Clinical
Type 2 diabetes impairs venous, but not arterial smooth muscle cell function: Possible role of differential RhoA activity

https://doi.org/10.1016/j.carrev.2014.02.005Get rights and content

Abstract

Background/purpose

Coronary heart disease is the leading cause of morbidity in patients with type 2 diabetes mellitus (T2DM), frequently resulting in a requirement for coronary revascularization using the internal mammary artery (IMA) or saphenous vein (SV). Patency rates of SV grafts are inferior to IMA and further impaired by T2DM whilst IMA patencies appear similar in both populations. Smooth muscle cells (SMC) play a pivotal role in graft integration; we therefore examined the phenotype and proliferative function of IMA- and SV-SMC isolated from non-diabetic (ND) patients or those diagnosed with T2DM.

Methods/materials

SMC were cultured from fragments of SV or IMA. Morphology was analyzed under light microscopy (spread cell area measurements) and confocal microscopy (F-actin staining). Proliferation was analyzed by cell counting. Levels of RhoA mRNA, protein and activity were measured by real-time RT-PCR, western blotting and G-LISA respectively.

Results

IMA-SMC from T2DM and ND patients were indistinguishable in both morphology and function. By comparison, SV-SMC from T2DM patients exhibited significantly larger spread cell areas (1.5-fold increase, P < 0.05), truncated F-actin fibers and reduced proliferation (33% reduction, P < 0.05). Furthermore, lower expression and activity of RhoA were observed in SV-SMC of T2DM patients (37% reduction in expression, P < 0.05 and 43% reduction in activity, P < 0.01).

Conclusions

IMA-SMC appear impervious to phenotypic modulation by T2DM. In contrast, SV-SMC from T2DM patients exhibit phenotypic and functional changes accompanied by reduced RhoA activity. These aberrancies may be epigenetic in nature, compromising SMC plasticity and SV graft adaptation in T2DM patients.

Summary

The internal mammary artery (IMA) is the conduit of choice for bypass grafting and is generally successful in all patients, including those with type 2 diabetes (T2DM). By contrast, saphenous vein (SV) is inferior to IMA and furthermore patients with T2DM suffer strikingly poorer outcomes than their non-diabetic (ND) counterparts. We discovered that SV-SMC from T2DM patients exhibit altered persistent morphology and function compared to ND SV-SMC, with differential expression and activity of the small GTPase RhoA, yet ND and T2DM IMA-SMC were indistinguishable. These data offer an explanation for the superior patency of IMA grafting independent of the presence of diabetes.

Introduction

Type 2 diabetes mellitus (T2DM) is an escalating global epidemic, and in the UK alone the number of patients with diagnosed T2DM has almost doubled over the past 15 years (diabetes.org.uk). Importantly, treatment of patients with T2DM and its resultant complications now accounts for approximately 10% of the entire UK National Health Service budget. One of the leading causes of morbidity and mortality in patients with T2DM is accelerated atherosclerosis and coronary heart disease [1] that often precedes clinical diagnosis of T2DM [2].

The surgical approach to revascularizing atherosclerotic coronary arteries is coronary artery bypass grafting (CABG) using autologous internal mammary artery (IMA) or saphenous vein (SV) to restore blood supply to the ischemic heart. Whilst the IMA is known to be a superior conduit with patency rates significantly higher than SV [3], due to its limited availability and the need for multiple grafts the SV is routinely the conduit of choice in many patients. Furthermore, patients with T2DM have poorer SV graft outcomes compared to their non-diabetic counterparts [4]. Interestingly, this is not the case with IMA, in which patency rates are comparable between patients with or without T2DM (reviewed recently in [5]).

IMA and SV are structurally distinct vessels [3] that respond differently to alterations in pressure and cyclical stretching that are evident when vessels are implanted following CABG. This leads to changes in vessel structure and function, e.g. altered distensibility and stiffness (reviewed in [6]) and intimal abnormalities [7], due at least in part to the orientation and behaviour of smooth muscle cells (SMC). Successful integration of grafts early after implantation requires efficient adaptive remodeling [8], a process that involves phenotypic switching of SMC in terms of co-ordinated migration, proliferation and cytoskeletal rearrangement. This functional capacity to adapt is temporally distinct from the subsequent neointimal thickening that underlies narrowing and restenosis. The signaling cascades regulating these processes are complex and include mitogen activated protein kinases (MAPK) such as extracellular signal regulated kinase (ERK) and p38 MAPK [9], amongst others. Small GTPases and in particular RhoA/Rho kinase are also well recognized effectors of such adaptive changes [10].

RhoA is an archetypal member of the Rho family of small GTPases, activation of which promotes formation of F-actin stress fibers and focal adhesions which link stress fibers to the plasma membrane, thereby affecting SMC contractility and adhesion [11]. RhoA regulates many cellular functions including migration and proliferation [11], dysregulation of which are implicated in cardiovascular disorders such as hypertension, coronary artery vasospasm and neointimal hyperplasia (reviewed in [10]). RhoA is reportedly activated by hyperglycemia, and accordingly aberrant RhoA activity has been demonstrated in rodent models of diabetic nephropathy [12] and myocardial fibrosis [13].

We have previously reported inherent differences in the morphology and function of SV-SMC from non-diabetic (ND) and T2DM patients; specifically that SV-SMC from patients with diabetes exhibited rhomboid-like morphology, altered cytoskeletal arrangement and impaired proliferative capacity compared to those isolated from patients without diabetes [14]. Of particular interest was our observation that the cellular disparities were maintained throughout culture and serial passaging and not influenced by glucose concentration. We therefore speculated that through prior exposure to the metabolic milieu, SMC from diabetic patients show evidence of “memory”. The aim of this study was therefore to investigate any influence of T2DM on IMA-SMC phenotype and to determine a potential role for RhoA.

Section snippets

SMC isolation and culture

SMC were obtained from IMA and SV fragments from a total of 63 different patients undergoing CABG at Leeds General Infirmary, UK, and were cultured by explant technique as described previously [15]. Local ethical committee approval and informed patient consent were obtained. This study conformed to the principles outlined in the Declaration of Helsinki. Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS), 1% L-Glutamine and 1%

Morphology of IMA and SV-SMC from ND and T2DM patients

Differences between IMA and SV-SMC were evident soon after explant (typically between 3 and 8 days). IMA-SMC presented a wide distribution of spread cell area but ND and T2DM IMA-SMC were not significantly different (9586 ± 814 μm2 versus 11969 ± 2728 μm2, respectively) (Fig. 1A and B).

In ND patients, venous cells were ~ 33% smaller in area than arterial cells (6454 ± 947 μm2 versus 9586 ± 814 μm2). However, when comparing SV-SMC between ND and T2DM patients, the latter were significantly larger (1.5-fold

Discussion

In this study we demonstrated that IMA-SMC from T2DM patients were morphologically indistinguishable from those of ND patients. In contrast, SV-SMC from T2DM patients displayed a distinct phenotype (aberrant morphology, cytoskeletal disarray and impaired proliferative capacity) that was accompanied by reduced levels of RhoA expression/activity. Effective adaptation to arterial environments early after implantation is a key determinant of the long-term patency of SV grafts [8]. The ability of

Conclusions

It is unequivocal that in vivo, the structural and mechanical differences between the SV and IMA, together with significant endothelial dysfunction impact vascular function. The novelty of this study is the observation of a distinct SV-SMC phenotype in T2DM whereas IMA-SMC do not appear vulnerable to T2DM-induced phenotypic alterations (cell morphology, cytoskeleton and proliferation). In contrast, SV-SMC isolated from patients with T2DM exhibit distinct aberrancies accompanied by reduced

Acknowledgments

We are grateful to Jean Kaye for cell culture expertise.

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