Background
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by acute and chronic inflammation of various tissues such as kidneys, brain, heart and vessels, often accompanied by hypertension and endothelial dysfunction among other alterations [
1‐
3]. Numerous studies suggest that the endothelium is prominently affected during SLE, as demonstrated by the high risk of development of atherosclerosis [
4,
5]. Endothelial cell dysfunction represents the earliest indicator of development of cardiovascular disease and is also a key element of SLE [
6]. SLE is considered per se responsible for direct detrimental effects on the vasculature, beyond the concomitance with traditional cardiovascular risk factors, including obesity, hypertension, dyslipidemia and diabetes mellitus [
7,
8]. The endothelial dysfunction in SLE is of unknown etiology.
Endothelial dysfunction is characterized by impaired nitric oxide (NO) availability and concomitant increased reactive oxygen species (ROS) generation [
9]. A key mechanism of endothelial dysfunction involves the vascular production of ROS, particularly O
2
.-, which reacts rapidly with and inactivates NO [
10]. Inflammatory responses in the endothelium induced by circulating autoantibodies and other inflammatory mediators are known to contribute to the pathogenesis of endothelial dysfunction, and numerous studies have implicated the release of cytokines in the progression of SLE [
11]. It is well-stablished that pro-inflammatory cytokines increase ROS production in endothelial cells. To our knowledge, the direct effects on human endothelial cells in plasma from patients with SLE have never been studied.
Previous studies implicated abnormal unfolded protein responses, termed endoplasmic reticulum (ER) stress, in oxidative stress and endothelial dysfunction [
12,
13]. ER stress has been described in peripheral blood leucocytes from patients with SLE [
14]. In addition, anti-double-stranded-DNA (anti-dsDNA), the hallmark autoantibodies in SLE, induce inflammation via ER stress in human mesangial cells [
15]. However, the role of ER stress in endothelial dysfunction in SLE remains undefined.
The peroxisome proliferator-activated receptors (PPARs) PPARα, PPARβ/δ, and PPARγ are members of the nuclear hormone receptor superfamily. PPARβ/δ is the least studied isoform of PPARs, and it is ubiquitously expressed in tissues [
16]. Pharmacological activation of PPARβ/δ has been shown to reduce hypertension, endothelial dysfunction, and organ damage in mice with severe lupus, which was associated with reduced plasmatic anti-dsDNA autoantibodies and anti-inflammatory and antioxidant effects in target tissues, identifying PPARβ/δ as a promising target for an alternative approach to the treatment of SLE and its associated vascular damage [
17]. However, it is unclear whether its protective effect on endothelial function is the indirect result of better control of both disease activity and metabolic parameters or, alternatively, a direct effect on the endothelial cells. Previous evidence indicates that PPARβ/δ agonists inhibit cytokine-induced ROS production in endothelial cells [
18], at least in part, by positively regulating the antioxidant genes and eliminating excessive production of ROS. More recently it has been described that PPARβ/δ-deficient mice exhibit increased ER stress in the heart [
19], and PPARβ/δ activation prevents palmitate-induced or thapsigargin-induced ER stress in the human cardiomyocyte cell line [
19], skeletal muscle cells [
20], and tunicamycin-induced ER stress in mouse aortic endothelial cells [
12]. We hypothesized that PPARβ/δ activation might exert protective effects on human endothelial cells exposed to plasma from patients with SLE by preventing ER stress. The aim of the present study was to analyze whether plasma from patients with SLE with active and/or inactive nephritis produces endothelial dysfunction by altering human umbilical endothelial cell (HUVEC) function, and to analyze the beneficial effects of PPARβ/δ activation and the role of ER stress.
Discussion
Activation of PPARβ/δ has been previously reported to have beneficial effects on endothelial function in lupus mice [
17]. However, in this in vivo study it is not clear if PPARβ/δ activation occurs primarily in immune cells or in the vascular endothelium, or if it is a combination of both. Herein we provide the first evidence that PPARβ/δ activation restores the impairment of NO production, induced by plasma from patients with SLE acting directly on endothelial cells via inhibition of ER stress. Moreover, this study points to PPARβ/δ and ER stress as a novel therapeutic target in human endothelial dysfunction in SLE. In addition, PPARβ/δ activation also improved endothelial dysfunction induced by plasma from patients with APS.
This study clearly demonstrates that NO production is impaired by plasma from patients with AN, stimulated by two agents, calcium ionophore A23187 and insulin, which stimulated eNOS by different pathways. The calcium ionophore A23187 activates eNOS by a calcium-dependent mechanism. However, insulin has calcium-independent vasodilator actions that are mediated by a phosphatidylinositol 3-kinase-dependent mechanism involving phosphorylation of eNOS by Akt [
25,
27]. However, plasma from patients with SLE with IN did not reduce NO production stimulated by both agents. This is in contrast with data on NZBWF1 mice. In this genetic model of SLE the impaired endothelium-dependent relaxant response to acetylcholine begins before the development of proteinuria and the increase in antinuclear antibodies [
28]. However, the reduced production of NO by HUVECs correlates with higher plasma cytokines (interferon gamma (IFN-γ), IL-6, IL-12) and anti-ds-DNA content in patients with SLE with AN as compared to that in patients without AN. Inflammatory responses in the endothelium induced by circulating autoantibodies and other inflammatory mediators are known to contribute to the pathogenesis of endothelial dysfunction, and numerous studies have implicated the release of cytokines in the progression of SLE [
11]. It is well-established that these cytokines induce endothelial dysfunction [
29,
30]. High concentrations of proinflammatory cytokines increase oxidative stress and downregulate eNOS bioactivity [
31]. In fact, concentrations of IFN-γ higher than those found in SLE plasma were needed to reduce NO production in HUVECs [
32], suggesting a cooperative effect among cytokines and anti-ds-DNA as responsible for endothelial dysfunction induced by plasma from patients with SLE.
aPL mediated vascular abnormalities in patients with primary APS. Patients with APS had endothelial dysfunction, as evidenced by decreased brachial artery endothelium-dependent flow-mediated dilation. Plasma samples from patients with APS revealed decreased NO availability and a pro-oxidative, proinflammatory, and pro-thrombotic state, and mice injected with aPL exibited decreased mesenteric endothelium-dependent relaxation [
33]. However, aPL did not contribute to endothelial dysfunction induced by plasma from patients with SLE in our study as aPL was absent in plasma from these patients. However, incubation of HUVECs with plasma from patients with APS also reduced A23187-stimulated NO production, showing that this effect is not SLE-specific.
Interestingly, GW0742, a highly potent and selective PPAR-β/δ agonist with 200-fold higher affinity toward PPAR-β/δ than other PPAR isotypes [
16], prevented the reduced NO production induced by SLE plasma, confirming a direct effect on endothelial cells. The effect of GW0742 reported herein was inhibited by GSK0660, a selective inhibitor of PPARβ/δ, confirming the specificity of this drug for this nuclear receptor. In addition, the involvement of PPARβ/δ in the protective effects of GW0742 was confirmed by silencing PPARβ/δ. In these conditions, GW0742 was unable to restore NO production stimulated by the calcium ionophore A23187. Moreover, we described for the first time that PPARβ/δ activation also increased the reduced NO production induced by plasma from patients with APS.
A key mechanism of endothelial dysfunction involves the vascular production of ROS, particularly O
2
.-, which reacts rapidly with and inactivates NO [
10]. ROS levels are increased in the aorta [
17,
34] and mesenteric arteries [
35] in mice with SLE. Increased ROS levels are involved in SLE endothelial dysfunction, as in vitro incubation with the antioxidant ascorbic acid [
35] or tempol [
34] and in vivo treatment with tempol plus apocynin [
34] normalizes endothelium-dependent relaxant responses. In our experiments, plasma from patients with SLE with AN also increased intracellular ROS production in HUVECs and GW0742 restored ROS content. This effect is derived from PPARβ/δ activation because inhibition of PPARβ/δ with GSK0660 and PPARβ/δ silencing suppressed the effect of GW0742.
The activity of the NADPH oxidase, considered the major source of O
2
.- in the vascular wall, was markedly increase in mice with SLE, accompanied with an increase in mRNA level of their catalytic subunits [
17,
34]. NADPH-oxidase-driven ROS production is a key event in endothelial dysfunction in SLE [
17,
34]. We found that increased ROS production in HUVECs induced by SLE plasma was suppressed by incubation by both the NADPH oxidase inhibitors apocynin and VAS2870 and the ER stress inhibitor 4-PBA, involving both NADPH oxidase and ER stress as sources of intracellular ROS. Increased ROS levels were also found in HUVECs incubated with plasma from patients with APS, and seem to be involved in endothelial dysfunction evoked by this plasma, as ROS reduction with NADPH oxidase inhibitors increased A23187-stimulated NO production. However, ER stress inhibition did not alter either NO production or the ROS level in HUVECs incubated with APS plasma, suggesting different intracellular pathways involved in endothelial dysfunction evoked by plasma from patients with SLE and APS.
NADPH oxidase activity has been described as an intermediate for ER stress in vascular endothelial dysfunction [
13]. In agreement with that, we also found that NADPH oxidase activity, which was increased by incubation of HUVECs with SLE plasma, was reduced by ER stress inhibition with 4-PBA. GW0742 treatment also inhibited the upregulation of NADPH oxidase subunits NOX2 and NOX4 found in HUVECs exposed to SLE plasma, suggesting ER stress inhibition. ER stress seems to be involved in the impaired A23187-stimulated and insulin-stimulated NO production induced by plasma from patients with SLE with AN, because 4-PBA improved NO levels.
To explore if GW074 inhibits ER stress in our experimental conditions we used tunicamycin to induce ER stress. This compound is a potent inhibitor of glycoprotein synthesis [
36] and promotes significant ER stress in HUVECs [
37]. We found that tunicamycin reduced A23187-stimulated NO production and increased ROS content. Both effects were restored by ER-stress inhibition, NADPH inhibition, and PPARβ/δ activation. These results confirm those from Cheang et al. [
12] showing that the PPARβ/δ agonist GW1516 reversed tunicamycin-induced ER stress, oxidative stress, and impairment of endothelium-dependent relaxation in the mouse aorta, and NO production in mouse aortic endothelial cells.
ER stress is mediated by three ER membrane-associated proteins, PERK, ATF6, and IRE, which engage complex downstream signaling pathways, including cleavage of ATF4, activation of the eIF2α/ATF3 pathway, and splicing of X-box binding protein 1. Bone marrow mesenchymal cells from patients with SLE showed ER stress [
38] evidenced by increased expression of PERK and IRE-1 [
39]. However, when ER stress-related genes were analyzed in peripheral blood leucocytes from patients with SLE and compared to healthy controls, an abnormal unfolded protein response was found in patients with SLE, especially IRE-1/XBP1 and PERK/CHOP axes, with no significant change in ATF6 [
14]. In our experiments, we found that SLE plasma induces ER stress in HUVECs involving only the PERK and ATF-6 pathways, without significant modification of IRE-1. PPARβ/δ activation reduced both ER stress pathways stimulated by SLE plasma. However, further investigation is needed to uncover which PPARβ/δ-responsive genes are related to protein degradation and thus regulate ER stress in the vasculature.