Antoine Geinoz

Heterogeneity of Smooth Muscle Cell Populations Cultured From Pig Coronary Artery

Objective — Heterogeneous smooth muscle cell (SMC) populations have been described in the arteries of several species. We have investigated whether SMC heterogeneity is present in the porcine coronary artery, which is widely used as a model of restenosis. Methods and Results — By using 2 isolation methods, distinct medial populations were identified: spindle-shaped SMCs (S-SMCs) after enzymatic digestion, with a “hill-and-valley” growth pattern, and rhomboid SMCs (R-SMCs) after explantation, which grow as a monolayer. Moreover, the intimal thickening that was induced after stent implantation yielded a large proportion of R-SMCs. R-SMCs exhibited high proliferative and migratory activities and high urokinase activity and were poorly differentiated compared with S-SMCs. Heparin and transforming growth factor-β2 inhibited proliferation and increased differentiation in both populations, whereas fibroblast growth factor-2 and platelet-derived growth factor-BB had the opposite effect. In addition, S-SMCs treated with fibroblast growth factor-2 or platelet-derived growth factor-BB or placed in coculture with coronary artery endothelial cells acquired a rhomboid phenotype. This change was reversible and was also observed with S-SMC clones, suggesting that it depends on phenotypic modulation rather than on selection. Conclusions — Our results show that 2 distinct SMC subpopulations can be recovered from the pig coronary artery media. The study of these subpopulations will be useful for understanding the mechanisms of restenosis.

Intimal Smooth Muscle Cells of Porcine and Human Coronary Artery Express S100A4, a Marker of the Rhomboid Phenotype In Vitro

We reported that smooth muscle cell (SMC) populations isolated from normal porcine coronary artery media exhibit distinct phenotypes: spindle-shaped (S) and rhomboid (R). R-SMCs are recovered in higher proportion from stent-induced intimal thickening compared with media suggesting that they participate in intimal thickening formation. Our aim was to identify a marker of R-SMCs in vitro and to explore its possible expression in vivo. S- and R-SMC protein extracts were compared by means of 2-dimensional polyacrylamide gel electrophoresis followed by tandem mass spectrometry. S100A4 was found to be predominantly expressed in R-SMC extracts. Using a monoclonal S100A4 antibody we confirmed that S100A4 is highly expressed by R-SMCs and hardly detectable in S-SMCs. S100A4 was colocalized with &agr;-smooth muscle actin in stress fibers of several quiescent cells and upregulated during migration. PDGF-BB, FGF-2 or coculture with endothelial cells, which modulate S-SMCs to a R-phenotype, increased S100A4 expression in both S- and R-SMCs. Silencing of S100A4 mRNA in R-SMCs decreased cell proliferation, suggesting a functional role for this protein. In vivo S100A4 was absent in normal porcine coronary artery media, but highly expressed by SMCs of stent-induced intimal thickening. In humans, S100A4 was barely detectable in coronary artery media and markedly expressed in SMCs of atheromatous and restenotic coronary artery lesions. Our results indicate that S100A4 is a marker of porcine R-SMCs in vitro and of intimal SMCs during intimal thickening development. It is also a marker of a large population of human atheromatous and restenotic SMCs. Clarifying S100A4 function might be useful to understand the evolution of atherosclerotic and restenotic processes.

Selective Antibody Intervention of Toll-like Receptor 4 Activation through Fc γ Receptor Tethering

Background: Dysregulated leukocyte activation via Toll-like receptor 4 (TLR4) is central to numerous inflammatory disorders. Results: A novel mechanism of action involving Fc γ receptor tethering allows anti-TLR4 blocking antibodies to achieve increased potency on inflammatory leukocytes. Conclusion: This novel mechanism of action allows selective targeting of TLR4 activation during inflammation. Significance: The data provide a novel mechanism to dampen TLR4-mediated inflammatory disorders. Inflammation is mediated mainly by leukocytes that express both Toll-like receptor 4 (TLR4) and Fc γ receptors (FcγR). Dysregulated activation of leukocytes via exogenous and endogenous ligands of TLR4 results in a large number of inflammatory disorders that underlie a variety of human diseases. Thus, differentially blocking inflammatory cells while sparing structural cells, which are FcγR-negative, represents an elegant strategy when targeting the underlying causes of human diseases. Here, we report a novel tethering mechanism of the Fv and Fc portions of anti-TLR4 blocking antibodies that achieves increased potency on inflammatory cells. In the presence of ligand (e.g. lipopolysaccharide (LPS)), TLR4 traffics into glycolipoprotein microdomains, forming concentrated protein platforms that include FcγRs. This clustering produces a microenvironment allowing anti-TLR4 antibodies to co-engage TLR4 and FcγRs, increasing their avidity and thus substantially increasing their inhibitory potency. Tethering of antibodies to both TLR4 and FcγRs proves valuable in ameliorating inflammation in vivo. This novel mechanism of action therefore has the potential to enable selective intervention of relevant cell types in TLR4-driven diseases.

Extensive remodeling of DC function by rapid maturation-induced transcriptional silencing

The activation, or maturation, of dendritic cells (DCs) is crucial for the initiation of adaptive T-cell mediated immune responses. Research on the molecular mechanisms implicated in DC maturation has focused primarily on inducible gene-expression events promoting the acquisition of new functions, such as cytokine production and enhanced T-cell-stimulatory capacity. In contrast, mechanisms that modulate DC function by inducing widespread gene-silencing remain poorly understood. Yet the termination of key functions is known to be critical for the function of activated DCs. Genome-wide analysis of activation-induced histone deacetylation, combined with genome-wide quantification of activation-induced silencing of nascent transcription, led us to identify a novel inducible transcriptional-repression pathway that makes major contributions to the DC-maturation process. This silencing response is a rapid primary event distinct from repression mechanisms known to operate at later stages of DC maturation. The repressed genes function in pivotal processes—including antigen-presentation, extracellular signal detection, intracellular signal transduction and lipid-mediator biosynthesis—underscoring the central contribution of the silencing mechanism to rapid reshaping of DC function. Interestingly, promoters of the repressed genes exhibit a surprisingly high frequency of PU.1-occupied sites, suggesting a novel role for this lineage-specific transcription factor in marking genes poised for inducible repression.

Pregnancy-Associated Plasma Protein-A (PAPP-A) is a Specific Inhibitor of the Third Component of Human Complement

Pregnancy-associated plasma protein-A (PAPP-A) was discovered in the plasma of pregnant women by Gall and Halbert (1972). It was later shown by Lin et al. (1974a) that PAPP-A was indeed a new pregnancy protein, immunologically different from other known pregnancy proteins.