Vascular endothelial growth factor induces mRNA expression of pro-inflammatory factors in the uterine cervix of mice

Abstract

Inflammation is believed to play a role in uterine cervical remodeling and infection-induced preterm labor. One of the distinct features of remodeling uterine cervix is presence of prominent vascular events, such as angiogenesis, vasodilation, and vascular permeability. Although the functional significance of these features is not yet clear, we know that in most tissue types, vascular remodeling is intricately intertwined with inflammation. Since vascular endothelial growth factor (VEGF) is the major architect of vascular remodeling, we sought to examine and elucidate the potential relationship between VEGF and inflammation in the uterine cervix of non-pregnant mice. The animals used were divided into 4 treatment groups: A) negative control (vehicle only), B) positive control (lipopolysaccharide, LPS), C) recombinant VEGF-164 protein, and D) LPS + VEGF blocker (n = 3). After the appropriate treatments, the uterine cervices were harvested and analyzed using real-time PCR and confocal fluorescence microscopy. Results showed that exogenous VEGF upregulates expression of interleukin (IL)-6 and tumor necrosis factor (TNF)-α mRNAs, whereas VEGF blocker partially diminishes the LPS-induced expression of pro-inflammatory factors compared to the positive control group. We conclude that a positive feed-forward relationship likely exists between VEGF and inflammation in the uterine cervix, thus implicating VEGF in inflammation-induced preterm labor.Inflammation is believed to play a role in uterine cervical remodeling and infection-induced preterm labor. During the course of pregnancy, the remodeling uterine cervix is, among other events, characterized by distinct vascular changes, notably, angiogenesis, vasodilation, and vascular permeability (7,(26)(27)(28). Although the functional significance of these changes during uterine cervical remodeling and at birth is, as yet, unclear, we know that vascular remodeling and inflammation are intricately intertwined (4). For instance, three of the five cardinal signs of inflammation are dependent on vascular changes, i.e., redness and heat are dependent on angiogenesis and vasodilation, whereas swelling or edema is dependent on leaky vessels or vascular permeability (33). More importantly, and of relevance to the present study, is the fact that uterine cervical remodeling and the birth process are generally considered inflammatory-like responses (25), in part, due to accumulation of immune cells and expansion of the vascular network in the uterine and uterine cervical tissues. The extent of tissue infiltration by different immune cells varies over the course of pregnancy, during and after birth (4). Data from non-human primate model studies showing successful blockade of infection-induced preterm labor using toll-like receptor 4 antagonists appear to support the role of in-

Vascular endothelial growth factor induces growth of uterine cervix and immune cell recruitment in mice

“…We recently demonstrated that exogenous VEGF administered vaginally (intraluminally) induces an inflammatory response by upregulating mRNA expression of classical pro-inflammatory factors, including TNFa, IL6, and COX II, implying that VEGF likely plays a role in the inflammatory response of uterine cervical epithelial cells during pathogenic invasions (Nguyen et al 2012). These observations are also consistent with our earlier DNA microarray studies that observed a 4.70-fold decrease in genes associated with immune response and decrease in expression of an array of chemokine, interleukins, and LPS binding protein, when VEGF actions were blocked in the cervix of pregnant rats by VEGF inhibitor .…”

“…Animals with normal uterine size 7 days after ovariectomy were eliminated from the study. Appropriate treatments were administered by injecting either vehicle only (negative control: 0.1 M PBS mixed with saturated (25%) pluronic gel and kept in liquid form under ice) or VEGF agonist (mouse recombinant VEGF 164 protein (Calbiochem, La Jolla, CA, USA)) to nonpregnant ovariectomized mice (nZ3/treatment group), following dosage, frequency, and route optimization, as described below and previously (Nguyen et al 2012). Some mice were also treated with VEGF inhibitor (PTK 787/ZK22584; generously provided by Novartis Pharma AG):…”

“…and or intravaginally (via intraluminal administration) (data not shown), once in the morning (1000 h) and once in the evening (1600 h) respectively daily for 4 days. Two hundred nanograms (200 ng) of exogenous VEGF were found to be the optimal dosage (Nguyen et al 2012). After treatment, tissues were harvested and split into ecto-cervix (uterine cervix portion proximal to vagina) and endo-cervix (uterine cervix portion proximal to uterus) by cutting the tissue transversely midway.…”

“…The tissues were then immediately fixed in 2.5% glutaraldehyde (dissolved in 0.1 M PBS) for SEM analysis (morphology). In later experiments of this study, we eliminated chronic treatments (O24 h treatments) for nonpregnant ovariectomized animals, following results of a detailed optimization study of exogenous VEGF treatment by another project in our laboratory (Nguyen et al 2012). …”

“…We have previously characterized the presence and profile of VEGF, the key architect of vascular events, and its key receptors in the uterine cervix of rodents (mice and rats) and delineated VEGF-related genes using DNA microarray analysis , Newell et al 2008, Nguyen et al 2012. The effects of VEGF are mediated by two tyrosinekinase receptors, namely VEGFR-1 (Flt1) and VEGFR-2 (Kdr/Flk1), which have different signaling properties and are largely expressed by endothelial cells (Ferrara 2009).…”