Association between oxidative-stress-related markers and calcified femoral artery in type 2 diabetes patients

https://doi.org/10.1016/j.jpba.2020.113535Get rights and content

Highlights

  • The calcified femoral artery proteome contains an important antioxidant module.

  • Low levels of SOD2 could be associated with a vascular atherosclerotic process.

  • Further efforts to harmonize assays for SOD2 as therapeutic target are needed.

Abstract

Currently, there are not many in-depth studies focusing on the protein analysis of antioxidants involved in the calcification of the femoral artery. In this context, this study aimed to increase the knowledge of the molecular redox mechanisms involved in this process. Samples from calcified femoral artery sections of seven patients diagnosed with type 2 diabetes (T2D) and critical ischemia were analyzed. The isolated proteins were identified using liquid chromatography and mass–mass spectrometry and were used to generate a protein–protein interaction (PPI) network. Subsequently, highly interconnected regions within the PPI network were identified to obtain a representative module linked to oxidative stress. The proteins of this module with a higher degree of centrality (hubs) were selected to validate them by datamining, transcriptomic and proteomic assays. The analysis of modules of the femoral PPI network showed a module with mainly antioxidant function in which superoxide dismutase 2 (SOD2) was reported as the most important hub. SOD2 was validated at transcriptomic and proteomic level and confirmed by datamining. These results indicate that SOD activity is highly linked to the atherosclerotic process. We suggest that SOD2 could be a potential therapeutic target to prevent the calcification of the femoral artery. The maintenance of optimal SOD2 levels and its cofactors could be used as a preventive strategy for vascular calcification and the related cardiovascular complications in T2D patients.

Introduction

According to World Health Organization, the number of people with diabetes mellitus has quadrupled over the last 30 years, representing the seventh major cause of death worldwide. Type 2 diabetes mellitus (T2D) constitute 90% of diabetes mellitus and cardiovascular disease (CVD) is the leading cause of mortality in the T2D population [1]. Among CVD, peripheral arterial disease (PAD) is highly prevalent in T2D patients. This occurs when part of the arterial internal wall develops extra-skeletal ossification resulting in atherosclerosis. Currently, there is an under-diagnosis of PAD since it is often asymptomatic [2]. This results in a progressive worsening of the atherosclerotic process leading to serious clinical implications such as the diabetic foot and / or the critical ischemia of the lower limbs. The presence of calcified plaques in the femoral artery can affect up to 44.6% of T2D patients [3]. Moreover, there is recent evidence that reports that the presence of femoral plaques is a better indicator of cardiovascular risk than carotid calcification [4], and it remarkably increases cardiovascular morbidity and mortality [5]. However, as compared to the other large vascular arteries, the femoral artery is one of the vascular territories that has fewer in-depth studies at the proteomic level.

Vascular calcification involves an imbalance between pro- and anti-calcifying factors. Oxidative stress, derived from impairment of reactive oxygen species (ROS) production and cellular antioxidant capacity, is one of the most important metabolic factors inducing the development of vascular calcification. It also actively participates in the osteogenic/chondrogenic trans-differentiation of vascular smooth muscle cells (VSMCs) by activating downstream effectors or by reducing the levels of nitric oxide [6].

Proteomic analysis is a hypothesis-free procedure that integrates genetic and epigenetic influences by assessing protein expression profiles with no bias due to prior knowledge. The main biomarker search studies are performed at the serum level, due to their easy acquisition by non-invasive techniques. However, some proteins are not released into the bloodstream but are expressed specifically in the affected tissues. Nonetheless, obtaining tissue samples from healthy donors is often difficult, which greatly limits the performance of comparative proteome case–control studies.

The recent incorporation of bioinformatics tools into the scientific field has helped overcome these limitations. Bioinformatics combines information from multiple sources and generates new knowledge from existing data. The use of resources aimed at the analysis and construction of biological networks allows one to obtain and identify the most important proteins in the network (hubs) by applying computational algorithms. The proteins categorized as hubs are essential for the structure of the biological network, indicating that these proteins play a key role in signaling and regulation processes [7].

In this context, the use of proteomic and bioinformatic approaches allows one to perform directed experiments to deepen our knowledge of the molecular mechanisms involved in oxidative stress, which lead to mineralization of vascular wall. The identification of hub proteins involved in vascular calcification compounding the atherosclerotic process could be an interesting study subject for the design of new preventive and therapeutic strategies to reduce morbi-mortality in the T2D population.

Section snippets

Study population

The study included 7 participants diagnosed with T2D according to the criteria established in the American Diabetes Association (2011). All the studied patients had suffered from critical ischemia according to the consensus document on peripheral disease TASC II [8] with an indication of lower limb amputation due to not being candidates for revascularization or due to it failing. All T2D patients were males (mean age 74 ± 10 years) with mean HbA1c values of 7.63 ± 2.28% and all of them were

Proteomics result and enrichment analysis

The proteomic analysis of the calcified femoral artery using nLC-MS/MS identified 751 proteins corresponding to the cytosolic fraction. The enrichment analysis by Bingo determined the main GO-slim categories in the protein set. Within the biological processes, the ontological terms were grouped into metabolic and cellular processes, response to stimulus, and regulation of biological process and transport. Regarding molecular functions, the most represented terms were grouped into binding,

Discussion

Despite advances in medicine in recent years, to date, there are no effective therapeutic strategies to prevent critical ischemia of the lower limbs in patients with advanced T2D to avoid amputation surgery. Searching for potential therapeutic targets that provide an alternative to performing disabling surgeries is crucial to improve the quality of life of the affected patients.

Our results, combining bioinformatic, genetic, and proteomic approaches revealed that the endogenous antioxidant

Financial support

This research was funded by by the Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía, Spain [grant numbers PI-0207-2016, PI-0268-2019] and by Instituto de Salud Carlos III, Madrid, Spain [grant through the project “PI18-00803” (co-funded by European Regional Development Fund “away to make Europe”)].

CRediT authorship contribution statement

Francisco Andújar-Vera: Methodology, Software, Formal analysis, Investigation, Writing - original draft, Visualization, Writing - review & editing. Cristina García-Fontana: Methodology, Software, Formal analysis, Investigation, Writing - original draft, Visualization, Writing - review & editing. Silvia Lozano-Alonso: Resources, Writing - review & editing. Sheila González-Salvatierra: Investigation, Resources. Iván Iglesias-Baena: Writing - review & editing, Validation. Manuel Muñoz-Torres:

Declaration of Competing Interest

The authors report no declarations of interest.

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