Angiotensin II upregulates the expression of placental growth factor in human vascular endothelial cells and smooth muscle cells
- Pingxi Pan†1,
- Hua Fu†2,
- Lingjun Zhang†1,
- He Huang†2,
- Fengming Luo3,
- Wenchao Wu1,
- Yingqiang Guo1, 4Email author and
- Xiaojing Liu1Email author
© Pan et al; licensee BioMed Central Ltd. 2010
Received: 2 November 2009
Accepted: 26 May 2010
Published: 26 May 2010
Atherosclerosis is now recognized as a chronic inflammatory disease. Angiotensin II (Ang II) is a critical factor in inflammatory responses, which promotes the pathogenesis of atherosclerosis. Placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family cytokines and is associated with inflammatory progress of atherosclerosis. However, the potential link between PlGF and Ang II has not been investigated. In the current study, whether Ang II could regulate PlGF expression, and the effect of PlGF on cell proliferation, was investigated in human vascular endothelial cells (VECs) and smooth muscle cells (VSMCs).
In growth-arrested human VECs and VSMCs, Ang II induced PlGF mRNA expression after 4 hour treatment, and peaked at 24 hours. 10-6 mol/L Ang II increased PlGF protein production after 8 hour treatment, and peaked at 24 hours. Stimulation with Ang II also induced mRNA expression of VEGF receptor-1 and -2(VEGFR-1 and -2) in these cells. The Ang II type I receptor (AT1R) antagonist blocked Ang II-induced PlGF gene expression and protein production. Several intracellular signals elicited by Ang II were involved in PlGF synthesis, including activation of protein kinase C, extracellular signal-regulated kinase 1/2 (ERK1/2) and PI3-kinase. A neutralizing antibody against PlGF partially inhibited the Ang II-induced proliferation of VECs and VSMCs. However, this antibody showed little effect on the basal proliferation in these cells, whereas blocking antibody of VEGF could suppress both basal and Ang II-induced proliferation in VECs and VSMCs.
Our results showed for the first time that Ang II could induce the gene expression and protein production of PlGF in VECs and VSMCs, which might play an important role in the pathogenesis of vascular inflammation and atherosclerosis.
Atherosclerosis is now considered to be a chronic inflammatory process which may ultimately lead to acute myocardial infarction, cerebrovascular and peripheral vascular diseases [1, 2]. Plenty of data suggest that the rennin-angiotensin system (RAS) plays an important role in the development of many cardiovascular diseases, including the pathophysiological process of atherosclerosis [3, 4]. Many studies have shown that inhibition of the RAS could reduce inflammation and oxidative stress . Angiotensin II (Ang II), one of the major effectors of the RAS, is a cytokine that regulates cell growth, inflammation and fibrosis contributing to the progression of vascular damage [4, 6, 7]. Ang II participates in atherosclerosis pathogenesis by inducing inflammation and apoptosis, facilitating absorption of oxidative low density lipoprotein, generating oxygenic radicals and impacting fibrinolysis function . The physiological actions of Ang II are mediated via its type 1 receptor (AT1R) and type 2 receptor (AT2R), which are expressed under different developmental, tissue-specific, and disease-specific conditions [8, 9]. It has been shown that Ang II activates NF-κB, a key component of inflammation, in vascular smooth muscle cells (VSMCs). However, the exact mechanism of Ang II-mediated inflammation in vascular endothelial cells (VECs) or VSMCs is still largely unclear.
Recently, Placenta growth factor (PlGF) has emerged as a key factor in vascular inflammation and progression of atherosclerosis [11–13]. PlGF is a member of the vascular endothelial growth factor (VEGF) family cytokines and is associated with inflammation and with pathologic angiogenesis [14–16]. It is a polypeptide growth hormone that binds to Flt-1-receptor (VEGFR-1), neuropilin-1 (NRP1) and neuropilin-2 (NRP2) receptors, but not to VEGF-receptor type 2 (VEGFR-2). Recent study has shown that PlGF is required for macrophage infiltration in early atherosclerotic lesions in apolipoprotein E-deficient mice . PlGF has atherogenic properties including recruitment and adhesion of monocytes, induction the production of proteinase, and thrombus formation through stimulating tissue factor secretion . It is up-regulated in early and advanced atherosclerotic lesions, acting as a primary inflammatory instigator of atherosclerotic plaque instability. Moreover, it has been recognized as an independent biomarker of adverse outcome in patients with acute coronary syndromes (ACS) [13, 17]. As a more specific marker of vascular inflammation, PlGF might be considered for risk stratification of patients with ACS. The PlGF expression can be induced by hypoxia and various pro-inflammatory stimuli . This induction is mediated via NF-kappa B and metal response transcription factor-1(MTF-1). However, the regulation of PlGF expression in vascular cells, and its mechanisms of action have received little attention in atherosclerosis research.
Because PlGF plays a role in initiation and progression of atherosclerosis, it is interesting to examine the potential interaction between Ang II and PlGF. However, the connection between Ang II and PlGF expression in vascular cells has not been studied. In this study, we examined the effect of Ang II on the PlGF expression in both human VECs and VSMCs. Previously, the HUVEC-derived endothelial cell line (EA.Hy 926) [20, 21] and human umbilical artery smooth muscle cells (HUASMCs)[22, 23] have been characterized as models of investigating the functions of VECs and VSMC, respectively. EA.Hy 926 endothelial cells and HUASMCs were used in the present study.
Angiotensin II increases PlGF mRNA and protein levels in both EA.Hy 926 cells and HUASMCs
The human umbilical vein endothelial cell-derived cell line EA.Hy 926 was used in this study. As an established cell line, EA.Hy 926 cell is homogenous compare to the variable primary cells from individual donors.
Next, we examined the Ang II- induced change of PlGF protein level in EA.Hy 926 endothelial cells and HUASMCs. Cells were exposed to Ang II treatment for indicated times. Subsequently, the secretion of PlGF in the culture media was measured by ELISA. Under serum-starvation condition, EA.Hy 926 cells secreted low level of PlGF (~ 12 pg/mL). Treatment with Ang II for 8, 24 or 48 hours evoked significant secretion of PlGF, as compared to the untreated cells (Figure 1C). Similarly, the growth-arrested HUASMCs released low level of PlGF protein in the cell culture supernatant. When HUASMCs were treated with Ang II for 24 or 48 hrs, the PlGF production was significantly higher than that in untreated cells (Figure 1C).
The expression of PlGF was also analyzed by immunofluorence technique. Only weak PlGF fluorescence intensity was observed in growth-arrested EA.Hy 926 cells and HUASMCs. Ang II stimulation for 24 hours significantly increased the cytoplasmic PlGF fluorescence intensity in both cell types (Figure 1D).
Taken together, our data indicated that Ang II increased PlGF mRNA and protein production in EA.Hy 926 cells and HUASMCs.
Angiotensin II increases VEGFR-1 and VEGFR-2 mRNA expression in both EA.Hy 926 cells and HUASMCs
Angiotensin II increases PlGF expression via AT1R in both EA.Hy 926 cells and HUASMCs
Signaling mechanisms involved in Angiotensin II-induced PlGF gene and protein production
Role of PlGF in the Angiotensin II-induced proliferation of EA.Hy 926 cells and HUASMCs
In order to examine if PlGF or VEGF played a role in Ang II-induced proliferation, we used MTT incorporation assay [29, 30] to study the proliferation of growth-arrested EA.Hy 926 cells and HUASMCs followed by (1) Ang II treatment, or (2) co-treatment of Ang II and a neutralizing antibody against to PlGF or VEGF (10 or 20 μg/mL), or (3) administration of the blocking antibody for PlGF or VEGF alone for 24 h.
Discussion and Conclusions
In the present study, the potential correlation between Ang II and PlGF was investigated in cultured EA.Hy 926 endothelial cells and in HUASMCs. The major findings of this study are: (1) Ang II, via AT1R, induces PlGF gene expression and protein secretion in both VECs and VSMCs; (2) Ang II increases VEGFR-1 and -2 gene expression in these vascular cells; (3) multiple signaling pathways, including PKC, ERK1/2 and PI3-K, are involved in this Ang II-induced PlGF upregulation; (4) blockade of PlGF results in the inhibition of Ang II-induced proliferation in these cells, whereas blockade of VEGF leads to inhibition of both basal and Ang II-elicited proliferation. Our observations established a role of PlGF in mediating Ang II-induced proliferation in vascular endothelial cells and vascular smooth muscle cells. In addition, our results suggested that inhibition of PlGF or VEGF might be useful in preventing abnormal VEC or VSMC proliferation evoked by Ang II.
Numerous studies suggest that the renin-angiotensin system (RAS) contributes to the pathogenesis of atherosclerosis [4, 7]. Angiotensin II, the principal effecter of the RAS, is not only a vasoactive hormone, but also a cytokine that regulates cell proliferation, inflammation and fibrosis. Ang II elicits the inflammatory response by stimulating the production of chemokines, cytokines, and adhesion molecules . Recent study demonstrated that Ang II induced vascular endothelial cell proliferation by increasing the expression of the angiogenic factor VEGF . Consistent with previous reports [24, 25, 31], we observed that Ang II increased VEGF and its two receptors expression in our experimental model. Meanwhile, our data provided direct evidence that, in vascular endothelial cell and smooth muscle cells, Ang II, via its AT1 receptor, could up-regulate PlGF expression. These results suggested that Ang II might participate in the regulation of pathological angiogenesis.
Ang II acts through binding to its specific AT1 and AT2 receptors, which are seven transmembrane glycoproteins with 30% sequence similarity. The AT1R is a classical G protein-coupled receptor, whereas AT2R often antagonizes the effects of signaling through the AT1R. Many AT1R-induced growth responses are mediated by transactivation of growth factor receptors. AT1 receptor regulates cell proliferation, cytokines production and some pathological processes, including Ang II-induced hypertension and cardiac hypertrophy. Although AT1 receptor mediates most of the recognized cardiovascular effects of Ang II, the AT2 receptor contributes to the regulation of blood pressure and renal function. Our data demonstrated that, in cultured EA.Hy 926 endothelial cells and HUASMCs, Ang II increased PlGF expression and synthesis via its AT1 receptor.
Several pathways, e.g. PKC , ERK1/2 [7, 26, 33], and PI3K/Akt , involved in AT1R activation. Using specific inhibitors, the present study showed that Ang II activated PKC, ERK1/2 and PI-3K pathways. The activation of all these pathways contributed to PlGF up-regulation in EA.Hy 926 endothelial cells and HUASMCs.
The induction of PlGF gene expression by Ang II may be of considerable clinical significance, especially in vascular inflammation and atherosclerosis. Pro-inflammatory cytokines play a crucial role of in the development of atherosclerosis and plaque instability. Quiescent vascular endothelial cells only release minimal amounts of PlGF. In contrast, activated endothelial cells could produce large amounts of PlGF. Previous studies demonstrated that PlGF activated monocytes and increased the expression of tumor necrosis factor-α(TNF-α), interleukin-1β (IL-1β), and monocyte chemotactic protein-1(MCP-1) in monocytes[36, 37]. Consequently, when stimulated by Ang II, vascular endothelial cells produce PlGF, which activates neutrophils and monocytes, results in their adherence to endothelial cells. This might trigger the pathophysiological changes observed in atherosclerosis. Our results indicated that, the administration of PlGF-neutralizing antibody significantly inhibited the Ang II-dependent proliferation of vascular endothelial cells, suggested that PlGF might be a down-stream angiogenic mediator of RAS. The neutralizing antibodies of PlGF or VEGF are less effective in inhibiting cell proliferation than the small molecule inhibitor of AT1R, since other effects are involved besides VEGF/PlGF production. It has been largely demonstrated that the Ang II-induced VSMC proliferation is mediated by PDGF and Egr-1 [38, 39]. And the AT1R blocker might be useful as an anti-angiogenic agent.
It has been recognized that VSMC proliferation within the vessel wall is an important pathogenic feature in the development of atherosclerosis [40, 41]. Ang II has been implicated to play an important role in this cellular mechanism. When exposed to hypoxia (3% O2), the proliferation and contraction of VSMC were enhanced by PlGF treatment . Furthermore, recent study has shown that PlGF expression in human atherosclerotic carotid plaques is related to inflammation and clinical plaque instability. Present observations showed that Ang II induced PlGF expression in VSMC, suggested a role of PlGF in mediating VSMC proliferation induced by Ang II.
It has been shown that Ang II could stimulate the expression of hypoxia inducible factor-1α (HIF-1α) , and HIF-1α seems to be involved in the enhanced PlGF expression stimulated by hypoxia . The hypoxia-inducible PlGF expression is mediated through NF-κB, metal-regulatory transcription factor-1(MTF-1) and the interaction between them . Ang II-induced PlGF expression might be mediated through the HIF-1α pathways. However, future studies in vascular cells are necessary to determine the role of Ang II receptors and PlGF in atherosclerosis.
In conclusion, PlGF might be one of the downstream effectors up-regulated by Ang II in vascular diseases. Several pathways, such as PKC and ERK 1/2 activation, seem to be involved in the Ang II-induced PlGF expression in vascular cells. PlGF also mediates Ang II-induced cell proliferation in VECs and HUASMCs. Thus, our study provides new insights into PlGF as one of the Ang II-inducible genes. The link of PlGF to Ang II might be a novel molecular mechanism to target cardiovascular diseases.
A human endothelial cell line (EA.Hy 926) was grown in RPMI 1640 (GIBCO-BRL) supplemented with 10% FBS, 1% penicillin/streptomycin, 2 mmol/L L-glutamine and 1% HAT (Sigma, USA).
Primary cultures of HUASMCs were isolated from freshly delivered umbilical cords by tissue explanting method [22, 23] and maintained in DMEM medium (GIBCO-BRL) supplemented with 20% fetal bovine serum (FBS) (Hyclone), 2 mmol/L L-glutamine, and 1% penicillin-streptomycin. All cell cultures were maintained in a humidified 5% CO2/95% air incubator at 37°C. HUASMCs were identified by the specific marker of vascular smooth muscle cell (α-smooth muscle actin, α-SMA) immunofluorescence. The study was approved by the medical ethics committee of the West China Hospital, Sichuan University.
In all experiments, confluent EA.Hy 926 cells or HUASMC cells at passage 4 to 8 were washed and incubated with serum-free media for 24 hours. These cells were treated with human angiotensin II (Ang II, 10-6 mol/L) for different time points. In some experiments, cells were pre-treated for 30 minutes with 10-7 mol/L Wortmannin, 10-6 mol/L Calphostin C, 10-6 mol/L PD98059, 10-6 mol/L Losartan (AT1R antagonist) or 10-6 mol/L PD123319(AT2R antagonist), and then stimulated with 10-6 mol/L Ang II for different time points.
Quantitative reverse transcription PCR (Real-time RT-PCR)
The expression of PlGF gene was identified by quantitative RT-PCR (Q-PCR) as reported earlier . Total RNA was extracted from HUASMCs or EA.Hy 926 cells using TRIZOL reagent (Invitrogen, USA). Q-PCR was carried out on an ABI Prism 7300 PCR Detection System (Applied Biosystems, USA) with fluorescence dye SYBR Green (SYBR Green Real-time PCR Master Mix, TOYOBO, Japan). The sequences of the primers were as follows: PlGF-F: 5'-GTT CAG CCC ATC CTG TGT CT-3'; PlGF-R: 5'-CTT CAT CTT CTC CCG CAG AG-3'. GAPDH-F: 5'-ACC ACA GT CCA TGC CAT CAC-3'; GAPDH-R: 5'-TCC ACC ACC CTG TTG CTG TA-3'. VEGFR-1-F: 5'-ATC ATT CCG AAG CAA GGT GTG-3'; VEGFR-1-R: 5'-AAA CCC ATT TGG CAC ATC TGT-3'. VEGFR-2-F: 5'-CAC CAC TCA AAC GCT GAC ATG TA-3'; VEGFR-2-R: 5'-GCT CGT TGG CGC ACT CTT-3'. The thermal cycling conditions were as following: 95°C 60 seconds, 40 cycles of 95°C for 15 seconds, 56°C for 15 seconds, 72°C for 45 seconds (data collection). Data analysis was carried out by ABI sequence detection software using relative quantification. For quantification, the target sequence was normalized to the GAPDH mRNA levels.
Quantification of PlGF and VEGF by ELISA
The PlGF or VEGF levels in the cell culture supernatant were measured by a "sandwich" enzyme immunoassay (Quantikine ELISA, R&D Systems, USA) according to manufacture's instructions. Briefly, the samples were added to 96-well plates coated with a specific monoclonal antibody to PlGF or VEGF. The unbound protein was washed three times, and an enzyme-linked polyclonal antibody specific to PlGF or VEGF was added. The plates were washed again for three times, and substrate solution was added to the wells. After 30 min of incubation, stop solution was added to each well. The amount of PlGF or VEGF was determined by optical density of the samples by comparison with the standards at 450 nm using an ELISA reader (Bio-Rad Model 680, USA).
Detection PlGF protein expression by immunofluorescence
EA.Hy 926 cells or HUASMCs were plated onto coverslips in 6-well plates, growth arrested and treated with Ang II at 10-6 mol/L concentration with or without other compounds. After that, the cells on the glass slides were then fixed and blocked as described before , followed by exposed to the primary anti-PlGF antibody. The second antibody was FITC-conjugated antibody and the cell nuclei were stained with DAPI (Sigma-Aldrich). Images were collected using an Eclipse TE2000-U fluorescent microscope system (Nikon, Japan) and analyzed with ImageJ software from NIH Image to semi-quantitatively determine the expression of PlGF protein in the cytoplasm of the cells.
Assessment of cell proliferation
The effect of Ang II and antagonists to PlGF or VEGF on cell proliferation was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as described previously. Briefly, EA.Hy 926 cells or HUASMC cells were subcultured in 96-well plates and incubated with serum-free medium for 24 hours. Quiescent cultures were treated with the Ang II (10-6 mol/L), Ang II (10-6 mol/L) and Losartan (10-6 mol/L), Ang II (10-6 mol/L) and the specific PlGF or VEGF neutralizing antibody (10-20 μg/mL) for 24 hours. Twenty microliters of MTT (15 mg/mL, Sigma, USA) was added to each well and incubated for 4 h at 37°C. The culture supernatant was discarded by aspiration and 150 μL of dimethyl sulfoxide (DMSO, Sigma) was added for 10 min. The light absorbance at 570 nm was detected using ELISA reader (Bio-Rad Model 680, USA).
The experimental data were expressed as means ± SD. Group means were compared by One-way ANOVA using the statistical software program SPSS 10.0 for Windows (Chicago, IL, USA), and P value < 0.05 was considered significant in all cases.
This work was supported by grants from the National Natural Science Foundation of China, No. 30470437, No. 30870596 (Xiaojing Liu), No. 30700149(Yingqiang Guo), No.30500222, No. 30871118 and No. 30971325 (Fengming Luo). We would like to acknowledge the assistance and critical advice provided by Dr. Jue Lin (University of California, San Francisco) and Dr. Rui Lin (Exelixis, Inc.) in the preparation of this manuscript.
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