Treatment of atherosclerosis through transplantation of endothelial progenitor cells overexpressing dimethylarginine dimethylaminohydrolase (DDAH) in rabbits

https://doi.org/10.1016/j.ijcard.2021.01.036Get rights and content

Highlights

  • EPC treatment effectively reduces the size of atherosclerotic plaque.

  • DDAH2 overexpression increases the endothelial protection function of EPCs.

  • Both EPCs and DDAH2 gene-transfected EPCs ameliorate endothelial defects by increasing eNOS activity.

  • DDAH2 gene-transfected EPCs are a potentially valuable tool for the treatment of atherosclerosis.

Abstract

Background

Endothelial dysfunction is a key event in the development of vascular diseases, including atherosclerosis. Endothelial progenitor cells (EPCs) play an important role in vascular repair. Decreased dimethylarginine dimethylaminohydrolase (DDAH) activity is observed in several pathological conditions, and it is associated with an increased risk of vascular disease. We hypothesized that bone marrow-derived EPCs and combination therapy with DDAH2-EPCs could reduce plaque size and ameliorate endothelial dysfunction in an atherosclerosis rabbit model.

Method

Four groups of rabbits (n = 8 per group) were subjected to a hyperlipidemic diet for a month. After establishing the atherosclerosis model, rabbits received 4 × 106 EPC, EPCs expressing DDAH2, through femoral vein injection, or saline (the control group with basic food and the untreated group). One month after transplantation, plaque thickness, endothelial function, oxidative stress, and inflammatory mRNAs, DDAH, and eNOS function were assessed.

Results

DDAH2-EPCs transplantation (p < 0.05) and EPCs transplantation (p < 0.05) were both associated with a reduction in plaque size compared to the control saline injection. The antiproliferative and antiatherogenic effects of EPCs were further enhanced by the overexpression of DDAH2 (p < 0.05, DDAH2-EPCs vs. EPCs). Furthermore, DDAH2-EPCs transplantation significantly increased endothelium integrity compared to the EPCs transplantation.

Conclusion

Transplantation of EPCs overexpressing DDAH2 may enhance the repair of injured

endothelium by reducing inflammation and restoring endothelial function. Therefore, pCMV6-mediated DDAH2 gene-transfected EPCs are a potentially valuable tool for the treatment of atherosclerosis.

Introduction

Atherosclerosis is a chronic progressive inflammatory systemic disease caused by hyperlipidemia, hypertension, hyperglycemia, and other associated risk factors, such as inflammation and dysfunction in endothelial cells [[1], [2], [3]]. Therefore, directed migration of inflammatory cells by chemokines, such as monocyte chemoattractant protein-1 (MCP-1), is involved in accumulating low-density lipoprotein-cholesterol (LDL-C) in the artery wall, loss of collagen, and the development of atherosclerotic plaques [[2], [3], [4]]. Increased levels of LDL particles lead to its entry into the arterial wall, where it may be modified through various processes such as oxidation by reactive oxygen species and aggregation. Modified forms of LDL trigger adaptive immune responses (presence of both activated T cells and antibody-producing B cells) and act as ligands for macrophage pattern recognition receptors, including Toll-like receptors (TLRs) and directly activate the expression of proinflammatory signalling pathways. The TLR activity leads to the production of cytokines and chemokines, amplification of the inflammatory process and intracellular formation of cholesterol crystals, may lead to an increase in inflammatory gene expression [5,6]. Intimal hemorrhage and plaque rupture is the leading cause of atherothrombosis, caused by damage to the endothelium and the release of atherosclerotic lipid depositions that comprise: macrophages, lymphocytes, smooth muscle cells, and fibroblasts, into the bloodstream [2,7]. Although anti-platelet and lipid-lowering drugs are relatively effective for treatment, these interventions are not very effective in stabilizing vulnerable plaques, and ultimately reducing the burden of the disease [2].

Endothelial cells are integral components of the normal intima, providing an interface between the circulation and the tissue surrounding the vessel [8]. In the face of endothelial dysfunction, a small population of cells, known as the endothelial progenitor cells (EPCs), which are a type of adult bone marrow-derived precursor cells (less than 1% of all bone marrow cells [9]), contribute to endothelial replacement by homing to sites of injury and rapidly differentiating into mature ECs locally, possibly as a compensatory mechanism [2,7,10,11]. Therefore, EPCs are proposed as crucial cells in the stabilization of atherosclerotic plaques and the regeneration of damaged endothelial cells [2,12] . The respective growth potential of EPCs involves inhibiting neointima formation following vascular injury, and reducing the occurrence of restenosis and cardiovascular complications [13,14]. Although atherosclerosis remains a major cause of morbidity and mortality worldwide, little research has been carried out on the pivotal role of cell-based therapy, including EPCs, in this disease due to difficulty in the attachment of the reseeded EPCs onto the injured endothelial layer and the risk factors that hinder EPC function [14,15].

An elevated level of asymmetric dimethylarginine (ADMA) is a useful diagnostic biomarker for cardiovascular events, such as endothelial dysfunction which is an early stage in the development of atherosclerosis [[16], [17], [18]]. This cysteine hydrolase enzyme, i.e., dimethylarginine dimethylaminohydrolase (DDAH), is responsible for catalyzing the hydrolysis of endogenous ADMA. However, reduced expression of DDAH or decreased activity of DDAH can contribute to local accumulation of ADMA that could inhibit all types of nitric oxide synthase (NOS), and impair endothelium-dependent relaxation in experimental models of cardiovascular diseases [[18], [19], [20]]. Isoform 2 DDAH (DDAH2), as an atheroprotective factor in the regulation of ADMA concentration in endothelial cells, is somewhat related to NOS isoforms [21,22]. Since there are no effective drugs for upregulating DDAH2 activity, gene delivery of DDAH2 to the damaged vascular wall might be an applicable approach for improving atherosclerosis [22].

As a potential method, EPCs can serve both as a tissue-engineering tool and a vehicle for gene transfer to reconstruct the heart's vasculature [23,24]. In this study, we studied the effect of combination therapy with human DDAH2 and bone marrow-derived EPCs in repairing endothelium dysfunction. We showed that the overexpression of DDAH2 gene in rabbit EPCs could reduce aortic plaque size, and potentially improve the oxidative stress and inflammatory parameters in the atherosclerosis rabbit model. These findings suggest that transplantation of engineered EPCs may be an effective alternative therapeutic strategy for cardiovascular complications.

Section snippets

Materials and methods

We have previously [25] identified rabbit bone marrow EPCs and constructed a hybrid structure with rabbit EPC and PCMV6 expressing GFP-DDAH2. Moreover, the function of this transfection has been confirmed in vitro.

Establishment of the atherosclerosis rabbit model

After feeding the rabbits a high fat diet for a month, mild to moderate atherosclerosis, characterized by local thickening of the intima-media on aorta, was observed (in our pilot study, 1% cholesterol diet for one month was confirmed to result in the accumulation of a small numbers of foam cells and smooth muscle cells under microscope in rabbits; data not shown). Furthermore, the serum levels of TC, TG, and LDLC were significantly increased while HDL levels were reduced in the high fat

Discussion

Atherosclerosis is an inflammatory disease that can be characterized by endothelial damage in the artery wall [14,30]. Dramatic reduction of EPCs, can lead to the progression of atherosclerotic damage from the development of the disease to systemic complications [31]. Since DDAH2 also plays a dominant role in the performance of cardiac endothelial cells [32,33], the effects of EPC-DDAH2 hybrid were investigated in an atherosclerosis animal model in the present study. Several studies have shown

Conclusions

EPC treatment effectively reduced the size of atherosclerotic plaque in the aorta. Furthermore, DDAH2 overexpression could increase the endothelial protection function of EPCs. These findings suggest that transplantation of genetically engineered EPCs (EPC-DDAH2 hybrid) can be considered as an effective therapeutic strategy for regulating endothelial cell function as well as treating atherosclerosis.

Acknowledgement

This work was conducted as a PhD thesis supported by a grant from Mashhad University of Medical Sciences, Mashhad, Iran.

Authorships

  • Participated in research design: Dr. Sara Shoeibi and Dr. Elahe Mahdipour

  • Participated in the writing of the paper: Dr. Sara Shoeibi

  • Participated in the performance of the research: Dr. Sara Shoeibi, Dr. Shabnam Mohammadi, and Dr. Elahe Mahdipour

  • Participated in data analysis: Dr. Sara Shoeibi, and Dr. Majid Ghayour-Mobarhan

Participated in the editing of the paper: Dr. Sara Shoeibi, Dr. Elahe Mahdipour, Dr. Majid Ghayour-Mobarhan, Dr. Shabnam Mohammadi, and Dr. Mohsen Moohebati.

Disclosure

The authors declare no conflicts of interest.

Funding

Mashhad University of Medical Sciences, Mashhad, Iran.

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