Daling Zhu

Chronic Hypoxia Activates Lung 15-Lipoxygenase, Which Catalyzes Production of 15-HETE and Enhances Constriction in Neonatal Rabbit Pulmonary Arteries

Abstract— Hypoxia causes localized pulmonary arterial (PA) constriction to divert blood flow to optimally ventilated regions of the lung. The biochemical mechanisms for this have remained elusive, especially during prolonged exposures to reduced Po2. We have evidence that subacute hypoxia activates 15-lipoxygenase (15-LO) in small PAs of neonatal rabbits maintained for 9 days in hypoxic environments (Fio2=0.12) compared with siblings raised under normoxia. PA microsomal products of 15-LO, 15-hydroxyeicosatetraenoic acid (HETE), 11,14,15-trihydroxyeicosatrienoic acid (THETA), and 11,12,15-THETA were identified by gas chromatography/mass spectrometry. Increased amounts of these products are synthesized in vivo and in vitro by the lungs of animal raised in hypoxic versus normoxic environments. 15-HETE formation is attenuated by lipoxygenase, but not cytochrome P450 or cyclooxygenase inhibitors. Activation of 15-LO is associated with translocation of the enzyme from the cytosol to membrane as seen by Western immunoblotting. Immunohistochemical analysis demonstrates that 15-LO expression is clearly localized in vascular cells in lungs from normoxic and hypoxic kits. 15-HETE causes concentration-dependent constriction of PA rings from animals exposed to hypoxic but not normoxic environments. In addition, lipoxygenase inhibitors reduce phenylephrine-induced constriction of PA rings. Therefore, subacute hypoxia increases expression of and activates 15-LO, and enhances sensitivity of pulmonary arteries to its product, 15-HETE. Because 15-HETE is a constrictor in this vascular bed, it may play an important role in hypoxia-induced pulmonary vasoconstriction in rabbit kits. Although a clear causal relationship remains to be demonstrated, these data suggest a previously unrecognized role for 15-LO in hypoxic vasoconstriction in neonatal mammals.

The MicroRNA-328 Regulates Hypoxic Pulmonary Hypertension by Targeting at Insulin Growth Factor 1 Receptor and L-Type Calcium Channel-α1C

Chronic hypoxia is the most common cause of secondary pulmonary hypertension, for which the mechanisms are still unclear. Recent studies implicated an important role for microRNAs (miRNAs) in hypoxia-mediated responses in various cellular processes, including cell apoptosis and proliferation. Therefore, we hypothesized that these regulatory molecules might be implicated in the etiology of hypoxic pulmonary hypertension. Here we show that miRNA-328, a posttranscriptional regulator, was drastically downregulated in the pulmonary artery (PA) after a hypoxic assault. PA rings, Western blot, quantitative real-time PCR, in situ hybridization, and luciferase assay were used to investigate the role of miRNA-328 in hypoxic pulmonary hypertension. We found that hypoxia produced a significant inhibition of miRNA-328 expression, which was involved in PA vasoconstriction and remodeling. Overexpressing miRNA-328 in the transgenic mice remarkably decreased the right ventricular systolic pressure and PA wall thickness under both normoxia and hypoxia. MiRNA-328 inhibited L-type calcium channel-&agr;1C expression through a miRNA-328 binding site within the 3′ untranslational region of L-type calcium channel-&agr;1C. The L-type calcium channel-&agr;1C inhibition attenuated the PA response to KCl. Furthermore, miRNA-328 suppressed the insulin growth factor 1 receptor, ultimately leading to apoptosis of pulmonary arterial smooth muscle cells. The posttranscriptional repression of L-type calcium channel-&agr;1C and insulin growth factor 1 receptor was further confirmed by luciferase reporter assay. These results showed that miRNA-328, an important protecting factor, plays a significant role in PA constriction and remodeling by regulating multiple gene targets in hypoxic pulmonary hypertension.

Key role of 15-lipoxygenase/15-hydroxyeicosatetraenoic acid in pulmonary vascular remodeling and vascular angiogenesis associated with hypoxic pulmonary hypertension.

We have found that 15-hydroxyeicosatetraenoic acid (15-HETE) induced by hypoxia was an important mediator in the regulation of hypoxic pulmonary hypertension, including the pulmonary vasoconstriction and remodeling. However, the underlying mechanisms of the remodeling induced by 15-HETE are poorly understood. In this study, we performed immunohistochemistry, pulmonary artery endothelial cells migration and tube formation, pulmonary artery smooth muscle cells bromodeoxyuridine incorporation, and cell cycle analysis to determine the role of 15-HETE in hypoxia-induced pulmonary vascular remodeling. We found that hypoxia induced pulmonary vascular medial hypertrophy and intimal endothelial cells migration and angiogenesis, which were mediated by 15-HETE. Moreover, 15-HETE regulated the cell cycle progression and made more smooth muscle cells from the G0/G1 phase to the G2/M+S phase and enhanced the microtubule formation in cell nucleus. In addition, we found that the Rho-kinase pathway was involved in 15-HETE–induced endothelial cells tube formation and migration and smooth muscle cell proliferation. Together, these results show that 15-HETE mediates hypoxia-induced pulmonary vascular remodeling and stimulates angiogenesis via the Rho-kinase pathway.

20-Hydroxyeicosatetraenoic acid inhibits the apoptotic responses in pulmonary artery smooth muscle cells.

20-Hydroxyeicosatetraenoic acid (20-HETE), a omega-hydroxylation product of arachidonic acid catalyzed by cytochrome P450 4A (CYP4A), plays a role in vascular smooth muscle remodeling. Although its effects on angiogenic responses are known, it remains unclear whether 20-HETE acts on apoptosis of pulmonary arterial smooth muscle cells (PASMC), an important step in PASMC remodeling, and what pathways are involved in the process. Here we show evidence for the missing information. The effect of 20-HETE on PASMC apoptosis and the apoptosis-associated signaling pathways were determined with cell viability assay, Annexin V and propidium idodide binding, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), mitochondrial potentials assay, caspase activity assay and Western blots. We found that exogenous 20-HETE suppressed the serum deprivation-induced loss of bovine PASMCs and prevented Annexin V binding, DNA nick end labeling and chromatin condensation. The effect was worsened by 17-octadecynoic acid (17-ODYA), which inhibited the production of endogenous 20-HETE. Furthermore, 20-HETE induced the expression of bcl-2, maintained the stability of mitochondria membrane, and relieved the activation of caspase-9 and caspase-3. Such effects were reversed in the presence of 17-ODYA. Thus, these findings indicate that 20-HETE protects PASMCs against apoptosis by acting on, at least in part, the intrinsic apoptotic pathway.

ROCK pathway participates in the processes that 15-hydroxyeicosatetraenoic acid (15-HETE) mediated the pulmonary vascular remodeling induced by hypoxia in rat.

15‐Hydroxyeicosatetraenoic acid (15‐HETE), a product of arachidonic acid (AA) catalyzed by 15‐lipoxygenase (15‐LO), plays an essential role in hypoxic pulmonary arterial hypertension. We have previously shown that 15‐HETE inhibits apoptosis in pulmonary artery smooth muscle cells (PASMCs). To test the hypothesis that such an effect is attributable to the hypoxia‐induced pulmonary vascular remodeling (PVR), we performed these studies. We found subtle thickening of proximal media/adventitia of the pulmonary arteries (PA) in rats that had been exposed to hypoxia. This was associated with an up‐regulation of the anti‐apoptotic Bcl‐2 expression and down‐regulation of pro‐apoptotic caspase‐3 and Bax expression in PA homogenates. Nordihydroguaiaretic acid (NDGA), which inhibits the generation of endogenous 15‐HETE, reversed all the alterations following hypoxia. In situ hybridization histochemistry and immunocytochemistry showed that the 15‐LO‐1 mRNA and protein were localized in pulmonary artery endothelial cells (PAECs), while the 15‐LO‐2 mRNA and protein were localized in both PAECs and PASMCs. Furthermore, the Rho‐kinase (ROCK) pathway was activated by both endogenous and exogenous 15‐HETE, alleviating the serum deprivation (SD)‐induced PASMC apoptosis. Thus, these findings indicate that 15‐HETE protects PASMC from apoptosis, contributing to pulmonary vascular medial thickening, and the effect is, at least in part, mediated via the ROCK pathway. J. Cell. Physiol. 222:82–94, 2010.

microRNA-138 plays a role in hypoxic pulmonary vascular remodelling by targeting Mst1

Unbalanced apoptosis is a major cause of structural remodelling of vasculatures associated with PAH (pulmonary arterial hypertension), whereas the underlying mechanisms are still elusive. miRNAs (microRNAs) regulate the expression of several proteins that are important for cell fate, including differentiation, proliferation and apoptosis. It is possible that these regulatory RNA molecules play a role in the development of PAH. To test this hypothesis, we studied the effect of several miRNAs on the apoptosis of cultured PASMCs (pulmonary artery smooth muscle cells) and identified miR-138 to be an important player. miR-138 was expressed in PASMCs, and its expression was subjected to regulation by hypoxia. Expression of exogenous miR-138 suppressed PASMC apoptosis, prevented caspase activation and disrupted Bcl-2 signalling. The serine/threonine kinase Mst1, an amplifier of cell apoptosis, seemed to be a target of miR-138, and the activation of the Akt pathway was necessary for the anti-apoptotic effect of miR-138. Therefore the results of the present study suggest that miR-138 appears to be a negative regulator of PASMC apoptosis, and plays an important role in HPVR (hypoxic pulmonary vascular remodelling).

Multi-therapeutic effects of human adipose-derived mesenchymal stem cells on radiation-induced intestinal injury

Radiation-induced intestinal injuries (RIII) commonly occur in patients who suffer from pelvic or abdominal cancer. However, current management of these injuries is ineffective. Recently, mesenchymal stem cells (MSCs) have been extensively used in regenerative medicine and have achieved a high level of efficacy. In the present study, we hypothesised that human adipose-derived mesenchymal stem cells (hAd-MSCs) could be used as potential tools to heal RIII. We observed that adult Sprague–Dawley rats that received whole-abdominal irradiation benefitted from hAd-MSC injection. hAd-MSCs had RIII-healing effects, including anti-inflammation, neovascularisation and maintenance of epithelium homeostasis, as indicated by elevated serum IL-10, upregulation of vascular endothelial growth factor, basic fibroblast growth factor and epidermal growth factor in irradiated intestine, mobilisation of CD31-positive haematopoietic stem cells or haematopoietic progenitor cells, and the prolonged presence of Bmi1-positive cells within crypts. Consequently, after hAd-MSC treatment, irradiated rats survived longer than non-treated animals. These results suggest that hAd-MSCs have therapeutic potential for RIII management.

Structural and functional alterations in the rat lung following whole thoracic irradiation with moderate doses: injury and recovery

Purpose: To characterize structural and functional injuries following a single dose of whole-thorax irradiation that might be survivable after a nuclear attack/accident. Methods: Rats were exposed to 5 or 10 Gy of X-rays to the whole thorax with other organs shielded. Non-invasive measurements of breathing rate and arterial oxygen saturation, and invasive evaluations of bronchoalveolar lavage fluid, (for total protein, Clara cell secretory protein), vascular reactivity and histology were conducted for at least 6 time points up to 52 weeks after irradiation. Results: Irradiation with 10 Gy resulted in increased breathing rate, a reduction in oxygen saturation, an increase in bronchoalveolar lavage fluid protein and attenuation of vascular reactivity between 4–12 weeks after irradiation. These changes were not observed with the lower dose of 5 Gy. Histological examination revealed perivascular edema at 4–8 weeks after exposure to both doses, and mild fibrosis beyond 20 weeks after 10 Gy. Conclusions: Single-dose exposure of rat thorax to 10 but not 5 Gy X-irradiation resulted in a decrease in oxygen uptake and vasoreactivity and an increase in respiratory rate, which paralleled early pulmonary vascular pathology. Vascular edema resolved and was replaced by mild fibrosis beyond 20 weeks after exposure, while lung function recovered.

15-HETE suppresses K + channel activity and inhibits apoptosis in pulmonary artery smooth muscle cells

Abstract15-Hydroxyeicosatetraenoic acid (15-HETE) is an important hypoxic product from arachidonic acid (AA) in the wall of pulmonary vessels. Although its effects on pulmonary artery constriction are well known, it remains unclear whether 15-HETE acts on the apoptotic responses in pulmonary artery smooth muscle cells (PASMCs) and whether K+ channels participate in this process. These hypothesises were validated by cell viability assay, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, mitochondrial potentials assay, caspase activity assay and western blot. We found that 15-HETE enhanced cell survival, suppressed the expression and activity of caspase-3, upregulated bcl-2 and attenuated mitochondrial depolarization, prevented chromatin condensation and partly reversed K+ channel opener-induced apoptosis in PASMCs under serum-deprived conditions. Our data indicated that 15-HETE inhibits the apoptosis in PASMCs through, at least in part, inactivating K+ channels.

Subacute hypoxia suppresses Kv3.4 channel expression and whole-cell K+ currents through endogenous 15-hydroxyeicosatetraenoic acid in pulmonary arterial smooth muscle cells

We have previously reported that subacute hypoxia activates lung 15-lipoxygenase (15-LOX), which catalyzes arachidonic acid to produce 15-HETE, leading to constriction of neonatal rabbit pulmonary arteries. Subacute hypoxia suppresses Kv3.4 channel expression and results in an inhibition of whole-cell K(+) currents (I(K)). Although the Kv channel inhibition is likely to be mediated through 15-HETE, direct evidence is still lacking. To reveal the role of the 15-LOX/15-HETE pathway in the hypoxia-induced down-regulation of Kv3.4 channel expression and inhibition of I(K), we performed studies using 15-LOX blockers, whole-cell patch-clamp, semi-quantitative PCR, ELISA and Western blot analysis. We found that Kv3.4 channel expression at the mRNA and protein levels was greatly up-regulated in pulmonary arterial smooth muscle cells after blockade of 15-LOX by CDC or NDGA. The 15-LOX blockade also partially restored I(K). In comparison, 15-HETE had a stronger effect than 12-HETE on the expression of Kv3.4 channels. 5-HETE had no noticeable effect on Kv3.4 channel expression. These data indicate that the 15-LOX pathway via its metabolite, 15-HETE, seems to play a role in the down-regulation of Kv3.4 expression and I(K) inhibition after subacute hypoxia.