Duas enzimas têm-se destacado como possíveis marcadores biológicos periféricos na esquizofrenia: a fosfolipase A2 (PLA2) e a glicogênio sintase quinase-3 beta (GSK-3B). Essas moléculas exercem importante influência na arquitetura e plasticidade celulares, na regulação de vias metabólicas comuns (metabolismo de fosfolípides e via Wnt), em fatores de transcrição, na regulação de genes e na sobrevivência celular. Tais aspectos tornam pertinentes as investigações a respeito da possível participação dessas enzimas na fisiopatologia da esquizofrenia. Foi verificada a atividade de subtipos de PLA2 (método radioenzimático: iPLA2, cPLA2 e sPLA2) e os níveis de GSK-3B total (GSK-3Bt) e fosforilada [p(Ser9)-GSK-3B] (ELISA) em plaquetas de pacientes com esquizofrenia inicialmente livres de tratamento medicamentoso com média de 5 anos de doença (D+5a) (n=10), aos quais foi prescrita a olanzapina. Foi avaliado também um grupo de pacientes livres de tratamento medicamentoso com menos de 6 meses de sintomas psicóticos (D-6m) (n=6) aos quais foi posteriormente prescrito o haloperidol. Esses pacientes foram comparados com um grupo controle (n = 20) e avaliados longitudinalmente após o tratamento descrito por 8 semanas. Um grupo de 40 pacientes com esquizofrenia (tempo médio da doença: 17 anos) com pelo menos 6 meses de tratamento com antipsicótico (clozapina, olanzapina ou haloperidol) foi ainda avaliado. Os sintomas clínicos foram avaliados por meio da Escala de Avaliação das Síndromes Positiva e Negativa (PANSS). Quando comparado com o grupo controle, os pacientes D+5a apresentaram aumento da atividade de iPLA2 (p<0,01) e os pacientes D-6m apresentaram aumento da atividade de sPLA2 (p<0,05). Na avaliação longitudinal, somente a olanzapina diminuiu a atividade de iPLA2, cPLA2 e sPLA2 (p<0,01). Quando comparada a atividade dos subtipos de PLA2 entre os pacientes medicados a pelo menos 6 meses e o grupo controle, não foram observadas diferenças significativas. Quando comparado com o grupo controle, os pacientes D+5a apresentaram diminuição dos níveis de GSK-3Bt e p(Ser9)-GSK-3B (p<0,05). Na avaliação longitudinal, foi observado que somente a olanzapina aumentou os níveis de GSK-3Bt e p(Ser9)-GSK-3B (p < 0,01). Quando comparados os níveis de GSK-3Bt e p(Ser9)-GSK-3B entre os pacientes medicados a pelo menos 6 meses e o grupo controle não foram observadas diferenças significativas. Para os pacientes medicados a pelo menos 6 meses foram observadas correlações entre a sub-escala negativa da PANSS e os níveis de p(Ser9)-GSK-3B (r=,53, p<0,001). Sugere-se que a medicação module PLA2 e GSK-3B, independente do antipsicótico utilizado. Esses resultados apontam para uma futura aplicação dessas enzimas na verificação de adesão ao tratamento e estabilização do quadro clínico / The enzymes phospholipases A2 (PLA2) and glycogen synthase kinase-3 beta (GSK-3B) are thought to play a role in schizophrenia by influencing cellular architecture and plasticity, common signaling pathways (phospholipids metabolism and Wnt pathway), gene transcription, regulation factors and apoptosis. These aspects motivated the investigation of both these enzymes in schizophrenia. The activities of PLA2 subtypes (iPLA2, cPLA2 and sPLA2 by radio enzymatic method) and the levels of total GSK-3B and phosphorylated GSK-3B [p(Ser9)-GSK-3B] (by immune enzyme assay) were performed in platelets of drug free patients with schizophrenia for average 5 years of disease (D+5y) (n=10), who was lately prescribed with olanzapine and in drug naïve patients with less than 6 months of psychotic symptoms (D-6m) who was lately prescribed with haloperidol. These patients were compared to a control group (n=20) and were longitudinally evaluated after 8 weeks of monotherapy treatment with the prescribed antipsychotic. These enzymes were also investigated in a group of 40 patients with schizophrenia (mean duration of disease: 17 years) who were at least 6 months treated with antipsychotic (clozapine; olanzapine or haloperidol). Psychopathology was assessed with the Positive and Negative Syndrome Scale (PANSS). Patients D+5y presented higher iPLA2 activity than control group (p < 0.01) and patients D-6m presented higher sPLA2 activity than control group (p < 0.05). On longitudinal evaluation, only olanzapine decreased iPLA2, cPLA2 and sPLA2 activities (p < 0.01). In the long-term medicated patients group compared to the control group, no differences regarding PLA2 subtype activity were found. Patients D+5y presented lower GSK-3Bt and p(Ser9)-GSK-3B levels than control group (p<0.05). On longitudinal evaluation, only olanzapine increased GSK-3Bt and p(Ser9)-GSK-3B levels (p < 0.01). In the long-term medicated patients group compared to the control group, no differences regarding GSK-3B levels were found. For long-term medicated patients, it was observed correlation between p(Ser9)-GSK-3B and the PANSS negative syndrome subscale score (r = .53, p < 0.001). It was suggested that antipsychotic treatment modulated PLA2 and GSK-3B, in spite of the drug used. The results pointed to a future use of these enzymes to verify drug treatment compliance and clinical stabilization

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine protein kinase which plays a key role in several signaling pathways and its homologues have been identified in most eukaryotes. Since GSK3£]is an essential protein kinase that regulates numerous functions within the cell, an effort to survey possible GSK3£]- interacting proteins from a human testis cDNA library using the yeast two-hybrid system is made. Two interesting candidates are chosen to characterize their functions in this study. One is a centrosomal protein, hNinein, and the other is a novel inhibitor of GSK3£], designated as GSKIP (GSK3£] interaction protein). In the first part of the present thesis we describe the identification of four diverse CCII-termini of human hNinein isoforms, including a novel isoform 6, by differential expression in a tissue-specific manner. In a kinase assay, the CCII region of hNinein isoforms provides a differential phosphorylation site by GSK3£]. In addition, either N-terminal or CCIIZ domain disruption may cause hNinein conformational change which recruits £^-tubulin to centrosomal or non-centrosomal hNinein-containing sites. Further, depletion of all hNinein isoforms caused a significant decrease in the £^-tubulin signal in the centrosome. In domain swapping, it clearly shows that the CCIIX-CCIIY region provides docking sites for £^-tubulin. Moreover, nucleation of microtubules from the centrosome is significantly affected by the overexpression of either the full-length hNinein or CCIIX-CCIIY region. Taken together, these results show that the centrosomal targeting signals of hNinein have a role not only in regulating hNinein conformation, resulting in localization change, but also provide docking sites to recruit £^-tubulin at centrosomal and non-centrosomal sites. In the second part of the thesis we describe another candidate, GSK3£]interaction protein (GSKIP), to characterize its functions in neuron differentiation. We use human neuroblastoma SH-SY5Y cells as a model of neuronal cell differentiation. When overexpression of GSKIP prevents neurite outgrowth from RA-mediated differentiation, this result is similar to the presence of LiCl or SB415286, an inhibitor of GSK3£]. Further, GSKIP regulates the activity of GSK3£] through protein-protein interactions rather than post-modulation and GSKIP may affect GSK3£] on neurite outgrowth via inhibiting the specific phosphorylation site of tau. In addition to inhibition of neurite outgrowth, GSKIP overexpressed in SH-SY5Y cells also promotes cell cycle progression by analyzing cell proliferation with cell growth and MTT assay. Furthermore, GSKIP raises the level of £]-catenin and cyclin D1 through inhibition of GSK3£] activity in RA-mediated differentiation SH-SY5Y cells. Taken together, the data suggest that GSKIP, a dual functional molecule, is able to inhibit neurite outgrowth and promote cell proliferation via negative regulation of GSK3£] activity in RA-mediated differentiation of SH-SY5Y cells.

Alzheimer¡¦s disease (AD) is a neurodegenerative disorder characterized by progressive deterioration of cognitive functions and the presence of intracellular neurofibrillary tangles (NFT) and extraneuronal senile plaques (SP). The major component of NFT is the hyperphosphorylated microtubules-associated protein tau. SP is consistent of extracellular deposition of £]-amyloid (A£]), mainly A£]1-42 peptide (A£]42). Given the need of tools for early and accurate diagnosis and prediction of disease progression and monitoring the efficacy of therapeutic agents for AD, development of cerebrospinal fluid (CSF) biomarkers have become a rapidly growing research field. In our study, patients with AD (n=28), non-AD dementia (n=16), other neurological disorder (OND, n=14) and healthy controls (HC, n=21) were included. Our results revealed that AD patients have significant higher CSF total tau (t-tau) and lower A£]42 levels than HC and OND groups. There is no significant difference of both CSF t-tau and A£]42 levels between AD and non-AD dementia groups. These results suggest that both CSF t-tau and A£]42 are good biomarkers for distinguishing AD from non-dementia control subjects but demonstrate less discriminating power in differentiating AD from non-AD dementia. Moreover, our results show only CSF t-tau level but not A£]42 has an inverse correlation with the score of short-term memory patients with AD (spearman: r = -0.444; p=0.018). These data indicate the higher CSF t-tau level is associated with much NFT pathology and more severe impairment of short-term memory in AD patients. In the study of the moleacular mechanism of tau¡¦s hyperphosphorylation by glycogen synthase kinase 3b (GSK3b), we show that the T231 is the primary phosphorylation site for GSK3b and the tau227-237 (AVVRTPPKSPS) derived from tau containing T231P232 motif is identified as the GSK3b binding site with high affinity of a Kd value 0.82 ¡Ó 0.16 mM. Our results suggest that direct binding and phosphorylation of T231P232 motif by GSK3b induces conformational change of tau and consequentially alters the inhibitory activity of its N-terminus that allows the sequential phosphorylation of C-terminus of tau by GSK3b. Furthermore, hyperphosphorylation reduces tau¡¦s ability to promote tubulin assembly and to form bundles in N18 cells. T231A mutant completely abolishes tau phosphorylation by GSK3b and retains the ability to promote tubulin polymerization and bundle formation. Taken together, these results suggest that phosphorylation of T231 by GSK3b may play an important role in tau¡¦s hyperphosphorylation and functional regulation.

Immunosenescence results in reduced immune response to infections with Streptococcus pneumoniae as well as to pneumococcal polysaccharide vaccines. The antibody response to the capsular polysaccharide (CPS) provides protection against S. pneumoniae infection. CPS immunoresponse is T cell independent and needs the macrophage-derived cytokines such as IL-12, IL-6 and IL-1β to elicit an antibody response. We showed a cytokine dysregulation, i.e. a decrease in IL-12, IL-6 and TNF-α but an increase in IL-10, in the aged (18-24 months old comparable to >65 years in human) compared to young adult mouse (8-12 weeks less than 65 years old) splenic macrophages (SM) or bone marrow derived macrophages (BMDM) activated via TLR4, TLR2 or TLR9 as well as heat killed Streptococcus pneumoniae (HKSP). There is also an age-associated defect in splenic B cells in the production of IgG3 upon stimulation with these ligands. A microarray analysis in SM followed by validation by both qt-RTPCR and western blots indicated that this age-associated defect in aged SM, BMDM and B cells was due to a heightened activity of the PI3K-Akt signaling pathway. We hypothesized that the senescence of immune responses in macrophages and B cells is due to an increase in activity of PI3K/Akt and decrease in the activity of GSK-3, the downstream kinase. Inhibition of the PI3-kinase with either LY294002 or Wortmannin restored the TLR2, 4, 9 and HKSP induced cytokine phenotype of the aged to that of the young adult in both the SM and BMDM and an enhanced IgG3 production in aged mice. We also showed that inhibition of glycogen synthase kinase-3 (GSK-3) the downstream target of the PI3K-Akt signaling pathway with SB216763 in SM, BMDM and B cells resulted in an enhancement in production of IL-10, IL-6 and IL-1β by macrophages and in B cell activation. Treatment of B cells with SB216763 in the presence of ligands for TLR-1/2, 4 or 9 as well as HKSP under in vitro conditions led to enhanced production of IgG3 and IgA, plasma cell formation and a slight increase in the proliferation of the B-cells with no adverse effects on the viability of the cells. Therefore, targeting the PI3K-AKT-GKS-3 signaling pathway could rescue the intrinsic signaling defect in the aged macrophages, increase IL-12 and IL-6, and enhance anti-CPS antibody responses.

Increasingly, cell-based therapy with autologous progenitor populations, such as endothelial progenitor cells (EPC), are being utilized for treatment of vascular diseases. However, both the number and functional capacity are diminished when cells are derived from patients with established risk factors for coronary artery disease (CAD). Herein, we report that inhibition of glycogen synthase kinase 3 (GSK) can improve both the number and function of endothelial progenitor cells in patients with CAD or diabetes mellitus (DM) leading to greater therapeutic benefit. Specifically, use of various small molecule inhibitors of GSK (GSKi) results in a 4-fold increased number of EPCs. Moreover, GSKi treatment improves the functional profile of EPCs through reductions in apoptosis, improvements in cell adhesion through up-regulation of very-late antigen-4 (VLA-4), and by increasing paracrine efficacy by increasing vascular endothelial growth factor (VEGF)secretion. Therapeutic improvement was confirmed in vivo by increased reendothelialization(RE) and reductions of neointima (NI) formation achieved when GSKi-treated cells were administered following vascular injury to CD-1 nude mice. Because cell-based therapy is technically challenging, we also tested a strategy of local delivery of GSKi at the site of arterial injury through GSKi-eluting stents. In vitro, GSKi elution increased EPC attachment to stent struts. In vivo, GSKi-eluting stents deployed in rabbit carotid arteries resulted in systemic mobilization of EPCs, improved local RE, and important reductions in in-stent NI formation. Finally, we tested the ability of GSKi to improve EPC-mediated arterial repair in patients with DM. As in patients with CAD, GSKi treatment improved EPC yield and diminished in vitro apoptosis. Utilizing a proteomics approach, we identified Cathepsin B (catB) as a differentially regulated protein necessary for reductions in apoptosis. Indeed, antagonism of catB prevented GSKi improvements in GSKi treated EPC mediated arterial repair in a xenotransplant wire injury model. Thus, our data demonstrates that GSKi treatment results in improvements in EPC number and function in vitro and in vivo resulting in enhanced arterial repair following mechanical injury. Accordingly, GSK antagonism is an effective cell enhancement strategy for autologous cell-based therapy with EPCs from high risk patients such as CAD or DM.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from first page of PDF file (viewed on June 8, 2009). Includes bibliographical references.

Thesis (Ph.D.)--Georgetown University, 2008. / Includes bibliographical references.

Apesar da elevada prevalência dos transtornos depressivos em idosos, os mecanismos fisiopatológicos subjacentes a estes quadros são pouco conhecidos. Atualmente, o principal foco dos estudos sobre a fisiopatologia da depressão geriátrica são as alterações cerebrovasculares associadas a estes quadros. Outros mecanismos fisiopatológicos têm sido estudados, como as alterações em cascatas neurotróficas e inflamatórias. A enzima glicogênio sintase quinase 3 beta (GSK-3B) tem sido implicada na patogênese de diversos transtornos mentais, em especial os transtornos afetivos (i.e. depressão maior e o transtorno afetivo bipolar) e doenças neurodegenerativas (i.e. doença de Alzheimer). Entretanto, não há estudos que avaliam o papel desta enzima nos pacientes idosos com depressão maior. Desta maneira, o objetivo principal deste trabalho é avaliar a atividade da GSK-3B em pacientes idosos com depressão maior. A hipótese deste estudo é que a atividade enzimática está aumentada nos pacientes idosos deprimidos em relação a idosos saudáveis. Para este estudo, recrutamos 40 idosos com depressão maior (de acordo com os critérios diagnósticos do DSM-IV) e que não estavam em uso de antidepressivos. O grupo comparativo foi constituído por 13 idosos saudáveis, sem evidências de transtornos cognitivos ou do humor. A gravidade da sintomatologia depressiva foi avaliada pela escala de depressão de Hamilton de 21 itens (HAM-D); o desempenho cognitivo dos pacientes e controles foi avaliado pelo teste cognitivo de Cambridge (CAMCOG) e pelo mini-exame do estado mental (MEEM). A expressão da GSK-3B foi determinada em plaquetas através de ensaio imunoenzimático (EIA), sendo estabelecido os níveis totais da GSK-3B (T-GSK-3B) e de sua forma fosforilada (P-GSK-3B), inativa. A atividade enzimática foi inferida indiretamente pela razão P-GSK- 3B / T-GSK-3B. Nos pacientes idosos com depressão maior, observou-se uma redução significante dos níveis P-GSK-3B (p=0,03) e da razão da GSK- 3B (p=0,03). Os pacientes com sintomatologia depressiva mais grave (HAMD > 21) e déficits cognitivos mais intensos (CAMCOG < 86) apresentaram maior atividade enzimática (p=0,03 e teste, p=0,01, respectivamente). Os resultados deste trabalho sugerem que a atividade da GSK-3B está significantemente aumentada em pacientes idosos com depressão maior e que está alteração é mais pronunciada nos pacientes com sintomatologia depressiva e déficits cognitivos mais graves. Neste contexto, a atividade da GSK-3B pode ser considerada um marcador de estado em pacientes idosos com episódios depressivos mais graves e ser um importante alvo para o desenvolvimento de estratégias terapêuticas para estes quadros / Despite the high prevalence of depressive disorders in the elderly, its main physiopathological mechanisms are largely unknown. In the recent years, most of the research efforts focused on the association between cerebrovascular changes and geriatric depression. Nonetheless, other mechanisms have been studied, such as changes in neurotrophic and inflammatory cascades. The enzyme glycogen synthase kinase 3 beta (GSK- 3B) has been implicated in many mental disorders, in particular affective disorders (i.e. major depression and bipolar disorder) and neurodegenerative disorders (i.e. Alzheimers disease). However, there is no study so far that addressed the role of this enzyme in elderly patients with major depression. Therefore, the main objective of this study was to evaluate if GSK-3B activity is changed in elderly patients with major. The working hypothesis is that enzyme activity is significantly increased in elderly patients with major depression as compared to elderly controls. We recruited 40 elderly patients with current major depressive episode (according to the DSM-IV criteria) that was not under antidepressant treatment. The comparison group included 13 healthy elderly subjects with no evidence of cognitive impairment or major psychiatric disorder. The severity of depressive symptoms was assessed by the Hamilton Depression Scale 21 items; cognitive performance was assessed by the Cambridge Cognitive test (CAMCOG) and the Mini-mental State Examination (MMSE). The levels of total and phosphorylated GSK-3B (T-GSK-3B and P-GSK-3B, respectively) levels were determined in platelets by immunoenzymatic assay (EIA). Enzyme activity was indirectly inferred by the ratio P-GSK-3B / T-GSK-3B. Elderly patients with major depression had a significant reduction in the P-GSK-3B levels (p = 0.03) and GSK-3B ratio (p= 0.03). The patients with severe depressive episode (HAM-D scores above 21 points) and cognitive impairment (CAMCOG scores below 86 points) presented the more significant reduction of GSK-3B ratio (p = 0.03 and p = 0.01, respectively). These data altogether suggest that GSK-3B activity is significantly increased in elderly patients with major depression, in particular in those with more severe depressive episode and worse cognitive performance. In this context, the increased enzyme activity may be regarded as a state marker of severe depressive episodes and may an important target to the development of therapeutic strategies to this disorder

Duas enzimas têm-se destacado como possíveis marcadores biológicos periféricos na esquizofrenia: a fosfolipase A2 (PLA2) e a glicogênio sintase quinase-3 beta (GSK-3B). Essas moléculas exercem importante influência na arquitetura e plasticidade celulares, na regulação de vias metabólicas comuns (metabolismo de fosfolípides e via Wnt), em fatores de transcrição, na regulação de genes e na sobrevivência celular. Tais aspectos tornam pertinentes as investigações a respeito da possível participação dessas enzimas na fisiopatologia da esquizofrenia. Foi verificada a atividade de subtipos de PLA2 (método radioenzimático: iPLA2, cPLA2 e sPLA2) e os níveis de GSK-3B total (GSK-3Bt) e fosforilada [p(Ser9)-GSK-3B] (ELISA) em plaquetas de pacientes com esquizofrenia inicialmente livres de tratamento medicamentoso com média de 5 anos de doença (D+5a) (n=10), aos quais foi prescrita a olanzapina. Foi avaliado também um grupo de pacientes livres de tratamento medicamentoso com menos de 6 meses de sintomas psicóticos (D-6m) (n=6) aos quais foi posteriormente prescrito o haloperidol. Esses pacientes foram comparados com um grupo controle (n = 20) e avaliados longitudinalmente após o tratamento descrito por 8 semanas. Um grupo de 40 pacientes com esquizofrenia (tempo médio da doença: 17 anos) com pelo menos 6 meses de tratamento com antipsicótico (clozapina, olanzapina ou haloperidol) foi ainda avaliado. Os sintomas clínicos foram avaliados por meio da Escala de Avaliação das Síndromes Positiva e Negativa (PANSS). Quando comparado com o grupo controle, os pacientes D+5a apresentaram aumento da atividade de iPLA2 (p<0,01) e os pacientes D-6m apresentaram aumento da atividade de sPLA2 (p<0,05). Na avaliação longitudinal, somente a olanzapina diminuiu a atividade de iPLA2, cPLA2 e sPLA2 (p<0,01). Quando comparada a atividade dos subtipos de PLA2 entre os pacientes medicados a pelo menos 6 meses e o grupo controle, não foram observadas diferenças significativas. Quando comparado com o grupo controle, os pacientes D+5a apresentaram diminuição dos níveis de GSK-3Bt e p(Ser9)-GSK-3B (p<0,05). Na avaliação longitudinal, foi observado que somente a olanzapina aumentou os níveis de GSK-3Bt e p(Ser9)-GSK-3B (p < 0,01). Quando comparados os níveis de GSK-3Bt e p(Ser9)-GSK-3B entre os pacientes medicados a pelo menos 6 meses e o grupo controle não foram observadas diferenças significativas. Para os pacientes medicados a pelo menos 6 meses foram observadas correlações entre a sub-escala negativa da PANSS e os níveis de p(Ser9)-GSK-3B (r=,53, p<0,001). Sugere-se que a medicação module PLA2 e GSK-3B, independente do antipsicótico utilizado. Esses resultados apontam para uma futura aplicação dessas enzimas na verificação de adesão ao tratamento e estabilização do quadro clínico / The enzymes phospholipases A2 (PLA2) and glycogen synthase kinase-3 beta (GSK-3B) are thought to play a role in schizophrenia by influencing cellular architecture and plasticity, common signaling pathways (phospholipids metabolism and Wnt pathway), gene transcription, regulation factors and apoptosis. These aspects motivated the investigation of both these enzymes in schizophrenia. The activities of PLA2 subtypes (iPLA2, cPLA2 and sPLA2 by radio enzymatic method) and the levels of total GSK-3B and phosphorylated GSK-3B [p(Ser9)-GSK-3B] (by immune enzyme assay) were performed in platelets of drug free patients with schizophrenia for average 5 years of disease (D+5y) (n=10), who was lately prescribed with olanzapine and in drug naïve patients with less than 6 months of psychotic symptoms (D-6m) who was lately prescribed with haloperidol. These patients were compared to a control group (n=20) and were longitudinally evaluated after 8 weeks of monotherapy treatment with the prescribed antipsychotic. These enzymes were also investigated in a group of 40 patients with schizophrenia (mean duration of disease: 17 years) who were at least 6 months treated with antipsychotic (clozapine; olanzapine or haloperidol). Psychopathology was assessed with the Positive and Negative Syndrome Scale (PANSS). Patients D+5y presented higher iPLA2 activity than control group (p < 0.01) and patients D-6m presented higher sPLA2 activity than control group (p < 0.05). On longitudinal evaluation, only olanzapine decreased iPLA2, cPLA2 and sPLA2 activities (p < 0.01). In the long-term medicated patients group compared to the control group, no differences regarding PLA2 subtype activity were found. Patients D+5y presented lower GSK-3Bt and p(Ser9)-GSK-3B levels than control group (p<0.05). On longitudinal evaluation, only olanzapine increased GSK-3Bt and p(Ser9)-GSK-3B levels (p < 0.01). In the long-term medicated patients group compared to the control group, no differences regarding GSK-3B levels were found. For long-term medicated patients, it was observed correlation between p(Ser9)-GSK-3B and the PANSS negative syndrome subscale score (r = .53, p < 0.001). It was suggested that antipsychotic treatment modulated PLA2 and GSK-3B, in spite of the drug used. The results pointed to a future use of these enzymes to verify drug treatment compliance and clinical stabilization

Increasingly, cell-based therapy with autologous progenitor populations, such as endothelial progenitor cells (EPC), are being utilized for treatment of vascular diseases. However, both the number and functional capacity are diminished when cells are derived from patients with established risk factors for coronary artery disease (CAD). Herein, we report that inhibition of glycogen synthase kinase 3 (GSK) can improve both the number and function of endothelial progenitor cells in patients with CAD or diabetes mellitus (DM) leading to greater therapeutic benefit. Specifically, use of various small molecule inhibitors of GSK (GSKi) results in a 4-fold increased number of EPCs. Moreover, GSKi treatment improves the functional profile of EPCs through reductions in apoptosis, improvements in cell adhesion through up-regulation of very-late antigen-4 (VLA-4), and by increasing paracrine efficacy by increasing vascular endothelial growth factor (VEGF)secretion. Therapeutic improvement was confirmed in vivo by increased reendothelialization(RE) and reductions of neointima (NI) formation achieved when GSKi-treated cells were administered following vascular injury to CD-1 nude mice. Because cell-based therapy is technically challenging, we also tested a strategy of local delivery of GSKi at the site of arterial injury through GSKi-eluting stents. In vitro, GSKi elution increased EPC attachment to stent struts. In vivo, GSKi-eluting stents deployed in rabbit carotid arteries resulted in systemic mobilization of EPCs, improved local RE, and important reductions in in-stent NI formation. Finally, we tested the ability of GSKi to improve EPC-mediated arterial repair in patients with DM. As in patients with CAD, GSKi treatment improved EPC yield and diminished in vitro apoptosis. Utilizing a proteomics approach, we identified Cathepsin B (catB) as a differentially regulated protein necessary for reductions in apoptosis. Indeed, antagonism of catB prevented GSKi improvements in GSKi treated EPC mediated arterial repair in a xenotransplant wire injury model. Thus, our data demonstrates that GSKi treatment results in improvements in EPC number and function in vitro and in vivo resulting in enhanced arterial repair following mechanical injury. Accordingly, GSK antagonism is an effective cell enhancement strategy for autologous cell-based therapy with EPCs from high risk patients such as CAD or DM.