Oligogalacturonic Acid Inhibits Vascular Calcification by Two Mechanisms: Inhibition of Vascular Smooth Muscle Cell Osteogenic Conversion and Interaction With Collagen.

OBJECTIVE: Cardiovascular diseases constitute the leading cause of mortality worldwide. Calcification of the vessel wall is associated with cardiovascular morbidity and mortality in patients having many diseases, including diabetes mellitus, atherosclerosis, and chronic kidney disease. Vascular calcification is actively regulated by inductive and inhibitory mechanisms (including vascular smooth muscle cell adaptation) and results from an active osteogenic process. During the calcification process, extracellular vesicles (also known as matrix vesicles) released by vascular smooth muscle cells interact with type I collagen and then act as nucleating foci for calcium crystallization. Our primary objective was to identify new, natural molecules that inhibit the vascular calcification process. APPROACH AND RESULTS: We have found that oligogalacturonic acids (obtained by the acid hydrolysis of polygalacturonic acid) reduce in vitro inorganic phosphate-induced calcification of vascular smooth muscle cells by 80% and inorganic phosphate-induced calcification of isolated rat aortic rings by 50%. A specific oligogalacturonic acid with a degree of polymerization of 8 (DP8) was found to inhibit the expression of osteogenic markers and, thus, prevent the conversion of vascular smooth muscle cells into osteoblast-like cells. We also evidenced in biochemical and immunofluorescence assays a direct interaction between matrix vesicles and type I collagen via the GFOGER sequence (where single letter amino acid nomenclature is used, O=hydroxyproline) thought to be involved in interactions with several pairs of integrins. CONCLUSIONS: DP8 inhibits vascular calcification development mainly by inhibition of osteogenic marker expression but also partly by masking the GFOGER sequence-thereby, preventing matrix vesicles from binding to type I collagen.

MR1B, a natural spliced isoform of the MHC-related 1 protein, is expressed as homodimers at the cell surface and activates MAIT cells.

The MHC-related 1 (MR1) protein is a monomorphic, evolutionarily conserved MHC class I-like molecule, which is necessary for the development and functions of mucosal-associated invariant T (MAIT) cells, a new subset of innate-like lymphocytes. Multiple isoforms of the MR1 gene are naturally transcribed, but only the full-length MR1A has been analyzed so far. Using transfected cell lines expressing an alternative spliced transcript, MR1B, characterized by the absence of the α3 extracellular domain, we show that MR1B is transcribed and glycosylated but remains in an immature (endoglycosidase H-sensitive) state. MR1B mostly accumulates in the ER, without interacting with proteins of the peptide-loading complex such as tapasin. Interestingly, it is nevertheless found expressed at the cell surface, independently of ß2-microglobulin, in a homodimeric form. MR1B is functional as its overexpression induces MAIT cell activation in vitro in the presence of bacteria. Altogether, these data show that MR1B displays several remarkable features, and probably plays a physiological role complementary to MR1A with respect to MAIT cell development and/or function.

Biochemical characterization of spontaneous mutants of rat VKORC1 involved in the resistance to antivitamin K anticoagulants.

Antivitamin K anticoagulants have been commonly used to control rodent pest all over the world for more than 50 years. These compounds target blood coagulation by inhibiting the vitamin K epoxide reductase (VKORC1), which catalyzes the reduction of vitamin K 2,3-epoxide to vitamin K. Resistance to anticoagulants has been reported in wild rat populations from different countries. From these populations, several mutations of the rVkorc1 gene have been reported. In this study, rat VKORC1 and its most frequent mutants L120Q-, L128Q-, Y139C-, Y139S- and Y139F-VKORC1 were expressed as membrane-bound proteins in Pichia pastoris and characterized by the determination of kinetic and inhibition parameters. The recombinant rVKORC1 showed similar properties than those of the native proteins expressed in the rat liver microsomes, validating the expression system as a good model to study the consequences of VKORC1 mutations. The determination of the inhibition parameters towards various antivitamin K anticoagulants demonstrated that mutations at Leu-120, Leu-128 and Tyr-139 confer the resistance to the first generation AVKs observed in wild rat populations.

Factors involved in CLL pathogenesis and cell survival are disrupted by differentiation of CLL B-cells into antibody-secreting cells.

Recent research has shown that chronic lymphocytic leukemia (CLL) B-cells display a strong tendency to differentiate into antibody-secreting cells (ASCs) and thus may be amenable to differentiation therapy. However, the effect of this differentiation on factors associated with CLL pathogenesis has not been reported. In the present study, purified CLL B-cells were stimulated to differentiate into ASCs by phorbol myristate acetate or CpG oligodeoxynucleotide, in combination with CD40 ligand and cytokines in a two-step, seven-day culture system. We investigated (i) changes in the immunophenotypic, molecular, functional, morphological features associated with terminal differentiation into ASCs, (ii) the expression of factors involved in CLL pathogenesis, and (iii) the expression of pro- and anti-apoptotic proteins in the differentiated cells. Our results show that differentiated CLL B-cells are able to display the transcriptional program of ASCs. Differentiation leads to depletion of the malignant program and deregulation of the apoptosis/survival balance. Analysis of apoptosis and the cell cycle showed that differentiation is associated with low cell viability and a low rate of cell cycle entry. Our findings shed new light on the potential for differentiation therapy as a part of treatment strategies for CLL.

New insights into the catalytic mechanism of vitamin K epoxide reductase (VKORC1) - The catalytic properties of the major mutations of rVKORC1 explain the biological cost associated to mutations.

The systematic use of antivitamin K anticoagulants (AVK) as rodenticides caused the selection of rats resistant to AVKs. The resistance is mainly associated to genetic polymorphisms in the Vkorc1 gene encoding the VKORC1 enzyme responsible for the reduction of vitamin K 2,3-epoxide to vitamin K. Five major mutations, which are responsible for AVK resistance, have been described. Possible explanations for the biological cost of these mutations have been suggested. This biological cost might be linked to an increase in the vitamin K requirements. To analyze the possible involvement of VKORC1 in this biological cost, rVKORC1 and its major mutants were expressed in Pichia pastoris as membrane-bound proteins and their catalytic properties were determined for vitamin K and 3-OH-vitamin K production. In this report, we showed that mutations at Leu-120 and Tyr-139 dramatically affect the vitamin K epoxide reductase activity. Moreover, this study allowed the detection of an additional production of 3-hydroxyvitamin K for all the mutants in position 139. This result suggests the involvement of Tyr-139 residue in the second half-step of the catalytic mechanism corresponding to the dehydration of vitamin K epoxide. As a consequence, the biological cost observed in Y139C and Y139S resistant rat strains is at least partially explained by the catalytic properties of the mutated VKORC1 involving a loss of vitamin K from the vitamin K cycle through the formation of 3-hydroxyvitamin K and a very low catalytic efficiency of the VKOR activity.