endothelial cell function and angiogenesis

Background The negative feedback system is an important physiological regulatory mechanism controlling angiogenesis. Soluble vascular endothelial growth factor (VEGF) receptor-1 (sFlt-1), acts as a potent endogenous soluble inhibitor of VEGF- and placenta growth factor (PlGF)-mediated biological function and can also form dominant-negative complexes with competent full-length VEGF receptors. Methods and results Systemic overexpression of VEGF-A in mice resulted in significantly elevated circulating sFlt-1. In addition, stimulation of human umbilical vein endothelial cells (HUVEC) with VEGF-A, induced a five-fold increase in sFlt-1 mRNA, a time-dependent significant increase in the release of sFlt-1 into the culture medium and activation of the flt-1 gene promoter. This response was dependent on VEGF receptor-2 (VEGFR-2) and phosphoinositide-3'-kinase signalling. siRNA-mediated knockdown of sFlt-1 in HUVEC stimulated the activation of endothelial nitric oxide synthase, increased basal and VEGF-induced cell migration and enhanced endothelial tube formation on growth factor reduced Matrigel. In contrast, adenoviral overexpression of sFlt-1 suppressed phosphorylation of VEGFR-2 at tyrosine 951 and ERK-1/-2 MAPK and reduced HUVEC proliferation. Preeclampsia is associated with elevated placental and systemic sFlt-1. Phosphorylation of VEGFR-2 tyrosine 951 was greatly reduced in placenta from preeclamptic patients compared to gestationally-matched normal placenta. Conclusion These results show that endothelial sFlt-1 expression is regulated by VEGF and acts as an autocrine regulator of endothelial cell function.


Background
Vascular endothelial growth factor-A (VEGF-A) is a multifunctional cytokine induced by hypoxic stress [1]. It plays a pivotal role in many aspects of embryonic cardiovascular development, including formation of blood vessels, cardiac morphogenesis, and development of the nervous system [2][3][4][5][6]. Loss and gain of function studies in mice indicate that VEGF-A levels have to be maintained within a narrow range to ensure proper cardiovascular development and embryo survival [7][8][9]. It has been shown that the effects of VEGF-A can be deleterious if uncontrolled. Over-expression of VEGF in experimental animals increases the leakiness of blood vessels, which may lead to severe edema, loss of limb and death [10,11]. Excess VEGF-A expression in skeletal muscle results in the induction of vascular tumors (hemangiomas) [12][13][14], whereas loss of VEGF-A activity due to increased production of its natural antagonist, sFlt-1 (soluble VEGF receptor-1/sVEGFR-1), as in preeclampsia, reduces angiogenesis [15]. Thus, homeostasis requires mechanisms to regulate the functional activity of VEGF-A.
Soluble Flt-1 is generated by alternative splicing of the fms-like tyrosine kinase (flt-1) gene [16], and binds to all isoforms of VEGF-A and placenta growth factor (PlGF) with high affinity [16,17]. It acts as a potent soluble inhibitor of both VEGF-A and PlGF-mediated biological activities [18] and can also form dominant-negative complexes with competent full-length VEGF receptors [16]. In pregnancies complicated with preeclampsia, sFlt-1 levels are elevated [15,[19][20][21]. Maternal serum levels of sFlt-1 are elevated five weeks prior to the onset of preeclampsia [22], supporting the premise that sFlt-1 is a key factor responsible for the clinical manifestation of this disorder [23]. The demonstration that sFlt-1 is fundamental to the clinical onset of preeclampsia [24] highlights the importance of understanding the intracellular mechanism underlying its regulation and release in endothelial cells. Recently it was shown that autocrine VEGF signaling is required for vascular homeostasis [25]. Here we demonstrate that endothelial sFlt-1 expression is regulated by VEGF and sFlt-1 is an autocrine regulator of endothelial cell function.

Placental tissues
Human placental tissue was obtained from normal pregnancies and gestationally-matched pregnancies complicated by preeclampsia. Preeclampsia was defined as blood pressure > 140/90 mm Hg on at least two consecutive measurements and proteinuria of at least 300 mg per 24 hours. Informed consent was obtained from the patients and the study had the approval of the South Birmingham Ethical Committee (Birmingham, UK).

Cell Culture
Primary human umbilical vein endothelial cells (HUVEC) were isolated and cultured as described [26]. Cells were used at passage two or three for experiments and serum-starved in endothelial cell serum-free medium (Gibco-BRL, UK) supplemented with 0.2% bovine serum albumin for 24 hours prior to stimulation.

Quantitative Real-Time PCR
Sample preparation and real-time PCR was performed as described previously [30]. Briefly, mRNA was prepared using TRIzol and DNase-1 digestion/purification on RNAeasy columns (Qiagen), and reverse transcribed with the cDNA Synthesis Kit (Promega). Triplicate cDNA samples and standards were amplified in SensiMix containing SYBR green (Quantace) with primers specific for sFlt-1 [31]. The mean threshold cycle (CT) was normalized to β-actin and expressed relative to control.

Transduction of chimeric VEGF Receptors in HUVEC
A chimeric VEGF/epidermal growth factor (EGF) receptor comprising the intracellular and transmembrane domains of VEGFR-2 fused to the extracellular domain of the human EGF receptor [33]. EGF does not bind to VEGF receptors, therefore, it does not activate the endogenous VEGF receptors. EGDR and its tyrosine-to-phenylalanine mutants (EGDR-Y951F) were generated and cloned into the pMMP retroviral vector, and retrovirus-containing cell supernatant was harvested and used immediately to infect HUVEC [33]. Following 16 hours of incubation, the medium was replaced with fresh growth medium and the HUVEC were used 48 hours after infection.

Nitric oxide (NO) Release
Total NO in conditioned media was assayed as nitrite, the stable breakdown product of NO, using a Sievers NO chemiluminescence analyzer (Analytix, Sunderland, UK) as described previously [33].

Tube Formation Assay
The formation of capillary-like structures was examined on growth factor-reduced Matrigel in 24-well plates as described previously [33]. Tube formation was quantified by measuring the total tube length in five random x200 power fields per well using a Nikon phase-contrast inverted microscope with Image ProPlus image analysis software (Media Cybernetics, Silver Spring, USA). Mean total tube length was calculated from three independent experiments performed in duplicate.
flt-1 gene promoter activity assay A 1.3 Kb fragment of the human flt-1 gene corresponding to -1214 to +155 bp relative to the first exon in the pGL2 luciferase vector (Promega) was used to determine flt-1 promoter activity [34]. Briefly, porcine aortic endothelial cells (PAEC) were transfected with the flt-1 promoter-reporter construct using Exgen 500 (Fermentas, UK) and the cell lysates assayed as described previously [34].

Statistical analysis
All data are expressed as mean ± SEM. Statistical comparisons were performed using one-way ANOVA followed by the Student-Newman-Keuls test as appropriate. Statistical significance was set at a value of p < 0.05.

VEGF-A stimulates sFlt-1 release
To evaluate the capacity of VEGF-A to regulate the secretion of its negative regulator, sFlt-1, HUVEC were incubated with VEGF-A and the conditioned media assayed for sFlt-1 by ELISA. VEGF-A stimulated a concentration and time dependent increase in the release of sFlt-1 from HUVEC that reached a maximum at 20 ng/ ml and 48 hours, respectively (Figure 1a and 1b). Consistent with these findings, qPCR revealed a greater than five-fold increase in sFlt-1 mRNA after 22 hours of VEGF-A stimulation (Figure 1c). In addition, VEGF-A induced flt-1 gene promoter activity in porcine aortic endothelial cells transfected with a flt-1 promoter luciferase construct (Figure 1d). Incubation of cells with cycloheximide abrogated the VEGF-A induced response (Figure 1e), which coupled with the fact that there is a negligible release with VEGF-A after two hours of stimulation, indicates that sFlt-1 secretion is due to de novo protein synthesis and not release from intracellular vesicles. Adenoviral-mediated overexpression of VEGF-A in mice caused an eight-fold increase in circulating sFlt-1 levels (Figure 1f), demonstrating, in vivo, that an increase in VEGF-A results in a concomitant rise in circulating sFlt-1 levels, presumably to compensate for elevated VEGF bioactivity.
VEGF stimulated sFlt-1 production, is mediated via PI3K To investigate the role of the PI3K pathway in VEGF-Ainduced sFlt-1 release, PI3K activity was inhibited through overexpression of PTEN (Phosphatase and Tensin homolog deleted on chromosome Ten), which dephosphorylates phosphatidylinositol 3,4,5-triphosphate and has been shown to reduce VEGF-mediated signaling and cellular function [28,35].  with VEGF-A for 24 hours. Inhibition of PI3K activity by PTEN overexpression led to a significant decrease in sFlt-1 release (Figure 2c). Furthermore, pre-incubation of HUVEC with LY294002, a pharmacological PI3K inhibitor, also attenuated the VEGF mediated release of sFlt-1 (Figure 2d) and of flt-1 gene promoter activity (Figure 2e).

Loss of sFlt-1 promotes angiogenesis
Adenoviral-mediated overexpression of sFlt-1 in HUVEC inhibited endothelial cell proliferation ( Figure  3a) and MAP kinase ERK-1/-2 phosphorylation (Figure  3a insert). Subsequently, to test whether knockdown of sFlt-1 would promote endothelial cell proliferation, HUVEC were transfected with two synthetic siRNA sequences targeted to the unique carboxyl-terminus region of sFlt-1. sFlt-1 siRNA transfection resulted in a substantial reduction in the release of sFlt-1 from HUVEC after 24 hours (Figure 3b). Endothelial cell proliferation was significantly increased (Figure 3c) and interestingly, sFlt-1 knockdown also led to a concomitant increase in VEGFR-2 phosphorylation at tyrosine 951 (Y951) (Figure 3d). In addition, sFlt-1 siRNA increased both basal and VEGF-A-mediated endothelial cell migration (Figure 4a) and tube formation on Matrigel (Figure 4b and 4c). VEGF stimulates eNOS activity and NO release [23,36] to mediate angiogenesis [33,37], thus we predicted that loss of sFlt-1 would increase eNOS phosphorylation in HUVEC. Phosphorylation of eNOS (ser1177) was significantly increased in cells lacking sFlt-1 (Figure 4d). These data provide direct evidence that sFlt-1 is itself a negative regulator of endothelial function. It is likely that sFlt-1 sequesters VEGF and PlGF to maintain a physiological steady state until angiogenesis is required, at which point this system must be overridden.

Excess sFlt-1 inhibits VEGFR-2 Y951 phosphorylation
Activation of VEGFR-2 leads to an increase in eNOS expression and activation, [38] which is essential for neovascularisation [37]. A recent study showed that mutation of VEGFR-2 Y951 to phenylalanine caused a significant reduction in VEGF-induced angiogenesis [33]. As preeclampsia is associated with elevated placental [15] and circulating [22] sFlt-1 and placental sFlt-1 inhibits angiogenesis [15], we speculated that elevated free sFlt-1 would lead to a reduction of VEGFR-2 phosphorylation in preeclamptic placenta. Using the EGFR-chimeric receptor system we show that mutation of Y951 to phenylalanine resulted in over 50% reduction in NO release (Figure 5a) and overexpression of sFlt-1 in endothelial cells abrogated phosphorylation of VEGFR-2 Y951 (Figure 5b). To assess whether VEGFR-2 phosphorylation was reduced in preeclamptic placenta, that express elevated sFlt-1, we undertook immunohistochemical analysis for phospho-VEGFR-2 Y951. Overall, phosphorylation of VEGFR-2 Y951 was greatly reduced in the preeclamptic placenta compared to gestationally-matched, normal placenta (Figure 5c). Together, these findings indicate that increased levels of sFlt-1 have a negative effect on VEGFR-2 tyrosine phosphorylation, which in turn would lead to concomitant inhibition of downstream function and signaling and compromise maternal vascular homeostasis and placental angiogenesis.

Conclusions
Endothelial cell sFlt-1 expression is regulated by VEGF and sFlt-1 is an autocrine regulator of endothelial cell function.