A direct comparison of endothelial progenitor cell dysfunction in rat metabolic syndrome and diabetes

doi:10.1016/j.atherosclerosis.2012.09.029

Atherosclerosis

Volume 226, Issue 1, January 2013, Pages 58-66

https://doi.org/10.1016/j.atherosclerosis.2012.09.029 Get rights and content

Abstract

Objectives

Diabetes mellitus (DM) is associated with impairment of endothelial progenitor cells (EPCs), but the effects of metabolic syndrome (MS) on EPCs have been less well characterized. We hypothesized that in the presence of MS, the number and functionality of EPCs would be markedly reduced, and would be similar to DM.

Methods

Mononuclear cells were isolated from the bone-marrow (BM) and peripheral blood of lean Zucker, obese Zucker, a model of MS, and Zucker diabetic fatty rats. Cultured BM-EPCs underwent in vitro functional testing and the ability of BM-EPCs to promote neovascularization in vivo was assessed in a model of hindlimb ischemia in athymic mice.

Results

While circulating EPC numbers were similarly reduced in both MS and DM rats, BM-derived EPC numbers were less affected. In vitro testing of cultured BM-EPCs from obese Zucker demonstrated a marked reduction in EPC differentiation, a greater propensity to apoptosis, a reduced migratory response and matrigel tubule formation, similar to findings in Zucker diabetic fatty rats. When delivered to the ischemic hindlimb of athymic mice, the recovery of perfusion using both BM-EPCs from obese Zucker and Zucker diabetic fatty rats were diminished, as compared to lean Zuckers.

Conclusion

In the presence of the MS, BM-derived EPCs develop marked functional impairment, resulting in severely reduced angiogenic capacity in vivo. Similar to DM, EPC dysfunction may play a prominent role in the pathogenesis of vascular complications in the MS, and may potentially limit the use of BM-derived EPCs for therapeutic angiogenesis.

Highlights

► We study endothelial progenitor cells (EPC) in metabolic syndrome (MS) and diabetes. ► EPC number and functionality are reduced in metabolic syndrome, similar to diabetes. ► Reduced EPC number in MS may be related to defects in mobilization. ► In MS, EPCs have markedly reduced capacity for angiogenic repair in acute ischemia.

Introduction

Endothelial progenitor cells (EPCs) represent one subset of progenitor cells that originate in the bone-marrow (BM) and are mobilized to the post-natal circulation. These cells can differentiate into mature endothelial cells, and home to sites of tissue injury where they play an important role in facilitating vascular repair and tissue regeneration [1]. Prior studies have demonstrated a clear link between reduced numbers and functionality of circulating EPCs and increased cardiovascular risk [2], [3]. Indeed, EPC dysfunction may not only be a marker of cardiovascular risk, but may also represent a pathophysiologic link between cardiovascular risk factors and the development of atherosclerosis [4], [5].

Abnormal EPC biology has been most characterized in diabetes mellitus (DM), where the number of circulating EPCs is reduced, and EPC proliferation, adhesion, and angiogenic properties are impaired [6], [7], [8]. Metabolic syndrome (MS), a cluster of well-defined cardiovascular risk factors [9], notably dysglycemia, hypertension, elevated triglyceride levels, low high-density lipoprotein cholesterol levels, and obesity (particularly central adiposity), is associated with an increased risk of coronary artery disease and myocardial infarction [10]. While emerging evidence now exists for decreased EPC number and impaired functionality in individuals with MS [11], [12], [13], [14], [15], [16], direct comparisons to EPCs in DM have not been performed. In this study, we hypothesized that in the presence of MS, BM-derived EPCs would have similar reduced angiogenic potential to that seen in DM. In order to test our hypothesis, we investigated the in vitro and in vivo angiogenic potential of BM-derived EPCs from rodent models of MS, Obese Zucker (OZ), and type II DM, the Zucker Diabetic Fatty (ZDF) rats.

Section snippets

Animal preparation

The study protocol was approved by the Animal Care Committee at St. Michael's Hospital Research Institute. Male obese Zucker (OZ) (mean weight 440 g), Zucker Diabetic fatty (ZDF) (mean weight 350 g), and lean Zucker (LZ) (mean weight 280 g) rats (n = 20 per group, 12–14 weeks of age, Charles River Laboratories) were used as EPC-donor animals for in vitro (n = 8–10 per group) and in vivo (n = 8 per group) studies. The Obese Zucker (OZ) rat develops central obesity, insulin resistance, impaired

Baseline biochemistry

Serum biochemistry from all animal groups is shown in Table 1. As expected, serum fasting blood glucose (FBG), total cholesterol (TC), triglycerides (TG), plasma insulin levels, fructosamine and HOMA index were normal in LZ rats. OZ rats displayed significant elevations in TC and TG. Compared to LZ, OZ rats also had marked elevations in fasting insulin levels and HOMA index, without significant elevations in FBG or fructosamine, in keeping with marked insulin resistance without frank

Discussion

In our present study, we demonstrate that in the presence of metabolic syndrome BM-EPCs develop marked functional impairment identical to that seen in DM, with defects in their migratory and proliferative potential which severely limits their ability to promote endothelial repair and angiogenesis.

Accumulating evidence has demonstrated a clear link between reduced circulating numbers of EPCs and increased cardiovascular risk [2], [3], with the impairment of EPC functionality best characterized

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Given the strong association between impaired renal microcirculation and disease states, significant attention has been directed toward understanding renal vascular repair. Endothelial progenitor cells (EPCs) have been studied in different cardiovascular models, including preclinical models of kidney disease. However, as this field of study has evolved over the last 20 years, it has been hampered by inconsistent definitions surrounding the term EPC.
This chapter attempts to provide relevant background regarding vascular injury and impaired regenerative responses in the setting of either acute kidney injury (AKI) or chronic kidney disease (CKD). We provide an updated discussion on different types of cells previously classified broadly as EPCs and discuss potential interactions of these cells in vascular repair. Using this framework, we present a hypothesis by which the activity of intrinsic vascular progenitors represents a central element of disease progression. Finally, we summarize evidence that cells with vasculoreparative properties may represent potential therapies in disease states.
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The repair and regeneration of adult tissues is accomplished through the existence and function of stem or progenitor cell populations. With regards to vascular repair, this is accomplished through the use of heterogeneous populations of proangiogenic cells, often called the endothelial progenitor cells (EPCs). In response to hypoxic or inflammatory signals, EPCs are mobilized from storage niches such as the bone marrow, home to site of vascular damage and participate in repair through terminal differentiation or paracrine effects. While the existence of these endothelial reparative cells has been recognized for almost 20 years, an accurate phenotypic description of these cells still remains controversial and complex analytical strategies are required for their identification and functional analysis. Multiple animal and human studies have demonstrated the physiological importance of EPCs, and evidence suggests a paucity of these cells or their dysfunction is correlated with poor vascular health and an increased risk of developing or exacerbating cardiovascular disease. Moreover, the levels and functionality of these cells are particularly sensitive to toxicological exposures and external influences, defining a mechanistic connection between such stimuli and resulting pathology.
FGF-2-mediated FGFR1 signaling in human microvascular endothelial cells is activated by vaccarin to promote angiogenesis
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The endothelial cell dysfunction plays critical roles in metabolic syndrome, diabetes mellitus and atherosclerosis [1–3].
Angiogenesis is a complex physiological process involving the growth of new capillaries. The impaired angiogenesis plays important roles in chronic wounds and ischaemic heart disease. Fibroblast growth factor 2 (FGF-2) exerts pro-angiogenic actions via activation of fibroblast growth factor receptor 1 (FGFR-1). We have identified that vaccarin increased the angiogenic activity of endothelial cells. In this study, we investigated whether FGF-2-mediated FGFR1 signaling pathway participated in vaccarin-mediated neovascularization formation. Human microvascular endothelial cells (HMEC)‐1 were incubated with various doses of vaccarin. Our results showed that vaccarin dose-dependently up-regulated FGF-2 levels and phosphorylation of FGFR-1. Neutralization of FGF-2 with anti-FGF-2 antibody also abolished the proliferation, migration and tube formation of HMEC‐1 cells induced by vaccarin. Both FGFR-1 inhibitor SU5402 and FGFR-1 siRNA blocked vaccarin-induced cell cycle progression and angiogenesis. The mouse Matrigel model study further unveiled that vaccarin stimulated the neovascularization and microvessel density in vivo, which was prevented by FGFR-1 inhibitor SU5402. Taken together, our results demonstrated for the first time that vaccarin was a novel inducer for FGF-2 expression, followed by phosphorylation of FGFR-1 and subsequent angiogenic behaviors in endothelial cells. Vaccarin may be a promising candidate of angiogenesis activator for neurovascular repair or therapy.
An association of serum vistafin level and number of circulating endothelial progenitor cells in type 2 diabetes mellitus patients
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However, the metabolic crosstalk between endothelium integrity, vascular function and the endothelial repair systems is largely unknown and innate molecular mechanisms regarding cooperation of above-mentioned factors remain incompletely understood [6]. Visceral adipose tissue (VAT) communicates with other organs by the synthesis and release of the wide spectrum of adipocytokines that have a critical role in inflammation, insulin resistance, obesity, T2DM and CV disease [7,8]. Visfatin (also known as nicotinamide phosphorybosil transferase) is an adipocytokine, which widely expressed in liver, skeletal muscles, bone marrow and predominantly secreted by white VAT [9].
The decreased number and impaired functions of endothelial progenitor cells (EPCs) may associate with cardiovascular disease (CV) including atherosclerosis. However, the role of vistafin in regulation of angiogenic EPC subset maturation in T2DM patients without known atherosclerosis is still not fully understood.
To investigate an association of serum vistafin level and number of circulating EPCs in T2DM patients beyond known CV disease.
This case–control observational investigation was evolved 54 subjects with T2DM and 35 healthy volunteers. The flow cytometry was used for predictably distinguishing cell subsets, which depend on expression of CD45, CD34, CD14, Tie-2, and VEGFR2. Biomarkers were measured at baseline of the study.
All T2DM patients were divided depending median of vistafin level (5.88 ng/mL) in to two cohorts with low vistafin level (<5.88 ng/mL; n = 29) and high vistafin level (≥5.88 ng/mL; n = 25) respectively. Logistic regression analysis has shown that visfatin, hs-CRP, age and BMI were the best variables in the prediction of EPC number labeled as CD14⁺CD309⁺ and CD14⁺CD309⁺Tie²⁺ cells. After adjustment of the model to age and BMI elevated visfatin level remained the best predictor for both CD14⁺CD309⁺ and CD14⁺CD309⁺Tie²⁺ EPCs (OR 0.92, 95% CI: 0.88–0.95; P = 0.001 and OR 0.90, 95% CI: 0.87–0.96; P = 0.001 respectively).
We found that elevated level of vistafin was an independent predictor for declined numerous of non-classical EPCs labeled as CD14⁺CD309⁺ and CD14⁺CD309⁺Tie²⁺, whereas CD34⁺ subsets of EPCs did not associate with vistafin level in T2DM individuals.
Low concentration of ethanol favors progenitor cell differentiation and neovascularization in high-fat diet-fed mice model
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It is known that BM is altered by changes in metabolism and metabolic diseases. Indeed, in the presence of metabolic syndrome, BM-derived cells develop marked functional impairment with defects in their differentiation, migratory capacity and proliferative potential (Kuliszewski et al., 2013). The HFD-fed rodent is one of the most widely used models for studying the metabolic defects caused by obesity in humans, including glucose intolerance, insulin resistance and type 2 diabetes mellitus (Park et al., 2005; Strissel et al., 2007).
Endothelial progenitor cells (EPCs) and monocytic cells from bone marrow (BM) can be recruited to the injured endothelium and contribute to its regeneration. During metabolic diseases such as obesity and diabetes, progenitor cell function is impaired. Several studies have shown that moderate alcohol consumption prevents the development and progression of atherosclerosis in a variety of animal/mouse models and increases mobilization of progenitor cells. Along with these studies, we identify ethanol at low concentration as therapeutic tool to in vitro expand progenitor cells in order to obtain an adequate number of cells for their use in the treatment of cardiovascular diseases.
We evaluated the effects of ethanol on the phenotype of BM-derived cells from mice fed with high-fat diet (HFD). HFD did not induce changes in weight of mice but induced metabolic alterations. HFD feeding increased the differentiation of monocytic progenitors but not EPCs. Whereas ethanol at 0.6% is able to increase monocytic progenitor differentiation, 1% ethanol diminished it. Furthermore, ethanol at 0.6% increased the ability of progenitor cells to promote in vivo angiogenesis as well as secretome of BM-derived cells from mice fed with HFD, but not in mice fed normal diet. In conclusion, ethanol at low concentration is able to increase angiogenic abilities of progenitor cells from animals with early metabolic alterations.
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It seems agreed that the number of blood EPCs or their function is affected in the age-related diseases or events that are associated with re-endothelialization. For example, the number of EPCs is reduced in patients with diabetes (Loomans et al., 2004; Togliatto et al., 2010; Kuliszewski et al., 2013). EPCs from patients with essential hypertension exhibit a decreased migratory and proliferative activity (Yang et al., 2010).
Advanced age is an independent risk factor for ageing-related complex diseases, such as coronary artery disease, stroke, and hypertension, which are common but life threatening and related to the ageing-associated vascular dysfunction. On the other hand, patients with progeria syndromes suffer from serious atherosclerosis, suggesting that the impaired vascular functions may be critical to organismal ageing, or vice versa. However, it remains largely unknown how vascular cells, particularly endothelial cell, become senescent and how the senescence impairs the vascular functions and contributes to the age-related vascular diseases over time. Here, we review the recent progress on the characteristics of vascular ageing and endothelial cell senescence in vitro and in vivo, evaluate how genetic and environmental factors as well as autophagy and stem cell influence endothelial cell senescence and how the senescence contributes to the age-related vascular phenotypes, such as atherosclerosis and increased vascular stiffness, and explore the possibility whether we can delay the age-related vascular diseases through the control of vascular ageing.

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^☆: Supported by an Operating Grant from the Heart and Stroke Foundation of Canada, and a Team Grant from the Canadian Institutes of Health Research, Ottawa, Ontario, Canada. Dr. Kutryk is supported by a Clinician Scientist Phase II Award from the Heart and Stroke Foundation, Ontario, Canada. Dr. Leong-Poi holds the Brazilian Ball Chair in Cardiovascular Research, St. Michael's Hospital, University of Toronto, and is supported by an Early Researcher Award from the Ministry of Research and Innovation, Ontario, Canada.

¹: Both authors contributed equally to this work.

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A direct comparison of endothelial progenitor cell dysfunction in rat metabolic syndrome and diabetes☆

Abstract

Objectives

Methods

Results

Conclusion

Highlights

Introduction

Section snippets

Animal preparation

Baseline biochemistry

Discussion

Am Heart J

Atherosclerosis

J Am Coll Cardiol

Atherosclerosis

Atherosclerosis

Atherosclerosis

Atherosclerosis

Mol Ther

Atherosclerosis

Am J Cardiol

Lancet

Atherosclerosis

Am Heart J

Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization

Circ Res

Circulating endothelial progenitor cells, vascular function, and cardiovascular risk

N Engl J Med