Protection of thrombin receptor expression under hypoxia

Thrombin receptor (ThR) plays a significant role in myocyte contractility and hypertrophy. Heart myocyte ischemic damage, caused by insufficient blood supply, is the leading cause of heart infarction. Here we demonstrate that when primary myocyte cultures are subjected to hypoxic stress, ThR mRNA levels are reduced markedly. This takes place also in vivo in a model of ischemic pig heart, exhibiting reduced levels of ThR compared with normal heart sections. Prior activation of ThR however, by either thrombin receptor-activating peptide (TRAP) or by alpha-thrombin resulted in full protection of ThR mRNA levels under hypoxia. The effect appeared specific to ThR because the addition of TRAP did not affect the hypoxic damage as shown by the levels of lactic dehydrogenase release and up-regulated GLUT-1, a glucose transporter gene. This protection effect took place not only in primary myocytes but also in NIH3T3 fibroblasts. ThR protection occurs via specific cell signaling events because activation of the receptor by TRAP, following interruption of the signaling cascade by calphostin C, a protein kinase C inhibitor, resulted in loss of ThR mRNA protection. Because Ras and Src are part of the ThR signaling cascade, the introduction of either dominant ras or src oncogenes to NIH3T3 murine fibroblasts gave rise to similar protection of ThR mRNA levels under hypoxic conditions without the exogenous addition of TRAP. Likewise, ThR mRNA protection was obtained after transfection with proto-oncogene vav. The 95-kDa protein Vav undergoes tyrosine phosphorylation after ThR activation, serving thus as part of the receptor machinery cascade. We therefore conclude that the initiation of the signaling cascades either exogenously by TRAP or within the cell via src or ras, as well as via vav oncogene interconnecting G-binding protein to the tyrosine kinase pathway, ultimately results in ThR protection under hypoxia. We present hereby, a novel concept of activated receptors, which under minimal oxygen tension protect their otherwise decaying mRNA. Maintaining the level of ThR that plays an active role in normal myocyte function may provide a significant repair mechanism in ischemic tissue, assisting in the regaining of normal myocyte functions.     

Down-regulation of ARC contributes to vulnerability of hippocampal neurons to ischemia/hypoxia

ARC is a caspase recruitment domain-containing molecule that plays an important role in the regulation of apoptosis. We examined ARC expression during neuronal cell death following ischemic injury in vivo and in vitro. After exposure to transient global ischemic conditions, the expression of ARC was substantially reduced in the CA1 region of hippocampus in a time-dependent manner with concomitant increase of TUNEL-positive cells. Quantitative analysis using Western blotting exhibited that most of ARC protein disappeared in the cultured hippocampal neurons exposed to hypoxia for 12 h and showing 60% cell viability. Forced expression of ARC in the primary cultures of hippocampal neurons or B103 neuronal cells significantly reduced hypoxia-induced cell death. Further, the C-terminal P/E rich region of ARC was effective to attenuate hypoxic insults. These results suggest that down-regulation of ARC expression in hippocampal neurons may contribute to neuronal death induced by ischemia/hypoxia.     

Induction of the proliferative phenotype in differentiated myogenic cells by hypoxia

The effect of cellular differentiation on the response of cells to hypoxic stress has been evaluated using the myogenic cell line BC3H1. Aerobic myocytes were predominantly in G0/G1 of the cell cycle and could be induced into S and G2/M of the cell cycle only by replating in high serum-containing medium at subconfluent cell density. In contrast, hypoxic myocytes demonstrated marked progression into S and G2/M upon reoxygenation without replating in the presence of serum. This modulation of myocytes by hypoxia was suggested further by the induction of 100-kDa and 9-kDa proteins (PSP 100 and PSP 9) which were otherwise only detectable in myoblasts. Two-dimensional gel analysis of newly synthesized proteins demonstrated that the five major glucose/oxygen-regulated proteins (GRP/ORP 260, 150, 100, 80, and 33) were induced in hypoxic myogenic cells independent of their state of differentiation. In addition to the GRP/ORPs, synthesis of 20 and 23 other major proteins was influenced in myocytes and myoblasts, respectively. The bulk of these alterations in myoblasts (70%) were inhibitions. In contrast, 75% of the alterations in myocyte protein synthesis were either enhancements or inductions. The data show that hypoxia can modulate the myocyte phenotype and invoke proliferative characteristics. Moreover, the data suggest that ischemia will have a different effect on and prognosis for tissues with a high mitotic index compared with differentiated tissues.     

Cardioprotective effects of adenosine A1 and A 3 receptor activation during hypoxia in isolated rat cardiac myocytes

Adenosine (ADO) is a well-known regulator of a variety of physiological functions in the heart. In stress conditions, like hypoxia or ischemia, the concentration of adenosine in the extracellular fluid rises dramatically, mainly through the breakdown of ATP. The degradation of adenosine in the ischemic myocytes induced damage in these cells, but it may simultaneously exert protective effects in the heart by activation of the adenosine receptors. The contribution of ADO to stimulation of protective effects was reported in human and animal hearts, but not in rat hearts. The aim of this study was to evaluate the role of adenosine A₁ and A₃ receptors (A₁R and A₃R), in protection of isolated cardiac myocytes of newborn rats from ischemic injury. The hypoxic conditions were simulated by exposure of cultured rat cardiomyocytes (4–5 days in vitro), to an atmosphere of a N₂ (95%) and CO₂ (5%) mixture, in glucose-free medium for 90 min. The cardiotoxic and cardioprotective effects of ADO ligands were measured by the release of lactate dehydrogenase (LDH) into the medium. Morphological investigation includes immunohistochemistry, image analysis of living and fixed cells and electron microscopy were executed. Pretreatment with the adenosine deaminase considerably increased the hypoxic damage in the cardiomyocytes indicating the importance of extracellular adenosine. Blocking adenosine receptors with selective A₁ and A₃ receptor antagonists abolished the protective effects of adenosine. A₁R and A₃R activation during the hypoxic insult delays onset of irreversible cell injury and collapse of mitochondrial membrane potential as assessed using DASPMI fluorochrom. Cardioprotection induced by the A₁R agonist, CCPA, was abolished by an A₁R antagonist, DPCPX, and was not affected by an A₃R antagonist, MRS1523. Cardioprotection caused by the A₃R agonist, Cl-IB-MECA, was antagonized completely by MRS1523 and only partially by DPCPX. Activation of both A₁R and A₃R together was more efficient in protection against hypoxia than by each one alone. Our study indicates that activation of either A₁ or A₃ adenosine receptors in the rat can attenuate myocyte injury during hypoxia. Highly selective A₁R and A₃R agonists may have potential as cardioprotective agents against ischemia or heart surgery.     

Viability and angiogenic activity of mesenchymal stromal cells from adipose tissue and bone marrow under hypoxia and inflammation in vitro

Progenitor stromal cells derived from adipose tissue (ADSC) and bone marrow (BMDSC) hold great promise for use in the cell-based therapy of ischemic diseases. It was demonstrated that these cells secrete a number of angiogenic cytokines that stimulate vascularization. It was demonstrated that ADSC or BMDSC injected intramuscularly or intravenously into the animals with experimental hind-limb ischemia improve vascularization. However, low oxygen levels and inflammation may impair the viability and functional activity of transplanted cells. We have examined ADSC and BMDSC properties in vitro under hypoxic and inflammatory conditions. ADSC and BMDSC derived from Balb/c mice have been cultivated under hypoxia or in the presence of inflammatory cytokines. The viability of cells assessed by annexin V-PE binding and 7AAD storage, as well as by the quantitative TUNEL method, was not changed under hypoxic conditions Cell exposure to inflammatory cytokines induced apoptosis in 70% of cells. Inflammatory cytokines did not stimulate gene expression of angiogenic growth factors. Under hypoxia conditions up-regulation of genes for pro-angiogenic factors and down-regulation of anti-angiogenic genes were more apparent in ADSC. Using angiogenesis models in vitro and in vivo, we demonstrated that stromal cell maintenance under hypoxic conditions increased their ability to stimulate the growth of blood vessels.     

MCP-1/CCL2 protects cardiac myocytes from hypoxia-induced apoptosis by a G(alphai)-independent pathway

Chemokines, in addition to their chemotactic properties, act upon resident cells within a tissue and mediate other cellular functions. In a previous study, we demonstrated that CCL2 protects cultured mouse neonatal cardiac myocytes from hypoxia-induced cell death. Leukocyte chemotaxis has been shown to contribute to ischemic injury. While the chemoattractant properties of CCL2 have been established, the protective effects of this chemokine suggest a novel role for CCL2 in myocardial ischemia/reperfusion injury. The present study examined the cellular signaling pathways that promote this protection. Treatment of cardiac myocyte cultures with CCL2 protected them from hypoxia-induced apoptosis. This protection was not mediated through the activation of G(alphai) signaling that mediates monocyte chemotaxis. Inhibition of the ERK1/2 signaling pathway abrogated CCL2 protection. Caspase 3 activation and JNK/SAPK phosphorylation were decreased in hypoxic myocytes co-treated with CCL2 as compared to hypoxia only-treated cultures. Expression of the Bcl-2 family proteins, Bcl-xL and Bag-1, was increased in CCL2-treated myocytes subjected to hypoxia. There was also downregulation of Bax protein levels as a result of CCL2 co-treatment. These data suggest that CCL2 cytoprotection and chemotaxis may occur through distinct signaling mechanisms.     

The effect of graded hypoxia on the embryonic chick heart

Embryonic ventricular function in the chick was measured in response to graded levels of hypoxia. Myocardial contractility, as measured by cinephotoanalysis and expressed as shortening fraction, was significantly depressed after 1 hour of moderate hypoxia (6% O2) and after 5 hours of milder (16% O2 and 11% O2) levels of hypoxia (P less than .05). Microscopy confirmed associated myocyte damage with cell death noted after 5 hours of moderate hypoxic stress. Heart rate change was not related to the severity of hypoxia. The greatest level of tachycardia was noted with conditions of mildest hypoxia (16% O2). The data confirm that cardiac contractility, as measured by shortening fraction, is depressed on exposure to hypoxia, with impairment of function related to the severity of the hypoxic conditions.     

Intermittent hypoxia attenuates ischemia／reperfusion induced apoptosis in cardiac myocytes via regulating Bcl-2／Bax expression

Intermittent hypoxia has been shown to provide myocardial protection against ishemiaJreperfusion-induced injury.Cardiac myocyte loss through apoptosis has been reported in ischemia/reperfusion injury. Our aim was to investigate whether intermittent hypoxia could attenuate ischemia/reperfusion-induced apoptosis in cardiac myocytes and its potential mechanisms. Adult male Sprague-Dawley rats were exposed to hypoxia simulated 5000 m in a hypobaric chamber for 6 h/day, lasting 42 days. Normoxia group rats were kept under normoxic conditions. Isolated perfused hearts from both groups were subjected to 30 min of global ischemia followed by 60 min reperfusion.Incidence of apoptosis in cardiac myocytes was determined by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and DNA agarose gel electrophoresis. Expressions of apoptosis related proteins,Bax and Bcl-2, in cytosolic and membrane fraction were detected by Western Blotting. After ischemia/reperfusion,enhanced recovery of cardiac function was observed in intermittent hypoxia hearts compared with normoxia group.Ischemia/reperfusion-induced apoptosis, as evidenced by TUNEL-positive nuclei and DNA fragmentation, was significantly reduced in intermittent hypoxia group compared with normoxia group. After ischemia/reperfusion,expression of Bax in both cytosolic and membrane fractions was decreased in intermittent hypoxia hearts comparedwith normoxia group. Although ischemia/reperfusion did not induce changes in the level of Bcl-2 expression in cytosolic fraction between intermittent hypoxia and normoxia groups, the expression of Bcl-2 in membrane fraction was upregulated in intermittent hypoxia group compared with normoxia group. These results indicated that the cardioprotection of intermittent hypoxia against ischemia/reperfusion injury appears to be in part due to reducemyocardial apoptosis. Intermittent hypoxia attenuated ischemia/reperfusion-induced apoptosis via increasing the ratio of Bcl-2/Bax, especially in membrane fraction.     

Hypoxic stress activates chaperone-mediated autophagy and modulates neuronal cell survival

Autophagy is a conserved mechanism responsible for the continuous clearance of unnecessary organelles or misfolded proteins in lysosomes. Three types of autophagy have been reported in the difference of substrate delivery to lysosome: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). Among these types, CMA is a unique autophagy system that selectively degrades substrates detected by heat shock cognate protein 70 (HSC70). Recently, autophagic cell death has been reported to be involved in neuronal death following brain ischemia; however, the contribution of CMA to neuronal death/survival after ischemic stress has not been addressed. In the present study, we determined whether quantitative alterations in LAMP-2A, which is the key molecule in CMA, would modulate neuronal cell survival under hypoxic conditions. Incubation of Neuro2A cells in a hypoxic chamber (1% O(2), 5% CO(2)) increased the level of LAMP-2A and induced accumulation of LAMP-2A-positive lysosomes in the perinuclear area, which is a hallmark of CMA activation. The activation of CMA in response to hypoxia was also confirmed by the GAPDH-HaloTag CMA indicator system at the single cell level. Next, we asked whether CMA was involved in cell survival during hypoxia. Blocking LAMP-2A expression with siRNA increased the level of cleaved caspase-3 and the number of propidium iodide-positive cells after hypoxic stress regardless of whether macroautophagy could occur, whereas the administration of mycophenolic acid, a potent CMA activator, rescued hypoxia-mediated cell death. Finally, we asked whether CMA was activated in the neurons after middle cerebral artery occlusion in vivo. The expression of LAMP-2A was significantly increased in the ischemic hemisphere seven days after brain ischemia. These results indicate that CMA is activated during hypoxia and contributes to the survival of cells under these conditions.     

Progressive hypoxia-on-a-chip: An in vitro oxygen gradient model for capturing the effects of hypoxia on primary hepatocytes in health and disease

Oxygen is vital to the function of all tissues including the liver and lack of oxygen, that is, hypoxia can result in both acute and chronic injuries to the liver in vivo and ex vivo. Furthermore, a permanent oxygen gradient is naturally present along the liver sinusoid, which plays a role in the metabolic zonation and the pathophysiology of liver diseases. Accordingly, here, we introduce an in vitro microfluidic platform capable of actively creating a series of oxygen concentrations on a single continuous microtissue, ranging from normoxia to severe hypoxia. This range approximately captures both the physiologically relevant oxygen gradient generated from the portal vein to the central vein in the liver, and the severe hypoxia occurring in ischemia and liver diseases. Primary rat hepatocytes cultured in this microfluidic platform were exposed to an oxygen gradient of 0.3–6.9%. The establishment of an ascending hypoxia gradient in hepatocytes was confirmed in response to the decreasing oxygen supply. The hepatocyte viability in this platform decreased to approximately 80% along the hypoxia gradient. Simultaneously, a progressive increase in accumulation of reactive oxygen species and expression of hypoxia-inducible factor 1α was observed with increasing hypoxia. These results demonstrate the induction of distinct metabolic and genetic responses in hepatocytes upon exposure to an oxygen (/hypoxia) gradient. This progressive hypoxia-on-a-chip platform can be used to study the role of oxygen and hypoxia-associated molecules in modeling healthy and injured liver tissues. Its use can be further expanded to the study of other hypoxic tissues such as tumors as well as the investigation of drug toxicity and efficacy under oxygen-limited conditions.     

Paracrine effects of hypoxic fibroblast-derived factors on the MPT-ROS threshold and viability of adult rat cardiac myocytes

Cardiac fibroblasts contribute to multiple aspects of myocardial function and pathophysiology. The pathogenetic relevance of cytokine production by these cells under hypoxia, however, remains unexplored. With the use of an in vitro cell culture model, this study evaluated cytokine production by hypoxic cardiac fibroblasts and examined two distinct effects of hypoxic fibroblast-conditioned medium (HFCM) on cardiac myocytes and fibroblasts. Hypoxia caused a marked increase in the production of tumor necrosis factor (TNF)-alpha by cardiac fibroblasts. HFCM significantly enhanced the susceptibility of cardiac myocytes to reactive oxygen species (ROS)-induced mitochondrial permeability transition (MPT), determined by high-precision confocal line-scan imaging following controlled, photoexcitation-induced ROS production within individual mitochondria. Furthermore, exposure of cardiac myocytes to HFCM for 5 h led to loss of viability, as evidenced by change in morphology and annexin staining. HFCM also decreased DNA synthesis in cardiac fibroblasts. Normoxic fibroblast-conditioned medium spiked with TNF-alpha at 200 pg/ml, a concentration comparable to that in HFCM, promoted loss of myocyte viability and decreased DNA synthesis in cardiac fibroblasts. These effects of HFCM are similar to the reported effects of hypoxia per se on these cell types, showing that hypoxic fibroblast-derived factors may amplify the distinct effects of hypoxia on cardiac cells. Importantly, because both hypoxia and oxidant stress prevail in a setting of ischemia and reperfusion, the effects of soluble factors from hypoxic fibroblasts on the MPT-ROS threshold and viability of myocytes may represent a novel paracrine mechanism that could exacerbate ischemia-reperfusion injury to cardiomyocytes.     

Histidine button engineered into cardiac troponin I protects the ischemic and failing heart

The myofilament protein troponin I (TnI) has a key isoform-dependent role in the development of contractile failure during acidosis and ischemia. Here we show that cardiac performance in vitro and in vivo is enhanced when a single histidine residue present in the fetal cardiac TnI isoform is substituted into the adult cardiac TnI isoform at codon 164. The most marked effects are observed under the acute challenges of acidosis, hypoxia, ischemia and ischemia-reperfusion, in chronic heart failure in transgenic mice and in myocytes from failing human hearts. In the isolated heart, histidine-modified TnI improves systolic and diastolic function and mitigates reperfusion-associated ventricular arrhythmias. Cardiac performance is markedly enhanced in transgenic hearts during reperfusion despite a high-energy phosphate content similar to that in nontransgenic hearts, providing evidence for greater energetic economy. This pH-sensitive 'histidine button' engineered in TnI produces a titratable molecular switch that 'senses' changes in the intracellular milieu of the cardiac myocyte and responds by preferentially augmenting acute and long-term function under pathophysiological conditions. Myofilament-based inotropy may represent a therapeutic avenue to improve myocardial performance in the ischemic and failing heart.     

Specific inhibition of hypoxia inducible factor 1 exaggerates cell injury induced by in vitro ischemia through deteriorating cellular redox environment

Hypoxia inducible factor 1 (HIF-1) has been suggested to play a critical role in the fate of cells exposed to hypoxic stress. However, the mechanism of HIF-1-regulated cell survival is still not fully understood in ischemic conditions. Redox status is critical for decisions of cell survival, death and differentiation. We investigated the effects of inhibiting HIF-1 on cellular redox status in SH-SY5Y cells exposed to hypoxia or oxygen and glucose deprivation (OGD), coupled with cell death analyses. Our results demonstrated that inhibiting HIF-1α expression by HIF-1α specific small interfering RNA (siRNA) transfection increased reactive oxygen species generation, and transformed the cells to more oxidizing environments (low GSH/GSSG ratio, low NADPH level) under either hypoxic or OGD exposure. Cell death increased dramatically in the siRNA transfected cells, compared to non-transfected cells after hypoxic/OGD exposures. In contrast, increasing HIF-1α expression by desferrioxamine, a metal chelator and hydroxylase inhibitor, induced a more reducing environment (high GSH/GSSG ratio, high NADPH level) and reduced cell death. Further studies showed that HIF-1 regulated not only glucose transporter-1 expression, but also the key enzymes of the pentose phosphate pathway such as glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. These enzymes are important in maintaining cellular redox homeostasis by generating NADPH, the primary reducing agent in cells. Moreover, catalase significantly decreased cell death in the siRNA-transfected cells induced by hypoxia and OGD. These results suggest that maintenance of cellular redox status by HIF-1 protects cells from hypoxia and ischemia mediated injuries.     

Metabolic profiling of hypoxic cells revealed a catabolic signature required for cell survival

Hypoxia is one of the features of poorly vascularised areas of solid tumours but cancer cells can survive in these areas despite the low oxygen tension. The adaptation to hypoxia requires both biochemical and genetic responses that culminate in a metabolic rearrangement to counter-balance the decrease in energy supply from mitochondrial respiration. The understanding of metabolic adaptations under hypoxia could reveal novel pathways that, if targeted, would lead to specific death of hypoxic regions. In this study, we developed biochemical and metabolomic analyses to assess the effects of hypoxia on cellular metabolism of HCT116 cancer cell line. We utilized an oxygen fluorescent probe in anaerobic cuvettes to study oxygen consumption rates under hypoxic conditions without the need to re-oxygenate the cells and demonstrated that hypoxic cells can maintain active, though diminished, oxidative phosphorylation even at 1% oxygen. These results were further supported by in situ microscopy analysis of mitochondrial NADH oxidation under hypoxia. We then used metabolomic methodologies, utilizing liquid chromatography-mass spectrometry (LC-MS), to determine the metabolic profile of hypoxic cells. This approach revealed the importance of synchronized and regulated catabolism as a mechanism of adaptation to bioenergetic stress. We then confirmed the presence of autophagy under hypoxic conditions and demonstrated that the inhibition of this catabolic process dramatically reduced the ATP levels in hypoxic cells and stimulated hypoxia-induced cell death. These results suggest that under hypoxia, autophagy is required to support ATP production, in addition to glycolysis, and that the inhibition of autophagy might be used to selectively target hypoxic regions of tumours, the most notoriously resistant areas of solid tumours.     

The characteristics of the cardioprotective action of fructose-1,6-diphosphate]

The cardioprotective effects of fructose-1,6-diphosphate (FDP) were investigated in infarcted rats and in conscious rabbits with myocardial ischemia. The influence of FDP on metabolic acidosis was studied in isolated hypoxic rat hearts. It was shown that FDP did not change the threshold of the initiation of ischemia in conscious rabbits, but decreased necrotic zone in infarcted rat hearts. After administration of FDP the myocardial contractility was prolonged significantly as compared with control under conditions of severe metabolic acidosis. However, FDP was not effective in hypoxic hearts with compensated metabolic acidosis. It was considered, that FDP influenced only ischemic myocytes with the changes in sarcolemmal permeability.     

Regional hypoxia elicits regional changes in chorioallantoic membrane vascular density in alligator but not chicken embryos

Hypoxic incubation increases vascularization in the chick chorioallantoic membrane (CAM). The effect of regional hypoxia on the vascular density of American alligator (Alligator mississippiensis) and chicken (Gallus gallus) CAMs was studied to determine if hypoxic proliferation of blood vessels is localized or global across the CAM. Eggs were incubated under normoxic conditions with a portion of the eggshell covered with non-toxic beeswax to induce external regional hypoxia. CAMs were examined under a microscope with a 'bulls eye' coverslip and a vascular density index (VDI) was determined. The hypoxic portions of the alligator CAMs were more vascular than the normoxic portions (VDI = 200.9 versus 157. 8, respectively). Presumably this response is maladaptive by causing increased blood flow to the poorly oxygenated portions, i.e. increased 'shunt'. Thus, we hypothesize increased vascularity due to hypoxic incubation is due to local release and subsequent rapid local breakdown or uptake of angiogenic factors. In contrast, the hypoxic and normoxic portions of the chick CAMs exhibited virtually no difference in VDI (VDI= 211.5 versus 217.9, respectively). We suggest the air cell and air space of the chicken eggs allows for circulation of gas in ovo, eliminating the possibility of regional internal hypoxia.