Figure 1.
Sortilin immunostaining of grossly normal human young and old and atherosclerotic aorta and carotid vessels.
Normal young vessels (A) do not display appreciable sortilin immunodetection; the latter is observed in old vessel intimal thickening and fatty streak and, more markedly, in fibroatheromatous plaque. Representative images (B) of serial sections of human fibroatheromatous plaque stained with Haematoxylin-Eosin (H-E), α-smooth muscle actin (α-SMA), CD68, sortilin, p75NTR and proNGF. Diaminobenzidine as chromogen, Haematoxylin counterstaining. Scale bar = 50 µm.
Figure 2.
Sortilin expression and apoptosis in normal and post-injury rat aortas.
Anti-sortilin and anti-p75NTR immunostainings (A) do not reveal detectable positivity in normal tunica media. Intimal thickening appears markedly sortilin, p75NTR and proNGF positive fifteen days after ballooning, but not after forty-five days; α-SMA immunodetection goes in the opposite direction. TUNEL+ cells are evident in the neointima 15 days after ballooning (arrow heads). Bar graphs (B) showing sortilin+, p75NTR+ and TUNEL+ apoptotic intimal cell percentages; *p<0.05. Scale bar = 50 µm.
Figure 3.
Sortilin expression in rat aortic smooth muscle cell populations.
Representative blot analysis of (A) sortilin protein expression and transcript levels (B) in normal rat aortic medial tissue and 15 days after ballooning. Sortilin and p75NTR (C) transcripts accumulate in intimal cells obtained 15 days after injury (IT cells). The latter and normal media SMCs (mSMCs) were harvested after 3 and 6 days in sparse and confluent cultures, respectively. Representative blots and densitometric analysis (D and E) after normalization to CMR1 expression; data are mean ± SEM of three experiments. Sortilin and p75NTR immunofluorescence (F) documents a higher intracellular sortilin and p75NTR level in IT cells compared to mSMCs; right panel: merged images showing the prevalent co-localization of sortilin with p75NTR; *p<0.05. Scale bar = 25 µm.
Figure 4.
ProNGF is a potent apoptotic inducer of IT cells.
IT cells (A) express TrkB and TrkC but not TrkA transcripts, while all Trk receptors are present in mSMCs. ProNGF (B and C) is a potent apoptotic inducer of IT cells and induces a dose-dependent increase of cultured rat aortic IT cell apoptosis as measured by TUNEL in serum-free medium after 24 and 48 hours; flow cytometry (D) of rat aortic intimal cells in sub-G1 (DNA content<2N) calculated as percentages of total events (10,000 cells). Agarose gel under UV light (E) after staining with ethidium bromide showing the ladder production after blunt end linker ligation confirms the dose-dependent and higher proNGF apoptotic DNA fragmentation compared to control IT cells. Representative immunofluorescence (F) of IT cells after 24 h of proNGF (10 ng/mL) treatment shows intracellular distribution of sortilin somehow more evident in the cell membrane compartment, whereas p75NTR localization is almost unchanged. Blot analysis (G and H) shows that proNGF (10 ng/mL) induces the increase of sortilin protein content only after 48 h. EMSA analysis (I) in IT cells after 24 h and 48 h of treatment with proNGF shows a significant reduction of NF-κB activity (upper arrow, p65/50 heterodimer; lower arrow, p50/50 homodimer). Data are reported as mean ± SEM of three independent experiments. *p<0.05. Scale bar = 25 µm.
Figure 5.
Silencing of sortilin partially prevents proNGF-induced apoptosis of IT cells.
Representative immunofluorescence (A) and densitometric analysis of blots (B) showing reduced sortilin expression in sortilin-silenced IT cells. Sortilin silencing (C) reduces 24 h proNGF-induced apoptosis. Graph (D) shows that SN50-induced NF-κB inhibition increases apoptosis of IT cells but not of mSMCs, that doesn’t further increase after successive 24 h of proNGF treatment. Data as mean ± SEM of three independent experiments. *p<0.05. Scale bar = 25 µm.