Follicle-stimulating hormone receptor expression in advanced atherosclerotic plaques

Abstract

Experimental evidence indicates that follicle-stimulating hormone (FSH), an essential hormone for reproduction, can act directly on endothelial cells inducing atherosclerosis activation and development. However, it remains unknown whether the FSH-receptor (FSHR) is expressed in human atherosclerosis plaques. To demonstrate the FSHR presence, we used immunohistochemical and immunoelectron microscopy involving a specific monoclonal antibody FSHR1A02 that recognizes an epitope present in the FSHR-ectodomain. In all 55 patients with atherosclerotic plaques located in carotid, coronary, femoral arteries, and iliac aneurysm, FSHR was selectively expressed in arterial endothelium covering atherosclerotic plaques and endothelia lining intraplaque neovessels. Lymphatic neovessels were negative for FSHR. M1-macrophages, foam cells, and giant multinucleated cells were also FSHR-positive. FSHR was not detected in normal internal thoracic artery. Immunoelectron microscopy performed in ApoEKO/hFSHRKI mice with atherosclerotic plaques, after injection in vivo with mouse anti-hFSHR monoclonal antibody FSHR1A02 coupled to colloidal gold, showed FSHR presence on the luminal surface of arterial endothelial cells covering atherosclerotic plaques. Therefore, FSHR can bind, internalize, and deliver into the plaque circulating ligands to FSHR-positive cells. In conclusion, we report FSHR expression in endothelial cells, M1-macrophages, M1-derived foam cells, giant multinucleated macrophages, and osteoclasts associated with human atherosclerotic plaques.

Keywords: Atherosclerosis; Atherosclerotic plaque; FSH; FSHR; FSHR1 isoform; FSHR1A02 antibody; Splenic aneurysm.

Figure 1

FSHR1 expression by endothelial cells in human carotid atherosclerotic plaques. A carotid atherosclerotic plaque showed strong staining of arterial and microvascular endothelial cells (Panel A), whereas endothelial cells of normal internal thoracic artery and its associated microvessels were negative for FSHR1 (Panel B). In serial paraffin sections of plaque tissue, the lymphatic neovessels stained for podoplanin with D2-40 antibody (Panel C) did not express FSHR1 (Panel D). Double immunofluorescence on carotid plaque tissue confirmed the identity of cells expressing FSHR1 (Panels E through H). An antibody against the vascular endothelial-cell marker von Willebrand factor, followed by a green-labeled secondary antibody (Panel F), overlapped with the signal from the mouse anti-hFSHR1A02 detected by a secondary red labeled antibody (Panel E). Merging of the two antibody signals with DAPI a staining fluorescent compound of nuclei (blue color) is shown in Panel H. Scale bar represents 50 µm in Panels A through D, and 25 µm in Panels E through H.

Figure 2

FSHR1-positive neovessels in human atherosclerotic plaques are immature blood vessels. Double immunofluorescence on atherosclerotic plaque tissue confirmed that vWF-positive blood vessels (asterisk, red staining) associated with atherosclerotic plaques lacked pericyte coverage and therefore are immature (no green staining for αSMA on mural cells). Scale bar: 50 μm. Inset: mature venule with pericyte investment (green staining). Scale bar: 50 μm.

Figure 3

Expression of FSHR1 in other cells associated with carotid atherosclerotic plaques. While FSHR1 was detectable in arterial endothelium covering the atherosclerotic plaques (Panel A), endothelia lining the blood microvessels (Panels C, D), M1-macrophages (Panel A, inset 1), foam cells (Panel A, inset 2), and in giant multinucleated macrophages (Panels D, E) no staining for FSHR1 was visible in M2-macrophages expressing CD163 (Panel B, inset 3, red color) and lymphocytes (Panel C). BV blood vessels, asterisks giant multinucleated macrophages. Scale bar in all panels represents: 50 µm).

Figure 4

FSHR1 expression in the coronary atherosclerotic plaques. Consistent and generalized expression of endothelial FSHR1 (brown staining) in myocardium and arteries of a patient who suffered fatal myocardial infarction (Scale bar: 2 mm). Inset: Positive staining for FSHR1 in ECs associated with a small muscular artery and the myocardial microvasculature, (BV blood vessels, Cap capillary, Ven venule. Scale bar: 100 µm).

Figure 5

FSHR1 expression in the atherosclerotic plaques located in femoral artery and splenic artery aneurysm. (Panel A) Atherosclerotic plaque located in femoral artery: consistent expression of FSHR1 by endothelial cells, macrophages, and giant multinucleated macrophages (Scale bar: 50 μm). Inset: Arterial endothelial cells covering the femoral atherosclerotic plaques. (Scale bar: 50 µm). (Panel B) Iliac artery aneurysm (80-year old female, no smoker with no comorbidities). The aneurysmal wall examination indicates an atrophy of the muscular media caused by the atherosclerotic plaque (Scale bar: 1 mm). Inset: FSHR1-positive ECs in capillaries (arrowheads) and venules (arrows) located between the collagen fibers (Scale bar: 50 μm).

Figure 6

Binding and routes of transport of FSHR1-A02 antibody-colloidal gold particles by the arterial endothelial cells covering atherosclerotic plaques located in the aortic arch of ApoEKO/hFSHR1KI mice. (Panels A through D) After 30 min of circulation in vivo, the tracer particles (black dots) bound to the luminal plasma membrane and clathrin-coated pits were internalized via clathrin-coated vesicles in endosomes, and delivered into the subendothelial space. (Panel E) In contact with macrophages the FSHR1-A02 antibody-colloidal gold particles were internalized via a pathway involving clathrin-coated pits, clathrin-coated vesicles and concentrated in endosomes. (EC, endothelial cell. Scale bar: 200 nm).