In this study we observed that supplementation of gT to pregnant ewes during late gestation increased IL-6, IL-8, KDR and VEGF mRNA abundances in uterus, caruncle and cotyledon whereas the mRNA expression of IL-1b, sFlt1 was suppressed compared to placebo treated ewes. In contrast, supplementation of aT suppressed mRNA expression of IL-1b, IL-6 and KDR in both cotyledon and caruncle, and IL-8 in cotyledon, compared to placebo treated ewes (P < 0.05).
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 . 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 . Protein tyrosine kinases are a further downstream target of IL-8 signaling responses in endothelial cells . 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 . 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 . It appears that lower IL-6 levels may induce a deregulation leading to an exacerbated inflammation, a poor angiogenesis and fetal death by ischemia . 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 . 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 . 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) . IL-1b induced a pattern of gene expression to favor vascular permeability involving the HIF-VEGF axis . 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 . 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  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 . 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 . 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) .
The aT exerts an inhibitory effect on the release of the proinflammatory cytokine, IL-1b, via inhibition of the 5-lipoxygenase pathway  and aT supplementation significantly lowered levels of C-reactive protein and interleukin-6 . Also, aT inhibits of IL-8 synthesis from endothelial cells . 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].