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 Sca1+ progenitor stem cell involvement during pathologic arterial remodeling. Methods and Results Partial ligation or sham-operation of the left carotid artery was performed in transgenic Sca1-eGFP mice gavaged with or without ‘daily moderate’ EtOH. The EtOH group had reduced adventitial thickening and less neo-intimal formation, compared to ligated controls. There was expansion of eGFP expressing (i.e., Sca1+) cells in remodeled vessels post-ligation (14d), especially in the neo-intima. Ethanol 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 (Ptch1) 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 hedgehog 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.

Disease-relevant single cell photonic signatures identify S100β stem cells and their myogenic progeny in vascular lesions

“…Moreover, while we have previously reported that local perivascular knockdown of the Ptch1 receptor attenuates intimal thickening 32 , systemic inhibition of Hh signalling, using a smoothened inhibitor, also significantly decreases arterial Ptch1 and Gli expression concomitant with a reduction in Sca1 + cells and lesion formation in vivo 84 .…”

“…were analyzed using SPOT Advanced imaging software. Assuming a circular structure in vivo, the circumference of the lumen was used to calculate the lumen area, the intimal area was defined by the luminal surface and internal elastic lamina (IEL), the medial area was defined by the IEL and external elastic lamina (EEL), and the adventitial area was the area between the EEL and the outer edge, essentially as described by us previously 8,84 .…”

“…Immunostaining essentially as previously described 8,78,84 . Carotid artery cryosections were air-dried for 1 h at room temperature, followed by incubation with blocking buffer (5% donkey serum, 0.1% Triton X-100 in PBS) for 30 minutes at room temperature and then incubated with primary antibody overnight at 4 0 C in antibody solution (2.5% BSA, 0.3M Glycine and 1% Tween in DPBS).…”

“…Transforming growth factor (TGF-β1) is a key cytokine regulating myogenic differentiation that is released by both inflammatory cells and vascular cells [endothelial (EC) and SMC] [15,16] within the vessel wall and has been implicated in the etiology of atherosclerosis [17], vessel restenosis [18] and the development of the neointima [19]. Similarly, Notch and hedgehog signalling components are secreted as pro-atherogenic stimuli to promote lesion formation [20][21][22][23].…”

The calcium binding protein S100β marks hedgehog-responsive resident vascular stem cells within vascular lesions

“…Several vascular progenitors have been identified within the vessel wall in both humans and rodents . In particular, the adventitia, which displays increased cell proliferation and inflammation following injury (Seidelmann et al, 2014), contains a heterogeneous population of progenitor/precursor cells with the majority of them expressing Sca1 (Fitzpatrick et al, 2017;Passman et al, 2008;Shikatani et al, 2016;Wan et al, 2012).…”