Use of 2-dimensional cell monolayers and 3-dimensional microvascular networks on microfluidic devices shows that iron increases transendothelial adiponectin flux via inducing ROS production

https://doi.org/10.1016/j.bbagen.2020.129796Get rights and content

Highlights

  • Iron is known to have widespread impact on cardiovascular system.

  • The circulating hormone adiponectin has cardioprotective effects.

  • We examined transendothelial adiponectin flux (2-D and 3-D microvessels).

  • Iron increased endothelial permeability and thus adiponectin flux.

  • This effect was mediated via stimulation of ROS production.

Abstract

Background

Iron excess is a risk factor for cardiovascular diseases and it is important to understand the effect of iron on vascular permeability, particularly for the transport of large metabolic hormones such as adiponectin.

Methods

We used 2-dimensional monolayers of cultured human dermal microvascular endothelial cells (HDMEC) and human umbilical vein endothelial cells (HUVEC) as well as 3-dimensional microvascular networks to measure transendothelial flux.

Results

Iron supplementation reduced transendothelial electric resistance (TEER). Flux analysis indicated that under control conditions permeability of 70 kDa dextran and oligomeric forms of adiponectin were restricted in comparison with a 3 kDa dextran, however upon iron treatment permeability of the larger molecules was increased. The increased permeability and size-dependent trans-endothelial movement in response to iron was also observed in 3-dimensional microvascular networks. Mechanistically, the alteration in barrier functionality was associated with increased oxidative stress in response to iron since alterations in TEER and permeability were rescued when reactive oxygen species production was attenuated by pre-treatment with the antioxidant N-acetyl cysteine.].

Conclusions

Iron supplementation induced ROS production resulting in increased transendothelial permeability.

General significance

Altogether, this suggests that the oxidative stress associated with iron excess could play an important role in the regulation of endothelial functionality, controlling hormone action in peripheral tissues by regulating the first rate-limiting step controlling hormone access to target tissues.

Introduction

The endothelium plays a critical physiological role in various ways, such as acting as the primary physical barrier for passage of blood-borne factors to and from the interstitial space in tissues. Both transcellular and paracellular transport routes exist, the latter dependent on proteins forming tight and adherens junction complexes [1]. Not only has it structural importance, the endothelium also makes an important contribution to modulating vascular tone and thus blood flow, as well as formation of atherosclerotic plaques [2,3]. Endothelial dysfunction is one important contributor to cardiovascular complications associated with the metabolic syndrome (MetS), a pathologic state characterized by abdominal obesity, insulin resistance, dyslipidemia and hypertension.

Adiponectin is an important metabolic regulatory hormone which elicits many beneficial responses, including insulin sensitization, cardioprotection and anti-inflammatory effects [4,5]. We and others have recently proposed that transendothelial flux from vasculature to interstitial space is a potentially important regulatory node of adiponectin action [[6], [7], [8], [9]]. The factors which are able to restrict or enhance adiponectin flux, and thus likely attenuate or facilitate its beneficial physiological effects, are still to be fully characterized. We showed recently that glucocorticoids restrict, whereas elevated glucose levels increase, adiponectin flux across endothelial cells or intact blood vessels [7,8].

Excess iron is established as a risk factor for diabetes, cardiomyopathy and other cardiovascular diseases [10,11]. For example, many previous clinical studies have provided correlative associations between iron excess and insulin resistance [12,13]. It has also been shown that high iron levels exacerbate atherosclerosis [14]. These outcomes are at least in part due to direct effects of labile iron on endothelial cells [15]. Studies have demonstrated that iron overload-induced oxidative stress is a primary driver of cellular dysfunction, including insulin resistance [16,17]. Thus, an iron excess-oxidative stress axis plays a causal role in diabetes and cardiovascular complications through mediating effects in a variety of tissues [18].

Although relatively little is known, there are clear indications of crosstalk between iron status and adiponectin. Interestingly, increased serum ferritin levels were associated with decreased adiponectin expression and its plasma level, likely due to adipocyte dysfunction mediated by oxidative stress [[19], [20], [21]]. On the other hand, overexpression of adiponectin attenuated accumulation of iron in the myocardium [22]. We contend that the significance of physiological crosstalk between iron status and adiponectin action are still underappreciated.

The impact of iron on endothelial permeability, and specifically the movement of adiponectin, was investigated in this study. To do so we established a model of iron supplementation in cultured endothelial cell monolayers and also tested our findings in 3-dimensional microvascular networks of human endothelial cells. The underlying mechanism, and in particular the role of oxidative stress, was also investigated. Our study thus provides new knowledge to integrate the ability of iron to produce reactive oxidative species in endothelial cells with consequent changes in endothelial permeability and transendothelial movement of adiponectin.

Section snippets

Cell culture (conventional)

Human dermal microvascular endothelial cells (HDMECs) were cultured in vascular cell basal medium (ATCC) with 10% fetal bovine serum (FBS) in 75cm2 flask at 37 °C, 5% CO2. Once cells were confluent, they were trypsinized and seeded for further experiments with 2% FBS. Human umbilical vascular endothelial cells (HUVECs) (ATCC, Manassas, VA, USA) were grown in microvascular endothelial growth medium 2 (EGM2-MV, Lonza, Cat# cc-3202, ON, CA) with 5% fetal bovine serum (FBS) at 37 °C, and 5% CO2.

Increased intracellular iron alters transendothelial electrical resistance (TEER) and molecular permeability of HUVECs

To monitor cellular iron uptake, both iron-sensitive fluorescent probe and a ratiometric FRET probe were used to assess the intracellular content of ferrous iron (Fe2+). HUVECs were pretreated with the fluorophore Phen Green SK (PGSK), fluorescence of which is quenched upon formation of a complex with Fe2+. Here we confirmed that upon exposure to iron supplementation conditions, the fluorescent signal was significantly reduced after 70 min of iron treatment (Fig. 1A,B). The FRET approach was

Discussion

Here we examined the influence of iron on endothelial function and barrier integrity, an area of fundamental importance in endocrine regulation [29], and with a particular focus on adiponectin flux [9]. Despite this, we know very little about the direct effect of iron on endothelial permeability changesand the subsequent physiological implications of this effect. We first established a model of iron excess by supplementation in a 2-dimensional system of cultured human endothelial cells.

Declaration of Competing Interest

The authors declare no conflict of interest.

Acknowledgements

This work was funded via a Discovery Grant from Natural Sciences and Engineering Research Council (NSERC) to GS. NY acknowledges support from a MITACS Glabalink Exchange Award to perform some of this research with JSJ. GS also acknowledges support via a Career Investigator Award from Heart & Stroke Foundation Ontario. This research was also supported by the Korea Basic Science Institute Grant (D010300), and the Korea Evaluation Institute of Industiral Technology (KEIT) grant funded by the Korea

References (37)

  • Y. Liu et al.

    Examining the potential of developing and implementing use of adiponectin-targeted therapeutics for metabolic and cardiovascular diseases

    Front. Endocrinol.

    (2019)
  • Z.V. Wang et al.

    Adiponectin, the past two decades

    J. Mol. Cell Biol.

    (2016)
  • J.M. Rutkowski et al.

    Differential transendothelial transport of adiponectin complexes

    Cardiovasc. Diabetol.

    (2014)
  • N. Yoon et al.

    Tracking adiponectin biodistribution via fluorescence molecular tomography indicates increased vascular permeability after streptozotocin-induced diabetes

    Am. J. Physiol-Endocrinol. Metab.

    (2019)
  • T.Q. Dang et al.

    Transendothelial movement of adiponectin is restricted by glucocorticoids

    J. Endocrinol.

    (2017)
  • N. Yoon et al.

    Altered transendothelial transport of hormones as a contributor to diabetes

    Diabetes Metab. J.

    (2014)
  • M. Kobayashi et al.

    Pathological roles of iron in cardiovascular disease

    Curr. Drug Targets

    (2018)
  • R. Gordan et al.

    Involvement of cytosolic and mitochondrial iron in iron overload cardiomyopathy: an update

    Heart Fail. Rev.

    (2018)
  • View full text