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. 2015 Aug;241(2):772-82.
doi: 10.1016/j.atherosclerosis.2015.05.011. Epub 2015 May 19.

Natural progression of atherosclerosis from pathologic intimal thickening to late fibroatheroma in human coronary arteries: A pathology study

Fumiyuki Otsuka  1 , Miranda C A Kramer  2 , Pier Woudstra  2 , Kazuyuki Yahagi  1 , Elena Ladich  1 , Aloke V Finn  3 , Robbert J de Winter  2 , Frank D Kolodgie  1 , Thomas N Wight  4 , Harry R Davis  1 , Michael Joner  1 , Renu Virmani  5
Affiliations

Natural progression of atherosclerosis from pathologic intimal thickening to late fibroatheroma in human coronary arteries: A pathology study

Fumiyuki Otsuka et al. Atherosclerosis. 2015 Aug.

Abstract

Objective: Smooth muscle cells, macrophage infiltration and accumulation of lipids, proteoglycans, collagen matrix and calcification play a central role in atherosclerosis. The early histologic changes of plaque progression from pathologic intimal thickenings (PIT) to late fibroatheroma lesions have not been fully characterized.

Methods: A total of 151 atherosclerotic coronary lesions were collected from 67 sudden death victims. Atherosclerotic plaques were classified as PIT without macrophage infiltration, PIT with macrophages, and early and late fibroatheromas. Presence of macrophages and proteoglycans (versican, decorin and biglycan) were recognized by specific antibodies while hyaluronan was detected by affinity histochemistry. Lipid deposition was identified by oil-red-O, and calcification was assessed following von Kossa and alizarin red staining.

Results: Lesion progression from PIT to late fibroatheroma was associated with increase in macrophage accumulation (p < 0.001) and decreasing apoptotic body clearance by macrophages (ratio of engulfed-to-total apoptotic bodies) (p < 0.001). Lipid deposition in lipid pool of PIT had a microvesicular appearance whereas those in the necrotic core were globular in nature. Overall, the accumulation of hyaluronan (p < 0.001), and proteoglycan versican (p < 0.001) and biglycan (p = 0.013) declined along with lesion progression from PIT to fibroatheromas. Microcalcification was first observed only within areas of lipid pools and its presence and size increased in lesions with necrotic core.

Conclusions: PIT to fibroatheroma lesions are accompanied by early lipid accumulation, followed by macrophage infiltration with defective clearance of apoptotic bodies along with decrease in proteoglycan and hyaluronan in lipid pools that convert to necrotic cores. Calcification starts in PIT and increases with plaque progression.

Keywords: Apoptosis; Atherosclerosis; Calcification; Extracellular matrix; Macrophage; Pathology; Proteoglycans.

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Conflict of interest statement

Disclosures: Dr. Virmani receives research support from Abbott Vascular, BioSensors International, Biotronik, Boston Scientific, Medtronic, MicroPort Medical, OrbusNeich Medical, SINO Medical Technology, and Terumo Corporation; has speaking engagements with Merck; receives honoraria from Abbott Vascular, Boston Scientific, Lutonix, Medtronic, and Terumo Corporation; and is a consultant for 480 Biomedical, Abbott Vascular, Medtronic, and W.L. Gore. Dr. Joner is a consultant for Biotronik and Cardionovum, and has received speaking honorarium from Abbott Vascular, Biotronik, Medtronic, and St. Jude. Dr. Otsuka has received speaking honorarium from Abbott Vascular and Merck. The other authors report no conflicts of interest relevant to the topic of this manuscript.

This paper is an original unpublished work and is not being considered for publication elsewhere. All authors have contributed substantially in various aspects of the work including: 1) conception and design or analysis and interpretation of data, or both; 2) drafting of the manuscript or revising it critically for important intellectual content; and 3) final approval of the manuscript submitted. We wish to draw the attention of the Editor to the following facts which may be considered as potential conflicts of interest.

Figures

Figure 1
Figure 1
Representative histologic sections showing pathologic intimal thickening (PIT) without macrophage (mac) infiltration, PIT with macrophages, early fibroatheroma (EFA), and late fibroatheroma (LFA). The left two columns show low and high power images of sections stained with Movat pentachrome, and the right three columns show high power images of immunohistochemical stains for macrophages (CD68), T-lymphocytes (CD45RO), and vasa vasorum (CD31/34).
Figure 2
Figure 2
Bar graphs and box-and-whisker plots showing the results of morphometric analysis and quantitative assessment of macrophage and apoptotic body density along with percentage of engulfed apoptotic bodies in 60 human coronary lesions. Bars represent mean values and T-bars indicate SD. Lines within boxed represent median values; the upper and lower lines of the boxes represent the 75th and 25th percentiles, respectively; and the upper and lower bars outside the boxes represent the 90th and 10th percentiles, respectively. IEL=internal elastic lamina.
Figure 3
Figure 3
(A) Representative histologic sections (low power images = Movat pentachrome, high power images = immunostaining) showing increase in macrophage infiltration and apoptotic bodies in association with plaque progression. The middle column shows single immunostaining for macrophages (CD68, brown), whereas the right column shows dual staining for macrophages (CD68, blue) and apoptotic bodies (red). (B) Dual immunostaining for macrophages (CD68, blue) and apoptotic bodies (red) demonstrated that most apoptotic bodies co-localized with macrophages and were engulfed in PIT with macrophages (a [×400] and b [×1000]), whereas the proportion of free apoptotic bodies increased in late fibroatheroma (c [×400] and d [×1000]) where engulfed apoptotic bodies decreased.
Figure 4
Figure 4
(A) Immunohistochemical identification of extracellular matrix (ECM) molecules hyaluronan and proteoglycan (versican, biglycan and decorin) in human coronary plaques. Movat pentachrome staining show lipid pool (LP) with or without macrophage infiltration in pathologic intimal thickening (PIT) and necrotic core (NC) formation in early (EFA) and late fibroatheromas (LFA). Immunohistochemistry shows intense staining for hyaluronan in LPs of PIT whereas early NC shows partial loss of staining and late NC exhibits almost complete loss of hyaluronan. Gradual decrease in versican was also noted from PIT without macrophages to LFA where the staining was almost absent in late NC. Immunohistochemical reaction to biglycan and decorin were relatively mild as compared with other two ECM molecules; however, the staining for biglycan in LFA was significantly less as compared to PIT and EFA. (B) Quantitative assessment of hyaluronan and proteoglycan (versican, biglycan, and decorin) showed a significant decrease in hyaluronan, versican, and biglycan from PIT to EFA and LFA.
Figure 4
Figure 4
(A) Immunohistochemical identification of extracellular matrix (ECM) molecules hyaluronan and proteoglycan (versican, biglycan and decorin) in human coronary plaques. Movat pentachrome staining show lipid pool (LP) with or without macrophage infiltration in pathologic intimal thickening (PIT) and necrotic core (NC) formation in early (EFA) and late fibroatheromas (LFA). Immunohistochemistry shows intense staining for hyaluronan in LPs of PIT whereas early NC shows partial loss of staining and late NC exhibits almost complete loss of hyaluronan. Gradual decrease in versican was also noted from PIT without macrophages to LFA where the staining was almost absent in late NC. Immunohistochemical reaction to biglycan and decorin were relatively mild as compared with other two ECM molecules; however, the staining for biglycan in LFA was significantly less as compared to PIT and EFA. (B) Quantitative assessment of hyaluronan and proteoglycan (versican, biglycan, and decorin) showed a significant decrease in hyaluronan, versican, and biglycan from PIT to EFA and LFA.
Figure 5
Figure 5
Representative histologic frozen sections showing lipid accumulation as assessed by oil red O (ORO) stain.
Figure 6
Figure 6
Representative histologic non-decalcified sections showing progression of microcalcification as assessed by alizarin red and von Kossa stains.
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