Vascular Adhesion Protein-1 Blockade Research Articles

Vascular adhesion protein-1 blockade in primary sclerosing cholangitis: Open-label, multicenter, single-arm, phase II trial.

Primary sclerosing cholangitis is a progressive inflammatory liver disease characterized by biliary and liver fibrosis. Vascular adhesion protein-1 (VAP-1) is important in the inflammatory process driving liver fibrosis. We evaluated the safety and efficacy of VAP-1 blockade with a monoclonal antibody (timolumab, BTT1023) in patients with primary sclerosing cholangitis. BUTEO was a prospective, single-arm, open-label, multicenter, phase II trial, conducted in 6 centers in the United Kingdom. Patients with primary sclerosing cholangitis aged 18-75 years had an alkaline phosphatase value of >1.5 times the upper limit of normal. The dose-confirmatory stage aimed to confirm the safety of timolumab through the incidence of dose-limiting toxicity and sufficient trough levels of circulating antibody to block VAP-1 function. The primary outcome of the dose-expansion portion of the trial was patient's response to timolumab at day 99, as measured by a reduction in serum alkaline phosphatase by 25% or more from baseline to day 99. Twenty-three patients were recruited: 7 into the initial dose-confirmatory stage and a further 16 into an expansion stage. Timolumab (8mg/kg) was confirmed to be safe for the duration of administration with sufficient circulating levels. Only 2 of the 18 evaluable patients (11.1%) achieved a reduction in alkaline phosphatase levels of 25% or more, and both the proportion of circulating inflammatory cell populations and biomarkers of fibrosis remained unchanged from baseline. The BUTEO trial confirmed 8mg/kg timolumab had no short-term safety signals and resulted in sufficient circulating levels of VAP-1 blocking timolumab. However, the trial was stopped after an interim assessment due to a lack of efficacy as determined by no significant change in serum liver tests.

Inhibition of Vascular Adhesion Protein-1 for Treatment of Graft-Versus-Host Disease in Mice.

Graft-versus-host disease (GVHD) is a potentially lethal complication of hematopoietic stem cell transplantation (HSCT). GVHD comprises acute and chronic forms. To date, several approaches to treat acute GVHD or chronic GVHD have been reported. However, there is no literature precedent regarding all-in-one methods to address the 2 GVHD types. Severe inflammation in organs affected by GVHD is highly problematic, and vascular adhesion protein-1 (VAP-1) is known to be detrimentally involved in various inflammatory diseases. Based on the previous reports, we envisaged that there would be a link between GVHD and VAP-1, and we strived to create effective therapies for the 2 types of GVHD using a mouse model of GVHD. Our investigation indicated that expression of VAP-1 was elevated in organs disordered by GVHD. Hence, we subsequently attempted to block VAP-1 by using a novel inhibitor. Our results indicate that systemic injection of the inhibitor prevented aberrant influx of inflammatory cells into tissues and thereby mitigate GVHD-elicited inflammation and fibrosis. Collectively, our study suggests that the increased expression of VAP-1 is detrimentally associated with the development of GVHD and that the blockade of VAP-1 could be a promising medical modality to combat the acute and chronic variants.-Mukai, S., Ogawa, Y., Kawakami, Y., Mashima, Y., Tsubota, K. Inhibition of vascular adhesion protein-1 for treatment of graft-versus-host disease in mice.

Pharmacologic blockade of vascular adhesion protein-1 lessens neurologic dysfunction in rats subjected to subarachnoid hemorrhage

Aneurysmal subarachnoid hemorrhage (SAH) is a potentially devastating clinical problem. Despite advances in the diagnosis and treatment of SAH, outcome remains unfavorable. An increased inflammatory state, one that is characterized by enhanced leukocyte trafficking has been reported to contribute to neuronal injury in association with multiple brain insults, including hemorrhagic and ischemic stroke. This study was designed to investigate, in rats, the neuropathologic consequences of heightened leukocyte trafficking following SAH, induced via endovascular perforation of the anterior cerebral artery. Experiments focused on the initial 48h post-SAH and sought to establish whether blockade of vascular adhesion protein-1 (VAP-1), with LJP-1586, was able to provide dose-dependent neuroprotection. Treatment with LJP-1586 was initiated at 6h post-SAH. An intravital microscopy and closed cranial window system, that permitted examination of temporal patterns of rhodamine-6G-labeled leukocyte adhesion/extravasation, was used. Effects of LJP-1586 on neurologic outcomes and leukocyte trafficking at 24h and 48h post-SAH were examined. In VAP-1-inhibited vs control rats, results revealed a significant attenuation in leukocyte trafficking at both 24h and 48h after SAH, along with an improvement in neurologic outcome. In conclusion, our findings support the involvement of an amplified inflammatory state, characterized by enhanced leukocyte trafficking, during the first 48h after SAH. VAP-1 blockade yielded neuroprotection that was associated with an attenuation of leukocyte trafficking and improved neurologic outcome.

Ischemia‐reperfusion injury is attenuated in VAP‐1‐deficient mice and by VAP‐1 inhibitors

Neutrophils mediate the damage caused by ischemia-reperfusion both at the site of primary injury and in remote organs. Vascular adhesion protein-1 (VAP-1) is an ectoenzyme expressed on endothelial cells and it has been shown to regulate leukocyte extravasation. Here we show for the first time using VAP-1-deficient mice that VAP-1 plays a significant role in the intestinal damage and acute lung injury after ischemia-reperfusion. Separate inhibition of VAP-1 by small molecule enzyme inhibitors and a function-blocking monoclonal antibody in WT mice revealed that the catalytic activity of VAP-1 is responsible for its pro-inflammatory action. The use of transgenic humanized VAP-1 mice also showed that the enzyme inhibitors alleviate both the ischemia-reperfusion injury in the gut and neutrophil accumulation in the lungs. These data thus indicate that VAP-1 regulates the inflammatory response in ischemia-reperfusion injury and suggest that blockade of VAP-1 may have therapeutic value.

Vascular adhesion protein‐1 blockade suppresses choroidal neovascularization

Vascular adhesion protein-1 (VAP-1) is an endothelial cell adhesion molecule involved in leukocyte recruitment. Leukocytes and, in particular, macrophages play an important role in the development of choroidal neovascularization (CNV), an integral component of age-related macular degeneration (AMD). Previously, we showed a role for VAP-1 in ocular inflammation. Here, we investigate the expression of VAP-1 in the choroid and its role in CNV development. VAP-1 was expressed in the choroid, exclusively in the vessels, and colocalized in the vessels of the CNV lesions. VAP-1 blockade with a novel and specific inhibitor significantly decreased CNV size, fluorescent angiographic leakage, and the accumulation of macrophages in the CNV lesions. Furthermore, VAP-1 blockade significantly reduced the expression of inflammation-associated molecules such as tumor necrosis factor (TNF) -alpha, monocyte chemoattractant protein (MCP) -1, and intercellular adhesion molecule (ICAM) -1. This work provides evidence for an important role of VAP-1 in the recruitment of macrophages to CNV lesions, establishing a novel link between VAP-1 and angiogenesis. Inhibition of VAP-1 may become a new therapeutic strategy in the treatment of AMD.

Vascular Adhesion Protein‐1 as a Potential Therapeutic Target in Liver Disease

Vascular adhesion protein-1 (VAP-1) is a homodimeric, transmembrane sialoglycoprotein, and amine-oxidase enzyme constitutively expressed by hepatic endothelial cells, and as a soluble protein in serum (sVAP-1). VAP-1 mediates leukocyte adhesion and migration in an enzyme activity-dependent manner. We wished to determine whether VAP-1 blockade reduces leukocyte recruitment in inflammatory liver disease, and the mechanism by which this occurs. Our results show that VAP-1 is upregulated in the liver and serum of patients with inflammatory liver disease. Expression is maintained on hepatic sinusoidal endothelial cells (HSECs) isolated from explanted livers. Blockade of VAP-1 activity modestly decreases migration of normal lymphocytes across HSECs but has significant effects on the migration of peripheral blood lymphocytes (PBLs) and liver-derived lymphocytes across HSECs. Engagement of VAP-1 results in PI3-kinase-dependent NF-kappaB activation and increased chemokine and adhesion molecule expression. Thus complex mechanisms regulate VAP-1-mediated recruitment of leukocytes. Direct binding to endothelial VAP-1 protein, indirect enzyme-dependent activation of other endothelial adhesive pathways, and activation of leukocyte by VAP-1 ligand occupancy all contribute to adhesion. The restricted expression of VAP-1 and increased production of sVAP-1 in inflammatory liver disease confirm the validity of this molecule as a therapeutic target.

Function-blocking antibodies to human vascular adhesion protein-1: A potential anti-inflammatory therapy

Human vascular adhesion protein-1 (VAP-1) is a homodimeric 170-kDa sialoglycoprotein that is expressed on the surface of endothelial cells and functions as a semicarbazide-sensitive amine oxidase and as an adhesion molecule. Blockade of VAP-1 has been shown to reduce leukocyte adhesion and transmigration in in vivo and in vitro models, suggesting that VAP-1 is a potential target for anti-inflammatory therapy. In this study we have constructed mouse-human chimeric antibodies by genetic engineering in order to circumvent the potential problems involved in using murine antibodies in man. Our chimeric anti-VAP-1 antibodies, which were designed to lack Fc-dependent effector functions, bound specifically to cell surface-expressed recombinant human VAP-1 and recognized VAP-1 in different cell types in tonsil. Furthermore, the chimeric antibodies prevented leukocyte adhesion and transmigration in vitro and in vivo. Hence, these chimeric antibodies have the potential to be used as a new anti-inflammatory therapy.

Prolonged therapeutic window associated with pharmacologic blockade of vascular adhesion protein-1 (VAP-1)-related post-ischemic leukocyte adhesion in diabetic, ovariectomized (OVX) female rats given chronic estrogen replacement

Endothelial VAP-1 facilitates post-ischemic leukocyte adhesion and infiltration in cerebral venules (abstract-this meeting). Evidence suggests that this role of VAP-1 relates, in part, to its function as an amine oxidase and the formation of H2O2 and aldehydes. In the present study, we examined whether treatment with a specific inhibitor of the enzymatic function of VAP-1 (LJP-1207) was capable of reducing the neuropathology accompanying transient forebrain ischemia (TFI). We used a model associated with a high level of post-ischemic leukocyte adhesion and infiltration—diabetic (6–8 wks post-streptozotocin) OVX female rats given 1 week of estrogen replacement therapy (ERT). The rats were allowed to recover over 3 days following 20 min TFI. We compared rats treated, either at the onset or at 6 h reperfusion, with saline or LJP-1207 (30 mg/kg iv). Neurologic function was scored each day over the 3-day reperfusion period. The daily scores could range from 0 (no dysfunction) to 18 (severe dysfunction). In controls (n=10), the 3-day score was 13 2; while in the VAP-1-inhibited rats, the scores were 2 1 (LJP-1207 treatment @0 h reperfusion [n=7]) and 6 1 (LJP-1207 treatment at 6 h reperfusion [n=5]) (see figure 1). Both treatment groups were statistically different from control. Findings from our laboratory have shown that leukocyte infiltration, in diabetic OVX females given ERT, commences at >6 h reperfusion (Stroke 35: 1974–8, 2004), and that this transformation is largely prevented by LJP-1207 (abstract-this meeting). These findings, therefore, indicate that VAP-1-mediated post-TFI leukocyte adhesion/infiltration, in diabetic OVX females given chronic ERT, contributes substantially to neuropathology. One intriguing implication of these findings is that preventing leukocyte infiltration may provide a substantial measure of neuroprotection. This could explain the finding of LJP-1207 having at least a 6-hour therapeutic window, in this model.

Blockade of Vascular Adhesion Protein-1 Inhibits Lymphocyte Infiltration in Rat Liver Allograft Rejection

Vascular adhesion protein-1 (VAP-1) has been shown to mediate lymphocyte adhesion to endothelia at sites of inflammation, but its functional role in vivo has not been tested in any rodent model. Here we report the effects of VAP-1 blockade on rat liver allograft rejection. BN recipients of PVG liver allografts (known to develop acute rejection by day 7) were treated with 2 mg/kg anti-VAP-1 (a new anti-rat VAP-1 mAb 174-5) or isotype-matched irrelevant antibody (NS1) every other day (n = 6/group) and one group with anti-VAP-1 2 mg/kg daily (n = 7). On day 7, samples were collected for transplant aspiration cytology, histology, and immunohistochemistry. Lymphocyte infiltration to the graft was clearly affected by VAP-blockade. The total inflammation, mainly the number of active lymphoid cells, in transplant aspiration cytology was significantly decreased in animals treated with anti-VAP-1 (4.7 +/- 1.0 and 2.4 +/- 1.0 corrected increment units, respectively) compared to control (6.6 +/- 1.0) (P < 0.05). In histology, the intensity of portal inflammation was significantly decreased (P < 0.05). The amount of T cells expressing activation markers diminished. This is the first demonstration in any prolonged in vivo model that VAP-1 plays an important role in lymphocyte infiltration to sites of inflammation, and, in particular, liver allograft rejection.

Vascular adhesion protein 1 (VAP-1) functions as a molecular brake during granulocyte rolling and mediates recruitment in vivo.

Granulocyte extravasation from the blood into tissues is a prerequisite for a proper inflammatory response. It is regulated by a multistep adhesion cascade consisting of successive contacts between leukocyte surface receptors and their endothelial ligands on vessels. Vascular adhesion protein 1 (VAP-1) is an endothelial surface glycoprotein with two functions. It is an enzyme (monoamine oxidase) and an adhesion molecule for lymphocytes. Its function in binding of granulocytes or in leukocyte trafficking into sites of inflammation in vivo has remained unknown. Here we show that treatment of rabbits with anti-VAP-1 monoclonal antibodies abrogates approximately 70% of granulocyte extravasation into a site of an experimental inflammation. Using intravital microscopy, VAP-1 blockade is shown to increase the velocity of the rolling granulocytes and the frequency of their jerky skippings during the rolling. In addition, the number of firmly bound leukocytes decreased by 44% when VAP-1 was rendered nonfunctional. Our results suggest that VAP-1 functions as a molecular brake early in the adhesion cascade and consequently decreases the firm adherence; it may also directly influence the transmigration step. These data elucidate a new interplayer in the granulocyte extravasation process and provide a novel physiological function for a member of the monoamine oxidase family.