Multiple factors contribute to the peripheral induction of cerebral ß-amyloidosis.

Deposition of aggregated amyloid-ß (Aß) peptide in brain is an early event and hallmark pathology of Alzheimer's disease and cerebral Aß angiopathy. Experimental evidence supports the concept that Aß multimers can act as seeds and structurally corrupt other Aß peptides by a self-propagating mechanism. Here we compare the induction of cerebral ß-amyloidosis by intraperitoneal applications of Aß-containing brain extracts in three Aß-precursor protein (APP) transgenic mouse lines that differ in levels of transgene expression in brain and periphery (APP23 mice, APP23 mice lacking murine APP, and R1.40 mice). Results revealed that beta-amyloidosis induction, which could be blocked with an anti-Aß antibody, was dependent on the amount of inoculated brain extract and on the level of APP/Aß expression in the brain but not in the periphery. The induced Aß deposits in brain occurred in a characteristic pattern consistent with the entry of Aß seeds at multiple brain locations. Intraperitoneally injected Aß could be detected in blood monocytes and some peripheral tissues (liver, spleen) up to 30 d after the injection but escaped histological and biochemical detection thereafter. These results suggest that intraperitoneally inoculated Aß seeds are transported from the periphery to the brain in which corruptive templating of host Aß occurs at multiple sites, most efficiently in regions with high availability of soluble Aß.

Survivin and YM155: how faithful is the liaison?

Survivin belongs to the family of apoptosis inhibitors (IAPs), which antagonizes the induction of cell death. Dysregulated expression of IAPs is frequently observed in cancers, and the high levels of survivin in tumors compared to normal adult tissues make it an attractive target for pharmacological interventions. The small imidazolium-based compound YM155 has recently been reported to block the expression of survivin via inhibition of the survivin promoter. Recent data, however, question that this is the sole and main effect of this drug, which is already being tested in ongoing clinical studies. Here, we critically review the current data on YM155 and other new experimental agents supposed to antagonize survivin. We summarize how cells from various tumor entities and with differential expression of the tumor suppressor p53 respond to this agent in vitro and as murine xenografts. Additionally, we recapitulate clinical trials conducted with YM155. Our article further considers the potency of YM155 in combination with other anti-cancer agents and epigenetic modulators. We also assess state-of-the-art data on the sometimes very promiscuous molecular mechanisms affected by YM155 in cancer cells.

Membrane-anchored Aß accelerates amyloid formation and exacerbates amyloid-associated toxicity in mice.

Pathological, genetic, and biochemical hallmarks of Alzheimer's disease (AD) are linked to amyloid-ß (Aß) peptide aggregation. Especially misfolded Aß42 peptide is sufficient to promote amyloid plaque formation. However, the cellular compartment facilitating the conversion of monomeric Aß to aggregated toxic Aß species remains unknown. In vitro models suggest lipid membranes to be the driving force of Aß conversion. To this end, we generated two novel mouse models, expressing either membrane-anchored or nonanchored versions of the human Aß42 peptide. Strikingly, membrane-anchored Aß42 robustly accelerated Aß deposition and exacerbated amyloid-associated toxicity upon crossing with Aß precursor protein transgenic mice. These in vivo findings support the hypothesis that Aß-membrane interactions play a pivotal role in early-onset AD as well as neuronal damage and provide evidence to study Aß-membrane interactions as therapeutic targets.

Quantitative mapping of averaged focal adhesion dynamics in migrating cells by shape normalization.

The spatially ordered formation and disassembly of focal adhesions is a basic requirement for effective cell locomotion. Because focal adhesions couple the contractile actin-myosin network to the substrate, their distribution determines the pattern of traction forces propelling the cell in a certain direction. In the present study, we quantitatively analyzed the spatial patterning of cell-substrate adhesion in migrating cells by mapping averaged focal adhesion growth dynamics to a standardized cell coordinate system. These maps revealed distinct zones of focal adhesion assembly, disassembly and stability and were strongly interrelated with corresponding actin flow and traction force patterns. Moreover, the mapping technique enables precise detection of even minute responses of adhesion dynamics upon targeted signaling perturbations. For example, the partial inhibition of vinculin phosphorylation was followed by the reduced number of newly formed adhesions, whereas growth dynamics of existing adhesions remained unaffected.

Overexpression of Orai1 and STIM1 proteins alters regulation of store-operated Ca2+ entry by endogenous mediators.

Orai1 and STIM1 have been identified as the main determinants of the store-operated Ca(2+) entry (SOCE). Their specific roles in SOCE and their molecular interactions have been studied extensively following heterologous overexpression or molecular knockdown and extrapolated to the endogenous processes in naïve cells. Using molecular and imaging techniques, we found that variation of expression levels of Orai1 or STIM1 can significantly alter expression and role of some endogenous regulators of SOCE. Although functional inhibition of Ca(2+)-independent phospholipase A(2) ß (iPLA(2)ß or PLA2g6A), or depletion of plasma membrane cholesterol caused a dramatic loss of endogenous SOCE in HEK293 cells, these effects were attenuated significantly when either Orai1 or STIM1 were overexpressed. Molecular knockdown of iPLA(2)ß impaired SOCE in both control cells and cells overexpressing STIM1. We also discovered important cross-talk between expression of Orai1 and a specific plasma membrane variant of iPLA(2)ß but not STIM1. These data confirm the role of iPLA(2)ß as an essential mediator of endogenous SOCE and demonstrate that its physiological role can be obscured by Orai1 and STIM1 overexpression.

Role of molecular determinants of store-operated Ca(2+) entry (Orai1, phospholipase A2 group 6, and STIM1) in focal adhesion formation and cell migration.

BACKGROUND: Store-operated Ca(2+) entry is important for cell migration. RESULTS: This study presents characterization of localization and roles of Orai1, STIM1, and PLA2g6 in adhesion dynamics during cell migration. CONCLUSION: Orai1 and PLA2g6 are involved in adhesion formation at the front, whereas STIM1 participates in both adhesion formation and disassembly. SIGNIFICANCE: Results uncovered new parameters of Orai1, STIM1, and PLA2g6 involvement in cell migration. Store-operated Ca(2+) entry and its major determinants are known to be important for cell migration, but the mechanism of their involvement in this complex process is unknown. This study presents a detailed characterization of distinct roles of Orai1, STIM1, and PLA2g6 in focal adhesion (FA) formation and migration. Using HEK293 cells, we discovered that although molecular knockdown of Orai1, STIM1, or PLA2g6 resulted in a similar reduction in migration velocity, there were profound differences in their effects on number, localization, and lifetime of FAs. Knockdown of STIM1 caused an increase in lifetime and number of FAs, their redistribution toward lamellae region, and an increase in cell tail length. In contrast, the number of FAs in Orai1- or PLA2g6-deficient cells was significantly reduced, and FAs accumulated closer to the leading edge. Assembly rate and Vinculin phosphorylation of FAs was similarly reduced in Orai1, PLA2g6, or STIM1-deficient cells. Although Orai1 and PLA2g6 accumulated and co-localized at the leading edge, STIM1 distribution was more complex. We found STIM1 protrusions in lamellipodia, which co-localized with FAs, whereas major accumulation could be seen in central and retracting parts of the cell. Interestingly, knockdown of Orai1 and PLA2g6 produced similar and non-additive effect on migration, whereas knockdown of STIM1 simultaneously with either Orai1 or PLA2g6 produced additional inhibition. Together these data suggest that although Orai1, PLA2g6, and STIM1 play major roles in formation of new FAs at the leading edge, STIM1 may also be involved in Orai1- and PLA2g6-independent disassembly of FAs in the back of cells.

Modeling familial Danish dementia in mice supports the concept of the amyloid hypothesis of Alzheimer's disease.

Familial Danish dementia (FDD) is a progressive neurodegenerative disease with cerebral deposition of Dan-amyloid (ADan), neuroinflammation, and neurofibrillary tangles, hallmark characteristics remarkably similar to those in Alzheimer's disease (AD). We have generated transgenic (tg) mouse models of familial Danish dementia that exhibit the age-dependent deposition of ADan throughout the brain with associated amyloid angiopathy, microhemorrhage, neuritic dystrophy, and neuroinflammation. Tg mice are impaired in the Morris water maze and exhibit increased anxiety in the open field. When crossed with TauP301S tg mice, ADan accumulation promotes neurofibrillary lesions, in all aspects similar to the Tau lesions observed in crosses between beta-amyloid (Abeta)-depositing tg mice and TauP301S tg mice. Although these observations argue for shared mechanisms of downstream pathophysiology for the sequence-unrelated ADan and Abeta peptides, the lack of codeposition of the two peptides in crosses between ADan- and Abeta-depositing mice points also to distinguishing properties of the peptides. Our results support the concept of the amyloid hypothesis for AD and related dementias, and suggest that different proteins prone to amyloid formation can drive strikingly similar pathogenic pathways in the brain.

Human Acellular Dermal Matrix (Epiflex®) in Immediate Implant-Based Breast Reconstruction after Skin- and Nipple-Sparing Mastectomy and Treatment of Capsular Fibrosis: Results of a Multicenter, Prospective, Observational NOGGO-AWOGyn Study.

BACKGROUND: Over the last decades, the number of acellular dermal matrix (ADM)-assisted implant-based breast reconstructions (IBBR) has substantially increased. However, there is still a lack of prospective data on complication rates. METHODS: We performed a non-interventional, multicenter, prospective cohort study to evaluate complication rates of a human ADM in patients undergoing an IBBR after skin- and nipple-sparing mastectomies. Patients with primary reconstruction (cohort A) and patients undergoing a secondary reconstruction after capsular fibrosis (cohort B) using the human ADM Epiflex® (DIZG gGmbH, Berlin, Germany) were enrolled in this study. Patients were followed-up for 12 months after surgery. RESULTS: Eighty-four eligible patients were included in this study of whom 28 women underwent a bilateral breast reconstruction, leading to 112 human ADM-assisted reconstructions in total (cohort A: 73, cohort B: 39). In 33.0% of the reconstructed breasts at least one of the complications of primary interest occurred, including implant loss 7.1%, seroma 15.2%; infection 5.4%, rash 8.0%, and Baker grade III/IV capsular fibrosis 2.7%, with no statistically significant differences between the cohorts. Previous radiation therapy was significantly associated with occurrence of any postoperative complication (OR 20.41; p value 0.027). CONCLUSION: The rates of most complications were comparable to the rates reported for other ADMs with relatively low rates of capsular fibrosis and infections. The rate of seroma was increased in our study. Prior radiation therapy increased the risk of any postoperative complications. Therefore, the use of ADM in these patients should be considered carefully.

One step ahead: role of filopodia in adhesion formation during cell migration of keratinocytes.

Cell adhesion is an essential prerequisite for cell function and movement. It depends strongly on focal adhesion complexes connecting the extracellular matrix to the actin cytoskeleton. Especially in moving cells focal adhesions are highly dynamic and believed to be formed closely behind the leading edge. Filopodia were thought to act mainly as guiding cues using their tip complexes for elongation. Here we show for keratinocytes a strong dependence of lamellipodial adhesion sites on filopodia. Upon stable contact of the VASP-containing tip spot to the substrate, a filopodial focal complex (filopodial FX) is formed right behind along the filopodia axis. These filopodial FXs are fully assembled, yet small adhesions containing all adhesion markers tested. Filopodial FXs when reached by the lamellipodium are just increased in size resulting in classical focal adhesions. At the same time most filopodia regain their elongation ability. Blocking filopodia inhibits development of new focal adhesions in the lamellipodium, while focal adhesion maturation in terms of vinculin exchange dynamics remains active. Our data therefore argue for a strong spatial and temporal dependence of focal adhesions on filopodial focal complexes in keratinocytes with filopodia not permanently initiated via new clustering of actin filaments to induce elongation.

Recovery index, attentiveness and state of memory after xenon or isoflurane anaesthesia: a randomized controlled trial.

BACKGROUND: Performance of patients immediately after anaesthesia is an area of special interest and so a clinical trial was conducted to compare Xenon with Isoflurane anaesthesia. In order to assess the early cognitive recovery the syndrome short test (SST) according to Erzigkeit (Geromed GmbH) was applied. METHODS: ASA I and II patients undergoing long and short surgical interventions were randomised to receive either general anaesthesia with Xenon or Isoflurane. The primary endpoint was the validated SST which covering memory disturbances and attentiveness. The test was used on the day prior to intervention, one and three hours post extubation. The secondary endpoint was the recovery index (RI) measured after the end of the inhalation of Xenon or Isoflurane. In addition the Aldrete score was evaluated up to 180 min. On the first post-operative day the patients rated the quality of the anaesthetic using a scoring system from 1-6. RESULTS: The demographics of the groups were similar. The sum score of the SST delivered a clear trend one hour post extubation and a statistically significant superiority for Xenon three hours post extubation (p < 0.01). The RI likewise revealed a statistically significant superiority of Xenon 5 minutes post extubation (p < 0.01). The Aldrete score was significantly higher for 45 min. The scoring system results were also better after Xenon anaesthesia (p < 0.001). CONCLUSIONS: The results show that recovery from anaesthesia and the early return of post-operative cognitive functions are significantly better after Xenon anaesthesia compared to Isoflurane. The results of the RI for Xenon are similar with the previously published results. TRIAL REGISTRATION: The trial was registered with the number ISRCTN01110844 http://www.controlled-trials.com/isrctn/pf/01110844.

The breast feeding mother and xenon anaesthesia: four case reports. Breast feeding and xenon anaesthesia.

BACKGROUND: Four nursing mothers consented to anaesthesia for urgent surgery only on condition that their ability to breast feed would not be impaired. METHODS: Following induction of general anaesthesia with propofol and remifentanil, 65-69% xenon supplemented with remifentanil was used as an inhalational anaesthetic for maintenance. RESULTS: After finishing surgery the women could be extubated between 2:52 and 7:22 minutes. The women were fully alert just minutes after extubation and spent about 45 minutes in the recovery room before discharge to a regular ward. They resumed regular breast feeding some time later. The propofol concentration in the blood was measured after 0, 30, 90, and 300 minutes and in the milk after 90 and 300 minutes. Just 90 minutes after extubation, the concentration of propofol in the milk was limited (> 3 mg/l) so that pharmacological effects on the babies were excluded after oral intake. Also, no traces of xenon gas were found in the maternal milk at any time. After propofol induction and maintenance of anaesthesia with xenon in combination with a water-soluble short-acting drug like remifentanil, the concentration of propofol in maternal milk is low (> 3 mg/l 90 min after anesthesia) and harmless after oral intake. CONCLUSIONS: These results, as well as the rapid elimination and absence of metabolism of xenon, are of great interest to nursing mothers. General anaesthesia with propofol for induction only, combined with remifentanil and xenon for maintenance, has not yet been described in breast feeding mothers.

Becoming stable and strong: the interplay between vinculin exchange dynamics and adhesion strength during adhesion site maturation.

The coordinated formation and release of focal adhesions is necessary for cell attachment and migration. According to current models, these processes are caused by temporal variations in protein composition. Protein incorporation into focal adhesions is believed to be controlled by phosphorylation. Here, we tested the exchange dynamics of GFP-vinculin as marker protein of focal adhesions using the method of Fluorescence Recovery After Photobleaching. The relevance of the phosphorylation state of the protein, the age of focal adhesions and the acting force were investigated. For stable focal adhesions of stationary keratinocytes, we determined an exchangeable vinculin fraction of 52% and a recovery halftime of 57 s. Nascent focal adhesions of moving cells contained a fraction of exchanging vinculin of 70% with a recovery halftime of 36 s. Upon maturation, mean saturation values and recovery halftimes decreased to levels of 49% and 42 s, respectively. Additionally, the fraction of stably incorporated vinculin increased with cell forces and decreased with vinculin phosphorylation within these sites. Experiments on a nonphosphorylatable vinculin mutant construct at phosphorylation site tyr1065 confirmed the direct interplay between phosphorylation and exchange dynamics of adhesion proteins during adhesion site maturation.

TRA-1-60-positive/CD45low cells found in the peripheral blood of prostate cancer patients with metastatic disease - A proof-of-concept study.

PURPOSE: Over 90% of all cancer related deaths are due to metastasis. However, current diagnostic tools can't reliably discriminate between invasive and localized cancers. PATIENTS AND METHODS: In this proof-of-concept study, we employed the embryonic stem cell marker TRA-1-60 (TRA+) to identify TRA + cells within the blood of prostate cancer patients and searched for TRA + cells in men with metastatic and localized cancers. We isolated whole peripheral blood mononuclear cells from 26 metastatic prostate cancer patients, from 13 patients with localized prostate cancer and from 17 healthy controls. Cells were stained for DAPI, CD45 and TRA + by immunofluorescence and imaged by epi-fluorescence microscopy. Imaged-based software was used both to identify TRA + cells, and to analyze CD45 levels in TRA+ and negative cells. RESULTS: We found high numbers of TRA + cells within the blood of metastatic cancer patients, whereas healthy individuals or men with localized prostate cancer showed none or very low numbers of TRA + cells. Further analysis of the CD45 levels of TRA + cells revealed a small population of TRA + cells with almost undetectable CD45 levels that were found frequently in metastatic prostate cancer patients. By excluding CD45 positive cells from the TRA + cell pool, we were able to refine the assay to be highly specific in identifying men with metastatic disease. In fact, the difference of CD45 levels between TRA+ and negative cells was a robust measure to distinguish between men with localized and metastatic prostate cancers in this small patient cohort. CONCLUSIONS: The data suggest that metastatic prostate cancer patient have significant numbers of TRA+/CD45low cells which might represent a potential tool for diagnostic assessment in the future.

Patient reported outcome and cosmetic evaluation following implant-based breast-reconstruction with a titanized polypropylene mesh (TiLOOP® Bra): A prospective clinical study in 269 patients.

INTRODUCTION: Implant-based or expander-supported breast reconstruction is an established surgical method after mastectomies due to cancer or to prophylactic reasons. Patient reported outcome (PRO) and cosmetic outcome after breast reconstruction with a synthetic surgical mesh was investigated in a prospective, single-arm, multi-center study. MATERIAL AND METHODS: Primary or secondary implant-based breast reconstruction with support of TiLOOP® Bra was performed in 269 patients during the PRO-BRA study. PRO 12 months after breast reconstruction was evaluated using Breast-Q questionnaire. Cosmetic outcome was evaluated by two independent experts by means of pictures taken preoperatively and at the follow-up visits. RESULTS: Breast-Q and 12 months FU were completed by 210 women. Patients without adverse event had a significantly higher Breast-Q score for "sexual well-being" (p = 0.001); "psychosocial well-being" was negatively influenced by prior therapies (p < 0.01), and older patients had significantly lower scores at 12 months FU compared to pre-OP for "satisfaction with breasts" (p < 0.01) while the opposite was true for younger patients. Unilateral surgery resulted in reduced "satisfaction with breast" at 12 months FU (p < 0.01). Radiotherapy negatively influenced "satisfaction with breast", "sexual well-being" and "physical well-being chest". The cosmetic evaluation showed a significant difference (p < 0.001) in the evaluation by the patients and experts with the patients' assessment being worse compared to experts' assessment. CONCLUSION: Our study showed that two years after implant-based breast reconstruction with support of TiLOOP® Bra PRO is influenced by different factors. This information can be used to improve the decision-making process for women who chose implant-based breast reconstruction.

The role of poly(ADP-ribose) polymerase (PARP) in the autonomous proliferative response of endothelial cells to hypoxia.

OBJECTIVE: The autonomous proliferative response of endothelial cells to hypoxia has been shown to be dependent on activation of NAD(P)H oxidase, on the cytosolic Ca2+ load, and, consequently, on nuclear translocation of extracellular signal-regulated kinase (ERK)1/2 during transient hypoxia. The aim of the present study was to investigate whether poly(ADP-ribose) polymerase (PARP) is a downstream signal of NAD(P)H oxidase, mediating cytosolic Ca2+ load and hence nuclear translocation of ERK1/2 and endothelial cell proliferation. METHODS: Porcine aortic endothelial cells were incubated under hypoxic conditions for 40 min. Cytosolic [Ca2+] and reactive oxygen species (ROS) formation were measured in fura-2- and DCF-loaded cells, respectively. PARP activation was detected by immunocytochemistry, and endothelial cell proliferation was determined 24 h after 60 min of transient hypoxia. RESULTS: Inhibition of NAD(P)H oxidase with antisense oligonucleotide against the p22(phox) subunit, MEK/ERK signalling with UO 126 (30 microM), or PARP with PJ 34 (10 microM) leads to a marked reduction in hypoxia-induced cytosolic Ca2+ load and activation of PARP. Hypoxia-induced translocation of ERK1/2 and endothelial cell proliferation were also prevented when NAD(P)H oxidase or PARP were inhibited; however, hypoxic ROS formation was not affected in the presence of PARP inhibitor. CONCLUSION: PARP represents a downstream effector of NADP(H) oxidase and acts as a necessary intermediate step for the hypoxic proliferative response of endothelial cells.

Ischemic acidosis causes apoptosis in coronary endothelial cells through activation of caspase-12.

OBJECTIVE: Myocardial ischemia has been shown to induce apoptosis of endothelial cells (EC). However, the mechanism of this endothelial injury is still poorly understood. To analyse the signaling pathway of ischemia-induced EC apoptosis was the aim of the present study. METHODS: The primary culture of rat coronary EC was exposed to simulated ischemia (glucose-free anoxia at pH(o) 6.4). Apoptosis was defined by staining of nuclei with Hoechst-33342 and TUNEL. Cytosolic Ca2+ and pH were measured with Fura-2 and BCECF, respectively. RESULTS: Apoptosis (29.2+/-1.7% of cells) induced by exposure to simulated ischemia for 2 h was accompanied by cytosolic Ca2+ overload (1090+/-52 nmol/l) and acidosis (pHi = 6.52+/-0.13). Simulated ischemia had no significant effect on caspase-8 cleavage, but induced cleavage of caspase-3 and caspase-12 and led to a slight release of cytochrome C. Prevention of cytosolic acidosis (anoxia at pH(o) 7.4) had no effect on cytochrome C release, but significantly reduced apoptosis, attenuated cytosolic Ca2+ overload, and prevented cleavage of caspase-12. A similar effect was achieved by inhibition of Ca2+ release channels in the endoplasmic reticulum with ryanodine and xestospongin C. Knock-down of caspase-12 with small interfering RNA suppressed caspase-3 activation and reduced apoptotic cell number by about 70%. CONCLUSION: Acidosis, rather than anoxia, is an important trigger of apoptosis in EC under simulated ischemia. The main pathway of the simulated ischemia-induced apoptosis consists of the Ca2+ leak from the ER followed by activation of caspase-12 and caspase-3.

Breast Implant-Associated Lymphoma.

BACKGROUND: There has been increasing evidence in recent years that breast implants can, in rare cases, be associated with the development of an anaplastic large-cell lymphoma (ALCL). METHODS: This review is based on relevant publications retrieved by a selective search in PubMed for articles that appeared from the time of the initial description of breast-implant-associated ALCL onward (1997 to January 2018), and by a further search in German nationwide databases. RESULTS: 516 pathologically confirmed cases of breast-implant-associated (BIA) ALCL were documented around the world until February 2018; seven of these arose in Germany and were reported to the Federal Institute for Drugs and Medical Devices (Bundesinstitut für Arzneimittel und Medizinprodukte, BfArM). In approximately 80% of the affected women, the BIA-ALCL manifested itself as a late-developing seroma at the implant site; in the rest, as a solid tumor with or without an accompanying seroma. The mean implant exposure time ranged from 7 to 13 years on average. 16 fatalities have been reported worldwide. Among the 7 cases reported in Germany, four women had undergone breast reconstruction with implants after breast cancer surgery, and two had undergone breast augmentation surgery. In all patients, the entire capsule-and-implant unit was resected. One patient underwent chemotherapy and one further patient underwent chemotherapy and adjuvant radiotherapy. CONCLUSION: The risk that a woman with breast implants will develop a primary anaplastic large-cell lymphoma is estimated at 0.35 to 1 case per million persons per year. The incidence of implant-associated ALCL is thus very low, yet nevertheless markedly higher than that of other primary lymphomas of the breast. Because of the low case numbers, recommendations for the diagnostic evaluation and treatment of this entity have not been adequately evaluated. Treatment with primary curative intent for BIA-ALCL confers a much better prognosis than when performed for a systemic ALCL. Whenever a patient with a breast implant presents with a late-developing seroma, BIA-ALCL should be included in the differential diagnosis. This diagnosis is reportable.

Contribution of PI 3-kinase isoforms to angiotensin II- and alpha-adrenoceptor-mediated signalling pathways in cardiomyocytes.

OBJECTIVE: Angiotensin II stimulation increases the formation of reactive oxygen species (ROS), the phosphorylation of p38 mitogen-activated protein kinase (MAPK), and the expression of transforming growth factor beta (TGFbeta) in adult cardiomyocytes. The aim of this study was to determine the involvement of PI 3-kinase and to specify the participation of different isoforms in the angiotensin II-induced formation of ROS in comparison to the hypertrophic pathway triggered by alpha-adrenoceptor stimulation. METHODS: Freshly isolated myocytes were used to examine formation of ROS via H(2)DCF fluorescence. p38 MAPK phosphorylation, p70(S6)-kinase phosphorylation, PI 3-kinase, and TGFbeta expression were measured by Western blotting. Sense and antisense oligonucleotides were used to down-regulate diverse PI 3-kinase isoforms. Hypertrophy was measured by (14)C-phenylalanine incorporation and cell volume. RESULTS: Inhibition of PI 3-kinase by Ly294002 or wortmannin, two inhibitors, decreased formation of ROS, phosphorylation of p38 MAPK, and TGFbeta expression. Down-regulation of the p110beta isoform by antisense oligonucleotides inhibited the angiotensin II-induced signalling pathway but not the alpha-adrenoceptor-mediated hypertrophic growth of cardiomyocytes. In contrast, down-regulation of the p110alpha isoform decreased the alpha-adrenoceptor-mediated hypertrophic growth of cardiomyocytes but did not affect the angiotensin II-mediated signalling pathway. CONCLUSION: Thus, our study identifies an involvement of PI 3-kinase in the angiotensin II-induced formation of ROS and provides a biochemical basis for ligand-specific responses for angiotensin II and alpha-adrenoceptor stimulation as relates to hypertrophy.

Accumulation of extracellular ATP protects against acute reperfusion injury in rat heart endothelial cells.

OBJECTIVE: Ischemia-reperfusion provokes barrier failure of the coronary microvasculature, leading to myocardial edema development that jeopardizes functional recovery of the heart during reperfusion. Here, we tested whether adenosine 5'-triphosphate (ATP), either exogenously applied or spontaneously released during reperfusion, protects the endothelial barrier against an imminent reperfusion injury and whether interventions preventing ATP breakdown augment this protective ATP effect. METHODS: Cultured microvascular coronary endothelial monolayers and isolated-perfused hearts of rat were used. RESULTS: After ischemic conditions were induced, reperfusion of endothelial monolayers activated the endothelial contractile machinery and caused intercellular gap formation. It also led to the release of ATP. When its breakdown was inhibited by 6-N,N-diethyl-beta,gamma-dibromomethylene-D-ATP (ARL 67156; 100 microM), a selective ectonucleotidase inhibitor, contractile activation and gap formation were significantly reduced. Reperfusion in the presence of exogenously added ATP (10 microM) plus ARL caused an additional reduction of both aforementioned effects. In contrast, elevation of ATP degradation by apyrase (1 U/ml), a soluble ectonucleotidase, or addition of adenosine (10 microM) provoked an increase in gap formation during reperfusion that could be completely inhibited by 8-phenyltheophylline (8-PT; 10 microM), an adenosine receptor antagonist. In Langendorff-perfused rat hearts, the reperfusion-induced increase in water content was significantly reduced by ARL plus ATP. Under conditions favouring ATP degradation, an increase in myocardial edema was observed that could be blocked by 8-PT. CONCLUSION: ATP, either released from cells or exogenously applied, protects against reperfusion-induced failure of the coronary endothelial barrier. Inhibition of ATP degradation enhances the stabilizing effect of ATP on barrier function.