Transient postoperative vascular endothelial growth factor (VEGF)-neutralisation improves graft survival in corneas with partly regressed inflammatory neovascularisation.

BACKGROUND: High-risk keratoplasties are usually performed after an uninflamed and quiescent interval in corneas with partly regressed blood and lymphatic vessels. We analysed whether the inhibition of post-keratoplasty revascularisation in mice with partly regressed corneal vessels ("intermediate-risk") improves graft survival. METHODS: Three interrupted stromal sutures (11-0) in corneas of Balb/c mice (6-8 weeks old) were placed for 6 weeks. Six months after suture removal, penetrating keratoplasty was performed with C57BL/6 donors. The treatment group received a vascular endothelial growth factor-A specific cytokine trap (VEGF Trap) intraperitoneally at days 0, 4, 7 and 14 after keratoplasty (25 mg/kg per mouse; controls received equal amounts of Fc protein). Pathological haemangiogenesis and lymphangiogenesis prior to as well as 3 days or 8 weeks after keratoplasty and graft survival were analysed. RESULTS: Three days after keratoplasty corneal revascularisation was sufficiently reduced by VEGF Trap (haem-vascularised areas 42.7% reduction; lymph-vascularised areas 54.7% reduction). Survival proportions 8 weeks after keratoplasty were 36% in the treatment group compared with 9% in the control group (n = 11; p<0.05). At that time no differences in haemangiogenesis or lymphangiogenesis were observed between the two groups. CONCLUSION: Early transient postoperative induction of haemangiogenesis and lymphangiogenesis and reformation of regressed corneal blood and lymphatic vessels are important for transplant rejections after "intermediate-risk" corneal transplantation.

Effects of inhibition of vascular endothelial growth factor at time of selection on follicular angiogenesis, expansion, development and atresia in the marmoset.

This study determined the effects of inhibiting vascular endothelial growth factor (VEGF) at follicle selection. Marmosets were given an injection of VEGF antagonist, the VEGF Trap on Day 5 of the follicular phase and ovaries were evaluated on Day 10 or 15. Ovaries from controls were assessed on Day 5 (time of selection), Day 10 (peri-ovulatory) and Day 15 (luteal phase). At Day 10, ovaries of four of the five controls contained dominant follicles, while one had ovulated. VEGF Trap-treated ovaries also contained large follicles on Day 10, but VEGF inhibition had suppressed endothelial cell proliferation, leading to reductions in the thecal vascularization and plasma estradiol relative to controls. By Day 15, ovaries of controls contained active corpora lutea whereas ovaries of four of the five treated animals still contained large antral follicles similar in size to pre-ovulatory follicles, and one had small, avascular corpora lutea. However, these follicles had a restricted vasculature, increased incidence of activated caspase-3 staining and morphological features indicating they would become degenerative non-functional cysts. These results show that after follicle selection, VEGF is essential for angiogenesis and the generation of healthy ovulatory follicles and corpora lutea, but fluid accumulation can still occur in selected follicles in the absence of VEGF.

Delta-like ligand 4 (Dll4) is induced by VEGF as a negative regulator of angiogenic sprouting.

Genetic deletion studies have shown that haploinsufficiency of Delta-like ligand (Dll) 4, a transmembrane ligand for the Notch family of receptors, results in major vascular defects and embryonic lethality. To better define the role of Dll4 during vascular growth and differentiation, we selected the postnatal retina as a model because its vasculature develops shortly after birth in a highly stereotypic manner, during which time it is accessible to experimental manipulation. We report that Dll4 expression is dynamically regulated by VEGF in the retinal vasculature, where it is most prominently expressed at the leading front of actively growing vessels. Deletion of a single Dll4 allele or pharmacologic inhibition of Dll4/Notch signaling by intraocular administration of either soluble Dll4-Fc or a blocking antibody against Dll4 all produced the same set of characteristic abnormalities in the developing retinal vasculature, most notably enhanced angiogenic sprouting and increased endothelial cell proliferation, resulting in the formation of a denser and more highly interconnected superficial capillary plexus. In a model of ischemic retinopathy, Dll4 blockade also enhanced angiogenic sprouting and regrowth of lost retinal vessels while suppressing ectopic pathological neovascularization. Our data demonstrate that Dll4 is induced by VEGF as a negative feedback regulator and acts to prevent overexuberant angiogenic sprouting, promoting the timely formation of a well differentiated vascular network.

VEGF trap as a novel antiangiogenic treatment currently in clinical trials for cancer and eye diseases, and VelociGene- based discovery of the next generation of angiogenesis targets.

The concept that tumors can be controlled by directly targeting their vascular supply has finally come of age, because clinical trials using a humanized monoclonal antibody that blocks VEGF have demonstrated exciting efficacy in cancer patients, as well as in vascular eye diseases that can lead to blindness. However, data suggest that these current regimens may not provide complete VEGF inhibition and, thus, that the maximum therapeutic potential of VEGF blockade has not yet been achieved. We describe the status of a very potent and high-affinity VEGF blocker, termed the VEGF Trap, that may provide the opportunity to maximize the potential of VEGF blockade in cancer as well as in vascular eye diseases. We also describe use of the VEGF Trap as a research tool, when coupled to high-throughput mouse genetics approaches such as VelociGene that can be exploited in strategies to discover and validate the next generation of angiogenesis targets.

Ciliary neurotrophic factor improves diabetic parameters and hepatic steatosis and increases basal metabolic rate in db/db mice.

Obesity plays a central role in the development of insulin resistance and type 2 diabetes. We therefore examined the effects of a modified form of ciliary neurotrophic factor [Axokine, which is hereafter referred to as ciliary neurotrophic factor (CNTF)Ax15], which uses a leptin-like mechanism to reduce body weight, in the db/db murine model of type 2 diabetes. In previous studies, weight loss produced by CNTF treatment could largely be attributed to its effects on food intake. In contrast, CNTFAx15 treatment of db/db mice caused significantly greater weight loss and marked improvements in diabetic parameters (e.g., levels of glucose, insulin, triglyceride, cholesterol, and nonesterified free fatty acids) than could be accounted for by reduced caloric intake alone. These beneficial effects, above and beyond those seen in animals controlled for either food restriction or body weight, correlated with the ability of CNTFAx15 to increase metabolic rate and energy expenditure and reduce hepatic steatosis while enhancing hepatic responsiveness to insulin. The hepatic effects were linked to rapid alterations in hepatic gene expression, most notably reduced expression of stearoyl-CoA desaturase 1, a rate-limiting enzyme in the synthesis of complex lipids that is also markedly suppressed by leptin in ob/ob mice. These observations further link the mechanisms of CNTF and leptin action, and they suggest important, beneficial effects for CNTF in diabetes that may be distinct from its ability to decrease food intake; instead, these effects may be more related to its influence on energy expenditure and hepatic gene expression.

Activation of the hypothalamic arcuate nucleus predicts the anorectic actions of ciliary neurotrophic factor and leptin in intact and gold thioglucose-lesioned mice.

Similar to leptin, ciliary neurotrophic factor (CNTF) suppresses appetite and selectively reduces body fat in leptin-deficient ob/ob mice. To assess the relative importance of specific regions of the hypothalamus in mediating these effects, we administered a CNTF analogue (CNTFAx15) or leptin to mice made obese by administration of gold thioglucose (GTG), which destroys a well-defined portion of the medial basal hypothalamus. CNTFAx15 treatment reduced appetite and body weight in obese GTG-lesioned C57BL/6 mice, whereas leptin failed to effect similar changes regardless of whether treatment was initiated before or after the lesioned mice had become obese. Because leptin does not reduce food intake or body weight in most forms of obesity (a condition termed 'leptin resistance'), we also investigated the actions of leptin in GTG-lesioned leptin-deficient (ob/ob) mice. By contrast to C57BL/6 mice, leptin treatment reduced food intake and body weight in GTG-lesioned ob/ob mice, although the effect was attenuated. To further compare the neural substrates mediating the anorectic actions of leptin and CNTF, we determined the patterns of neurone activation induced by these proteins in the hypothalamus of intact and GTG-lesioned mice by staining for phosphorylated signal transducer and activator of transcription 3 (pSTAT3). CNTFAx15 stimulated robust pSTAT3 signalling in neurones of the medial arcuate nucleus in both intact and lesioned C57BL/6 and ob/ob mice. Leptin administration stimulated pSTAT3 signalling in only a few neurones of the medial arcuate nucleus in intact or lesioned C57BL/6 mice, but elicited a robust response in intact or lesioned ob/ob mice. By contrast to CNTFAx15, leptin treatment also resulted in prominent activation of STAT3 in several areas of the hypothalamus outside the medial arcuate nucleus. This leptin-induced pSTAT3 signal was at least as prominent in intact and GTG-lesioned C57BL/6 mice as it was in ob/ob mice, and thus was not correlated with appetite suppression or weight loss. These results indicate that the medial arcuate nucleus is a key mediator of appetite suppression and weight loss produced by CNTF and leptin, whereas GTG-vulnerable regions play a role only in leptin-induced weight loss. Other regions of hypothalamus in which pSTAT3 signal is induced by leptin may regulate energy metabolism through mechanisms other than appetite reduction.

VEGF-initiated blood-retinal barrier breakdown in early diabetes.

PURPOSE: The objectives of this study were to (1) determine whether endogenous vascular endothelial growth factor (VEGF) triggers diabetic blood-retinal barrier breakdown, and (2) identify the site as well as phenotype of the hyperpermeable diabetic retinal vessels. METHODS: Retinal VEGF mRNA levels were quantified in 1-week diabetic rats using the RNase protection assay. VEGF bioactivity was blocked via the systemic administration of a highly specific VEGF-neutralizing soluble Flt/F(c) construct (VEGF TrapA(40)). An inactive IL6 receptor/F(c) construct (IL6R Trap) was used as an isotype control. Blood-retinal barrier breakdown was quantified using the Evans blue technique and was spatially localized with fluorescent microspheres. RESULTS: Retinal VEGF mRNA levels in 1-week diabetic animals were 3.2-fold higher than in nondiabetic controls (P < 0.0001). Similarly, retinal vascular permeability in 8-day diabetic animals was 1.8-fold higher than in normal nondiabetic controls (P < 0.05). Diabetes-induced blood-retinal barrier breakdown was dose-dependently inhibited with VEGF TrapA(40), with 25 mg/kg producing complete inhibition of the diabetes-induced increases (P < 0.05). Blood-retinal barrier breakdown in diabetic animals treated with solvent alone or IL6R Trap did not differ significantly from untreated diabetic animals (P > 0.05). Spatially, early blood-retinal barrier breakdown was localized to the retinal venules and capillaries of the superficial retinal vasculature. CONCLUSIONS: Early blood-retinal barrier breakdown in experimental diabetes is VEGF dependent and is restricted, in part, to the venules and capillaries of the superficial inner retinal vasculature. VEGF inhibition should prove a useful therapeutic approach in the treatment of early diabetic blood-retinal barrier breakdown.

Infusion of brain-derived neurotrophic factor into the lateral ventricle of the adult rat leads to new neurons in the parenchyma of the striatum, septum, thalamus, and hypothalamus.

The findings that brain-derived neurotrophic factor (BDNF) promotes in vitro the survival and/or differentiation of postnatal subventricular zone (SVZ) progenitor cells and increases in vivo the number of the newly generated neurons in the adult rostral migratory stream and olfactory bulb prompted us to investigate whether the infusion of BDNF influences the proliferation and/or differentiation of cells in other regions of the adult forebrain. We examined the distribution and phenotype of newly generated cells in the adult rat forebrain 16 d after intraventricular administration of BDNF in conjunction with the cell proliferation marker bromodeoxyuridine (BrdU) for 12 d. BDNF infusion resulted in numerous BrdU(+) cells, not only in the SVZ lining the infused lateral ventricle, but moreover, in specific parenchymal structures lining the lateral and third ventricles, including the striatum and septum, as well as the thalamus and hypothalamus, in which neurogenesis had never been demonstrated previously during adulthood. In each region, newly generated cells expressed the neuronal marker microtubule-associated protein-2, or neuron-specific tubulin, identified by the antibody TuJ1. The percentage of the newly generated cells expressing TuJ1 ranged from 27 to 42%, suggesting that the adult forebrain has a more profound capacity to produce neurons than recognized previously. The extent of cell proliferation after BDNF infusion was correlated with the level of expression of full-length TrkB, the high-affinity receptor for BDNF, despite the fact that the BrdU(+) cells were not themselves TrkB(+). Collectively, our results demonstrate that the adult brain parenchyma may recruit and/or generate new neurons, which could replace those lost as a result of injury or disease.

Luteal angiogenesis: prevention and intervention by treatment with vascular endothelial growth factor trap(A40).

The possibility of stimulating or inhibiting paracrine factors regulating angiogenesis may lead to new approaches for the treatment of pathological conditions of the female reproductive tract. We examined the effects of a clinical candidate, a soluble truncated form of the Flt-1 receptor, vascular endothelial growth factor trap(A40) (VEGF trap), in a primate model to determine its ability to prevent the onset of luteal angiogenesis or intervene with the on-going process. Marmosets were treated from the day of ovulation until luteal day 3 (prevention regimen) or on luteal day 3 for 1 day (intervention regimen). Effects of VEGF inhibition were studied by obtaining a proliferation index using bromodeoxyuridine incorporation, quantifying endothelial cell area using CD31, and assessing luteal function by plasma progesterone. After both treatments, intense luteal endothelial proliferation was suppressed, a concomitant decrease in endothelial cell area confirmed the inhibition of vascular development, and a marked fall in plasma progesterone levels showed that luteal function was compromised. In situ hybridization was used to localize and quantify compensatory effects on the expression of angiogenic genes. VEGF messenger ribonucleic acid (mRNA) expression in luteal cells was increased, whereas expression of its receptor, Flt, was decreased. Inhibition of VEGF resulted in localized increased expression of angiopoietin-2 mRNA and its receptor, Tie-2. The results show that the VEGF trap can prevent luteal angiogenesis and inhibit the established process with resultant suppression of luteal function. Luteal Flt mRNA expression is dependent upon VEGF, and VEGF inhibition results in abortive increases in expression of VEGF, angiopoietin-2, and Tie-2.

Angiogenesis during follicular development in the primate and its inhibition by treatment with truncated Flt-1-Fc (vascular endothelial growth factor Trap(A40)).

The aims of this study were to 1) quantify changes in angiogenesis during follicular growth in a primate model; 2) investigate the molecular regulation using in situ hybridization of vascular endothelial growth factor (VEGF), its receptor, Flt-1, the angiopoietins (Ang-1 and Ang-2), and their receptor, Tie-2; 3) elucidate the role of VEGF in follicular angiogenesis by blocking its action by treatment with a soluble truncated form of the Flt-1 receptor, (VEGF Trap(A40)). Changes in angiogenesis were quantified using bromodeoxyuridine to obtain a proliferation index, and CD31 immunocytochemistry to visualize endothelial cell area. Percentage of proliferating endothelial cells was calculated by double labeling for bromodeoxyuridine and CD31. Vascularization was first observed in follicles containing four granulosa cell layers. A significant increase in proliferation in the thecal layer was observed from the early to late secondary stage, and dual staining showed that 25% of proliferating cells were of endothelial cell origin. VEGF messenger RNA (mRNA) was expressed in granulosa cells with an increase of grain density from late secondary to tertiary follicles. Ang-1 was weakly expressed in the theca of tertiary follicles. Ang-2 mRNA was not detected in any follicles. The mRNA for the Flt-1 and Tie-2 receptors was localized in endothelial cells of the theca. Unexpectedly, Tie-2 mRNA was also found in granulosa cells of early follicular stages and its translation was confirmed by immunocytochemistry. VEGF trap treatment for 3 days resulted in an 87% decrease of proliferation in the theca of secondary and tertiary follicles, a reduction in endothelial cell area and a marked decline in Flt-1 mRNA expression. Granulosa cell proliferation also decreased. These results show that onset and establishment of the follicle vasculature takes place early during follicular development. The ability of VEGF trap treatment to severely restrict follicular angiogenesis establishes that VEGF is the major regulator of this process in the primate ovary.

Ciliary neurotrophic factor activates leptin-like pathways and reduces body fat, without cachexia or rebound weight gain, even in leptin-resistant obesity.

Ciliary Neurotrophic Factor (CNTF) was first characterized as a trophic factor for motor neurons in the ciliary ganglion and spinal cord, leading to its evaluation in humans suffering from motor neuron disease. In these trials, CNTF caused unexpected and substantial weight loss, raising concerns that it might produce cachectic-like effects. Countering this possibility was the suggestion that CNTF was working via a leptin-like mechanism to cause weight loss, based on the findings that CNTF acts via receptors that are not only related to leptin receptors, but also similarly distributed within hypothalamic nuclei involved in feeding. However, although CNTF mimics the ability of leptin to cause fat loss in mice that are obese because of genetic deficiency of leptin (ob/ob mice), CNTF is also effective in diet-induced obesity models that are more representative of human obesity, and which are resistant to leptin. This discordance again raised the possibility that CNTF might be acting via nonleptin pathways, perhaps more analogous to those activated by cachectic cytokines. Arguing strongly against this possibility, we now show that CNTF can activate hypothalamic leptin-like pathways in diet-induced obesity models unresponsive to leptin, that CNTF improves prediabetic parameters in these models, and that CNTF acts very differently than the prototypical cachectic cytokine, IL-1. Further analyses of hypothalamic signaling reveals that CNTF can suppress food intake without triggering hunger signals or associated stress responses that are otherwise associated with food deprivation; thus, unlike forced dieting, cessation of CNTF treatment does not result in binge overeating and immediate rebound weight gain.

Vascular growth factors in cerebral ischemia.

During the past decade, there has been a surge of interest in growth factors (GFs) that act selectively on vascular endothelium and perivascular cells. Studies employing mutant mice or the administration of recombinant proteins have suggested that these factors not only mediate the proliferation of endothelial cells, but also regulate vascular differentiation, regression, and permeability. During and after cerebral ischemia, brain vasculature becomes leaky and unstable, and the normally impermeable blood-brain barrier breaks down. Several days after the ischemic insult, endothelial cells begin to proliferate, and angiogenesis occurs. Expression studies have shown that key vascular GFs are regulated, during these processes, in a complex and coordinated manner. The distinct pattern of regulation exhibited by each vascular GF suggests a unique role for each factor during the initial vascular destabilization and subsequent angiogenesis that occurs after cerebral ischemia. Data from studies in other biological systems support these suggested roles. Thus, manipulation of vascular GFs may prove to be an effective means of stabilizing or enriching brain vasculature after ischemia, and ameliorating the detrimental effects of blood-brain barrier breakdown and vessel regression after stroke.

Molecular events implicated in brain tumor angiogenesis and invasion.

We have conducted studies designed to help elucidate the molecular mechanisms involved in brain tumor invasion and angiogenesis, which are critical in the growth of malignant tumors of the central nervous system. A variety of molecular factors have been implicated in these processes. Here we focus on three that are of particular importance in the progression of brain tumors. Angiopoietins are involved in the regulation of vascular development. Hypoxia inducible factor-1 is a transcription factor that up-regulates genes, including genes encoding vascular endothelial growth factor under hypoxic conditions. Focal adhesion kinase is associated with infiltration of tumor cells and angiogenesis.

Vascular-specific growth factors and blood vessel formation.

A recent explosion in newly discovered vascular growth factors has coincided with exploitation of powerful new genetic approaches for studying vascular development. An emerging rule is that all of these factors must be used in perfect harmony to form functional vessels. These new findings also demand re-evaluation of therapeutic efforts aimed at regulating blood vessel growth in ischaemia, cancer and other pathological settings.

Vascular apoptosis and involution in gliomas precede neovascularization: a novel concept for glioma growth and angiogenesis.

Vascular changes in gliomas were analyzed by implanting fluorescent-labeled glioma 261 cells in the brains of 28 mice. Seven animals were killed each week for 4 weeks. We investigated the expression of angiopoietin-2 (Ang-2) by in situ hybridization and compared it with the distribution of apoptotic cells identified by DNA strand breaks (using the terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end labeling [TUNEL] method) and transmission electron microscopy (TEM). As early as 1 week after implantation, tumor cells accumulated around vessels, which expressed Ang-2 and were TUNEL negative. TEM showed tumor cells adjacent to the vascular cells "lifting up" the normal astrocytic feet processes away from the endothelial cells and disrupting normal pericytic cuffing. After 2 weeks the number of perivascular glioma cells had increased. No increase in the number of blood vessels was detected at this time. Vascular cells remained positive for Ang-2 and rare ones were TUNEL positive. TEM showed closely packed proliferating perivascular tumor cells. After 3 weeks, there was vascular involution with scant zones of tumor necrosis. Ang-2 was still detected in vascular cells, but now numerous vascular cells were TUNEL positive. In addition, TEM showed apoptotic vascular cells. After 4 weeks, there were extensive areas of tumor necrosis with pseudopalisading and adjacent angiogenesis. Ang-2 was detected in vascular cells at the edge of the tumors in the invaded brain and in vessels surrounded by tumor cells. At both 3 and 4 weeks, most of the TUNEL-positive tumor cells lacked morphological features characteristic of apoptosis and displayed features consistent with necrotic cell death as determined by TEM. Only rare tumor cells appeared truly apoptotic. In contrast, the TUNEL-positive endothelial cells and pericytes were round and shrunken, with condensed nuclear chromatin by TEM, suggesting that vascular cells were undergoing an apoptotic cell death. These results suggest that vascular cell apoptosis and involution preceded tumor necrosis and that angiogenesis is a later event in tumor progression in experimental gliomas. Moreover, Ang-2 is detected prior to the onset of apoptosis in vascular cells and could be linked to vascular involution.

Ciliary neurotrophic factor and stress stimuli activate the Jak-STAT pathway in retinal neurons and glia.

Ciliary neurotrophic factor (CNTF) is pleiotrophic for central, peripheral, and sensory neurons. In the mature retina, CNTF treatment enhances survival of retinal ganglion and photoreceptor cells exposed to otherwise lethal perturbation. To understand its mechanism of action in vivo, the adult rat retina was used as a model to investigate CNTF-mediated activation of Janus kinase/signal transducer and activator of transcription (Jak-STAT) and ras-mitogen activated protein kinase (ras-MAPK). Intravitreal injection of Axokine, an analog of CNTF, phosphorylates STAT3 and MAPK and produces delayed upregulation of total STAT3 and STAT1 protein in rat retina. Activated STAT3 is predominantly localized in nuclei of retinal Müller (glial) cells, ganglion cells, and astrocytes, but not in photoreceptors. Although CNTF alpha-receptor (CNTFRalpha) mRNA and protein are localized predominantly if not exclusively in retinal neurons, coincident CNTF-mediated STAT3 signaling was observed in both glia and neurons. CNTF-induced activation of Jak-STAT signaling prompted us to investigate STAT3 phosphorylation after a variety of stress-mediated, conditioning stimuli. We show that STAT3 is activated in the retina after exposure to subtoxic bright light, mechanical trauma, and systemic administration of the alpha(2)-adrenergic agonist xylazine, all of which have been shown previously to condition photoreceptors to resist light-induced degeneration. These results demonstrate that CNTF directly stimulates Jak-STAT and ras-MAPK cascades in vivo and strongly suggest that STAT3 signaling is an underlying component of neural responsiveness to stress stimuli. The observation that CNTF activates STAT3 in ganglion cells, but not in photoreceptors, suggests that Jak-STAT signaling influences neuronal survival via both direct and indirect modes of action.

The ciliary neurotrophic factor and its receptor, CNTFR alpha.

Ciliary neurotrophic factor (CNTF) is expressed in glial cells within the central and peripheral nervous systems. CNTF stimulates gene expression, cell survival or differentiation in a variety of neuronal cell types such as sensory, sympathetic, ciliary and motor neurons. In addition, effects of CNTF on oligodendrocytes as well as denervated and intact skeletal muscle have been documented. CNTF itself lacks a classical signal peptide sequence of a secreted protein, but is thought to convey its cytoprotective effects after release from adult glial cells by some mechanism induced by injury. Interestingly, mice that are homozygous for an inactivated CNTF gene develop normally and initially thrive. Only later in adulthood do they exhibit a mild loss of motor neurons with resulting muscle weakness, leading to the suggestion that CNTF is not essential for neural development, but instead acts in response to injury or other stresses. The CNTF receptor complex is most closely related to, and shares subunits with the receptor complexes for interleukin-6 and leukemia inhibitory factor. The specificity conferring alpha subunit of the CNTF complex (CNTFR alpha), is extremely well conserved across species, and has a distribution localized predominantly to the nervous system and skeletal muscle. CNTFR alpha lacks a conventional transmembrane domain and is thought to be anchored to the cell membrane by a glycosyl-phosphatidylinositol linkage. Mice lacking CNTFR alpha die perinatally, perhaps indicating the existence of a second developmentally important CNTF-like ligand. Signal transduction by CNTF requires that it bind first to CNTFR alpha, permitting the recruitment of gp130 and LIFR beta, forming a tripartite receptor complex. CNTF-induced heterodimerization of the beta receptor subunits leads to tyrosine phosphorylation (through constitutively associated JAKs), and the activated receptor provides docking sites for SH2-containing signaling molecules, such as STAT proteins. Activated STATs dimerize and translocate to the nucleus to bind specific DNA sequences, resulting in enhanced transcription of responsive genes. The neuroprotective effects of CNTF have been demonstrated in a number of in vitro cell models as well as in vivo in mutant mouse strains which exhibit motor neuron degeneration. Intracerebral administration of CNTF and CNTF analogs has also been shown to protect striatal output neurons in rodent and primate models of Huntington's disease. Treatment of humans and animals with CNTF is also known to induce weight loss characterized by a preferential loss of body fat. When administered systemically, CNTF activates downstream signaling molecules such as STAT-3 in areas of the hypothalamus which regulate food intake. In addition to its neuronal actions, CNTF and analogs have been shown to act on non-neuronal cells such as glia, hepatocytes, skeletal muscle, embryonic stem cells and bone marrow stromal cells.

Peptide immunoreactivity in aged rat cortex and hippocampus as a function of memory and BDNF infusion.

Brain-derived neurotrophic factor (BDNF) modulates neuropeptide levels in hippocampus and cortex of young adult rats. Neuropeptide levels are altered in some age-related disorders, such as Alzheimer's and Parkinson's Disease. BDNF may be able to rectify peptide abnormalities but, because plasticity decreases with age, BDNF may not alter peptide levels as readily in aged animals. To determine if BDNF would regulate peptide levels in aged rats, young, aged memory-impaired, and unimpaired rats were infused with BDNF or vehicle into hippocampus and cortex. Cell profile counts, cell profile areas, fiber counts, and/or fiber terminal densities were measured for sections immunostained for neuropeptide Y (NPY), somatostatin (SOM), cholecystokinin-8 (CCK), and dynorphin A(1-8) (DYN). Results showed that BDNF upregulated cortical NPY-immunoreactivity (ir) and SOM-ir, upregulated hippocampal NPY-ir, and downregulated hippocampal DYN-ir in both aged and young rats. In addition, BDNF significantly and selectively normalized the areas of atrophied deep cortical CCK-ir cell profiles in aged-impaired rats. Finally, decreased CCK-ir fiber density was found in the hippocampal formation of aged memory-impaired rats.

In situ expression of angiopoietins in astrocytomas identifies angiopoietin-2 as an early marker of tumor angiogenesis.

Angiopoietin-1 (Ang-1) and its naturally occurring antagonist angiopoietin-2 (Ang-2) are novel ligands that regulate tyrosine phosphorylation of the Tie2/Tek receptor on endothelial cells. Proper regulation of Tie2/Tek is absolutely required for normal vascular development, seemingly by regulating vascular remodeling and endothelial cell interactions with supporting pericytes/smooth muscle cells. We investigated the expression of Ang-1 and Ang-2 in human astrocytomas by in situ hybridization and compared them to the distribution of pericytes/smooth muscle cells by immunohistochemistry for alpha-smooth muscle actin (SMA). Ang-1 mRNA was localized in tumor cells and Ang-2 mRNA was detected in endothelial cells of hyperplastic and nonhyperplastic tumor vessels. Ang-2 was also expressed in partially sclerotic vessels and in vascular channels surrounded by tumor cells in brain adjacent to the tumor. Neither Ang-1 nor Ang-2 was detected in normal brain. Dynamic changes in SMA expression during glioma tumorigenesis appear to progress from fragmentation in early vascular hyperplasia to subsequent reassociation and enhanced expression in later stages of vascular proliferation in hyperplastic complexes in high-grade gliomas. All these vessels displaying dynamic changes in SMA immunoreactivity also expressed Ang-2 mRNA. Moreover, SMA immunoreactive intratumoral vascular channels lacking morphological evidence of hyperplasia also showed upregulation of Ang-2. These results suggest that angiopoietins are involved in the early stage of vascular activation and in advanced angiogenesis, and they identify Ang-2 as an early marker of glioma-induced neovascularization. The association between Ang-2 expression and alterations in SMA immunoreactivity suggests a role for Ang-2 in tumor-associated activation of pericytes/smooth muscle cells.