Hypoxia-Independent Upregulation of Placental Hypoxia Inducible Factor-1α Gene Expression Contributes to the Pathogenesis of Preeclampsia
Accumulation of hypoxia inducible factor-1α (HIF-1α) is commonly an acute and beneficial response to hypoxia, whereas chronically elevated HIF-1α is associated with multiple disease conditions, including preeclampsia, a serious hypertensive disease of pregnancy. However, the molecular basis underlying the persistent elevation of placental HIF-1α in preeclampsia and its role in the pathogenesis of preeclampsia are poorly understood. Here we report that Hif-1α mRNA and HIF-1α protein were elevated in the placentas of pregnant mice infused with angiotensin II type I receptor agonistic autoantibody, a pathogenic factor in preeclampsia. Knockdown of placental Hif-1α mRNA by specific siRNA significantly attenuated hallmark features of preeclampsia induced by angiotensin II type I receptor agonistic autoantibody in pregnant mice, including hypertension, proteinuria, kidney damage, impaired placental vasculature, and elevated maternal circulating soluble fms-like tyrosine kinase-1 levels. Next, we discovered that Hif-1α mRNA levels and HIF-1α protein levels were induced in an independent preeclampsia model with infusion of the inflammatory cytokine tumor necrosis factor superfamily member 14 (LIGHT). SiRNA knockdown experiments also demonstrated that elevated HIF-1α contributed to LIGHT-induced preeclampsia features. Translational studies with human placentas showed that angiotensin II type I receptor agonistic autoantibody or LIGHT is capable of inducing HIF-1α in a hypoxia-independent manner. Moreover, increased HIF-1α was found to be responsible for angiotensin II type I receptor agonistic autoantibody or LIGHT-induced elevation of Flt-1 gene expression and production of soluble fms-like tyrosine kinase-1 in human villous explants. Overall, we demonstrated that hypoxia-independent stimulation of HIF-1α gene expression in the placenta is a common pathogenic mechanism promoting disease progression. Our findings reveal new insight to preeclampsia and highlight novel therapeutic possibilities for the disease.
Preeclampsia is a life-threating hypertensive complication of pregnancy and is a leading cause of maternal and neonatal morbidity and mortality.1,2 Despite intensive research efforts and several large clinical trials, current strategies for managing preeclampsia remain inadequate and are limited to symptomatic therapy or the termination of pregnancy. Thus, uncovering novel factors and signaling pathways that contribute to the pathogenesis of preeclampsia are needed for the establishment of mechanism-based preventative and therapeutic strategies to improve the prognosis of the disease.
Hypoxia inducible factor-1 (HIF-1) is a key transcription factor that plays a central role in the cellular response to low oxygen tension under physiological and pathological conditions.3,4 HIF-1 is a heterodimer consisting of 2 subunits, α and β. Although HIF-1β is constitutively expressed, HIF-1α levels are precisely regulated by post-translational modification depending on oxygen tension. HIF-1α is rapidly degraded under normoxic conditions, but quickly stabilized when oxygen availability is reduced. Thus, hypoxia-induced HIF-1α is usually transient and brief at the protein level.4 A variety of studies have shown that women with preeclampsia are characterized by persistently elevated placental HIF-1α levels that promote enhanced transcription of genes encoding soluble fms-like tyrosine kinase-1 (sFlt-1), soluble endoglin (sEng), and endothelin-1 (ET-1), all known to contribute to preeclampsia.5–7 However, the molecular basis underlying prolonged elevated placental HIF-1α in preeclampsia and the pathological role of sustained elevated HIF-1α in preeclampsia are largely unknown.
Numerous recent studies have shown that HIF-1α levels can be regulated by means that are independent of hypoxia.8 For example, angiotensin II and the inflammatory cytokines tumor necrosis factor (TNF) and interleukin (IL)-6 induce HIF-1α gene expression in vascular smooth muscle cells, kidney cells, and hepatocytes, respectively.9–11 Multiple studies have revealed that inflammatory cytokines and autoantibodies are elevated in preeclampsia patients and contribute to pathophysiology preeclampsia.12–14 For example, earlier studies showed that injection or infusion of pathogenic autoantibodies, such as the angiotensin II type 1 receptor agonistic autoantibody (AT1-AA) or the inflammatory cytokine tumor necrosis factor superfamily member 14 (LIGHT), into pregnant mice results in features of preeclampsia, including hypertension, proteinuria, placental abnormalities, and increased circulating sFlt-1, soluble endoglin, and endothelin-1.14–16 Thus, we hypothesized that the pathogenic autoantibody, AT1-AA, and the inflammatory cytokine, LIGHT, stimulate placental HIF-1α production and in this way contribute to features of preeclampsia. Here we conducted both mouse and human studies to assess these hypotheses.
For detailed descriptions, refer to the Methods section in the online-only Data Supplement.
Increased Placental HIF-1α Contributes to the Development of Preeclampsia Features in an Autoantibody-Injection Model of Preeclampsia in Pregnant Mice
To examine a potential role of elevated HIF-1α in preeclampsia, we took advantage of an experimental model of preeclampsia in mice induced by the injection of patient-derived-IgG (PE-IgG) known to contain the pathogenic autoantibodies, AT1-AA.17 We found that Hif-1α gene expression was induced significantly in the placentas of mice injected with PE-IgG compared with the pregnant mice injected with IgG from normotensive pregnant women (NT-IgG; Figure 1A). In contrast, no significant difference was observed in the kidneys between PE-IgG- and NT-IgG-injected pregnant mice (Figure S1 in the online-only Data Supplement). We also confirmed that PE-IgG induced the elevation of placental HIF-1α expression at the protein level by immunoblotting (Figure 1B). Additionally, immunohistochemical analysis revealed that the PE-IgG induced elevation of HIF-1α protein expression throughout the placenta (Figure 1C and 1D). In addition, the PE-IgG-induced elevation of placental HIF-1α expression was almost completely inhibited when PE-IgG was coinjected with losartan, an angiotensin II type 1 receptor (AT1R) blocker, or with the autoantibody-neutralizing 7 amino acid epitope peptide (Figure 1A–1D). These results indicate that the elevation of placental HIF-1α resulting from injection of PE-IgG was because of the activation of AT1Rs by AT1-AA.
Global HIF-1α-deficient mice die in midgestation from cardiac and vascular malformation.18 This embryonic lethality makes it difficult to examine the in vivo role of HIF-1α. To determine the pathophysiologic significance for PE-IgG-induced placental HIF-1α expression, we conducted siRNA-induced in vivo knockdown of Hif-1α mRNA. Briefly, siRNA-encapsulated nanoparticles were injected into pregnant mice on embryonic days 13.5 and 14.5, together with PE-IgG to specifically knockdown Hif-1α mRNA levels. As shown in Figure 2A, placental Hif-1α mRNA levels were successfully downregulated in Hif-1α siRNA-injected mice compared with control scrambled siRNA-injected mice. We also confirmed the reduction of HIF-1α protein expression levels in the placentas of Hif-1α siRNA-injected mice by immunoblotting (Figure S2). As a result of in vivo knockdown of Hif-1α mRNA, we found that PE-IgG-induced diagnostic features of preeclampsia, hypertension and proteinuria, were significantly reduced compared with control siRNA-injected mice (Figure 2B and 2C). Histological analysis of mouse kidneys revealed that PE-IgG-induced pathological changes seen in the glomeruli of control siRNA-injected mice (ie, swollen glomeruli with narrowed capillary and Bowman’s spaces) were attenuated in the kidneys of Hif-1α siRNA-injected mice (Figure 2D). Additionally, we found that placentas of Hif-1α siRNA-injected mice displayed significantly less tissue damage, including placental calcifications, a hallmark of placental distress observed in placentas of preeclampsia patients, as compared with those of control siRNA-injected mice (Figure S3). Moreover, we examined placental vasculature using CD31 staining. As a result, PE-IgG-induced disorganized and impaired vasculature in the labyrinthine zone of control siRNA-injected mice (low density of CD31-positive vessels and narrowed capillary spaces) was attenuated in the placentas of Hif-1α siRNA-injected mice (Figure 2E).
The Flt-1 gene is a direct transcriptional target of HIF-1α.19 A splice variant encodes sFlt-1, an antiangiogenic factor secreted by the placenta into the maternal circulation, that is believed to contribute to the development of systemic endothelial dysfunction, hypertension, and multiorgan damage, including the kidneys in preeclampsia patients.20 As such, we also found that Hif-1α mRNA knockdown in vivo suppressed the PE-IgG-induced elevation of Flt-1 mRNA in the placenta, as well as circulating sFlt-1 levels (Figure 2F and 2G). These results provide in vivo evidence that the induction of HIF-1α in the placenta contributes to the development of pathogenic autoantibody-induced features of preeclampsia and is also involved in the increased sFlt-1 production.
Elevated HIF-1α Contributes to the Development of LIGHT-Induced Preeclampsia Features
Emerging evidence indicates that an increased inflammatory response is involved in preeclampsia.12,13 Supporting this concept, a recent study showed that a member of the TNF superfamily, LIGHT, is elevated in the circulation and placentas of preeclampsia patients and that the injection of LIGHT into pregnant mice induces features of preeclampsia, including the overproduction of sFlt-1.15 The following experiments were conducted to determine whether elevated HIF-1α contributes to LIGHT-induced preeclampsia features in pregnant mice. We found that LIGHT injection into pregnant mice resulted in increased levels of Hif-1α mRNA in placentas (Figure 3A) but not in kidneys (Figure S1). Next, we found that LIGHT-induced placental Hif-1α mRNA levels were significantly reduced by neutralizing antibodies specific for LIGHT receptors: lymphotoxin β receptor and herpes virus entry mediator (Figure 3A). These results indicated that LIGHT signaling via its receptors induced placental Hif-1α gene expression.
To assess whether elevated HIF-1α in the placenta plays a detrimental role in the LIGHT-induced preeclampsia development as it does in PE-IgG-injected mice, we conducted in vivo knockdown of Hif-1α mRNA by injecting Hif-1α siRNA-encapsulated nanoparticles, together with LIGHT, into pregnant mice. Hif-1α siRNA injection successfully reduced the levels of placental Hif-1α mRNA compared with those of control siRNA-injected mice (Figure 3A). Moreover, preeclamptic features induced by LIGHT injection (hypertension and proteinuria) were attenuated significantly in Hif-1α siRNA-injected pregnant mice compared with control siRNA-injected pregnant mice (Figure 3B and 3C). Additionally, we found that Hif-1α mRNA knockdown attenuated the LIGHT-induced elevation of Flt-1 mRNA levels, as well as circulating sFlt-1 protein (Figure 3D and 3E). These findings indicate that placental elevated HIF-1α contributes to the development of preeclampsia features in the LIGHT-induced experimental model of preeclampsia.
HIF-1α Is Elevated in Placentas of Preeclampsia Patients and AT1-AA or LIGHT Directly Induces HIF-1α Expression in Cultured Human Placental Villous Explants Independent of Hypoxia
To extend our mouse findings to humans, we determined that the expression of HIF-1α was elevated in placentas of preeclampsia patients at both mRNA and protein levels compared with those of normotensive pregnant women (Figure 4A–4C). To determine whether AT1-AA or LIGHT can directly induce HIF-1α gene expression in the human placenta independent of hypoxia, we used primary human placental villous explants isolated from normotensive pregnant women. We cultured human villous explants treated with PE-IgG, NT-IgG, or LIGHT under ambient oxygen levels. We found that PE-IgG significantly induced HIF-1α mRNA levels compared with the NT-IgG-treated human villous explants, and the induction was significantly reduced by cotreatment with losartan to inhibit AT1R activation or 7 amino acid epitope peptide to neutralize AT1-AA (Figure 4D). Similarly, we found that treatment of cultured villous explants with LIGHT resulted in increased HIF-1α mRNA levels compared with the controls (Figure 4E). Additionally, we also confirmed that PE-IgG or LIGHT induced the elevation of HIF-1α protein (Figure 4F). Thus, these results indicate that AT1-AA and LIGHT are capable of directly inducing HIF-1α gene expression in cultured human villous explants independent of hypoxia.
AT1-AA- or LIGHT-Induced HIF-1α Promotes Flt-1 Gene Expression and Subsequent sFlt-1 Secretion in Human Villous Explants Independent of Hypoxia
We examined whether PE-IgG- or LIGHT-induced HIF-1α is capable of promoting Flt-1 gene expression and subsequent sFlt-1 production in human placentas independent of hypoxia. To test this possibility, we treated human villous explants under ambient oxygen levels with PE-IgG or LIGHT in the presence or absence of CAY10585, a specific HIF-1α inhibitor. First, we confirmed that the elevation of HIF-1α induced by PE-IgG or LIGHT was suppressed by the treatment of CAY10585 (Figure 5A). We found that Flt-1 mRNA levels and the amount of secreted sFlt-1 were increased by the treatment of human villous explants with PE-IgG or LIGHT (Figure 5B and 5C). In contrast, treatment with a HIF-1α inhibitor, CAY10585, significantly reduced PE-IgG- or LIGHT-induced Flt-1 gene expression and sFlt-1 production (Figure 5B and 5C). To further validate the role of HIF-1α for Flt-1 gene induction and subsequent sFlt-1 production induced by PE-IgG or LIGHT, we conducted HIF-1α mRNA knockdown in human villous explants. We confirmed that the elevation of HIF-1α induced by PE-IgG or LIGHT was successfully downregulated by the treatment of HIF-1α siRNA compared with the control-scrambled siRNA-treated group (Figure 5D). The increase in Flt-1 mRNA levels and the amount of secreted sFlt-1 induced by PE-IgG or LIGHT were significantly suppressed by the knockdown of HIF-1α mRNA in human villous explants (Figure 5E and 5F). These results indicate that PE-IgG or LIGHT directly induces Flt-1 gene expression in a HIF-1α-dependent manner in cultured human villous explants independent of hypoxia.
Here we report that Hif-1α mRNA and HIF-1α protein levels were elevated in the placentas of 2 independent animal models of preeclampsia, based on the injection with AT1-AA or LIGHT. We also showed that specific siRNA knockdown of Hif-1α mRNA attenuated hallmark features of preeclampsia, including hypertension, proteinuria, kidney damage, impaired placental vasculature, and maternal elevated circulating sFlt-1 in both preeclampsia mouse models. These results indicate that increased HIF-1α gene expression is a common pathogenic factor contributing to preeclampsia. Extending animal studies to humans, we confirmed that HIF-1α mRNA and HIF-1α protein levels were elevated in the placentas of preeclampsia patients. Using human villous explant cultures under nonhypoxic conditions, we showed that AT1-AA or LIGHT induced HIF-1α mRNA and HIF-1α protein levels, resulting in elevated Flt-1 mRNA levels and increased sFlt-1 secretion in a hypoxia-independent manner. Overall, we provide both in vivo animal studies and in vitro human evidence showing the pathogenic role of elevated HIF-1α gene expression in preeclampsia and hypoxia-independent mechanisms underlying its elevation in the placentas.
Numerous early studies showed that HIF-1α can be induced by nonhypoxic stimuli in various cell types.6 For example, studies in vascular smooth muscle cells showed that angiotensin II stimulates HIF-1α production by a protein kinase C–mediated transcriptional activation of the Hif-1α gene expression and by a reactive oxygen species–dependent mechanism, leading to enhanced translation of Hif-1α mRNA.10,21 In this case, angiotensin II induces HIF-1α protein, leading to an increase in vascular endothelial growth factor gene expression. Other studies have shown that angiotensin II induces Hif-1α mRNA production in renal glomerular cells.22 Women with preeclampsia harbor autoantibodies (AT1-AA) that mimic the action of angiotensin II and activate the major angiotensin receptor, AT1R. These pathogenic autoantibodies may serve as hypoxia-independent factors, leading to the increased production of HIF-1α observed in placentas of women with preeclampsia. Supporting our hypothesis, we have provided human in vitro studies showing that purified PE-IgG, which contains AT1-AA, activates AT1Rs, resulting in the induction of HIF-1α gene expression under nonhypoxic conditions. Additionally, we demonstrated that Hif-1α expression is induced significantly in the placentas of mice injected with PE-IgG. We realize that the PE-IgG used in our experiment is a complex mixture of immunoglobulins that is likely to contain other autoantibodies. However, we have shown in prior publications that the same effects are seen when AT1-AA is specifically purified by affinity chromatography.17 Furthermore, the PE-IgG or affinity-purified AT1-AA-induced preeclampsia features are blocked by losartan or the 7aa epitope peptide, indicating that the effects are mediated by interaction with the specific epitope on the AT1 receptor. Our results are also in good agreement with those of Wenzel et al, who showed that pregnant rats infused with a rabbit antibody that activates AT1Rs resulted in elevated HIF-1α in rat placentas.23 Our current study has provided human and mouse evidence that the activation of AT1Rs by AT1-AA induces HIF-1α gene expression in the placenta, thereby contributing to the development of preeclampsia.
A growing body of studies indicate that preeclampsia is characterized by increased circulating levels of proinflammatory cytokines, such as TNF-α, IL-1β, IL-6, IL-17, and LIGHT.12,13,15 A pathological role for these cytokines is supported by experimental evidence showing that infusion of these cytokines into pregnant rodents produces features of preeclampsia, including the production of AT1-AA.15,16 These results suggest that the elevation of inflammatory cytokines is an early event in the development of preeclampsia and functions upstream of AT1-AA production. Additional evidence shows that inflammatory cytokines induce HIF-1α under nonhypoxic conditions.6 For example, TNF-α and IL-1β activate HIF-1α gene expression in human hepatoma cells.24 TNF-α increases HIF-1α mRNA levels, as well as the expression of HIF-1 target genes glucose transporter type 1 and type 3 in human embryonic kidney cells.9 IL-6 induces both HIF-1α mRNA and HIF-1α protein levels and the expression of the HIF-1α target gene, erythropoietin, in hepatocytes.11 IL-1β induces HIF-1α-mediated VEGF secretion in trophoblast cells.25 Thus, a pathogenic role for HIF-1α in response to elevated inflammatory cytokines associated with preeclampsia may be an important contributor to disease pathogenesis. However, a role for HIF-1α in increased inflammatory cytokine-induced features in preeclampsia was not recognized before the results of our experiments reported here with the inflammatory cytokine, LIGHT. As a member of the TNF superfamily, LIGHT is known to be elevated and contributes to preeclampsia features in pregnant mice.15 As with AT1-AA, we have shown here that infusion of LIGHT stimulates production of HIF-1α in placentas of pregnant mice and in human villous explants independent of hypoxia. The increased levels of HIF-1α stimulate the transcriptional activation of the Flt-1 gene, thereby providing for excessive sFlt-1 production, leading to the features of preeclampsia. Our data represent the first in vivo animal evidence showing the pathological role of HIF-1α in inflammatory cytokine-induced preeclampsia pathophysiology.
Hypoxia is known to induce HIF-1α transiently, largely as a result of protein stabilization.26 However, how HIF-1α remains persistently elevated in the placenta in the setting of preeclampsia was previously unknown, and the role of HIF-1α in preeclampsia remained unclear. Here we provide both in vivo animal and in vitro human evidence that AT1-AA and LIGHT are 2 hypoxia-independent factors that stimulate Hif-1α gene expression, resulting in subsequent HIF-1α-mediated activation of Flt-1 gene expression. Our studies support a working model (Figure 5G) in which AT1-AA or LIGHT-induced HIF-1α expression in placental trophoblasts is followed by HIF-1α-mediated induction of Flt-1 gene expression and subsequent sFlt-1 production independent of hypoxia. Considerable evidence now indicates that elevated production of sFlt-1 plays a pathogenic role in preeclampsia.20 Thus, interfering with persistently elevated HIF-1α is likely to reduce the overproduction of sFlt-1 and slow the progression of the disease (Figure 5G).
In conclusion, our current studies have added significant new insight to the pathogenesis of preeclampsia by identifying the detrimental role of chronic elevated placental HIF-1α initially triggered by hypoxia-independent factors, a pathogenic autoantibody and an inflammatory cytokine. Chronically elevated placental HIF-1α promotes excessive sFlt-1 production and disease progression. Supporting this working model, we demonstrated that reducing elevated HIF-1α by siRNA-induced mRNA knockdown successfully halted HIF-1α-induced sFlt-1 production and prevented disease development in 2 independent mouse models of preeclampsia in vivo. Altogether, these findings suggest that HIF-1α suppression may serve as a target for pharmacological intervention for preeclampsia.
Sources of Funding
This work was supported by
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Novelty and Significance
What Is New?
Hypoxia inducible factor-1α (HIF-1α) gene expression and protein levels were induced in the placentas of 2 independent animal models of preeclampsia infused with angiotensin II type I receptor agonistic autoantibody or tumor necrosis factor superfamily member 14 (LIGHT).
In vivo knockdown of HIF-1α gene expression using siRNA attenuated hallmark features of preeclampsia, including hypertension, proteinuria, kidney damage, impaired placental vasculature, and maternal elevated circulating soluble fms-like tyrosine kinase-1 in both preeclampsia mouse models.
Using human villous explant culture, we found that angiotensin II type I receptor agonistic autoantibody or LIGHT directly induced HIF-1α gene expression and upregulated HIF-1α was responsible for angiotensin II type I receptor agonistic autoantibody or LIGHT-induced elevation of Flt-1 gene expression and the production of soluble fms-like tyrosine kinase-1 independent of hypoxia.
What Is Relevant?
Our current studies have provided new insight to the pathogenesis of preeclampsia by identifying the detrimental role of chronically elevated placental HIF-1α initially triggered by hypoxia-independent factors. Additionally, our discoveries indicate therapeutic possibilities targeting HIF-1α.
We have provided both mouse and human evidence that increased HIF-1α in the placenta plays a general pathological role in the pathogenesis of preeclampsia induced by a pathogenic autoantibody or an inflammatory cytokine. Our findings highlight novel therapeutic possibilities for preeclampsia.