Effect of tocopherol supplementation during last trimester of pregnancy on mRNA abundances of interleukins and angiogenesis in ovine placenta and uterus

Angiogenesis refers to the formation of new vascular beds, and is a critical process for normal placental growth and development [1‐3]. Although numerous factors have been implicated in angiogenesis, recent observations have led to the identification of the major factors regulating the angiogenic process, including those that occur during placental vascularization. These angiogenic factors include the vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and the angiopoietin (ANG) protein families, as well as their respective receptors [4‐6]. The VEGF is the major angiogenic growth factor of the placenta because this protein probably accounts for most of the angiogenic activity produced by placental tissues [7‐11]. The VEGF acts as specific mitogen for endothelial cells through specific membrane receptors, kinase domain-containing receptor (KDR), and the fin-like tyrosine kinase 1(Flt-1) [12]. When VEGF binds to its receptors, it initiates auto-phosphorylation, induces tyrosine kinase activity, and subsequently stimulates cellular responses in normal and abnormal angiogenesis.

Several inflammatory interleukins have been linked with angiogenesis. These interleukins include IL-1, IL-6, and IL-8 [13‐28]. Interleukin (IL)-8 acts as a potent chemoattractant for neutrophils, the major cellular component of acute inflammatory infiltrates [13, 14]. In addition to this proinflammatory function, there is growing evidence that IL-8 exerts effects on nonimmune cells, including the vascular endothelium [14]. The receptors for IL-8 are widely expressed on normal and various tumor cells and bind IL-8 receptors with high affinity [15]. IL-8 expression correlates with vascularity and microvessel counts [16‐18]. Similarly IL-6, a multifunctional cytokine and is a critical factor in various physiological conditions including immune regulation, hematopoiesis and inflammation by modulating a variety of events, such as cell proliferation, differentiation and apoptosis [19‐21]. Studies have demonstrated that immunostaining for IL-6 was present in both syncytiotrophoblasts and extra-villous trophoblasts [22]. IL-6 stimulates VEGF synthesis and release, and promotes angiogenesis occurrence in vivo and in vitro [23‐26]. These cytokines could play a role in vascular remodeling associated with placentation. The cytokine IL-1 mainly affects inflammatory processes but also possesses various immune, degradative, and growth promoting properties. There are two IL-1 agonistic proteins, IL-1α and IL-1β. Both were shown to contribute to tumor angiogenesis and invasiveness, but the role of IL-1β is more evident in these processes [27].

Tocopherol has immunomodulatory effects, besides its antioxidant properties. Vitamin E is implicated in placental and embryonic development, whereas its deficiency affects embryo survival, and placental and fetal development. Alternative effects of Vitamin E have been recently reported as unrelated to its antioxidant capacity [28]. It induces the expression of VEGF promoter [28]. Vitamin E (15 mg/day) has been able to decrease abortion rate and to increase IL-6 placental levels, while both treatments increased placental levels of VEGF [29]. We have shown that oral supplementation of tocopherol induces angiogenesis in placental vascular network in late pregnant ewes [10]. The hypothesis of the study is that the tocopherol's angiogenic effect following oral supplementation during late gestation in ewes is via promoting interleukins.

The objective of this study is to investigate whether tocopherol's angiogenic activity in placental network is enhanced via promoting interleukins in pregnant ewes during late gestation.

The mRNA expressions of IL-6, IL-8, IL-1b, TNF-α, VEGF, sFlt1 and KDR in placenta and uterus in tocopherol supplemented groups relative to CON (where CON group is 1), are given in Figures 1, 2 and 3. In cotyledon (Figure 1) and caruncle (Figure 2), the IL-6, IL-8, VEGF and KDR in ewes supplemented with gT were significantly higher in abundance compared to the CON group (P < 0.05). The IL-1b, sFlt1 mRNA abundances in cotyledon (Figure 1) and sFlt1 mRNA abundance in caruncle (Figure 2) were lower (P < 0.05) in gT group compared to the CON groups. The supplementation of aT suppressed mRNA expression of IL-8, IL-1b, TNF-α and KDR in cotyledon (Figure 1), and IL-1b and TNF-α in caruncle (Figure 2) compared to placebo treated ewes (P < 0.05).

In the uterus, the IL-6 and IL-8, VEGF and KDR mRNA expressions in ewes supplemented with gT were significantly higher in abundance, where as IL-1b and sFlt1 was significantly lower in abundance compared to the CON group (Figure 3). The IL-6, IL-8, IL-1b and KDR and TNF-α mRNA abundance in ewes supplemented with aT were significantly lower compared to the CON group (Figure 3).

Oral supplementation of gT increased IL-6, IL-8, KDR and VEGF mRNA expression whereas sFlt1 mRNA expression was suppressed in cotyledon (Figure 1), caruncle (Figure 2) and uterus (Figure 3) compared to aT treated ewes (P < 0.05). The mRNA abundances of both TNF-α and IL-1b was suppressed in uterus, caruncle and cotyledon but TNF-α was different between gT and aT treatment groups whereas IL-1b was similar between treatment groups (P > 0.1).

In our previous study, we showed that the tocopherol concentrations of uterus and placentome increased following oral supplementation of tocopherol during late gestation in pregnant ewes [10]. IL-8 signaling has been shown to promote the transactivation of the epidermal growth factor receptor in vascular endothelial cells [31, 32], promoting downstream activation of MAPK signaling. In addition, IL-8 signaling has been shown to induce the phosphorylation of the KDR, (VEGFR-2) in endothelial cells, regulating the permeability of the endothelial barrier [33]. Protein tyrosine kinases are a further downstream target of IL-8 signaling responses in endothelial cells [31]. In our previous study, we demonstrated increased fractal dimension and lowered lacunarity, signs for improved angiogenesis, in placental vascular network following gT supplementation compared to aT supplemented and control ewes during late gestation [10]. In this study, we demonstrated increased IL-8, KDR and VEGF mRNA abundances in gT supplemented ewes. The stimulatory effect of IL-8 might plausibly be regulated by increased VEGF mRNA expression, which is enhanced via both VEGFR1 (low sFlt1 mRNA abundance) and VEGFR2 (high KDR mRNA abundance).

Fan et al. (2008) showed that interleukin (IL)-6 increased endothelial progenitor cell proliferation, migration and in vitro angiogenic-like tubulogenesis [22]. It appears that lower IL-6 levels may induce a deregulation leading to an exacerbated inflammation, a poor angiogenesis and fetal death by ischemia [29]. However, it should be noted that the underlying angiogenic mechanism is that IL-6 upregulates KDR. IL-6 triggers VEGF-induced angiogenic activity through increasing VEGF release, up-regulates KDR expression and phosphorylation through activating ERK1/2 signaling, and stimulates MMP-9 overexpression [34, 35]. It is evident, in this study, that ewes supplemented with gT had increased IL-6, VEGF and KDR causing enhanced angiogenesis in the placental vascular network in that group.

Numerous studies demonstrated a role for IL-6 in increased blood pressure. For example, plasma levels of IL-6 are strongly associated with hypertension in humans and can be reduced by administration of Ang II receptor antagonists [36‐38]. Animal studies showed that infusion of IL-6 induces hypertension in pregnant rats [39, 40]. Trophoblast cells are capable of responding to IL-6 [41‐43]. Trophoblast cells have a JAK/STAT-signaling pathway that functions downstream of IL-6R activation. JAK/STAT signaling, downstream of IFN-γ stimulation, is responsible for increased sFlt-1 production by human corneal fibroblasts [44]. The authors speculated that the IL-6 stimulates sFlt-1 and sEng production by trophoblast cells in an autocrine manner by JAK-STAT signaling, and that increased IL-6 contributes to antibody-mediated hypertension in women with PE via IL-6-induced stimulation of sFlt-1 production by trophoblasts [44]. Interestingly in this study, sFlt-1 mRNA expression is suppressed in tocopherol treated groups plausibly explaining that ischemic necrosis is prevented by such treatment thus favoring angiogenesis.

IL-1b is a potent immunoregulatory and proinflammatory cytokine secreted by a variety of activated immune cells. Several studies provide evidence that angiogenesis and VEGF is IL-1 dependent [45‐48] and the major transcriptional activator of the VEGF gene is HIF (Hypoxia Inducible Factor) [49]. IL-1b induced a pattern of gene expression to favor vascular permeability involving the HIF-VEGF axis [50]. Activation of the ERK1/2 pathway by IL-1b may result in the accumulation of HIF-1a protein, which initiates VEGF secretion in normal human cytotrophoblast cells. In the previous study, we observed that VEGF, HIF-1a and HIF-2a mRNA expressions in caruncle and uterus were greater in ewes supplemented with gT compared to the ewes in the CON group which is coincided with improved angiogenesis in the placental vascular network in gT group [10]. In the present study, IL-1b mRNA abundance was suppressed in gT treated ewes compared to ewes in the CON group. It is plausible that the IL-1b mRNA abundance is lower during late gestation. Studies on immunohistochemical localization of IL-1a and IL-1b in normal human placenta showed that both IL-1a and IL-1b forms are localized to villous syncytiotrophoblast and to extravillous trophoblast [51] and there is a gradual decrease of IL-1 reactivity with increasing gestational age. Also, IL-1a contributes to angiogenesis in hypoxia, when IL-1b secretion is reduced to suboptimal levels [51]. In addition, Molvarec et al., (2011) did not find association between serum levels of leptin and pro-inflammatory cytokines in preeclampsia, which might be explained--at least partly--by the fact that the latter (especially TNF-α and IL-1β) have a very short-half life in the maternal circulation [52]. In dogs, the expression was absent in uterus during diestrus (10-12 days post ovulation), and preimplantation (10-12 dpb) and placentation sites (day 20-35 dpb) [53].

The aT exerts an inhibitory effect on the release of the proinflammatory cytokine, IL-1b, via inhibition of the 5-lipoxygenase pathway [54] and aT supplementation significantly lowered levels of C-reactive protein and interleukin-6 [55]. Also, aT inhibits of IL-8 synthesis from endothelial cells [56]. It is evident that findings from these studies showed that supplementation of aT suppressed the expression of interleukins. In this study, the supplementation of aT suppressed mRNA expression of IL-1b, IL-6 and KDR in placentome, and IL-8 in caruncle compared to placebo treated ewes.

It should be noted that the protein expressions work was not performed in this study. However, protein expressions of proinflammatory cytokines and angiogenic markers in sheep placental tissues were reported in mid and late gestation [57, 58].

Gamma tocopherol supplementation increased IL-6, IL-8 and KDR mRNA expression, and suppressed sFlt1 mRNA expression in uterus, caruncle and cotyledon during late gestation. In addition, the VEGF mRNA expression in uterus, caruncle and cotyledon is also increased following supplementation during late gestation. The increase in VEGF mRNA abundance is enhanced via both VEGFR1 (low sFlt1) and VEGFR2 (high KDR) mRNA abundances. Taken findings from this study together, it is conceivable that the angiogenic effect of gamma tocopherol in placental vascular network is exerted via an alternate path by enhancing IL-6 and IL-8 mRNA.