Elevated arterial shear rate increases indexes of endothelial cell autophagy and nitric oxide synthase activation in humans

Abstract

Continuous laminar shear stress increases the process of autophagy, activates endothelial nitric oxide (NO) synthase phosphorylation at serine 1177 (p-eNOS^S1177), and generates NO in bovine and human arterial endothelial cells (ECs) compared with static controls. However, the translational relevance of these findings has not been explored. In the current study, primary ECs were collected from the radial artery of 7 men using sterile J-wires before (Pre) and after (Post) 60 min of rhythmic handgrip exercise (HG) performed with the same arm. After ECs were identified by positive costaining for vascular endothelial cadherin and 4',6'-diamidino-2-phenylindole, immunofluorescent antibodies were used to assess indices of autophagy, NO generation, and superoxide anion (O₂^·-) production. Commercially available primary human arterial ECs were stained and processed in parallel to serve as controls. All end points were evaluated using 75 ECs from each subject. Relative to Pre-HG, HG elevated arterial shear rate ( P < 0.05) ~3-fold, whereas heart rate, arterial pressure, and cardiac output were not altered. Compared with values obtained from ECs Pre-HG, Post-HG ECs displayed increased ( P < 0.05) expression of p-eNOS^S1177, NO generation, O₂^·- production, BECLIN1, microtubule-associated proteins 1A/1B light chain 3B, autophagy-related gene 3, and lysosomal-associated membrane protein 2A and decreased ( P < 0.05) expression (i.e., enhanced degradation) of the adaptor protein p62/sequestosome-1. These novel findings provide evidence that elevated arterial shear rate associated with functional hyperemia initiates autophagy, activates p-eNOS^S1177, and increases NO and O₂^·- generation in primary human ECs. NEW & NOTEWORTHY Previously, our group reported in bovine arterial and human arterial endothelial cells (ECs) that shear stress initiates trafficking of the autophagosome to the lysosome and increases endothelial nitric oxide (NO) synthase phosphorylation at serine 1177, NO generation, and O₂^·- production. Here, the translational relevance of these findings is documented. Specifically, functional hyperemia induced by rhythmic handgrip exercise elevates arterial shear rate to an extent that increases indices of autophagy, NO generation, and O₂^·- production in primary arterial ECs collected from healthy men.

Keywords: blood vessel; exercise; immunofluorescence; shear stress.

Fig. 1.

A: subject instrumentation. BA, brachial artery; BP, blood pressure; ECG, electrocardiogram; RA, radial artery. B: rhythmic handgrip exercise (HG) increased arterial shear rate. BL, baseline, i.e., Pre-HG. Values are means ± SE from 7 subjects. *P < 0.01 vs. BL.

Fig. 2.

Elevated arterial shear rate increases endothelial nitric oxide (NO) synthase phosphorylation at serine 1177 (p-eNOS^S1177), NO generation, and O₂^·− production. Relative to values obtained before rhythmic handgrip exercise (Pre-HG), Post-HG endothelial cells (ECs) exhibited elevated p-eNOS^S1177 (A and C), NO generation (E), and O₂^·− production (F), whereas eNOS (B and D) protein expression was not altered by HG. Each panel presents a representative EC from each staining protocol: bottom left, vascular endothelial cadherin (VE-cadherin; green); bottom right, 4′,6′-diamidino-2-phenylindole (DAPI; blue); top left, protein of interest; and top right, merge. Mean intensity of the protein of interest (top left) is documented below each representative image. For A–D, n = 7 subjects. Note: two representative images are shown for p-eNOS^S1177 and eNOS. For E and F, n = 3 subjects. *P < 0.05 vs. Pre-HG. Scale bar represents 10 µm. Magnification = ×60. Symbols aligned with the histogram indicate individual subject data points. AU, arbitrary units; DAF, diaminofluorescein diacetate; DHE, dihydroethidium; HAEC, human arterial EC.

Fig. 3.

Elevated arterial shear rate increases indices of autophagy in primary arterial endothelial cells (ECs). Relative to values obtained before rhythmic handgrip exercise (Pre-HG), Post-HG ECs exhibited increased expression of BECLIN1 (A), autophagy-related gene 3 (ATG3; B), microtubule-associated proteins 1A/1B light chain 3B [LC3B; C; quantification of LC3 puncta from figures of each group are analyzed by mean LC3 puncta per cell (n = 30 cells per subject)], and lysosomal-associated membrane protein 2A (LAMP2A; E) and decreased expression (i.e., enhanced degradation) of the adaptor protein p62/sequestosome-1 (D). The 4 quadrants of each panel are described in the legend of Fig. 2. Data are reported as the ratio of Pre-HG or Post-HG EC staining intensity divided by human arterial EC (HAEC) staining intensity. Mean intensity of the protein of interest (top left) is documented to the right of each representative image. For A–E, n = 7 subjects. *P < 0.05 vs. Pre-HG. Scale bar represents 10 µm. Magnification = ×60. Symbols aligned with the histogram indicate individual subject data points. AU, arbitrary units; DAPI, 4′,6′-diamidino-2-phenylindole; VE-cadherin, vascular endothelial cadherin.