Juan C. Ibla

Coordinated adenine nucleotide phosphohydrolysis and nucleoside signaling in posthypoxic endothelium: role of ectonucleotidases and adenosine A2B receptors.

Limited oxygen delivery to tissues (hypoxia) is common in a variety of disease states. A number of parallels exist between hypoxia and acute inflammation, including the observation that both influence vascular permeability. As such, we compared the functional influence of activated polymorphonuclear leukocytes (PMN) on normoxic and posthypoxic endothelial cells. Initial studies indicated that activated PMN preferentially promote endothelial barrier function in posthypoxic endothelial cells (>60% increase over normoxia). Extension of these findings identified at least one soluble mediator as extracellular adenosine triphosphate (ATP). Subsequent studies revealed that ATP is coordinately hydrolyzed to adenosine at the endothelial cell surface by hypoxia-induced CD39 and CD73 (>20-and >12-fold increase in mRNA, respectively). Studies in vitro and in cd39-null mice identified these surface ecto-enzymes as critical control points for posthypoxia-associated protection of vascular permeability. Furthermore, insight gained through microarray analysis revealed that the adenosine A2B receptor (AdoRA2B) is selectively up-regulated by hypoxia (>5-fold increase in mRNA), and that AdoRA2B antagonists effectively neutralize ATP-mediated changes in posthypoxic endothelial permeability. Taken together, these results demonstrate transcription coordination of adenine nucleotide and nucleoside signaling at the vascular interface during hypoxia.

Crucial Role for Ecto-5′-Nucleotidase (CD73) in Vascular Leakage during Hypoxia

Extracellular adenosine has been widely implicated in adaptive responses to hypoxia. The generation of extracellular adenosine involves phosphohydrolysis of adenine nucleotide intermediates, and is regulated by the terminal enzymatic step catalyzed by ecto-5′-nucleotidase (CD73). Guided by previous work indicating that hypoxia-induced vascular leakage is, at least in part, controlled by adenosine, we generated mice with a targeted disruption of the third coding exon of Cd73 to test the hypothesis that CD73-generated extracellular adenosine functions in an innate protective pathway for hypoxia-induced vascular leakage. Cd73 −/− mice bred and gained weight normally, and appeared to have an intact immune system. However, vascular leakage was significantly increased in multiple organs, and after subjection to normobaric hypoxia (8% O2), Cd73 −/− mice manifested fulminant vascular leakage, particularly prevalent in the lung. Histological examination of lungs from hypoxic Cd73 −/− mice revealed perivascular interstitial edema associated with inflammatory infiltrates surrounding larger pulmonary vessels. Vascular leakage secondary to hypoxia was reversed in part by adenosine receptor agonists or reconstitution with soluble 5′-nucleotidase. Together, our studies identify CD73 as a critical mediator of vascular leakage in vivo.

Identification of Ectonucleotidases CD39 and CD73 in Innate Protection during Acute Lung Injury

Acute lung injury (ALI), such as that which occurs with mechanical ventilation, contributes to morbidity and mortality of critical illness. Nonetheless, in many instances, ALI resolves spontaneously through unknown mechanisms. Therefore, we hypothesized the presence of innate adaptive pathways to protect the lungs during mechanical ventilation. In this study, we used ventilator-induced lung injury as a model to identify endogenous mechanisms of lung protection. Initial in vitro studies revealed that supernatants from stretch-induced injury contained a stable factor which diminished endothelial leakage. This factor was subsequently identified as adenosine. Additional studies in vivo revealed prominent increases in pulmonary adenosine levels with mechanical ventilation. Because ectoapyrase (CD39) and ecto-5′-nucleotidase (CD73) are rate limiting for extracellular adenosine generation, we examined their contribution to ALI. In fact, both pulmonary CD39 and CD73 are induced by mechanical ventilation. Moreover, we observed pressure- and time-dependent increases in pulmonary edema and inflammation in ventilated cd39−/− mice. Similarly, pharmacological inhibition or targeted gene deletion of cd73 was associated with increased symptom severity of ventilator-induced ALI. Reconstitution of cd39−/− or cd73−/− mice with soluble apyrase or 5′-nucleotidase, respectively, reversed such increases. In addition, ALI was significantly attenuated and survival improved after i.p. treatment of wild-type mice with soluble apyrase or 5′-nucleotidase. Taken together, these data reveal a previously unrecognized role for CD39 and CD73 in lung protection and suggest treatment with their soluble compounds as a therapeutic strategy for noninfectious ALI.

Ketamine Activates Cell Cycle Signaling and Apoptosis in the Neonatal Rat Brain

Background:Prolonged exposure to ketamine results in accelerated neurodegeneration and neurocognitive deficits in the neonatal rats. Experimental models of neurodegeneration have implicated reentry of postmitotic neurons into the cell cycle, leading to cell death. The authors hypothesize that the ketamine-induced neuroapoptosis is partially due to aberrant cycle cell reentry. To explore this hypothesis, the authors characterized the effect of ketamine on the cell cycle signaling pathway in the developing rodent brain in vivo and in vitro. Methods:Postnatal day 7 rat pups and primary neurons were used for the experiments. Each rat pup received five intraperitoneal doses of either saline or ketamine (5, 10, and 20 mg/kg/dose) at 90-min intervals over 6 h. Primary neurons were exposed to varying concentrations of ketamine to determine the dose and duration effects. The expression of cell cycle proteins (cyclin D1, cyclin-dependent kinase 4, and E2F1), Bcl2-interacting mediator of cell death (Bim), and activated caspase-3 was determined. The effect of cyclin D1 knockdown by small interfering RNA was also examined in primary neurons incubated in ketamine. Results:Ketamine mediated a dose- and time-dependent increase in expression of cell cycle proteins and activated caspase-3. Cyclin D1, cyclin-dependent kinase 4, E2F1, Bim, and cleaved caspase-3 expression increased at 12 h and peaked at 24 h in vitro. Knockdown of cyclin D1 by small interfering RNA attenuated Bim and cleaved caspase-3 expression. Conclusion:These findings support a model in which ketamine induces aberrant cell cycle reentry, leading to apoptotic cell death in the developing rat brain.

Antiinflammatory adaptation to hypoxia through adenosine-mediated cullin-1 deneddylation

A major adaptive pathway for hypoxia is hypoxic preconditioning (HPC), a form of endogenous protection that renders cells tolerant to severe challenges of hypoxia. We sought to define the antiinflammatory properties of HPC. cDNA microarray analysis of lung tissue from mice subjected to hypoxia or HPC identified a cluster of NF-kappaB-regulated genes whose expression is attenuated by HPC. Studies using an NF-kappaB luciferase reporter assay confirmed a significant suppression of NF-kappaB activation during HPC. HPC-elicited activity was conferrable, as a soluble supernatant from HPC-treated cells, and the active fraction was purified and identified as adenosine (Ado). Guided by recent studies demonstrating bacterial inhibition of NF-kappaB through cullin-1 (Cul-1) deneddylation, we found a dose-dependent deneddylation of Cul-1 by Ado receptor stimulation predominantly mediated by the Ado A2B receptor subtype. Further, siRNA-mediated repression of CSN5, a subunit of the COP9 signalosome responsible for deneddylation of Cul-1, partially reversed HPC-mediated inhibition of NF-kappaB. Cul-1 deneddylation was evident in a murine model of HPC and lost in animals lacking extracellular Ado (Cd73-/- mice). Taken together, these results demonstrate that HPC induces extracellular accumulation of Ado and suppresses NF-kappaB activity through deneddylation of Cul-1. These results define a molecular regulatory pathway by which Ado provides potent antiinflammatory properties.

University of Wisconsin cerebroplegia in a piglet survival model of circulatory arrest

BACKGROUND Previous acute studies in immature piglets at our institution have demonstrated improved recovery of cerebral blood flow, intracellular pH, and high-energy phosphates with the administration of multidose University of Wisconsin solution as cerebroplegia during a period of deep hypothermic circulatory arrest (HCA). In an effort to define further the clinical applicability of this technique, we have developed a survival model of swine cardiopulmonary bypass (CPB) and HCA. METHODS 12 Yorkshire pigs (age 4 to 5 weeks) were placed on CPB via the right femoral artery and right atrium. Animals were cooled to a rectal temperature of 15 degrees C and submitted to 90 minutes of HCA. Group UW (n = 6) received a single infusion of 50 mL/kg of 4 degrees C University of Wisconsin solution delivered antegrade to the cerebral circulation. The control group (n = 6) received no intervention. Animals were reperfused, rewarmed to 35 degrees C, and weaned from CPB. Neurologic assessments using neurologic deficit scoring (0 = normal, 500 = brain death) and overall performance categories (1 = normal, 5 = brain death) were performed at 24-hour intervals for 5 days. On the 5th postoperative day all brains were perfusion-fixed and examined for histologic evidence of neuronal injury (0 = normal, 5 = severe injury). RESULTS All animals were extubated 18 to 20 hours postoperatively. There was no significant difference between the mean neurologic score of the two groups. The mean day 5 neurologic deficit score was 108 for the UW group and 68 for the control group (p > 0.05). The day 5 overall performance category was 2.8 for the UW group and 2.0 for the control group (p > 0.05). Three of the UW animals but none of the control animals experienced generalized seizures. Histologic examination revealed more severe damage in UW animals, primarily in the cerebral cortex. Injury was more widespread in UW animals, involving cerebellum and hippocampus. The mean histologic injury score was 3.8 for UW animals and 2.4 for the control group (p = 0.06). CONCLUSIONS A clinically relevant survival model of CPB with HCA in immature swine is feasible. Cold UW solution as single-dose cerebroplegia is not beneficial, and may be injurious to the immature swine brain subjected to CPB and HCA. Further studies are indicated to determine optimal composition and administration of cerebroplegic solutions.

Prolonged Exposure to Ketamine Increases Brain Derived Neurotrophic Factor Levels in Developing Rat Brains

Prolonged exposure to ketamine, a NMDA receptor antagonist, results in accelerated neurodegeneration and attenuated weight gain in neonatal rats. Suppression of the NMDA receptors by non-competitive antagonists has resulted in conflicting reports of both increased and decreased expression of BDNF. To examine the effect of prolonged ketamine exposure on BDNF expression, we administered saline or ketamine (20 mg/kg) at 90-minute intervals over 9 hours to postnatal day 7 (P7) rat pups. The ketamine-treated rat pups had increased neurodegeneration, BDNF and TrkB cDNA products and protein levels. This increased expression of BDNF may be a response to ketamine-induced injury.

Adenosine Signaling Mediates SUMO-1 Modification of IκBα during Hypoxia and Reoxygenation

Small ubiquitin-like modifier 1 (SUMO-1) modification of IκBα has been described to actively participate in NFκB regulation. Following proteosomal degradation of IκBα, an auto-regulatory loop consisting of transcriptional activation of IκBα gene and SUMO-1 modification of newly synthesized IκBα proceeds. The SUMOylated IκBα form is resistant to signal-induced degradation, consequently halting NFκB activation. We describe a mechanistic model by which adenosine (Ado) signaling results in significant accumulation of SUMO-1 modified IκBα with subsequent attenuation of NFκB activation. Using models of hypoxia followed by reoxygenation (H/R), we have documented an H/R cycle-dependent increase in extracellular Ado correlating with increases in the cytoplasmic pool of IκBα/SUMO-1. We demonstrate a dose-dependent increase in IκBα/SUMO in cells treated with the general Ado receptor agonist NECA and abolished by Ado receptor antagonists. Experiments in cells exposed to cycles of H/R followed by hypoxia demonstrated differential patterns of SUMOylation and phosphorylation of IκBα, greatly impacting its proteosomal degradation by the 26 S proteasome. Assays targeting knockdown and overexpression of SUMO-1 demonstrated significant regulation of NFκB activation and NFκB-mediated gene transcription (interleukin-6). These results were confirmed in vivo using wild type and cd73 null mouse lung tissue. In summary, we present an endogenous mechanism by which cells and tissues acquire anti-inflammatory properties by recruiting a nondegradable form of IκBα, a major control point for NFκB activation via Ado signaling.

Adenosine signaling mediates small ubiquitin-like protein (sumo-1) modification of IκBα during hypoxia and re-oxygenation

Small ubiquitin-like modifier 1 (SUMO-1) modification of IκBα has been described to actively participate in NFκB regulation. Following proteosomal degradation of IκBα, an auto-regulatory loop consisting of transcriptional activation of IκBα gene and SUMO-1 modification of newly synthesized IκBα proceeds. The SUMOylated IκBα form is resistant to signal-induced degradation, consequently halting NFκB activation. We describe a mechanistic model by which adenosine (Ado) signaling results in significant accumulation of SUMO-1 modified IκBα with subsequent attenuation of NFκB activation. Using models of hypoxia followed by reoxygenation (H/R), we have documented an H/R cycle-dependent increase in extracellular Ado correlating with increases in the cytoplasmic pool of IκBα/SUMO-1. We demonstrate a dose-dependent increase in IκBα/SUMO in cells treated with the general Ado receptor agonist NECA and abolished by Ado receptor antagonists. Experiments in cells exposed to cycles of H/R followed by hypoxia demonstrated differential patterns of SUMOylation and phosphorylation of IκBα, greatly impacting its proteosomal degradation by the 26 S proteasome. Assays targeting knockdown and overexpression of SUMO-1 demonstrated significant regulation of NFκB activation and NFκB-mediated gene transcription (interleukin-6). These results were confirmed in vivo using wild type and cd73 null mouse lung tissue. In summary, we present an endogenous mechanism by which cells and tissues acquire anti-inflammatory properties by recruiting a nondegradable form of IκBα, a major control point for NFκB activation via Ado signaling.

Effects of the fibroblast activation protein on the invasion and migration of gastric cancer.

OBJECTIVE Cancer-associated fibroblasts (CAFs) is one of the most important components of tumor microenvironment. CAFs are believed to play an important role in tumor invasion and metastasis. Recently, fibroblast activation protein (FAP), a type II integral membrane glycoprotein belonging to the serine protease family, has emerged as a specific marker of CAFs. FAP was overexpressed in stromal fibroblasts of solid malignancies, however, the role of FAP on the process of invasion and metastasis of gastric carcinomas are still unknown. METHODS Expression of FAP level was detected by immunohistochemistry in 60 gastric cancers surgical specimens (28 with omentum metastasis and 32 without), 20 normal human gastric tissues and omentum of 10 non-neoplastic gastric diseases. Fibroblasts were isolated from patients tissues in the distal normal zones and tumor zones respectively, which were correspondingly designated as normal zone fibroblasts (NFs) and cancer-associated fibroblasts (CAFs). To explore the effects of FAP on NFs or CAFs, fibroblasts were co-cultured with human gastric cancer cell line MGC-803 cells. The ability of invasion and migration of MGC-803 cells was evaluated after transfecting FAP siRNA into CAFs of gastric carcinomas. RESULTS We investigated the level of expression of FAP in surgical specimens, and found overexpressed in CAFs and non- expressed in NFs. Expression of FAP level in CAFs is significantly associated with Lauren classification, the degree of differentiation, depth of tumor invasion and TNM stage, but it is not correlated to age and gender in gastric carcinoma patients. There was positive correlation between the FAP level with metastasis to the omentum (p<0.05, R2=0.2736, p<0.05, R2=0.1479). In addition, the invasion and migration abilities of MGC-803 cells were significantly increased when cells were co-cultured with CAFs. On the other hand, invasion and migration abilities were significantly decreased by 46.9 and 50.3%, respectively, after knocking down FAP in CAFs. Further, NFs did not have appreciable effect on the invasion and migration of MGC-803 cells. CONCLUSIONS Our findings showed that FAP was overexpressed in CAFs of gastric carcinomas, and siRNA-mediated knock down of FAP significantly suppressed invasion and migration of MGC-803 cells. FAP may be an important regulator in the invasion and migration of gastric cancer and may be provide a novel therapeutic target in gastric carcinomas.