Inhibition of Intimal Thickening By PRH (Proline-Rich Homeodomain) in Mice

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.


Immunocytochemistry
To detect bound cleaved caspase-3 antibody, cells were incubated with biotinylated

Collagen Contraction Assay
VSMC contractile capability was assessed using Cell Biolabs' Collagen-based Contraction Assay Kit (Cell Biolabs, CBA-201) following the manufacturer's instructions. Briefly, VSMCs were seeded into tissue culture wells and infected with recombinant adenoviruses for 18h. VSMCs were detached using 0.05% (w/v) Trypsin-Ethylenediaminetetraacetic acid (EDTA) at 37°C for 5 minutes and resuspended in 10% (v/v) FBS/DMEM. Cold collagen gel working solution was prepared by mixing the following components in this order collagen solution, 5 x PBS and neutralizing solution. The collagen gel working solution and VSMCs in suspension were mixed on ice at a ratio of 1:4. Following this, 500 μl of the final mixture was cast into each well of a 24-well culture plate. The solution was then allowed to polymerize for 30 min at RT, then 1.0 ml of culture medium was added atop each collagen gel lattice. After polymerization for 48 hours, the gels were gently released from the plates using a sterile needle to allow contraction.

qPCR
Relative quantification of mRNA was conducted using the comparative 2−ΔΔCt method with GAPDH and 36B4 as the reference gene, in which ΔCt = Ct(test) − Ct(control), ΔΔCt = ΔCt(Target gene)− ΔCt(GAPDH/36B4). The quality of qPCR was demonstrated by the detection of a single peak melt curve corresponding to single PCR product.

Next Generation Sequencing
HSV-VSMCs were subjected to infection with 5 x 10 8 pfu/ml of adenovirus encoding either empty vector or c-myc-tagged PRH S163C:S177C, for 18 hours. HSV-ECs were subjected to infection with 1 x 10 8 pfu/ml of adenovirus encoding c-myc-tagged PRH S163C:S177C combined with 4 x 10 8 pfu/ml of adenovirus encoding empty vector or 5 x 10 8 pfu/ml of adenovirus encoding 5 x 10 8 pfu/ml empty vector, for 18 hours. Subsequently, cells were incubated for another 24 hours after replenishing the culture medium. Total RNA was extracted, purified, and quantified prior to NGS by QIAGEN Genomic Services (Germany). Samples were subjected to highthroughput RNA sequencing to perform genome wide analysis of transcriptional diversity and regulation using the TruSeq stranded mRNA kit (QIAGEN) and Illumina NextSeq 550. All analysis was performed using CLC Genomics Workbench (version 12.0.2) and CLC Genomics Server (version 11.0.2). In addition, the human genome version used was hg38 with an annotation of ENSEMBL Homo_sapiens.GRCh38.97.
The samples have been run on an Illumina NextSeq 550 and the aim was to acquire an average of 30 million reads per sample. The parameters were: 100 ng input RNA, 15 cycles of PCR in the fragment enrichment step and the loading molarity used was 1.5 pM.

Bioinformatic Analysis
IPA was performed to identify enriched canonical pathways, diseases and functions, and prioritise the differentially regulated genes (DEGs) identified by NGS and to categorize differentially expressed transcription factors in specific diseases and functions. For an in-depth literature retrieval, 'vascular smooth muscle cell', 'cell proliferation', 'cell contractility', 'phenotypic switching', 'cell migration', 'endothelial cell', and 'inflammation' was used as the keywords to search for the association of DEGs to vein graft failure.

Species
Vendor or Source Background Strain Sex Persistent ID / URL Mouse Charles River C57BL/6J M/F C57BL/6 Mice | Charles River (criver.com)