Autophagy inhibitor

HypoXia-inducible factor-1: Regulatory mechanisms and drug development in stroke

Zirong Pan, Guodong Ma, Linglei Kong *, Guanhua Du *
Beijing Key Laboratory of Drug Target Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, PR China


Stroke is an acute cerebrovascular disease caused by sudden rupture of blood vessels in the brain or blockage of blood vessels, which has now become one of the main causes of adult death. During stroke, hypoXia-inducible factor-1 (HIF-1), as an important regulator under hypoXia conditions, is involved in the pathological process of stroke by regulating multi-pathways, such as glucose metabolism, angiogenesis, erythropoiesis, cell survival. However, the roles of HIF-1 in stroke are still controversial, which are related with ischemic time and degree of ischemia. The regulatory mechanisms of HIF-1 in stroke include inflammation, autophagy, oXidative stress, apoptosis and energy metabolism. The potential drugs targeting HIF-1 have attracted more attention, such as HIF-1 inhibitors, HIF-1 stabilizers and natural products. Based on the role of HIF-1 in stroke, HIF-1 is expected to be a potential target for stroke treatment. Resolving when and what interventions for HIF-1 to take during stroke will provide novel strategies for stroke treatment.

Stroke HypoXia-inducible factor 1 Drug development

1. Introduction

Stroke is an acute cerebrovascular disease caused by sudden rupture of blood vessels in the brain or blockage of blood vessels, including ischemic and hemorrhagic stroke. Stroke is characterized by high morbidity, mortality and disability. In the past 30 years, the incidence, prevalence and death rate of stroke in China have been on the rise, and it has now become one of the main causes of adult death, imposing a heavy economic burden on families and society [1,2].
After the occurrence of stroke, the blood supply to the brain tissue is reduced and the oXygen and nutrient supply to the brain tissue is insufficient, causing local ischemia and hypoXia [3]. After stroke, a variety of proteins and molecules in the brain are altered, which are associated with inflammation, apoptosis, oXidative stress and energy disorder. HypoXia-inducible factor-1 (HIF-1) is a key transcription factor that maintains oXygen homeostasis under hypoXia conditions, which involved in the pathological process of stroke by regulating multi-pathways, such as glucose metabolism, angiogenesis, erythropoi- esis and cell survival.
The roles of HIF-1 in stroke are still controversial. EXperimental studies have found that hypoXic preconditioning (HPC) and HIF-1 in- ducers (feroXamine and cobalt chloride) exert neuroprotective effect by increasing HIF-1 level after cerebral ischemia [4]. However, other studies have also proved that inhibition of HIF-1 plays anti-cerebral ischemia effect [5]. In this review, we summarize the roles and regula- tory mechanisms of HIF-1 in stroke, and drug development targeting HIF-1, which are important for understanding the pathogenesis of stroke and conducting the development of related therapeutic drugs.

2. The regulation of HIF-1 under physiological conditions

HIF-1 is a transcriptional activator that regulates gene expression in mediated the expression of a series of genes. During stroke, HIF-1 is response to changes of intracellular oXygen concentration, which consists of a heterodimer of the oXygen regulatory subunit HIF-1α (120 kDa) and the structural subunit HIF-1β (91 kDa). The expression of HIF-1 is regulated by oXygen concentration, as well as the phosphatidylinositol 3 kinase (PI3K) and ERK mitogen-activated protein kinase pathways [6, 7]. HIF-1β is constitutively expressed in cells, whereas the expression of HIF-1α is regulated by oXygen concentration. Both subunits are mem- bers of the bHLH-PAS protein family, and their amino-terminal bHLH and PAS structural domains are associated with dimerization, DNA binding and signal transduction [8]. In addition to the bHLH and PAS domains, HIF-1α has a C-terminal trans-activation domain-C (C-TAD), an N-terminal trans-activation domain-N (N-TAD), oXygen-dependent degradation (ODD) domain and inhibitory domains (ID).
The C-TAD domain regulates gene expression by binding to tran- scriptional activators CBP and p300, and the asparagine (Asn803) in C- TAD is hydroXylated under normoXic conditions by the factor inhibiting HIF-1 (FIH-1), which inhibits HIF-1 transcriptional activity by blocking the interaction between C-TAD and the p300/CBP transcriptional co- activator [9]. In addition, ODD domain can be hydroXylated by pro- line hydroXylase-2 (PHD-2) in normoXia, leading to degradation of the α-subunit [10]. The ID can repress the transcriptional activity of C-TAD under normoXic conditions [11] (Fig. 1).
Under normoXic conditions, the activity of HIF-1α is inhibited through two pathways. Firstly, HIF-1α is prolylhydroXylated in its degradation domain via HIF-prolyl hydroXylase, leading to HIF-1α spe- cifically recognized by VHL protein (VHL protein is an E3 ubiquitin- linked enzyme that targets HIF-1α for proteasome degradation), then degraded by the proteasome through ubiquitination. Secondly, FIH-1 acts as an asparaginyl hydroXylase to activate the asparagine residue (N803) at the TAD-C terminus of HIF-1α to block the interaction of HIF- 1α with transcriptional co-activators (p300/CBP), thus inhibiting the transactivation activity of HIF-1 [12].
When cells are in hypoXic conditions, both oXygen dependent hy- droXylation and degradation of HIF-1α are prevented, resulting in the decrease of FIH-1 activity and the recovery of the transactivation ac- tivity of HIF-1 [12]. Then HIF-1α, with improved stability and tran- scriptional activity, translocates to the nucleus and interacts with HIF-1β to form a heterodimer. HIF-1 binds to the target DNA sequences con- taining the hypoXia response elements via its coactivator p300/CBP and induces the expression of downstream genes [10] (Fig. 2).

3. The expression of HIF-1 in stroke

3.1. The expression of HIF-1 with ischemic time

In 1996, Wiener et al. firstly reported that when rats or mice were exposed to hypoXia for 30–60 min, HIF-1α and β subunits were both induced in brain [13]. Subsequently, the spatial and temporal expression of HIF-1 have been investigated. In the areas around the infarction, HIF-1α mRNA was induced at 7.5 h after ischemia, and increased further at 19 and 24 h [14]. Marti et al. and Matrone et al. found that in mice permanent middle cerebral artery occlusion (pMCAO) model, HIF-1α mRNA peaked at 24 h after ischemia in the area around the infarction [15,16].
EXcept for the area around the infarction, the expression of HIF-1α has also been found in other ischemic regions. In MCAO model, HIF-1α expression was observed in ischemic caudoputamen and cortex, which began to increase at 1 h, peaked at 12 h, and declined at 24 h after reperfusion [17]. In neonatal hypoXia-ischemia model, the expression of HIF-1α increased at 4 h, peaked at 8 h, and declined at 24 h after ischemia in the cortex and hippocampus [18]. Mu et al. also revealed that HIF-1α protein in injured cortex peaked at 8 h, and declined sub- sequently at 24 h in neonatal MCAO model [19]. In addition, in neonatal hippocampal, HIF-1α mRNA increased at 2 h after ischemia and peaked at 24 h [20]. These results suggest that HIF-1α can be induced after ischemia in different brain regions and the expression of HIF-1α is related to ischemic time. HIF-1α rapidly increases after ischemia, peaks within 8–24 h and finally returns to the original level. HIF-1α in different brain regions may play different roles in stroke. Therefore, the spatial and temporal expression of HIF-1α in the brain should be considered at different periods after stroke, which may bring break- throughs in the treatment of stroke.

3.2. The expression of HIF-1 in different cells

The correlation between the expression of HIF-1 in different cells of brain tissue and ischemic time has been also investigated. The pathological role of HIF-1α could be associated with cell type in central ner- vous system. Vangeison et al. suggested that the loss of HIF-1α function in neuron reduced neuronal viability during hypoXia, while selective loss of HIF-1α function in astrocytes protected neurons from hypoXic- induced neuronal death [21]. In a mouse MCAO model, HPC with 15 min caused a rapid and transient increase of HIF-1α in neuron, whereas HIF-1α in astrocytes increased slowly but lasted for a long time. The increase of neuronal HIF-1α depended on the inhibition of HIF-1α degradation enzymes, while astrocytes induced persistent HIF-1α expression by P2X7 receptor-dependent mechanism [22]. Hirayama et al. found that astrocytic HIF-1α induced by HPC was involved in ischemic tolerance through the regulation of P2X7 receptor, while increased neuronal HIF-1α was not involved in ischemic tolerance [23]. In addition, Yan et al. showed that the expression of HIF-1 was both induced in neurons and brain endothelial cells, and inhibition of HIF-1 by YC-1 played different roles on infarct volume and BBB perme- ability. The further study suggested that YC-1-mediated exaggeration of brain damage may be related with the inhibition of HIF-1 expression in neurons, and BBB protection may be associated with the inhibition of HIF-1 in brain endothelial cells [24]. Bok et al. reported that in microglia of myeloid-specific Hif-1α or Hif-2α knockout mice, HIF-1α may pro- mote microglial functions including chemotaxis, phagocytosis, ROS production, and TNF-α production to disrupt adult neurogenesis in the acute phase of ischemic stroke [25]. Above results indicate that HIF-1 expressed in specific cells exerts different effects after stroke. There- fore, future studies need to focus on specific types of cells to better un- derstand the roles of HIF-1 in stroke. For example, transcriptomics can be used to explore the relevance of HIF-1 in the dysregulation of hypoXia-related gene products in different cells of brain, and the addi- tional regulatory mechanisms associated with stroke. Single cell sequencing can be defined the transcriptional landscape of specific cells to reveal cell heterogeneity and identify the expression and role of HIF-1 in specific cells.

4. Roles of HIF-1 in stroke

The roles of HIF-1 in stroke have not been determined. HIF-1 plays different roles in different conditions, which are related to the devel- opment process and severity of stroke. For example, early inhibition of HIF-1α protected against ischemic brain injury in neonates, which was associated with maintaining blood-brain barrier (BBB) integrity and reducing brain edema and neuronal death [4]. In a stroke model of Hif1α/Hif2α-deficient mice with lacking anti-survival factor, early acute neuronal cell death and neurological damage were improved. At 24 h after stroke, cell death and edema were significantly decreased, but it was obviously impaired after 72 h, companied with increased apoptosis and reduced angiogenesis [26]. In addition, silencing HIF-1α after 0.5 h of ischemia in rats attenuated brain edema and apoptosis, whereas silencing HIF-1α after 8 h of ischemia increased neuronal injury and decreased vascular endothelial growth factor (VEGF) expression [27].
When mild and moderate hypoXia occurs, HIF-1 promotes angiogenesis and cell survival by increasing VEGF expression [28], but during severe and sustained hypoXia, VEGF increases substantially and leads to BBB disruption [29]. Therefore, the roles of HIF-1 are related with ischemic time and severity of ischemia (Fig. 3).

4.1. The protective effects of HIF-1 in stroke

The protective effects of HIF-1 in the brain may be related to the regulation of a variety of genes that promote cellular adaptation to hypoXia after ischemic stroke [30]. These genes are associated with the control of vasodilation and contraction, angiogenesis, erythropoiesis, cell proliferation and energy metabolism [31–33]. HIF-1 promotes the expression of erythropoietin (EPO), which increases erythropoiesis to enhance oXygen delivery and protects cells from hypoXic/ischemic injury [34]. In MCAO model of Sv129 mice, HPC for 180 or 300 min induced relative tolerance to cerebral ischemia and reduced infarct volume, while the protective effect was significantly reduced after blocking endogenously EPO [35,36]. Li et al. found that HIF‑1α attenuated neuronal apoptosis in cerebral ischemic rat partially through upregulating EPO [34]. Yan et al. reported that the expression of EPO and GLUT-3 was induced in neurons after ischemia and was suppressed by YC-1, which may be associated with YC-1-mediated exaggeration of brain damage caused by ischemia [24].
Glucose transporter and glycolytic enzymes, as key genes in energy metabolism, are regulated by HIF-1 and play important roles in cell survival. Activation of HIF-1 leads to enhanced glucose transport and glycolytic pathway, thereby promoting cell survival [37,38]. Under hypoXic conditions, HIF-1 dynamically regulates glucose fluX through the glycolytic pathway to defend against the risk of increased ROS production [39,40]. In pMCAO model, the expression of HIF-1α mRNA, glucose transporter protein-1, and several glycolytic enzymes in the infarct penumbra were upregulated by ischemia, which might improve tissue survival in the ischemic penumbra by increasing glucose transport and glycolysis [41]. Wang et al. found that in cobalt chloride treatment astrocytes, both protein and mRNA levels of GLUT‑1 and GLUT‑3 were elevated in a time‑dependent manner, followed by accumulation of HIF‑1α, leading to reduced energy production and cell viability [42].
After hypoXia, increased HIF-1 promotes the expression of neuronal sodium-calcium exchanger 1(NCX1), a key mediator in maintaining sodium and calcium homeostasis, and silencing HIF-1α before HPC prevents NCX1 upregulation and neuroprotective effects, indicating that HIF-1 regulates sodium-calcium homeostasis in neurons by upregulating NCX1 to maintain the stability of the intracellular environment [43]. Valsecchi et al. also reported that overexpression of NCX1 induced by HIF-1 in the brain might be partially responsible for neuroprotection in an animal model of cerebral ischemic preconditioning [44,45].
HIF-1 increases the supply of oXygen and nutrients to ischemic brain tissue by upregulating the expression of VEGF, thereby promoting neovascularization [46,47]. In response to hypoXia, HIF-1 increases the expression of VEGF to facilitate the cells’ adaptation to hypoXia [48]. In pMCAO model of mice, a substantial increase of newly formed vessels at the infarct border was observed from 48 h to 72 h after the occlusion, and the expression of VEGF and VEGFR was also significantly induced by HIF-1 and HIF-2, which suggested that VEGF-mediated angiogenesis was regulated by HIF-1 and HIF-2 [15]. Rania et al. found that, empa- gliflozin, a sodium-glucose cotransporter 2 inhibitor, significantly reduced infarct size and enhanced neurobehavioral functions in cerebral ischemic rats via the HIF-1α/VEGF pathway [49]. Therefore, in the re- covery period of stroke, HIF-1 plays a protective effect by regulating VEGF-mediated angiogenesis.

4.2. The harmful effects of HIF-1 in stroke

The harmful effects of HIF-1 in the brain after stroke involve several pathways. HIF-1 aggravates brain injury by regulating apoptotic genes, such as p53 and Caspase-3. At 1 h after ischemia, the inhibition of HIF- 1α by using HIF-1α-siRNA in a rat MCAO model significantly reduced infarct volume and mortality, improved neurological deficits. Further experiments revealed that treatment with HIF-1α siRNA protected neurons by inhibiting HIF-1α, VEGF and apoptosis-related proteins such as p53 and Caspase-3 [50]. In addition, HIF-1 can induce BNIP3, causing mitochondrial dysfunction, depolarization of mitochondrial membrane, and eventually lead to apoptosis and necrosis [51]. In a rat MCAO model and oXygen glucose deprivation (OGD) model of primary cortical increases the BBB permeability [24]. In vitro hypoXia-reoXygenation model of adult rat brain endothelial cell, the BBB permeability increased, while the inhibition of HIF-1α could protect the BBB by inhibiting the accumulation of HIF-1α and the expression of VEGF [29, 52]. Chen et al. reported that inhibition of HIF-1 could reduce BBB damage after stroke in adult and neonatal rats, which may be partially due to blocking HIF-1α signaling pathway in the neuronal apoptosis and down-regulating VEGF activity to protect the BBB [53].
HIF-1-mediated neuroinflammation exacerbates brain injury. HIF-1 induces the expression of several cytokines and chemokines to aggra- vate inflammatory injury [54,55]. In the acute phase of ischemic stroke, HIF-1 promotes microglia chemotaxis and the release of inflammatory factors [25], and mediates neuroinflammation [56]. Knockdown of HIF-1 inhibits neutrophil infiltration and improves neurological func- tion [57]. The relationship between HIF-1 and inflammation will be discussed in detail below.
In conclusion, the beneficial or detrimental effects of HIF-1 in stroke depends on the duration and severity of hypoXia [58] and cell type [21]. According to the duration and severity of hypoXia, HIF-1α regulates the expression of adaptive and pro-death genes in the brain in different time-dependent manner [59]. It is necessary to combine the ischemic time and severity of ischemia, and analyze specific cell types to clarify the roles of HIF-1.

5. Regulatory mechanisms of HIF-1 in stroke

As mentioned above, HIF-1 plays different roles under different conditions, but how does HIF-1 participate in the pathological process of stroke? In this section, we reviewed the regulatory mechanisms of HIF-1 in stroke (Fig. 4).

5.1. HIF-1 and inflammation

After ischemic stroke, nerve cells die due to ischemia and hypoXia, activates the innate immune response in the brain and promotes the production of neurotoXic substances, such as inflammatory cytokines, chemokines, reactive oXygen species, and nitrogen oXide (NO), which mediate a series of inflammatory cascade reactions, leading to BBB disruption and neurological deficits [60]. HIF-1 is involved in hypoXia-induced neuroinflammatory response via regulating inflammatory factor release and inflammatory cell infiltration [52–54].
Some studies have shown that HIF-1 induces the expression of cy- tokines and chemokines, such as IL-20, MCP-1 and MCP-5 to aggravate inflammatory injury [54,55]. In the acute phase of ischemic stroke, HIF-1 promotes microglia chemotaxis, ROS and TNF-α production via CD36 and MFG-E8 pathways, thus interfering with neuro regeneration [25]. In a mouse cerebral hypoXia-ischemia model, knockdown of HIF-1 inhibited neutrophil infiltration and improved neurological function [57]. Toll-like receptor 4 (TLR4) is an important receptor mediated neuroinflammation, and closely associated with microglia activation and neutrophil infiltration [61]. TLR4 deficiency inhibits microglia polarization, neutrophil infiltration and inflammatory factor release, and ameliorates the pathological process of stroke and cerebral hem- orrhage [62]. Under hypoXic conditions, HIF-1 regulates TLR4 expres- sion by directly binding to the promoter of TLR4 gene, while HIF-1 expression can be also stimulated by TLR4 through NF-κB pathway [56].
In a cerebral hemorrhage model, HIF-1 activated the TLR4 pathway through the upregulation of Tim3, which was involved in neuro- inflammation and microglia polarization [63]. In hypoXia-stimulated BV2 cells in vitro, inhibition of HIF-1 reduced TLR4 expression and neuron, treatment with 2-methoXyestradiol (2ME) at 0.5 h after ischemia or pretreatment with HIF-1α siRNA decreased brain injury, brain edema and the number of apoptotic cells [27]. These results showed that early inhibition of HIF-1α reduced apoptosis to attenuate brain injury.
In the early stage of stroke, HIF-1 induces the expression of VEGF and attenuated neuroinflammation [64].

5.2. HIF-1 and autophagy

Autophagy is a self-protection mechanism developed by eukaryotic cells during long-term evolution. Autophagy has both pro- and anti- survival effects on cells, and its effects are related to the endoplasmic reticulum stress and the duration and degree of autophagy [65]. The role of autophagy in ischemic stroke is bidirectional, the activation of autophagy helps to remove accumulated proteins and is beneficial for nerve cell survival, while excessive activation of autophagy may lead to nerve cell death [65]. Some studies have shown that HPC plays pro- tective effects in ischemic stroke, possibly through HIF-1α/Beclin1-regulated autophagy [66]. In the OGD model of SH-SY5Y cells, HIF-1 enhanced autophagy and led to ischemic/hypoXic brain injury [67].
HIF-1 can regulate autophagy through multiple pathways. BNIP3 is one of the most important target genes of HIF-1α. BNIP3 promotes the dissociation of Beclin1 from Bcl-2 and activates autophagy by competing with beclin-1 to bind Bcl-2 [66]. BNIP3 also activates autophagy by inhibiting Rheb (an upstream activator of mTOR/S6 K/4E-BP signaling) and mTOR [68,69]. HIF-1α-induced upregulation of p53 also contrib- utes to autophagy activation. In a rat model of global cerebral ischemia, the upregulation of HIF-1α induced the stabilization of p53 [70], and NO. The oXygen radicals during ischemic stroke are mainly pro- duced by mitochondria, which produce superoXide anion radicals in the process of electron transfer. Increased superoXide anion radicals cause PHD inactivation, resulting in stabilization and accumulation of HIF-1α [75]. However, this phenomenon is not observed in the mitochondrial DNA deficient cells model, suggesting that ROS produced by mito- chondria is necessary for the stabilization of HIF-1α [76]. Furthermore, enhanced ROS can activate the NF-κB pathway, which in turn induces HIF-1α through a novel NF-κB binding site in the HIF-1α promoter, resulting in elevated expression of HIF-1α [77].
It has also found that inducible nitric oXide synthase (iNOS) protein is induced by HIF-1 in the ischemic core region, peri-ischemic region and ipsilateral hippocampus in pMCAO model [16]. Under hypoXic condi- tions, HIF-1 binds to the iNOS promoter and induces iNOS expression. Therefore, activation of HIF-1 may be associated with cell survival or death induced by iNOS after cerebral ischemia [16]. In addition, Li et al. also reported that isoflurane post-conditioning led to accumulation of HIF-1α and iNOS, HIF-1α transcriptional activity enhancement and mediating excitotoXicity through apoptosis and autophagy [71]. co-localization of HIF-1α and iNOS. Silencing of HIF-1α in the primary
Meanwhile, in vitro experiments also revealed that inhibition of p53 activation reduced TNF-α-induced apoptosis and autophagy [72]. In addition, HIF-1 induces mitochondrial autophagy by promoting the expression of BNIP3 and Beclin-1/Atg5 complexes during hypoXia, thereby reducing the production of ROS [73]. Gong et al. also found thatthe upregulation of HIF-1α protein induced mitochondrial autophagy in primary cortical cell through the inhibition of the mTOR pathway [74].

5.3. HIF-1 and oxidative stress

Numerous preclinical and clinical observations have shown that the formation of free radicals increases after stroke. The free radicals involved in stroke include superoXide anion radicals, hydroXyl radicals cortical neurons reduced the accumulation of iNOS and the protective effects of isoflurane post-conditioning, suggesting the involvement of HIF-1α in the regulation of iNOS during tolerance against cerebral ischemia induced by isoflurane post-conditioning [78].

5.4. HIF-1 and apoptosis

Apoptosis is a form of programmed cell death that occurs in multi- cellular organisms and leads to characteristic cellular changes and death. In stroke, HIF-1 also has a biphasic regulatory effect on apoptosis. Inhibition of HIF-1α in the early stage of ischemia reduces brain damage and the number of apoptotic cells [27]. In contrast, silencing HIF-1α in the later stage increases neuronal damage and apoptotic cell numbers [34].
HIF-1 activates apoptotic signaling pathways by upregulating pro- apoptotic molecules. The Bcl-2 family includes pro-apoptotic protein molecules such as Bax, Bak, BNIP3L, Nip3 and BNIP3. During hypoXia, the expression of BNIP3L was induced by HIF-1, then interacts with anti- apoptotic proteins, such as Bcl-2, Bcl-X (L), and E1B19K, to inhibit the function of anti-apoptotic proteins, thereby promoting apoptosis [79, 80]. In addition, HIF-1α regulates apoptosis by inhibiting Mdm2 (p53 ubiquitin ligase) mediated p53 degradation and nuclear translocation, increasing p53 stability [81]. However, HIF-1α can also attenuate

5.5. HIF-1 and energy metabolism

The brain is the organ that consumes the most oXygen and glucose in human body. It relies on the oXidative glucose metabolism to produce energy under normoXic conditions. After stroke, the balance of energy and redoX homeostasis is disrupted by ischemia [82]. An important function of glucose metabolism is maintaining a cytoreductive environment through oXidative phosphorylation, glycolysis and pentose phosphate pathways, by which the damaged brain tissue can be resistant to cerebral ischemia and hypoXia. HIF-1 enhances glucose uptake by neuronal cells through regulating glucose transport proteins, glycolytic enzymes and enhancing the glycolytic pathway [83,84]. In vitro OGD model of SH-SY5Y cells, knockdown of HIF-1α inhibited the key enzymes of glucose metabolism and the pentose phosphate pathway, such as glucose transporter-1, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, which were important in maintaining cellular redoX homeostasis. Therefore, the cells with HIF-1α knockdown were more sensitive to hypoXia and ischemia exposure than normal cells. Maintaining cellular redoX homeostasis is one of the [118–120]

6. Potential therapeutic drugs targeting HIF-1 in stroke

At present, the treatment strategies of stroke are mainly focused on improving cerebral blood circulation and neuroprotection. The drugs used in the treatment of stroke mainly include antiplatelet aggregation drugs, anticoagulant drugs, thrombolytic drugs, neuroprotective drugs, dependent activation of NF-κB and HIF-1α and reduces reactive oXygen species production
Inhibits HIF-1-VEGF-Src pathway and protects against cerebral ischemia/reperfusion injury vasodilators and anti-inflammatory drugs. However, the clinically application of these drugs is limited by side effects and narrow thera- peutic window. For example, tissue plasminogen activator (t-PA) can restore cerebral blood flow by thrombolysis in the early stage of stroke, but the effective treatment time window is only within 4.5 h after onset.
Over 4.5 h, the risk of hemorrhagic transformation increases by more than 10-fold, and the incidence and mortality of ischemic stroke by 10–40%, and only 3–5% of patients receive treatment [85]. In addition, the development and clinical transformation of neuroprotective agents have been a hot topic in the field of ischemic stroke treatment, but several neuroprotective agents have failed in clinical trials, which may be related with the choice of the treatment time window, the effective therapeutic concentrations in the ischemic brain tissue and the indi- vidual differences in patients. Therefore, drug development for the treatment of ischemic stroke is still a worldwide problem [86].

6.1. HIF-1 inhibitors

To date, a series of HIF-1 inhibitors have been reported. They affect the pathological process of stroke by interfering with the HIF-1α pathway to inactivate HIF-1.

6.1.1. 2-ME

2-MethoXyestradiol (2-ME), a natural metabolite of estradiol and 2- hydroXyestradiol, is a high-affinity agonist of the G-protein-coupled estrogen receptor. 2-ME binds to the colchicine binding site of micro- tubule and disrupts interstitial tumor microtubules [10], thereby affecting the localization of the HIF-1α mRNA complex to the microtu- bule cytoskeleton and inhibiting HIF-1α translation [87]. In a hypoXia-ischemic brain injury model of neonatal rats, 2-ME at different doses (1.5 mg/kg, 15 mg/kg and 150 mg/kg) was injected intraperito- neally 5 min after hypoXia. The results showed that 2-ME reduced infarct volume and brain edema with dose-dependent manner at 48 h after hypoXia. In contrast, injection with 2-ME at 3 h after hypoXia had no neuroprotective effect [4]. In vitro, 2-ME was applied at 8 h after hypoXia, increased neuron damage and decreased VEGF expression were observed. These results suggested that the application of 2-ME exerted different effects at different times after hypoXia [27].

6.1.2. YC-1

3-(5’-hydroXymethyl-2’-furanyl)-1-phenylmethylindazole (YC-1) was first discovered to have anti-platelet activity in 1994 [88]. In vitro experiments revealed that YC-1 reduced hypoXia-induced HIF-1α accu- mulation and the expression of HIF-1 target genes [89]. Another study found that YC-1 degraded HIF-1α independently of the oXygen and proteasome pathway, while the C-terminal of HIF-1α was sensitively degraded by YC-1 [90]. Additionally, YC-1 inhibits HIF-1α via the FIH-dependent CAD inactivation [91] and the PI3K/Akt/mTOR/4E-BP pathway, which regulates HIF-1α expression at the translational step [92]. In rat ischemic stroke model, it was found that the inhibition of HIF-1 and its downstream genes by pretreatment with YC-1 significantly increased mortality and infarct volume, but improved BBB disruption. This may be because VEGF induced by HIF-1 increased BBB perme- ability, while HIF-1-induced EPO and GLUT played a neuroprotective role in ischemic stroke [24]. Yeh et al. also found that in hypoXia/r- eoXygenation model of adult rat brain endothelial cells, YC-1 inhibited HIF-1α accumulation and VEGF production, thereby protecting the BBB [29]. In addition, our recent study indicated that pretreatment with YC-1 inhibited the expression of HIF-1, protected BBB integrity and alleviated t-PA-induced hemorrhagic transformation, which may be associated with inhibition of HMGB1/TLR4/NF-κB-mediated neutrophil infiltration [93].

6.1.3. Geldanamycin

Molecular chaperone Hsp90 interacts with HIF-1α to maintain its stability in the expression of HIF-1α. Geldanamycin (GA) is a natural Hsp90 inhibitor, which interferes with the function of Hsp90 to cause protein ubiquitination and degradation by the proteasome [94]. In renal cell carcinoma [95] and prostate cancer cell lines [96], GA induced HIF-1α degradation in a VHL-independent manner. In MCAO model, injection of GA into the lateral ventricle reduced infarct volume and brain edema, improved behavioral outcome [97]. Wen et al. also found that lateral ventricle injection with GA in MCAO model protected against focal ischemia, which may be related to the overexpression of Hsp90 [98].

6.2. HIF-1 stabilizer

6.2.1. PHD inhibitors

As previously mentioned, PHD plays an important role in HIF-1 degradation. Inhibition of PHD activity stabilizes HIF-1α, and induces the expression of VEGF and other genes. The hydroXylation reaction in PHD-catalyzed HIF-1 degradation requires Fe2+, 2-oXoglutarate (2-OG) and oXygen as reactants [99]. There are two pathways to inhibit PHD and stabilize HIF-1α. The first pathway is iron chelators, and the use of iron chelators can reduce the free divalent amount of iron, thus stabi- lizing HIF-1α. DeferoXamine (DFO) is a high-affinity iron chelator used to treat iron overload. Preclinical studies have shown that systemically administered DFO prevents and treats ischemic stroke and cerebral hemorrhage [100]. In addition, lipid-soluble ferrous iron chelator 2, 2′-dipyridyl (DP) has been reported to alleviate brain damage in different rodent models of cerebral ischemia and intracerebral hemor- rhage [101].
The use of divalent metal ions that compete with Fe2+ in PHD-mediated HIF-1 hydroXylation can also help stabilize HIF-1α. Cobalt chloride is the most common divalent metal ion used to inhibit PHD. It has found that HIF-1α and HIF-1β protein levels were significantly increased after injection of cobalt chloride and DFX into neonatal rats.
Compared with control group, pretreatment with cobalt chloride or DFX 24 h before stroke reduced the incidence of cerebral infarction by 75% or 56%, respectively [14].
The second pathway involves the use of 2-OG analogs to inhibit prolyl 4-hydroXylase [102]. 3,4-DHB is a 2-OG analog and has been shown to stabilize HIF-1α, activate HIF-1α-dependent gene expression and protect cortical neurons in cerebral ischemic model in vitro and in vivo [103]. In addition, there are some molecules that bind directly to the active site of PHDs to inhibit their activity, such as the PHD inhibitor FG-4497, which induces the HIF-1 signaling pathway and promotes neuronal survival. Pretreatment with FG-4497 improves the prognosis of ischemic stroke by increasing the expression of VEGF and EPO and preventing vascular leakage and edema formation, suggesting that FG-4497 may be a potential drug for the treatment of stroke [104].

6.2.2. Proteasome inhibitors

The ubiquitin-proteasome pathway is responsible for HIF-1 degra- dation, some proteasome inhibitors can stabilize HIF-1α. Badawi et al. reported that proteasome inhibitors increased HIF-1α stabilization and cell viability in OGD model of primary cortical neuron better than PHD inhibitors [105]. In a mouse model of transient intracranial cerebral artery occlusion, intraperitoneal injection of the proteasome inhibitor Bsc2118 after stroke exerted neuroprotective effects by decreasing infarction volume and promoting neurological recovery, and inhibition of HIF-1α reversed Bsc2118-induced neuroprotection [106]. Additionally, inhibition of immunoproteasome LMP2 in rats after MCAO could promote angiogenesis and facilitate neurological functional recovery by enhancing HIF-1α abundance [107].

6.2.3. Folic acid

Folic acid is a type of B vitamin naturally found in some foods. Clinical trials revealed that folic acid treatment could reduce the risk of first stroke in hypertensive males [108]. Meta-analyses and umbrella review also showed evidence for preventive benefits of folic acid for stroke [109,110]. Folic acid has been found to stabilize HIF-1 by binding to PHD2, FIH and VHL protein in molecular dynamics experiments. Further study in vitro OGD model of SH-SY5Y cells demonstrated that pretreatment with folic acid promoted cell survival after OGD injury, suggesting that the post-ischemic neuroprotective effect of folic acid may be partly attributed to its stabilizing effect on HIF-1α and pro-angiogenic properties [111].

6.3. Natural products

6.3.1. Berberine

Berberine is a major component of the antibacterial activity of Phellodendron. In a rat model of ischemia-reperfusion, berberine was found to promote angiogenesis and neurological function recovery by activating HIF-1/VEGF signaling pathway [112]. Pretreatment with berberine in MCAO model can activate endogenous neuroprotective mechanisms related to the S1P/HIF-1 pathway [113]. In cobalt chloride-induced PC12 cell injury model, pretreatment with berberine promoted PC12 cells survival and inhibited apoptosis by suppressing HIF-1α and the followed apoptotic pathway [114].

6.3.2. Ligustilide

Ligustilide is the main bioactive component of the rhizome of Chuanxiong, which has been widely used in the treatment of cerebro- vascular diseases. It has been reported that ligustilide plays neuro- protective effect by promoting angiogenesis after cerebral ischemia [115]. Wu et al. showed that ligustilide could inhibit the HIF-1α/VEGF pathway and aquaporin-4 to reduce OGD-induced BBB permeability, thus exerting a protective effect on cerebral vasculature and improving stroke prognosis [116]. In OGD/R-treated SH-SY5Y cells, ligustilide reduced the increase of iron partly by inhibiting HIF-1α-induced in- crease of transferrin-bound iron uptake and iron accumulation, decreasing iron-mediated free radical formation, oXidative stress and apoptosis [117].

6.3.3. Andrographolide

Andrographolide is the main active ingredient of Andrographis pan- iculata. Andrographolide possesses diverse biological effects including regulation of cytochrome c/caspase-3 signaling in cortical ischemic penumbra [138].
In conclusion, the effects of the above natural products on stroke are all related to the regulation of HIF-1-related pathways. Through structure-activity relationship analysis, we have found that these com- pounds all have ring structures with molecular weight under 500 g/mol and less than 10 hydrogen bond acceptors, which may be the main active groups. In addition, most of them have high lipid-water partition coefficient and may pass through the BBB, thereby exerting an anti- cerebral ischemia effect. Therefore, natural products may have better development prospects.

6.4. Other drugs

Minocycline is a broad-spectrum tetracyclic antibiotic and provides anti-inflammatory, anti-oXidative, and anti-apoptotic benefits. Several studies have reported the effects of minocycline for the treatment of anti-inflammatory, antioXidant, and antineoplastic properties [118, stroke, multiple sclerosis, and cancers [139–141]. In cancer, minocy-119]. In MCAO model, treatment with andrographolide 1 h after ischemia could reduce ischemia-reperfusion injury in mice. In combi- nation with in vitro microglia experiments, andrographolide was found to reduce the expression of iNOS and NOX2 by limiting PI3K/AKT-dependent activation of NF-κB and HIF-1α, thereby inhibit- ing the production of ROS, alleviating neurological deficits and infarct volume [120].

6.3.4. Salvianolic acid A

Salvianolic acid A (SAA) is a component of the root of Salvia mil- tiorrhiza, which has been used for the treatment of cerebrovascular diseases [121]. Some studies have revealed that SAA can protect against cerebral ischemia/reperfusion injury [122]. In an autologous thrombus stroke model, pretreatment with SAA (10 mg/kg) significantly improved the neurological deficits, BBB disruption, and vascular endothelial dysfunction, which may be associated with the inhibition of HIF-1-VEGF-Src signaling pathway [123]. Xie et al. found that in human umbilical vein endothelial cells, pretreatment with SAA could protect against hypoXia-induced endothelial ER stress and apoptosis via inhib- iting recruitment of HIF-1α to very low-density lipoprotein receptor gene promoter [124].

6.3.5. Catalpol

Catalpol is a compound from the herbal drug Rehmannia glutinosa, which has been reported to exert protective effect in ischemic stroke [125]. Previous studies have shown that catalpol promotes angiogenesis
in infarcted regions of the brain and alleviates brain capillary endo- thelial cell edema [126–128]. Wang et al. found that in cerebral ischemic rats and OGD-exposed brain microvascular endothelial cells, catalpol reduced pathological changes during ischemia and promoted angiogenesis both in vivo and in vitro. Further study indicated that the HIF-1α/VEGF pathway was activated by catalpol both in the brains and brain microvascular endothelial cells after ischemia, suggesting that catalpol protected vascular structure and promoted angiogenesis in ce- rebral ischemia via HIF-1α/VEGF pathway [129].

6.3.6. Other natural drugs

Bu Yang Huan Wu (BYHW) decoction and Houshiheisan are traditional Chinese medicine formulas which has been used to treat acute stroke [130–133]. BYHW protected against cerebral ischemia in MCAO rats through inhibiting the activation of the HIF-1α/VEGF pathway [134]. In rat pMCAO model, Houshiheisan promoted angiogenesis via HIF-1α/VEGF and SDF-1/CXCR4 pathways for stroke recovery [135]. Angelica sinensis extract from the dry root of Angelica sinensis, plays neuroprotective effect on cerebral ischemia [136,137]. Cheng et al. showed that the angiogenesis and anti-apoptotic effects of Angelica sinensis extract can be attributed to the activation of p38/HIF-1/VEGF-A/vWF and p38/HIF-1/VEGF-A/p-Bad-related cline has been found to accelerate HIF-1α degradation and inhibit angiogenesis in vitro [142,143]. In stroke, minocycline inhibits HIF-1α-mediated high vascular permeability and protects the BBB integrity via the SIRT-3/PHD-2 pathway, making it a potential agent for the prevention and treatment of stroke [144].
Dexmedetomidine is a potent α2-adrenoceptor agonist and has been shown to reduce ischemia/reperfusion injury [145,146]. In a rat model of ischemia/reperfusion, dexmedetomidine was found to inhibit neuronal apoptosis by inhibiting the HIF-1α pathway [147].
Glycine, as a non-essential amino acid, is an inhibitory neurotransmitter in the brain and plays neuroprotective effect in cerebral ischemia [148]. Treatment with glycine reduces brain damage and neuronal death in ischemic stroke [149]. Clinical trials suggest that glycine treatment improves the prognosis of patients with ischemic stroke [150]. Glycine has been found to indirectly reduce ischemia-induced neuronal death by regulating microglia polarization and promoting anti-inflammatory effects through the PTEN/AKT/NF-κB/HIF-1α signaling pathway [151].
N-acetylcysteine (NAC), a commonly used antioXidant, can effec- tively protect the brain against ischemic injury [152,153]. In a mouse ischemia-reperfusion model, pretreatment with NAC significantly reduced brain infarct volume. Further analysis revealed that NAC increased the expression of Hsp90 and the interaction between Hsp90 and HIF-1α in ischemic brain, thereby enhancing the stability of HIF-1α to exert neuroprotective effects [154].

7. Summary

After stroke, HIF-1 is activated and plays important roles in the pathological process of stroke by regulating its target genes, involving multi-mechanisms such as inflammation, apoptosis, autophagy, oXida- tive stress and energy metabolism. The roles of HIF-1 in stroke depend on the duration and the degree of ischemia. In the early stage of ischemic stroke, inhibition of HIF-1α reduces brain damage, brain edema and apoptosis, mainly associated with the suppression of pro-apoptotic genes. However, in the recovery phase of stroke, pro-apoptotic genes are not significantly elevated, whereas angiogenic genes continue to be highly expressed [155]. Therefore, inhibition of HIF-1α promotes neuronal damage [27]. Furthermore, stabilization of HIF-1 prior to cerebral ischemia is neuroprotective, whereas stabilization of HIF-1 immediately after ischemia is detrimental [156].
To date, drug development targeting HIF-1 has attracted more attention. However, there are no approved drugs targeting HIF-1 for clinical application. Among these potential drugs, natural products may have greater prospects due to its multiple pharmacological activities and safety. With the further development on the roles and regulatory mechanisms of HIF-1, HIF-1 is expected to be a potential target for stroke treatment. Therefore, resolving when and what interventions for HIF-1 to take during stroke will provide novel therapeutic strategies for ischemic stroke.


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