Pulmonary hypertension (PH) is a life-threatening disorder that is characterized by pulmonary arterial smooth muscle cell (PASMC) hyperplasia. Until now, little was been known about early changes that underlie the manifestation of PH. To characterize these early changes, we performed whole-genome microarray analysis of lungs from mice exposed to either 24 hours hypoxia or normoxia. TrkB, a member of the tyrosine kinase receptor family, and its ligand, brain-derived neurotrophic factor (BDNF), were strongly up-regulated in hypoxic mouse lungs, as well as in arteries of patients suffering from idiopathic pulmonary arterial hypertension (IPAH). BDNF stimulation of PASMC in vitro resulted in increased proliferation, TrkB and ERK1/2 phosphorylation, and nuclear translocation of the transcription factor early growth response factor 1 (Egr-1). In addition, increased Egr-1 expression was observed in idiopathic PAH lungs. The pro-proliferative effect of BDNF was attenuated by TrkB kinase inhibitor (K252a) or ERK1/2 inhibitor (U0126) pretreatment, and by knocking down Egr-1. Consequently, we have identified the BDNF-TrkB-ERK1/2 pathway as a proproliferative signaling pathway for PASMC in PH. Interference with this pathway may thus serve as an attractive reverse remodeling approach.

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Background: Small biological samples obtained from biopsies or laser microdissection often do not yield sufficient RNA for successful microarray hybridization; therefore, RNA amplification is performed before microarray experiments. We compared 2 commonly used techniques for RNA amplification. Methods: We compared 2 commercially available methods, Arcturus RiboAmp for in vitro transcription (IVT) and Clontech BD SMART TM for PCR, to preamplify 50 ng of total RNA isolated from mouse livers and kidneys. Amplification factors of 3 sequences were determined by real-time PCR. Differential expression profiles were compared within and between techniques as well as with unamplified samples with 10K 50mer oligomerspotted microarrays (MWG Biotech). The microarray results were validated on the transcript and protein levels by comparison with public expression databases. Results: Amplification factors for specific sequences were lower after 2 rounds of IVT than after 12 cycles of SMART. Furthermore, IVT showed a clear decrease in amplification with increasing distance of the amplified sequences from the polyA tail, indicating generation of smaller products. In the microarray experiments, reproducibility of the duplicates was highest after SMART. In addition, SMART-processed samples showed higher correlation when compared with unamplified samples as well as with expression databases. Conclusions: Whenever 1 round of T7-IVT does not yield sufficient product for microarray hybridization, which is usually the case when <200 ng of total RNA is