Paola Bianciardi

Carbamylated erythropoietin ameliorates the metabolic stress induced in vivo by severe chronic hypoxia

Ischemia and chronic hypoxia (CH) trigger a variety of adverse effects arising from metabolic stress that injures cells. In response to reduced O2, hypoxia-inducible factor 1α (HIF-1α) activates erythropoietin (Epo) as well as many other target genes that counteract the effects of O2 deficiency. Epo produced by the kidney stimulates erythrocyte production, leading to decreased HIF-1α production by improved tissue O2 delivery. However, Epo is produced by many other tissues, and it is currently unclear to what extent, if any, locally produced Epo modulates HIF-1α expression. Derivatives of Epo that possess tissue-protective activities but do not stimulate erythropoiesis [e.g., carbamylated Epo (CEpo)] are useful tools with which to determine whether exogenous Epo modulates HIF-1α in the absence of changes in hemoglobin concentration. We compared the effects of CH (6.5% O2 for 10 days) with or without CEpo administered by daily s.c. injection (10 μg/kg of body weight). CEpo administration did not alter the survival rate, weight loss, or increased hemoglobin concentration associated with CH. Therefore, CEpo does not directly suppress HIF-mediated erythropoiesis. CEpo does, however, prevent CH-induced neuronal increases of HIF-1α and Epo receptor-associated immunoreactivity (a measure of stress) while reducing the apoptotic index. In contrast, the myocardium did not exhibit increased HIF-1α expression during CH, although CEpo did reduce the apoptotic index. These observations therefore demonstrate that CEpo administration reduces the metabolic stress caused by severe CH, resulting in improved cellular survival independent of erythrocyte production.

Phosphodiesterase-5 Inhibition Abolishes Neuron Apoptosis Induced by Chronic Hypoxia Independently of Hypoxia-Inducible Factor-1α Signaling

Exposure to hypoxia triggers a variety of adverse effects in the brain that arise from metabolic stress and induce neuron apoptosis. Overexpression of the hypoxia-inducible factor-1α (HIF-1α) is believed to be a major candidate in orchestrating the cell’s defense against stress. To test the impact of HIF-1α on apoptosis during chronic hypoxia in vivo, we examined the protective effect of modulating the nitric oxide (NO)/cGMP pathway by sildenafil, a selective inhibitor of phosphodiesterase-5 (PDE-5). Male ICR/CD-1 mice were divided into 3 groups (n = 6/group): normoxic (21% O2), hypoxic (9.5% O2), and hypoxic with sildenafil (1.4-mg/kg intraperitoneal injections daily). At the end of the 8-day treatment period, the mice were euthanized and cerebral cortex biopsies were harvested for analyses. We found that sildenafil: (1) did not significantly alter the hypoxia-induced weight loss and hemoglobin increase, but did augment plasma nitrates+nitrites and the tissue content of cGMP and phosphorylated (P) NO synthase III; (2) reversed the hypoxia-induced neuron apoptosis (terminal deoxynucleotidyl transferase positivity and double-staining immunofluorescence, P = 0.009), presumably through increased bcl-2/Bax (P = 0.0005); and (3) did not affect HIF-1α, but rather blunted the hypoxia-induced increase in P-ERK1/2 (P = 0.0002) and P-p38 (P = 0.004). We conclude that upregulating the NO/cGMP pathway by PDE-5 inhibition during hypoxia reduces neuron apoptosis, regardless of HIF-1α, through an interaction involving ERK1/2 and p38.

Antitumour activity of melatonin in a mouse model of human prostate cancer: relationship with hypoxia signalling.

Melatonin is known to exert antitumour activity in several types of human cancers, but the underlying mechanisms as well as the efficacy of different doses of melatonin are not well defined. Here, we test the hypothesis whether melatonin in the nanomolar range is effective in exerting antitumour activity in vivo and examine the correlation with the hypoxia signalling mechanism, which may be a major molecular mechanism by which melatonin antagonizes cancer. To test this hypothesis, LNCaP human prostate cancer cells were xenografted into seven‐wk‐old Foxn1nu/nu male mice that were treated with melatonin (18 i.p. injections of 1 mg/kg in 41 days). Saline‐treated mice served as control. We found that the melatonin levels in plasma and xenografted tissue were 4× and 60× higher, respectively, than in control samples. Melatonin tended to restore the redox imbalance by increasing expression of Nrf2. As part of the phenotypic response to these perturbations, xenograft microvessel density was less in melatonin‐treated animals, indicative of lower angiogenesis, and the xenograft growth rate was slower (P < 0.0001). These changes were accompanied by a reduced expression of Ki67, elevated expression of HIF‐1α and increased phosphorylation of Akt in melatonin than saline‐treated mice. We conclude that the beneficial effect of melatonin in reducing cancer growth in vivo was evident at melatonin plasma levels as low as 4 nm and was associated with decreased angiogenesis. Higher HIF‐1α expression in xenograft tissue indicates that the antitumour effect cannot be due to a postulated antihypoxic effect, but may stem from lower angiogenesis potential.

Chronic systemic hypoxia promotes LNCaP prostate cancer growth in vivo

Solid tumors contain underperfused regions where hypoxia‐inducible factor‐1α (HIF‐1α) over‐expression induces hypoxia adaptation and cell proliferation. We test the hypothesis that systemic hypoxia promotes prostate cancer growth in vivo and examine HIF‐1α centrality in this effect.

Heart HIF-1α and MAP Kinases During Hypoxia: Are They Associated In Vivo?:

To study the in vivo dynamics of hypoxia-inducible factor 1α (HIF-1α), master regulator of O2-dependent gene expression, and mitogen-activated protein kinases (MAPKs) in the hypoxic myocardium, Sprague-Dawley rats (n = 4 to 6 per group) were exposed to 1-hr hypoxia (10% O2), 23-hr hypoxia, and 23-hr hypoxia, followed by reoxygenation. HIF-1α increased 15-fold after 1-hr hypoxia, remained constant for 23 hrs, and returned to baseline on reoxygenation. Extracellular signal–regulated kinases (ERK1/2) were unchanged throughout. Phosphorylated p38 increased 4-fold after 1-hr hypoxia and returned to baseline within 23-hr hypoxia. The activity of stress-activated protein kinases/c-Jun NH2-terminal kinases (JNKs), measured as phosphorylated c-Jun, increased 3-fold after 1-hr hypoxia and remained sustained afterward. Furthermore, HIF-1α was halved in rats that were administered with the p38 inhibitor SB202190 and made hypoxic for 1 hr. In conclusion, although very sensitive to the reoxygenation, HIF-1α is overexpressed in vivo in the hypoxic myocardium, and its acute induction by hypoxia is correlated with that of p38.

Myocardial impairment in chronic hypoxia is abolished by short aeration episodes: involvement of K+ATP channels

In vivo exposure to chronic hypoxia is considered to be a cause of myocardial dysfunction, thereby representing a deleterious condition, but repeated aeration episodes may exert some cardioprotection. We investigated the possible role of ATP-sensitive potassium channels in these mechanisms. First, rats (n = 8/group) were exposed for 14 days to either chronic hypoxia (CH; 10% O2) or chronic hypoxia with one episode/day of 1-hr normoxic aeration (CH+A), with normoxia (N) as the control. Second, isolated hearts were Langendorff perfused under hypoxia (10% O2, 30 min) and reoxygenated (94% O2, 30 min) with or without 3 μM glibenclamide (nonselective K+ATP channel-blocker) or 100 μM diazoxide (selective mitochondrial K+ATP channel-opener). Blood gasses, hemoglobin concentration, and plasma malondialdehyde were similar in CH and CH+A and in both different from normoxic (P < 0.01), body weight gain and plasma nitrate/nitrite were higher in CH+A than CH (P < 0.01), whereas apoptosis (number of TUNEL-positive nuclei) was less in CH+A than CH (P < 0.05). During in vitro hypoxia, the efficiency (ratio of ATP production/pressure x rate product) was the same in all groups and diazoxide had no measurable effects on myocardial performance, whereas glibenclamide increased end-diastolic pressure more in N and CH than in CH+A hearts (P < 0.05). During reoxgenation, efficiency was markedly less in CH with respect to N and CH+A (P < 0.0001), and rate x pressure product remained lower in CH than N and CH+A hearts (P < 0.001), but glibenclamide or diazoxide abolished this difference. Glibenclamide, but not diazoxide, decreased vascular resistance in N and CH (P < 0.005 and < 0.001) without changes in CH+A. We hypothesize that cardioprotection in chronically hypoxic hearts derive from cell depolarization by sarcolemmal K+ATP blockade or from preservation of oxidative phosphorylation efficiency (ATP turnover/myocardial performance) by mitochondrial K+ATP opening. Therefore K+ATP channels are involved in the deleterious effects of chronic hypoxia and in the cardioprotection elicited when chronic hypoxia is interrupted with short normoxic aeration episodes.

In vivo up-regulation of the unfolded protein response after hypoxia.

BACKGROUND Low oxygen (O2) availability, a condition called hypoxia, has different and profound consequences in tissues and organs. Besides the hypoxia-inducible response, mammalian cells induce a coordinated cytoprotective pathway called Unfolded Protein Response (UPR). We studied the molecular basis of UPR and apoptosis in animal models exposed to different hypoxic stresses and assessed the ability of liver and myocardium to respond to low oxygen by activating different arms of the UPR according to the severity of the insults in a tissue specific manner. METHODS We assessed the levels of several UPR markers in hypoxic animals by Real Time PCR and Western blotting. RESULTS While the hepatocytes activate the apoptotic pathway mediated, in part, by CHOP and p-JNK, we could not detect an UPR-dependent apoptosis in myocytes. Moreover, severe hypoxia results in ATF4 translation, and induction of CHOP and GADD34 transcripts in liver, by contrast in the myocardium, the ATF4-CHOP-GADD34 signaling pathway is not detectably activated. GENERAL SIGNIFICANCE Comparison of several UPR markers in liver and myocardium enabled to underscore the ability of hepatocytes and myocites to selectively activate and fine tune the UPR signaling pathway during hypoxia in vivo.

Supplementation of Creatine and Ribose Prevents Apoptosis in Ischemic Cardiomyocytes

Background/Aims. To alleviate ischemia-induced injury in the myocardium, a tissue that depends critically on energy-yielding processes, creatine may be used to enhance energy metabolism, whereas D-ribose may provide building blocks for ATP synthesis. We test the hypothesis that simultaneous supplementation of creatine+D-ribose protects non-irreversibly injured ischemic cardiomyocytes by reducing apoptosis. Results. When H9c2 cardiomyocytes were exposed to 24-h ischemia (1% O2 with glucose deprivation), viability was severely compromised, but administration of 2.5 mM creatine + 5 mM D-ribose alleviated the fall in viability, whereas 2.5 mM creatine or 5 mM D-ribose did not. These findings correlated with up-regulation of protein kinase B (Akt) phosphorylation. Creatine+D-ribose also blunted adenosine monophosphate-activated protein kinase (AMPK) and down-regulated apoptosis by reducing caspase-3 activation and poly (ADP-ribose) polymerase (PARP) cleavage. Conclusions. Simultaneous administration of creatine+D-ribose confers anti-ischemic protection that was absent when treating cardiomyocytes with either creatine or D-ribose. The involved mechanisms stem from the Akt and AMPK signaling pathways. These findings may form the basis of a paradigm whereby re-energization of non-irreversibly damaged cardiomyocytes is a critical step to counteract apoptosis.

Expression of carbohydrate-antigen sialyl-Lewis a on colon cancer cells promotes xenograft growth and angiogenesis in nude mice.

We investigated the role of carbohydrate antigen sialyl-Lewis a (sLea), an E-selectin ligand and epitope of tumor marker CA19.9, in the development of xenografts in nude mice. To this end, animals were inoculated with the human colon cancer cell line HCT-15, expressing no Lewis antigens, or with a clone expressing sLea (HCT-15-T5). The size of HCT-15-T5 xenografts appeared larger than those of HCT-15 and their average weight was over twice bigger. In both xenografts the mitotic index was found elevated, as determined by Ki-67 assay, and no apoptosis was detected in the tumor cells by both caspase 8 or TUNEL assays. Some apoptotic signals were instead detected in the vessels. Conversely, microvessel density, determined through CD-31 immunohistochemistry, was found 3.2-folds bigger in HCT-15-T5 xenografts (p<0.012). Only the membranes of HCT-15-T5 cells grown as xenografts reacted intensively with the anti CA19.9 antibody 1116-NS-19-9 by immunofluorescence, but not by immunohistochemistry. Unknown structures were instead stained by such technique in both xenografts, as were in mouse tissues not expressing the antigen and in human colon adenocarcinoma. We conclude that expression of sLea on the surface of colon cancer cells improves xenograft growth and is associated with enhanced angiogenesis, while immunohistochemistry with 1116-NS-19-9 antibody appears not suitable to determine CA19.9 expression.

Phosphodiesterase-5 inhibition mimics intermittent reoxygenation and improves cardioprotection in the hypoxic myocardium.

Although chronic hypoxia is a claimed myocardial risk factor reducing tolerance to ischemia/reperfusion (I/R), intermittent reoxygenation has beneficial effects and enhances heart tolerance to I/R. Aim of the study: To test the hypothesis that, by mimicking intermittent reoxygenation, selective inhibition of phosphodiesterase-5 activity improves ischemia tolerance during hypoxia. Adult male Sprague-Dawley rats were exposed to hypoxia for 15 days (10% O2) and treated with placebo, sildenafil (1.4 mg/kg/day, i. p.), intermittent reoxygenation (1 h/day exposure to room air) or both. Controls were normoxic hearts. To assess tolerance to I/R all hearts were subjected to 30-min regional ischemia by left anterior descending coronary artery ligation followed by 3 h-reperfusion. Whereas hypoxia depressed tolerance to I/R, both sildenafil and intermittent reoxygenation reduced the infarct size without exhibiting cumulative effects. The changes in myocardial cGMP, apoptosis (DNA fragmentation), caspase-3 activity (alternative marker for cardiomyocyte apoptosis), eNOS phosphorylation and Akt activity paralleled the changes in cardioprotection. However, the level of plasma nitrates and nitrites was higher in the sildenafil+intermittent reoxygenation than sildenafil and intermittent reoxygenation groups, whereas total eNOS and Akt proteins were unchanged throughout. Conclusions: Sildenafil administration has the potential to mimic the cardioprotective effects led by intermittent reoxygenation, thereby opening the possibility to treat patients unable to be reoxygenated through a pharmacological modulation of NO-dependent mechanisms.