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Inhibition of Intimal Thickening By PRH (Proline-Rich Homeodomain) in Mice
Lien M Reolizo1, Helen Williams1, Kerry Wadey1
1Bristol Heart Institute, University of Bristol, UK (L.M.R., H.W., K.W., A.F., Z.L., J.L.J., S.J.G.).
Insights
Nonphosphorylatable proline-rich homeodomain (PRH) S163C:S177C protein inhibits vascular smooth muscle cell proliferation and migration, promoting vein graft patency. This novel therapeutic approach preserves endothelial function while reducing intimal thickening.
Area of Science:
- Vascular Biology
- Regenerative Medicine
- Biotechnology
Background:
- Late vein graft failure stems from intimal thickening, driven by endothelial cell (EC) damage and inflammation.
- This process promotes vascular smooth muscle cell (VSMC) dedifferentiation, migration, and proliferation.
- Nonphosphorylatable proline-rich homeodomain (PRH) S163C:S177C protein offers enhanced stability and sustained antimitotic effects.
Purpose of the Study:
- To investigate if adenovirus-delivered PRH S163C:S177C protein can attenuate intimal thickening.
- To assess the protein's effect on VSMC phenotype modification.
- To determine if PRH S163C:S177C has detrimental effects on ECs.
Main Methods:
- Adenovirus-mediated expression of PRH S163C:S177C in human saphenous vein-VSMCs, human saphenous vein-ECs, and ligated mouse carotid arteries.
- Quantification of proliferation, migration, and apoptosis; assessment of VSMC phenotype.
- Measurement of EC inflammation markers (VCAM-1, ICAM-1, IL-6, MCP-1) and monocyte adhesion.
- Next Generation Sequencing to identify downstream mediators of PRH action.
Main Results:
- PRH S163C:S177C inhibited VSMC proliferation, migration, and apoptosis, promoting a contractile phenotype.
- In ECs, PRH S163C:S177C reduced apoptosis and TNF-α-induced VCAM-1/ICAM-1 expression and monocyte adhesion, while suppressing IL-6 and MCP-1.
- In vivo, PRH S163C:S177C significantly impaired neointimal proliferation and thickening in ligated mouse carotid arteries without affecting endothelial coverage.
- STAT-1 and HDAC-9 were identified as key mediators of PRH action.
Conclusions:
- PRH S163C:S177C attenuates VSMC proliferation and migration while enhancing differentiation, partly via STAT-1 and HDAC-9 signaling.
- The protein promotes endothelial repair and exhibits anti-inflammatory properties.
- PRH S163C:S177C holds potential for preserving endothelial function, suppressing intimal thickening, and reducing late vein graft failure.
Background:
Late vein graft failure is caused by intimal thickening resulting from endothelial cell (EC) damage and inflammation which promotes vascular smooth muscle cell (VSMC) dedifferentiation, migration, and proliferation. Nonphosphorylatable PRH (proline-rich homeodomain) S163C:S177C offers enhanced stability and sustained antimitotic effect. Therefore, we investigated whether adenovirus-delivered PRH S163C:S177C protein attenuates intimal thickening via VSMC phenotype modification without detrimental effects on ECs.
Methods:
PRH S163C:S177C was expressed in vitro (human saphenous vein-VSMCs and human saphenous vein-ECs) and in vivo (ligated mouse carotid arteries) by adenoviruses. Proliferation, migration, and apoptosis were quantified and phenotype was assessed using Western blotting for contractile filament proteins and collagen gel contraction. EC inflammation was quantified using VCAM (vascular cell adhesion protein)-1, ICAM (intercellular adhesion molecule)-1, interleukin-6, and monocyte chemotactic factor-1 measurement and monocyte adhesion. Next Generation Sequencing was utilized to identify novel downstream mediators of PRH action and these and intimal thickening were investigated in vivo.
Results:
PRH S163C:S177C inhibited proliferation, migration, and apoptosis and promoted contractile phenotype (enhanced contractile filament proteins and collagen gel contraction) compared with virus control in human saphenous vein-VSMCs. PRH S163C:S177C expression in human saphenous vein-ECs significantly reduced apoptosis, without affecting cell proliferation and migration, while reducing TNF (tumor necrosis factor)-α-induced VCAM-1 and ICAM-1 and monocyte adhesion and suppressing interleukin-6 and monocyte chemotactic factor-1 protein levels. PRH S163C:S177C expression in ligated murine carotid arteries significantly impaired carotid artery ligation-induced neointimal proliferation and thickening without reducing endothelial coverage. Next Generation Sequencing revealed STAT-1 (signal transducer and activator of transcription 1) and HDAC-9 (histone deacetylase 9) as mediators of PRH action and was supported by in vitro and in vivo analyses.
Conclusions:
We observed PRH S163C:S177C attenuated VSMC proliferation, and migration and enhanced VSMC differentiation at least in part via STAT-1 and HDAC-9 signaling while promoting endothelial repair and anti-inflammatory properties. These findings highlight the potential for PRH S163C:S177C to preserve endothelial function whilst suppressing intimal thickening, and reducing late vein graft failure.
