Classification of Pulmonary Hypertension

The aim of this study was to investigate the influence of two-pore channels mediated receptor-operated Ca²⁺ entry on pulmonary arterial smooth muscle cell (PASMC) under hypoxia conditions. PASMCs were separated using the direct adherent culture method. The cultured cells were observed under optic microscope and the phenotypes of cells were identified by immunohistochemistry. The expression of NAADP was examined by ELISA. CaN, TPC1, TPC2 and NFATc3 protein levels were examined using Western blotting. Real-time PCR was utilized to detect the level of TPC1 and TPC2 mRNA. Fluorescent probe technique was used to explore the [Ca²⁺]i in PASMCs. Proliferation and migration of PASMCs were examined by MTT assay and Transwell, respectively. The results showed that cells displayed a typical “peak-valley” growth pattern and positive for α-actin staining. Expression of NAADP, CaN, NFATc3, TPC1 and TPC2 under PASMCs exposed to hypoxia after 24 h and 48 h were higher than control, however, cells treated with Ned-19 were significantly decreased compared with control. Levels of CaN and NFATc3 protein collected from RPASMCs transfected with TPCs siRNA were observably decreased than scrambled siRNA. Under hypoxia condition for 12 h, 24 h and 48 h, TPC1 and TPC2 mRNA levels were higher in PASMCs compared as control. The [Ca²⁺]i evoked by hypoxia significantly increased than normoxia group. Nevertheless, the [Ca²⁺]i of the groups treated with Ned-19 and transfected with TPCs siRNA were markedly lower compared with control. In conclusion, the TPCs influence on function of pulmonary artery smooth muscle cells by mediated Ca²⁺ Signals under hypoxia condition.

Within human pulmonary artery, neurotrophin growth factors [NTs; e.g. brain-derived neurotrophic factor (BDNF)] and their high-affinity receptors (tropomyosin-related kinase; Trk) and low-affinity receptors p75 neurotrophin receptor (p75NTR) have been reported, but their functional role is incompletely understood. We tested the hypothesis that BDNF is produced by human pulmonary artery endothelial cells (PAECs). In the context of hypoxia as a risk factor for pulmonary hypertension, we examined the effect of hypoxia on BDNF secretion and consequent autocrine effects on pulmonary endothelium. Initial ELISA analysis of circulating BDNF in 30 healthy human volunteers showed that 72 h exposure to high altitude (~ 11,000 ft, alveolar PO₂ = 100 mm Hg) results in higher BDNF compared to samples taken at sea level. Separately, in human PAECs exposed for 24 h to normoxia vs. hypoxia (1–3% O₂), ELISA of extracellular media showed increased BDNF levels. Furthermore, quantitative PCR of PAECs showed 3-fold enhancement of BDNF gene transcription with hypoxia. In PAECs, BDNF induced NO production (measured using an NO-sensitive fluorescent dye DAF2-DA) that was significantly higher under hypoxic conditions, an effect also noted with the TrkB agonist 7,8-DHF. Importantly, hypoxia-induced NO was blunted by neutralization of secreted BDNF using the chimeric TrkB-Fc. Both hypoxia and BDNF increased iNOS (but not eNOS) mRNA expression. In accordance, BDNF enhancement of NO in hypoxia was not blunted by 50 nM l-NAME (eNOS inhibition) but substantially lower with 100 μM l-NAME (eNOS and iNOS inhibition). Hypoxia and BDNF also induced expression of hypoxia inducible factor 1 alpha (HIF-1α), a subunit of the transcription factor HIF-1, and pharmacological inhibition of HIF-1 diminished hypoxia effects on BDNF expression and secretion, and NO production. These results indicate that human PAECs express and secrete BDNF in response to hypoxia via a HIF-1-regulated pathway.