Effect of chronic hypoxia on lung levels of BH₄ and BH₂.

<p>(<i>A</i>) BH₄ levels, (<i>B</i>) BH₂ levels, and (<i>C</i>) BH₄/BH₂ ratio in lung from mice exposed to normoxia (NX) or 10, 21 and 40 days of hypoxia (HX 10d, HX 21d and HX 40d, respectively). Results are expressed as mean ± SEM from 6 experiments. * p<0.05, ** p<0.01, compared to NX.</p

Effect of chronic hypoxia on expression of GTPCH-1, DHFR and eNOS in lungs and intrapulmonary arteries.

<p>Graphs showing quantified data normalized to β-actin and representative Western-blotting for (<i>A</i> and <i>B</i>) GTPCH-1, (<i>C</i> and <i>D</i>) DHFR, and (<i>E</i> and <i>F</i>) total eNOS, in lungs (ACE) and intrapulmonary arteries (BDF), from mice exposed to normoxia (NX) or 10, 21 and 40 days of hypoxia (HX 10d, HX 21d and HX 40d, respectively). (<i>G</i>) Expression of eNOS at the mRNA level by quantitative RT-PCR in lung from mice exposed to normoxia (NX) or 21 days of hypoxia (HX 21d). Results are expressed as mean ± SEM from 5 (Western-blotting) to 9 (RT-PCR) experiments. * p<0.05, ** p<0.01, *** p<0.001, compared to NX.</p

Effect of eNOS gene deletion on hypoxia-induced alterations of contraction to phenylephrine, remodeling of pulmonary arterioles, right ventricular pressure and hypertrophy.

<p>(<i>A</i>) Contractile responses to phenylephrine in intrapulmonary arteries, (<i>B</i>) wall thickness of pulmonary arterioles, (<i>C</i>) right ventricular systolic pressure and (<i>D</i>) weight ratio right ventricle / (left ventricle + septum) in wild-type (WT) and <i>eNOS</i>^<i>-/-</i> mice exposed to normoxia (NX) or 21 days of hypoxia (HX 21d). Results are expressed as mean ± SEM from 8-18 experiments for reactivity to phenylephrine, 4-7 mice for wall thickness, 6-14 mice for right ventricular systolic pressure and from 41-94 experiments for weight ratio right ventricle / (left ventricle + septum). * p<0.05, ** p<0.01, *** p<0.001, compared to respective NX (WT or <i>eNOS</i>^<i>-/-</i>), ^# p<0.05 compared to WT exposed to 21 days of hypoxia.</p

Effect of sepiapterin and L-NAME on hypoxia-induced alterations of relaxation to acetylcholine in intrapulmonary arteries.

<p>Relaxant response of acetylcholine in intrapulmonary arteries from mice exposed to (<i>A</i>) normoxia (NX), (<i>B</i>) 10 days, (<i>C</i>) 21 days, and (<i>D</i>) 40 days of hypoxia (HX 10d, HX 21d and HX 40d, respectively), in the presence or absence of sepiapterin (10^-4 M) or L-NAME (3.10^-4 M). Results are expressed as mean ± SEM from 4 to 6 experiments, 0% relaxation corresponding to the level of precontraction induced by 3.10^-6 M phenylephrine * p<0.05 compared to control.</p

Effect of chronic hypoxia on eNOS dimer / monomer ratio in intrapulmonary arteries and cardiac ventricles.

<p>(<i>A</i>) Graphs showing quantified data for eNOS dimer / monomer ratio and representative Western-blots in intrapulmonary arteries from mice exposed to normoxia or 10, 21 and 40 days of hypoxia and treated or not with sepiapterin (10^-4 M). (<i>B</i>) Graphs showing quantified data for eNOS dimer / monomer ratio and representative Western-blots in left ventricles (LV) and right ventricles (RV) from mice exposed to normoxia (NX) or 21 days of hypoxia (HX 21d). Results are expressed as mean ± SEM from 3 to 4 experiments. * p<0.05, compared to NX, ^# p<0.05 compared to 21 days hypoxia.</p

Effect of sepiapterin and L-NAME on hypoxia-induced alterations of contraction to phenylephrine in intrapulmonary arteries.

<p>Contractile response to phenylephrine in intrapulmonary arteries from mice exposed to (<i>A</i>) normoxia (NX), (<i>B</i>) 10 days, (<i>C</i>) 21 days, and (<i>D</i>) 40 days of hypoxia (HX 10d, HX 21d and HX 40d, respectively), in the presence or absence of sepiapterin (10^-4 M) or L-NAME (3.10^-4 M). Results are expressed as mean ± SEM from 6 to 9 experiments. * p<0.05 compared to control.</p

Effect of sepiapterin on hypoxia-induced remodeling of pulmonary arterioles, right ventricular pressure and hypertrophy.

<p>(<i>A</i>) Representative pictures, and (<i>B</i>) quantified data of wall thickness of pulmonary arterioles in mice exposed to normoxia (NX) or in mice exposed to 21 days of hypoxia (HX 21d) which were treated or not with sepiapterin (30 mg/kg, one day before exposure to hypoxia and every other day during hypoxia). (<i>C</i>) Right ventricular systolic pressure and (<i>D</i>) weight ratio right ventricle / (left ventricle + septum) in mice exposed to normoxia (NX) or in mice exposed to 21 days of hypoxia (HX 21d) which were treated or not with sepiapterin (30 mg/kg, one day before exposure to hypoxia and every other day during hypoxia). Results are expressed as mean ± SEM from all the arteries on each lung section from 4 to 7 mice for wall thickness, from 6-14 mice for right ventricular systolic pressure and from at least 8 experiments for weight ratio right ventricle / (left ventricle + septum). * p<0.05, *** p<0.001, compared to NX ; ^#p<0.05, ^###p<0.001 compared to 21 days hypoxia.</p

Absolute numbers of LDGs, CD14⁺ cells and white blood cells.

<p>The white blood cell counts were determined after Ficoll using trypan blue exclusion and are expressed as cell number per ml of blood.</p

L-arginine levels and CD3ζ expression in CD4⁺ and CD8⁺ T cells.

<p>Plasma was isolated by centrifugation of the blood of controls (n = 11), VL patients (n = 11) and treated VL patients (n = 11) and (A) the levels of L-arginine were measured by HPLC. PBMCs were isolated by density gradient from the blood of controls (n = 10), VL patients (n = 10) and treated VL patients (n = 10) and the mean fluorescence intensity of CD3ζ was determined in CD4⁺ T cells (B) and CD8⁺ T cells (C). Box = interquartile range and median; whiskers = range. Statistical significance was determined by a two-tailed Mann-Whitney test. NS = not significant. Patients = VL patients before treatment; treated patients = VL patients after 3–4 weeks treatment.</p

Haematological data.

<p>Hct = hematocrit; Hb = haemoglobin.</p><p>Normal range: platelets (×10³) = 150–450; white blood cells (×10³) = 4.5–10.5; Hct (%) = 35–60; hb (g/dl) = 11–18.</p><p>The white blood cells were counted in total blood before Ficoll using a COULTER Ac•T diff Hematology Analyzer and are expressed as number of WBC (×10³) per µl of blood.</p