Fluid shear stress upregulates placental growth factor in the vessel wall via NADPH oxidase 4

Abstract

Placental growth factor (PLGF), a potent stimulator of arteriogenesis, is upregulated during outward arterial remodeling. Increased fluid shear stress (FSS) is a key physiological stimulus for arteriogenesis. However, the role of FSS in regulating PLGF expression is unknown. To test the hypothesis that FSS regulates PLGF expression in vascular cells and to identify the signaling pathways involved, human coronary artery endothelial cells (HCAEC) and human coronary artery smooth muscle cells were cultured on either side of porous Transwell inserts. HCAEC were then exposed to pulsatile FSS of 0.07 Pa ("normal," mimicking flow through quiescent collaterals), 1.24 Pa ("high," mimicking increased flow in remodeling collaterals), or 0.00 Pa ("static") for 2 h. High FSS increased secreted PLGF protein ∼1.4-fold compared with static control (n = 5, P < 0.01), while normal FSS had no significant effect on PLGF. Similarly, high flow stimulated PLGF mRNA expression nearly twofold in isolated mouse mesenteric arterioles. PLGF knockdown using siRNA revealed that HCAEC were the primary source of PLGF in cocultures (n = 5, P < 0.01). Both H2O2 and nitric oxide production were increased by FSS compared with static control (n = 5, P < 0.05). N(G)-nitro-l-arginine methyl ester (100 μM) had no significant effect on the FSS-induced increase in PLGF. In contrast, both catalase (500 U/ml) and diphenyleneiodonium (5 μM) attenuated the effects of FSS on PLGF protein in cocultures. Diphenyleneiodonium also blocked the effect of high flow to upregulate PLGF mRNA in isolated arterioles. Further studies identified NADPH oxidase 4 as a source of reactive oxygen species for this pathway. We conclude that FSS regulates PLGF expression via NADPH oxidase 4 and reactive oxygen species signaling.

Keywords: arteriogenesis; collateral circulation; endothelium; hemodynamics; vascular endothelial growth factors.

Fig. 1.

A: coculture system with the cone in place. Transwell inserts were placed inside 6-well plates. Human coronary artery endothelial cells (HCAEC) were seeded on the top side of the insert, while human coronary artery smooth muscle cells (HCASMC) were seeded on the underside. The 0.4-μm pore size prevented either cell type from migrating completely through the membrane, which was readily permeable to soluble factors and allowed some direct cell-cell contact. B and C: shear stress waveforms, shown over 1 cardiac cycle (0.9 s). B: waveform representing “normal” shear stress in a collateral arising from a coronary artery free of stenosis. C: waveform representing “high” shear stress in a collateral arising from a coronary artery with 60% stenosis.

Fig. 2.

Effect of fluid shear stress (FSS) exposure on placental growth factor (PLGF) protein expression in cocultures and arterioles. A: cocultures were exposed to static control conditions (no FSS), normal FSS (0.07 Pa), or high FSS (1.24 Pa). PLGF protein in medium was assessed prior to and up to 24 h after shear exposure. A: neither waveform had a significant effect on PLGF protein levels after 1 h of exposure (n = 5). B: 2 h of exposure to high, but not normal, FSS induced a significant increase in PLGF protein compared with static control (n = 5). *P < 0.001. C: mesenteric arterioles (∼150 μm ID) were cannulated and perfused with 1% BSA-PSS for 2 h. Perfusion with a pressure gradient of 50 mmHg significantly increased PLGF mRNA compared with perfusion with a pressure gradient of 20 mmHg (n = 5). *P < 0.05.

Fig. 3.

Effect of FSS on PLGF mRNA expression in HCAEC (EC) and HCASMC (SMC). A: HCAEC demonstrated an immediate increase in PLGF gene expression in response to high FSS, which was maintained for up to 4 h after shear exposure (n = 5). *P < 0.001. Normal FSS had no significant effect on PLGF mRNA in HCAEC (n = 5). B: HCASMC showed no significant change in PLGF gene expression, regardless of the level of shear exposure received by the coculture (n = 5). HCASMC were not directly exposed to FSS.

Fig. 4.

Contribution of HCAEC and HCASMC to basal and FSS-stimulated PLGF expression in cocultures. PLGF expression was knocked down using siRNA in HCAEC or HCASMC. PLGF knockdown was confirmed by real-time PCR in HCAEC (A; n = 5) and HCASMC (B; n = 5). *P < 0.01. Medium was sampled immediately prior to exposure to 2 h of high FSS and 24 h thereafter. WT, wild-type; siSCR, negative control scrambled siRNA; siPLGF, PLGF siRNA. C: in cocultures containing siPLGF-treated HCAEC (siEC) but wild-type HCASMC (wtSMC), PLGF was significantly decreased compared with levels in untreated cocultures (n = 5) and failed to increase in response to FSS. *P < 0.001. Cocultures with negative control scrambled siRNA-treated HCAEC showed a slight, but significant, decrease in preshear PLGF levels compared with untreated cells (n = 5); however, exposure to high FSS significantly increased PLGF as expected (n = 5). *P < 0.001; †P < 0.05. D: in cocultures containing wild-type HCAEC and siPLGF-treated HCASMC (siSMC), there was a significant increase in total PLGF protein in the medium compared with untreated cocultures (n = 5). *P < 0.001. PLGF was further increased by exposure to high FSS in both negative control scrambled siRNA- and siPLGF-treated groups compared with static untreated controls (n = 5) and the corresponding preshear values for each treatment condition (n = 5). *P < 0.001; †P < 0.05.

Fig. 5.

Effect of FSS exposure on PLGF levels in HCAEC monocultures. A: baseline (unstimulated) level of PLGF protein was significantly lower in medium of HCAEC monocultures than in medium of HCAEC-HCASMC cocultures (n = 5). *P < 0.001. B: HCAEC monocultures were exposed to static conditions, normal FSS, or high FSS for 2 h as described for cocultures. Culture medium was sampled before and after shear exposure. High FSS and normal FSS induced a significant increase in PLGF protein, but only at 8 h after exposure (n = 5). *P < 0.05. Thus the time course and intensity dependence of the effect were altered in the absence of HCASMC.

Fig. 6.

Role of nitric oxide (NO) in the FSS-induced increase in PLGF protein. A: high, but not normal, FSS significantly increased nitrate/nitrite levels immediately after shear exposure compared with static control (n = 5). *P < 0.01. B: the NO synthase inhibitor N^G-nitro-l-arginine methyl ester (l-NAME, 100 μM) had no effect on the shear-induced increase in PLGF protein (n = 5). *P < 0.05.

Fig. 7.

Role of H₂O₂ in the FSS-induced increase in PLGF protein. A: high and normal FSS significantly increased H₂O₂ in cocultures immediately after shear exposure compared with static control (n = 5). *P < 0.05. B: catalase (500 U/ml) blocked the effect of FSS to upregulate PLGF (n = 5). *P < 0.01. C: diphenyleneiodonium (DPI, 5 μM) also prevented the increase in PLGF protein in response to FSS and further decreased PLGF protein below static control levels (n = 5). *P < 0.01. D: DPI (5 μM) also inhibited the flow-induced increase in PLGF mRNA in cannulated arterioles [20 mmHg (n = 6), 50 mmHg (n = 5), and DPI + 50 mmHg (n = 4)]. *P < 0.01.

Fig. 8.

Effect of FSS on NADPH oxidase 4 (Nox4) gene expression in HCAEC and HCASMC. A: in HCAEC, high FSS significantly increased Nox4 mRNA compared with static control at 4 h after exposure, while normal FSS had no significant effect on Nox4 mRNA (n = 5). *P < 0.01. B: high FSS increased Nox4 mRNA only at 24 h after exposure in HCASMC (n = 5). *P < 0.05.

Fig. 9.

Role of endothelial Nox4 in the FSS-induced increase in PLGF protein. A and B: real-time PCR confirmed knockdown of Nox4 in HCAEC (n = 5) and HCASMC (n = 5). siNox4, Nox4 siRNA. *P < 0.01. C: in cocultures containing siNox4-treated HCAEC but wild-type HCASMC, the FSS-induced increase in PLGF protein was inhibited and PLGF was decreased to below static control levels (n = 5). *P < 0.01. PLGF remained responsive to shear in cocultures with negative control scrambled siRNA-treated HCAEC (n = 5). †P < 0.05. D: response of PLGF to FSS was unaffected in cocultures containing wild-type HCAEC and siNox4-treated HCASMC (n = 5). *P < 0.01 vs. static. †P < 0.05 vs. preshear. E: knockdown of Nox4 in HCAEC prevented the effect of high FSS to increase H₂O₂, identifying endothelial Nox4 as a key source of FSS-induced H₂O₂ production in the coculture system (n = 5). *P < 0.05.