Regenerative medicine for the treatment of heart disease.

Heart failure is a major cause of mortality worldwide with a steady increase in prevalence. There is currently no available cure beyond orthotopic heart transplantation, which for a number of reasons is an option only for a small fraction of all patients. Considerable hope has therefore been placed on the possibility of treating a failing heart by replacing lost cardiomyocytes, either through transplantation of various types of stem cells or by boosting endogenous regenerative mechanisms in the heart. Here, we review the current status of stem and progenitor cell-based therapies for heart disease. We discuss the pros and cons of different stem and progenitor cell types that can be considered for transplantation and describe recent advances in the understanding of how cardiomyocytes normally differentiate and how these cells can be generated from more immature cells ex vivo. Finally, we consider the possibility of activation of endogenous stem and progenitor cells to treat heart failure.

Non-canonical Notch signaling activates IL-6/JAK/STAT signaling in breast tumor cells and is controlled by p53 and IKKα/IKKß.

Notch signaling is frequently hyperactivated in breast cancer, but how the enhanced signaling contributes to the tumor process is less well understood. In this report, we identify the proinflammatory cytokine interleukin-6 (IL-6) as a novel Notch target in breast tumor cells. Enhanced Notch signaling upregulated IL-6 expression, leading to activation of autocrine and paracrine Janus kinase/signal transducers and activators of transcription signaling. IL-6 upregulation was mediated by non-canonical Notch signaling, as it could be effectuated by a cytoplasmically localized Notch intracellular domain and was independent of the DNA-binding protein CSL. Instead, Notch-mediated IL-6 upregulation was controlled by two proteins in the nuclear factor (NF)-κB signaling cascade, IKKα and IKKß (inhibitor of nuclear factor kappa-B kinase subunit alpha and beta, respectively), as well as by p53. Activation of IL-6 by Notch required IKKα/IKKß function, but interestingly, did not engage canonical NF-κB signaling, in contrast to IL-6 activation by inflammatory agents such as lipopolysaccharide. With regard to p53 status, IL-6 expression was upregulated by Notch when p53 was mutated or lost, and restoring wild-type p53 into p53-mutated or -deficient cells abrogated the IL-6 upregulation. Furthermore, Notch-induced transcriptomes from p53 wild-type and -mutated breast tumor cell lines differed extensively, and for a subset of genes upregulated by Notch in a p53-mutant cell line, this upregulation was reduced by wild-type p53. In conclusion, we identify IL-6 as a novel non-canonical Notch target gene, and reveal roles for p53 and IKKα/IKKß in non-canonical Notch signaling in breast cancer and in the generation of cell context-dependent diversity in the Notch signaling output.

Regenerative medicine: a 2009 overview.

The rapidly evolving field of regenerative medicine holds much promise for cell-based therapies for a range of debilitating conditions from spinal cord injury to haematological and neurological diseases. The groundbreaking discovery of induced pluripotent stem (iPS) cells has offered new perspectives on disease progression and the possibility of patient-specific cell transplants. In this review we first give a brief history of the field of regenerative medicine and then discuss the current state of regenerative medicine with a focus on embryonic stem cells and iPS cells. In order to keep abreast with this rapidly developing field, the Journal of Internal Medicine organized a 2-day Symposium, on March 12-13(th) 2009, at the Karolinska Insitute in Stockholm, which featured talks by 19 leading scientists in the field of regenerative medicine. In this review, we discuss the Symposium and introduce six accompanying review articles, by Symposium speakers, which focus on some of the topics discussed at the meeting.

Notch-1 associates with IKKalpha and regulates IKK activity in cervical cancer cells.

Notch-1 inhibits apoptosis in some transformed cells through incompletely understood mechanisms. Notch-1 can increase nuclear factor-kappa B (NF-kappaB) activity through a variety of mechanisms. Overexpression of cleaved Notch-1 in T-cell acute lymphoblastic leukemia cells activates NF-kappaB via interaction with the I kappa B kinase (IKK) signalosome. Concomitant activation of the Notch and NF-kappaB pathways has been described in a large series of cervical cancer specimens. Here, we show that wild-type, spontaneously expressed Notch-1 stimulates NF-kappaB activity in CaSki cervical cancer cells by associating with the IKK signalosome through IKKalpha. A significant fraction of tumor necrosis factor (TNF)-alpha-stimulated IkappaB kinase activity in CaSki cells is Notch-1-dependent. In addition, Notch-1 is found in the nucleus in association with IKKalpha at IKKalpha-stimulated promoters and is required for association of IKKalpha with these promoters under basal and TNF-alpha-stimulated conditions. Notch-1-IKKalpha complexes are found in normal human keratinocytes as well, suggesting that IKK regulation is a physiological function of Notch-1. Both Notch-1 and IKKalpha knockdown sensitize CaSki cells to cisplatin-induced apoptosis to equivalent extents. Our data indicate that Notch-1 regulates NF-kappaB in cervical cancer cells at least in part via cytoplasmic and nuclear IKK-mediated pathways.

Generation and phenotypic characterization of a galanin overexpressing mouse.

In most parts of the peripheral nervous system galanin is expressed at very low levels. To further understand the functional role of galanin, a mouse overexpressing galanin under the platelet-derived growth factor-B was generated, and high levels of galanin expression were observed in several peripheral tissues and spinal cord. Thus, a large proportion of neurons in autonomic and sensory ganglia were galanin-positive, as were most spinal motor neurons. Strong galanin-like immunoreactivity was also seen in nerve terminals in the corresponding target tissues, including skin, blood vessels, sweat and salivary glands, motor end-plates and the gray matter of the spinal cord. In transgenic superior cervical ganglia around half of all neuron profiles expressed galanin mRNA but axotomy did not cause a further increase, even if mRNA levels were increased in individual neurons. In transgenic dorsal root ganglia galanin mRNA was detected in around two thirds of all neuron profiles, including large ones, and after axotomy the percentage of galanin neuron profiles was similar in overexpressing and wild type mice. Axotomy reduced the total number of DRG neurons less in overexpressing than in wild type mice, indicating a modest rescue effect. Aging by itself increased galanin expression in the superior cervical ganglion in wild type and transgenic mice, and in the latter also in preganglionic cholinergic neurons projecting to the superior cervical ganglion. Galanin overexpressing mice showed an attenuated plasma extravasation, an increased pain response in the formalin test, and changes in muscle physiology, but did not differ from wild type mice in sudomotor function. These findings suggest that overexpressed galanin in some tissues of these mice can be released and via a receptor-mediated action influence pathophysiological processes.

Induction of astrocytic nestin expression by depolarization in rats.

Nestin is expressed in central nervous system (CNS) progenitor cells and its expression in mature cells represents transition to a less differentiated cellular state under cellular stress. This study was performed to corroborate the hypothesis that nestin synthesis is induced by depolarization and dependent on N-methyl-D-aspartate (NMDA)-receptor activation. Depolarization was induced with application of potassium chloride on the exposed rat cortex and nestin expression was evaluated by immunohistochemistry. Depolarization induced astrocytic nestin expression that was local, or evident in the entire ipsilateral cortex depending on the time of exposure. Nestin expression was NMDA-receptor-dependent since MK-801 treatment abolished the response. Understanding the mechanisms for nestin expression is important since this protein is expressed in reactive and less differentiated CNS cell states and also in neural stem cells. Insights into the control of nestin expression may also provide means for controlling differentiation of CNS cells either post-trauma/ischemia or in transplantation strategies.

Suppressed kindling epileptogenesis in mice with ectopic overexpression of galanin.

The neuropeptide galanin has been shown to suppress epileptic seizures. In cortical and hippocampal areas, galanin is normally mainly expressed in noradrenergic afferents. We have generated a mouse overexpressing galanin in neurons under the platelet-derived growth factor B promoter. RIA and HPLC analysis revealed up to 8-fold higher levels of galanin in transgenic as compared with wild-type mice. Ectopic galanin overexpression was detected especially in dentate granule cells and hippocampal and cortical pyramidal neurons. Galanin-overexpressing mice showed retardation of seizure generalization during hippocampal kindling, a model for human complex partial epilepsy. The high levels of galanin in mossy fibers found in the transgenic mice were further increased after seizures. Frequency facilitation of field excitatory postsynaptic potentials, a form of short-term synaptic plasticity assessed in hippocampal slices, was reduced in mossy fiber-CA3 cell synapses of galanin-overexpressing mice, indicating suppressed glutamate release. This effect was reversed by application of the putative galanin receptor antagonist M35. These data provide evidence that ectopically overexpressed galanin can be released and dampen the development of epilepsy by means of receptor-mediated action, at least partly by reducing glutamate release from mossy fibers.

Notch and the birth of glial cells.

Notch receptors play crucial roles in many cellular differentiation programs. In addition to a more classical role for Notch in keeping cells in an undifferentiated state, a recent paper has provided clear evidence that Notch signaling is a powerful means of turning adult CNS precursor cells into astrocytes. This work, combined with other reports from the developing CNS, retina and the PNS, demonstrates a role for Notch in gliogenesis. These findings can also be linked to novel insights into the function of proneural basic helix-loop-helix proteins in astrocytic differentiation.

A 1.8kb GFAP-promoter fragment is active in specific regions of the embryonic CNS.

The intermediate filament glial fibrillary acidic protein (GFAP) constitutes the major cytoskeletal protein in astrocytes (J. Neuroimmunol. 8 (1985) 203) and is traditionally referred to as a specific marker for astrocytes. To identify early glial precursors, we created GFAPpromoter-lacZ transgenic mice, using a 1.8kb 5' fragment of human GFAP. The expression of the transgene was first detected in the neuroepithelium at embryonic day 9.5. It was further found in the ventricular zone of the developing telencephalon, in the cerebellar primordium, trigeminal ganglia, and radial glia. Later, scattered beta-gal+ cells were seen in pons, brain stem and glia limitans. The results indicate that GFAP activity is regulated in a region-specific manner during central nervous system (CNS) development and that the gene is turned on in different cell types independently.

Microdialysis in freely moving mice: determination of acetylcholine, serotonin and noradrenaline release in galanin transgenic mice.

In the present study, we describe micro-surgical methods for simultaneous implantation of a microdialysis probe and an intraventricular injection cannula via their respective guide cannulas into the mouse brain. Basal and stimulated release of acetylcholine (ACh), serotonin (5-HT) and noradrenaline (NA) was determined in the ventral hippocampus of freely moving mice. NA and 5-HT were determined in one run by a newly developed HPLC method based on precolumn derivatization with benzylamine and fluorescence detection. The mice with a loss-of-function mutation of the galanin gene (KO) and the mice that over-expressed galanin (OE) were studied. No significant differences in basal, potassium-stimulated or scopolamine-induced extracellular ACh levels were observed in 4-month-old wild-type (WT) and KO mice. In the aged, 10-month-old animals, the basal extracellular ACh levels were significantly reduced in both WT and KO groups. Galanin (1 nmol i.c.v.) caused a significant reduction of basal extracellular NA by about 40% in both WT and galanin OE mice, however, in the latter group the effect was delayed by almost 2 h. A 10-min forced swimming stress caused a higher increase in release of NA and 5-HT in the OE group than in the corresponding WT mice. Finally, venlafaxin (10 mg/kg i.p.) increased extracellular NA to 400% of the control values in the CBA mice, but only to 250% in the C57BL mice. It is concluded that galanin may play an important role in the cholinergic mechanisms underlying cognitive disorders. Furthermore, modulation by galanin and by behavioral activation, of NA and 5-HT neurotransmission in galanin over-expressing mice indicates its possible role in the aetiology of mood disorders.

The Notch intracellular domain is ubiquitinated and negatively regulated by the mammalian Sel-10 homolog.

The Caenorhabditis elegans sel-10 protein is structurally similar to E3 ubiquitin ligases and is a negative regulator of Notch (lin-12) and presenilin signaling. In this report, we characterize the mammalian Sel-10 homolog (mSel-10) and analyze its effects on Notch signaling. We find that mSel-10 localizes to the cell nucleus, and that it physically interacts with the Notch 1 intracellular domain (IC) and reduces Notch 1 IC-mediated activation of the HES 1 promoter. Notch 1 IC is ubiquitinated by mSel-10, and ubiquitination requires the presence of the most carboxyl-terminal region of the Notch IC, including the PEST domain. In the presence of the proteasome inhibitor MG132, the amount of Notch 1 IC and its level of ubiquitination are increased. Interestingly, this accumulation of Notch 1 IC in the presence of MG132 is accompanied by decreased activation of the HES 1 promoter, suggesting that ubiquitinated Notch 1 IC is a less potent transactivator. Finally, we show that mSel-10 itself is ubiquitinated and degraded by the proteasome. In conclusion, these data reveal the importance of ubiquitination and proteasome-mediated degradation for the activity and turnover of Notch ICs, and demonstrate that mSel-10 plays a key role in this process.

The expression of intermediate filament protein nestin as related to vimentin and desmin in regenerating skeletal muscle.

Intermediate filament (IF) proteins show specific spatial and temporal expression during development of skeletal muscle. Nestin, the least known muscle IF, has an important role in neuronal regeneration. Therefore, we analyzed the expression pattern of nestin as related to that of vimentin and desmin during skeletal muscle regeneration. Nestin and vimentin appear at 6 h post-injury in myoblasts, with maximum expression around day 3-5 post-injury. Thereafter, vimentin expression ceases completely, whereas that of nestin is downregulated to remain only in the sarcoplasm next to neuromuscular and myotendinous junctions. Desmin appears at 6-12 h post-injury and becomes the predominant IF in myofibers simultaneously with the appearance of cross-striations. The expression pattern and colocalization of nestin and vimentin, known to form heteropolymers, suggests that they are essential during the early dynamic phase of the myofiber regeneration when migration, fusion, and structural modeling of myogenic cells occurs, whereas desmin is responsible for keeping myofibrils in register in mature myofibers. In conclusion, the expression of nestin is dynamically orchestrated with that of vimentin and desmin during skeletal muscle regeneration and recapitulates that seen during myogenesis, i.e. these IFs have key functional roles in the construction and restoration of skeletal myofibers.

The origin of the ankyrin repeat region in Notch intracellular domains is critical for regulation of HES promoter activity.

Notch signal transduction is mediated by proteolysis of the receptor and translocation of the intracellular domain (IC) into the nucleus, where it functions as a regulator of HES gene expression after binding to the DNA-binding protein RBP-J kappa. The mammalian Notch receptors are structurally very similar, but have distinct functions. Most notably, Notch 1 IC is a potent activator of the HES promoter, while Notch 3 IC is a much weaker activator and can repress Notch 1 IC-mediated HES activation in certain contexts. In this report we explore the molecular basis for this functional difference between Notch 1 and Notch 3 IC. We find that Notch 3 IC, like Notch 1 IC, can bind the SKIP and PCAF proteins. Furthermore, both Notch 1 and Notch 3 ICs displace the co-repressor SMRT from the DNA-binding protein RBP-J kappa on the HES promoter. The latter observation suggests that both Notch 3 IC and Notch 1 IC can access RBP-J kappa in vivo, and that the difference in activation capacity instead stems from structural differences in the two ICs when positioned on RBP-J kappa. We show that two distinct regions in the Notch IC are critical for the difference between the Notch 1 and Notch 3 IC. First, the origin of the ankyrin repeat region is important, i.e. only chimeric ICs containing a Notch 1-derived ankyrin repeat region are potent activators. Second, we identify a novel important region in the Notch IC. This region, named the RE/AC region (for repression/activation), is located immediately C-terminal to the ankyrin repeat region, and is required for Notch 1 IC's ability to activate and for Notch 3 IC's ability to repress a HES promoter. The interplay between the RE/AC region and the ankyrin repeat region provides a basis to understand the difference in HES activation between structurally similar Notch receptors.

MHC class I-Ly49 interactions shape the Ly49 repertoire on murine NK cells.

This study aims to determine how the interaction of Ly49 receptors with MHC class I molecules shapes the development of the Ly49 repertoire. We have examined the percentage of NK cells that expressed Ly49A, Ly49G2, and Ly49D in single and double Ly49A/C-transgenic mice on four different MHC backgrounds, H-2(b), H-2(d), H-2(b/d), and beta(2)-microglobulin(-/-). The results show that the total numbers of NK cells were not different among the strains. The prior expression of a Ly49 receptor capable of binding to self MHC class I altered the percentage of NK cells expressing endogenous Ly49A, Ly49G2, and Ly49D even in mice in which no MHC ligand was present for the latter receptors. The NK cells in the Ly49-transgenic mice expressed the same level of endogenous Ly49 receptors as wild-type mice of a similar MHC background. In contrast, the number of NK T cells was reduced in mice in which the Ly49 transgene could bind to a MHC class I molecule. The onset of Ly49 receptor expression on NK cells during ontogeny was not altered in the presence of transgenic Ly49 receptors. These data support a sequential model and argue against a selection model for Ly49 repertoire development on NK cells.

Mitotic reorganization of the intermediate filament protein nestin involves phosphorylation by cdc2 kinase.

The intermediate filament protein nestin is expressed during early stages of development in the central nervous system and in muscle tissues. Nestin expression is associated with morphologically dynamic cells, such as dividing and migrating cells. However, little is known about regulation of nestin during these cellular processes. We have characterized the phosphorylation-based regulation of nestin during different stages of the cell cycle in a neuronal progenitor cell line, ST15A. Confocal microscopy of nestin organization and (32)P in vivo labeling studies show that the mitotic reorganization of nestin is accompanied by elevated phosphorylation of nestin. The phosphorylation-induced alterations in nestin organization during mitosis in ST15A cells are associated with partial disassembly of nestin filaments. Comparative in vitro and in vivo phosphorylation studies identified cdc2 as the primary mitotic kinase and Thr(316) as a cdc2-specific phosphorylation site on nestin. We generated a phosphospecific nestin antibody recognizing the phosphorylated form of this site. By using this antibody we observed that nestin shows constitutive phosphorylation at Thr(316), which is increased during mitosis. This study shows that nestin is reorganized during mitosis and that cdc2-mediated phosphorylation is an important regulator of nestin organization and dynamics during mitosis.

Expression of the intermediate filament nestin in gastrointestinal stromal tumors and interstitial cells of Cajal.

It has recently been proposed that gastrointestinal stromal tumors (GISTs) originate from stem cells that differentiate toward a phenotype of interstitial cells of Cajal (ICCs). Nestin is a newly identified intermediate filament protein, and is predominantly expressed in immature cells, such as neuroectodermal stem cells and skeletal muscle progenitor cells, and tumors originating from these cells. In this study, we examined, using immunohistochemistry, the nestin expression in GISTs and ICCs to clarify the origin of GISTs. Strong immunoreactivity for nestin was observed in all 18 GISTs, and its expression was confirmed by Western blot and Northern blot analyses. In contrast, three leiomyomas and a schwannoma that developed in the gastrointestinal tract showed no apparent immunoreactivity for nestin. Among 17 mesenchymal tumors (seven leiomyosarcomas, five malignant peripheral nerve sheath tumors, and five fibrosarcomas) that occurred in sites other than the gastrointestinal tract, only two malignant peripheral nerve sheath tumors were moderately immunoreactive for nestin. Furthermore, with fluorescence double immunostaining of the normal small intestine, nestin expression was demonstrated in ICCs. These results show that nestin may be a useful marker for diagnosis of GISTs, and support the current hypothesis that GISTs are tumors of stem cells that differentiate toward an ICC phenotype.

Mice over-expressing galanin have elevated heat nociceptive threshold.

The neuropeptide galanin may have a role in modulation of nociceptive input at spinal level. Here we report that mice over-expressing galanin exhibit significant elevation of nociceptive threshold to thermal stimulation in comparison to wild-type mice as assessed by the tail flick and paw heat irradiation tests. No change in response to mechanical or cold stimulation was seen. The elevated heat nociceptive threshold in the galanin over-expressing mice was reversed by intrathecal application of the putative galanin receptor antagonist M-35, galanin-(1-12)-pro-bradykinin-(2-9). The results thus support that galanin has an inhibitory function in rodent spinal cord.

Oh no, Notch again!

The Notch receptor signaling pathway is important for morphogenesis and development of many organs and tissues in most if not all multicellular species. The classical view holds that Notch signaling keeps cells in an undifferentiated state. Recently, however, this notion has been challenged in the nervous system by two sets of observations: Notch plays an active role in the differentiation of glial cells,(1-4) and Notch influences the length and organisation of neuronal processes.(5-7) In this review, we analyse these recent data and discuss how Notch signaling may be able to perform such quite different tasks during nervous system development. BioEssays 23:3-7, 2001.