Vascular endothelial growth factor receptor 2 contributes to shear stress-induced endothelial nitric oxide synthase expression in human lung microvascular endothelial cells

Abstract

Introduction: Physiological shear stress (pSS) helps to maintain endothelial homeostasis via numerous intracellular signaling pathways. We previously showed that pSS upregulates mRNA and protein levels of endothelial nitric oxide synthase (eNOS) in primary human lung microvascular endothelial cells (hLMVECs), and that phosphorylation of Akt (p-Akt), a phosphatidylinositol 3-kinase (PI3K) target, is increased. Upstream regulatory mechanisms for these responses have not been fully studied. Since vascular endothelial growth factor receptor 2 (VEGFR2) is an integral component of the mechanosensory complex that transduces shear stress-mediated signaling pathways in endothelial cells, we hypothesized that VEGFR2 is required for shear stress-induced eNOS expression and PI3K activation in hLMVECs. Methods: We cultured early passage (p≤7) primary hLMVECs from 4 unique donor lots in standard 6-well culture plates using a fixed volume (depth) and viscosity of tissue culture media. We exposed cells to shear stress for 24 hours using an orbital shaker and calculated the magnitude of shear stress exerted on hLMVECs using the extended solution to Stokes’ second problem. We defined the magnitude of pSS as 12 dyn/cm2. In some experiments, we exposed cells to pSS in the presence of chemical inhibitors of VEGFR2 (SU5416; 5 or 10 μM) or PI3K (LY294002; 10 μM). We collected whole cell lysates and performed immunoblotting to probe for phosphorylated eNOS (p-eNOS; Serine 1177), total eNOS, p-Akt (Serine 473), total Akt and housekeeping protein β-tubulin. Potential interactions between chemical inhibitors and pSS on protein expression and phosphorylation were analyzed by two-way ANOVA. Results: After 24 hours of pSS exposure, total eNOS levels were significantly (p<0.05) increased in hLMVECs, although the p-eNOS/eNOS ratio was not changed (p=0.85). Conversely, 24 hours of pSS exposure increased the p-Akt/Akt ratio (p<0.05) but did not increase total Akt levels (p=0.88). Inhibition of VEGFR2 activity with SU5416 attenuated pSS-induced changes in total eNOS levels (p<0.05 for interaction) but did not attenuate pSS-induced increases in the p-Akt/Akt ratio (p=0.91 for interaction). Inhibition of PI3K activity using LY294002 did not modify the effects of pSS on total eNOS levels (p=0.25 for interaction) or p-Akt/Akt ratio (p=0.47 for interaction). Conclusions: We conclude that VEGFR2 activity is necessary for pSS-mediated increases in eNOS protein levels, but not PI3K activity, in hLMVECs. Furthermore, neither VEGFR2 nor PI3K activity appear to be required for pSS-mediated eNOS phosphorylation in these cells. Collectively, these data suggest that shear stress regulates hLMVEC eNOS signaling via multiple signaling pathways. This complex regulation may have important implications for local nitric oxide bioavailability under pathological conditions that modify shear stress (e.g., pulmonary embolism, pulmonary hypertension). This work is supported by NIH grants HL126514 and HL159906. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.