Gla-rich protein function as an anti-inflammatory agent in monocytes/macrophages: implications for calcification-related chronic inflammatory diseases

Calcification-related chronic inflammatory diseases are multifactorial pathological processes, involving a complex interplay between inflammation and calcification events in a positive feed-back loop driving disease progression. Gla-rich protein (GRP) is a vitamin K dependent protein (VKDP) shown to function as a calcification inhibitor in cardiovascular and articular tissues, and proposed as an anti-inflammatory agent in chondrocytes and synoviocytes, acting as a new crosstalk factor between these two interconnected events in osteoarthritis. However, a possible function of GRP in the immune system has never been studied. Here we focused our investigation in the involvement of GRP in the cell inflammatory response mechanisms, using a combination of freshly isolated human leucocytes and undifferentiated/differentiated THP-1 cell line. Our results demonstrate that VKDPs such as GRP and matrix gla protein (MGP) are synthesized and gamma-carboxylated in the majority of human immune system cells either involved in innate or adaptive immune responses. Stimulation of THP-1 monocytes/macrophages with LPS or hydroxyapatite (HA) up-regulated GRP expression, and treatments with GRP or GRP-coated basic calcium phosphate crystals resulted in the down-regulation of mediators of inflammation and inflammatory cytokines, independently of the protein gamma-carboxylation status. Moreover, overexpression of GRP in THP-1 cells rescued the inflammation induced by LPS and HA, by down-regulation of the proinflammatory cytokines TNF alpha, IL-1 beta and NFkB. Interestingly, GRP was detected at protein and mRNA levels in extracellular vesicles released by macrophages, which may act as vehicles for extracellular trafficking and release. Our data indicate GRP as an endogenous mediator of inflammatory responses acting as an anti-inflammatory agent in monocytes/macrophages. We propose that in a context of chronic inflammation and calcification-related pathologies, GRP might act as a novel molecular mediator linking inflammation and calcification events, with potential therapeutic application.Portuguese Science and Technology Foundation (FCT) [PTDC/SAU-ORG/117266/2010, PTDC/BIM-MEC/1168/2012, UID/Multi/ 04326/2013]; FCT fellowships [SFRH/BPD/70277/2010, SFRH/BD/111824/2015

Insulin-like growth factor II plays a central role in atherosclerosis in a mouse model.

Insulin-like growth factor II is a fetal promoter of cell proliferation that is involved in some forms of cancer and overgrowth syndromes in humans. Here, we provide two sources of genetic evidence for a novel, pivotal role of locally produced insulin-like growth factor II in the development of atherosclerosis. First, we show that homozygosity for a disrupted insulin-like growth factor II allele in mice lacking apolipoprotein E, a widely used animal model of atherosclerosis, results in aortic lesions that are approximately 80% smaller and contain approximately 50% less proliferating cells compared with mice lacking only apolipoprotein E. Second, targeted expression of an insulin-like growth factor II transgene in smooth muscle cells, but not the mere elevation of circulating levels of the peptide, causes per se aortic focal intimal thickenings. The insulin-like growth factor II transgenics presented here are the first viable mutant mice spontaneously developing intimal masses. These observations provide the first direct evidence for an atherogenic activity of insulin-like growth factor II in vivo

TAP1-Deficiency Does Not Alter Atherosclerosis Development in <em>Apoe</em>^−/− Mice

<div><p>Antigen presenting cells (APC) have the ability to present both extra-cellular and intra-cellular antigens via MHC class I molecules to CD8⁺ T cells. The cross presentation of extra-cellular antigens is reduced in mice with deficient Antigen Peptide Transporter 1 (TAP1)-dependent MHC class I antigen presentation, and these mice are characterized by a diminished CD8⁺ T cell population. We have recently reported an increased activation of CD8⁺ T cells in hypercholesterolemic <em>Apoe^−/−</em> mice. Therefore, this study included TAP1-deficient <em>Apoe^−/−</em> mice (<em>Apoe^−/−Tap1^−/−</em>) to test the atherogenicity of CD8⁺ T cells and TAP1-dependent cross presentation in a hypercholesterolemic environment. As expected the CD8⁺ T cell numbers were low in <em>Apoe^−/−Tap1^−/−</em> mice in comparison to <em>Apoe^−/−</em> mice, constituting ∼1% of the lymphocyte population. In spite of this there were no differences in the extent of atherosclerosis as assessed by <em>en face</em> Oil Red O staining of the aorta and cross-sections of the aortic root between <em>Apoe^−/−Tap1^−/−</em> and <em>Apoe^−/−</em> mice. Moreover, no differences were detected in lesion infiltration of macrophages or CD3⁺ T cells in <em>Apoe^−/−Tap1^−/−</em> compared to <em>Apoe^−/−</em> mice. The CD3⁺CD4⁺ T cell fraction was increased in <em>Apoe^−/−Tap1^−/−</em> mice, suggesting a compensation for the decreased CD8⁺ T cell population. Interestingly, the fraction of CD8⁺ effector memory T cells was increased but this appeared to have little impact on the atherosclerosis development.</p> <p>In conclusion, <em>Apoe^−/−Tap1^−/−</em> mice develop atherosclerosis equal to <em>Apoe^−/−</em> mice, indicating a minor role for CD8⁺ T cells and TAP1-dependent antigen presentation in the disease process.</p> </div