Endothelial nitric oxide synthase mediates protective effects of hypoxic preconditioning in lungs

To elucidate the protective mechanism of whole-body hypoxic preconditioning (WHPC) on pulmonary ischemia-reperfusion injury focussing on nitric oxide synthases (NOS), mice were placed in a hypoxic chamber (FIO(2)=0.1) for 4h followed by 12h of normoxia. Then, pulmonary ischemia for 1h followed by 5h of reperfusion was performed by clamping the left hilum in vivo (I/R). WHPC protected WT mice from pulmonary leukocyte infiltration as assessed by myeloperoxidase (MPO) activity, associated with a mild further increase in endothelial permeability (Evans Blue extravasation). When all NOS isoforms were inhibited during WHPC by L-NAME, mortality and MPO activity after I/R markedly increased. To determine the responsible NOS isoform, quantitative RT-PCR was performed for eNOS and iNOS mRNA, showing that only eNOS was upregulated in response to WHPC. While eNOS total protein expression remained unchanged, the amount of phosphorylated eNOS also increased. The WHPC/IR experiments were then repeated with eNOS knockout mice. Here, we found that the protective effect of WHPC on pulmonary leukocyte sequestration was abrogated, and endothelial leakage was further exacerbated. We conclude that WHPC limits neutrophil sequestration via an eNOS-dependent mechanism, and that eNOS helps preserve endothelial permeability during hypoxia and I/R.     

Role of O(2)-sensitive K(+) and Ca(2+) channels in the regulation of the pulmonary circulation: potential role of caveolae and implications for high altitude pulmonary edema

High altitude pulmonary edema (HAPE) is a potentially fatal complication in response to exposure to low O(2) at high altitudes. Hypoxia, by causing pulmonary vasoconstriction, increases pulmonary vascular resistance and pulmonary arterial pressure, both of which are features in the pathogenesis of HAPE. Uneven hypoxic pulmonary vasoconstriction is thought to be responsible for increased capillary pressure and leakage, resulting in edema. O(2)-sensitive ion channels are known to play pivotal roles in determining vascular tone in response to hypoxia. K(+), Ca(2+) and Na(+) channels are ubiquitously expressed in both endothelial and smooth muscle cells of the pulmonary microvasculature, subfamilies of which are regulated by local changes in P(O(2)). Hypoxia reduces activity of voltage-gated K(+) channels and down-regulates their expression leading to membrane depolarization, Ca(2+) influx in pulmonary artery smooth muscle cells (by activating voltage-dependent Ca(2+) channels) and vasoconstriction. Hypoxia up-regulates transient receptor potential channels (TRPC) leading to enhanced Ca(2+) entry through receptor- and store-operated Ca(2+) channels. Altered enrichment of ion channels in membrane microdomains, in particular in caveolae, may play a role in excitation-contraction coupling and perhaps in O(2)-sensing in the pulmonary circulation and thereby may contribute to the development of HAPE. We review the role of ion channels, in particular those outlined above, in response to low O(2) on vascular tone and pulmonary edema. Advances in the understanding of ion channels involved in the physiological response to hypoxia should lead to a greater understanding of the pathogenesis of HAPE and perhaps in the identification of new therapies.     

缺氧和酵母多糖对清醒山羊肺动脉压和肺内液体的影响

作者采用山羊慢性肺淋巴瘘模型,观察了缺氧和酵母多糖活化的血浆对清醒山羊肺动脉压、肺血管壁通透性和肺组织间液形成的影响。缺氧时肺动脉压显著升高,这可能是肺毛细血管前肌性小动脉收缩的结果。为缺氧动物输入酵母多糖活化的血浆,肺动脉压在缺氧增高的基础上又有短暂升高,接着出现肺血管扩张,缺氧性肺动脉增压反应受抑。酵母多糖活化的血浆可致山羊肺微血管壁受损,缺氧(模拟4000米高原)并未加重其损伤。     

缺氧和空气栓塞对清醒山羊肺动脉增压反应及肺内液体交换的影响

作者采用山羊慢性肺淋巴瘘方法,观察缺氧、空气栓塞单独和复合作用,对清醒山羊肺动脉压、肺微血管壁通透性和肺内液体交换的影响。实验结果表明:①缺氧所致的成年山羊肺动脉压增高,可能是毛细血管前阻力血管收缩的结果。成年山羊在模拟4000m高原缺氧1小时,未致肺微血管壁通透性增高;②肺血管空气栓塞可致肺动脉压和微血管壁通透性增高;③缺氧和空气栓塞复合作用时,肺动脉增压反应的幅度并非两者单独作用的叠加;④空气栓塞引起的肺血管壁通透性增高未为缺氧所加重。     

The protective effect of hepatocyte growth-promoting factor (pHGF) against hydrogen peroxide-induced acute lung injury in rats

To examine the protective effect of hepatocyte growth-promoting factor (pHGF) in hydrogen peroxide (H(2)O(2))-induced acute lung injury in rats, we observed the pathological changes in lung tissue by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and by light and electron microscopy. We also measured the serum levels of lipid peroxide (LPO). At 6 to 24 h after H(2)O(2) injection, the level of LPO was significantly higher in the H(2)O(2) group than in the H(2)O(2) + pHGF-treated group. This finding indicated that pHGF protected against cell membrane damage in H2O2-induced acute lung injury. Positive TUNEL signals were found in capillary endothelial cells, alveolar epithelial cells, and inflammatory cells. In the H(2)O(2) + pHGF-treated group, TUNEL-positive signals were reduced compared with those in the H(2)O(2) group. This finding indicated that pHGF acts to suppress apoptosis. In the H(2)O(2) group, severe pulmonary edema was seen 3 h after H(2)O(2) injection, and at 24 h, severe atelectasis was seen. In the H(2)O(2) + pHGF-treated group, pulmonary edema was scarcely seen and severe atelectasis was not found. This finding indicated that pHGF acts to suppress both severe pulmonary edema and atelectasis. In the H(2)O(2) group, the formation of subendothelial blebs and disruption of endothelial cells was observed. Edema and disruption were seen in type I epithelial cells. In type II lung epithelial cells, mitochondria were swollen and microvilli had disappeared. In the H(2)O(2) + pHGF-treated group, the formation of subendothelial blebs was seen, but no severe subendothelial blebs were observed. Disruption of capillary endothelial cells and type I epithelial cells was not evident, nor was there damage to type II lung epithelial cells. These findings indicated that pHGF protects the progression of H(2)O(2)-induced acute lung injury, and showed that pHGF acts to stabilize the cell membrane in capillary endothelial cells and lung epithelial cells.     

Lung overexpression of angiostatin aggravates pulmonary hypertension in chronically hypoxic mice

Exposure to hypoxia leads to the development of pulmonary hypertension (PH) as a consequence of pulmonary smooth muscle hyperplasia. Hypoxia concomitantly stimulates lung expression of angiogenic factors. To investigate the role of angiogenesis processes in development of hypoxic PH, we examined the effects of lung overexpression of angiostatin, an angiogenesis inhibitor, on development of hypoxic PH and lung endothelial cell (EC) density. Angiostatin delivery was achieved by a defective adenovirus expressing a secretable angiostatin K3 molcule driven by the cytomegalovirus promoter (Ad.K3). Comparison was made with a control vector containing no gene in the expression cassette (Ad.CO1). Treatment with Ad.K3 (300 plaque-forming units [pfu]/cell) inhibited cultured human pulmonary artery EC migration by 100% and proliferation by 50%, but was without effects on human pulmonary artery smooth muscle cells. After intratracheal administration of Ad.K3 (109 pfu) to mice, angiostatin protein became detectable in bronchoalveolar lavage fluid. Mice pretreated with Ad.K3 1 d before a 2-wk exposure to hypoxia (10% O2) showed more severe pulmonary hypertension than Ad.CO1-pretreated controls, as assessed by higher right ventricular systolic pressure (36.5 +/- 2.4 versus 30.2 +/- 1.4, respectively), aggravation of right ventricular hypertrophy (P < 0.05), and muscularization of distal vessels (P < 0.01). Lung factor VIII, CD31 immunostaining, as well as eNOS expression were significantly increased after exposure to hypoxia in Ad.CO1-pretreated controls, but decreased in both normoxic and hypoxic animals after treatment with Ad.K3. The results show that inhibition of hypoxia-induced stimulation of lung angiogenic processes aggravates development of hypoxic PH. This suggests that endogenous lung angiogenesis counteracts development of hypoxic PH.     

高原低氧对大鼠心肺组织超微结构的影响

背景：研究表明快速进入高原地区时,机体不可避免地会受到不同程度的损伤,以心肺损伤较显著。 目的：观察低氧习服对高原低氧大鼠心肺组织的超微结构影响。 方法：将SD大鼠分别为进行高原低氧干预1,3和30 d,并设置对照组。3个高原低氧组由海拔5 m的西安途中耗时1 d带到海拔2 700 m的青海格尔木地区、途中耗时3,30 d分别带到海拔4 500 m的西藏那曲地区,观察各时间点心肺标本的组织学变化。 结果与结论：急性高原低氧1,3 d组肺组织显微和超微结构出现明显的间质性肺水肿和肺泡性肺水肿,其心脏组织光镜下大鼠各室壁心肌细胞均可见不同程度的浊肿、空泡变性、溶解坏死及间质水肿等,电镜下可见心肌细胞线粒体肿胀,肌浆网扩张,肌原纤维溶解,细胞内外水肿等,急性高原低氧3 d上述改变右室壁较左室壁明显,而低氧习服后高原低氧30 d组间质性水肿和则肺水肿明显减轻。结果证实,高原急性缺氧可造成大鼠间质性肺水肿和肺泡型肺水肿,并引起以右心室为主的全心性损伤,经过高原低氧习服后心肺组织病变明显减轻。     

慢性低氧小鼠肺组织糖蛋白的变化

目的 :研究在慢性常压低氧下的小鼠肺组织多糖、粘蛋白的适应性变化。方法 :低氧 (1 0 % )第 1、3、5、7天分别取左肺中部组织作冰冻切片 ,以高碘酸Schiff反应 (PAS)显示糖原及粘蛋白并进行光镜观察。结果 :与动脉平滑肌比较 ,低氧小鼠支气管平滑肌糖原合成一过性增强 ;静脉内皮细胞糖代谢的敏感性显著高于动脉 ;低氧能刺激支气管粘膜分泌细胞分泌粘蛋白 ,反应随低氧作用时间而变化。结论 :小鼠肺组织糖原、粘蛋白代谢在慢性低氧下产生适应性变化。杯状细胞与管壁内腺体的分泌物在上皮表面共同构成的粘液性屏障有利于小鼠的低氧适应     

Effect of wood combustion smoke inhalation on angiotensin-1-converting enzyme in the dog

One lung of each dog was exposed to smoke from burning pine wood, while the other was subjected to acute hypoxia. Angiotensin-1-converting enzyme (ACE) activity in biopsied tissue of the smoke-exposed lung was markedly increased immediately after the injury and even higher 30 minutes later. No change in ACE activity was observed in the hypoxic contralateral lung. Serum ACE activity did not change significantly following anesthesia and before smoke inhalation. Serum aldosterone and cortisol levels increased at this juncture. Smoke inhalation caused intra-alveolar hemorrhages and edema. Pulmonary and systolic, diastolic and mean pressures, pulmonary capillary, wedge pressure, cardiac output and systemic and pulmonary arteriolar resistances remained unchanged throughout the experiment. The changes of ACE activity are presumably a direct effect of smoke inhalation. They are seen as an early response of the lung endothelial cells to many types of injury (chronic hypoxia, bleomycin or monocrotaline administration) and may represent an important step in the development of the organ's response to the injury.     

Hypoxic induction of cox-2 regulates proliferation of human pulmonary artery smooth muscle cells

Chronic hypoxia-induced pulmonary hypertension results partly from proliferation of smooth muscle cells in small peripheral pulmonary arteries. Therefore, we examined the effect of hypoxia on growth of pulmonary artery smooth muscle cells (PASMCs) from human distal pulmonary arteries. Initial studies identified that serum-induced proliferation of explant-derived PASMCs was inhibited under hypoxic conditions (3-4 kPa in medium). However, selection of hypoxia-stimulated cells was achieved by culturing cells at low density under conditions of prolonged hypoxia (1-2 wk). In hypoxia-inhibited and -stimulated cells, Western blotting revealed hypoxic induction of cyclooxygenase (COX)-2, which was dependent on the activation of p38(MAPK), but not COX-1, inducible nitric oxide synthase (iNOS), or hemoxygenase-1 (HO-1). Hypoxic induction of COX-2 was also observed in the media of pulmonary arteries in lung organ culture. Hypoxia induced a 4- to 5-fold increase (P < 0.001) in prostaglandin (PG)E(2), PGD(2), PGF(2alpha), and 6-keto-PGF(1alpha) release from PASMCs. Hypoxic inhibition of proliferation was attenuated by incubation with indomethacin (10 micro M), or the COX-2 antagonist, NS398 (10 micro M), but not by the COX-1 antagonist, valeryl salicylate (0.5 mM). In conclusion, we have isolated cells from human peripheral pulmonary arteries that are either inhibited or stimulated by culture under hypoxic conditions. In both cell types hypoxia modulates cell proliferation by induction of COX-2 and production of antiproliferative prostaglandins. Induction of COX-2 may contribute to the inhibition of hypoxia-induced pulmonary vascular remodeling.     

细胞对缺氧的感受和信号转导

缺氧是最常见的病理过程，急性缺氧在数秒或数分钟即可通过改变蛋白质的活性引起细胞的兴奋、抑制、运动、分泌、代谢等变化；数小时或数天的慢性缺氧可通过相关基因转录的变化导致细胞的增殖、分化、凋亡及代谢、机能等变化。     

Role of O2-sensitive K and Ca channels in the regulation of the pulmonary circulation: Potential role of caveolae and implications for high altitude pulmonary edema

High altitude pulmonary edema (HAPE) is a potentially fatal complication in response to exposure to low O₂ at high altitudes. Hypoxia, by causing pulmonary vasoconstriction, increases pulmonary vascular resistance and pulmonary arterial pressure, both of which are features in the pathogenesis of HAPE. Uneven hypoxic pulmonary vasoconstriction is thought to be responsible for increased capillary pressure and leakage, resulting in edema. O₂-sensitive ion channels are known to play pivotal roles in determining vascular tone in response to hypoxia. K⁺, Ca²⁺ and Na⁺ channels are ubiquitously expressed in both endothelial and smooth muscle cells of the pulmonary microvasculature, subfamilies of which are regulated by local changes in P_O2. Hypoxia reduces activity of voltage-gated K⁺ channels and down-regulates their expression leading to membrane depolarization, Ca²⁺ influx in pulmonary artery smooth muscle cells (by activating voltage-dependent Ca²⁺ channels) and vasoconstriction. Hypoxia up-regulates transient receptor potential channels (TRPC) leading to enhanced Ca²⁺ entry through receptor- and store-operated Ca²⁺ channels. Altered enrichment of ion channels in membrane microdomains, in particular in caveolae, may play a role in excitation–contraction coupling and perhaps in O₂-sensing in the pulmonary circulation and thereby may contribute to the development of HAPE. We review the role of ion channels, in particular those outlined above, in response to low O₂ on vascular tone and pulmonary edema. Advances in the understanding of ion channels involved in the physiological response to hypoxia should lead to a greater understanding of the pathogenesis of HAPE and perhaps in the identification of new therapies.     

Ventilatory chemosensitivity in the chick embryo

In the externally pipped chicken embryo, oxygen consumption through the chorioallantoic membrane (VO2CAM) ranged between 2 and 55% (mean approximately 24%) of that through the lungs (VO2lung). Hypercapnia (5'-10' of 2, 5, or 8% CO2) or mild hypoxia (15% O2) had minor effects on VO2, whereas moderate or severe hypoxia (10-5% O2) caused large drops of VO2. Hypoxia or hypercapnia delivered through the lungs increased pulmonary ventilation (VE), largely through increases in tidal volume (VT). Exposures of the whole embryo provoked VE responses either similar, or significantly higher, than with exposures of the lungs alone, because of greater increases in VT. The larger the VO2CAM, the smaller were the VE and VT responses when hypoxia or hypercapnia were delivered through the lungs. Hypoxic or hypercapnic exposures for longer periods of time (30'-40') gave qualitatively similar results. We conclude that in the externally pipped chick embryo (1) the increase in VT is the primary means of expressing the hypoxic or hypercapnic VE chemosensitivity, (2) hypometabolism contributes to the hypoxic hyperventilation, and (3) CAM gas exchange lowers the ventilatory effects of lung hypoxia or hypercapnia.     

牛磺酸抗缺氧及防治肺动脉高压的研究及其细胞机制初探

目的通过动物模型观察牛磺酸对缺氧性肺动脉高压的治疗作用,同时观察其对体外培养牛肺动脉平滑肌细胞(PASMC)和内皮细胞(PAEC)增殖的影响。方法采用模拟高原5 000 m制作缺氧大鼠模型,缺氧2周。设平原(C组)及缺氧对照组(H组),观察牛磺酸治疗后(T组)的肺动脉压(mPAP)、血浆乳酸脱氢酶(LDH)活性、脂质过氧化产物丙二醛(MDA)、肺匀浆一氧化氮(NO)含量、右心室肥大指数的变化。体外培养PASMC和PAEC用3H-TdR掺入法比较牛磺酸对缺氧PASMC和PAEC增殖的影响。结果H组大鼠LDH活性升高为C的10.1倍(P<0.01);肺匀浆NO含量降低为C组的32%(P<0.01);血浆MDA含量显著升高为C组的1.64倍(P<0.01);mPAP显著增高,约为C组的2.74倍(P<0.01);右心室肥大指数是C组的1.56倍(P<0.01)。T组与H组相比较:LDH活性、血浆MDA、右心室肥大指数均显著降低(P<0.01);mPAP显著降低(P<0.05)。高浓度(10～20 mmol/L)的牛磺酸抑制缺氧内皮及平滑肌细胞的3H-TdR掺入,而低浓度的牛磺酸促进缺氧时PAEC的3H-TdR掺入(P<0.05),抑制缺氧时PASMC的3H-TdR掺入(P<0.05)。结论牛磺酸有抗缺氧及防治肺动脉高压的作用。缺氧抑制内皮细胞的增殖而促进平滑肌细胞的增殖,适当剂量的牛磺酸可以对抗缺氧对PAEC和PASMs的作用:减弱缺氧对PAEC的增殖抑制作用,抑制缺氧的促PASMC增殖作用,使之接近常氧水平。这可能是牛磺酸防治肺动脉高压的细胞机制。提示牛横酸对于高山病缺氧性肺血管收缩和血管结构改建的预防和治疗,可能具有广阔的应用前景。     

高原低氧大鼠肺组织超微结构及其低氧诱导因子1α的表达

背景：低氧诱导因子1α可介导哺乳动物细胞适应低氧环境。 目的：观察高原低氧对大鼠肺组织超微结构的影响及其低氧诱导因子1α表达变化。 方法：将SD大鼠分别为进行高原低氧干预1,2,3和30 d,并设置对照组。4个高原低氧组由海拔5 m的西安地区途中耗时1 d带到海拔2 700 m的青海格尔木地区、途中耗时2 d带到海拔5 000 m的唐古拉地区,途中耗时3,30 d分别带到海拔4 500 m的西藏那曲地区。 结果与结论：光镜及电镜观察显示,急性高原低氧2 d组肺组织出现明显的高原肺水肿,急性高原低氧30 d组低氧诱导因子1α mRNA的表达明显增高(P < 0.01),高原肺水肿现象则明显减轻。结果证实,低氧习服后肺组织低氧诱导因子1α mRNA表达的提高有利于减轻高原肺水肿。     

Hypoxic suppression of K+ currents in type I carotid body cells: selective effect on the Ca2(+)-activated K+ current.

Whole-cell K+ currents were recorded in isolated type I carotid body cells using the patch-clamp technique. Hypoxia (pO2 25 torr) reversibly suppressed K+ currents in a voltage-dependent manner: maximal effects were seen at low, positive test potentials, where the Ca2(+)-activated component of K+ currents was greatest. Enhancing this component with 5 microM BAY K 8644 exaggerated the effects of hypoxia, and when the component was inhibited (100 microM Cd2+ or 5 microM nifedipine) hypoxic effects were abolished. As hypoxia does not affect Ca2+ currents directly, these data indicate the suppressive effect of hypoxia is selective for the Ca2(+)-activated component of K+ currents in type I cells.     

Ischemic preconditioning of the lower extremity attenuates the normal hypoxic increase in pulmonary artery systolic pressure

Ischemic pre-condition of an extremity (IPC) induces effects on local and remote tissues that are protective against ischemic injury. To test the effects of IPC on the normal hypoxic increase in pulmonary pressures and exercise performance, 8 amateur cyclists were evaluated under normoxia and hypoxia (13% F(I)O(2)) in a randomized cross-over trial. IPC was induced using an arterial occlusive cuff to one thigh for 5 min followed by deflation for 5 min for 4 cycles. In the control condition, the resting pulmonary artery systolic pressure (PASP) increased from a normoxic value of 25.6±2.3 mmHg to 41.8±7.2 mmHg following 90 min of hypoxia. In the IPC condition, the PASP increased to only 32.4±3.1 mmHg following hypoxia, representing a 72.8% attenuation (p=0.003). No significant difference was detected in cycle ergometer time trial duration between control and IPC conditions with either normoxia or hypoxia. IPC administered prior to hypoxic exposure was associated with profound attenuation of the normal hypoxic increase of pulmonary artery systolic pressure.     

Different patterns of pulmonary vascular disease induced by type 1 diabetes and moderate hypoxia in rats

Although type 1 and type 2 diabetes are strongly associated with systemic cardiovascular morbidity, the relationship with pulmonary vascular disease had been almost disregarded until recent epidemiological data revealed that diabetes might be a risk factor for pulmonary hypertension. Recent experimental studies suggest that diabetes induces changes in lung function insufficient to elevate pulmonary pressure. The aim of this study was to assess the effects of diabetes on the sensitivity to other risk factors for pulmonary hypertension. We therefore analysed the effects of the combination of diabetes with exposure to moderate hypoxia on classical markers of pulmonary hypertension. Control (saline-treated) and diabetic (70 mg kg(-1) streptozotocin-treated) male Wistar-Kyoto rats were followed for 4 weeks and exposed to normoxia or moderate normobaric hypoxia (14%) for another 2 weeks. Hypoxia, but not diabetes, strongly reduced voltage-gated potassium currents, whereas diabetes, but not hypoxia, induced pulmonary artery endothelial dysfunction. Both factors independently induced pulmonary vascular remodelling and downregulated the lung bone morphogenetic protein receptor type 2. However, diabetes, but not hypoxia, induced pulmonary infiltration of macrophages, which was markedly increased when both factors were combined. Diabetes plus hypoxia induced a modest increase in diastolic and mean pulmonary artery pressure and right ventricular weight, while each of the two factors alone had no significant effect. The pattern of changes in markers of pulmonary hypertension was different for moderate hypoxia and diabetes, with no synergic effect except for macrophage recruitment, and the combination of both factors was required to induce a moderate elevation in pulmonary arterial pressure.     

Hypoxia decreases cellular ATP demand and inhibits mitochondrial respiration of a549 cells

Hypoxia inhibits activity and expression of transporters involved in alveolar Na reabsorption and fluid clearance. We studied whether this represents a mechanism for reducing energy consumption or whether it is the consequence of metabolic dysfunction. Oxygen consumption (JO2) of A549 cells and primary rat alveolar type II cells was measured by microrespirometry during normoxia, hypoxia (1.5% O2), and reoxygenation. In both cell types, acute and 24-h hypoxia decreased total JO2 significantly and reoxygenation restored JO2 after 5 min but not after 24 h of hypoxia in A549 cells, whereas recovery was complete in type II cells. In A549 cells under normoxia Na/K-ATPase accounted for approximately 15% of JO2, whereas Na/K-ATPase-related JO2 was decreased by approximately 25% in hypoxia. Inhibition of other ion transporters did not affect JO2. Protein synthesis-related JO2 was not affected by acute hypoxia, but decreased by 30% after 24-h hypoxia. Acute and 24-h hypoxia decreased JO2 of A549 cell mitochondrial complexes I, II, and III by 30-40%. Reoxygenation restored complex I activity after acute hypoxia but not after 24-h hypoxia. ATP was decreased 30% after 24-h hypoxia, but lactate production rate was not affected. Reduced nicotinamine adenine dinucleotide was slightly elevated in acute hypoxia. Our findings indicate that inhibition of the Na/K-ATPase by hypoxia contributes little to energy preservation in hypoxia. It remains unclear to what extent hypoxic inhibition of mitochondrial metabolism affects ATP-consuming processes.