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Contenido archivado el 2024-05-28

Comparative Study And Mechanisms Of Calcification Heterogeneity In Atherosclerotic Plaques

Final Report Summary - CALTHERO (Comparative Study And Mechanisms Of Calcification Heterogeneity In Atherosclerotic Plaques)

Arterial calcifications in atherosclerotic plaques contribute to morbidity and are predictors of premature death. It is estimated that more than 60% of the population above 60 years-old develop arterial calcification. This question represents therefore a major clinical challenge. Despite exposure to the same risk factors, studies have reported the heterogeneity of atheroma according to the arterial beds. These differences have a major clinical impact on the occurrence of cardiovascular events (embolism, thrombosis, dissection, restenosis), and recurrence of the disease. Our work recently showed different types and contents of calcification among vascular beds, from amorphous mineral deposits, to authentic bone structures often found in femoral arteries. The mechanisms leading to calcification of atherosclerotic lesions remain unclear, but shares many similarities with bone remodeling.

Objectives and impact
The general objective is to identify mechanisms involved in atheromatous calcification and its heterogeneity among vascular beds. To characterize cellular and molecular pathways implicated in this process, we will take advantage of two biocollections, ECLA and ECLAGEN that include lesions from several arterial beds (carotid, thoracic, abdominal, femoral, lower limb). An extensive characterization of lesion composition, in vitro work using atheromatous cells from various vascular beds, and establishment of experimental models of mice developing accelerated calcification in atherosclerotic lesions will contribute to the understanding of calcification mechanisms in vascular beds. Finally, this project will assess the predictive clinical impact of the nature of the plaque (calcified or lipid-rich) on intra-stent restenosis, a major complication following intravascular treatment for PAD. The understanding of the cellular and molecular mechanisms responsible for arterial calcification heterogeneity figures among the most critical questions in cardiovascular research, due to its implications in CVD complications. Beyond its direct impact on cardiovascular diseases, the work presented here could also be valuable in the field of bone metabolism. Indeed, common mechanisms are probably responsible for calcification remodeling in atherosclerotic, diseased arteries and in cartilage and bones. Paradoxically, aging patients developing calcification in arteries can also suffer from osteoporosis or arthritis, characterized by a progressive resorption of the cartilages and bones. Understanding the mechanisms underlying pathological calcification/bone formation in arteries could therefore give clues on the molecular basis of osteoporosis or other bone disorders (i.e. bone tumors).

Work performed
During the course of this funding, we analyzed thoroughly the extent and nature calcification in lesions in each arterial bed, and their potential association with atheromatous and inflammatory cells. Arterial beds develop different lesion and calcification types depending on their anatomical location. Aorta and Carotid artery develop mostly lipid-rich lesions, with predominant amorphous and diffuse calcification (microcalcification, sheet like). Plaques from lower limb (femoral and infrapopliteal arteries) are fibrotic with extensive calcification, sheet-like together with more condensed calcification (nodules). Osteoid metaplasia, actual bone structure, is found mainly in femoral arteries, with scarce presence or complete absence in any other vascular location, suggesting that this arterial bed is specifically predisposed for OM, reinforcing our hypothesis that specific local and/or molecular determinants orient calcification process in each vascular bed.
While quantitatively, at the level of the whole plaque, there was no clear correlation between endothelial cell (EC), smooth muscle cell (SMC), macrophage or pericyte content with any of the calcification type, consistent spatial proximity of SMC with sheet-like calcification, and macrophage (especially M2a subtype) with nodule suggest a role of these cells in their formation. The presence of multiple calcification types in lesions accounts for the difficulty to associate quantitatively cell content and calcification.
To assess the question of biological heterogeneity and molecular pathways involved in calcification heterogeneity, we started the analysis of ECLAGEN microarray. More than 163 samples (atherosclerotic and healthy arteries from 5 locations) were studied. Arteries from different vascular beds present specific transcriptomic profiles. Interestingly, even healthy arteries exhibit specific profiles. Comparable to what was observed in atherosclerotic arteries by histology, carotid arteries and aortas are close in term of genes expression, with specific gene clusters express differentially between these locations. Femoral and popliteal arteries show a drastic difference in gene expression compared to carotid and aorta, but also present distinct signatures. Close analysis of gene specific of femoral arteries, more prone to develop OM, allowed us to identify genes related to bone biology/tissue mineralization. We also selected genes that were not associated with bone biology but showed similar expression pattern. Altogether, this strategy allowed us to focus on 79 genes. Bibliographic analysis and follow-up qRT-PCR validation highlighted a potential role of 4 genes of interest in calcification process.
In vitro, we established that among cell types present in lesions, SMC and pericytes are the ones able to undergo direct osteoblastic differentiation, and are likely to play the role of mineralizing cells in plaque calcification, at least as early to mid-stage of plaque development. We can’t exclude that progenitor cells could also play a role in this process. Interestingly, SMC mineralizing properties vary depending on their anatomical location, further supporting biological differences among SMCs, and the heterogeneous propensity to develop calcification between arterial beds. Macrophages can modulate this process. M2a cells were found in direct contact with calcified nodules in lesions. We confirmed the supporting role of this subset in cell mineralization by demonstrating that M2a-derived supernatant increased SMC mineralization.
Reciprocally, supernatants from vascular cells, especially pericytes, strongly orient macrophages into an M2 phenotype, suggesting that vascular cell and macrophage interactions are major regulators of calcification process, as shown in recent publications from E. Aikawa’s group.
Given our microarray and in vitro results, we tested whether TGFb signaling could be actively implicated in cell mineralization. Inhibition with SD208 of TGFbR1 was sufficient to block femoral SMC mineralization, but did not impact carotid-derived SMC mineralization. This result is consistent with qPCR data that indicated a stronger basal expression of TGF signaling members in femoral compared to carotid arteries. Furthermore, TGF signaling blockade was sufficient to inhibit macrophage-induced mineralization of SMC. Altogether, our data support an important role of TGF signaling in both heterogeneous intrinsic capacities of mineralization between arterial beds, as well as in the paracrine inducers of calcification via its release by M2 macrophage.
To assess the functional impact of TGF and our additional genes of interest in plaque calcification in peripheral arteries, we needed to establish an experimental model of atherosclerotic mice with accelerated calcification. We generated 2 murine colonies, double deficient for apoE and rankl, and for apoE and opg. We first created apoE-/- rankl-/- mutant mice, infertile and much smaller than apoE-/- rankl+/- littermates. More importantly, they develop important membrane bone defect, and a limited life expectancy, making them incompatible for atherosclerosis studies. This bone phenotype was absent in apoE-/- and in rankl-/- single mutants, so we could anticipate this result. This model will be used nonetheless to further characterize the important and unknown link between apoE and rankl/rank signaling, and could lead to important findings on bone remodeling biology.
We next generated apoE-/- opg-/- model, that have been previously described. The apoE-/- opg-/- mutants were however infertile and poor breeding between apoE-/- opg+/- during the last year of funding delayed further characterization of their vascular phenotype (probably accentuated by environmental stress and progressive aging of breeders). Young heterozygous breeders are currently mating and numerous litters are coming of age, and more are expected in the next months. 8 week males will be fed an atherogenic diet for 4-12 weeks for the study of plaque calcification in aortic root, innominate artery, and femoral artery.
This tool will be instrumental in the validation of molecular pathway of interest identified through our transcriptomic, histological and in vitro analysis.

• Objectives and impact can be found on the Webpage in the Pathophysiology of Bone Resorption and Therapy of Primary Bone Tumors laboratory’s (LPRO) website