Alcohol Reduces Arterial Remodeling by Inhibiting Sonic Hedgehog-Stimulated Stem Cell Antigen-1 Positive Progenitor Stem Cell Expansion

Abstract

Background: Cell and molecular mechanisms mediating the cardiovascular effects of alcohol are not fully understood. Our aim was to determine the effect of moderate ethanol (EtOH) on sonic hedgehog (SHh) signaling in regulating possible stem cell antigen-1 positive (Sca1⁺ ) progenitor stem cell involvement during pathologic arterial remodeling.

Methods: Partial ligation or sham operation of the left carotid artery was performed in transgenic Sca1-enhanced green fluorescent protein (eGFP) mice gavaged with or without "daily moderate" EtOH.

Results: The EtOH group had reduced adventitial thickening and less neointimal formation, compared to ligated controls. There was expansion of eGFP-expressing (i.e., Sca1⁺ ) cells in remodeled vessels postligation (day 14), especially in the neo intima. EtOH treatment reduced the number of Sca1⁺ cells in ligated vessel cross-sections concomitant with diminished remodeling, compared to control ligated vessels. Moreover, EtOH attenuated SHh signaling in injured carotids as determined by immunohistochemical analysis of the target genes patched 1 and Gli2, and RT-PCR of whole-vessel Gli2 mRNA levels. Intraperitoneal injection of ligated Sca1-eGFP mice with the SHh signaling inhibitor cyclopamine diminished SHh target gene expression, reduced the number of Sca1⁺ cells, and ameliorated carotid remodeling. EtOH treatment of purified Sca1⁺ adventitial progenitor stem cells in vitro inhibited SHh signaling, and their rSHh-induced differentiation to vascular smooth muscle cells.

Conclusions: EtOH reduces SHh-responsive Sca1⁺ progenitor cell myogenic differentiation/expansion in vitro and during arterial remodeling in response to ligation injury in vivo. Regulation of vascular Sca1⁺ progenitor cells in this way may be an important novel mechanism contributing to alcohol's cardiovascular protective effects.

Keywords: Alcohol; Atherosclerosis; Hedgehog; Progenitor Cells; Stem Cell Antigen-1; Stem Cells.

Figure 1. Daily moderate EtOH inhibits ligation-induced arterial remodeling

Partial carotid ligation was performed in Sca1-eGFP transgenic mice treated with or without ‘moderate’ amounts of EtOH (0.8 g/kg in 200 μL volume by oral gavage, once daily 1 wk prior and each day after ligation, resulting in a peak BAC of 15 mmol/L). Control animals received an isocaloric cornstarch solution. Vessels harvested 14 days post ligation were used for morphological analysis. (a) Ligation injury increased adventitial (Adv) and medial (Med) compartments, and stimulated neo-intimal (NI) hyperplasia, with a decrease in lumen. Daily moderate EtOH inhibited adventitial and neo-intimal formation, with no significant effect on the media or lumen, compared to ligated controls. Data are given as Mean ± SEM, n=5 animals, (5 section analyzed per animal) S= Sham-operated, L= Ligated, L+E= Ligated + EtOH. *P<0.05 vs Sham; #P<0.05 vs Ligated control. (b) Intima/media ratio was calculated for vessels from ligated mice +/- EtOH. N=5, * P< 0.05 vs control. (c) Representative Van Gieson stained carotid cross sections also shown. Scale bars 50 μM.

Figure 2

SMC α-actin expression in Sca1-eGFP transgenic mice carotid cross sections from sham-operated (Sham), ligated (cornstarch), and ligated treated with Ethanol (Ligated + EtOH) groups. Immunohistochemistry for smooth muscle specific α-actin was performed on sections from vessels harvested 14 days post ligation or sham-operation, using an anti alpha-smooth muscle actin (α-SMA) antibody (Abcam ab5694). Representative images shown; blue = Dapi nuclear stain, Green = eGFP (i.e., Sca1⁺), red = α-actin. ×20 magnification on left (scale bars 50 μM); ×60, of boxed portion, shown on right. Width of Adventitia ‘A’ denoted by yellow line, Media: ‘M’ white line, Neo-intima: ‘NI’ red line.

Figure 3. Daily moderate EtOH consumption reduces the number of Sca1⁺ cells in ligated carotids

Partial carotid ligation or sham-operation was performed in Sca1-eGFP transgenic mice treated daily with or without ‘moderate’ amounts of EtOH (0.8 g/kg, oral gavage). Control ligated animals received an isocaloric cornstarch solution. Vessels harvested on day 14 were fixed, sectioned and imaged for eGFP, indicative of Sca1 expression. Daily moderate EtOH inhibited adventitial and intimal formation concomitant with a reduction in the number of Sca1⁺ cells per section as analyzed by Fiji ImageJ software. (a) Representative images (×20 magnification, scale bar 50 μM.); Verhoeff-Van Gieson stained sections (top) and corresponding confocal immunofluorescence pics (bottom) for sham, ligated, and ligated + EtOH groups. Thickness of Adventitia: ‘A’ yellow line, Media: ‘M’ white line, Neo-intima: ‘NI’ red line. (b) Higher magnification view (×60) of sham-operated carotid with a Sca1⁺ cells present in adventitia and intimal layer indicated by arrows. (c) Bar graph shows cumulative data for Sca1⁺ cells as a percentage of total cells (determined by DAPI); Sham n=5, Ligated n=6, Ligated + EtOH, n=6, *p<0.05 vs sham, #p<0.05 vs ligated).

Figure 4. EtOH inhibits Patched1 expression in ligated carotids

The protein expression of Sonic hedgehog target gene Patched 1 (Ptch1) in carotid cross sections from the different experimental groups [sham-operated, ligated (cornstarch control), ligated + EtOH] was assessed by immunohistochemistry using a rabbit polyclonal anti-Ptch1 primary antibody and an Alexa fluor 594-conjugated secondary antibody. Representative images (×20 magnification, scale bar 50 μM) shown. DAPI (blue); Ptch1 (red), eGFP (green, i.e., Sca1⁺), merged image (bottom). Dotted lines trace internal and external elastic laminae.

Figure 5. EtOH inhibits Gli2 expression in ligated carotids

(a) The protein expression of Sonic hedgehog target gene Gli2 in carotid cross sections from the different experimental groups [sham-operated, ligated (cornstarch control), ligated + EtOH] was assessed by immunohistochemistry using a rabbit polyclonal anti-Gli2 primary antibody and an Alexa fluor 594-conjugated secondary antibody. Representative images (×20 magnification, scale bar 50 μM) shown. DAPI (blue); Gli2 (red), eGFP (green, i.e., Sca1⁺), merged image (bottom). Dotted lines trace internal and external elastic laminae. (b) Higher magnification view (×60) of boxed portions from Figure 4 and 5. Arrows indicate sample cells co-expressing (i) Ptch1 and Sca1 or (ii) Gli2 and Sca1. (iii) Quantitative RT-PCR analysis of Gli2 mRNA isolated 2 wks post-ligation or sham operation from whole carotid arteries of control (cornstarch) or EtOH-gavaged C57Bl/6 mice. N=5, p<0.05 vs sham, #P<0.05 vs ligated.

Figure 5. EtOH inhibits Gli2 expression in ligated carotids

(a) The protein expression of Sonic hedgehog target gene Gli2 in carotid cross sections from the different experimental groups [sham-operated, ligated (cornstarch control), ligated + EtOH] was assessed by immunohistochemistry using a rabbit polyclonal anti-Gli2 primary antibody and an Alexa fluor 594-conjugated secondary antibody. Representative images (×20 magnification, scale bar 50 μM) shown. DAPI (blue); Gli2 (red), eGFP (green, i.e., Sca1⁺), merged image (bottom). Dotted lines trace internal and external elastic laminae. (b) Higher magnification view (×60) of boxed portions from Figure 4 and 5. Arrows indicate sample cells co-expressing (i) Ptch1 and Sca1 or (ii) Gli2 and Sca1. (iii) Quantitative RT-PCR analysis of Gli2 mRNA isolated 2 wks post-ligation or sham operation from whole carotid arteries of control (cornstarch) or EtOH-gavaged C57Bl/6 mice. N=5, p<0.05 vs sham, #P<0.05 vs ligated.

Figure 6. The Hedgehog inhibitor Cyclopamine inhibits Patched 1 (Ptch1) expression in ligated carotids

The protein expression of Sonic hedgehog target gene Ptch1 in carotid cross sections from Ligated (HβCD vehicle control) and Ligated + Cyclopamine (10 mg/kg, IP, every other day) was assessed using a rabbit polyclonal anti-Ptch1 primary antibody and an Alexa fluor 594-conjugated secondary antibody. Representative images (×20 magnification, scale bar 50 μM) shown. DAPI (blue); Ptch1 (red), eGFP (green, i.e., Sca1⁺), merged image (bottom). Dotted lines trace internal and external elastic laminae.

Figure 7. Cyclopamine attenuates ligation-induced carotid remodeling concomitant with inhibition of Sca1⁺ cell expansion

Ligation was performed in Sca1-eGFP transgenic mice treated with or without the SHh signaling inhibitor Cyclopamine (10 mg/kg) or the vehicle control 2-hydropropyl-β-cyclodextrin (HβCD). Carotids harvested 14 d post ligation were assessed for morphology and imaged for enhanced green fluorescent protein (eGFP), indicative of Sca1 expression. Thickness of Adventitia: ‘A’ yellow line, Media: ‘M’ white line, Neo-intima: ‘NI’ red line. Cyclopamine reduced the number of eGFP expressing (Sca1⁺) cells per cross-section, compared to controls and inhibited neo-intima formation. Representative images (×20, scale bars 50 μM) (Verhoeff-Van Gieson stained sections (top) and corresponding confocal immunofluorescence pics showing eGFP (bottom).

Figure 8. EtOH inhibits SHh-induced myogenic differentiation of Sca1⁺ adventitial progenitor cells in vitro

(a) Rat Sca1⁺ adventitial progenitor cells in maintenance media were treated in vitro with or without rShH (0.5 μg/ml), in the absence or presence of EtOH (25 mM), or cyclopamine (15 μM), for 7 days before mRNA levels of the SHh target gene Gli2, and smooth muscle specific markers Myosin heavy chain 11 (Myh11) and Calponin (Cnn1) were assessed by RT-PCR. Data are from a representative experiment of 3 with similar results. (b) rSHh treatment of rat Sca1⁺ APC cells increased the fraction expressing Cnn1 as determined by immunocytochemistry, an effect markedly attenuated by either EtOH (25 mM), or cyclopamine (15 μM) (Figure 8). Representative images shown, together with cumulative data showing fraction of Cnn1⁺ cells in each experimental group. Mean ± SEM, n= 3.