PURPOSE To determine whether systemic treatment with AMG 386, a selective angiopoietin 1/2-neutralizing peptibody, inhibits neovascular processes in animal models of ocular disease. METHODS AMG 386 was tested in a laser-induced choroidal neovascularization (CNV) model in monkeys using fluorescein angiography. The biodistribution of (125)I-AMG 386 was determined in cynomolgus monkeys by whole-body autoradiography and radioanalysis of ocular tissues. A murine retinopathy of prematurity (ROP) model was used to examine the effect of AMG 386 on established and newly formed retinal vessels, either as a single agent or when combined with VEGF inhibition.AMG 386 pharmacokinetics were evaluated in each model. RESULTS In the CNV model, AMG 386 significantly decreased fluorescent angiographic leakage and reduced fibroplasia, indicating an impaired healing response consistent with angiogenesis blockade. Radiolabeled AMG 386 was widely distributed across ocular tissues, with highest concentrations in the choroid, cornea, retinal pigmented epithelium, iris/ciliary body, and sclera. In the ROP model, AMG 386 prevented pathologic retinal angiogenesis when administered from P8 to P16 but transiently impeded regression of these abnormal vessels when administered from P17 to P23. Combining AMG 386 with VEGF inhibition led to cooperative prevention of retinal angiogenesis in this model. No AMG 386-related ocular toxicities occurred, and no treatment-related clinical observations were made in any of the studies. CONCLUSIONS In this study, AMG 386 inhibited angiogenesis in animal models of CNV and ROP, supporting investigation of AMG 386 for the treatment of ocular neovascular diseases in the clinical setting.

Slowed lymphocyte rolling along high endothelial venules (HEV) is initiated by the L selectin: PNAd interaction, as an essential first step in a cascade of reactions leading to extravasation of lymphocytes across HEV and into the lymphoid tissues. CHST4, a golgi resident carbohydrate sulfotransferase, has been described as an HEV restricted sulfotransferase in part responsible for the sulfation step required for L selectin: PNAd interaction. The MECA79 antibody which recognizes sulfated PNAd provides a useful tool for monitoring sulfotransferase activity. The presence of both CHST4 and the MECA79 sulfated epitope have been described in HEV as well as lymphoid aggregates and tertiary lymphoid tissues at the sites of chronic inflammation. For the studies described, we investigated the expression of CHST4 with real time PCR, In Situ Hybridization (ISH), Immunohistochemistry (IHC) as well as MECA79 IHC. Expression patterns were examined in an extensive collection of normal human tissue panels as well as diseased tissue collected from several inflammatory conditions. Resulting data, in addition to confirming an HEV and lymphoid aggregate restricted expression profile also revealed expression of the gene in epithelial and ductal epithelial cells in a small set of diverse organ tissues. The functional consequences of such expression and the co-localized MECA79 epitope expression are currently unknown.

Rats with collagen-induced arthritis (CIA) were necropsied on 14 occasions from 4 days after induction to 27 days after disease onset to evaluate the kinetics of local (joint protein extracts) and systemic (serum) levels of inflammatory and pro-erosive factors. Systemic increases in alpha1 acid glycoprotein and KC/GRO together with systemic and local enrichment of interleukin (IL)-1beta, IL-6, CCL2, transforming growth factor (TGF)-beta and elevated IL-1alpha and IL-18 in joint extracts preceded the onset of clinical disease. Systemic upregulation of IL-1beta, IL-6, TGF-beta CCL2, RANKL and prostaglandin E(2) (PGE(2)) during acute and/or chronic CIA coincided with systemic leukocytosis and a CD4+ T-cell increase in blood and spleen. In contrast, progression of joint erosions during clinical CIA was associated with intra-articular increases in IL-1alpha/beta, IL-6, IL-18, CCL2, KC/GRO and RANKL, and a dramatic decline in osteoprotegerin (OPG). These data indicate that systemic and local events in inflammatory arthritis can be discrete processes, driven by multiple cellular and humoral mediators with distinct temporospatial profiles.

The interleukin (IL)-1 family members IL-1alpha, -1beta, and -18 are potent inflammatory cytokines whose activities are dependent on heterodimeric receptors of the IL-1R superfamily, and which are regulated by soluble antagonists. Recently, several new IL-1 family members have been identified. To determine the role of one of these family members in the skin, transgenic mice expressing IL1F6 in basal keratinocytes were generated. IL1F6 transgenic mice exhibit skin abnormalities that are dependent on IL-1Rrp2 and IL-1RAcP, which are two members of the IL-1R family. The skin phenotype is characterized by acanthosis, hyperkeratosis, the presence of a mixed inflammatory cell infiltrate, and increased cytokine and chemokine expression. Strikingly, the combination of the IL-1F6 transgene with an IL1F5 deficiency results in exacerbation of the skin phenotype, demonstrating that IL-1F5 has antagonistic activity in vivo. Skin from IL1F6 transgenic, IL1F5(-/-) pups contains intracorneal and intraepithelial pustules, nucleated corneocytes, and dilated superficial dermal blood vessels. Additionally, expression of IL1RL2, -1F5, and -1F6 is increased in human psoriatic skin. In summary, dysregulated expression of novel agonistic and antagonistic IL-1 family member ligands can promote cutaneous inflammation, revealing potential novel targets for the treatment of inflammatory skin disorders.

Mutations affecting the activity of the Wnt co-receptors LRP5 and LRP6 that cause alterations in skeletal biology confirmed the involvement of Wnt signaling in bone formation. We evaluated the potential role of Dkk1, an inhibitor of LRP5/6 activity, in bone formation by examining the normal expression pattern of Dkk1 in normal young mice and by assessing the consequences of osteoblast overexpression of Dkk1 in transgenic mice. Endogenous Dkk1 expression was detected primarily in osteoblasts and osteocytes. Transgenic over-expression of Dkk1 using two different rat collagen 1A1 promoters resulted in distinct bone phenotypes. More widespread Dkk1 expression (driven by the Col1A1 3.6 kb promoter) yielded osteopenia with forelimb deformities and hairlessness, while expression restricted to osteoblasts (driven by the Col1A1 2.3 kb promoter) induced severe osteopenia without limb defects or alopecia. The decrease in bone mass in vivo resulted from a significant 49% reduction in osteoblast numbers and was reflected in a 45% reduction in serum osteocalcin concentration; an in vitro study revealed that Dkk1 caused a dose-dependent suppression of osteoblast matrix mineralization. These data indicate that Dkk1 may directly influence bone formation and suggest that osteopenia develops in mice over-expressing Dkk1 at least in part due to diminished bone formation resulting from reduced osteoblast numbers.

Angiopoietin-2 (Ang2) exhibits broad expression in the remodeling vasculature of human tumors but very limited expression in normal tissues, making it an attractive candidate target for antiangiogenic cancer therapy. To investigate the functional consequences of blocking Ang2 activity, we generated antibodies and peptide-Fc fusion proteins that potently and selectively neutralize the interaction between Ang2 and its receptor, Tie2. Systemic treatment of tumor-bearing mice with these Ang2-blocking agents resulted in tumor stasis, followed by elimination of all measurable tumor in a subset of animals. These effects were accompanied by reduced endothelial cell proliferation, consistent with an antiangiogenic therapeutic mechanism. Anti-Ang2 therapy also prevented VEGF-stimulated neovascularization in a rat corneal model of angiogenesis. These results imply that specific Ang2 inhibition may represent an effective antiangiogenic strategy for treating patients with solid tumors.

Initially identified in high-grade gliomas, mutations in the PTEN tumor-suppressor are also found in many sporadic cancers and a few related autosomal dominant hamartoma syndromes. PTEN is a 3′-specific phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P 3 ) phosphatase and functions as a negative regulator of PI3K signaling. We generated a tissue-specific deletion of the mouse homolog Pten to address its role in brain function. Mice homozygous for this deletion ( Pten loxP/loxP ; Gfap-cre ), developed seizures and ataxia by 9 wk and died by 29 wk. Histological analysis showed brain enlargement in Pten loxP/loxP ; Gfap-cre mice as a consequence of primary granule-cell dysplasia in the cerebellum and dentate gyrus. Pten mutant cells showed a cell-autonomous increase in soma size and elevated phosphorylation of Akt. These data represent the first evidence for the role of Pten and Akt in cell size regulation in mammals and provide an animal model for a human phakomatosis condition, Lhermitte-Duclos disease (LDD).