Identification of IgG1 isotype phosphorylcholine antibodies for the treatment of inflammatory cardiovascular diseases.

BACKGROUND: Phosphorylcholine (PC) is an important pro-inflammatory damage-associated molecular pattern. Previous data have shown that natural IgM anti-PC protects against cardiovascular disease. We aimed to develop a monoclonal PC IgG antibody with anti-inflammatory and anti-atherosclerotic properties. METHODS: Using various techniques PC antibodies were validated and optimized. In vivo testing was performed in a femoral artery cuff model in ApoE3*Leiden mice. Safety studies are performed in rats and cynomolgus monkeys. RESULTS: A chimeric anti-PC (PC-mAb(T15), consisting of a human IgG1 Fc and a mouse T15/E06 Fab) was produced, and this was shown to bind specifically to epitopes in human atherosclerotic tissues. The cuff model results in rapid induction of inflammatory genes and altered expression of genes associated with ER stress and choline metabolism in the lesions. Treatment with PC-mAb(T15) reduced accelerated atherosclerosis via reduced expression of endoplasmic reticulum stress markers and CCL2 production. Recombinant anti-PC Fab fragments were identified by phage display and cloned into fully human IgG1 backbones creating a human monoclonal IgG1 anti-PC (PC-mAbs) that specifically bind PC, apoptotic cells and oxLDL. Based on preventing macrophage oxLDL uptake and CCL2 production, four monoclonal PC-mAbs were selected, which to various extent reduced vascular inflammation and lesion development. Additional optimization and validation of two PC-mAb antibodies resulted in selection of PC-mAb X19-A05, which inhibited accelerated atherosclerosis. Clinical grade production of this antibody (ATH3G10) significantly attenuated vascular inflammation and accelerated atherosclerosis and was tolerated in safety studies in rats and cynomolgus monkeys. CONCLUSIONS: Chimeric anti-PCs can prevent accelerated atherosclerosis by inhibiting vascular inflammation directly and through reduced macrophage oxLDL uptake resulting in decreased lesions. PC-mAb represents a novel strategy for cardiovascular disease prevention.

An unexpected intriguing effect of Toll-like receptor regulator RP105 (CD180) on atherosclerosis formation with alterations on B-cell activation.

OBJECTIVE: In atherosclerosis, Toll-like receptors (TLRs) are traditionally linked to effects on tissue macrophages or foam cells. RP105, a structural TLR4 homolog, is an important regulator of TLR signaling. The effects of RP105 on TLR signaling vary for different leukocyte subsets known to be involved in atherosclerosis, making it unique in its role of either suppressing (in myeloid cells) or enhancing (in B cells) TLR-regulated inflammation in different cell types. We aimed to identify a role of TLR accessory molecule RP105 on circulating cells in atherosclerotic plaque formation. APPROACH AND RESULTS: Irradiated low density lipoprotein receptor deficient mice received RP105(-/-) or wild-type bone marrow. RP105(-/-) chimeras displayed a 57% reduced plaque burden. Interestingly, total and activated B-cell numbers were significantly reduced in RP105(-/-) chimeras. Activation of B1 B cells was unaltered, suggesting that RP105 deficiency only affected inflammatory B2 B cells. IgM levels were unaltered, but anti-oxidized low-density lipoprotein and anti-malondialdehyde-modified low-density lipoprotein IgG2c antibody levels were significantly lower in RP105(-/-) chimeras, confirming effects on B2 B cells rather than B1 B cells. Moreover, B-cell activating factor expression was reduced in spleens of RP105(-/-) chimeras. CONCLUSIONS: RP105 deficiency on circulating cells results in an intriguing unexpected TLR-associated mechanisms that decrease atherosclerotic lesion formation with alterations on proinflammatory B2 B cells.

T-cell co-stimulation by CD28-CD80/86 and its negative regulator CTLA-4 strongly influence accelerated atherosclerosis development.

OBJECTIVE: T-cells are central to the immune response responsible for native atherosclerosis. The objective of this study is to investigate T-cell contribution to post-interventional accelerated atherosclerosis development, as well as the role of the CD28-CD80/86 co-stimulatory and Cytotoxic T-Lymphocyte Antigen (CTLA)-4 co-inhibitory pathways controlling T-cell activation status in this process. METHODS AND RESULTS: The role of T-cells and the CD28-CD80/86 co-stimulatory and CTLA-4 co-inhibitory pathways were investigated in a femoral artery cuff mouse model for post-interventional remodeling, with notable intravascular CTLA-4+ T-cell infiltration. Reduced intimal lesions developed in CD4(-/-) and CD80(-/-)CD86(-/-) mice compared to normal C57Bl/6J controls. Systemic abatacept-treatment, a soluble CTLA-4Ig fusion protein that prevents CD28-CD80/86 co-stimulatory T-cell activation, prevented intimal thickening by 58.5% (p=0.029). Next, hypercholesterolemic ApoE3*Leiden mice received abatacept-treatment which reduced accelerated atherosclerosis development by 78.1% (p=0.040) and prevented CD4 T-cell activation, indicated by reduced splenic fractions of activated KLRG1+, PD1+, CD69+ and CTLA-4+ T-cells. This correlated with reduced plasma interferon-γ and elevated interleukin-10 levels. The role of CTLA-4 was confirmed using CTLA-4 blocking antibodies, which strongly increased vascular lesion size by 66.7% (p=0.008), compared to isotype-treated controls. CONCLUSIONS: T-cell CD28-CD80/86 co-stimulation is vital for post-interventional accelerated atherosclerosis development and is regulated by CTLA-4 co-inhibition, indicating promising clinical potential for prevention of post-interventional remodeling by abatacept.

Annexin A5 prevents post-interventional accelerated atherosclerosis development in a dose-dependent fashion in mice.

BACKGROUND: Activated cells in atherosclerotic lesions expose phosphatidylserine (PS) on their surface. Annexin A5 (AnxA5) binds to PS and is used for imaging atherosclerotic lesions. Recently, AnxA5 was shown to inhibit vascular inflammatory processes after vein grafting. Here, we report a therapeutic role for AnxA5 in post-interventional vascular remodeling in a mouse model mimicking percutaneous coronary intervention (PCI). METHODS AND RESULTS: Associations between the rs4833229 (OR = 1.29 (CI 95%), p(allelic) = 0.011) and rs6830321 (OR = 1.35 (CI 95%), p(allelic) = 0.003) SNPs in the AnxA5 gene and increased restenosis-risk in patients undergoing PCI were found in the GENDER study. To evaluate AnxA5 effects on post-interventional vascular remodeling and accelerated atherosclerosis development in vivo, hypercholesterolemic ApoE(-/-) mice underwent femoral arterial cuff placement to induce intimal thickening. Dose-dependent effects were investigated after 3 days (effects on inflammation and leukocyte recruitment) or 14 days (effects on remodeling) after cuff placement. Systemically administered AnxA5 in doses of 0.1, 0.3 and 1.0mg/kg compared to vehicle reduced early leukocyte and macrophage adherence up to 48.3% (p = 0.001) and diminished atherosclerosis development by 71.2% (p = 0.012) with a reduction in macrophage/foam cell presence. Moreover, it reduced the expression of the endoplasmic reticulum stress marker GRP78/BiP, indicating lower inflammatory activity of the cells present. CONCLUSIONS: AnxA5 SNPs could serve as markers for restenosis after PCI and AnxA5 therapeutically prevents vascular remodeling in a dose-dependent fashion, together indicating clinical potential for AnxA5 against post-interventional remodeling.

Matrix metalloproteinases 2 and 3 gene polymorphisms and the risk of target vessel revascularization after percutaneous coronary intervention: Is there still room for determining genetic variation of MMPs for assessment of an increased risk of restenosis?

OBJECTIVE: Mixed results have been reported of matrix metalloproteinases (MMP) and their association with restenosis after percutaneous coronary intervention (PCI). The current study examines whether multiple single nucleotide polymorphisms (SNPs), covering the full genomic region of MMP2 and MMP3, were associated with restenosis in the GENDER study population. METHODS AND RESULTS: The GENetic DEterminants of Restenosis (GENDER) study enrolled 3104 consecutive patients after successful PCI. The primary endpoint was clinical restenosis, defined as target vessel revascularization (TVR), occurring in 9.8% of the patients. From the Hapmap database, 19 polymorphisms of MMP2 and 11 of MMP3 were selected. Furthermore, in a subpopulation, a genome-wide association analysis (GWA) was performed. No significant association was found with any of the investigated SNPs, including the previously reported 5A/6A polymorphism (rs3025058), with regard to TVR using single SNP analysis or haplotype analysis. CONCLUSION: We found no significant association of MMP2 or MMP3 with TVR with this SNP-broad gene approach. Although we did not test all the known polymorphisms of these genes, using tagging analyses we examined those SNPs covering all known haplotypes of MMP2 and MMP3 to conclude that these genes do not correlate with a genetic risk of coronary restenosis after successful PCI.

Differential cellular regulation of the mitochondrial permeability transition in an in vitro model of 1,3-dinitrobenzene-induced encephalopathy.

Exposure to 1,3-dinitrobenzene (DNB) is associated with neuropathologic changes in specific brainstem nuclei, mediated by oxidative stress and mitochondrial dysfunction. The expression of Bcl-2-family proteins as a function of sensitivity to 1, 3-dinitrobenzene (DNB)-induced mitochondrial permeability transition (MPT) was examined in C6 glioma and SY5Y neuroblastoma cells. Neuroblastoma cells were 10-fold more sensitive than glioma cells to DNB-induced decreases in mitochondrial reducing potential, measured by reduction of the tetrazolium compound, 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). The IC(50) values for DNB-related inhibition of MTT reduction were 107+/-25 microM in SY5Y cells and 1047+/-101 microM in C6 cells. Levels of reactive oxygen species (ROS) were increased in both SY5Y and C6 cells following DNB exposure by 4.6- and 6.0-fold above control, respectively. DNB caused abrupt depolarization of mitochondria in both neuroblastoma and glioma cells that was inhibited by trifluoperazine. The first order rate constants for mitochondrial depolarization were: C6, k=0.31+/-0.02 min(-1); SY5Y, k=0.14+/-0.01 min(-1). Onset of MPT occurred at 10-fold lower concentration of DNB in SY5Y cells than in C6 cells. The antioxidants, deferoxamine and alpha-tocopherol, effectively prevented DNB-induced MPT in C6 and SY5Y cells, suggesting involvement of ROS in the initiation of MPT. Exposure to DNB resulted in decreased cellular ATP content in SY5Y cells and efflux of mitochondrial calcium in both SY5Y and C6 cells, concurrent with onset of MPT. The expression of Bcl-2, Bcl-X(L), and Bax was evaluated in both cell types by Western blot analysis. C6 glioma cells strongly expressed Bcl-X(L) and only weakly expressed Bcl-2 and Bax, whereas SY5Y neuroblastoma cells expressed lower levels of Bcl-X(L) and higher levels of both Bcl-2 and Bax. Collectively, these results suggest that higher constitutive expression of Bcl-X(L), rather than Bcl-2, correlates with resistance to DNB-induced MPT in SY5Y and C6 cells and that differential regulation of the permeability transition pore may underlie the cell-specific neurotoxicity of DNB.