Humanin, a cytoprotective peptide, is expressed in carotid atherosclerotic [corrected] plaques in humans

Abstract

Objective: The mechanism of atherosclerotic plaque progression leading to instability, rupture, and ischemic manifestation involves oxidative stress and apoptosis. Humanin (HN) is a newly emerging endogenously expressed cytoprotective peptide. Our goal was to determine the presence and localization of HN in carotid atherosclerotic plaques.

Methods and results: Plaque specimens from 34 patients undergoing carotid endarterectomy were classified according to symptomatic history. Immunostaining combined with digital microscopy revealed greater expression of HN in the unstable plaques of symptomatic compared to asymptomatic patients (29.42±2.05 vs. 14.14±2.13% of plaque area, p<0.0001). These data were further confirmed by immunoblot (density of HN/β-actin standard symptomatic vs. asymptomatic 1.32±0.14 vs. 0.79±0.11, p<0.01). TUNEL staining revealed a higher proportion of apoptotic nuclei in the plaques of symptomatic patients compared to asymptomatic (68.25±3.61 vs. 33.46±4.46% of nuclei, p<0.01). Double immunofluorescence labeling revealed co-localization of HN with macrophages (both M1 and M2 polarization), smooth muscle cells, fibroblasts, and dendritic cells as well as with inflammatory markers MMP2 and MMP9.

Conclusions: The study demonstrates a higher expression of HN in unstable carotid plaques that is localized to multiple cell types within the plaque. These data support the involvement of HN in atherosclerosis, possibly as an endogenous response to the inflammatory and apoptotic processes within the atheromatous plaque.

Figure 1. Histological immunoreactivity of HN is greater in symptomatic patients.

HN expression is greater in carotid plaques from TIA (n = 12) and stroke (n = 10) symptomatic patients than in asymptomatic patients (n = 12; †p<0.001).

Figure 2. Representative immunostaining of HN.

HN can be seen in carotid plaques (top row) from asymptomatic (left), TIA (middle), and stroke (right) patients. Increased magnification of these slides (bottom row) demonstrates intracellular localization (arrows indicate examples of staining).

Figure 3. HN protein blotting is greater in symptomatic patients.

Western Blot demonstrates amount of HN is greater in carotid plaques of symptomatic patients than in asymptomatic patients (combined symptomatic not depicted, p<0.01). Representative immunoblot shown above. Results expressed as the ratio of HN and β-actin densitometric signals (†p<0.05 vs. asymptomatic).

Figure 4. Cellular apoptosis is greater in plaques of symptomatic patients.

Representative TUNEL staining can been seen in carotid plaques from (A) asymptomatic and (B) symptomatic patients. Inserts show magnification of the black boxes in the main figure and arrows indicate brown apoptotic nuclei (original magnification 200×). Proportion of apoptotic nuclei was greater in plaques of symptomatic patients compared to asymptomatic (p<0.01).

Figure 5. HN is localized to multiple cells of the atheromatous plaque.

Immunofluorescence of the carotid plaque to detect the presence of (A) HN with (B) CD68. (C) Nuclei can be visualized using a DAPI counterstain. (D) Merging of these images demonstrates co-localization of HN with plaque macrophages, (E) seen better with increased magnification. Merged images below demonstrate co-localization of HN with (F) smooth muscle α-actinin, (G) fibroblast vimentin, and (H) dendritic cell fascin.

Figure 6. HN is expressed in M1 phase macrophages.

Immunofluorescence reveals the presence of more HN-secreting macrophages in the M1 polarization phase (indicated by iNOS) in symptomatic patients (*p<0.05 vs. asymptomatic, #p<0.05 vs. TIA).

Figure 7. HN is expressed in M2 phase macrophages.

HN-secreting macrophages in the M2 polarization phase (indicated by arginase-1) were present, but less in number than HN-secreting M1 macrophages. No differences were seen between groups.

Figure 8. HN co-localizes with MMP2 and MMP9 in the plaque.

(Left) HN (green) co-localizing with MMP2 (red) in the intima with DAPI counterstain (blue). (Right) Similar staining can be seen with MMP9 (red) in the media.