Effects of pathological flow on pulmonary artery endothelial production of vasoactive mediators and growth factors

Abstract

Background: Alterations in pulmonary blood flow are often associated with the initiation and progression of pulmonary vascular disease. However, the cellular mechanisms involved in mediating flow effects in the pulmonary circulation remain unclear. Depending on the disease condition, flow may be extremely low or high. We therefore examined effects of pathologically low and high flow on endothelial production of factors capable of affecting pulmonary vascular tone and structure as well as on potential underlying mechanisms.

Methods: Flow effects on pulmonary endothelial release of NO, PGF(1a), ET-1 and TxB(2), on expression of total and phosphorylated eNOS as well as Akt, and on VEGF were examined. Additionally, in a coculture system, effects of flow-exposed endothelial cells on smooth muscle (SM) proliferation and contractile protein were studied.

Results: Compared to physiological flow, pathologically high and low flow attenuated endothelial release of NO and PGF(1a), and enhanced release of ET-1. Physiological flow activated the Akt/eNOS pathway, while pathological flow depressed it. Pathologically high flow altered VE-cadherin expression. Pathologically high flow on the endothelium upregulated alpha-SM-actin and SM-MHC without affecting SM proliferation.

Conclusion: Physiological flow leads to production of mediators which favor vasodilation. Pathological flow alters the balance of mediator production which favors vasoconstriction.

Figure 1. Pathological shear stresses reduce NO and PGF_1α release and promote ET-1 release

(a) Nitric oxide release as measured by the total content of nitrite in flow media. (b) EIA measurement results of the 6-keto-prostaglandin F_1α (PGF_1α) content in the flow media. (c) ELISA measurement results of the endothelin-1 (ET-1) content in the flow media; (d) EIA measurement results of the thromboxane B₂ (TxB₂) content in the flow media. The media was collected from the flow circulation after cells were exposed to different shear stresses. Data represent means ± SEM (n=3–6). The conditions marked with ‘*’ are significantly different from 20 dynes/cm² shear stress (P ≤ 0.05).

Figure 2. Shear stresses affect expression and activation of eNOS through PI3K/Akt signaling pathway

Western blot images and quantitative measurements of proteins expressed by PAECs: (a) total eNOS (T-eNOS) expressed as the ratio of T-eNOS to β-actin; (b) phosphorylated eNOS^er1177 (P-eNOS) expressed as the ratio of P-eNOS to T-eNOS; and (c) Phosphorylated Akt^Ser473 (P-Akt) expressed as the ratio of P-Akt to total Akt (T-Akt). Protein expressions were measured with PAECs exposed to shear stresses of 0, 5, 20, 60, 90, 120 dynes/cm². (d) Total and phosphorylated eNOS expression under flow of 20 dynes/cm² with or without wortmannin (100nM). Data represent means ± SEM, n=4–6 for each group. The conditions marked with ‘*’ are significantly different from shear stress at 20 dynes/cm² (P ≤ 0.05).

Figure 3. Pathological shear stresses reduce VEGF expression

Western blot images and quantitative measurements of VEGF expression. Results expressed as ratio of VEGF to β-actin. Data represent means ± SE (n=3–4). The conditions marked with ‘*’ are significantly different from 20 dynes/cm² (P ≤ 0.05).

Figure 4. Shear stresses affect endothelial structure

Subcellular structures of PAECs were stained with different colors: F-actin (green), VE-cadherin (red) and nuclei (blue). The numbers indicates the level of flow shear stresses (dynes/cm²) that cells were exposed to.

Figure 5. High shear stresses increase α-SM-actin and SM-MHC expression, but not DNA synthesis in co-cultured PASMC

Quantitative measurements of α-SM-actin (a), and SM-MHC (b) in PASMCs co-cultured with PAECs. (c) Western blot images of α-SM-actin, SM-MHC, and PCNA expressions in PASMCs co-cultured with PAECs. Data represent means ± SE (n=3–4). The conditions marked with ‘*’ are significantly different from 20 dynes/cm² (P ≤ 0.05).