Pre-Clinical Assessment of Composition of Chronic Venous Occlusion Formed in a Pig

Venous thrombosis that transition to chronic occlusions that are difficult to address with current interventions. Histotripsy is a focused ultrasound therapy that ablates tissue through bubble cloud activity. Pre-clinical studies indicate acute thrombosis is sensitive to histotripsy, but outcomes for chronic disease require further investigation. This study developed a porcine model to test the hypothesis that histotripsy will disrupt disorganized extracellular matrix within chronic venous occlusions. Thrombi were formed in the femoral vein of female mixed breed pigs via selective catheterization. The occlusion was monitored for 30 days with ultrasound imaging, after which the femoral bundle was resected for analysis with bulk RNA-seq (N = 3). Changes to the occlusion appearance were noted throughout aging. At 30 days, obstructed venous tissue was composed primarily of collagen (mean: 39.7% by area) with activated macrophages (mean: 22.2% of cells) and fibroblasts (mean: 21.6% of cells). Overall design: Thrombi were formed in the femoral vein of female mixed breed pigs via selective catheterization. After 30 days, the vascular bundle was resected and embedded in parafin. Histological regions of interest on control chronic occlusions were identified and annotated on H&E-stained images. The corresponding tissue areas were dissected and processed for RNA isolation using the RNeasy FFPE kit (QIAGEN Cat#73504) according to the manufacturer instructions. Selected regions were deparaffinized, digested with Proteinase K and crosslinks were reversed before RNA purification. The resulting purified product was subsequently used for RNA sequencing. Strand-specific RNA-seq libraries were prepared using an TruSEQ total RNA-SEQ library protocol (Illumina provided). Library quality and quantity was assessed using an Agilent bio-analyzer and libraries were sequenced using an Illumina NovaSEQ-X2. The data were processed to count tables using the nf-core/rnaseq pipeline. Reads were trimmed using Trim Galore and then mapped to the Sscrofa11.1 genome assembly using STAR. Quantification of gene counts was done using Salmon. FastQC and MultiQC were used for data quality checking. The online version of CYBERSORTx was used for deconvolution. The safeTME matrix was exported from the spatialDecon R package and used as the signature matrix in CYBERSORTx. The raw count table was normalized by library size, scaled by gene length, and log2 transformed to be used as the mixture file in CYBERSORTx. The "Impute Cell Fractions" analysis module was run with the following parameters: Batch correction - disabled, Disable quantile normalization - true, Run mode (relative or absolute) - relative, Permutations - 1. The top 200 highly expressed genes in the RNAseq sample were overlapped with the safeTME signature genes to verify the deconvoluted major cell types. Two sections were processed for each control chronic occlusion with residual thrombus (N = 6 sections total from 3 venous occlusions).