Negative pressure wound therapy affects circulating plasma microRNAs in patients with diabetic foot ulceration

Abstract

Aims

Negative pressure wound therapy (NPWT) is commonly used in diabetic foot ulceration (DFU). The molecular mechanisms of NPWT action, particularly outside of the wound site, have not been described. We assessed NPWT’s effect on circulating miRNA expression levels in type 2 diabetes (T2DM) patients with DFU.

Methods

We examined 34 T2DM patients treated with either NPWT (n = 24) or standard therapy (ST, n = 10). The group assignment was based on clinical criteria and local practice. Next-generation sequencing-based microRNA expression was determined on the patient’s plasma collected before therapy and after 8 days.

Results

NPWT patients were similar to the ST group in terms of age, BMI, and HbA1c level; however, they differed by mean wound area (12.6 cm² vs. 1.1 cm² p = 0.0005). First, we analyzed the change of miRNA after NPWT or ST and observed an upregulation of let-7f-2 only in the NPWT group. Then, we analyzed the differential expression between NPWT and ST groups, looking at possible wound size effects. We found 12 differentially expressed miRNAs in pre-treatment comparison, including let-7f-2, while in post-treatment analysis we identified 28 miRNAs. The pathway enrichment analysis suggests that identified miRNAs may be involved in wound healing, particularly through angiogenesis.

Conclusion

We found initial evidence that NPWT in T2DM patients with DFU affects miRNA expression in plasma. Additionally, some differences in plasma miRNA expression may be related to wound size.

Introduction

Poorly controlled diabetes mellitus predisposes patients to different clinical manifestations of atherosclerosis, microvascular abnormalities, and peripheral neuropathy [1]. One of the most common and severe complications of this disease is diabetic foot syndrome, which is defined as ulceration or destruction of deep tissues of the foot [2]. Diabetic foot ulceration (DFU) represents a major risk factor for intractable infection, foot deformity, lower extremity amputations and mortality [3]. The standard management for DFU treatment includes glycemic control, surgical wound debridement, pressure off-loading, revascularization, infection management and different topical dressings [4]. Despite this standard approach, many patients still suffer from delayed wound healing and concomitant complications; therefore, new treatment options such as negative pressure wound therapy (NPWT) have emerged as adjunctive therapy for DFU. NPWT involves the application of air-tight occlusive dressings and local subatmospheric pressure to a wound in order to accelerate wound healing [5] by different local molecular mechanisms promoting pro-angiogenic and anti-inflammatory effects in the wound bed as reviewed by Borys et al. [6]. It is, however, worth considering that NPWT may also stimulate cellular mechanisms resulting in profile changes to circulating microRNAs (miRNAs).

MicroRNA (miRNA), highly conserved sequences of small RNAs (~22 nucleotides), are regulators of a diverse range of cellular functions, acting mainly via translational inhibition after binding to mRNA transcripts [7]. Thereby, miRNAs regulate many biological processes, such as cellular differentiation, proliferation, inflammation and apoptosis [8]. Some miRNAs are released into the circulation either passively upon cell injury and death, or actively for intercellular communication [9]. These extracellular miRNAs are protected from degradation by RNAses because they are packaged into vesicles, such as exosomes, microparticles and/or apoptotic bodies, or linked to RNA-binding proteins [10]. Although the assessment of circulating miRNA cell origin is not yet possible, it is likely that miRNAs profiles in the circulation are influenced by processes in organs and tissues whose functions are altered in human diseases [11]. MiRNAs present in the blood, isolated either from plasma or serum, have been described as novel biomarkers of the onset and progression of diabetes mellitus [12], [13], [14]. Additionally, circulating miRNAs may also contribute to the development of diabetic complications [15], [16]. Interestingly, there is also some evidence that therapies, such as NPWT, may act, at least partially, by impacting miRNA expression [17]. The possible effect of NPWT on plasma miRNA signatures in DFU is unknown. Therefore, we investigated the impact of NPWT on circulating miRNA patterns in patients with type 2 diabetes mellitus (T2DM) with DFU and compared it with standard therapy (ST).