Fibrosis Distinguishes Critical Limb Ischemia Patients from Claudicants in a Transcriptomic and Histologic Analysis

Abstract

Most patients with critical limb ischemia (CLI) from peripheral arterial disease (PAD) do not have antecedent intermittent claudication (IC). We hypothesized that transcriptomic analysis would identify CLI-specific pathways, particularly in regards to fibrosis. Derivation cohort data from muscle biopsies in PAD and non-PAD (controls) was obtained from the Gene Expression Omnibus (GSE120642). Transcriptomic analysis indicated CLI patients (N = 16) had a unique gene expression profile, when compared with non-PAD controls (N = 15) and IC (N = 20). Ninety-eight genes differed between controls and IC, 2489 genes differed between CLI and controls, and 2783 genes differed between CLI and IC patients. Pathway enrichment analysis showed that pathways associated with TGFβ, collagen deposition, and VEGF signaling were enriched in CLI but not IC. Receiver operating curve (ROC) analysis of nine fibrosis core gene expression revealed the areas under the ROC (AUC) were all >0.75 for CLI. Furthermore, the fibrosis area (AUC = 0.81) and % fibrosis (AUC = 0.87) in validation cohort validated the fibrosis discrimination CLI from IC and control (all n = 12). In conclusion, transcriptomic analysis identified fibrosis pathways, including those involving TGFβ, as a novel gene expression feature for CLI but not IC. Fibrosis is an important characteristic of CLI, which we confirmed histologically, and may be a target for novel therapies in PAD.

Interventional‐ and amputation‐stage muscle proteomes in the chronically threatened ischemic limb

“…Development of fibrosis has been previously reported in muscle biopsy specimens from PAD patients 45,46 . Uniformity in expanded extracellular matrix protein abundance across the CLTI population supports the histological characterization of the limb muscles from these patients as pro‐fibrotic 7,47 . The expansion of non‐contractile tissues in response to limb ischemia is not limited to humans, as this is also a hallmark of inbred strains of mice that do not regenerate and recover limb muscle perfusion or function after hindlimb ischemia 48–51 .…”

“…Amputation outcomes in CLTI are defined by a failing cellular microenvironment that may occur despite technically successful surgical intervention. The underlying mechanisms contributing to the unacceptably high amputation rates in this patient population are still somewhat unclear, however this work contributes critical information to a recently expanding book on these patients 6–8,10,11,44,47,55 . This study, in particular, builds on transcriptomics data to highlight a deficit in limb muscle mitochondrial function and the corresponding mitochondrial proteome that defines the non‐salvageable ischemic limb.…”

“… 45 , 46 Uniformity in expanded extracellular matrix protein abundance across the CLTI population supports the histological characterization of the limb muscles from these patients as pro‐fibrotic. 7 , 47 The expansion of non‐contractile tissues in response to limb ischemia is not limited to humans, as this is also a hallmark of inbred strains of mice that do not regenerate and recover limb muscle perfusion or function after hindlimb ischemia. 48 , 49 , 50 , 51 Fibrosis, in general, is a common occurrence in muscles that fail to adequately respond to a degenerative insult.…”

“… 3 In fact, major adverse limb events remain unacceptably high, 4 , 5 highlighting the dire need to better understand CLTI pathobiology. Recent work using wide‐ranging “omics” and screening technologies have revealed tissue characteristics unique to the CLTI presentation (compared to non‐PAD or mild claudicants), including the transcriptome, 6 , 7 , 8 cytokine profile, 9 metabolome, 10 , 11 mitochondrial function, 6 , 12 , 13 and lipid profiles 11 of the limb muscle. Each of these studies has provided new insight into the biological underpinnings of the affected CLTI limb, a necessity to develop new ideas to improve lower limb tissue level perfusion and/or alleviate the severe myopathy that persists in these patients.…”

Peripheral artery disease affects the legs of claudicating patients in a diffuse manner irrespective of the level of the arterial tree primarily involved

“…Arterial blockages and the ischemia and ischemia/reperfusion they generate in the muscles they affect, produce an ischemic myopathy in the legs of PAD patients. At the histological level the principal characteristics of the myopathy are myofiber degeneration and fibrosis of the extracellular matrix and microvessels of the affected muscles [20]- [25], [64]. At the biochemical level the myopathy is characterized by oxidative damage, mitochondrial dysfunction, cytoskeletal disintegration and a characteristic upregulation of cytokines [20], [22], [26]- [31].…”