Development, preclinical evaluation and validation of a novel quick vascular closure device for transluminal, cardiac and radiological arterial catheterization.

Following percutaneous coronary intervention, vascular closure devices (VCDs) are increasingly used to reduce time to ambulation, enhance patient comfort, and reduce potential complications compared with traditional manual compression. Newer techniques include complicated, more or less automated suture devices, local application of pads or the use of metal clips and staples. These techniques often have the disadvantage of being time consuming, expensive or not efficient enough. The VCD failure rate in association with vascular complications of 2.0-9.5%, depending on the type of VCD, is still not acceptable. Therefore, the aim of this study is to develop a self-expanding quick vascular closure device (QVCD) made from a bioabsorbable elastic polymer that can be easily applied through the placed introducer sheath. Bioabsorbable block-co-polymers were synthesized and the chemical and mechanical degradation were determined by in vitro tests. The best fitting polymer was selected for further investigation and for microinjection moulding. After comprehensive haemocompatibility analyses in vitro, QVCDs were implanted in arterial vessels following arteriotomy for different time points in sheep to investigate the healing process. The in vivo tests proved that the new QVCD can be safely placed in the arteriotomy hole through the existing sheath instantly sealing the vessel. The degradation time of 14 days found in vitro was sufficient for vessel healing. After 4 weeks, the remaining QVCD material was covered by neointima. Overall, our experiments showed the safety and feasibility of applying this novel QVCD through an existing arterial sheath and hence encourage future work with larger calibers.

The plasminogen receptor, Plg-RKT, is essential for mammary lobuloalveolar development and lactation.

Essentials Plg-RKT-/- female mice give birth, but no offspring of Plg-RKT-/- female mice survive to weaning. Causal mechanisms of potential lactational failure in Plg-RKT-/- mice are unknown. Plg-RKT regulates extracellular matrix remodeling, cell proliferation, apoptosis, fibrin surveillance. Plg-RKT is essential for lactogenesis and mammary lobuloalveolar development. SUMMARY: Background Lactational competence requires plasminogen, the zymogen of the serine protease, plasmin. Plg-RKT is a unique transmembrane plasminogen receptor that promotes plasminogen activation to plasmin on cell surfaces. Plg-RKT-/- mice are viable, but no offspring of Plg-RKT-/- female mice survive to weaning. Objectives We investigated potential lactational failure in Plg-RKT-/- mice and addressed causal mechanisms. Methods Fibrin accumulation, macrophage infiltration, processing of extracellular matrix components, effects of genetic deletion of fibrinogen, expression of fibrosis genes, and proliferation and apoptosis of epithelial cells were examined in lactating mammary glands of Plg-RKT-/- and Plg-RKT+/+ mice. Results Milk was not present in the stomachs of offspring of Plg-RKT-/- female mice and the pups were rescued by foster mothers. Although the mammary ductal tree developed normally in Plg-RKT-/- glands, lobuloalveolar development was blocked by a hypertrophic fibrotic stroma and infiltrating macrophages were present. A massive accumulation of fibrin was also present in Plg-RKT-/- alveoli and ducts. Although this accumulation was decreased when Plg-RKT-/- mice were made genetically heterozygous for fibrinogen, defects in lobuloalveolar development were not rescued by fibrinogen heterozygosity. Transcriptional profiling revealed that EGF was downregulated 12-fold in Plg-RKT-/- glands. Furthermore, proliferation of epithelial cells was not detectable. In addition, the pro-survival protein, Mcl-1, was markedly downregulated and apoptosis was observed in Plg-RKT-/- but not Plg-RKT+/+ glands. Conclusions Plg-RKT is essential for lactogenesis and functions to maintain the appropriate stromal extracellular matrix environment, regulate epithelial cell proliferation and apoptosis, and, by regulating fibrinolysis, preserve alveolar and ductal patency.

Evaluation of platelet adhesion and activation on polymers: Round-robin study to assess inter-center variability.

The regulatory agencies provide recommendations rather than protocols or standard operation procedures for the hemocompatibility evaluation of novel materials e.g. for cardiovascular applications. Thus, there is a lack of specifications with regard to test setups and procedures. As a consequence, laboratories worldwide perform in vitro assays under substantially different test conditions, so that inter-laboratory and inter-study comparisons are impossible. Here, we report about a prospective, randomized and double-blind multicenter trial which demonstrates that standardization of in vitro test protocols allows a reproducible assessment of platelet adhesion and activation from fresh human platelet rich plasma as possible indicators of the thrombogenicity of cardiovascular implants. Standardization of the reported static in vitro setup resulted in a laboratory independent scoring of the following materials: poly(dimethyl siloxane) (PDMS), poly(ethylene terephthalate) (PET) and poly(tetrafluoro ethylene) (PTFE). The results of this in vitro study provide evidence that inter-laboratory and inter-study comparisons can be achieved for the evaluation of the adhesion and activation of platelets on blood-contacting biomaterials by stringent standardization of test protocols.

Preclinical evaluation of the thrombogenicity and endothelialization of bare metal and surface-coated neurovascular stents.

BACKGROUND AND PURPOSE: Stent-assisted coiling is routinely used for the endovascular treatment of complex or wide-neck intracranial aneurysms. However, in-stent thrombosis, thromboembolic events, and ischemic complications remain a major concern associated with stent implants. Therefore, a novel low-profile neurovascular stent with a bare metal surface was investigated with regard to thrombogenicity and endothelialization and compared with the same stent coated with albumin or heparin. MATERIALS AND METHODS: The bare metal and heparin- or albumin-coated stents were loaded in heparin-coated tubing, which was then filled with heparinized human blood (n = 5) and circulated at 150 mL/min and 37°C for 60 minutes. Before and after circulation, measurement of various inflammation and coagulation markers and scanning electron microscopy were performed. Endothelialization of the stents was investigated in an in vitro model including human umbilical vascular endothelial cells. RESULTS: Our results showed that platelet loss and platelet activation and activation of the coagulation cascade, which are induced by the bare metal stents, were significantly reduced by heparin or albumin coating. Adverse effects on erythrocytes, leukocytes, and the complement cascade were not induced by the bare metal or coated stents. Moreover, the bare metal and albumin-coated stents showed good endothelialization properties. CONCLUSIONS: Albumin and heparin coatings clearly improve the thrombogenicity of the stents and thus may represent fundamental progress in the treatment of intracranial aneurysms. Moreover, preclinical evaluation of neurovascular stents under physiologic conditions supports and accelerates the development of more biocompatible neurovascular stents.

Cytocompatibility evaluation of different biodegradable magnesium alloys with human mesenchymal stem cells.

In the last few years, the use of biodegradable magnesium (Mg) alloys has evoked great interest in the orthopedic field due to great advantages over long-term implant materials associated with various side effects like allergy and sensitization and consequent implant removal surgeries. However, degradation of these Mg alloys results in ion release, which may cause severe cytotoxicity and undesirable complications after implantation. In this study, we investigated the cytological effects of various Mg alloys on cells that play an important role in bone repair. Eight different magnesium alloys containing varying amounts of Al, Zn, Nd and Y were either incubated directly or indirectly with the osteosarcoma cell line Saos-2 or with uninduced and osteogenically-induced human mesenchymal stem cells (MSCs) isolated from bone marrow specimens obtained from the femoral shaft of patients undergoing total hip replacement. Cell viability, cell attachment and the release of ions were investigated at different time points in vitro. During direct or indirect incubation different cytotoxic effects of the Mg alloys on Saos-2 cells and osteogenically-induced or uninduced MSCs were observed. Furthermore, the concentration of degradation products released from the Mg alloys differed. Overall, Mg alloys MgNd2, MgY4, MgAl9Zn1 and MgY4Nd2 exhibit good cytocompatibility. In conclusion, this study reveals the necessity of cytocompatibility evaluation of new biodegradable magnesium alloys with cells that will get in direct contact to the implant material. Furthermore, the use of standardized experimental in vitro assays is necessary in order to reliably and effectively characterize new Mg alloys before performing in vivo experiments.

Short-acting P2Y12 blockade to reduce platelet dysfunction and coagulopathy during experimental extracorporeal circulation and hypothermia.

BACKGROUND: Extracorporeal circulation (ECC) and hypothermia are routinely used in cardiac surgery to maintain stable circulatory parameters and to increase the ischaemic tolerance of the patient. However, ECC and hypothermia cause platelet activation and dysfunction possibly followed by a devastating coagulopathy. Stimulation of the adenosinediphosphate (ADP) receptor P(2)Y(12) plays a pivotal role in platelet activation. This experimental study tested P(2)Y(12) receptor blockade as an approach to protect platelets during ECC. METHODS: Human blood was treated with the short-acting P(2)Y(12) blocker cangrelor (1 µM, t(1/2)<5 min) or the P(2)Y(12) inhibitor 2-MeSAMP (100 µM) and circulated in an ex vivo ECC model at normothermia (37°C) and hypothermia (28°C). Before and after circulation, markers of platelet activation and of coagulation (thrombin-antithrombin complex generation) were analysed. During hypothermic ECC in pigs, the effect of reversible P(2)Y(12) blockade on platelet function was evaluated by cangrelor infusion (0.075 µg kg(-1) min(-1)). RESULTS: During ex vivo hypothermic ECC, P(2)Y(12) blockade inhibited platelet granule release (P<0.01), platelet-granulocyte binding (P<0.05), and platelet loss (P<0.001), whereas no effects on platelet-ECC binding, platelet CD42bα expression, glycoprotein IIb/IIIa activation, or thrombin-antithrombin complex generation were observed. During hypothermic ECC in pigs, cangrelor inhibited platelet-fibrinogen binding (P<0.05) and ADP-induced platelet aggregation (P<0.001). Platelet function was rapidly restored after termination of cangrelor infusion. CONCLUSIONS: P(2)Y(12) blockade by cangrelor prevents platelet activation during ECC and hypothermia. Owing to its short half-life, platelet inhibition can be well controlled, thus potentially reducing bleeding complications. This novel pharmacological strategy has the potential to reduce complications associated with ECC and hypothermia.

Forebrain projections of arcuate neurokinin B neurons demonstrated by anterograde tract-tracing and monosodium glutamate lesions in the rat.

Neurokinin B (NKB) and kisspeptin receptor signaling are essential components of the reproductive axis. A population of neurons resides within the arcuate nucleus of the rat that expresses NKB, kisspeptin, dynorphin, NK3 receptors and estrogen receptor alpha (ERalpha). Here we investigate the projections of these neurons using NKB-immunocytochemistry as a marker. First, the loss of NKB-immunoreactive (ir) somata and fibers was characterized after ablation of the arcuate nucleus by neonatal injections of monosodium glutamate. Second, biotinylated dextran amine was injected into the arcuate nucleus and anterogradely labeled NKB-ir fibers were identified using dual-labeled immunofluorescence. Four major projection pathways are described: (1) local projections within the arcuate nucleus bilaterally, (2) projections to the median eminence including the lateral palisade zone, (3) projections to a periventricular pathway extending rostrally to multiple hypothalamic nuclei, the septal region and BNST and dorsally to the dorsomedial nucleus and (4) Projections to a ventral hypothalamic tract to the lateral hypothalamus and medial forebrain bundle. The diverse projections provide evidence that NKB/kisspeptin/dynorphin neurons could integrate the reproductive axis with multiple homeostatic, behavioral and neuroendocrine processes. Interestingly, anterograde tract-tracing revealed NKB-ir axons originating from arcuate neurons terminating on other NKB-ir somata within the arcuate nucleus. Combined with previous studies, these experiments reveal a bilateral interconnected network of sex-steroid responsive neurons in the arcuate nucleus of the rat that express NKB, kisspeptin, dynorphin, NK3 receptors and ERalpha and project to GnRH terminals in the median eminence. This circuitry provides a mechanism for bilateral synchronization of arcuate NKB/kisspeptin/dynorphin neurons to modulate the pulsatile secretion of GnRH.

Effects of ovariectomy on the neuroendocrine axes regulating reproduction and energy balance in young cynomolgus macaques.

Degeneration of the ovary in middle-aged women results in castrate levels of ovarian steroids and increased gonadotropin secretion from the anterior pituitary gland. Ageing in women is also accompanied by significant changes in energy homeostasis. We have observed alterations in hypothalamic morphology and gene expression in older women, including hypertrophy and increased gene expression of neurokinin B (NKB) neurones, elevated levels of gonadotropin releasing-hormone (GnRH) mRNA and decreased numbers of neurones expressing pro-opiomelanocortin (POMC) mRNA. To determine if loss of ovarian steroids could produce comparable changes in gene expression in young primates, we measured the effects of ovariectomy on NKB, GnRH and POMC gene expression in young cynomolgus monkeys. We also measured serum leptin and body weight to examine the consequences of ovariectomy on energy balance. NKB neurones in the infundibular nucleus of ovariectomized monkeys were larger, more numerous and displayed increased levels of NKB mRNA compared to those of intact controls. Moreover, ovariectomy increased the number of neurones expressing GnRH gene transcripts and elevated serum luteinizing hormone. By contrast, several parameters related to energy balance, including POMC gene expression, serum leptin and body weights, were unchanged by ovariectomy. Thus, the rise in NKB and GnRH gene expression in older women was simulated by ovariectomy in monkeys, but the changes in POMC gene expression and energy balance were not. This study provides strong support for the hypothesis that ovarian failure contributes to the increased NKB and GnRH gene expression observed in postmenopausal women.

Specific caspase interactions and amplification are involved in selective neuronal vulnerability in Huntington's disease.

Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder resulting in selective neuronal loss and dysfunction in the striatum and cortex. The molecular pathways leading to the selectivity of neuronal cell death in HD are poorly understood. Proteolytic processing of full-length mutant huntingtin (Htt) and subsequent events may play an important role in the selective neuronal cell death found in this disease. Despite the identification of Htt as a substrate for caspases, it is not known which caspase(s) cleaves Htt in vivo or whether regional expression of caspases contribute to selective neuronal cells loss. Here, we evaluate whether specific caspases are involved in cell death induced by mutant Htt and if this correlates with our recent finding that Htt is cleaved in vivo at the caspase consensus site 552. We find that caspase-2 cleaves Htt selectively at amino acid 552. Further, Htt recruits caspase-2 into an apoptosome-like complex. Binding of caspase-2 to Htt is polyglutamine repeat-length dependent, and therefore may serve as a critical initiation step in HD cell death. This hypothesis is supported by the requirement of caspase-2 for the death of mouse primary striatal cells derived from HD transgenic mice expressing full-length Htt (YAC72). Expression of catalytically inactive (dominant-negative) forms of caspase-2, caspase-7, and to some extent caspase-6, reduced the cell death of YAC72 primary striatal cells, while the catalytically inactive forms of caspase-3, -8, and -9 did not. Histological analysis of post-mortem human brain tissue and YAC72 mice revealed activation of caspases and enhanced caspase-2 immunoreactivity in medium spiny neurons of the striatum and the cortical projection neurons when compared to controls. Further, upregulation of caspase-2 correlates directly with decreased levels of brain-derived neurotrophic factor in the cortex and striatum of 3-month YAC72 transgenic mice and therefore suggests that these changes are early events in HD pathogenesis. These data support the involvement of caspase-2 in the selective neuronal cell death associated with HD in the striatum and cortex.

Bifunctional apoptosis inhibitor (BAR) protects neurons from diverse cell death pathways.

The bifunctional apoptosis regulator (BAR) is a multidomain protein that was originally identified as an inhibitor of Bax-induced apoptosis. Immunoblot analysis of normal human tissues demonstrated high BAR expression in the brain, compared to low or absent expression in other organs. Immunohistochemical staining of human adult tissues revealed that the BAR protein is predominantly expressed by neurons in the central nervous system. Immunofluorescence microscopy indicated that BAR localizes mainly to the endoplasmic reticulum (ER) of cells. Overexpression of BAR in CSM 14.1 neuronal cells resulted in significant protection from a broad range of cell death stimuli, including agents that activate apoptotic pathways involving mitochondria, TNF-family death receptors, and ER stress. Downregulation of BAR by antisense oligonucleotides sensitized neuronal cells to induction of apoptosis. Moreover, the search for novel interaction partners of BAR identified several candidate proteins that might contribute to the regulation of neuronal apoptosis (HIP1, Hippi, and Bap31). Taken together, the expression pattern and functional data suggest that the BAR protein is involved in the regulation of neuronal survival.

Contribution of caspase-8 to apoptosis of axotomized rat retinal ganglion cells in vivo.

We have investigated the role of caspase-8 and its mode of activation during apoptosis of adult rat retinal ganglion cells (RGCs) in vivo. Retinal pro-caspase-8 expression was almost completely restricted to RGCs. Although caspase-8 is known to be involved in death-receptor-dependent apoptosis, measurable caspase-8 activity or even RGC death could be induced by neither tumor necrosis factor-alpha nor Fas ligand injections into unlesioned eyes. However, substantial caspase-8 activation could be detected after optic nerve transection as shown by a fluorogenic activity assay and Western blot analysis. Intravitreal injection of caspase-8 inhibitors significantly attenuated degeneration of RGCs and reduced the number of RGCs showing caspase-3 activation. A late peak of caspase-8 activity and additive protective effects of caspase-8 and -9 inhibition on axotomized RGCs place caspase-8 in our model rather late in the apoptosis cascade, possibly after the onset of mitochondrial dysfunction.

Mild traumatic brain injury induces apoptotic cell death in the cortex that is preceded by decreases in cellular Bcl-2 immunoreactivity.

Although mild traumatic brain injury is associated with behavioral dysfunction and histopathological alterations, few studies have assessed the temporal pattern of regional apoptosis following mild brain injury. Anesthetized rats were subjected to mild lateral fluid-percussion brain injury (1.1-1.3 atm), and brains were evaluated for the presence of in situ DNA fragmentation (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling, TUNEL) and morphologic characteristics of apoptotic cell death (nuclear and cytoplasmic condensation, presence of apoptotic bodies). Significant numbers of apoptotic TUNEL(+) cells were observed in the injured parietal cortex and underlying white matter up to 72 h post-injury (P<0.05 compared to sham-injured-injured), with maximal numbers present at 24 h. Apoptosis was confirmed by the presence of 180-200 bp nuclear DNA fragments in tissue homogenates. The appearance of apoptotic TUNEL(+) cells in the injured cortex was preceded by a marked decrease in immunoreactivity for the anti-cell death protein, Bcl-2, as early as 2 h post-injury. This decrease in cellular Bcl-2 staining was not accompanied by a concomitant loss of staining for the pro-cell death Bax protein, suggesting that post-traumatic neuronal death in the cortex may be dependent on altered cellular ratios of Bcl-2:Bax. In the hippocampus, no significant increase in apoptotic TUNEL(+) cells was observed compared to sham-injured-injured animals. However, selective neuronal loss was evident in the CA3 region at 24 h post-injury, that was preceded by an overt loss of neuronal Bcl-2 immunoreactivity at 6 h. No changes in either cellular Bcl-2 or Bax expression were observed in the thalamus or white matter at any time post-injury. Taken together from these data, we suggest that apoptosis contributes to cell death in both gray and white matter, and that decreases in cellular Bcl-2 may, in part, be associated with both apoptotic and non-apoptotic cell death following mild brain trauma.

Bag1 is a regulator and marker of neuronal differentiation.

Bag 1 acts as a co-chaperone for Hsp70/Hsc70. We report here that stable over-expression of Bag1 in immortalized neuronal CSM14.1 cells prevents death following serum deprivation. Bag1 over-expression slowed the proliferative rate of CSM14.1 cells, resulted in increased levels of phospo-MAP kinases and accelerated neuronal differentiation. Immunocytochemistry revealed mostly nuclear localization of Bag1 protein in these cells. However, during differentiation in vitro, Bag1 protein shifted from predominantly nuclear to mostly cytosolic in CSM14.1 cells. To explore in vivo parallels of these findings, we investigated Bag1 expression in the developing mouse nervous system using immunohistochemical methods. Early in brain development, Bag1 was found in nuclei of neuronal precursor cells, whereas cytosolic Bag1 staining was observed mainly after completion of neuronal precursor migration and differentiation. Taken together, these findings raise the possibility that the Bag1 protein is expressed early in neurogenesis in vivo and is capable of modulating neuronal cell survival and differentiation at least in part from a nuclear location.

Expression of apoptotic regulators and their significance in cervical cancer.

Insufficient apoptosis is implicated in many human cancers, including cervical carcinoma. The objectives of this study were to explore changes of apoptosis-regulating gene expression and their clinical significance in cervical cancer. The expression of apoptosis-regulating genes, including five Bcl-2 family and two caspase family members, was evaluated in 43 cervical invasive squamous cell carcinomas, using immunohistochemistry. Specimens in which >or=10% of the neoplastic cells showed cytosolic immunoreactivity were considered to be immunopositive. Results were correlated with clinico-pathologic characteristics of the subjects. All seven apoptotic regulators examined were positive in a proportion of the tumors. The percentage of cases expressing Bax was higher in the patients without evidence of disease after treatment than in the patients alive with disease or who died of disease (P<0.05). A significant difference in disease-free survival was detected between Bax-positive and -negative groups (P<0.05), and in overall survival between Mcl-1-positive and -negative groups (P<0.05). Significant association between the seven markers tested was only found for caspase 3 and Bak immunoreactivity in cervical carcinoma (P<0.05). The results demonstrate expression of multiple apoptosis-modulating proteins in cervical cancer. There appears to be complex regulation of apoptosis protein levels in association with clinical behavior of cervical squamous cell carcinoma.

Dynamics of expression of apoptosis-regulatory proteins Bid, Bcl-2, Bcl-X, Bax and Bak during development of murine nervous system.

We have used immunohistochemistry and immunoblotting to examine the expression of Bid and four other Bcl-2 family proteins (Bcl-2, Bcl-X, Bax and Bak) in the developing and adult murine central nervous system (CNS). Bid protein is widespread in embryonic and postnatal brain, and its expression is maintained at a high level late into the adulthood. Bid is expressed both in the germ disc, early neural tube, proliferating stem cells of ventricular zones, and in postmitotic, differentiated neurons of the developing central and peripheral nervous system. As the differentiation proceeds, the neurons express higher levels of Bid than the stem cells of the paraventricular zone. Both in embryonic and postnatal life, Bid protein is present in the most vital regions of brain, such as the limbic system, basal ganglia, mesencephalic tectum, Purkinje cells in cerebellum, and the ventral columns of spinal cord. The p15 cleaved form of Bid was detectable in the brain specimens at fetal stages of development, consistent with caspase-mediated activation of this pro-apoptotic Bcl-2 family protein. Among the Bcl-2 family proteins only Bid and Bcl-XL continue to be expressed at high levels in the adult brain.

Distinct expression pattern of microtubule-associated protein-2 in human oligodendrogliomas and glial precursor cells.

Microtubule-associated protein 2 (MAP2), a protein linked to the neuronal cytoskeleton in the mature central nervous system (CNS), has recently been identified in glial precursors indicating a potential role during glial development. In the present study, we systematically analyzed the expression of MAP2 in a series of 237 human neuroepithelial tumors including paraffin-embedded specimens and tumor tissue microarrays from oligodendrogliomas, mixed gliomas, astrocytomas, glioblastomas, ependymomas, as well as dysembryoplastic neuroepithelial tumors (DNT), and central neurocytomas. In addition, MAP2-immunoreactive precursor cells were studied in the developing human brain. Three monoclonal antibodies generated against MAP2A-B or MAP2A-D isoforms were used. Variable immunoreactivity for MAP2 could be observed in all gliomas with the exception of ependymomas. Oligodendrogliomas exhibited a consistently strong and distinct pattern of expression characterized by perinuclear cytoplasmic staining without significant process labeling. Tumor cells with immunoreactive bi- or multi-polar processes were mostly encountered in astroglial neoplasms, whereas the small cell component in neurocytomas and DNT was not labeled. These features render MAP2 immunoreactivity a helpful diagnostic tool for the distinction of oligodendrogliomas and other neuroepithelial neoplasms. RT-PCR, Western blot analysis, and in situ hybridization confirmed the expression of MAP2A-C (including the novel MAP2+ 13 transcript) in both oligodendrogliomas and astrocytomas. Double fluorescent laser scanning microscopy showed that GFAP and MAP2 labeled different tumor cell populations. In embryonic human brains, MAP2-immunoreactive glial precursor cells were identified within the subventricular or intermediate zones. These precursors exhibit morphology closely resembling the immunolabeled neoplastic cells observed in glial tumors. Our findings demonstrate MAP2 expression in astrocytic and oligodendroglial neoplasms. The distinct pattern of immunoreactivity in oligodendrogliomas may be useful as a diagnostic tool. Since MAP2 expression occurs transiently in migrating immature glial cells, our findings are in line with an assumed origin of diffuse gliomas from glial precursors.

The AKT kinase is activated in multiple myeloma tumor cells.

Immunohistochemistry (IHC) was performed on archived bone marrow (BM) with a phosphospecific anti-AKT antibody. IHC on 26 BM biopsies from patients with multiple myeloma (MM) demonstrated phospho-AKT staining of malignant plasma cells in a cell membrane-specific pattern, whereas nonmalignant hematopoietic cells did not stain. Preabsorption of the antibody with phosphorylated AKT peptide, but not nonphosphorylated peptide, abrogated staining. Frequency of plasma cell staining in BMs of patients with stage I or smoldering MM was significantly less than that of stage III MM marrows. Plasma cells in 10 patients with monoclonal gammopathy of undetermined significance were not stained by the antibody. To investigate the significance of AKT activation, 2 cell lines initiated from cultures of primary MM cells were also studied. Both demonstrated constitutive AKT activation. Interruption of AKT activation and activity, achieved by either exposure to wortmannin or by ectopic expression of a dominant negative AKT mutant, resulted in inhibition of MM cell growth in vitro. These results indicate that activation of the AKT kinase is a characteristic of MM cells and suggest that AKT activity is important for MM cell expansion.

Caspases 3 and 9 send a pro-apoptotic signal from synapse to cell body in olfactory receptor neurons.

Caspase-9, an initiator caspase, and caspase-3, an effector caspase, have been suggested to mediate the terminal stages of neuronal apoptosis, but little is known about their activation in vivo. We examined temporal and spatial aspects of caspase-9 and -3 activation in olfactory receptor neurons (ORNs) undergoing apoptosis after target removal in vivo. After removal of the olfactory bulb, enhanced expression of procaspase-9 and -3 is observed in ORNs, followed by activation initially at the level of the lesion, then in axons, and only later in the ORN soma. We established the amyloid precursor-like protein-2 (APLP2) as a caspase substrate that is cleaved in an identical spatiotemporal pattern, suggesting its cleavage is the result of retrograde propagation of a pro-apoptotic signal in a caudorostral wave from the synapse through the axon to the ORN cell body. A null mutation in caspase-3 causes a change in axonal patterning indicative of an overall developmental expansion of the ORN population, and mature ORNs of caspase-3 knock-outs do not undergo caspase-dependent terminal dUTP nick end labeling-positive apoptosis after olfactory bulb removal. These results demonstrate that ORNs require caspase-3 activation to undergo normal developmental and mature target-deprived apoptosis. In addition, we demonstrate an axonal site of action for caspase-3 and -9 and show that regulation and activation of caspase-3 and -9 leading to apoptosis is a highly ordered process that occurs initially at the presynaptic level and only later at the cell body after deafferentation.

Temporal and spatial profile of caspase 8 expression and proteolysis after experimental traumatic brain injury.

Recent studies have demonstrated that the downstream caspases, such as caspase 3, act as executors of the apoptotic cascade after traumatic brain injury (TBI) in vivo. However, little is known about the involvement of caspases in the initiation phase of apoptosis, and the interaction between these initiator caspases (e.g. caspase 8) and executor caspases after experimental brain injuries in vitro and in vivo. This study investigated the temporal expression and cell subtype distribution of procaspase 8 and cleaved caspase 8 p20 from 1 h to 14 days after cortical impact-induced TBI in rats. Caspase 8 messenger RNA levels, estimated by semiquantitaive RT-PCR, were elevated from 1 h to 72 h in the traumatized cortex. Western blotting revealed increased immunoreactivity for procaspase 8 and the proteolytically active subunit of caspase 8, p20, in the ipsilateral cortex from 6 to 72 h after injury, with a peak at 24 h after TBI. Similar to our previous studies, immunoreactivity for the p18 fragment of activated caspase 3 also increased in the current study from 6 to 72 h after TBI, but peaked at a later timepoint (48 h) as compared with proteolyzed caspase 8 p20. Immunohistologic examinations revealed increased expression of caspase 8 in neurons, astrocytes and oligodendrocytes. Assessment of DNA damage using TUNEL identified caspase 8- and caspase 3-immunopositive cells with apoptotic-like morphology in the cortex ipsilateral to the injury site, and immunohistochemical investigations of caspase 8 and activated caspase 3 revealed expression of both proteases in cortical layers 2-5 after TBI. Quantitative analysis revealed that the number of caspase 8 positive cells exceeds the number of caspase 3 expressing cells up to 24 h after impact injury. In contrast, no evidence of caspase 8 and caspase 3 activation was seen in the ipsilateral hippocampus, contralateral cortex and hippocampus up to 14 days after the impact. Our results provide the first evidence of caspase 8 activation after experimental TBI and suggest that this may occur in neurons, astrocytes and oligodendrocytes. Our findings also suggest a contributory role of caspase 8 activation to caspase 3 mediated apoptotic cell death after experimental TBI in vivo.