The use of HAART has made a significant impact on the lives of HIV-1 infected individuals. However, poor penetration of the CNS by these therapies has led to the need for adjunct treatments to address the growing number of individuals affected by neurologic consequences of HIV infection. We previously reported the presence of increased circulating levels of sCD40L in HIV-1 infected individuals with cognitive impairment as compared to infected individuals without cognitive impairment (14
). Furthermore, in the same report, we demonstrated that CD40L potentiates the ability of HIV-1 Tat to activate microglia and monocytes (14
). Therefore, attenuation of sCD40L levels may prove beneficial in the control of this aspect of the disease by helping to ameliorate the harmful effects of the virus within the CNS. Here we demonstrate that VPA is able to reduce plasma levels of sCD40L in both HIV infected individuals and in an in vivo
mouse model. Considering the current widespread clinical use of VPA for conditions such as bipolar disorder and epilepsy, these results have important implications for the potential of VPA or similar drugs as adjunctive therapies for treatment of HAND.
HAND is widely believed to be an inflammatory disease; so it is fitting that elevated levels of CD40L have been implicated in numerous other inflammatory diseases, including cardiovascular disease (4
) and ischemia/reperfusion injury (8
). In these instances the interaction of platelet-derived CD40L with endothelial cells is believed to induce inflammation. CD40, the receptor for CD40L, is constitutively expressed on endothelial cells and, upon ligation by CD40L, these cells become more conducive to monocytes that are being recruited in response to the inflammatory signals (7
). We speculate that the elevated levels of sCD40L observed in HIV-1 infected, cognitively impaired individuals are contributing to this inflammatory disorder in a similar fashion. Therapeutic targeting of CD40L is an attractive approach for the treatment of inflammatory disorders (39
); however, CD40L is an important co-stimulatory molecule expressed on T cells and interfering with it could alter immune competence. Current strategies for targeting CD40L include cyclosporine A, an inhibitor of calcineurin that results in decreased CD40L expression in T cells, and anti-CD40L monoclonal antibodies; both of which have the potential to confer immunosuppression and thus would not be well-suited as adjunct therapies in HAND. The ability of VPA to decrease abnormally high plasma levels of sCD40L, without directly interfering with CD40 signaling, would therefore be advantageous in that desirable humoral immune responses would not be negatively affected. This further highlights the potential of VPA as a candidate adjunct therapy for HAND.
Some reports indicate that patients receiving VPA treatment may experience VPA-induced thrombocytopenia (28
); however, any thrombocytopenia observed appears to depend on variables such as gender, age, dosage, or low baseline platelet counts (28
). Other published reports indicate that the dosage of VPA required to significantly increase risk of developing thrombocytopenia are above 40 mg/kg of body weight per day (41
), considered a high dose. In our animal studies, mice received concentrations well above 40 mg/kg of body weight per day, however they had no evidence of thrombocytopenia. This discrepancy could be explained by the fact that mice possess higher platelet counts than humans, resulting in higher baseline platelet levels (42
). The apparent risk of thrombocytopenia seems to vary depending on risk factors, and while this should be considered clinically, the widespread clinical use of this drug, demonstrating safety and tolerability, still makes it an attractive adjunct therapeutic candidate.
In order to determine the mechanism by which VPA reduces sCD40L release from platelets, we focused on GSK3β, which is involved in numerous signaling pathways and known to be inhibited by VPA. GSK3β has been implicated in HAND previously, as it was shown to be activated by PAF in neurons (20
). Consistent with this notion, we now show here that activation of GSK3β is also induced by cPAF in platelets. Several reports have indicated that potent platelet activators such as thrombin, ADP, and collagen lead to the inhibition, rather than activation, of GSK3β (18
). This phosphorylation-dependent inhibition of GSK3β involves activation of the upstream kinases phosphoinositide 3-kinase (PI3K) and protein kinase B (PKB/Akt) (19
). Consistent with these findings, we observed an increase in levels of phosphorylated GSK3β (indicative of inhibition) in response to thrombin treatment in platelets (data not shown), however, treatment with cPAF results in a significant decrease in phospho-GSK3β, suggesting that PAF activates a different signaling pathway to induce activation, rather than inhibition, of this kinase. Interestingly, we also see a significant increase in sCD40L release in response to thrombin, which would seemingly contradict our hypothesis, that active GSK3β is playing a role in sCD40L release. However, this paradox may be explained by the fact that there is a great deal of complexity in the regulatory pathways of this kinase. For example, reduced phosphorylation of GSK3β at serine 9 is usually associated with a 30–50% increase in kinase activity, which is apparently sufficient to induce biological effects (such as neuronal apoptosis) (44
). Numerous signaling mechanisms target only a specific pool of the GSK3β present in the cells because of the sub-cellular distribution of both GSK3β and each regulatory molecule. Although GSK3β is traditionally considered a cytosolic protein, it is also present in other cellular compartments such as nuclei, mitochondria, and membrane lipid rafts (44
). An activity status of GSK3β is different in each compartment, such that the kinase moiety present in the nuclei, mitochondria and lipid raft is highly active (dephosphorylated at serine 9), and in contrast, cytosolic GSK3β is largely inactive. Thus, complete inhibition or activation of GSK3β in response to regulatory signaling events is highly unlikely. Indeed, we do not see complete inhibition of GSK3β in response to thrombin (data not shown). As previously mentioned, HIV-1 infection is associated with an increase in PAF (21
), suggesting that platelets could be activated during infection in a manner that would allow aberrant GSK3β activation and therefore facilitate excess sCD40L release.
The data presented herein suggest that VPA is able to inhibit the release of sCD40L from platelets due to attenuated cytoskeletal rearrangement via GSK3β inhibition. This data is consistent with previous work that indicates that cytoskeletal rearrangement is indeed necessary for sCD40L release (46
). While it is still unclear whether GSK3β is acting directly on CD40L to inhibit it's trafficking to the cellular membrane (which occurs prior to its release), or rather, if its inhibition blocks the formation or movement of other CD40L containing vesicles to the surface prior to its cleavage, it is clear that platelet shape change is a necessary component of this process and inhibiting this can lead to altered CD40L solubilization.
The present report is also consistent with the previous findings by Barry and coworkers (19
) that demonstrated an inhibition of platelet activity in vitro
by short-term exposure of platelets to several GSK3β inhibitors (including lithium). In addition, Hayashi and Sudo (48
) showed that treatment of platelets with various agents that elevate cAMP levels inhibit GSK3β thereby blocking platelet activity. Our study, as well as those of Barry et al. (19
) and Hayashi and Sudo (48
), contrast somewhat with the findings reported by Li et al. (18
), who found that GSK3β works as a negative regulator of platelet function and thrombosis. In their report, they demonstrate that GSK3β+/− platelets exhibit agonist-dependent aggregation, ATP secretion, and fibrinogen binding, compared with GSK3β+/+ platelets, suggesting that GSK3β suppresses platelet function in vitro
). There are, however, important differences between our experiments and those conducted by Li and coworkers. These include the fact that we have examined the effect of pharmacologic inhibition of GSK3β on platelet-derived sCD40L levels, while Li and colleagues determined the effect of genetic deletion of this molecule on thrombotic events (18
). In addition, we have focused on the effect of VPA on platelet activation (and GSK3β) by effector molecules associated with HIV-1-infection (mainly Tat and PAF). In contrast, Li et al. studied platelet function in the context of non-pathogenic regulators of GSK3β. Thus, we hypothesize that the pathologic upregulation of GSK3β activity may lead to quite different effects on inflammatory mediators released by platelets.
Our group was the first to report the potential of VPA as an adjunct therapy for HIV-associated cognitive impairment (15
), demonstrating not only a trend toward improved cognitive performance but also improvements in measures of brain metabolism when tested in a controlled pilot patient study (15
). Along these same lines, neuroprotective effects of VPA in a murine model of HIV encephalitis were also previously reported (16
). The results presented here demonstrate that VPA is able to reduce plasma levels of sCD40L in HIV infected individuals and indicate that this action is linked to the ability of VPA to inhibit GSK3β in platelets. The use of VPA in this context may confer therapeutic benefits not only for HAND, but also other inflammatory diseases, such as stroke, that are linked to platelet activation.