We have previously shown that a Taiwanese cohort of HIV-uninfected individuals was associated with the significantly elevated levels of serum beta-chemokines, macrophage inflammatory protein (MIP-1)-alpha and MIP-beta, and RANTES. In the present study, we report that the members of this cohort have significantly greater numbers of lower buoyant-density neutrophils in their blood, which leads to further investigation of the effects of beta-chemokines on neutrophils. By electron and confocal microscopic techniques and FACScan, the results demonstrated that MIP-1alpha, MIP-beta, and/or RANTES readily activated the cells to release a large quantity of alpha-defensins in vitro through the degranulation process, which was the cause of low-buoyant-density neutrophil production. The purified neutrophils underwent chemotaxis and increased phagocytic capability when beta-chemokines were present. Only when using all 3 neutralizing antibodies for CCR1, CCR3, and CCR5 could the chemotaxis of neutrophils be inhibited completely, suggesting that these receptors are involved in transducing activating signals. Because neutrophils are the most abundant white blood cells that can be activated simultaneously to release alpha-defensins and because these proteins are antiviral, including anti-HIV, our results support the hypothesis that in addition to beta-chemokines, the innate immunity of the cohort plays a role in inhibiting the transmission of HIV.
Two chemokine (C-X3-C) receptor 1 (CX3CR1) gene polymorphisms, V249I and T280M, and 10 CC chemokine receptor 5 (CCR5) promoter haplotypes, P1-P10, have recently been reported to influence the progression of acquired immune-deficiency syndrome (AIDS). As these studies were performed mainly with Caucasian and African-American subjects, we determined the distribution of these alleles in Chinese people for the purpose of predicting possible clinical responses to the human immunodeficiency virus type 1 (HIV) epidemics in countries with significant Chinese populations, as well as to establish their effects on the expression of surface CCR5. Ninety-six HIV-negative Chinese individuals in Taiwan were subjected to genotyping, and we thus determined that the allelic frequencies of CX3CR1V249I and T280M changes were 2.6% and 2.1%, respectively, which were lower than found in Caucasians (25.5% and 14.0%, respectively). Unlike the previous reports, we only detected CCR5P1 and P4 haplotypes in Taiwanese people, and the P1/P1, P1/P4 and P4/P4 genotype frequencies were 21.0%, 41.1% and 37.9%, respectively. The sequencing data confirmed the results of previous studies, showing that CCR5P1 exhibited a complete linkage disequilibrium with a polymorphic allele 59029A present in the CCR5 promoter. Furthermore, fluorescence-activated cell sorter analysis revealed that, in the absence of the CCR2-64I mutation, individuals carrying CCR5P1 tended to express more surface CCR5 on monocytes and CD4+ cells. Therefore, this study not only reports the frequencies for the CX3CR1 and CCR5 promoter haplotypes in a Chinese population living in Taiwan, but also identifies a statistical link between the P1/P1 haplotype and the elevated CCR5 expression levels in the study group.
SummaryThe molecular mechanism mediated by multiple forms of angiostatin via acting on proliferating vascular endothelium remains elusive. To address whether three forms of angiostatin, K1-3, K1-4 or K1-4.5, utilized similar or distinct pathways to mediate anti-angiogenesis, we adopted an adenoviral expression system to express secretable angiostatin molecules for CM collection. The anti-angiogenic activity of K1-3, K1-4 or K1-4.5 was confirmed by using proliferation, migration, tube formation and apoptotic assays of human endothelial cells. These angiostatin molecules at comparable expression level inhibited various in vitro angiogenesis assays with some variations. Furthermore, K1-3, K1-4 or K1-4.5 increased the expression of p53 protein and its downstream effectors, enhanced FasL-mediated signaling pathways, and decreased activation of AKT. At least three different receptors, Fas, integrin αvβ3 and ATP synthase, were involved in the anti-angiogenic action of angiostatin molecules. Besides, the expression of 189 genes at mRNA level was significantly altered by K1-3, K1-4 or K1-4.5. More than 70% of these genes participate in growth, inflammation, apoptosis, migration and extracellular matrix. Taken together, K1-3, K1-4 and K1-4.5, regardless of the number of kringles in the angiostatin molecules, mediated anti-angiogenesis via mostly similar pathways. We are the first to demonstrate the involvement of DAPK1 in the mediation of anti-angiogenesis by angiostatin.
-W. Angiostatin K1-3 induces E-selectin via AP1 and Ets1: a mediator for anti-angiogenic action of K1-3. J Thromb Haemost 2008; 6: 1953-61. Summary. Background: Angiostatin, a circulating angiogenic inhibitor, is an internal fragment of plasminogen and consists of several isoforms, K1-3 included. We previously showed that K1-3 was the most potent angiostatin to induce E-selectin mRNA expression. The purpose of this study was to identify the mechanism responsible for K1-3-induced E-selectin expression and investigate the role of E-selectin in the anti-angiogenic action of K1-3. Methods and results: Quantitative real time RT-PCR and Western blotting analyses confirmed a timedependent increase of E-selectin mRNA and protein induced by K1-3. Subcellular fractionation and immunofluorescence microscopy showed the co-localization of K1-3-induced Eselectin with caveolin 1 (Cav1) in lipid rafts in which E-selectin may behave as a signaling receptor. Promoter-driven reporter assays and site-directed mutagenesis showed that K1-3 induced E-selectin expression via promoter activation and AP1 and Ets-1 binding sites in the proximal E-selectin promoter were required for E-selectin induction. The in vivo binding of both protein complexes to the proximal promoter was confirmed by chromatin immunoprecipitation (ChIP). Although K1-3 induced the activation of ERK1/2 and JNK, only repression of JNK activation attenuated the induction of E-selectin by K1-3. A modulatory role of E-selectin in the anti-angiogenic action of K1-3 was manifested by both overexpression and knockdown of E-selectin followed by cell proliferation assay. Conclusions: We show that K1-3 induced E-selectin expression via AP1 and Ets-1 binding to the proximal E-selectin promoter ()356/+1), which was positively mediated by JNK activation. Our findings also demonstrate E-selectin as a novel target for the anti-angiogenic therapy.
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