High Resolution Vascular Imaging of the Rat Spine using Liposomal Blood Pool MR Agent

High resolution, vascular magnetic resonance imaging of the spine region in small animals poses several challenges. The small anatomical features, extravascular diffusion, and the low signal-to-noise ratio limit the use of conventional contrast agents. We hypothesize that a long circulating, intravascular liposomal-encapsulated MR contrast agent (liposomal-Gd) would facilitate visualization of small anatomical features of the perispinal vasculature not visible with conventional contrast agent (Gd-DTPA).

High-resolution vascular imaging of the rat spine using liposomal blood pool MR agent.

BACKGROUND AND PURPOSE: High-resolution, vascular MR imaging of the spine region in small animals poses several challenges. The small anatomic features, extravascular diffusion, and low signal-to-noise ratio limit the use of conventional contrast agents. We hypothesize that a long-circulating, intravascular liposomal-encapsulated MR contrast agent (liposomal-Gd) would facilitate visualization of small anatomic features of the perispinal vasculature not visible with conventional contrast agent (gadolinium-diethylene-triaminepentaacetic acid [Gd-DTPA]). METHODS: In this study, high-resolution MR angiography of the spine region was performed in a rat model using a liposomal-Gd, which is known to remain within the blood pool for an extended period. The imaging characteristics of this agent were compared with those of a conventional contrast agent, Gd-DTPA. RESULTS: The liposomal-Gd enabled acquisition of high quality angiograms with high signal-to-noise ratio. Several important vascular features, such as radicular arteries, posterior spinal vein, and epidural venous plexus were visualized in the angiograms obtained with the liposomal agent. The MR angiograms obtained with conventional Gd-DTPA did not demonstrate these vessels clearly because of marked extravascular soft-tissue enhancement that obscured the vasculature. CONCLUSIONS: This study demonstrates the potential benefit of long-circulating liposomal-Gd as a MR contrast agent for high-resolution vascular imaging applications.

Role of NonO-histone interaction in TNFalpha-suppressed prolyl-4-hydroxylase alpha1.

Inflammation is a key process in cardiovascular diseases. The extracellular matrix (ECM) of the vasculature is a major target of inflammatory cytokines, and TNFalpha regulates ECM metabolism by affecting collagen production. In this study, we have examined the pathways mediating TNFalpha-induced suppression of prolyl-4 hydroxylase alpha1 (P4Halpha1), the rate-limiting isoform of P4H responsible for procollagen hydroxylation, maturation, and organization. Using human aortic smooth muscle cells, we found that TNFalpha activated the MKK4-JNK1 pathway, which induced histone (H) 4 lysine 12 acetylation within the TNFalpha response element in the P4Halpha1 promoter. The acetylated-H4 then recruited a transcription factor, NonO, which, in turn, recruited HDACs and induced H3 lysine 9 deacetylation, thereby inhibiting transcription of the P4Halpha1 promoter. Furthermore, we found that TNFalpha oxidized DJ-1, which may be essential for the NonO-P4Halpha1 interaction because treatment with gene specific siRNA to knockout DJ-1 eliminated the TNFalpha-induced NonO-P4Halpha1 interaction and its suppression. Our findings may be relevant to aortic aneurysm and dissection and the stability of the fibrous cap of atherosclerotic plaque in which collagen metabolism is important in arterial remodeling. Defining this cytokine-mediated regulatory pathway may provide novel molecular targets for therapeutic intervention in preventing plaque rupture and acute coronary occlusion.

MMP-9/TIMP-1 imbalance induced in human dendritic cells by Porphyromonas gingivalis.

Matrix metalloproteinase-9 (MMP-9) cleaves collagen, allowing leukocytes to traffic toward the vasculature and the lymphatics. When MMP-9 is unregulated by tissue inhibitor of metalloproteinase-1 (TIMP-1), this can lead to tissue destruction. Dendritic cells (DCs) infiltrate the oral mucosa increasingly in chronic periodontitis, characterized by infection with several pathogens including Porphyromonas gingivalis. In this study, human monocyte-derived DCs were pulsed with different doses of lipopolysaccharide of P. gingivalis 381 and of Escherichia coli type strain 25922, as well as whole live isogenic fimbriae-deficient mutant strains of P. gingivalis 381. Levels of induction of MMP-9 and TIMP-1, as well as interleukin-10 (IL-10), which reportedly inhibits MMP-9 induction, were measured by several approaches. Our results reveal that lipopolysaccharide of P. gingivalis, compared with lipopolysaccharide from E. coli type strain 25922, is a relatively potent inducer of MMP-9, but a weak inducer of TIMP-1, contributing to a high MMP-9/TIMP-1 ratio.Whole live P. gingivalis strain 381, major fimbriae mutant DPG-3 and double mutant MFB were potent inducers of MMP-9, but minor fimbriae mutant MFI was not. MMP-9 induction was inversely proportional to IL-10 induction. These results suggest that lipopolysaccharide and the minor and the major fimbriae of P. gingivalis may play distinct roles in induction by DCs of MMP-9, a potent mediator of local tissue destruction and leukocyte trafficking.

Targeted Multistage Delivery of Nanoparticles to the Bone Marrow

Bone marrow is a target organ site involved in multiple diseases including myeloproliferative disorders and hematologic malignancies and metastases from breast and prostate. Most of these diseases are characterized with poor quality of life, and the treatment options are only palliative due to lack of delivery mechanisms for systemically injected drugs which results in dose limitation to protect the healthy hematopoietic cells. Therefore, there is a critical need to develop effective therapeutic strategies that allow for selective delivery of therapeutic payload to the bone marrow. Nanotechnology-based drug delivery systems provide the opportunity to deliver drugs to the target tissue while decreasing exposure to normal tissues. E-selectin is constitutively expressed on the bone marrow vasculature, but almost absent in normal vessels, and therefore, E-selectin targeted drug delivery presents an ideal strategy for the delivery of therapeutic nanoparticles to the bone marrow. The objective of this study was to develop a novel bone marrow targeted multistage vector (MSV) via E-selectin for delivery of therapeutics and imaging agents. To achieve this goal, Firstly, an E-selectin thioaptamer (ESTA) ligand was identified through a two-step screening from a combinatorial thioaptamer library. Next, ESTA-conjugated MSV (ESTA-MSV) were developed and evaluated for their stability and binding to E-selectin expressing endothelial cells. Different types of nanoparticles including liposomes, quantum dots, and iron oxide nanoparticles were loaded into the porous structure of ESTA-MSV. In vivo targeting experiments demonstrated 8-fold higher accumulation of ESTA-MSV in the mouse bone marrow as compared to non-targeted MSV Furthermore, intravenous injection of liposomes loaded ESTA-MSV resulted in a significantly higher accumulation of liposome in the bone marrow space as compared to injection of non-targeted MSV or liposomes alone. Overall this study provides first evidence that E-selectin targeted multistage vector preferentially targets to bone marrow vasculature and delivers larger amounts of nanoparticles. This delivery strategy holds potential for the selective delivery of large amounts of therapeutic payload to the vascular niches in the bone marrow for the treatment of bone marrow associated diseases.

MODELLING β2AR REGULATION

The β2 adrenergic receptor (β2AR) regulates smooth muscle relaxation in the vasculature and airways. Long- and Short-acting β-agonists (LABAs/SABAs) are widely used in treatment of chronic obstructive pulmonary disorder (COPD) and asthma. Despite their widespread clinical use we do not understand well the dominant β2AR regulatory pathways that are stimulated during therapy and bring about tachyphylaxis, which is the loss of drug effects. Thus, an understanding of how the β2AR responds to various β-agonists is crucial to their rational use. Towards that end we have developed deterministic models that explore the mechanism of drug- induced β2AR regulation. These mathematical models can be classified into three classes; (i) Six quantitative models of SABA-induced G protein coupled receptor kinase (GRK)-mediated β2AR regulation; (ii) Three phenomenological models of salmeterol (a LABA)-induced GRK-mediated β2AR regulation; and (iii) One semi-quantitative, unified model of SABA-induced GRK-, protein kinase A (PKA)-, and phosphodiesterase (PDE)-mediated regulation of β2AR signalling. The various models were constrained with all or some of the following experimental data; (i) GRK-mediated β2AR phosphorylation in response to various LABAs/SABAs; (ii) dephosphorylation of the GRK site on the β2AR; (iii) β2AR internalisation; (iv) β2AR recycling; (v) β2AR desensitisation; (vi) β2AR resensitisation; (vii) PKA-mediated β2AR phosphorylation in response to a SABA; and (viii) LABA/SABA induced cAMP profile ± PDE inhibitors. The models of GRK-mediated β2AR regulation show that plasma membrane dephosphorylation and recycling of the phosphorylated β2AR are required to reconcile with the measured dephosphorylation kinetics. We further used a consensus model to predict the consequences of rapid pulsatile agonist stimulation and found that although resensitisation was rapid, the β2AR system retained the memory of prior stimuli and desensitised much more rapidly and strongly in response to subsequent stimuli. This could explain tachyphylaxis of SABAs over repeated use in rescue therapy of asthma patients. The LABA models show that the long action of salmeterol can be explained due to decreased stability of the arrestin/β2AR/salmeterol complex. This could explain long action of β-agonists used in maintenance therapy of asthma patients. Our consensus model of PKA/PDE/GRK-mediated β2AR regulation is being used to identify the dominant β2AR desensitisation pathways under different therapeutic regimens in human airway cells. In summary our models represent a significant advance towards understanding agonist-specific β2AR regulation that will aid in a more rational use of the β2AR agonists in the treatment of asthma.

Fibrillins: Sequence, phylogeny and carboxy terminal domain proteolytic processing

Fibrillin-1 and -2 are large secreted glycoproteins that are known to be components of extracellular matrix microfibrils located in the vasculature, basement membrane and various connective tissues. These microfibrils are often associated with a superstructure known as the elastic fiber. During the development of elastic tissues, fibrillin microfibrils precede the appearance of elastin and may provide a scaffolding for the deposition and crosslinking of elastin. Using RT/PCR, we cloned and sequenced 3.85Kbp of the FBN2 gene. Five differences were found between our contig sequence and that published by Zhang et al. (1995). Like many extracellular matrix proteins, the fibrillins are modular proteins. We compared analogous domains of the two fibrillins and also members of the latent TGF-$\beta$ binding protein (LTBP) family to determine their phylogenetic relationship. We found that the two families are homologous. LTBP-2 is the most similar to the fibrillin family while FBN-1 is the most similar to the LTBP family. The fibrillin-1 carboxy terminal domain is proteolytically processed. Two eukaryotic protein expression systems, baculoviral and CHO-K1, were developed to examine the proteolytic processing of the carboxy terminal domain of the fibrillin-1 protein. Both expression systems successfully processed the domain and both processed a mutant less efficiently. In the CHO-K1 cells, processing occurred intracellularly. ^

THE RELATIONSHIP BETWEEN THE RHEOENCEPHALOGRAM AND CEREBRAL BLOOD FLOW

The rheoencephalogram (REG) is the change in the electrical impedance of the head that occurs with each heart beat. Without knowledge of the relationship between cerebral blood flow (Q) and the REG, the utility of the REG in the study of the cerebral vasculature is greatly limited. The hypothesis is that the relationship between the REG and Q when venous outflow is nonpulsatile is^ (DIAGRAM, TABLE OR GRAPHIC OMITTED...PLEASE SEE DAI)^ where K is a proportionality constant and Q is the mean Q.^ Pulsatile CBF was measured in the goat via a chronically implanted electromagnetic flowmeter. Electrodes were implanted in the ipsilateral cerebral hemisphere, and the REG was measured with a two electrode impedance plethysmograph. Measurements were made with the animal's head elevated so that venous flow pulsations were not transmitted from the heart to the cerebral veins. Measurements were made under conditions of varied cerebrovascular resistance induced by altering blood CO(,2) levels and under conditions of high and low cerebrospinal fluid pressures. There was a high correlation (r = .922-.983) between the REG calculated from the hypothesized relationship and the measured REG under all conditions.^ Other investigators have proposed that the REG results from linear changes in blood resistivity proportional to blood velocity. There was little to no correlation between the measured REG and the flow velocity ( r = .022-.306). A linear combination of the flow velocity and the hypothesized relationship between the REG and Q did not predict the measured REG significantly better than the hypothesized relationship alone in 37 out of 50 experiments.^ Jacquy proposed an index (F) of cerebral blood flow calculated from amplitudes and latencies of the REG. The F index was highly correlated (r = .929) with measured cerebral blood flow under control and hypercapnic conditions, but was not as highly correlated under conditions of hypocapnia (r = .723) and arterial hypotension (r = .681).^ The results demonstrate that the REG is not determined by mean cerebral blood flow, but by the pulsatile flow only. Thus, the utility of the REG in the determination of mean cerebral blood flow is limited. ^

Vasculogenesis: A process of vessel formation and its role during Ewing's sarcoma development

Vasculogenesis is the process by which Endothelial Precursor Cells (EPCs) form a vasculature. This process has been traditionally regarded as an embryological process of vessel formation. However, as early as in the 60's the concept of postnatal vasculogenesis was introduced, with a strong resurface of this idea in recent years. Similarly, previous work on a mouse skin tumor model provided us with the grounds to consider the role of vasculogenesis during tumor formation. ^ We examined the contribution of donor bone marrow (BM)-derived cells to neovascularization in recipient nude mice with Ewing's sarcoma. Ewing's sarcoma is a primitive neuroectodermal tumor that most often affects children and young adults between 5 and 30 years of age. Despite multiple attempts to improve the efficacy of chemotherapy for the disease, the 2-year metastases-free survival rate for patients with Ewing's sarcoma has not improved over the past 15 years. New therapeutic approaches are therefore needed to reduce the mortality rate. ^ The contribution of BM endothelial precursor cells in the development of Ewing's sarcoma was examined using different strategies to track the donor-derived cells. Using a BMT model that takes advantage of MHC differences between donor and recipient mice, we have found that donor BM cells were involved in the formation of Ewing's sarcoma vasculature. ^ Cells responsible for this vasculogenesis activity may be located within the stem cell population of the murine BM. These stem cells would not only generate the hematopoietic lineage but they would also generate ECs. Bone marrow SP (Side Population) cells pertain to a subpopulation that can be identified using flow cytometric analysis of Hoechst 33342-stained BM. This population of cells has HSC activity. We have tested the ability of BM SP cells to contribute to vasculogenesis in Ewing's sarcoma using our MHC mismatched transplant model. Mice transplanted with SP cells developed tumor neovessels that were derived from the donor SP cells. Thus, SP cells not only replenished the hematopoietic system of the lethally irradiated mice, but also differentiated into a non-hematopoietic cell lineage and contributed to the formation of the tumor vasculature. ^ In summary, we have demonstrated that BM-derived cells are involved in the generation of the new vasculature during the growth of Ewing's sarcoma. The finding that vasculogenesis plays a role in Ewing's sarcoma development opens the possibility of using genetically modified BM-derived cells for the treatment of Ewing's sarcomas. ^

Targeted cancer diagnosis with radiolabeled endostatin

Nuclear imaging is used for non-invasive detection, staging and therapeutic monitoring of tumors through the use of radiolabeled probes. Generally, these probes are used for applications in which they provide passive, non-specific information about the target. Therefore, there is a significant need for actively-targeted radioactive probes to provide functional information about the site of interest. This study examined endostatin, an endogenous inhibitor of tumor angiogenesis, which has affinity for tumor vasculature. The major objective of this study was to develop radiolabeled analogues of endostatin through novel chemical and radiochemical syntheses, and to determine their usefulness for tumor imaging using in vitro and in vivo models of vascular, mammary and prostate tumor cells. I hypothesize that this binding will allow for a non-invasive approach to detection of tumor angiogenesis, and such detection can be used for therapeutic monitoring to determine the efficacy of anti-angiogenic therapy. ^ The data showed that endostatin could be successfully conjugated to the bifunctional chelator ethylenedicysteine (EC), and radiolabeled with technetium-99m and gallium-68, providing a unique opportunity to use a single precursor for both nuclear imaging modalities: 99mTc for single photon emission computed tomography and 68Ga for positron emission tomography, respectively. Both radiolabeled analogues showed increased binding as a function of time in human umbilical vein endothelial cells and mammary and prostate tumor cells. Binding could be blocked in a dose-dependent manner by unlabeled endostatin implying the presence of endostatin receptors on both vascular and tumor cells. Animal biodistribution studies demonstrated that both analogues were stable in vivo, showed typical reticuloendothelial and renal excretion and produced favorable absorbed organ doses for application in humans. The imaging data provide evidence that the compounds quantitate tumor volumes with clinically-useful tumor-to-nontumor ratios, and can be used for treatment follow-up to depict changes occurring at the vascular and cellular levels. ^ Two novel endostatin analogues were developed and demonstrated interaction with vascular and tumor cells. Both can be incorporated into existing nuclear imaging platforms allowing for potential wide-spread clinical benefit as well as serving as a diagnostic tool for elucidation of the mechanism of action of endostatin. ^

Ligand-directed targeting of malignant gliomas

The dramatic poor survival of patients diagnosed with glioblastoma multiforme (GBM) is a reflection of the struggles that accompany traditional treatments. Thus, the development of molecular-based targeted therapies represents new windows for intervention. In this study, we hypothesized that we could select peptide-ligands that selectively target GBM based on the idea that the glioma microenvironment may induce or modify the expression of cell surface receptors that could be accessed by circulating peptides. To select the peptides we employed two distinct in vivo screenings. First, a random phage-displayed peptide library was injected into mice bearing intracranial tumors. Phage that bound to tumor were recovered and sequenced. We found that the tumor-derived phage CLSYKGRC, CNKVSTKC and CQSSREKC were recovered with the highest frequencies and used for subsequent targeting experiments. Second, the phage peptide library was injected into mice without tumors and phage were recovered from brain and sequenced. A phage-displayed peptide (CRTIGPSVC) with homology to transferrin (Tf) was selected and injected into brain tumor-bearing mice. Results showed that after 6 hours of circulation, the CLSYKGRC, CNKVSTKC and CQSSREKC-phage selectively targeted GBM vasculature. In contrast, Tf-like phage accumulated outside the tumor blood vessels in the cytoplasm of cells located within GBM, suggesting it was internalized in vivo. However, after short periods of circulation this phage was restricted to the tumor vasculature. Importantly, none of the selected phage targeted normal brain cells in animals bearing intracranial tumors. An affinity column coupled to the CNKVSTKC zpeptide was used to identify receptors from GBM. Using mass-spectrometry Vimentin, a marker of glial malignancy, was identified as a potential receptor. Other studies showed that the Tf-like phage bound selectively to Apo-Tf (iron free), with no binding to Holo-Tf (iron loaded) or to Tf receptor (TfR). However, the binding of Tf-like phage to glioma cells that express TfR increased in the presence of Apo-Tf. Thus, the Tf-like phage could indirectly target TfR using the endogenous Tf pathway. We propose that the novel peptides identified in this study could be conjugated to therapeutic or imaging agents for use GBM. ^

Bone marrow-derived stem/progenitor cells play an important role in the growth of Ewing's sarcoma tumors

Both angiogenesis and vasculogenesis contribute to the formation and expansion of tumor neovasculature. We demonstrated that bone marrow (BM)-derived cells migrated to TC71 Ewing's tumors and differentiated into endothelial cells lining perfused, functional tumor neovessels. In addition, a substantial fraction of recruited, BM-derived cells resided in the vessel vicinity but did not demonstrate endothelial differentiation. Rather, these perivascular cells expressed desmin and PDGFR-β, implying pericyte-like/vascular smooth muscle cell differentiation. No defined, consensus set of markers exists for endothelial progenitor cells (EPCs) and the specific subsets of BM cells that participate in vessel formation are poorly understood. We used a functional in vivo assay to investigate the roles performed by specific human- and murine-derived stem/progenitor subpopulations within Ewing's sarcoma tumors. CD34 +45+, CD34+38-, VEGFR2 + and Sca1+Gr1+ cells were demonstrated to establish residence within the expanding tumor vascular network and differentiate into endothelial cells and pericytes. By constrast, CD34-45 + and Sca1-Gr1+ cells predominantly localized to sites outside the Ewing's tumor vasculature, and differentiated into macrophages. Cytokines, such as VEGF, influence the recruitment of BM cells and their incorporation into the tumor vasculature. VEGF165-inhibited TC/siVEGF7-1 Ewing's tumors showed delayed in vivo tumor growth, decreased vessel density, and reduced infiltration of BM progenitor cells. We tested whether another chemoattractant, Stromal Cell-Derived Factor-1 (SDF-1), could augment the growth of these VEGF165-inhibited TC/siVEGF 7-1 tumors by enhancing the recruitment of BM cells and stimulating neovasculature expansion. SDF-1 promoted progenitor cell chemotaxis and retainment of BM-derived pericyte precursors in close association with functional, perfused tumor blood vessels. Treatment of TC/siVEGF7-1 tumors with adenovirus-SDF-1α resulted in augmented tumor size, enhanced pericyte coverage of tumor neovessels, remodeling of vascular endothelium into larger, functional structures, and upregulation of PDGF-BB, with no effect on VEGF165. Taken together, these findings suggest that the recruitment of BM stem/progenitor cells plays an important role in the growth of Ewing's tumors. ^

In vivo longitudinal studies of spinal cord injury and vascular endothelial growth factor treatment

Approximately 12,000 new cases of spinal cord injury (SCI) are added each year to the estimated 259,000 Americans living with SCI. The majority of these patients return to society, their lives forever changed by permanent loss of sensory and motor function. While there are no FDA approved drugs for the treatment of SCI or a universally accepted standard therapy, the current though controversial treatment includes the delivery of high dosages of the corticosteroid methyliprednisolone sodium succinate, surgical interventions to stabilize the spinal column, and physical rehabilitation. It is therefore critically important to fully understand the pathology of injury and determine novel courses and rationally-based therapies for SCI. ^ Vascular endothelial growth factor (VEGF) is an attractive target for treating central nervous system (CNS) injury and disease because it has been shown to influence angiogenesis and neuroprotection. Preliminary studies have indicated that increased vasculature may be associated with functional recovery; therefore exogenous delivery of a pro-angiogenic growth factor such as VEGF may improve neurobehavioral outcome. In addition, VEGF may provide protection from secondary injury and result in increased survival and axonal sprouting. ^ In these studies, SCI rats received acute intraspinal injections of VEGF, the antibody to VEGF, or vehicle control. The effect of these various agents was investigated using longitudinalmulti-modal magnetic resonance imaging (MRI), neuro- and sensory behavioral assays, and end point immunohistochemistry. We found that rats that received VEGF after SCI had increased tissue sparing and improved white matter integrity at the earlier time points as shown by advanced magnetic resonance imaging (MRI) techniques. However, these favorable effects of VEGF were not maintained, suggesting that additional treatments with VEGF at multiple time points may be more beneficial, Histological examinations revealed that VEGF treatment may result in increased oligodendrogenesis and therefore may eventually lead to remyelination and improved functional outcome. ^ On the neurobehavioral studies, treatments with VEGF and Anti-VEGF did not significantly affect performance on tests of open-field locomotion, grid walk, inclined plane, or rearing. However, VEGF treatment resulted in significantly increased incidence of chronic neuropathic pain. This phenomenon could possibly be attributed to the fact that VEGF treatment may promote axonal sprouting and also results in tissue sparing, thereby providing a substrate for the growth of new axons. New connections made by these sprouting axons may involve components of pathways involved in the transmission of pain and therefore result in increased pain in those animals. ^

YKL -40, a vascular -associated serum marker for glioblastoma, correlates with chronic activation of AKT

YKL-40 is a secreted glycoprotein that has been reported to be expressed in pathologic conditions of extracellular matrix degradation and angiogenesis, such as rheumatoid arthritis, severe osteoarthritis, primary colorectal cancer, metastatic breast cancer, and recurrent ovarian cancer (Dehn, Hogdall et al. 2003). ^ We have identified YKL-40 as a serum marker for glioblastoma multiforme (GBM) using microarray analysis from samples of GBM. We compared the gene expression profile of 19 gliomas to pooled normal brain tissue using the Incyte 10,000 gene expression array. The most differentially expressed gene in this analysis was YKL-40; it was detected in GBM samples with a range of 3 to 62-fold elevation over normal brain. Western blot analysis of glioma samples for YKL-40 protein levels revealed substantial elevation in approximately 65% of GBMs, and undetectable levels in lower-grade gliomas and normal brain tissue. ELISA analysis on serum samples of glioma patients showed that YKL-40 levels were substantially elevated in many of the GBM patients. Statistical analysis indicated that in patients with glioma, serum YKL-40 levels correlate with tumor grade and potentially tumor burden in GBM. ^ Furthermore, we found that YKL-40 expression by in-situ hybridization on a brain tumor tissue array was limited to GBM's and gliosarcomas (GSA), and that YKL-40 expression was specific to the GBM component of GSA. Additional in-situ hybridization analysis, found it to be regionally associated with tumor vasculature as well as activated AKT expression in both human and mouse GBM's. Correlation of elevated YKL-40 with phospho-AKT was confirmed by Western blot analysis on a series of glioblastoma tumors, and inhibition of PI3 Kinase signaling by addition of LY294002 also decreased secretion of YKL-40 over a 7-day period in U87 glioblastoma cell tine. Lastly, YKL-40 expression was induced in response to serum starvation and altered by interaction with specific extracellular matrix (ECM) modules. In summary, we have identified the first accurate serum marker for high-grade gliomas. Furthermore, our findings indicate that YKL-40 is a highly expressed vascular-related glycoprotein in human GBM tissue and that it is affected by the AKT signaling pathway and interaction with components of brain ECM proteins. ^