Laurence Dewachter

Cross Talk Between Endothelial and Smooth Muscle Cells in Pulmonary Hypertension: Critical Role for Serotonin-Induced Smooth Muscle Hyperplasia

Background— The mechanism of pulmonary artery smooth muscle cell (PA-SMC) hyperplasia in idiopathic pulmonary artery hypertension (iPH) may involve both an inherent characteristic of PA-SMCs and abnormal control by external stimuli. We investigated the role of pulmonary microvascular endothelial cells (P-ECs) in controlling PA-SMC growth. Methods and Results— Serum-free medium of quiescent P-ECs elicited marked PA-SMC proliferation, and this effect was greater with P-ECs from patients with iPH than from control subjects and greater with PA-SMCs from these patients than from control subjects. Fluoxetine, which inhibits serotonin-induced mitogenesis by blocking the serotonin transporter, and p-chlorophenylalanine, which inhibits serotonin synthesis by blocking tryptophan hydroxylase (TPH), caused a similar 60% reduction in the growth-promoting effect of P-EC media, whereas endothelin receptor blockers had no effect. Assays of TPH activity in P-EC medium based on p-chlorophenylalanine–sensitive 5-hydroxytryptophan accumulation or serotonin determination indicated serotonin synthesis by P-ECs and an increase in this TPH-dependent process in iPH. Expression of the tph1 gene encoding the peripheral form of the TPH enzyme was increased in lungs and P-ECs from patients with iPH. Lung TPH1 immunostaining was confined to the pulmonary vessel intima. Conclusions— P-ECs produce paracrine factors governing PA-SMC growth. Serotonin, the main P-EC–derived growth factor, is overproduced in iPH and contributes to PA-SMC hyperplasia.

Autocrine Fibroblast Growth Factor-2 Signaling Contributes to Altered Endothelial Phenotype in Pulmonary Hypertension

Pulmonary vascular remodeling is key to the pathogenesis of idiopathic pulmonary arterial hypertension (IPAH). We recently reported that fibroblast growth factor (FGF)2 is markedly overproduced by pulmonary endothelial cells (P-ECs) in IPAH and contributes significantly to smooth muscle hyperplasia and disease progression. Excessive FGF2 expression in malignancy exerts pathologic effects on tumor cells by paracrine and autocrine mechanisms.We hypothesized that FGF2 overproduction contributes in an autocrine manner to the abnormal phenotype of P-ECs, characteristic of IPAH. In distal pulmonary arteries (PAs) of patients with IPAH, we found increased numbers of proliferating ECs and decreased numbers of apoptotic ECs, accompanied with stronger immunoreactivity for the antiapoptotic molecules, B-cell lymphoma (BCL)2, and BCL extra long (BCL-xL) compared with PAs from control patients. These in situ observations were replicated in vitro, with cultured P-ECs from patients IPAH exhibiting increased proliferation and diminished sensitivity to apoptotic induction with marked increases in the antiapoptotic factors BCL2 and BCL-xL and levels of phosphorylated extracellular signal-regulated (ERK)1/2 compared with control P-ECs. IPAH P-ECs also exhibited increased FGF2 expression and an accentuated proliferative and survival response to conditioned P-EC media or exogenous FGF2 treatment. Decreasing FGF2 signaling by RNA interference normalized sensitivity to apoptosis and proliferative potential in the IPAH P-ECs. Our findings suggest that excessive autocrine release of endothelial-derived FGF2 in IPAH contributes to the acquisition and maintenance of an abnormal EC phenotype, enhancing proliferation through constitutive activation of ERK1/2 and decreasing apoptosis by increasing BCL2 and BCL-xL.

106 Contribution of the angiopoietin/Tie2 pathway to pulmonary artery smooth muscle hyperplasia in idiopathic pulmonary hypertension

Introduction Angiopoietins are involved in blood-vessel maturation and stabilization through activation of the selective endo-thelial-specific receptor Tie2, which leads to inhibition of endothelial cell (EC) proliferation and induces release of endothe-lial-derived growth factors. Recent studies suggest that alterations in the Ang 1/Tie2 pathway may contribute to pulmonary artery smooth muscle cell (PA-SMC) hyperplasia in idiopathic pulmonary hypertension (IPH). Methods In the present study, we used lung homogenates and cultured cells (PA-EC and PA-SMC) from patients with IPH and controls to examine expression of angiopoietins (Angl and Ang 2) and Tie2 receptors, as well as the consequences of Tie2 receptor activation on release of serotonin, endothelin-1, EGF, and PDGF-BB from PA-EC. Results We found that expression of Angl and Ang2 (quantitative RT/PCR and Western blot for determination of mRNA and protein, respectively) in lungs and PA-SMCs did not differ between IPH patients and controls. Tie2 receptor expression (mRNA and protein) was 3-4 fold higher in lungs and cultured PA-EC from patients with IPH than from controls (p Conclusion The results show that an increase in Angl-induced activation of Tie2 receptors in IPH is responsible for increased production of growth factors acting on PA-SMC.