We have identified a new role for the matrix enzyme lysyl oxidase-like-2 (LOXL2) in the creation and maintenance of the pathologic microenvironment of cancer and fibrotic disease. Our analysis of biopsies from human tumors and fibrotic lung and liver tissues revealed an increase in LOXL2 in disease-associated stroma and limited expression in healthy tissues. Targeting LOXL2 with an inhibitory monoclonal antibody (AB0023) was efficacious in both primary and metastatic xenograft models of cancer, as well as in liver and lung fibrosis models. Inhibition of LOXL2 resulted in a marked reduction in activated fibroblasts, desmoplasia and endothelial cells, decreased production of growth factors and cytokines and decreased transforming growth factor-beta (TGF-beta) pathway signaling. AB0023 outperformed the small-molecule lysyl oxidase inhibitor beta-aminoproprionitrile. The efficacy and safety of LOXL2-specific AB0023 represents a new therapeutic approach with broad applicability in oncologic and fibrotic diseases.

Expression of matrix metalloproteinase 9 (MMP9) is elevated in a variety of inflammatory and oncology indications, including ulcerative colitis and colorectal cancer. MMP9 is a downstream effector and an upstream mediator of pathways involved in growth and inflammation, and has long been viewed as a promising therapeutic target. However, previous efforts to target matrix metalloproteinases (MMPs), including MMP9, have utilized broad-spectrum or semi-selective inhibitors. While some of these drugs showed signs of efficacy in patients, all MMP-targeted inhibitors have been hampered by dose-limiting toxicity or insufficient clinical benefit, likely due to their lack of specificity. Here, we show that selective inhibition of MMP9 did not induce musculoskeletal syndrome (a characteristic toxicity of pan-MMP inhibitors) in a rat model, but did reduce disease severity in a dextran sodium sulfate-induced mouse model of ulcerative colitis. We also found that MMP9 inhibition decreased tumor growth and metastases incidence in a surgical orthotopic xenograft model of colorectal carcinoma, and that inhibition of either tumor- or stroma-derived MMP9 was sufficient to reduce primary tumor growth. Collectively, these data suggest that selective MMP9 inhibition is a promising therapeutic strategy for treatment of inflammatory and oncology indications in which MMP9 is upregulated and is associated with disease pathology, such as ulcerative colitis and colorectal cancer. In addition, we report the development of a potent and highly selective allosteric MMP9 inhibitor, the humanized monoclonal antibody GS-5745, which can be used to evaluate the therapeutic potential of MMP9 inhibition in patients.

Physiological responses to chronic hypoxia include polycythemia, pulmonary arterial remodeling, and vasoconstriction. Chronic hypoxia causes pulmonary arterial hypertension leading to right ventricular hypertrophy and heart failure. During pulmonary hypertension, pulmonary arteries exhibit increased expression of smooth muscle-␣-actin and -myosin heavy chain. NFATc3 (nuclear factor of activated T cells isoform c3), which is a Ca 2؉ -dependent transcription factor, has been recently linked to smooth muscle phenotypic maintenance through the regulation of the expression of ␣-actin. The aim of this study was to determine if: (a) NFATc3 is expressed in murine pulmonary arteries, (b) hypoxia induces NFAT activation, (c) NFATc3 mediates the up-regulation of ␣-actin during chronic hypoxia, and (d) NFATc3 is involved in chronic hypoxia-induced pulmonary vascular remodeling. NFATc3 transcript and protein were found in pulmonary arteries. NFAT-luciferase reporter mice were exposed to normoxia (630 torr) or hypoxia (380 torr) for 2, 7, or 21 days. Exposure to hypoxia elicited a significant increase in luciferase activity and pulmonary arterial smooth muscle nuclear NFATc3 localization, demonstrating NFAT activation. Hypoxia induced up-regulation of ␣-actin and was prevented by the calcineurin/NFAT inhibitor, cyclosporin A (25 mg/kg/day s.c.). In addition, NFATc3 knock-out mice did not showed increased ␣-actin levels and arterial wall thickness after hypoxia. These results strongly suggest that NFATc3 plays a role in the chronic hypoxia-induced vascular changes that underlie pulmonary hypertension.As altitude increases, the barometric pressure and atmospheric oxygen partial pressure decrease. This decrease in barometric pressure is the basic cause of all hypoxia-related problems in high altitude pathophysiology. Similar levels of hypoxia are present in patients with chronic bronchitis, emphysema, cystic fibrosis, asthma, and severe restrictive lung diseases (1). Chronic hypoxia (CH) 2 causes pulmonary hypertension due to pulmonary vasoconstriction, arterial remodeling, and polycythemia, which ultimately results in right ventricular (RV) hypertrophy and often heart failure (2). Pulmonary vasoconstriction is thought to be caused by elevated vascular tone through increased pulmonary arterial smooth muscle cell (PASMC) intracellular Ca 2ϩ ([Ca 2ϩ ] i ) (3-8) and increased sensitivity of the contractile apparatus to Ca 2ϩ (9 -12).Regardless of the cause of pulmonary hypertension, the structural change that is thought to underlie the increased vascular resistance is remodeling of small pulmonary arteries. A prominent feature of this vascular remodeling is medial thickening. In proximal pulmonary vessels, medial enlargement is caused by hypertrophy and hyperplasia of the pre-existing smooth muscle cells (reviewed in Ref.

Lysyl oxidase-like 2 (LOXL2), a secreted enzyme that catalyzes the cross-linking of collagen, plays an essential role in developmental angiogenesis. We found that administration of the LOXL2-neutralizing antibody AB0023 inhibited bFGF-induced angiogenesis in Matrigel plug assays and suppressed recruitment of angiogenesis promoting bone marrow cells. Small hairpin RNA-mediated inhibition of LOXL2 expression or inhibition of LOXL2 using AB0023 reduced the migration and network-forming ability of endothelial cells, suggesting that the inhibition of angiogenesis results from a direct effect on endothelial cells. To examine the effects of AB0023 on tumour angiogenesis, AB0023 was administered to mice bearing tumours derived from SKOV-3 ovarian carcinoma or Lewis lung carcinoma (LLC) cells. AB0023 treatment significantly reduced the microvascular density in these tumours but did not inhibit tumour growth. However, treatment of mice bearing SKOV-3-derived tumours with AB0023 also promoted increased coverage of tumour vessels with pericytes and reduced tumour hypoxia, providing evidence that anti-LOXL2 therapy results in the normalization of tumour blood vessels. In agreement with these data, treatment of mice bearing LLC-derived tumours with AB0023 improved the perfusion of the tumour-associated vessels as determined by ultrasonography. Improved perfusion and normalization of tumour vessels after treatment with anti-angiogenic agents were previously found to improve the delivery of chemotherapeutic agents into tumours and to result in an enhancement of chemotherapeutic efficiency. Indeed, treatment with AB0023 significantly enhanced the anti-tumourigenic effects of taxol. Our results suggest that inhibition of LOXL2 may prove beneficial for the treatment of angiogenic tumours.

This study suggests that LOX and LOXL2 may play an important role in the pathogenesis of AMD. Targeting LOXL2 could have a broader efficacy than targeting LOX, by reducing angiogenesis and inflammation, as well as fibrosis.