Yin and Yang of PDGF-mediated signaling pathway in the context of HIV infection and drug abuse.

The control and eradication of neurological complications associated with AIDS continues to be an important goal in efforts toward improving the well being of HIV-infected patients. Although combined antiretroviral therapies have contributed significantly to increasing the longevity of patients by suppressing the virus burden in the systemic compartments, the prevalence of HIV-associated neurological disorders continues to be on the rise. This in turn, leads to an impaired quality of life of the infected individuals who continue to suffer from mild to moderate cognitive decline and memory loss. Developing therapeutic interventions that reverse neuronal injury in the context of HIV infection, is thus of paramount importance in the field. Our previous studies have demonstrated that platelet-derived growth factor (PDGF) has a neuroprotective potential against HIV envelope protein gp120 and Tat. Paradoxically, PDGF is also a cerebrovascular permeant with deleterious effects on the blood-brain barrier resulting in increased influx of monocytes in the CNS. Herein, we review the opposing roles of PDGF in the context of HIV-associated neurodegenerative disorder (HAND).

Platelet-derived growth factor CC-mediated neuroprotection against HIV Tat involves TRPC-mediated inactivation of GSK 3beta.

Platelet-derived growth factor-CC (PDGF-CC) is the third member of the PDGF family, and has been implicated both in embryogenesis and development of the CNS. The biological function of this isoform however, remains largely unexplored in the context of HIV-associated dementia (HAD). In the present study, we demonstrate that exposure of human neuroblastoma cells SH-SY5Y to HIV transactivator protein Tat resulted in decreased intrinsic expression of PDGF-CC as evidenced by RT-PCR and western blot assays. Reciprocally, pretreatment of SH-SY5Y cells with PDGF-CC abrogated Tat-mediated neurotoxicity by mitigating apoptosis and neurite & MAP-2 loss. Using pharmacological and loss of function approaches we identified the role of phosphatidylinositol 3-kinase (PI3K)/Akt signaling in PDGF-CC-mediated neuroprotection. We report herein a novel role about the involvement of transient receptor potential canonical (TRPC) channel 1 in modulation of calcium transients in PDGF-CC-mediated neuroprotection. Furthermore we also demonstrated PDGF-CC-mediated inactivation of the downstream mediator--glycogen synthase kinase 3ß (GSK3ß) evidenced by its phosphorylation at Ser-9. This was further validated by gain and loss of function studies using cells transfected with either the wild type or mutant GSK3ß constructs. Intriguingly, pretreatment of cells with either the PI3K inhibitor or TRPC blocker resulted in failure of PDGF-CC to inactivate GSK3ß, thereby suggesting the intersection of PI3K and TRPC signaling at GSK3ß. Taken together our findings lead to the suggestion that PDGF-CC could be developed as a therapeutic target to reverse Tat-mediated neurotoxicity with implications for HAD.

Platelet-derived growth factor-mediated induction of the synaptic plasticity gene Arc/Arg3.1.

Platelet-derived growth factor (PDGF) is a pleiotropic protein with critical roles in both developmental as well as pathogenic processes. In the central nervous system specifically, PDGF is critical for neuronal proliferation and differentiation and has also been implicated as a neuroprotective agent. Whether PDGF also plays a role in synaptic plasticity, however, remains poorly understood. In the present study we demonstrated that in the rat hippocampal neurons PDGF regulated the expression of Arc/Arg3.1 gene that has been implicated in both synapse plasticity and long term potentiation. Relevance of these findings was further confirmed in vivo by injecting mice with intracerebral inoculations of PDGF, which resulted in a rapid induction of Arc in the hippocampus of the injected mice. PDGF induced long term potentiation in rat hippocampal slices, which was abolished by PDGF receptor-tyrosine kinase inhibitor STI-571. We also present evidence that PDGF-mediated induction of Arc/Arg3.1 involved activation of the MAPK/ERK (MEK) pathway. Additionally, induction of Arc/Arg3.1 also involved the upstream release of intracellular calcium stores, an effect that could be blocked by thapsigargin but not by EGTA. Pharmacological approach using inhibitors specific for either MAPK/ERK phosphorylation or calcium release demonstrated that the two pathways converged downstream at a common point involving activation of the immediate early gene Egr-1. Chromatin immunoprecipitation assays demonstrated the binding of Egr-1, but not Egr-3, to the Arc promoter. These findings for the first time, thus, suggest an additional role of PDGF, that of induction of Arc.

Cooperative induction of CXCL10 involves NADPH oxidase: Implications for HIV dementia.

With the increasing prevalence of HIV-associated neurocognititve disorders (HAND), understanding the mechanisms by which HIV-1 induces neuro-inflammation and subsequent neuronal damage is important. The hallmark features of HIV-encephalitis, the pathological correlate of HIV-associated Dementia (HAD), are gliosis, oxidative stress, chemokine dysregulation, and neuronal damage/death. Since neurons are not infected by HIV-1, the current thinking is that these cells are damaged indirectly by pro-inflammatory chemokines released by activated glial cells. CXCL10 is a neurotoxic chemokine that is upregulated in astroglia activated by HIV-1 Tat, IFN-gamma, and TNF-alpha. In this study we have demonstrated that HIV-1 Tat increases CXCL10 expression in IFN-gamma and TNF-alpha stimulated human astrocytes via NADPH oxidase. We have shown that the treatment of astrocytes with a mixture of Tat and cytokines leads to a respiratory burst that is abrogated by apocynin, an NADPH oxidase inhibitor. Pretreatment of Tat, IFN-gamma, and TNF-alpha stimulated astrocytes with apocynin also resulted in concomitant inhibition of CXCL10 expression. Additionally, apocynin was also able to reduce Tat and cytokine-mediated activation of the corresponding signaling molecules Erk1/2, Jnk, and Akt with a decrease in activation and nuclear translocation of NF-kappaB, important regulators of CXCL10 induction. Understanding the mechanisms involved in reducing both oxidative stress and the release of pro-inflammatory agents could lead to the development of therapeutics aimed at decreasing neuro-inflammation in patients suffering from HAD.

PDGF-mediated protection of SH-SY5Y cells against Tat toxin involves regulation of extracellular glutamate and intracellular calcium.

The human immunodeficiency virus (HIV-1) protein Tat has been implicated in mediating neuronal apoptosis, one of the hallmark features of HIV-associated dementia (HAD). Mitigation of the toxic effects of Tat could thus be a potential mechanism for reducing HIV toxicity in the brain. In this study we demonstrated that Tat-induced neurotoxicity was abolished by NMDA antagonist-MK801, suggesting the role of glutamate in this process. Furthermore, we also found that pretreatment of SH-SY5Y cells with PDGF exerted protection against Tat toxicity by decreasing extracellular glutamate levels. We also demonstrated that extracellular calcium chelator EGTA was able to abolish PDGF-mediated neuroprotection, thereby underscoring the role of calcium signaling in PDGF-mediated neuroprotection. We also showed that Erk signaling pathway was critical for PDGF-mediated protection of cells. Additionally, blocking calcium entry with EGTA resulted in suppression of PDGF-induced Erk activation. These findings thus underscore the role of PDGF-mediated calcium signaling and Erk phosphorylation in the protection of cells against HIV Tat toxicity.

Involvement of TRPC channels in CCL2-mediated neuroprotection against tat toxicity.

Chemokine (C-C motif) ligand 2 (CCL2), also known as monocyte chemoattractant protein-1, plays a critical role in leukocyte recruitment and activation. In the present study, we identify an additional role for CCL2 that of neuroprotection against HIV-1 transactivator protein (Tat) toxicity in rat primary midbrain neurons. Furthermore, we report the involvement of transient receptor potential canonical (TRPC) channels in CCL2-mediated neuroprotection. TRPC are Ca(2+)-permeable, nonselective cation channels with a variety of physiological functions. Blockage of TRPC channels resulted in suppression of both CCL2-mediated neuroprotection and intracellular Ca(2+) elevations. Parallel but distinct extracellular signal-regulated kinase (ERK)/cAMP response element-binding protein (CREB) and Akt/nuclear factor kappaB (NF-kappaB) pathways were involved in the CCL2-mediated neuroprotection. Blocking TRPC channels and specific downregulation of TRPC channels 1 and 5 resulted in suppression of CCL2-induced ERK/CREB activation but not Akt/NF-kappaB activation. In vivo relevance of these findings was further corroborated in wild-type and CCR2 knock-out mice. In the wild-type but not CCR2 knock-out mice, exogenous CCL2 exerted neuroprotection against intrastriatal injection of HIV-1 Tat. These findings clearly demonstrate a novel role of TRPC channels in the protection of neurons against Tat through the CCL2/CCR2 axis.

Proinflammatory cytokines and HIV-1 synergistically enhance CXCL10 expression in human astrocytes.

HIV encephalitis (HIVE), the pathologic correlate of HIV-associated dementia (HAD) is characterized by astrogliosis, cytokine/chemokine dysregulation, and neuronal degeneration. Increasing evidence suggests that inflammation is actively involved in the pathogenesis of HAD. In fact, the severity of HAD/HIVE correlates more closely with the presence of activated glial cells than with the presence and amount of HIV-infected cells in the brain. Astrocytes, the most numerous cell type within the brain, provide an important reservoir for the generation of inflammatory mediators, including interferon-gamma inducible peptide-10 (CXCL10), a neurotoxin and a chemoattractant, implicated in the pathophysiology of HAD. Additionally, the proinflammatory cytokines, IFN-gamma and TNF-alpha, are also markedly increased in CNS tissues during HIV-1 infection. In this study, we hypothesized that the interplay of host cytokines and HIV-1 could lead to enhanced expression of the toxic chemokine, CXCL10. Our findings demonstrate a synergistic induction of CXCL10 mRNA and protein in human astrocytes exposed to HIV-1 and the proinflammatory cytokines. Signaling molecules, including JAK, STATs, MAPK (via activation of Erk1/2, AKT, and p38), and NF-kappaB were identified as instrumental in the synergistic induction of CXCL10. Understanding the mechanisms involved in HIV-1 and cytokine-mediated up-regulation of CXCL10 could aid in the development of therapeutic modalities for HAD.

Mechanisms of platelet-derived growth factor-mediated neuroprotection--implications in HIV dementia.

Platelet-derived growth factor (PDGF) has been implicated in promoting survival and proliferation of immature neurons, and even protecting neurons from gp120-induced cytotoxicity. However, the mechanisms involved in neuroprotection are not well understood. In the present study we demonstrate the role of phosphatidylinositol 3-kinase (PI3K)/Akt signaling in PDGF-mediated neuroprotection. Pharmacological inhibition of PI3K greatly reduced the ability of PDGF-BB to block gp120 IIIB-mediated apoptosis and cell death in human neuroblastoma cells. The role of Akt in PDGF-mediated protection was further corroborated using a dominant-negative mutant of Akt, which was able to block the protective effect of PDGF. We next sequentially examined the signals downstream of Akt in PDGF-mediated protection in human neuroblastoma cells. In cells pretreated with PDGF prior to gp120 there was increased phosphorylation of both GSK-3beta and Bad, an effect that was inhibited by PI3-kinase inhibitor. Nuclear translocation of NF-kappaB, which lies downstream of GSK-3beta, however, remained unaffected in cells treated with PDGF. In addition to inducing phosphorylation of Bad, PDGF-mediated protection also involved down-regulation of the proapoptotic protein Bax. Furthermore, PDGF-mediated protection also involved the inhibition of gp120-induced release of mitochondrial cytochrome C. Our findings thus underscore the roles of both PI3K/Akt and Bcl family pathways in PDGF-mediated neuroprotection.

Molecular mechanism(s) involved in the synergistic induction of CXCL10 by human immunodeficiency virus type 1 Tat and interferon-gamma in macrophages.

Synergistic interactions between viral proteins and soluble host factors released from infected mononuclear phagocytes play a critical role in the pathogenesis of human immunodeficiency virus (HIV)-associated dementia (HAD). The chemokine CXCL10 has been found to be closely associated with the progression of HIV-1-related central nervous system (CNS) disease and its related neuropsychiatric impairment. In this report the authors demonstrate that the HIV-1 protein Tat can interact with the proinflammatory cytokine interferon (IFN)-gamma to dramatically induce the expression of CXCL10 in macrophages. Synergistic induction of CXCL10 by both Tat and IFN-gamma was susceptible to inhibition by the MEK1/2 inhibitor U0126 and the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580. In addition, JAK/STAT pathway plays a major role in Tat/gamma-mediated CXCL10 induction in macrophages because pretreatment of stimulated macrophages with JAK inhibitor completely abrogated the synergistic induction of the chemokine. Functionality of the synergistically induced CXCL10 was further demonstrated by its chemotactic activity for peripheral blood lymphocytes. Taken together, these findings demonstrate that the cooperative interaction of Tat and IFN-gamma results in enhanced chemokine expression, which in turn can amplify the inflammatory responses within the CNS of HAD patients by recruiting more lymphocytes in the brain.

Platelet-derived growth factor protects neurons against gp120-mediated toxicity.

The human immunodeficiency virus (HIV)-1 envelope glycoprotein gp120 has been implicated in mediating neuronal apoptosis, a hallmark feature of HIV-associated dementia (HAD). Mitigation of the toxic effects of gp120 could thus be a potential mechanism for reducing HIV toxicity in the brain. In this study the authors hypothesized that neurotrophic factor, such as platelet-derived growth factor (PDGF), could protect the neurons against gp120-mediated apoptosis. SH-SY5Y cells treated with gp120 exhibited increased cell death when measured by lactate dehydrogenase (LDH) and deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay, with concomitant loss of neurites and increased cell rounding. Pretreatment with PDGF-BB, however, reduced gp120-associated neurotoxicity and rescued the neurite outgrowth. Additionally, gp120-mediated activation of caspase-3 was also significantly reduced in cells pretreated with PDGF-BB. Antiapoptotic effects of PDGF-BB were also confirmed by monitoring levels of anti- and proapoptotic genes, Bcl-xL and Bax, respectively. Furthermore, PDGF-mediated protection against gp120 involved the phosphoinositide (PI) 3-kinase/Akt pathway. Taken together these findings lead us to suggest that PDGF-BB could be considered as a therapeutic agent that can mitigate gp120-mediated neurotoxicity in HAD.

Cocaine-mediated alteration in tight junction protein expression and modulation of CCL2/CCR2 axis across the blood-brain barrier: implications for HIV-dementia.

One of the hallmark features underlying the pathogenesis of HIV encephalitis is the disruption of blood-brain barrier (BBB). Cocaine, often abused by HIV-infected patients, has been suggested to worsen the HIV-associated dementia (HAD) via unknown mechanisms. The objective of the present study was to explore the effects of cocaine on BBB permeability using human brain microvascular endothelial cells (HBMECs). Additionally, because the chemokine CCL2 and its receptor CCR2 play a crucial role in the recruitment of inflammatory cells into the central nervous system in HAD brains, we tested for the effect of cocaine in modulating the CCL2/CCR2 axis. Our findings suggest that exposure of HBMECs to cocaine correlated with the breakdown of ZO-1 tight junction protein and reorganization of the cytoskeleton resulting in stress fiber formation. Furthermore, cocaine also modulated upregulation of the CCL2/CCR2 axis in monocytes. These findings conform to the multifaceted effects of cocaine leading to accelerated progression of HIV-1 neuropathogenesis.

Cocaine-mediated enhancement of virus replication in macrophages: implications for human immunodeficiency virus-associated dementia.

Injection drug use has been recognized as a major risk factor for acquired immunodeficiency syndrome (AIDS) from the outset of the epidemic. Cocaine, one of the most widely abused drugs in the United States, can both impair the functions of macrophages and CD4(+) lymphocytes and also activate human immunodeficiency virus (HIV)-1 expression in these cells. Because the brain is the target organ for both cocaine and HIV, the objective of the present study was to explore the effects of cocaine on virus replication in macrophages, the target cells for the virus in the central nervous system (CNS). Cocaine markedly enhanced virus production in simian human immunodeficiency virus (SHIV)-infected monocyte-derived macrophages (MDMs) and in U1 cells, a chronically infected promonocytic cell line as monitored by enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry. Cocaine treatment also resulted in the activation of nuclear factor (NF)-kappa B and transcriptional activation of the HIV-LTR (long terminal repeat) gag-GFP (green fluorescent protein). Analyses of chemokines in cocaine-treated macrophages by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and Luminex assays suggested increased expression of interleukin (IL)-10, a cytokine that is known to promote HIV replication in MDMs. In addition to enhancing IL-10 expression, cocaine also caused an up-regulation of the macrophage activation marker, human leukocyte antigen (HLA)-DR, in MDMs. The synergistic effect of cocaine on virus replication and its enhancement of host activation markers suggest that cocaine functions at multiple pathways to accelerate HIV-associated dementia (HAD).

Recombinant thymosin beta 4 can promote full-thickness cutaneous wound healing.

Human thymosin beta 4 (TB4) is a small acidic peptide involved in angiogenesis, wound healing, cancer metastasis and cardiac repair. Currently human TB4 is synthesized chemically for research and this is costly. In order to obtain sufficient biologically active human TB4 economically, we cloned and overexpressed this protein in an Escherichia coli system. We also developed a one-step affinity purification method to purify this fusion protein. After the fusion tag was removed from the fusion protein through autohydrolysis by dithiothreitol (DTT), the biological activity and function of this recombinant human TB4 was evaluated by cell proliferation assay using prepared spleen cells and wound assay using a mouse model, respectively. Our data demonstrated that human recombinant TB4 can promote lymphocyte proliferation and differentiation. Further, it can also promote full-thickness cutaneous wound healing in BALB/c mice. To our knowledge, this is the first report of recombinant human TB4 with the ability to promote wound healing.

PDGF synergistically enhances IFN-gamma-induced expression of CXCL10 in blood-derived macrophages: implications for HIV dementia.

There is increasing cumulative evidence that activated mononuclear phagocytes (macrophages/microglia) releasing inflammatory mediators in the CNS are a better correlate of HIV-associated dementia (HAD) than the actual viral load in the brain. Earlier studies on simian HIV/rhesus macaque model of NeuroAIDS confirmed that pathological changes in brains of macaques with encephalitis were associated with up-regulation of platelet-derived growth factor (PDGF) and the chemokine, CXCL10. Because the complex interplay of inflammatory mediators released by macrophages often leads to the induction of neurotoxins in HAD, we hypothesized that PDGF could interact with IFN-gamma to modulate the expression of CXCL10 in these primary virus target cells. Although PDGF alone had no effect on the induction of CXCL10 in human macrophages, in conjunction with IFN-gamma, it significantly augmented the expression of CXCL10 RNA & protein through transcriptional and posttranscriptional mechanisms. Signaling molecules, such as JAK and STATs, PI3K, MAPK, and NF-kappaB were found to play a role in the synergistic induction of CXCL10. Furthermore, PDGF via its activation of p38 MAPK was able to increase the stability of IFN-gamma-induced CXCL10 mRNA. Understanding the mechanisms involved in the synergistic up-regulation of CXCL10 could aid in the development of therapeutic modalities for HAD.

Human bocavirus infection, People's Republic of China.

A newly identified parvovirus, human bocavirus (HBoV), was found in 21 (8.3%) of 252 nasopharyngeal aspirates from hospitalized children with lower respiratory tract infection in Hunan Province, People's Republic of China. Viral loads were 10(4) to 10(10) copies/mL. Phylogenetic analysis of the VP1 gene showed a single genetic lineage of HBoV worldwide.

Purification of recombinant human interferon-epsilon and oligonucleotide microarray analysis of interferon-epsilon-regulated genes.

Recently identified interferon-epsilon (IFN-epsilon) belongs to type I interferons. IFN-epsilon is highly and constitutively expressed in the brain, but its biochemical and biological characteristics are poorly understood. In this study, full-length IFN-epsilon cDNA was cloned from human peripheral blood lymphocyte by RT-PCR, and was expressed in Escherichia coli (E. coli). Reverse phase high pressure liquid chromatography was used to purify recombinant human IFN-epsilon (rhIFN-epsilon) and to facilitate refolding of the protein. About 0.8mg of highly purified rhIFN-epsilon protein was obtained from 100ml of E. coli culture. Functional study of rhIFN-epsilon demonstrated that the antiviral activity of rhIFN-epsilon was 6+/-0.5x10(5)IU/mg, which was lower than that of rhIFN-alpha-2b in the WISH-VSV (WISH cells infected with vesicular stomatitis virus) assay system. As for the activity to promote NK cytotoxicity and antiproliferation activities, rhIFN-epsilon was about 60 times less potent than rhIFN-alpha-2b. However, oligonucleotide microarray analyses revealed dramatic differences in gene expression profiles of cultured human cells treated with IFN-epsilon and IFN-alpha-2b. Particularly, differential regulation of genes related to central nervous system by rhIFN-epsilon suggests a role for IFN-epsilon in maintenance of the structure and function of brain.

[Biological activities of recombinant human interferon Epsilon].

OBJECTIVE: To construct a novel recombinant rhIFN-epsilon155ser, and study its biological activities. METHODS: The whole sequence of rhIFN-epsilon was artificially synthesized and some codons were altered according to the preferred codon using of E.coli. The sequence was cloned into plasmid vector pBV220 to express in E.coli DH5alpha. After purification and re-folding of rhIFN-epsilon155ser inclusion body, the final product was tested for its biological activities, including anti-viral, anti-proliferative and NK cell enhancing activities. At the same time, by using DNA microarray biochips, the gene expression patterns in the rhIFN-epsilon155ser and rhIFN-alpha2b treated cells were compared and analyzed. RESULTS: The re-built rhIFN-epsilon155ser sequence was expressed in E.coli as a form of inclusion body. After purified and re-folded, the rhIFN-epsilon155ser protein reached a purity of above 95%. The rhIFN-epsilon155ser protein had a specific anti-viral activity of about 6 x 10(5) IU/mg in WISH/VSV system. Its anti-proliferative activity and NK cell enhancing activities in vitro seemed to be lower than that of rhIFN-alpha2b. Data obtained from microarray biochips indicated that there were 283 pieces increasing 2 folds and 1489 pieces decreasing 2 folds among totally 22,278 pieces of human genes were found in the rhIFN-epsilon155ser treated cells; more changes in gene expression pattern were detected in the rhIFN-alpha treated cells. CONCLUSION: A novel recombinant rhIFN-epsilon155ser was constructed, which belonged to type 1 interferon. The biological activities of rhIFN-epsilon155ser were compared with rhIFN-alpha2b. The changes of gene expression pattern in the interferon treated cells were detected, analyzed and discussed.

[Cloning, expression and purification of interferon-kappa, a novel human interferon, and its antiviral activity].

OBJECTIVE: To prepare human interferon-k (hIFN-kappa) and study its biological activities. METHODS: Whole length of hIFN-kappa's cDNA was cloned, and its sequence was chemically synthesized according to the optimized codons of E.coli, then was expressed in E.coli DH5alpha. After purified, the rhIFN-kappa protein was tested for its various kinds of biological activities. RESULTS: The purity of rhIFN-kappa was above 90%. In WHIS-VSV system, the antiviral activity of rhIFN-kappa was 2.0 x 10(6) IU/mg. Compared with rhIFN-alpha-2b, the biological activities of rhIFN-kappa were all feeble, including antiviral activity, promoting NK cell activity and anti-proliferation activity. CONCLUSION: Antiviral activities of rhIFN-kappa on cell lines of different species are different, different viruses show different sensitivity to rhIFN-kappa.