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Pregnancy-Associated Plasma Protein-A Is Markedly Expressed by Monocyte-Macrophage Cells in Vulnerable and Ruptured Carotid Atherosclerotic Plaques

A Link Between Inflammation and Cerebrovascular Events

Giuseppe Sangiorgi, MD, FESC^_*,_*, Alessandro Mauriello, MD, Elena Bonanno, MD, Claus Oxvig, PhD^¶, Cheryl A. Conover, MD, Michael Christiansen, MD^#, Santi Trimarchi, MD, Vincenzo Rampoldi, MD, David R. Holmes, Jr, MD, FACC^||, Robert S. Schwartz, MD, FACC^_** and Luigi Giusto Spagnoli, MD

^_* Department of Cardiovascular Disease, Istituto Policlinico San Donato, San Donato Milanese, Milan, Italy
Department of Vascular Surgery, Istituto Policlinico San Donato, San Donato Milanese, Milan, Italy
Department of Pathology, University of Rome Tor Vergata, Rome, Italy
Endocrinology Research Unit, Mayo Clinic and Foundation, Rochester, Minnesota
^|| Department of Cardiovascular Disease, Mayo Clinic and Foundation, Rochester, Minnesota
^¶ Department of Molecular and Structural Biology, University of Aarhus, Aarhus, Denmark
^# Department of Clinical Biochemestry, Staten Serum Institute, Copenhagen, Denmark
^_** Department of Cardiovascular Disease, Minneapolis Heart Foundation, Minneapolis, Minnesota

View larger version (13K): [in a new window]   Figure 1 Graphs showing linear regression between pregnancy-associated protein-A (PAPP-A) score versus plaque thickness (A) and PAPP-A score versus total cells’ plaque cap inflammatory infiltrate (B) for the 65 carotid atherosclerotic plaques submitted to histopathologic examination. A decrease in cap thickness and an increase in inflammatory infiltrate in the cap region of the plaque are both strongly correlated with increase in PAPP-A expression.

View larger version (75K): [in a new window]   Figure 2 Photomicrographs of a carotid atherosclerotic plaque removed by carotid endarterectomy from a patients who suffered a major stroke. A vulnerable plaque characterized by a large lipidic-necrotic core, a thin fibrous cap (A, Movat pentachrome, 4x) associated to a heavy inflammatory infiltrate constituted mainly by macrofagic foam cells, CD68-positive (B, 10x). A strong positive reaction for pregnancy-associated protein-A was observed (C, 20x). (D, 10x and G, 20x) show CD68 antibody revealed by streptavidin-Texas Red fluorescent conjugate. (E, 10x and H, 20x) Show pregnancy-associated protein-A reaction revealed by streptavidin-fluorescin conjugate. Two dimension confocal analysis of the two fluorochromes (F and I) clearly showed a strong and diffuse positive reaction for pregnancy-associated protein-A in macrophagic cells (concordant double positivity appears as a yellow stain). (K) Shows confocal analysis of the two fluorochromes, smooth muscle actin immunostain (red) and pregnancy-associated protein-A stain (green). Smooth muscle cells observed in this plaque gave rise to a strong positive reaction for pregnancy-associated protein-A (yellow stain). (L) Shows confocal analysis of the two fluorochromes, CD3 immunostain (red) and pregnancy-associated protein-A stain (green). Almost all T lymphocytes (red) observed were negative for pregnancy-associated protein-A in plaques where a diffuse positive reaction was observed (green). Only occasional T lymphocytes were pregnancy-associated protein-A-positive (double positivity, yellow stain, arrow).

View larger version (76K): [in a new window]   Figure 3 Photomicrographs of a stable carotid atherosclerotic plaque removed by carotid endarterectomy from an asymptomatic patient. In the shoulder of the plaque (A, Movat pentachrome, 4x), there is a moderate inflammatory infiltrate, constituted mainly by macrophagic foam cells, CD68-positive (B, a contiguous section of A). The expression of pregnancy-associated protein-A is very low (C, contiguous section of A). Only few cells are pregnancy-associated protein-A-positive (inset). (D) (20x) Shows CD68 antibody revealed by streptavidin-Texas Red fluorescent conjugate. (E) (20x) Pregnancy-associated protein-A reaction revealed by streptavidin-fluorescin conjugate. Two dimension confocal analysis of the two fluorochromes reported in F shows that monocytes/macrophages resident in this "stable" plaque were almost negative for pregnancy-associated protein-A. The pregnancy-associated protein-A reaction showed only slight and focal positivity. Smooth muscle cells were almost negative for pregnancy-associated protein-A. The pregnancy-associated protein-A reaction showed only slight and focal positivity (G, concordant double positivity appears as a yellow stain, arrow, 10x). (H) T lymphocytes and pregnancy-associated protein-A double immunostain (10x). Few T lymphocytes (CD3 antibody revealed by streptavidin-Texas Red fluorescent conjugate) were observed in this plaque, and the reaction for pregnancy-associated protein-A was negative (pregnancy-associated protein-A revealed by streptavidin-fluorescin conjugate).

View larger version (11K): [in a new window]   Figure 4 Bar graphs showing pregnancy-associated protein-A (PAPP-A) messenger ribonucleic acid (mRNA) expression in carotid plaques collected by surgical carotid endarterectomy from symptomatic patients relative to plaque from an asymptomatic patient. All samples had detectable PAPP-A mRNA from one- to four-fold greater than control. All data represent means of three samples from at least three separately performed experiments. Data are presented as means ± SD. S = stroke; T = transient ischemic attack.