RESUMO
Neuroimaging studies have documented reduced striatal dopamine D2/D3 receptor (D2/D3R) availability in cocaine abusers, which has been associated with impaired prefrontal activity and vulnerability for relapse. However, the mechanism(s) underlying the decreases in D2/D3R remain poorly understood. Recent studies have shown that sleep deprivation is associated with a downregulation of striatal D2/D3R in healthy volunteers. As cocaine abusers have disrupted sleep patterns, here we investigated whether reduced sleep duration mediates the relationship between cocaine abuse and low striatal D2/D3R availability. We used positron emission tomography with [(11)C]raclopride to measure striatal D2/D3R availability in 24 active cocaine abusers and 21 matched healthy controls, and interviewed them about their daily sleep patterns. Compared with controls, cocaine abusers had shorter sleep duration, went to bed later and reported longer periods of sleep disturbances. In addition, cocaine abusers had reduced striatal D2/D3R availability. Sleep duration predicted striatal D2/D3R availability and statistically mediated the relationship between cocaine abuse and striatal D2/D3R availability. These findings suggest that impaired sleep patterns contribute to the low striatal D2/D3R availability in cocaine abusers. As sleep impairments are similarly observed in other types of substance abusers (for example, alcohol and methamphetamine), this mechanism may also underlie reductions in D2/D3R availability in these groups. The current findings have clinical implications suggesting that interventions to improve sleep patterns in cocaine abusers undergoing detoxification might be beneficial in improving their clinical outcomes.
Assuntos
Transtornos Relacionados ao Uso de Cocaína/complicações , Transtornos Relacionados ao Uso de Cocaína/metabolismo , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D3/metabolismo , Transtornos do Sono-Vigília/complicações , Transtornos do Sono-Vigília/metabolismo , Adulto , Encéfalo/diagnóstico por imagem , Feminino , Humanos , Entrevistas como Assunto , Masculino , Tomografia por Emissão de Pósitrons , Fatores de TempoRESUMO
In alcohol-dependent (AD) patients, alcohol cues induce strong activations in brain areas associated with alcohol craving and relapse, such as the nucleus accumbens (NAc) and amygdala. However, little is known about the influence of depressive symptoms, which are common in AD patients, on the brain's reactivity to alcohol cues. The methylation state of the dopamine transporter gene (DAT) has been associated with alcohol dependence, craving and depression, but its influence on neural alcohol cue reactivity has not been tested. Here, we compared brain reactivity to alcohol cues in 38 AD patients and 17 healthy controls (HCs) using functional magnetic resonance imaging and assessed the influence of depressive symptoms and peripheral DAT methylation in these responses. We show that alcoholics with low Beck's Depression Inventory scores (n=29) had higher cue-induced reactivity in NAc and amygdala than those with mild/moderate depression scores (n=9), though subjective perception of craving was higher in those with mild/moderate depression scores. We corroborated a higher DAT methylation in AD patients than HCs, and showed higher DAT methylation in AD patients with mild/moderate than low depression scores. Within the AD cohort, higher methylation predicted craving and, at trend level (P=0.095), relapse 1 year after abstinence. Finally, we show that amygdala cue reactivity correlated with craving and DAT methylation only in AD patients with low depression scores. These findings suggest that depressive symptoms and DAT methylation are associated with alcohol craving and associated brain processes in alcohol dependence, which may have important consequences for treatment. Moreover, peripheral DAT methylation may be a clinically relevant biomarker in AD patients.
Assuntos
Alcoolismo , Tonsila do Cerebelo/fisiopatologia , Depressão , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Núcleo Accumbens/fisiopatologia , Adulto , Alcoolismo/complicações , Alcoolismo/metabolismo , Fissura/fisiologia , Sinais (Psicologia) , Depressão/diagnóstico , Depressão/etiologia , Depressão/fisiopatologia , Etanol/metabolismo , Etanol/farmacologia , Feminino , Humanos , Imageamento por Ressonância Magnética/métodos , Masculino , Metilação , Pessoa de Meia-Idade , Fatores de Proteção , Escalas de Graduação Psiquiátrica , Prevenção Secundária , Estatística como AssuntoRESUMO
OBJECTIVE: Nef was shown to be the predominant viral protein expressed in HIV-1-infected astrocytes in vivo and in vitro suggesting a distinct role of Nef in this cell type. Nef-induced activation of T cells is well described, whereas the functional activities of Nef in astrocytes are unknown. Our aim was to examine the effect of Nef on growth properties and activation of astrocytes. DESIGN: Human Nef-expressing astrocytic cell lines were established by stable transfection with different wild-type and mutant nef genes derived from laboratory isolates and brain tissue. METHODS: Nef-expressing astrocytes were characterized in terms of growth properties (proliferation, growth in soft agar, focus formation) and morphology. Apoptotic cell death and expression of activation markers were determined by fluorescent antibody cell sorting. RESULTS: Astrocytic cell lines revealed persistent Nef expression--detectable at the levels of mRNA and protein--and showed altered growth properties and morphology. Elevated expression of activation markers such as glial fibrillary acidic protein and CD88 (complement receptor C5a) was observed; these are regarded as markers for inflammatory processes in the brain. This effect was independent of the nef type or the expression level of the Nef protein. In contrast with previous reports no evidence for increased apoptotic cell death was found in astrocytes expressing Nef stably. CONCLUSIONS: Our findings suggest that Nef changes the cellular properties of astrocytes, thus contributing to astrocyte activation and induction of astrogliosis in the central nervous system of individuals with AIDS.
Assuntos
Astrócitos/patologia , Astrócitos/virologia , Genes nef , HIV-1/genética , HIV-1/patogenicidade , Apoptose , Astrócitos/fisiologia , Sequência de Bases , Divisão Celular , Linhagem Celular , Primers do DNA/genética , Expressão Gênica , Produtos do Gene nef/genética , Proteína Glial Fibrilar Ácida/fisiologia , Infecções por HIV/imunologia , Infecções por HIV/patologia , Infecções por HIV/virologia , HIV-1/imunologia , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/imunologia , Linfócitos T/virologia , Virulência/genética , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
To study the effects of HIV-1 Nef on CNS-derived cells in vivo, an expression system based on the murine neural stem cell line C17.2 was established. Stable expression of LAV-1(Bru)-nef in these cells induced a transformed phenotype and enhanced cell growth in soft agar. Further experiments using previously established nef-expressing human astrocytoma cell lines as well as nef-expressing murine fibroblasts suggested a brain cell-specific transforming activity of Nef. After implantation into syngeneic or nude mice both murine and human nef-expressing CNS-derived cells induced tumor development. Interestingly, human astrocytoma cells expressing a Nef mutant carrying a disrupted SH3-binding motif involved in protein-protein interactions failed to induce tumor formation. These in vivo data suggest that Nef promotes neoplastic transformation of immortalized murine neural stem cells and enhances malignancy of low-tumorigenic human astrocytoma cells. Nef may therefore be involved in the development of AIDS-associated brain tumors.
Assuntos
Transformação Celular Neoplásica/genética , Sistema Nervoso Central/citologia , Produtos do Gene nef/fisiologia , Genes nef , Infecções por HIV/complicações , HIV-1/genética , Animais , Astrocitoma/etiologia , Astrocitoma/patologia , Neoplasias Encefálicas/etiologia , Neoplasias Encefálicas/patologia , Sistema Nervoso Central/patologia , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Transferência de Genes , Infecções por HIV/genética , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Fenótipo , Células Tumorais Cultivadas , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
Low dopamine D2 receptor (D2R) levels in the striatum are consistently reported in cocaine abusers; inter-individual variations in the degree of the decrease suggest a modulating effect of genetic makeup on vulnerability to addiction. The PER2 (Period 2) gene belongs to the clock genes family of circadian regulators; circadian oscillations of PER2 expression in the striatum was modulated by dopamine through D2Rs. Aberrant periodicity of PER2 contributes to the incidence and severity of various brain disorders, including drug addiction. Here we report a newly identified variable number tandem repeat (VNTR) polymorphism in the human PER2 gene (VNTR in the third intron). We found significant differences in the VNTR alleles prevalence across ethnic groups so that the major allele (4 repeats (4R)) is over-represented in non-African population (4R homozygosity is 88%), but not in African Americans (homozygosity 51%). We also detected a biased PER2 genotype distribution among healthy controls and cocaine-addicted individuals. In African Americans, the proportion of 4R/three repeat (3R) carriers in healthy controls is much lower than that in cocaine abusers (23% vs 39%, P=0.004), whereas among non-Africans most 3R/4R heterozygotes are healthy controls (10.5% vs 2.5%, P=0.04). Analysis of striatal D2R availability measured with positron emission tomography and [(11)C]raclopride revealed higher levels of D2R in carriers of 4R/4R genotype (P<0.01). Taken together, these results provide preliminary evidence for the role of the PER2 gene in regulating striatal D2R availability in the human brain and in vulnerability for cocaine addiction.
Assuntos
Alelos , Transtornos Relacionados ao Uso de Cocaína/genética , Corpo Estriado/metabolismo , Marcadores Genéticos/genética , Predisposição Genética para Doença/genética , Variação Genética/genética , Proteínas Circadianas Period/genética , Receptores de Dopamina D2/genética , Adulto , Negro ou Afro-Americano/genética , Asiático/genética , Transtornos Relacionados ao Uso de Cocaína/etnologia , Computadores Moleculares , Corpo Estriado/diagnóstico por imagem , Feminino , Frequência do Gene/genética , Triagem de Portadores Genéticos , Genética Populacional , Genótipo , Hispânico ou Latino/genética , Homozigoto , Humanos , Masculino , Repetições Minissatélites/genética , Polimorfismo Genético/genética , Tomografia por Emissão de Pósitrons , População Branca/genéticaRESUMO
AKAPs (A-kinase anchoring proteins) are members of a diverse family of scaffold proteins that minimally possess a characteristic binding domain for the RI/RII regulatory subunit of protein kinase A and play critical roles in establishing spatial constraints for multivalent signalling assemblies. Especially for G-protein-coupled receptors, the AKAPs provide an organizing centre about which various protein kinases and phosphatases can be assembled to create solid-state signalling devices that can signal, be modulated and trafficked within the cell. The structure of AKAP250 (also known as gravin or AKAP12), based on analyses of milligram quantities of recombinant protein expressed in Escherichia coli, suggests that the AKAP is probably an unordered scaffold, acting as a necklace on which 'jewels' of structure-function (e.g. the RII-binding domain) that provide docking sites on which signalling components can be assembled. Recent results suggest that AKAP250 provides not only a 'tool box' for assembling signalling elements, but may indeed provide a basis for spatial constraint observed for many signalling paradigms. The spatial dimension of the integration of cell signalling will probably reflect many functions performed by members of the AKAP family.
Assuntos
Proteínas de Transporte/química , Proteínas de Ligação ao GTP/química , Proteínas de Ancoragem à Quinase A , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Escherichia coli/metabolismo , Humanos , Bicamadas Lipídicas/química , Microscopia de Fluorescência , Modelos Biológicos , Proteína Quinase C/metabolismo , Estrutura Terciária de Proteína , Proteínas/química , Proteínas/fisiologia , Proteínas Recombinantes/química , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
In T-cells HIV-1 Nef exerts various functions and interacts with actin. In astrocytes interaction of Nef with cellular proteins is poorly understood. Therefore, human astrocytic cell clones stably transfected with nef-genes derived from HIV-1 Bru and its myristoylation-defective TH-variant were investigated by confocal laser scanning microscopy for expression of Nef and cytoskeleton proteins actin and GFAP, a marker for activated astrocytes. Myristoylated Nef was detected in cytoplasm, Golgi and plasmamembrane, while non-myristoylated Nef was exclusively cytoplasmic. Nef co-localised with GFAP in the perinuclear region of astrocytes. In contrast, Nef did not interact with actin filaments in human astrocytes. Nef/GFAP interaction could contribute to changes in morphology and activation state of astrocytes shown previously which are both critical for development of astrogliosis in HIV-1 infected brain.
Assuntos
Astrócitos/metabolismo , Citoesqueleto/metabolismo , Produtos do Gene nef/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , HIV-1 , Actinas/análise , Actinas/biossíntese , Astrócitos/citologia , Linhagem Celular , Membrana Celular/metabolismo , Citoplasma/metabolismo , Expressão Gênica , Produtos do Gene nef/análise , Produtos do Gene nef/genética , Proteína Glial Fibrilar Ácida/análise , Complexo de Golgi/metabolismo , HIV-1/genética , Humanos , Microscopia Confocal , Ácidos Mirísticos/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
The cyclic AMP-dependent kinase-anchoring proteins (AKAPs) function as scaffolds for a wide-range of protein-protein interactions. The 250-kDa AKAP known as gravin plays a central role in organizing G-protein-coupled receptors to the protein kinases and phosphatases that regulate receptor function in desensitization, resensitization, and sequestration. Although gravin is critical for G-protein-linked receptor biology, the molecular features of the receptor necessary for interaction with this scaffold are not known. Herein, we map the regions of the beta(2)-adrenergic receptor that are required for binding to gravin. Intracellular loops 1, 2, and 3 appear not to participate in the binding of the receptor to the scaffold. In contrast, the C-terminal cytoplasmic region of the receptor (Arg-329 to Leu-413) competes readily for the binding of the beta(2)-adrenergic receptor by gravin, both using in vitro and in vivo assays. C-terminally truncated peptides with sequences ranging from Arg-329 to Leu-342 (13 aminoacyl residues), to Asn-352 (23 residues), to Tyr-366 (37 residues), to Asp-380 (51 residues), or to His-390 (61 residues), as well as N-terminally truncated peptides from Gln-391 to Leu-413 (23 residues) or Leu-381 to Leu-413 (33 residues) displayed no ability to block binding of receptor to gravin. The combination of Arg-329 to His-390 peptide and Gln-391 to Leu-413 peptide, however, reconstitutes a fragmented but full-length C-terminal region and also potently blocks the ability of gravin to bind the beta(2)-adrenergic receptor. The gravin-receptor interaction was examined in response to agonist by confocal microscopy. Remarkably, the association of the receptor with gravin was not disrupted during agonist-induced sequestration. The receptor-scaffold complex was maintained during agonist-induced sequestration. These data, in agreement with the biochemical data, reveal that gravin binds the receptor through the beta(2)-adrenergic receptor C-terminal cytoplasmic domain and that this interaction is maintained as the receptor is internalized. This is the first report of an AKAP scaffold protein translocating with its receptor, in this case a G-protein-coupled receptor.
Assuntos
Proteínas/metabolismo , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Proteínas de Ancoragem à Quinase A , Arginina/química , Arrestinas/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação para Baixo , Quinase 2 de Receptor Acoplado a Proteína G , Humanos , Leucina/química , Substâncias Macromoleculares , Microscopia de Fluorescência , Modelos Biológicos , Fragmentos de Peptídeos/farmacologia , Ligação Proteica , Estrutura Terciária de Proteína , Receptores Adrenérgicos beta 2/genética , Proteínas Recombinantes de Fusão/metabolismo , Células Tumorais Cultivadas , Quinases de Receptores Adrenérgicos beta , beta-ArrestinasRESUMO
The nonreceptor tyrosine kinase Src has been implicated in the switching of signaling of beta2-adrenergic receptors from adenylylcyclase coupling to the mitogen-activated protein kinase pathway. In the current work, we demonstrate that Src plays an active role in the agonist-induced desensitization of beta2-adrenergic receptors. Both the expression of dominant-negative Src and treatment with the 4-amine-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) inhibitor of Src kinase activity blocks agonist-induced desensitization. Agonist triggers tyrosine phosphorylation of the beta2-adrenergic receptor and recruitment and activation of Src. Because phosphorylation of the Tyr-350 residue of the beta2-adrenergic receptor creates a conditional, canonical SH2-binding site on the receptor, we examined the effect of the Y350F mutation on Src phosphorylation, Src recruitment, and desensitization. Mutant beta2-adrenergic receptors with a Tyr-to-Phe substitution at Tyr-350 do not display agonist-induced desensitization, Src recruitment, or Src activation. Downstream of binding to the receptor, Src phosphorylates and activates G-protein-linked receptor kinase 2 (GRK2), a response obligate for agonist-induced desensitization. Constitutively active Src increases GRK phosphorylation, whereas either expression of dominant-negative Src or treatment with the PP2 inhibitor abolishes tyrosine phosphorylation of GRK and desensitization. Thus, in addition to its role in signal switching to the mitogen-activated protein kinase pathway, Src recruitment to the beta2-adrenergic receptor and activation are obligate for normal agonist-induced desensitization.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores Adrenérgicos beta 2/fisiologia , Substituição de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Proteína Tirosina Quinase CSK , Carcinoma de Células Escamosas , Cricetinae , Genes Reporter , Proteínas de Fluorescência Verde , Humanos , Iodocianopindolol/farmacologia , Isoproterenol/farmacologia , Proteínas Luminescentes/análise , Proteínas Luminescentes/biossíntese , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Fosforilação , Fosfotirosina , Proteínas Tirosina Quinases/genética , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Transfecção , Células Tumorais Cultivadas , Quinases de Receptores Adrenérgicos beta , Domínios de Homologia de src , Quinases da Família srcRESUMO
Lithium is a monovalent cation used therapeutically to treat a range of affective disorders (1), although the cellular mechanisms of lithium regulation that might contribute to its therapeutic effects at the level of neurotransmitter receptors are not known. Herein we report the ability of lithium to stimulate the internalization of beta2-adrenergic receptors. Lithium treatment of A431 human epidermoid carcinoma cells resulted in a rapid, prominent desensitization and internalization of beta2-adrenergic receptors. The ability of these receptors to generate a cyclic AMP response was strongly inhibited by lithium, at concentrations therapeutic in humans. Receptors for the serotonin (5HT1c) and for opiates (mu-opioid), in sharp contrast, resisted the effects of lithium on internalization. These data provide the first receptor-based mechanism to be described for lithium that could explain, in part, the therapeutic effects of lithium on affective disorders.
Assuntos
Antagonistas de Receptores Adrenérgicos beta 2 , Antimaníacos/farmacologia , Lítio/farmacologia , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais/efeitos dos fármacos , AMP Cíclico/biossíntese , Endocitose , Humanos , Cinética , Microscopia de Fluorescência , Receptores Opioides mu/metabolismo , Receptores de Serotonina/metabolismo , Células Tumorais CultivadasRESUMO
In the human immunodeficiency virus type 1 (HIV-1)-infected brain, the virus does not replicate in astrocytes, but a synthesis of viral regulatory proteins occurs in these cells, leading to accumulation of Nef. As an approach to understand the effects of Nef on astrocyte functional activity, we analyzed whether intracellular Nef interferes with the expression and activation of the enzyme protein kinase C (PKC), which is an important regulator of astroglial functions and HIV-1 replication. Astrocytoma clones (U251 MG) not expressing Nef (Neo), or expressing wild-type Nef (Bru) or nonmyristoylated Nef (TH) were used to monitor the expression and activation of 10 PKC isoforms. The same clones were used to evaluate the effect of Nef on the viral long terminal repeat (LTR) promoter after activation of PKC with the phorbol ester 12-myristate 13-acetate (PMA). PKC intracellular distribution and activation were evaluated by Western blot analysis of cytosolic and membrane fractions of control and Nef-expressing clones. PMA-induced LTR activation was analyzed in clones transfected with a plasmid encoding for the CAT reporter gene controlled by the LTR promoter, by using an enzyme-linked immunosorbent assay to measure CAT expression. Nef selectively downregulated the expression and activation of betaII and epsilon PKC isoforms in astrocytoma cells. Such downregulation correlated with an inhibition of LTR activation after PMA stimulation. The myristoylation of Nef and its membrane localization were essential for these effects. These results suggest that Nef may alter astrocytic functions by interfering with PKC expression and activation and contribute to the restriction of HIV-1 replication in astrocytes.
Assuntos
Astrocitoma/patologia , Neoplasias Encefálicas/patologia , Regulação Neoplásica da Expressão Gênica , Produtos do Gene nef/fisiologia , HIV-1/fisiologia , Isoenzimas/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Proteína Quinase C/biossíntese , Sequências Repetidas Terminais , Complexo AIDS Demência/enzimologia , Complexo AIDS Demência/virologia , Acilação , Astrocitoma/enzimologia , Neoplasias Encefálicas/enzimologia , Cloranfenicol O-Acetiltransferase/biossíntese , Indução Enzimática/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Produtos do Gene nef/química , Genes Reporter , Humanos , Isoenzimas/genética , Ácido Mirístico/metabolismo , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteínas do Tecido Nervoso/genética , Regiões Promotoras Genéticas , Proteína Quinase C/genética , Proteína Quinase C beta , Proteína Quinase C-épsilon , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/biossíntese , Acetato de Tetradecanoilforbol/farmacologia , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacos , Replicação Viral , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
Alterations in the vascular system and the onset of angioproliferative lesions such as Kaposi's sarcoma (KS) are common traits of human immunodeficiency virus-1 (HIV-1)-infected patients. To investigate possible factors involved in acquired immunodeficiency syndrome (AIDS)-associated vasculopathy and vascular malfunction, expression of vascular endothelial cell growth factor-A (VEGF-A) was analyzed in HUT 78 T lymphocytes upon infection with HIV-1. VEGF-A was found to be increased in supernatants from infected cells as compared with uninfected cells. In addition, VEGF-A mRNA expression and protein secretion were significantly increased in HUT 78 cells incubated with conditioned medium (CM) derived from HIV-1 chronically infected HUT 78 cells (HIV-TCM) as compared with CM from uninfected cells (TCM). Increase of VEGF-A production in T cells was promoted by inflammatory cytokines (IC) present in HIV-TCM, including tumor necrosis factor alpha (TNFalpha), interferon gamma (IFNgamma), interleukin-1beta (IL-1beta), and IL-6. These IC that have been shown to be increased in sera of HIV-1-infected patients and to be increased by HIV-1 infection or cell activation in these individuals as well as HIV-TCM also increased VEGF-A expression in primary T lymphocytes. Consistent with this, VEGF-A concentrations were found to be higher in sera of HIV-1-infected patients with (mean, 357.1 +/- 197.9 pg/mL) and without KS (mean, 256.7 +/- 137.5 pg/mL) as compared with uninfected individuals (mean, 188.6 +/- 91.7 pg/mL). These data suggest that increased secretion of VEGF-A by T lymphocytes of HIV-1-infected individuals may induce vascular leakage and stimulate proliferation of vascular endothelial cells, which are hallmarks of AIDS-associated vasculopathy and especially of KS development.