Soluble Urokinase-Type Plasminogen Activator Receptor Plasma Concentration May Predict Susceptibility to High Altitude Pulmonary Edema.

Introduction. Acute exposure to high altitude induces inflammation. However, the relationship between inflammation and high altitude related illness such as high altitude pulmonary edema (HAPE) and acute mountain sickness (AMS) is poorly understood. We tested if soluble urokinase-type plasminogen activator receptor (suPAR) plasma concentration, a prognostic factor for cardiovascular disease and marker for low grade activation of leukocytes, will predict susceptibility to HAPE and AMS. Methods. 41 healthy mountaineers were examined at sea level (SL, 446 m) and 24 h after rapid ascent to 4559 m (HA). 24/41 subjects had a history of HAPE and were thus considered HAPE-susceptible (HAPE-s). Out of the latter, 10/24 HAPE-s subjects were randomly chosen to suppress the inflammatory cascade with dexamethasone 8 mg bid 24 h prior to ascent. Results. Acute hypoxic exposure led to an acute inflammatory reaction represented by an increase in suPAR (1.9 ± 0.4 at SL versus 2.3 ± 0.5 at HA, p < 0.01), CRP (0.7 ± 0.5 at SL versus 3.6 ± 4.6 at HA, p < 0.01), and IL-6 (0.8 ± 0.4 at SL versus 3.3 ± 4.9 at HA, p < 0.01) in all subjects except those receiving dexamethasone. The ascent associated decrease in PaO2 correlated with the increase in IL-6 (r = 0.46, p < 0.001), but not suPAR (r = 0.27, p = 0.08); the increase in IL-6 was not correlated with suPAR (r = 0.16, p = 0.24). Baseline suPAR plasma concentration was higher in the HAPE-s group (2.0 ± 0.4 versus 1.8 ± 0.4, p = 0.04); no difference was found for CRP and IL-6 and for subjects developing AMS. Conclusion. High altitude exposure leads to an increase in suPAR plasma concentration, with the missing correlation between suPAR and IL-6 suggesting a cytokine independent, leukocyte mediated mechanism of low grade inflammation. The correlation between IL-6 and PaO2 suggests a direct effect of hypoxia, which is not the case for suPAR. However, suPAR plasma concentration measured before hypoxic exposure may predict HAPE susceptibility.

Alveolar but not intravenous S-ketamine inhibits alveolar sodium transport and lung fluid clearance in rats.

BACKGROUND: S-ketamine is frequently used for analgosedation, especially during sepsis and cardiovascular instability. Because S-ketamine blocks voltage-gated sodium (Na+) channels in neurons and skeletal muscle, it is conceivable that S-ketamine also blocks alveolar epithelial Na+ channels that are crucial for alveolar fluid clearance (AFC). We studied the effects of alveolar and IV S-ketamine on transalveolar Na+ transport and AFC, and investigated whether IV S-ketamine enters the alveolar space in response to endotoxemia-induced pulmonary inflammation. METHODS: Cultured rat alveolar type II (ATII) cells were exposed to S-ketamine and/or the Na+ channel blocker amiloride (100 microM) and transepithelial transport indicated by short circuit current (ISC) was measured in Ussing chambers. AFC was measured in fluid-instilled lungs of anesthetized rats with or without amiloride added to the instillate. S-ketamine was either added to the instillate or injected IV. To induce mild lung injury that might favor the appearance of IV S-ketamine at the alveolar surface, endotoxemia was induced by IV lipopolysaccharide (7.5 mg/kg). RESULTS: In ATII cells, S-ketamine (25 microg/mL) caused a decrease of ISC regardless of apical (-18.9%+/- 1.4%; P < 0.001) or basolateral (-20.4% +/- 3.7%; P < 0.001) application. In ATII cells pretreated with amiloride, addition of apical or basolateral S-ketamine did not decrease ISC. AFC was approximately 8% per 30 minutes in control rats. S-ketamine (5 microg/mL) in the instillate reduced AFC to 1.1% +/- 1.5% (P = 0.04) by decreasing amiloride-sensitive transepithelial Na+ transport. Intravenous S-ketamine (20 mg/kg) did not affect AFC (P = 0.31). In the presence of lipopolysaccharide-induced inflammation, the concentration of IV-injected S-ketamine in bronchoalveolar lavage fluid remained below the concentration that inhibited AFC. CONCLUSIONS: Although exposure of the rat alveolar epithelium to S-ketamine decreases amiloride-sensitive transalveolar Na+ transport and AFC, IV S-ketamine at clinically relevant bolus concentrations does not affect AFC, even in the presence of mild lung injury.

The effect of endothelin-1 on alveolar fluid clearance and pulmonary edema formation in the rat.

BACKGROUND: Endothelin-1 (ET-1) is thought to play a pivotal role in pulmonary edema formation. The underlying mechanisms remain uncertain but may include alterations in capillary pressure and vascular permeability. There are no studies investigating whether ET-1 also affects alveolar fluid clearance which is the primary mechanism for the resolution of pulmonary edema. Therefore, we performed this study to clarify effects of ET-1 on alveolar reabsorption and fluid balance in the rat lung. METHODS: Alveolar fluid clearance was measured in fluid instilled rat lungs using a 5% albumin solution with or without ET-1 (10(-7) M) and/or amiloride (100 microM). Net alveolar fluid balance, time course of edema formation, pulmonary capillary pressure, and alveolar permeability to albumin were measured in the isolated, ventilated, constant pressure perfused rat lung with or without ET-1 (0.8 nM) added to the perfusate. RESULTS: In the fluid-instilled lung, ET-1 reduced alveolar fluid clearance by about 65%, an effect that was related to a decrease in amiloride-sensitive transepithelial Na(+) transport (P < 0.001). The ET-1-induced inhibition was completely prevented by the endothelin B receptor antagonist BQ788 (P = 0.006), whereas the endothelin A receptor antagonist BQ123 had no effect (P = 0.663). In the isolated, ventilated, perfused rat lung ET-1 caused a net accumulation of alveolar fluid by about 20% (P = 0.011 vs control), whereas lungs of control rats cleared about 20% of the instilled fluid. ET-1 increased pulmonary capillary pressure (+9.4 cm H(2)O), decreased perfusate flow (-81%), accelerated lung weight gain and reduced lung survival time (P < 0.001). Permeability to albumin was not significantly affected by ET-1 (P = 0.24). CONCLUSION: ET-1 inhibits alveolar fluid clearance of anesthetized rats by inhibition of amiloride-sensitive epithelial Na(+) channels. The inhibitory effect of ET-1 results from activation of the endothelin B receptor. These findings suggest a mechanism by which ET-1, in addition to increasing capillary pressure, contributes to pulmonary edema formation.

Altered Left Ventricular Geometry and Torsional Mechanics in High Altitude-Induced Pulmonary Hypertension: A Three-Dimensional Echocardiographic Study.

BACKGROUND: Changes in left ventricular (LV) torsion have been related to LV geometry in patients with concomitant long-standing myocardial disease or pulmonary hypertension (PH). We evaluated the effect of acute high altitude-induced isolated PH on LV geometry, volumes, systolic function, and torsional mechanics. METHODS: Twenty-three volunteers were prospectively studied at low altitude and after the second (D3) and third night (D4) at high altitude (4,559 m). LV ejection fraction, multidirectional strains and torsion, LV volumes, sphericity, and eccentricity were derived by speckle-tracking on three-dimensional echocardiographic data sets. Pulmonary pressure was estimated from the transtricuspid pressure gradient (TRPG), LV preload from end-diastolic LV volume, and transmitral over mitral annular E velocity (E/e'). RESULTS: At high altitude, oxygen saturation decreased by 15%-20%, heart rate and cardiac index increased by 15%-20%, and TRPG increased from 21 ± 2 to 37 ± 9 mm Hg (P < .01). LV volumes, preload, ejection fraction, multidirectional strains, and sphericity remained unaffected, but diastolic (1.04 ± 0.07 to 1.09 ± 0.09 on D3/D4, P < .05) and systolic (1.00 ± 0.06 to 1.08 ± 0.1 [D3] and 1.06 ± 0.07 [D4], P < .05) eccentricity slightly increased, indicating mild septal flattening. LV torsion decreased from 2.14 ± 0.85 to 1.34 ± 0.68 (P < .05) and 1.65 ± 0.54 (P = .08) degrees/cm on D3/D4, respectively. Changes in torsion showed a weak inverse relationship to changes in systolic (r = -0.369, P = .013) and diastolic (r = -0.329, P = .032) eccentricity but not to changes in TRPG, heart rate or preload. CONCLUSIONS: High-altitude exposure was associated with mild septal flattening of the LV and reduced ventricular torsion at unchanged global LV function and preload, suggesting a relation between LV geometry and torsional mechanics.

Exaggerated hypoxic pulmonary vasoconstriction without susceptibility to high altitude pulmonary edema.

BACKGROUND: Abnormally high pulmonary artery pressure (PAP) in hypoxia due to exaggerated hypoxic pulmonary vasoconstriction (HPV) is a key factor for development of high-altitude pulmonary edema (HAPE). It was shown that about 10% of a healthy Caucasian population has an exaggerated HPV that is comparable to the response measured in HAPE-susceptible individuals. Therefore, we hypothesized that those with exaggerated HPV are HAPE-susceptible. METHODS AND RESULTS: We screened 421 healthy Caucasians naïve to high altitude for HPV using Doppler echocardiography for assessment of systolic PAP in normobaric hypoxia (PASPHx; Po2 corresponding to 4500 m). Subjects with exaggerated HPV and matched controls were exposed to 4559 m with an identical protocol that causes HAPE in 62% of HAPE-S. Screening revealed 39 subjects with exaggerated HPV, of whom 33 (PASPHx 51±6 mmHg) ascended within 24 hours to 4559 m. Four (13%) of them developed HAPE during the 48 h-stay. This incidence is significantly lower than the recurrence rate of 62% previously observed in HAPE-S in the same setting. None of the control subjects (PASPHx 33±5 mmHg) developed HAPE. CONCLUSION: An exaggerated HPV cannot be considered a surrogate maker for HAPE-susceptibility although excessively elevated PAP is a hallmark in HAPE, while a normal HPV appears to protect from HAPE in this study.

Growth differentiation factor-15 deficiency inhibits atherosclerosis progression by regulating interleukin-6-dependent inflammatory response to vascular injury.

BACKGROUND: Growth differentiation factor (GDF)-15 is a distant and divergent member of the transforming growth factor-ß superfamily (TGF-ß) . There is growing evidence indicating the involvement of GDF-15 in various pathologies. Expression of GDF-15 is induced under conditions of inflammation and increased GDF-15 serum levels are suggested as a risk factor for cardiovascular diseases. METHODS AND RESULTS: We show here that GDF-15 and proinflammatory cytokine interleukin (IL)-6 levels are highly increased (5-fold) in cultured oxidized low-density lipoproteins-stimulated peritoneal macrophages derived from GDF-15(+/+)/apolipoprotein (apo) E(-/-), mice. Notably, IL-6 induction on oxidized low-density lipoproteins stimulation is completely abolished in the absence of GDF-15. Consistent with our in vitro data GDF-15 mRNA expression and protein levels are upregulated (2.5- to 6-fold) in the atherosclerotic vessel wall of GDF-15(+/+)/apoE(-/-) mice after a cholesterol-enriched diet. GDF-15 deficiency inhibits lumen stenosis (52%) and (18)FDG uptake (34%) in the aortic arch despite increased serum triglyceride/cholesterol levels and elevated body weight. Immunohistomorphometric investigations of atherosclerotic lesions reveal a decreased percentage of inflammatory CD11b(+) (57%) or IL-6(+), leukocytes, and apoptotic cells (74%) after 20 weeks. However, the total number of macrophages and cell density in atherosclerotic lesions of the innominate artery are increased in GDF-15(-/-)/apoE(-/-) mice. CONCLUSIONS: Our data suggest that GDF-15 is involved in orchestrating atherosclerotic lesion progression by regulating apoptotic cell death and IL-6-dependent inflammatory responses to vascular injury.

Characterization of human myoblast differentiation for tissue-engineering purposes by quantitative gene expression analysis.

Tissue engineering of skeletal muscle is an encouraging possibility for the treatment of muscle loss through the creation of functional muscle tissue in vitro from human stem cells. Currently, the preferred stem cells are primary, non-immunogenic satellite cells ( = myoblasts). The objective of this study was to determine the expression patterns of myogenic markers within the human satellite cell population during their differentiation into multinucleated myotubes for an accurate characterization of stem cell behaviour. Satellite cells were incubated (for 1, 4, 8, 12 or 16 days) with a culture medium containing either a low [ = differentiation medium (DM)] or high [ = growth medium (GM)] concentration of growth factors. Furthermore, we performed a quantitative gene expression analysis of well-defined differentiation makers: myogenic factor 5 (MYF5), myogenin (MYOG), skeletal muscle αactin1 (ACTA1), embryonic (MYH3), perinatal (MYH8) and adult skeletal muscle myosin heavy chain (MYH1). Additionally, the fusion indices of forming myotubes of MYH1, MYH8 and ACTA1 were calculated. We show that satellite cells incubated with DM expressed multiple characteriztic features of mature skeletal muscles, verified by time-dependent upregulation of MYOG, MYH1, MYH3, MYH8 and ACTA1. However, satellite cells incubated with GM did not reveal all morphological aspects of muscle differentiation. Immunocytochemical investigations with antibodies directed against the differentiation markers showed correlations between the gene expression and differentiation. Our data provide information about time-dependent gene expression of differentiation markers in human satellite cells, which can be used for maturation analyses in skeletal muscle tissue-engineering applications.

Evaluation of valid reference genes during stimulation with static magnetic fields in human myoblast cultures.

Investigation of gene expression using real-time PCR (qRT-PCR) requires normalization with genes that are continuously expressed (reference genes; RGs). For accurate measurements, it is exceedingly important that RG expression is invariant under the investigated experimental conditions. It has recently become evident that RG expression may vary considerably under different culture conditions, which results in inaccurate qRT-PCR measurements. Static magnetic fields (SMFs) have been shown to enhance myogenic cell differentiation in the rat cell line L6, and may also induce differentiation in human myoblast cultures. In order to perform precise qRT-PCR measurements in human myoblast cell cultures stimulated with SMFs, one prerequisite is to find the most suitable RG. In this study, qRT-PCR was used to investigate the gene expression of six widely used RGs in human myoblast cell cultures stimulated with SMFs, with the aim of identifying the most stable among them. The mRNA concentration of ß-actin (ACTB), ß-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), peptidylprolyl isomerase A (PPIA), TATA box binding protein (TBP) and ribosomal protein, large, P0 (RPLPO) were quantified, and the most suitable RGs were identified using the geNorm and NormFinder software programs. Results were verified by BestKeeper software. mRNA expression of the following genes of interest was analyzed: myosin, heavy chain 1, skeletal muscle, adult (MYH1); myosin, heavy chain 3, skeletal muscle, embryonic (MYH3); myosin, heavy chain 8, skeletal muscle, perinatal (MYH8), as well as the immunoreactivity of MYH1 (irMYH1). Using geNorm, PPIA and B2M were found to be the most stable genes, followed by GAPDH. NormFinder identified PPIA as the most stable gene, followed by B2M and GAPDH. Finally, BestKeeper revealed TBP and PPIA to be the most stable genes, while B2M was ranked third.

Dexamethasone prevents transport inhibition by hypoxia in rat lung and alveolar epithelial cells by stimulating activity and expression of Na+-K+-ATPase and epithelial Na+ channels.

Hypoxia inhibits Na and lung fluid reabsorption, which contributes to the formation of pulmonary edema. We tested whether dexamethasone prevents hypoxia-induced inhibition of reabsorption by stimulation of alveolar Na transport. Fluid reabsorption, transport activity, and expression of Na transporters were measured in hypoxia-exposed rats and in primary alveolar type II (ATII) cells. Rats were treated with dexamethasone (DEX; 2 mg/kg) on 3 consecutive days and exposed to 10% O(2) on the 2nd and 3rd day of treatment to measure hypoxia effects on reabsorption of fluid instilled into lungs. ATII cells were treated with DEX (1 muM) for 3 days before exposure to hypoxia (1.5% O(2)). In normoxic rats, DEX induced a twofold increase in alveolar fluid clearance. Hypoxia decreased reabsorption (-30%) by decreasing its amiloride-sensitive component; pretreatment with DEX prevented the hypoxia-induced inhibition. DEX increased short-circuit currents (ISC) of ATII monolayers in normoxia and blunted hypoxic transport inhibition by increasing the capacity of Na(+)-K(+)-ATPase and epithelial Na(+) channels (ENaC) and amiloride-sensitive ISC. DEX slightly increased the mRNA of alpha- and gamma-ENaC in whole rat lung. In ATII cells from DEX-treated rats, mRNA of alpha(1)-Na(+)-K(+)-ATPase and alpha-ENaC increased in normoxia and hypoxia, and gamma-ENaC was increased in normoxia only. DEX stimulated the mRNA expression of alpha(1)-Na(+)-K(+)-ATPase and alpha-, beta-, and gamma-ENaC of A549 cells in normoxia and hypoxia (1.5% O(2)) when DEX treatment was begun before or during hypoxic exposure. These results indicate that DEX prevents inhibition of alveolar reabsorption by hypoxia and stimulates the expression of Na transporters even when it is applied in hypoxia.