When cells are subjected to stress by changes in their extracellular environment, unfolded proteins accumulate in the endoplasmic reticulum (ER), causing ER stress. This initiates the unfolded protein response (UPR), a signal transduction cascade aiming at restoring cellular homeostasis. The UPR and angiogenesis are involved in the pathogenesis of many diseases such as cancer, pulmonary diseases and chronic liver diseases (CLDs) including alcoholic liver disease, non-alcoholic steatohepatitis and hepatitis B. This review summarizes the upcoming knowledge of the interaction between the UPR and angiogenesis in physiological angiogenesis and in different CLDs and other diseases.
5′ adenosine monophosphate-activated protein kinase
activating transcription factor 4
activating transcription factor 6
binding immunoglobulin protein
C/EBP homologous protein
chronic liver diseases
eukaryotic initiation factor 2
ER associated degradation
fibroblast growth factor
hepatitis B virus
hypoxia inducible factor
hepatic stellate cells
inositol-requiring enzyme 1α
mouse embryonic fibroblasts
non-alcoholic fatty liver disease
oxygen induced retinopathy
protein kinase RNA-like endoplasmic reticulum kinase
placental growth Factor
unfolded protein response
vascular endothelial growth factor A
spliced X box binding protein 1
Endoplasmic reticulum (ER) stress is defined as the accumulation of unfolded proteins in the ER which occurs when a cell is subjected to stress by changes in its environment. ER stress can be induced by physiological stimuli such as hypoxia, glucose and amino acid deprivation or chemical inducers such as thapsigargin, tunicamycin or dithiothreitol. Consequently, the unfolded protein response (UPR) is activated, trying to restore cellular homeostasis. The UPR is involved in many processes in the liver such as hepcidin induction and the acute phase response [1, 2]. In addition, the UPR and angiogenesis are also involved in the progression of different chronic liver diseases (CLDs). A clear interaction between the UPR and vascular endothelial growth factor (VEGF) is also revealed in multiple diseases such as cancer, diabetic retinopathy, atherosclerosis and ischaemic renal disease [3-6]. Chronic liver diseases such as alcoholic liver disease, non-alcoholic steatohepatitis and hepatitis B can progress to cirrhosis, leading to hepatocellular insufficiency, splanchnic vasodilatation, portal hypertension and hepatocellular carcinoma (HCC). The mechanisms by which these CLDs cause cirrhosis are not fully understood. However, ER stress and the UPR are known to be involved in the pathogenesis of many CLDs . In addition, angiogenesis plays a crucial role in the progression of these CLDs leading to an angioarchitecture distinctive for cirrhosis. Knowledge about the role of the UPR in angiogenesis could lead to new therapeutics targeting angiogenesis by inhibiting specific ER stress mediators. It would also be very interesting to investigate whether current anti-angiogenic therapies reduce ER stress.
The goal of this review is to give an overview of all upcoming knowledge on the interaction between the UPR and angiogenesis in different pathologies.
The unfolded protein response
The UPR consists of three pathways, with three different resident transmembrane proteins, responsible for sensing ER stress: (i) inositol-requiring enzyme 1α (Ire1α); (ii) protein kinase RNA-like endoplasmic reticulum kinase (PERK) and (iii) activating transcription factor 6 (ATF6). The chaperone binding immunoglobulin protein (BiP or glucose-regulated protein 78 kDa (Grp78)), present in the lumen of the ER, is bound to these proteins under basal conditions. However, when ER stress occurs, BiP will bind to the unfolded proteins, dissociating from Ire1α, PERK and ATF6 and thereby activating these sensor proteins [8, 9].
Upon dissociation from BiP, Ire1α undergoes transautophosphorylation which activates its endoribonuclease activity. Activated Ire1α induces unconventional splicing of the mRNA of X-box binding protein-1 (XBP1) (Fig. 1). The spliced XBP1 (XBP1s) is an active transcription factor of genes coding for ER chaperones, ER associated degradation (ERAD) and quality control [8, 9]. Ire1 has also been shown to induce rapid turnover of mRNA through regulated Ire1-dependent decay in the presence of ER stress .
Activation of a second sensor, PERK, a serine/threonine kinase, leads to dimerization and autophosphorylation of PERK, which phosphorylates the eukaryotic initiation factor 2 (eIF2α) (Fig. 1). Phosphorylated eIF2α reduces global translation but promotes translation of proteins such as activating transcription factor 4 (ATF4), which regulate ER chaperone genes, the transcription factor C/EBP homologous protein (CHOP), ERAD pathway genes and amino acid metabolism genes [11, 12]. (iii) Following Bip dissociation, ATF6 translocates to the Golgi apparatus where it is cleaved by regulated intramembrane proteolysis. The ATF6 fragment is translocated to the nucleus where it promotes transcription of chaperones and other UPR genes (Fig. 1) .
The three different UPR pathways can be assessed by measuring the expression of the signalling molecules eIF2a, ATF4, XBP1, XBP1s and the genes they activate (CHOP, GADD34, Grp94, BiP) at the mRNA level and the three sensor proteins (Ire1, PERK and ATF6) and their signalling molecules at protein level .
The UPR is initially cytoprotective, increasing the level of molecular chaperones that bind to the unfolded proteins, decreasing protein synthesis and driving the capacity of ER associated degradation of these proteins. However, severe or sustained UPR provokes apoptosis of the cell [13, 15]. The UPR is known to trigger different pathways that lead to apoptosis. ER stress causes conformational alteration of the pro-apoptotic proteins Bak and Bax at the ER membrane, leading to release of Ca2+ from the ER . The increase in Ca2+ concentration in the cytoplasm activates the calcium-dependent cysteine protease calpain in the cytosol, which cleaves procaspase-12 to mature caspase-12 in the ER . Activated caspase-12 then cleaves procaspase-9, which in turn forms the apoptosome with released cytochrome c and Apaf-1, to activate the caspase-3 and apoptosis [18, 19]. Down-regulation of the transcription of anti-apoptotic protein B-cell CLL/lymphoma 2 (Bcl-2) by CHOP may also be involved in the induction of apoptosis . A third pathway involved in apoptosis is the Ire1-TRAF2-ASK1-JNK pathway, in which the formation of an Ire1-TRAF2-ASK1 complex is essential for activation of JNK, which causes apoptosis via pro-apoptotic proteins BID and BIM [21, 22]. ER stress associated cell death is involved in the development of HCC . A link between deregulated UPR and cell death is also seen in a rat model of LPS-induced acute-on-chronic liver failure. Cirrhotic livers exhibit partial UPR activation and full UPR activation after LPS challenge. In cirrhotic livers, eIF2α phosphorylation is sustained but there is no LPS-induced accumulation of NF-κB-dependent anti-apoptotic proteins which sensitizes cirrhotic livers to LPS/TNFα-mediated apoptosis .
Angiogenesis refers to the sprouting, migration and remodelling of existing blood vessels and plays a crucial role in several physiological processes as well as in a number of diseases such as cancer, diabetic retinopathy, atherosclerosis and liver diseases [13, 25, 26]. Angiogenesis differs from vasculogenesis, which is the process of blood vessel formation occurring by de novo production of endothelial cells from mesoderm cell precursors. The first vessels during embryogenesis form through vasculogenesis, after which angiogenesis is responsible for most, if not all, blood vessel growth during development and disease [27, 28]. Angiogenesis is regulated by pro- and anti-angiogenic factors that are produced in steady-state . Pro-angiogenic factors such as VEGF, fibroblast growth factor (FGF), platelet derived growth factor and interleukin 8 (IL-8), bind to their specific receptors on endothelial cells, causing proliferation of these cells, release of matrix metalloproteinases and migration towards the angiogenic stimuli, inducing formation of new blood vessels .
The best studied and key pro-angiogenic factor is VEGFA [13, 30]. The regulation of VEGFA is of importance during pathogenesis and it is indispensable during embryogenesis as VEGFA knockout (KO) mice are not viable [31, 32]. The best characterized pathway regulating VEGF is the Hypoxia Inducible Factor (HIF) pathway which is activated by hypoxia (Fig. 2). HIF1 and HIF2 are heterodimeric transcription factors composed of an α and β subunit. The β subunit is constitutively expressed but the α subunit is labile in an oxygen rich environment. When hypoxia occurs the α subunit is stabilized, thereby activating the HIF complex, which binds to the promoters of pro-angiogenic factors such as VEGF .
Another member of the VEGF family which contributes to pathological angiogenesis is placental growth factor (PlGF). The role of PlGF during embryogenesis and physiological angiogenesis is however negligible, unlike VEGFA. PlGF deficient mice are viable and fertile and do not have any visible abnormalities [34, 35]. Members of the FGF family are also inducers of angiogenesis . They are primarily involved in arteriogenesis, while VEGF members mostly regulate angiogenesis .
Interleukin 8 (IL-8) has also been shown to participate in angiogenesis. The main receptors binding to IL-8 are Il-8 receptor alpha (CXCR1) and IL-8 receptor beta (CXCR2). IL-8 directly enhances endothelial cell proliferation, survival and MMP expression in CXCR1- and CXCR2-expressing endothelial cells .
There are a number of other pro-angiogenic factors, such as platelet derived growth factor, growth-regulated oncogene α (CXCL1), macrophage inflammatory protein 2-alpha (CXCL2) and several others, which will not be discussed in this review because the link with ER stress has not been described.
The UPR regulates angiogenesis in physiological conditions
VEGFA is of critical importance during gestation. VEGF null mice die during embryogenesis, by impaired vasculogenesis and blood-island formation. Angiogenesis during embryogenesis is VEGF dose dependent as loss of a single VEGF allele in mice leads to vascular deformities and embryonic death [31, 32]. The ER stress sensor Ire1α is indispensible during embryogenesis as Ire1α KO mice are not viable. Noteworthy, Ire1α KO embryos exhibit liver hypoplasia which might contribute to the observed prenatal lethality. Also XBP1 deletion is lethal in mice and can be rescued by a transgene encoding for XBP1s expressed specifically in hepatocytes [39, 40]. In mouse embryonic fibroblasts (MEFs) treated with ER stress inducers thapsigargin, tunicamycin or hypoxia, a 10-fold increase is seen in VEGFA transcription which was abolished in XBP1 KO MEFs , stressing the importance of the XBP1 pathway in angiogenesis. VEGF induces XBP1 mRNA splicing in endothelial cells. This induction of XBP1 seems specific for VEGF since FGF could not activate XBP1 mRNA splicing .
Ire1 is also highly activated in the placenta during mouse embryogenesis. Another study explains the mortality in Ire1 KO mice by the reduced VEGFA levels specifically in the placenta, leading to ischaemic injury and ER stress. The development of the liver would not be the cause of death since Ire1α conditional KO mice with functionally normal placentas were viable and showed no liver hypoplasia . Although it remains unclear what causes embryonic lethality in Ire1 KO mice, it seems that the XBP1 pathway is important for VEGF regulation and angiogenesis as seen in placental as well as liver development. No research has been done on the involvement of the other UPR pathways in physiological angiogenesis. Further research could address this issue and unravel the role of Ire1 in physiological angiogenesis by VEGF rescue in Ire1 KO mice.
Interaction between the UPR and angiogenesis during disease
Angiogenesis plays an important role in the progression of alcoholic and non-alcoholic steatohepatitis (NASH) and chronic hepatitis B [43-45]. It is closely associated with fibrosis development and is mainly located in the fibrotic areas of the liver . Although angiogenesis is present in most chronic inflammatory and fibrogenic disorders, the liver has some specific characteristics which make its process of angiogenesis different from others. These characteristics include the special role of activated hepatic stellate cells (HSCs), unique for the liver, and the presence of two different vascular structures: the sinusoids and the larger vessels (portal and hepatic veins and hepatic arteries). The activated HSCs seem to play an important role in VEGFA expression. Angiogenic inhibitors, such as monoclonal VEGFR antibodies, have been shown to reduce angiogenesis in experimental models of portal hypertension . The role of angiogenesis and ER stress as separate processes in the pathogenesis of many CLDs is well-described, however, the interaction between the UPR and angiogenesis is not. Here, we focus on what is known about the interaction of the different UPR pathways with angiogenesis in CLDs.
ER stress induces angiogenesis in chronic HBV and NASH
ER stress is shown to regulate angiogenesis in hepatitis B and NASH. Hepatocytes infected with the hepatitis B virus (HBV) contain large HBV surface antigen mutants with deletions at the pre-S region. These pre-S deletion mutant HBV surface antigens are accumulated in the endoplasmic reticulum and initiate ER stress . This ER stress has been observed in patients with untreated hepatitis C . VEGFA is upregulated by pre-S mutants in HuH-7 cells, a well-differentiated HCC cell line. This upregulation is suppressed by ER stress inhibitor vomitoxin, suggesting ER stress as a regulator of VEGF expression . It has, however, not been investigated which ER stress pathway is responsible for VEGF upregulation in hepatitis B infected hepatocytes.
In NASH, fat accumulation induces disruption of ER homeostasis which causes ER stress and subsequently apoptosis of the liver cells . This lipotoxicity activates cytokines, which subsequently induce the recruitment of inflammatory cells. Inflammation contributes to the formation of new vascular structures in the liver, through the production of cytokines such as VEGF, TNFα, nitric oxide by Kupffer cells and activated HSCs . Angiogenic factors play an early role in the disease progression from steatosis to NASH, as was shown by elevated VEGF levels after 1 week in mice fed a methionine choline-deficient diet. Mice treated for 6 weeks with an antibody against VEGF receptor 2 showed significantly less steatosis and inflammation compared with untreated mice. However, anti-PlGF did not significantly improve liver histology in mice models of NASH . Liver samples from patients with NAFLD and NASH showed increased eIF2α phosphorylation and BiP expression . The mechanism by which ER stress contributes to NAFLD is not entirely clear. NAFLD is related to insulin resistance and mice deficient in XBP-1 develop insulin resistance. Obesity is shown to cause ER stress which in turn leads to suppression of insulin receptor signalling . ER stress also alters hepatic lipogenesis. Enhanced lipogenesis is most likely the consequence of sterol regulatory element-binding protein activation by the PERK pathway [54, 55]. Reduction of ER stress by chemical chaperones 4-phenyl butyric acid and taurine-conjugated ursodeoxycholic acid results in resolution of fatty liver disease, stressing the importance of ER stress in NASH development [56, 57].
The XBP1 pathway regulates VEGF in cancer and retinopathy
Cancer cells are confronted with hypoxia and nutrient deprivation as the tumour grows. Hypoxia induces upregulation of pro-angiogenic factors through the HIF pathway. In addition to the HIF pathway, the UPR has been shown to induce positive regulators of angiogenesis. HIF1α measurements in different cancer cell lines (Daoy, NB1691, SKNAS, C6, and NIH3T3) treated with ER stress inducers thapsigargin, tunicamycin, glucose free medium or hypoxia, suggest that HIF1α is not the major regulator of VEGFA expression under ER stress conditions other than hypoxia (Fig. 2) . The XBP1 pathway, however, seems important for VEGF upregulation in cancer. XBP1s adheres to two binding sites at the rat VEGFA promoter after ER stress induction via thapsigargin or glucose deprivation treatment . In addition, tumour cells expressing a dominant negative Ire1 transgene as well as Ire1α−/− MEFs were unable to trigger VEGFA upregulation after oxygen or glucose deprivation . Mice implanted with a glioma and expressing a dominant-negative Ire1 showed avascular tumours, whereas wild-type gliomas showed a dense vascularization pattern, suggesting that Ire1 plays a major role in angiogenesis . Although earlier research had shown that XBP1 had no role in the angiogenic response in MEFs and human fibrosarcoma tumour cells . Although every cancer is different and the effect of XBP1 on angiogenesis was not tested in HCC, there is reason to suspect that the Ire1 pathway has an influence on angiogenesis in HCC.
ER stress is also present in the retina of animal models of diabetic retinopathy and oxygen induced retinopathy (OIR) and contributes to retinal angiogenesis, which is illustrated by treating the OIR mouse model with tunicamycin or thapsigargin. Pathological vasculature and VEGF expression are increased in the mice treated with these ER stress inducers compared with the controls [52, 61]. Intraperitoneal injections with 4-phenyl butyric acid, an ER chaperone, in the OIR mouse model and diabetic mice, decreased VEGF expression in the retina of these mice, demonstrating the importance of the UPR in VEGF expression . Angiogenesis was impaired in XBP1 endothelial cell-specific KO mice triggered by ischaemia. Reconstitution of XBP1 in the XBP1 KO mice significantly improved angiogenesis in the ischaemic tissue . This was confirmed in a later study, in which siRNA knockdown or pharmacological blockade of Ire1α or ATF6 resulted in a significant reduction of in vitro angiogenesis. This reduction was also seen by inhibiting Ire1α or ATF6 in the mouse model of OIR. The combination of VEGF neutralizing antibody and UPR inhibitors or siRNAs reduced retinal neovascularization even further .
The PERK pathway influences angiogenesis in alcohol-induced liver disease, atherosclerosis and acute ischaemic renal disease
Just like XBP1s, ATF4 binds to a regulatory site in the VEGF gene [13, 63]. The increase in VEGFA in ATF4 wild type MEFS is, however, not higher than in ATF4 KO MEFs, suggesting only a marginal role for ATF4 in VEGF upregulation . Although later research did see a significant decrease in VEGF expression in PERK KO MEFS and in ATF4 knocked down 81B cell lines compared with wild type cells . This is also illustrated by the reduction, but no complete resolution, in tumour vascularization in the PERK deficient insulinoma mouse model and in vivo tumour cells underexpressing PERK [60, 64].
In NASH and NAFLD the PERK pathways does not control VEGF regulation . Although ER stress does not seem to play a significant role in the early stage of alcohol-induced liver disease (ALD), it probably has a more significant role in late stage ALD [66, 67]. Intragastric ethanol feeding in mice, a model for ALD, induces the activation of the UPR through hyperhomocysteinaemia , a well-known trigger of ER stress in other diseases such as diabetic retinopathy [3, 9]. In diabetic retinopathy, homocysteine causes perturbations in the formation of disulfide bridges and in the folding of proteins in the ER, thereby causing ER stress, which in turn causes an increase in VEGF by a mechanism involving the ATF4 pathway . An independent study confirmed the role of ATF4 in VEGF production in the retina Müller cells, suggesting that VEGF upregulation is partially mediated by the ATF4 pathway . The ATF4 pathway may therefore be important in VEGF production in ALD.
In atherosclerosis, oxidized phospholipids upregulate VEGF via an ATF4 dependent mechanism, thereby inducing angiogenesis. Knock down of ATF4 in a human umbilical vein endothelial cell line and human aortic endothelial cell line is associated with loss of VEGF induction by oxidized phospholipids. Oxidized phospholipids stimulate binding of ATF4 to a regulatory site in the VEGFA promoter, but do not alter the stability of VEGF mRNA. Here, the role of HIF-1 dependent transactivation of VEGF was excluded [4, 5]. IL-8 was also upregulated in human aortic endothelial cells treated with ER stress inducers tunicamycin or dithiothreitol. ATF4 is required for this upregulation as shown by significant reduction in IL-8 in human aortic endothelial cells treated with siRNA targeting ATF4 .
In a rat model of acute ischaemic renal stress, the UPR is activated in parallel with VEGFA and basic FGF expression, independently of HIF-1α. Inhibition of PERK expression by siRNA in human renal endothelial cells decreases VEGFA both at the transcriptional and protein level. The secretion of FGF is also significantly reduced in these cells, however, the transcript expression is not altered, suggesting that the UPR activates FGF secretion only at the post-transcriptional level. Inhibition of Ire1α or ATF6 does not alter the VEGFA or FGF expression in a model for ischaemic renal failure . Earlier research had shown an increase in phosphorylated eIF2a in kidney homogenates following 10 min of cardiac arrest-induced ischaemia and 10 min reperfusion .
The role of the ATF6 pathway is not straightforward
The ATF6 pathway seems of less importance to VEGF regulation than the other pathways, as ATF6α did not seem to induce VEGFA transcription significantly in MEFs [13, 26]. However, later research rejected this by discovering that all three ER stress sensors are involved in the induction of VEGFA in the human HepG2 cancer cell line .
ER stress regulates angiogenesis by other mechanisms
The UPR regulates VEGFA expression at the transcriptional, as well as at the post-transcriptional and post-translational level. The UPR also increases the stability of VEGF mRNA via the 5′ adenosine monophosphate-activated protein kinase (AMPK), which was demonstrated by C6 glioma cells treated with a combination of compound C, an AMPK inhibitor, and UPR inducers hypoxia, thapsigargin or glucose free medium. There was a significant reduction in VEGFA transcripts in these cells compared with C6 cells treated with UPR inducers but no AMPK inhibitor, suggesting that AMPK plays a role in the UPR induced stabilization of VEGF mRNA. Activation of AMPK in cells activated by ER stress was confirmed by Western Blotting . At the post-translational level, ER stress induces the ER chaperone ORP150 which facilitates correct processing and secretion of VEGF .
The expression of the pro-angiogenic factor IL-8 is also increased after ER stress induction with thapsigargin, tunicamycin, glucose deprivation or glutamine deprivation in cancer cells [13, 72].
Conclusion and future perspectives
The UPR plays a significant role in VEGFA regulation in different diseases. The current literature on the interaction between the different UPR pathways and angiogenesis is summarized in Table 1. We have to emphasize that the influence of ER stress on angiogenesis is only tested in a minimal amount of CLD models. Moreover, many studies only investigated the effect of ER stress on VEGF expression, missing the effect on the blood vessels. The link between the UPR and VEGF does not systematically mean that there is a direct effect on endothelial cells. The direct effect on vessel formation is however studied in cancer models, in which the influence of the different UPR pathways is well-described [58-60]. There is evidence suggesting an interaction between the UPR and angiogenesis in CLDs, however, it is not very clear and needs to be investigated further. Although evidence is mostly circumstantial, the PERK pathway seems the most important pathway in the control of angiogenesis in CLDs. Although the PERK pathway is activated in NASH, it does not seem to influence VEGF regulation. This is contradictory with what we would expect regarding the data in the other diseases. Till now, most research was focused on the role of VEGF in angiogenesis. However, little is known about the relationship between the UPR and other pro- and anti-angiogenic factors. Moreover, non-specific ER stress inhibitors are often used. Future studies with Ire1- and PERK-inhibitors could refine the results. Further research about the specific role of the UPR in angiogenesis could lead to therapeutic ER stress modulators targeting pathological angiogenesis. However, the UPR is involved in many processes such as apoptosis and occurs in many organs so that pharmacological intervention at the UPR level probably also causes many side-effects. The use of current anti-angiogenic therapies is limited by many side-effects. In clinical trials with sunitinib, sorafenib and anti-VEGF antibody bevacizumab, a range of side-effects were observed in patients such as diarrhoea, hypertension, nausea, skin toxicities and mild thrombotic and bleeding events [73, 74]. The ideal treatment of CLDs has not been found yet, however, progress has been made over the years and future studies may support this progress.
Table 1. Role of the different unfolded protein response (UPR) pathways in angiogenesis in different diseases
The authors wish to thank the Fund for Scientific Research (FWO Flanders) for the material support (3G075112) and Ghent University for the financial support. Hans Van Vlierberghe is Senior Researcher of the FWO.
Financial support: Ghent University.
Conflict of interest: The authors do not have any disclosures to report.