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We examined the neuroprotective efficacy associated with post-ischemic vascular adhesion protein-1 (VAP-1) blockade in rats subjected to transient (1 h) middle cerebral artery occlusion (MCAo). We compared saline-treated control rats to rats treated with a highly selective VAP-1 inhibitor, LJP-1586 [Z-3-fluoro-2-(4-methoxybenzyl) allylamine hydrochloride]. Initial intraperitoneal LJP-1586 (or saline control) treatments were delayed until 6 h or 12 h reperfusion. At 72-h reperfusion, LJP-1586-treated rats displayed 51% and 33% smaller infarct volumes, relative to their controls, in the 6- and 12-h treatment groups, respectively. However, only in the 6-h treatment group was the infarct volume reduction significant (p < 0.05). On the other hand, we observed significantly improved neurologic functions in both 6- and 12-h treatment groups, versus their matched controls (p < 0.05). Also, the effect of 6-h LJP-1586 treatment on post-ischemic leukocyte trafficking in pial venules overlying the ischemic cortex was evaluated using intravital microscopy. These experiments revealed that: 1) LJP-1586 did not affect intravascular leukocyte (largely neutrophil) adhesion, at least out to 12-h reperfusion; and 2) the onset of neutrophil extravasation, which occurred between 6–8-h reperfusion in control rats, was prevented by LJP-1586-treatment. In conclusion, in rats subjected to transient MCAo, selective VAP-1 pharmacologic blockade provided neuroprotection, with a prolonged therapeutic window of 6–12-h reperfusion.
Stroke is a leading cause of death and disability worldwide. Yet, despite a substantial research effort, covering many years, effective therapies remain limited. One of the few treatment strategies arising from this work relates to the use of thrombolytics. This approach derives from the rationale that interventions promoting early recanalization of blocked cerebral arteries can yield benefits. Unfortunately, thrombolytic strategies have a temporally limited therapeutic utility of approximately 3–4.5 h and a potential for intracranial hemorrhage. Moreover, in patients subjected to thrombolysis, slow clinical recovery is often observed despite complete recanalization (Warach and Latour 2004). In addition, spontaneous recanalization of large cerebral arteries has been estimated to occur within hours of stroke onset in ~17% of patients (Kassem-Moussa and Graffagnino 2002). Initiation of post-ischemic reperfusion, whether occurring spontaneously or induced by thrombolysis, is often accompanied by inflammatory activity (Barone and Feuerstein 1999; Huang et al. 2006). One such inflammatory cascade involves increased leukocyte trafficking at venular sites within reperfused brain regions (e.g., Becker et al. 2001; Stevens et al. 2002; Xu et al. 2006). Although this may promote expansion of brain tissue damage, it also can provide opportunities for palliative interventions.
One largely ignored potential participant in post-ischemic, leukocyte-related inflammation is the endothelial protein, vascular adhesion protein-1 (VAP-1), which is also called semicarbazide-sensitive amine oxidase (SSAO). The VAP-1/SSAO protein is thought to play an important role in promoting adhesion as well as transmigration of multiple classes of leukocytes—i.e., polymorphonuclear leukocytes (PMNLs), monocytes, and lymphocytes. At sites of injury and inflammation, VAP-1/SSAO is acutely mobilized to the luminal surface of endothelial cells and chronically up-regulated. It can exacerbate and prolong the inflammatory process by attracting additional leukocytes to the site of injury (Salmi and Jalkanen 2001). It is important to note that mobilization and concentration of VAP-1/SSAO at the vascular luminal surface will render it more accessible to circulating drugs that selectively block SSAO/VAP-1 actions, thereby focusing the beneficial actions of such blockers to sites of inflammation (Salter-Cid et al. 2005). Furthermore, in VAP-1/SSAO-knockout mice, the inflammatory response to non-microbial stimuli is repressed, but antimicrobial reactivity remains generally intact (Stolen et al. 2005). The latter has strong potential clinical relevance, as a major contributing factor to acute post-stroke mortality in patients is the onset of immunoincompetence and an increased incidence of peripheral infection (e.g., pneumonia) (Meisel et al. 2004, 2005). Thus, by preserving antimicrobial function, blocking VAP-1/SSAO may be associated with less patient risk than more generalized anti-inflammatory interventions.
LJP-1586 [Z-3- fluoro-2-94-methoxybenzyl) allylamine hydrochloride] is a non-hydrazine compound that has substantial specificity for inhibition of VAP-1/SSAO. With oral administration, LJP-1586 was reported to sustain its pharmacodynamic effect for up to 72 h despite a fairly short elimination half-life (~90 min) (O'Rourke et al. 2008). Its ability to restrict inflammatory mediator production and leukocyte transmigration in the brain was recently revealed in a mouse intracerebral hemorrhage model (Ma et al. 2011).
To date, no investigations have examined the possible neuroprotective effects of selective VAP-1 inhibition in association with transient focal cerebral ischemia. In this study, we employed a reversible middle cerebral artery occlusion (MCAo) model. Present experiments were designed to examine the therapeutic window associated with post-ischemic VAP-1 inhibition, where initiation of LJP-1586 administration was delayed by up to 12 h following the onset of reperfusion.
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A key observation in this investigation was that post-MCAo treatment with a selective pharmacologic inhibitor of VAP-1 (LJP-1586), starting at 6-h reperfusion, yielded significant neuroprotection, with some benefit being seen even when the initiation of drug administration was delayed until 12 h of reperfusion. Additional, albeit circumstantial, evidence suggested that the neuroprotective action of LJP-1586 may have been related to, at least in part, limiting VAP-1-mediated PMNL/neutrophil extravasation that is initiated at 6–8-h reperfusion and continues to 12 h and probably beyond.
There is sufficient evidence to support the postulate that VAP-1 is a principal ‘convergence site’ in pathologies associated with both acute and chronic cerebral inflammation (Xu et al. 2006; O'Rourke et al. 2007; Unzeta et al. 2007; Mao 2008; Hernandez-Guillamon et al. 2010; Ma et al. 2011). Thus, it represents a potentially important target for therapeutic intervention. Indeed, direct VAP-1-blocking strategies avoid some of the limitations arising from treatments designed to interfere with other upstream mediators of intravascular leukocyte adhesion (e.g., P- and E-selectin), which may not necessarily possess a sufficient influence on transmigration (Engelhardt et al. 1997). Also, owing to the presence of multiple downstream effectors of the damage caused by leukocyte infiltration into the brain [e.g., matrix metalloproteinases (MMPs), reactive O2 species, cytokines, granzymes, and other pro-apoptotic substances], inhibiting just one of those effectors may not be nearly as effective as intervening at an upstream process (e.g., transmigration) common to the cytotoxicity caused by multiple leukocyte subsets.
The mechanisms through which VAP-1 acts to promote the transmigration of leukocytes remain unsettled. It is commonly thought that the products of VAP-1's amine oxidase function, especially hydrogen peroxide, contribute to the diapedesis process (Salmi and Jalkanen 2001). There has been some recent progress in the identification of leukocyte subset-specific counterreceptors for endothelial VAP-1, i.e., sialic acid-binding Ig-like lectins (Aalto et al. 2011; Kivi et al. 2009). The enzymatic function of VAP-1 has also been linked to up-regulation of E- and P-selectins and ICAM-1 on vascular endothelium (Jalkanen et al. 2007; Ma et al. 2011). However, the general role of those adhesion molecules in the transmigration process remains unclear and further study is warranted.
Previous findings from our laboratory (Xu et al. 2006) demonstrated the neuroprotective efficacy of pharmacologic blockade of VAP-1 in rats subjected to TFI. In that report, evidence was obtained indicating that the benefits of VAP-1 inhibitor treatment were largely the result of preventing post-ischemic neutrophil infiltration into the brain. The observation of an onset of neutrophil transmigration at ~6-h reperfusion provided the rationale, in that study, for waiting until the 6-h post-ischemic time point before initiating VAP-1 inhibitor treatment. This had the intended effect of maintaining the elevated levels of intravascular neutrophil adhesion, while preventing the appearance of neutrophils outside of cerebral venules, over the remainder of the 10-h post-ischemic observation period. The above strategy was effective in providing significant neuroprotection, giving us a therapeutic window of at least 6 h. However, an important limitation of these findings arises from the fact that the rats studied in that report (diabetic, ovariectomized females given estrogen replacement) are particularly susceptible to enhanced post-TFI neutrophil trafficking, perhaps permitting a more robust response to VAP-1 inhibition. Therefore, in this investigation, we sought to examine rats exposed to a more ‘conventional’ ischemic insult. To that end, we studied non-diabetic male rats subjected to temporary MCAo.
Whether or not neutrophils contribute to post-MCAo neuropathology has been the subject of debate. That is, some indicate an absence (Beray-Berthat et al. 2003b; Harris et al. 2005; Martin et al. 2006; Liesz et al. 2011), while others report the presence (Beray-Berthat et al. 2003a; Justicia et al. 2003; Morrison et al. 2011) of a contributory neutrophil role. Results obtained in this study suggest that infiltrating neutrophils contribute to the brain damage elicited by transient MCAo. That conclusion derives from a couple of key experimental findings. First, we showed that rhodamine 6G-labeled leukocyte trafficking (in pial venules), over the initial 12 h of reperfusion, is markedly repressed in association with anti-PMNL antibody treatment. This suggests that the rhodamine 6G-labeled adherent cells we observed post-MCAo were, indeed, mostly neutrophils. Second, the absence of any extravasation of these cells over 4–12-h reperfusion in the presence of VAP-1 blockade, as opposed to the extravasation seen in control rats subjected to MCAo, supports a key role for VAP-1 in promoting acute post-ischemic neutrophil infiltration.
An involvement of monocytes in neuropathologies arising during post-MCAo reperfusion has been proposed (Gelderblom et al. 2009). However, the nature of the participation of these cells appears to be complex, and may include contributions from resident macrophages and microglial cells during the early phase of reperfusion, as well as a role for blood-derived monocytes at much later stages (Stevens et al. 2002; Jin et al. 2010). In contrast, there is evidence against monocyte contributions in the neuropathology accompanying transient MCAo (Justicia et al. 2006). A possible benefit arising from VAP-1 inhibitor (LJP-1586) treatment on monocyte contributions to the neuropathology accompanying hemorrhagic stroke in mice was recently reported (Ma et al. 2011). This included suppression of macrophage/microglial activation and prevention of monocyte chemoattractant protein-1 (MCP-1) up-regulation. Whether a similar benefit can be realized in ischemic stroke models awaits additional experimentation.
In studies examining lymphocyte contributions, despite evidence to the contrary (Justicia et al. 2006), current information favors a significant role. This includes results from lymphocyte-deficient murine models (Hurn et al. 2007; Liesz et al. 2011) and mice treated with a blocking antibody toward lymphocyte α4-integrin (Liesz et al. 2011). On the other hand, the findings of recent reports indicated that not all interventions designed to limit lymphocyte trafficking following MCAo are beneficial. For example, while evidence indicates a neurotoxic influence arising from increased inflammatory T-cell trafficking in the post-ischemic brain, there is some disagreement regarding whether regulatory T cells are protective or not. Regulatory B cells, on the other hand, appear to play a neuroprotective role (Liesz et al. 2011; Ren et al. 2011a, b). Similarly, there is some evidence to indicate that natural killer (NK) lymphocytes may have a beneficial counterinflammatory influence in acute cerebral ischemia (Marsh et al. 2009). Nevertheless, little or nothing is known regarding whether VAP-1 plays any role in modulating interactions among these lymphocyte subsets. This needs to be addressed in future investigations.
In conclusion, present findings showed that treatment with a highly selective blocker of VAP-1 provided significant neuroprotection in rats subjected to temporary MCAo. Some protection was still evident even when initial treatment was delayed until 12 h after the onset of reperfusion. Thus, the neuropathology accompanying focal ischemia and reperfusion includes significant contributions from VAP-1. In the brain, this protein is reported to be primarily found in microvascular cells (endothelium and smooth muscle) (Jiang et al. 2008), but absent from neurons and glia (Unzeta et al. 2007), and converts primary amines into products (e.g., H2O2; aldehydes) that are thought to facilitate leukocyte trafficking and promote cytotoxicity in pro-inflammatory conditions (Salmi and Jalkanen 2001).