stress (FSS) is known to mediate multiple phenotypic changes in the endothelium. Laminar FSS (undisturbed flow) is known to promote endothelial alignment to flow, which is key to stabilizing the endothelium and rendering it resistant to atherosclerosis and thrombosis. The molecular pathways responsible for endothelial responses to FSS are only partially understood. In this study, we determine the role of PGC1α (peroxisome proliferator gamma coactivator-1α)-TERT (telomerase reverse transcriptase)-HMOX1 (heme oxygenase-1) during shear stress in vitro and in vivo. Approach and Results: Here, we have identified PGC1α as a flow-responsive gene required for endothelial flow alignment in vitro and in vivo. Compared with oscillatory FSS (disturbed flow) or static conditions, laminar FSS (undisturbed flow) showed increased PGC1α expression and its transcriptional coactivation. PGC1α was required for laminar FSS-induced expression of TERT in vitro and in vivo via its association with ERRα(estrogen-related receptor alpha) and KLF (Kruppel-like factor)-4 on the TERT promoter. We found that TERT inhibition attenuated endothelial flow alignment, elongation, and nuclear polarization in response to laminar FSS in vitro and in vivo. Among the flow-responsive genes sensitive to TERT status, HMOX1 was required for endothelial alignment to laminar FSS.

Conclusions: These data suggest an important role for a PGC1α-TERT-HMOX1 axis in the endothelial stabilization response to laminar FSS.

Keywords: atherosclerosis; heme oxygenase-1; peroxisome proliferators; telomerase; thrombosis.

Figure 1.. PGC1α regulates endothelial cell function during fluid shear stress.

A-B) PGC1α mRNA expression (A) or protein expression (B) from human aortic endothelial cells (HAECs) were measured by RT-qPCR or western blots after cells were subjected to static, laminar or oscillatory fluid shear stress (FSS) for 48 hours (n = 5). C) Bright-field image of mouse lung endothelial cells (MLECs) isolated from either wild-type (WT) or PGC1α-ECKO mice after exposure to laminar FSS. D) MLECs from WT and PGC1α-ECTG mice were exposed to laminar FSS, and RT-qPCR was performed for the indicated genes (n = 5). E) HAECs were treated with scrambled or PGC1α siRNA, and RT-qPCR was performed for the indicated genes after exposure to laminar FSS for 48 hours (n = 5). F-G) Aortae were isolated from either WT (F) or PGC1α-ECTG (G) mice, mRNA isolated, and RT-qPCR performed in the indicated regions reflecting disturbed or laminar flow (n = 5). (H) Sample sites of disturbed (oscillatory) vs. laminar fluid shear stress in mouse aorta. (I) En face staining with β-Catenin and DAPI in WT and PGC1α-ECKO aorta. Scale bar, 20 μm. (J) Composite data of length/width ratio of the endothelium in the thoracic region of mouse aortae (n = 18–24). All experiments were repeated 3–5 times. Statistically significant differences were measured by Student’s t-test or one-way ANOVA with post-hoc comparison as appropriate with control group. The data are mean ± SEM.

Figure 2.. PGC1α is required for endothelial functional responses to exercise.

A) Aortae were isolated from WT or PGC1α-ECKO mice and gene expression determined in total aortic tissue by RT-qPCR for the indicated genes before and after exercise; n= 7/group (PGC1α mRNA expression), n=18–32/group (TERT) and n=12–21/group (HMOX1). B) Reactive oxygen species (ROS) production was measured as indicated in WT and PGC1α-ECKO mice before and after exercise; n=8–12/group (MitoSOX), n=16–17/group (DHE) and n=10–20/group (AmplexRed). C) MLECs were isolated from WT mice with or without exercise, and PGC1α mRNA expression determined by RT-qPCR before and after exercise. D) Endothelial function measured as aortic isometric force in response to acetylcholine (Ach; n=10–12). E) Nitroglycerin-mediated smooth muscle cell function by treatment and genotype (NTG; n= 8–12;). Statistically significant differences were measured by Student’s t-test or either one-way or two-way ANOVA with post-hoc comparison as appropriate with control group. The data are mean ± SEM. P values vs. control mice (Black); vs. control + exercise mice (Red) and vs. PGC1α-ECKO mice (Blue).

Figure 3.. PGC1α regulates TERT expression.

A-B) HAECs were either treated with scrambled or ERRα siRNA (A) or scrambled or KLF4 siRNA (B), and RT-qPCR was performed for different shear stress-related genes after exposure of cells to laminar FSS for 48 hours (n = 5). C) HAECs were lysed and immunoprecipitation (IP) was performed with control IgG or ERRα antibody and immunoblotting was done with antibodies against PGC1α and KLF4. Lysates were examined by probing with GAPDH antibody. D-E) MLECs were isolated from control and PGC1α-ECTG mice, and either RT-qPCR (D) or immunoblot analysis (E) was performed with the probes and antibodies as indicated. F) Lysates prepared from HAECs treated with control shRNA or shRNA against PGC1α (48 hrs) were examined by immunoblot analysis using antibodies for TERT, PGC1α and GAPDH. G) MLECs were isolated from WT and PGC1α-ECKO mice and mRNA expression was measured by RT-qPCR for TERT gene before and after exercise (n = 6). H) Aortae were isolated, and mRNA expression was measured for TERT by RT-qPCR in the arch and thoracic region of WT mice (n = 5). I-J) ChIP-qPCR analysis of PGC1α recruitment to the TERT promoter region was performed in HAECs (n = 5). All the experiments were repeated 3–6 times. Statistically significant differences between groups are indicated. Statistically significant differences were measured by Student’s t-test or one-way ANOVA with post-hoc comparison as appropriate with control group. P values vs. control group (black) + exercise group (red) by Student’s t-test. The data are mean ± SEM.

Figure 4.. TERT is required for endothelial alignment to the flow.

A) Bright-field image of HAECs after exposure to laminar FSS in the presence of either control or TERT inhibitor. B) mRNA was isolated and PGC1α expression for cells treated with the vehicle control or TERT inhibitor was measured by RT-qPCR (n = 6). C) HAECs morphology was measured by staining for CD31/DAPI as a function of pharmacologic (iTERT) or genetic (siTERT) inhibition of TERT (Scale bar, 25 μm). D) Length to width ratio of CD31 stain in HAECs was measured after TERT inhibition in the presence of laminar FSS (n = 15–19). E) HAEC nuclear polarization towards the direction of laminar flow was measured with GOLPH4 (Golgi) and DAPI (nuclei) staining with and without TERT inhibitor (Scale bar, 10 μm). F) Compass plots of Golgi/nuclear angle as a function of TERT inhibition. Each ring represents an observation of an average of different fields of control or TERT inhibitor-treated cells. All the experiments were repeated 3 – 6 times. Statistically significant differences between groups are indicated (P values vs. control by Student’s t-test). The data are mean ± SEM.

Figure 5.. TERT regulates mitochondrial structure and function.

A) Mitochondrial morphology and mass (MitoTracker fluorescence) were measured in the presence of laminar flow with and without TERT inhibition (iTERT- BIBR1532) and compared with oscillatory flow (Scale bar, 5 μm). B) Quantification of mitochondrial mass (n = 5) and morphology during FSS (n = 22–25). C) ROS production in HAECs either treated with control or TERT inhibitor (n = 9–13). D) En face staining with β-Catenin and Hoechst 33342 in WT and TERT knockout aorta (Scale bar, 10 μm). E) Composite data of length/width ratio of the endothelium in WT and TERT knockout aortae. n = as indicated in each group. All experiments were repeated 3 – 6 times. Statistically significant differences were measured by Student’s t-test or one-way ANOVA with post-hoc comparison as appropriate with control group. The data are mean ± SEM.

Figure 6.. PGC1α-TERT regulates HMOX1 expression.

A) MLECs were isolated from control and PGC1α-ECTG mice and immunoblot analysis was performed with the antibodies as indicated. B) MLECs were isolated from WT and PGC1α-ECKO mice aortae, and mRNA expression was measured by RT-qPCR for the HMOX1 gene before and after exercise (n = 6). C) HUVECs were exposed to laminar or oscillatory FSS with or without TERT inhibitor and immunoblot analysis was performed with the antibodies as indicated. D) HAECs were either treated with scrambled or HMOX1 siRNA and RT-qPCR was performed for different genes related to endothelial function after exposure to laminar FSS for 48 hours (n = 6). E) Bright-field image of HAECs after exposure to laminar FSS in the presence of either control (CuPP 0.25 μM) or two different HMOX1 inhibitors (ZnPP 0.25 μM and OB 24 hydrochloride 0.25 μM). F) Cell and mitochondrial morphology (MitoRed fluorescence) were imaged in the presence of laminar flow with and without HMOX1 inhibition (Scale bar, 5 μm). G) Schematic diagram of LSS-induced PGC1α-TERT-HMOX1 pathway. n = 6 in each group. All the experiments were repeated 3 – 5 times. Statistically significant differences between groups are indicated. Statistically significant differences were measured by Student’s t-test or one-way ANOVA with post-hoc comparison as appropriate with control group. P values vs. control group (black) + exercise group (red) by Student’s t-test). The data are mean ± SEM.