Conjugated linoleic acid improves endothelial Ca2+ signaling by blocking growth factor and cytokine-mediated Cx43 phosphorylation
Introduction
The vascular endothelium is responsible for sensing blood borne signals and maintenance of homeostasis in the cardiovascular system. Endothelial dysfunction is characterized in part by a reduction in vasodilator production and a shift towards a pro-inflammatory profile, and can be caused by local and systemic changes in the growth factor and cytokine environment (Visser et al., 2007; Saito et al., 2007). There are a variety of diseases in which endothelial dysfunction plays a role, including hypertension, atherosclerosis, chronic kidney failure, and preeclampsia (PE) (Rajendran et al., 2013; Boeldt and Bird, 2017). Despite the widespread acknowledgement of the important role endothelial dysfunction plays in such disorders, therapeutic targeting of the endothelium remains underutilized.
Endothelial intracellular free Ca2+ concentration ([Ca2+]i) is an important determinant of vasodilator production (Tran et al., 2009). Such elevations in [Ca2+]i often take the form of periodic Ca2+ bursts, which correlate directly with vasodilator production (Yi et al., 2010; Yi et al., 2005; Krupp et al., 2013). Ca2+ bursting is dependent on functionally coupled Cx43 gap junctions (Yi et al., 2010; Boeldt et al., 2015; Boeldt et al., 2017). Cx43 gap junctions facilitate communication between adjacent cells through the passage of small molecules and ions, such as IP3 and likely Ca2+ (Bird et al., 2013; Solan and Lampe, 2009). The function of Cx43 gap junctions is highly influenced by growth factor and cytokine mediated kinase activation. Src activation results in direct phosphorylation of Cx43 at the inhibitory Y265 residue (Solan and Lampe, 2014). Phosphorylation of Y265 promotes downregulation of Cx43-mediated communication, most likely by destabilizing the Cx43 plaques and increasing the rate of protein turnover (Solan and Lampe, 2014). Because sustained endothelial Ca2+ signaling depends on functionally coupled Cx43 gap junctions, the net result of increased Y265 phosphorylation is reduced Ca2+ signaling capacity (Boeldt et al., 2015; Ampey et al., 2019).
Previous studies have shown that exposure of intact endothelium to 10 ng/ml Vascular Endothelial Growth Factor (VEGF)165 reduces ATP-stimulated Ca2+/nitric oxide (NO) production to a level that is not compensated for by the Ca2+/NO production attributable to VEGF165 itself (Boeldt et al., 2017; Yi et al., 2011). Many of the growth factors and cytokines that may mediate endothelial dysfunction can be sorted into groups/classes based on how their receptors couple directly (VEGF165, basic Fibroblast Growth Factor (bFGF), Epidermal Growth Factor (EGF), Tumor Necrosis Factor (TNF)α) (D'Angelo et al., 1995; Deo et al., 2002; Eliceiri et al., 1999; Huang et al., 2003; Nwariaku et al., 2002; Parenti et al., 1998; Tang et al., 2007; Waltenberger et al., 1994) or indirectly (Interleukin (IL)-6, IL-8, IL-1β) (Huang et al., 2013; Liu et al., 2016) to the same common post receptor kinase pathways. However, the literature has widely failed to address the effects of these growth factors and cytokines when used in combination, as they exist in pathophysiologic context, instead of individually. Therefore, there is a need to construct “cocktails” utilizing multiple growth factors and cytokines to bridge the gap from single agonist experiments to experimental conditions that are more representative of the in-vivo environment of endothelial dysfunction. Co-stimulation with multiple growth factors and cytokines may be dependent on an unappreciated signaling hierarchy, and because these kinases may present a common pathway for endocrine-mediated endothelial dysfunction, they may be a prime target for therapeutic intervention (Bird et al., 2013).
In this study, we use human umbilical vein endothelial cells (HUVECs) to model the combined actions of these growth factors and cytokines on endothelial cell function. We use PE as a model system to construct our cocktails as it has been described as a condition of cytokine-mediated endothelial dysfunction (Redman et al., 1999). As appropriate levels of growth factors and cytokines are necessary to support normal pregnancy, we also use a cocktail representative of normal pregnancy as a control. For this study we include TNFα, IL-6 and VEGF in our cocktails, using doses attributed to ‘normal’ and PE pregnancies for TNFα and IL-6 (Lau et al., 2013; Tosun et al., 2010; Xie et al., 2011), and varied doses of VEGF165 to mimic systemic and local concentrations. While PE is used as the model for which we construct our dysfunctional phenotype, the resulting findings will likely apply to a broad range of diseases that have an endothelial dysfunction component.
The pharmacological Src inhibitor PP2 has already proven successful in rescuing VEGF165 and TNFα pretreated endothelium from a dysfunctional Ca2+ signaling phenotype to one more congruent with normal function (Boeldt et al., 2015; Ampey et al., 2019). A pharmacological tool like PP2 is not used in humans due to significant toxicity and concerns of teratogenicity in pregnancy, and therefore offers no therapeutic value. A pair of isomers of conjugated linoleic acid (CLA) used at FDA recommended doses for treatment of inflammatory conditions including rheumatoid arthritis, Crohn's disease and hypertension may hold promise as a safe method for Src inhibition, even in pregnancy. The t10, c12 CLA isomer has been shown to be a Src inhibitor in cancer cells (Shahzad et al., 2018). When used in ovine endothelial cells, VEGF165-mediated inhibition of Ca2+ responses were rescued with as little as 5 μm t10, c12 CLA, and 50 μM t10, c12 CLA rescued TNFα-mediated Ca2+ response inhibition (Boeldt et al., 2015; Ampey et al., 2019). Herein, we examine the efficacy of individual c9, t11 and t10, c12 isomers, as well as a 1:1 mix to rescue Cx43-dependent Ca2+ signaling in HUVEC pretreated with individual growth factors or cytokines. We also examine the effect of combinations of a selection of growth factors and cytokines on Ca2+ signaling and the ability of CLA isomers alone or as a 1:1 mix, to restore function to the normal phenotype.
Section snippets
Materials
TP (disodium salt), Heparin sodium salt and all other chemicals were purchased from Sigma- Aldrich (St Louis, MO, USA) unless otherwise stated. Growth factors and cytokines (bFGF, EGF, VEGF165, TNFα, IL-1β, IL-6, IL-8) were purchased from R & D systems (Minneapolis, MN, USA). Glass bottom microwell dishes for Ca2+ imaging studies were from MatTek Corporation (Ashland, MA, USA). Minimum Essential Medium (MEM) was purchased from Invitrogen (Life Technologies Inc., Grand Island, NY). Serum used in
Effect of individual growth factors and cytokines on Ca2+ bursts and CLA rescue
Representative single cell tracings from Fura-2 loaded HUVECs are shown in Fig. 1. A short baseline precedes 100 μM ATP stimulation at 0 s. Upon stimulation, an immediate initial Ca2+ peak is seen followed by multiple transient Ca2+ bursts over the duration of the 30-min experiment. Fig. 1A–F shows individual ATP-stimulated cells before and after a pretreatment with Krebs buffer control (top panels A, B), 10 ng/ml VEGF165 (middle panels C, D), or 10 ng/ml TNFα (bottom panels E, F). Cells were
Discussion
The first goal of this study was to determine if individual growth factors or cytokines might induce endothelial dysfunction through inhibition of Ca2+ signaling mechanisms. Initially, we used a standard dose of 10 ng/ml for all growth factors and cytokines, which was based on previous published and unpublished studies from ovine and HUVEC models in which effects were related to Cx43 inhibitory phosphorylations via Src kinase activity (Boeldt et al., 2015; Khurshid, 2015). In this study, we
Financial support
NIH R21 HD069181, P01 HD038843, and R03 HD079865. Funding sources had no role in study design, data collection, data analysis, or manuscript preparation.
CRediT authorship contribution statement
Amanda K. Mauro: Methodology, Investigation, Formal analysis, Writing - original draft, Writing - review & editing. Danielle M. Berdahl: Methodology, Writing - original draft, Investigation, Formal analysis. Nauman Khurshid: Formal analysis, Investigation. Luca Clemente: Formal analysis, Investigation, Methodology. Amanda C. Ampey: Investigation, Writing - original draft, Formal analysis. Dinesh M. Shah: Conceptualization, Writing - review & editing. Ian M. Bird: Conceptualization, Funding
Declaration of competing interest
DSB and IMB have patent pending for the therapeutic use of t10, c12 CLA in preeclampsia. No other authors have conflicts to disclose.
Acknowledgement
This work was undertaken by NK and DMB as part of their MS, and AKM as part of her PhD in the University of Wisconsin-Madison Endocrinology and Reproductive Physiology Training program. Current institution of NK is Promedica Toledo Hospital, Toledo, OH. Current institution for DMB is Parkview Hospital System, Fort Wayne, IN. AKM was also supported by NIH T32 predoctoral training award HD041921.
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