Shear stress-induced mitochondrial biogenesis decreases the release of microparticles from endothelial cells

Abstract

The concept of enhancing structural integrity of mitochondria has emerged as a novel therapeutic option for cardiovascular disease. Flow-induced increase in laminar shear stress is a potent physiological stimulant associated with exercise, which exerts atheroprotective effects in the vasculature. However, the effect of laminar shear stress on mitochondrial remodeling within the vascular endothelium and its related functional consequences remain largely unknown. Using in vitro and in vivo complementary studies, here, we report that aerobic exercise alleviates the release of endothelial microparticles in prehypertensive individuals and that these salutary effects are, in part, mediated by shear stress-induced mitochondrial biogenesis. Circulating levels of total (CD31(+)/CD42a(-)) and activated (CD62E(+)) microparticles released by endothelial cells were significantly decreased (∼40% for both) after a 6-mo supervised aerobic exercise training program in individuals with prehypertension. In cultured human endothelial cells, laminar shear stress reduced the release of endothelial microparticles, which was accompanied by an increase in mitochondrial biogenesis through a sirtuin 1 (SIRT1)-dependent mechanism. Resveratrol, a SIRT1 activator, treatment showed similar effects. SIRT1 knockdown using small-interfering RNA completely abolished the protective effect of shear stress. Disruption of mitochondrial integrity by either antimycin A or peroxisome proliferator-activated receptor-γ coactivator-1α small-interfering RNA significantly increased the number of total, and activated, released endothelial microparticles, and shear stress restored these back to basal levels. Collectively, these data demonstrate a critical role of endothelial mitochondrial integrity in preserving endothelial homeostasis. Moreover, prolonged laminar shear stress, which is systemically elevated during aerobic exercise in the vessel wall, mitigates endothelial dysfunction by promoting mitochondrial biogenesis.

Keywords: endothelial microparticle; exercise; mitochondrial biogenesis; shear stress.

Fig. 1.

Effects of aerobic exercise training (AEXT) and shear stress on endothelial cell microparticle and nitric oxide production. A and B: plasma CD62⁺ endothelial cell microparticle (EMP), CD31⁺/CD42a⁻ EMP, and nitric oxide (NOx) levels from human subjects at before (pre) and after (post) 6-mo AEXT (n = 21). C and D: 7 separate human umbilical vein endothelial cell (HUVEC) cell lines from 7 different donors were exposed to either low (5 dyne/cm²) or high (20 dyne/cm²) laminar shear stress (LSS) for 24 h. Accumulated CD62⁺EMP and NOx were measured from the cell culture media. Each column represents means ± SE. *P < 0.05; **P < 0.01.

Fig. 2.

Shear stress induces mitochondrial biogenesis in human endothelial cells. A: phase-contrast images of HUVECs exposed to LSS at 5 to 20 dyne/cm² for 36 h; 0 dyne/cm² indicates static control (STT). Arrows indicate the direction of flow. Scale bars indicate 100 μm. B: cell lysates were analyzed by Western blotting using anti-SIRT1, anti-peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), and anti-transcription factor A (TFAM) antibodies. The bar graphs are results of densitometry analyses. C: mitochondrial content determined by abundance of porin. For both Western blots in B and C, α-Tubulin was used as a loading control. D: representative fluorescence microscopic images of MitoTracker Green FM staining of HUVECs under static or LSS (20 dyne/cm² for 36 h). Nu, nucleus. The bar graphs are results of densitometry analysis. E: mitochondrial DNA copy number calculated by relative mitochondrial DNA (mtDNA)-encoded genes (COX I or COX II) copy number normalized by 18S. Bar graphs in all panels are means ± SE from 3–5 independent experiments. *P < 0.05; **P < 0.01 vs. static control.

Fig. 3.

EMP release inversely correlates with LSS-induced mitochondrial biogenesis. Confluent monolayers of HUVECs were subjected to LSS (20 dyne/cm²) for 36 h. A: activated- (CD62E⁺) or total- (CD31⁺/CD42a⁻) EMP released from HUVECs under STT or LSS. B: Pearson's correlation plots between mitochondrial content vs. CD62E⁺ EMP or CD31⁺/CD42a⁻ EMP. C and D: cell surface expression of CD62E determined by flow cytometry. Bar graphs in all panels are means ± SE from 3–5 independent experiments. *P < 0.05; **P < 0.01 vs. static control.

Fig. 4.

EMP release inversely correlates with mitochondrial enrichment. Confluent monolayers of HUVECs were subjected to various conditions of resveratrol (RSV; 20 μM) or RSV + LSS (20 dyne/cm²) combination treatment for the indicated times. A and B: HUVECs were incubated with RSV at various concentrations for 24 h or at 20 μM for various times. Cell lysates were analyzed by Western blotting with specific antibodies. C: mitochondrial content. D: activated- (CD62E⁺) or total- (CD31⁺/CD42a⁻) EMPs. Bar graphs in all panels are means ± SE from 3–5 independent experiments. *P < 0.05; **P < 0.01 vs. static control. α-Tub, α-Tubulin; SIRT, sirtuin 1.

Fig. 5.

LSS reduces EMP release by SIRT1-mediated mitochondrial biogenesis. HUVECs were transiently transfected with either scrambled or SIRT1 small-interfering RNA (siRNA) and maintained in STT or LSS (20 dyne/cm² for 24 h). A: SIRT1, PGC-1α, and TFAM protein expressions. α-Tubulin was used as a loading control (CTR). The bar graphs are results of densitometry analyses. B: mitochondrial content. C: activated- (CD62E⁺) or total- (CD31⁺/CD42a⁻) EMP. Each column represents means ± SE from 3–5 independent experiments. *P < 0.05; **P < 0.01. N.S., not significant.

Fig. 6.

LSS normalizes increased EMP production in endothelial cells with defective mitochondria. HUVECs were treated with antimycin A (AA; 10 μM) or LSS (20 dyne/cm²) for 12 h. Antimycin A-preconditioned HUVECs were subjected to STT or LSS (20 dyne/cm²) for succeeding 24 h. A: mitochondrial content. B: activated- (CD62E⁺) or total- (CD31⁺/CD42a⁻) EMP. Each column represents means ± SE from 3–5 independent experiments. *P < 0.05.

Fig. 7.

Schematics of the proposed mechanism.