Upregulation of nitric oxide synthase in mice with severe hypoxia-induced pulmonary hypertension

BACKGROUND: The importance of nitric oxide (NO) in hypoxic pulmonary hypertension has been demonstrated using nitric oxide synthase (NOS) knockout mice. In that model NO from endothelial NOS (eNOS) plays a central role in modulating pulmonary vascular tone and attenuating hypoxic pulmonary hypertension. However, the normal regulation of NOS expression in mice following hypoxia is uncertain. Because genetically engineered mice are often utilized in studies of NO, we conducted the present study to determine how hypoxia alters NOS expression in wild-type mice. METHOD: Mice were exposed to sea level, ambient conditions (5280 feet) or severe altitude (17,000 feet) for 6 weeks from birth, and hemodynamics and lung NOS expression were assessed. RESULTS: Hypoxic mice developed severe pulmonary hypertension (right ventricular systolic pressure [RVsP] 60 mmHg) as compared with normoxic mice (27 mmHg). Using quantitative reverse-transcription PCR, it was found that expressions of eNOS and inducible NOS (iNOS) increased 1.5-fold and 3.5-fold, respectively, in the lung. In addition, the level of lung eNOS protein was increased, neuronal NOS (nNOS) protein was unchanged, and iNOS was below the limit of detection. Immunohistochemistry demonstrated no change in lung iNOS or nNOS staining in either central or peripheral areas, but suggested increased eNOS in the periphery following hypoxia. CONCLUSION: In mice, hypoxia is associated with increases in lung eNOS, possibly in iNOS, but not in nNOS; this suggests that the pattern of lung NOS expression following hypoxia must be considered in studies using genetically engineered mice

Serum can overcome contact inhibition in confluent human pulmonary artery smooth muscle cells.

Pulmonary artery endothelial cells (PAEC) in an intact vessel are continually exposed to serum, but unless injured, do not proliferate, constrained by confluence. In contrast, pulmonary artery smooth muscle cells (PASMC) attain, and maintain, confluence in the presence of minimal serum, protected from serum's stimulatory effects except when the endothelial barrier becomes more permeable. We hypothesized therefore, that confluent PASMC may be less constrained by contact inhibition in the presence of serum than PAEC and tested this idea by exposing confluent non-transformed human PAEC and PASMC to media containing increasing concentrations of fetal bovine serum (FBS) and determining cell growth over 7 days. PAEC that had attained confluence in low serum did not proliferate even when exposed to 5% serum, the highest concentration tested. In contrast, PASMC that attained confluence in low serum did proliferate once serum levels were increased, an effect that was dose dependent. Consistent with this observation, PASMC had more BrdU incorporation and a greater percentage of cells in S phase in 5% compared to 0.2% FBS, whereas no such difference was seen in PAEC. These results suggest that confluent human PAEC are resistant to the stimulatory effects of serum, whereas confluent PASMC can proliferate when serum levels are increased, an effect mediated in part by differences in phosphoinositide 3-kinase activation. This observation may be relevant to understanding the PASMC hyperplasia observed in humans and animals with pulmonary hypertension in which changes in endothelial permeability due to hypoxia or injury expose the underlying smooth muscle to serum

PASMC that attain confluence at high serum cannot maintain that cell density in low serum.

<p>Human PASMC were grown to confluence in 10% FBS and maintained in 10% serum for 7 days. Confluence was determined by light microscopy and by the demonstration of stable cell number over 7 days. Cells were then exposed to various concentrations of serum ranging from 0.1% to 10%: A) Cell counts done 7 days later. B) Cells were stained with the membrane impermeable dye ethidium homodimer-1 at different time points to determine cell viability. Ethidium homodimer-1 can only stain apoptotic or necrotic cells and is a marker of cell death. Cell death is increased by day 2 and returns to baseline by day 7. (n = 3 separate experiments, * indicates p<.05 compared with starting cell number for (A) or compared to day 0 at 10% serum for (B).</p

PI3-kinase inhibitors, LY294002 and wortmannin, inhibit rat PASMC proliferation, but only wortmannin inhibits AKT phosphorylation.

<p>Rat PASMC were grown to confluence, growth arrested in 0.1% serum for 72 hours, and then exposed to 10% or fresh 0.1% FBS in the presence or absence of wortmannin (W) or LY294002 (LY). Cells were examined 24 hours later for BrdU incorporation (A and B) and AKT phosphorylation (C), (n = 3 experiments, * indicates p<.05).</p

Increased serum phosphorylates retinoblastoma, Akt^Ser473, and AKT^Thr308 and decreases p27^KIP1 in confluent PASMC, but not in confluent PAEC.

<p>A and B) Human PAEC and PASMC attained confluence in low (0.2%) serum and were maintained in low serum for seven days. Cells were then exposed to 0.2% or 5% serum and 24 hours later, cell lysates were harvested for protein analysis. Representative Western blots are shown. C) Human PAEC and PASMC attained confluence in low (0.2%) serum and were maintained in low serum for seven days. Cells were infected for 2 hours at a multiple of infectivity of 200 with a replication-deficient adenovirus serotype 5 containing either a human p27^KIP1 (<i>Ad</i>p27) or alkaline phosphatase (<i>Ad</i>C) cDNA driven by a CMV promoter. Cells were then exposed to 5% serum and cell lysates harvested 24 hours later. A representative Western blot is shown. [pRB: hypophosphorylated retinoblastoma; ppRB: hyperphosphorylated retinoblastoma]; D) BrdU incorporation 24 hours after exposure to 5% serum in p27^KIP1-infected human PAEC and PASMC.</p

PI3-kinase inhibition prevents retinoblastoma hyperphosphorylation in confluent PASMC exposed to high serum concentrations.

<p>Human PASMC attained confluence in low (0.2%) serum and were maintained in low serum for seven days. Cells were then exposed to 0.1% or 5% serum in the presence or absence of LY294002 or wortmannin and cell lysates harvested 24 hours later for protein analysis. Representative Western blot is shown. [pRB: hypophosphorylated retinoblastoma; ppRB: hyperphosphorylated retinoblastoma].</p

Increased serum stimulates cell cycle progression/DNA synthesis in confluent PASMC, but not PAEC.

<p>Human PAEC and PASMC attained confluence in low (0.2%) serum and were maintained in low serum for seven days. Cells were then exposed to 0.2% or 5% serum and cell cycle profile (A and C) and BrdU incorporation (B and D) determined 24 hours later. (n = 3 experiments; * indicates p<.05).</p

The PI3-kinase inhibitors, LY294002 and wortmannin, block serum-induced proliferation in confluent PASMC.

<p>Human PASMC attained confluence in low (0.2%) serum and were maintained in low serum for seven days. Cells were then exposed to 0.2% or 5% serum in the presence or absence of the PI3-kinase inhibitors LY294002 (15 µmol) or wortmannin (50 nmol) and cell cycle profile determined 24 hours later. A) Representative plot of single experiment; B) Aggregate results from 3 separate experiments (n = 3 experiments, * indicates p<.05).</p

Exposure to increased serum stimulates growth in confluent PASMC, but not in confluent PAEC.

<p>Human PAEC (A) and PASMC (B) attained confluence in low (0.2%) serum as determined by light microscopy and the absence of proliferation. Cells were then exposed to increasing concentrations of serum and cell number determined in triplicate seven days later. (n = 3 experiments; * indicates p<.05 compared with starting cell number).</p