Proteomic analysis of hypoxia-induced tube breakdown of an in vitro capillary model composed of HUVECs: potential role of p38-regulated reduction of HSP27

We recently reported that hypoxia could induce the breakdown of capillary-like tubes formed by human umbilical vein endothelial cells (HUVECs) and that this breakdown was regulated by p38 and not by a caspase cascade, although the exact molecular mechanisms remain unknown. The aim of this study was to identify proteins that regulated hypoxia-induced tube breakdown through p38-regulated and caspase-independent mechanisms. The involvement of adhesion proteins, integrins, VE-cadherin, PECAM-1, and occludin was first investigated. Although some of these proteins decreased after hypoxia, none of them met the conditions of being quantitatively restored by p38 inhibition but not by caspase inhibition. We then conducted 2-D DIGE coupled with MALDI-TOF/TOF-MS to identify altered protein expression. The differential proteomic analysis of tube-forming HUVECs treated with normoxia or hypoxia and treated with hypoxia in the presence or absence of SB203580, a specific p38 inhibitor, revealed the involvement of heat shock proteins in this tube breakdown. We also confirmed that the amount of HSP27 and HSP70 changed in a p38-regulated and caspase-independent manner during hypoxia. Knocking down HSP27 expression using RNAi further augmented hypoxia-induced tube breakdown. Taken together, it was shown that p38-regulated and caspase-independent reduction of HSP27 plays an important role in hypoxia-induced tube breakdown.     

Gender-related differences in proliferative response of cardiac fibroblasts to hypoxia

Ischemic heart disease is more prevalent in men than in women. The remodeling of extracellular matrix, is a structural correlate of heart failure of ischemic origin and proliferation of cardiac fibroblasts is a key factor in this remodeling. We asked if proliferative response of male and female cardiac fibroblasts is differentially susceptible to hypoxia. DNA synthesis, using 3H-thymidine incorporation was compared under hypoxia (2% O₂) in cardiac fibroblasts obtained from adult, age-matched male and female rat heart. In female cells DNA synthesis remained unchanged under hypoxia and this resistance was dependent on tyrosine kinase activation, as it was abolished in the presence of genistein, a tyrosine kinase inhibitor. Male cells, on the other hand, were susceptible to hypoxia and their DNA synthesis was reduced significantly (70%, (p < 0.0001). This effect was partially reversed by inhibition of tyrosine kinase. Western analysis showed a higher abundance of tyrosine phosphorylated proteins in male cells compared to female cells as well as differences in molecular weight of basal and hypoxia-induced tyrosine-phosphorylated proteins between male and female cells. The presence of estrogen (17- estradiol, 10 nM) altered the response of both cells to hypoxia. In female cells the combined effect of hypoxia and estrogen led to inhibition of DNA synthesis, whereas in male cells estrogen partially reversed the hypoxia-induced inhibition of DNA synthesis (37% (p < 0.01) inhibition in the presence of estrogen vs. 70% (p < 0.0001) inhibition in the absence of estrogen). The effects of estrogen in male and female cells were mediated via estrogen receptors as they were reversed by the pure anti-estrogen, ICI 182,780. Western analysis of cell lysate showed hypoxia-induced increase in the level of estrogen receptor in both male and female cells. Gel shift analysis showed hypoxia-induced increase in cytoplasmic ERE (estrogen response element)-binding activity and decrease in nuclear ERE-binding in male cells. In female cells cytoplasmic and nuclear ERE-binding activities remained unchanged under hypoxia. Together, these data demonstrate that while female cells are resistant to hypoxia-induced inhibition in DNA synthesis, male cells are susceptible; intracellular pathways involving tyrosine phosphorylation are involved in the response of both cells; and estrogen, via estrogen-receptor-dependent mechanisms, differentially alters the response of male and female cells to hypoxia.     

Hypoxic Response of Synaptosomal Proteins in Term Guinea Pig Fetuses

Early events in the hypoxia-induced response trigger tyrosine phosphorylation cascades involving a large number of enzymes and substrates. The resolving power of advanced two-dimensional gel electrophoresis, followed by immunoblotting with specific antibodies to phosphotyrosine, has been used to analyze hypoxia-induced modifications in guinea pig brain synaptosomes. These procedures, in conjunction with computer-aided image analysis, are useful in the differential display of gene products, providing comparison at the level of posttranslationally modified products. Studies were performed in cerebral cortical synaptosomes from three normoxic and three hypoxic newborn guinea pigs. To filter off background noise consisting of nonreproducible migrating protein spots, only reproducible features of electrophoretic patterns were considered. Immunoreactivity patterns obtained with anti-phosphotyrosine antibodies proved to be different in normoxic and hypoxic synaptosomes: of a total of 130 immunoreactive spots, 49 were tyrosine-phosphorylated in hypoxic synaptosomes only and 20 in the normoxic ones only. Our data suggest that hypoxia extensively remodels the signaling pathway by switching off tyrosine phosphorylation of some cellular components (i.e., alpha-internexin) and switching on tyrosine phosphorylation of some other proteins (i.e., heat shock cognate 70, aconitase, 2',3'-cyclic nucleotide 3'-phosphodiesterase, and pyruvate kinase).     

Time-dependent changes of the susceptibility of cardiac contractile function to hypoxia-reoxygenation after myocardial infarction in rats

In this study we analyzed the susceptibility of contractile function of the myocardium to hypoxia-reoxygenation after infarction. For this purpose, the contractility of isolated papillary muscles from rats was studied at high oxygen tension (pO₂ 80 kPa) and during hypoxia (pO₂ 3 kPa) with subsequent reoxygenation at variable intervals between 15 h and 9 weeks after permanent ligation of the left coronary artery. Hypoxic exposure reduced the contractile performance of the preparations to a similar extent in both groups. Notably, the contractility and, in particular, the relaxation rates recovered more completely from hypoxia in the hypertrophied myocardium of rats with coronary artery ligation than in sham-operated (SO) animals. The recovery of contractile function was improved maximally between 6 and 9 weeks after myocardial infarction (MI). The lower sensitivity of the (post)ischemic myocardium to hypoxia-reoxygenation correlated with enhanced left ventricular glutathione peroxidase (GSH-Px) activity (15 h to 9 weeks post-MI) and 2–3-fold increased expression levels (15 h to 6 weeks post-MI) of the 72 kDa heat shock protein (HSP72) in the papillary muscles. These findings suggest that the greater antioxidant potential and, possibly, stimulation of HSPs contribute to the sustained tolerance of the myocardium to hypoxia-reoxygenation injury after infarction.     

Methyl group deficiency and guanidine production in uremia

Heat shock protein 70 (HSP70) has been reported to be involved in myocardial self-preservation system. This study shows direct evidence of the effect of HSP70 on lymphocytes during ischemia and reperfusion in CABG (coronary artery bypass graft) surgery. Lymphocytes were separated from the blood obtained from 10 patients undergoing CABG at different time intervals. (i) Baseline samples (drawn before onset of bypass), (ii) ischemic samples (30 min after cross-clamp) and (iii) reperfusion samples (10 min after the cross clamp removal) were incubated with recombinant HSP70 and the cells were harvested after 36 h. The effect of HSP70 was monitored by measuring second messengers such as intracellular calcium, protein kinase C (PKC) and inositol tri phosphate (IP₃). In addition CD69 expression was also measured. The results showed a significant decrease in intracellular calcium and CD69 expression in ischemia and further in reperfusion samples as compared to their respective un-triggered controls. PKC and IP₃ levels however remained unaffected. The protective effect of HSP70 during ischemia and reperfusion could thus be attributed to decreasing intracellular calcium and CD69 expression. This study could therefore provide a mechanism of cardioprotection afforded by HSP70.     

Transactivation of hsp70-1/2 in geldanamycin-treated human non-small cell lung cancer H460 cells: involvement of intracellular calcium and protein kinase C

Geldanamycin is an antitumor drug that binds HSP90 and induces a wide range of heat shock proteins, including HSP70s. In this study we report that the induction of HSP70s is dose-dependent in geldanamycin-treated human non-small cell lung cancer H460 cells. Analysis of the induction of HSP70s specific isoform using LC-ESI-MS/MS analysis and Northern blotting showed that HSP70-1/2 are the major inducible forms under geldanamycin treatment. Transactivation of hsp70-1/2 was determined by electrophoretic mobility-shift assay using heat shock element (HSE) as a probe. The signaling pathway mediators involved in hsp70-1/2 transactivation were screened by the kinase inhibitor scanning technique. Pretreatment with serine/threonine protein kinase inhibitors H7 or H8 blocked geldanamycin-induced HSP70-1/2, whereas protein kinase A inhibitor HA1004, protein kinase G inhibitor KT5823, and myosin light chain kinase inhibitor ML-7 had no effect. Furthermore, the protein kinase C (PKC)-specific inhibitor Ro-31-8425 and the Ca2+-dependent PKC inhibitor G?-6976 diminished geldanamycin-induced HSP70-1/2, suggesting an involvement of the PKC in the process. In addition, geldanamycin treatment causes a transient increase of intracellular Ca2+. Chelating intracellular Ca2+ with BAPTA-AM or depletion of intracellular Ca2+ store with A23187 or thapsigargin significantly decreased geldanamycin-transactivated HSP70-1/2 expression. Taken together, our results demonstrate that geldanamycin-induced specific HSP70-1/2 isoforms expression in H460 cells through signaling pathway mediated by Ca2+ and PKC.     

Endothelial cells downregulate expression of the 70 kDa heat shock protein during hypoxia

Hsp70 is induced by hypoxia in most mammalian cell types and contributes to their ability to survive hypoxic episodes. However, little is known about Hsp70 expression in the hypoxia-tolerant endothelial cells (ECs). We investigated the effect of hypoxia on Hsp70 in human microvascular endothelial HMEC-1 cells. Reduction of pO(2) to 2.5% of normal for 20 h stimulated lactate production and the activity of glycolytic enzymes. This metabolic adaptation to hypoxia was accompanied by a remarkable reduction of Hsp70 on the protein level and on the mRNA level. Approximately 12 h after the hypoxic period Hsp70 expression reached pre-hypoxia levels again. Since ECs are adapted to the low oxygen tension of the vasculature they are confronted with a supraphysiological oxygen level during in vitro culture. We suppose that the high Hsp70 under these conditions reflects a stress response which disappears at the more physiological reduced oxygen tension during hypoxia.     

Hypoxia induces epidermal keratinocyte matrix metalloproteinase-9 secretion via the protein kinase C pathway

Hypoxia promotes keratinocyte migration on wound bed connective tissues and is a profound biological signal that transforms a basal keratinocyte, destined to differentiate, into a motile cell that is essential for re-epithelialization. In this study, we examined the effect of hypoxia on keratinocyte-derived collagenases associated with keratinocyte migration. Cells plated on various connective tissue matrices under normoxic and hypoxic conditions, demonstrated a two-fold increase in the 92 kDa, type IV collagenase (MMP-9) when examined by quantitative zymography and ELISA. Western blotting and ELISA demonstrated a two-fold increase in tissue inhibitor of metalloproteinase (TIMP-1), an enzyme that binds to MMP-9 and inhibits its activity. The hypoxia-induced increase in cell motility could be inhibited by a neutralizing antibody to MMP-9. Northern blotting demonstrated that MMP-9 and TIMP-1 mRNA increased 2.5- to 4-fold, 2-12 h after the cells were made hypoxic. The hypoxia-induced changes in MMP-9 and TIMP-1 were inhibited by staurosporine and bisindolylmaleimide, inhibitors of protein kinase C (PKC), but not by inhibitors of tyrosine phosphorylation and the mitogen-activated protein kinase pathway. Inhibition of PKC also inhibited hypoxia-induced keratinocyte migration on type I collagen. These data provide evidence that hypoxia-induced keratinocyte migration is mediated by increased cellular secretion of MMP-9 via the PKC pathway.     

Chronic hypoxia stress-induced differential modulation of heat-shock protein 70 and presynaptic proteins

Chronic hypoxia exposure can cause neurobehavioral dysfunction, but the underlying cellular and molecular mechanisms remain unclear. Here, we found that adult Lymnaea stagnalis snails maintained in low O(2) (approximately 5%) for 4 days developed slowed reactions to light stimuli, and reduced righting movement. Semiquantitative immunoblotting analyses showed that hypoxia exposure induced increased expression of heat-shock protein (HSP)70 in ganglion preparations, and suppressed expression of the presynaptic proteins syntaxin I, synaptic vesicle protein 2 (SV2) and synaptotagmin I. Detailed time course analyses showed that an early moderate increase developed within 6 h, preceding a substantial up-regulation of HSP70 after 4 days; an early reduction of syntaxin I in the first 24 h; a delayed reduction of synaptotagmin I after 4 days; and a biphasic change in SV2. Using a double-stranded RNA interference approach, we demonstrated that preventing the hypoxia inducible HSP70 enhanced down-regulation of syntaxin and synaptotagmin, and aggravated motor and sensory suppression. Co-immunoprecipitation analysis revealed an interaction between HSP70 and syntaxin. We have thus provided the first evidence that early induction of HSP70 by chronic hypoxia is critical for maintaining expression levels of presynaptic proteins. These findings implicate a new molecular mechanism underlying chronic hypoxia-induced neurobehavioral adaptation and impairment.     

Tumor promoter phorbol ester reversibly modulates tyrosine dephosphorylatioin/ inactivation of protein kinase FA/GSK-3α in A431 cells

The signal transducrion mechanism of protein kinase F_A/GSK-3α by tyrosine phosphorylation in A431 cells was investigated. Kinase F_A/GSK-3α was found to exist in a highly tyrosine-phosphorylated/activated state in resting cells but could be tyrosine-dephosphorylated and inactivated to ～60% of the control level when cells were acutely treated with 1 μM tumor phorbol ester (TPA) at 37^oC for 30 min, as demonstrated by metabolic ³²P-labeling the cells, followed by immunoprecipitation and two-dimensional phosphoamino acid analysis and by immunodetection in an antikinase F_A/GSK-3α immunoprecipitate kinase assay. Conversely, when cells were chronically treated with 1 μM TPA at 37°C for 24 h and processed under identical condetions, kinase F_A/GSK-3α was found to be rephosphorylated on tyrosine residue and reactivated to ～130% of the original control level. Taken together, the results provide initial evidence that the phosphotyrosine content and cellular activity of kinase F_A/GSK-3α can be modulated in a reversible manner by short-term and long-term exposure of A431 cells to TPA. Since acute exposure of cells to TPA causes up-regulation of cellular protein kinase C (PKC) activity and prolonged exposure to TPA causes down-regulation of PKC, the results further suggest that the TPA-mediated modulation of PKC may play a role in the regulation of tyrosine phosphorylation and concurrent activation of kinase F_A/GSK-3α in cells, representing a new mode of signal transduction pathway for the regulation of this multisubstrate/multifunctional protein kinase in cells.     

Biomarkers of Dissolved Oxygen Stress in Oysters: A Tool for Restoration and Management Efforts

The frequency and intensity of anoxic and hypoxic events are increasing worldwide, creating stress on the organisms that inhabit affected waters. To understand the effects of low dissolved oxygen stress on oysters, hatchery-reared oysters were placed in cages and deployed along with continuously recording environmental data sondes at a reef site in Mobile Bay, AL that typically experiences low oxygen conditions. To detect and measure sublethal stress, we measured growth and survival of oysters as well as expression of three biomarkers, heat shock protein 70 (HSP70), hypoxia inducible factor (HIF) and phospho-p38 MAP kinase, in tissues from juvenile and adult oysters. Survival rates were high for both juvenile and adult oysters. Expression levels of each of the 3 isoforms of HSP 70 were negatively correlated to dissolved oxygen (DO) concentrations, suggesting that HSP 70 is useful to quantify sublethal effects of DO stress. Results for HIF and phospho-p38 MAP kinase were inconclusive. Test deployments of oysters to assess expression of HSP 70 relative to environmental conditions will be useful, in addition to measuring abiotic factors, to identify appropriate sites for restoration, particularly to capture negative effects of habitat quality on biota before lethal impacts are incurred.     

Hypoxia stimulates osteoclast formation from human peripheral blood

Active pathological bone destruction in humans often occurs in locations where oxygen tension (pO₂) is likely to be low, for example, at the sites of tumours, inflammation, infections and fractures, or the poorly vascularized yellow fatty marrow of the elderly. We examined the effect of pO₂ on formation of osteoclasts, the cells responsible for bone resorption, in 14‐day cultures of normal human peripheral blood mononuclear cells (hPBMCs) on ivory discs. Hypoxia (1–2% O₂) caused threefold increases in the number of osteoclasts formed, compared with 20% O₂. Hypoxia also caused a twofold increase in the number of nuclei per osteoclast, leading to stimulations of resorption pit formation of up to 10‐fold. Exposure to hypoxia led to stabilization of the hypoxia‐inducible factors, HIF1α and HIF2α, and upregulation of vascular endothelial growth factor and interleukin‐6 expression by hPBMCs. These findings help explain why extravasation of mononuclear precursors into relatively O₂‐deficient bone microenvironments could result in osteoclast formation and suggest a new mechanism for the bone loss associated with the pathophysiological conditions where hypoxia commonly occurs. Copyright © 2010 John Wiley & Sons, Ltd.     

Oxygen supply of the brain cortex in acute nitrite hypoxia in rodents with different ecological specialization

Microhemodynamics and oxygen tension (pO₂) in the brain cortex tissues as well as the heart rate were studied in rodents with different ecological specialization during hypoxia produced by subcutaneous injection of sodium nitrite (3 mg/100 g body mass). It was shown that the blood flow in animals with low (rats) and high (muskrats) resistance to hypoxia decreased by the 30th min of the nitrite action, with its subsequent restoration to 85% and 83% of the initial level by the 60th min. The interspecies difference consisted in an increase of the brain blood flow (by 24%) in muskrats and a decrease (by 33%) in rats 15 min after the injection. In rats, simultaneously with the blood-flow dynamics, a pO₂ increase was observed in some brain cortex microareas, while in others—a pO₂ decrease 15 min after the NaNO₂ injection: meanwhile, in muskrats, at this time period a significant pO₂ decrease was observed on the background of a blood flow increase. In both animal species, the pO₂ minimal value was reached by the 45th min, while restoration almost to the initial levels—by the 60th min of the nitrite action. Changes in the rats, synchronous and unidirectional with the heart rate frequency, of the brain blood-flow, as well as tachycardia developing throughout the whole experiment in rats allow suggesting that restoration of the oxygen regime in the brain cortex microareas is provided by activation of systemic mechanisms of regulation of circulation.     

Hypoxia modulates expression of the 70-kD heat shock protein and reduces<Emphasis Type="Italic">Leishmania</Emphasis> infection in macrophages

Hypoxia, a microenvironmental factor present in diseased tissues, has been recognized as a specific metabolic stimulus or a signal of cellular response. Experimental hypoxia has been reported to induce adaptation in macrophages such as differential migration, elevation of proinflammatory cytokines and glycolytic enzyme activities, and decreased phagocytosis of inert particles. In this study we demonstrate that although exposure to hypoxia (5% O₂, 5% CO₂, and balanced N₂) did not change macrophage viability, or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cleavage and proliferation, it significantly reduced expression of the 70-kD heat shock protein (HSP70), which was restored to prehypoxia levels after reoxygenation. The influence of low oxygen tension on macrophage functional activity was also studied, i.e. the ability of these cells to maintain or resist infection by a microorganism. We demonstrate that macrophages from two different sources (a murine cell line and primary cells) exposed to hypoxia were efficiently infected withLeishmania amazonensis, but after 24 h showed a reduction in the percentage of infected cells and of the number of intracellular parasites per macrophage, indicating that hypoxia induced macrophages to kill the intracellular parasites. These results support the notion that hypoxia, a microenvironmental factor, can modulate macrophage protein expression and functional activity.     

Functional alterations in macrophages after hypoxia selection

Regions of low oxygen tension are common features of inflamed and infected tissues and provide physiologic selective pressure for the expansion of cells with enhanced hypoxia tolerance. The aim of this study was to investigate whether macrophages resistant to death induced by hypoxia were accompanied by functional alterations. A mouse macrophage cell line (J774 cells) was used to obtain subpopulations of death-resistant macrophages induced by long-term exposure to severe hypoxia (<1% O(2)). The results indicated that exposing J774 macrophages to periods of severe hypoxia results in the selection of cells with phenotypes associated with the modulation of heat-shock protein 70 kDa (HSP70) expression, tumor necrosis factor-alpha (TNF-alpha), and nitric oxide (NO) production and reduced susceptibility to parasite Leishmania infection. Thus, we suggest that hypoxia-selected macrophages may influence the outcome of inflammation and infection.     

Hypoxia induces activation and subcellular translocation of focal adhesion kinase (p125(FAK)) in cultured rat cardiac myocytes.

We previously reported that hypoxia caused rapid activation of RAS/mitogen-activated protein kinase (MAPK) pathway, two other stress-activated MAPK family members, stress-activated protein kinase (SAPK) and p38MAPK, and Src family tyrosine kinases, p60(c-src) and p59(c-fyn) in cultured rat cardiac myocytes. In this study, to elucidate how hypoxia affects adhesive interaction between cardiac myocytes and extracellular matrix (ECM), we investigated the molecular mechanism of the activation of focal adhesion-associated tyrosine kinases p125(FAK) and paxillin. Here, we show that hypoxia induced tyrosine phosphorylation of p125(FAK) and paxillin and that hypoxia-induced activation of p125(FAK) was accompanied by its increased association with adapter proteins Shc and GRB2, and non-receptor type tyrosine kinase p60(c-src). Furthermore, hypoxia caused subcellular translocation of p125(FAK) from perinuclear sites to the focal adhesions. These results strongly suggest that p125(FAK) is one of the most important components in hypoxia-induced intracellular signaling in cardiac myocytes and may play a pivotal role in adhesive interaction between cardiac myocytes and ECM.     

Role of calpain in hypoxic inhibition of nitric oxide synthase activity in pulmonary endothelial cells

Pulmonary artery endothelial cells (PAEC) were exposed to normoxia or hypoxia (0% O(2)-95% N(2)-5% CO(2)) in the presence and absence of calpain inhibitor I or calpeptin, after which endothelial nitric oxide synthase (eNOS) activity and protein content were assayed. Exposure to hypoxia decreased eNOS activity but not eNOS protein content. Both calpain inhibitor I and calpeptin prevented the hypoxic decrease of eNOS activity. Incubation of calpain with total membrane preparations of PAEC caused dose-dependent decreases in eNOS activity independent of changes in eNOS protein content. Exposure of PAEC to hypoxia also caused time-dependent decreases of heat shock protein 90 (HSP90) that were prevented by calpain inhibitor I and calpeptin. Moreover, the HSP90 content in anti-eNOS antibody-induced immunoprecipitates from hypoxic PAEC lysates was reduced, and repletion of HSP90 reversed the decrease of eNOS activity in these immunoprecipitates. Incubation of PAEC with a specific inhibitor of HSP90 (geldanamycin) mimicked the hypoxic decrease of eNOS activity. These results indicate that the hypoxia-induced reduction in eNOS activity in PAEC is due to a decrease in HSP90 caused by calpain activation.