Intra-Section Analysis of Human Coronary Arteries Reveals a Potential Role for Micro-Calcifications in Macrophage Recruitment in the Early Stage of Atherosclerosis

Abstract

Background: Vascular calcification is associated with poor cardiovascular outcome. Histochemical analysis of calcification and the expression of proteins involved in mineralization are usually based on whole section analysis, thereby often ignoring regional differences in atherosclerotic lesions. At present, limited information is available about factors involved in the initiation and progression of atherosclerosis.

Aim of this study: This study investigates the intra-section association of micro-calcifications with markers for atherosclerosis in randomly chosen section areas of human coronary arteries. Moreover, the possible causal relationship between calcifying vascular smooth muscle cells and inflammation was explored in vitro.

Technical approach: To gain insights into the pathogenesis of atherosclerosis, we performed analysis of the distribution of micro-calcifications using a 3-MeV proton microbeam. Additionally, we performed systematic analyses of 30 to 40 regions of 12 coronary sections obtained from 6 patients including histology and immuno-histochemistry. Section areas were classified according to CD68 positivity. In vitro experiments using human vascular smooth muscle cells (hVSMCs) were performed to evaluate causal relationships between calcification and inflammation.

Results: From each section multiple areas were randomly chosen and subsequently analyzed. Depositions of calcium crystals at the micrometer scale were already observed in areas with early pre-atheroma type I lesions. Micro-calcifications were initiated at the elastica interna concomitantly with upregulation of the uncarboxylated form of matrix Gla-protein (ucMGP). Both the amount of calcium crystals and ucMGP staining increased from type I to IV atherosclerotic lesions. Osteochondrogenic markers BMP-2 and osteocalcin were only significantly increased in type IV atheroma lesions, and at this stage correlated with the degree of calcification. From atheroma area type III onwards a considerable number of CD68 positive cells were observed in combination with calcification, suggesting a pro-inflammatory effect of micro-calcifications. In vitro, invasion assays revealed chemoattractant properties of cell-culture medium of calcifying vascular smooth muscle cells towards THP-1 cells, which implies pro-inflammatory effect of calcium deposits. Additionally, calcifying hVSMCs revealed a pro-inflammatory profile as compared to non-calcifying hVSMCs.

Conclusion: Our data indicate that calcification of VSMCs is one of the earliest events in the genesis of atherosclerosis, which strongly correlates with ucMGP staining. Our findings suggest that loss of calcification inhibitors and/or failure of inhibitory capacity is causative for the early precipitation of calcium, with concomitant increased inflammation followed by osteochondrogenic transdifferentiation of VSMCs.

Fig 1. Regional differentiation of atherosclerotic lesion types.

An overview of a human coronary artery section, depicting atherosclerotic stages I to IV, is shown in the left panel, with enlargements of the selected regions at the right panel. The regions of coronary lesions classified as types I, II, III, and IV, based on CD68 positivity, are shown in 1A, with corresponding regions in the calcium yield scan in 1B.

Fig 2. Representative images of type I, II, III and IV regions.

Representative images of regions classified as type I, II, III and IV with the corresponding calcium yield scan and (immuno-)histochemical staining of von Kossa, αSMA, ucMGP, cMGP, BMP-2 and osteocalcin (OC) in adjacent sections. Arrows indicate calcification close to the internal elastic lamina. Scale bars are 200μm.

Fig 3. Correlation of degree of atherosclerotic lesion with micro-calcification and proteinous catalysts/inhibitors of calcification.

Intimal regions of coronary lesions were chosen and classified as types I, II, III, and IV based on CD68 positivity. Calcification was calculated in corresponding regions using the Calcium yield scan (S1 File). Immuno-histochemical analysis of proteinous catalysts and inhibitors of calcification was performed according to “Method one” for CD68, aSMA, OC and BMP2 (as described in S2 File), and according to “Method two” for cMGP and ucMGP (as described in S3 File). The number of observations ranged from 6 to 8 for type I lesions and between 9 and 12 for type II, III and IV lesions. *P < 0.05, **P < 0.001, ***P < 0.0001, significance was assessed by unpaired non-parametric t test (Mann-Whitney).

Fig 4. Correlation of amount of micro-calcification with expressions of proteinous catalysts and inhibitors of calcification.

Correlations of intimal calcification in atherosclerotic regions and expression of markers aSMA, CD68, ucMGP, cMGP, BMP2 and osteocalcin, irrespective of type of lesion. Calcification was calculated according to “Method one” on the calcium yield scan (S2 File) and represents all values obtained in the type I, II, III, IV regions. Regression analyses between calcification and expression of proteinous catalysts and inhibitors of calcification revealed significant correlations with CD68, ucMGP, cMGP, BMP2 and osteocalcin. Values (r²) for determining the degree of correlation between calcification and CD68 or ucMGP, was found to be 0.52 and 0.48, respectively. The degree of correlation between calcification and cMGP, BMP2 or osteocalcin was found to be substantially lower, i.e., 0.24, 0.20 and 0.24, respectively. Significance was determined using the Pearson correlation test.

Fig 5. In vitro model for chemoattractant properties of calcifying VSMCs on macrophages.

The effect of calcifying VSMCs on the attraction of inflammatory cells was tested via invasion assays using PMA-stimulated THP-1 cells (macrophages). Conditioned medium of both control and calcifying VSMCs was used and calcium was added to control medium to obtain equal concentration of calcium in both conditions. Medium from calcifying VSMCs increased the invasion of macrophages significantly, indicating that VSMCs that calcify produce chemoattractants for inflammatory cells. Results were normalised to cell number at start. *P < 0.05, **P < 0.001 significance was assessed unpaired non-parametric t-test (Mann-Whitney).

Fig 6. Calcifying VSMCs in Vitro display a pro-inflammatory and not an osteochondrogenic phenotype.

qPCR of calcifying human primary VSMCs show a significant decrease in MGP as compared to control VSMCs. No differences were found in the expression of the osteochondrogenic markers Runx2, BMP-2 and osteocalcin. The pro-inflammatory cytokines MCP1, IL1b and IFNy were significantly increased in calcifying VSMCs indicative that calcifying VSMCs can initiate local vascular inflammation and promote macrophage migration towards the vascular wall.

Fig 7. Model showing the potential mechanism of initiation and progression of calcification of the vascular wall.

1) Contractile VSMCs in the thickened intima change phenotype towards synthetic VSMCs. Synthetic VSMCs start secreting extracellular vesicles into the extracellular environment. In case of shortage of vitamin K, a vitamin required for the conversion of ucMGP into the active form cMGP, extracellular vesicles are loaded with ucMGP which is unable to prevent nucleation of calcium-phosphate. 2) Calcifying vesicles provide the first nidus for mineralisation and microcalcifications will be formed. These microcalcifications induce an inflammatory response in VSMCs. 3) VSMCs start secreting pro-inflammatory cytokines that will attract macrophages. 4) Macrophages start fueling the inflammation process by phagocytosing mcirocalcifications and secreting pro-inflammatory cytokines. 5) Pro-inflammatory macrophages affect synthetic VSMCs which will in turn produce BMP2. Synthetic VSMCs will transdifferentiate towards osteochondrogenic VSMCs that subsequently will produce bone-forming proteins such as osteocalcin. 6) Macrocalcifications are the final result of the osteochondrogenic environment in the atherosclerotic plaque.