Insights to Ang/Tie signaling pathway: another rosy dawn for treating retinal and choroidal vascular diseases

Abstract

Retinal neurovascular unit (NVU) is a multi-cellular structure that consists of the functional coupling between neural tissue and vascular system. Disrupted NVU will result in the occurrence of retinal and choroidal vascular diseases, which are characterized by the development of neovascularization, increased vascular permeability, and inflammation. This pathological entity mainly includes neovascular age-related macular degeneration (neovascular-AMD), diabetic retinopathy (DR) retinal vein occlusion (RVO), and retinopathy of prematurity (ROP). Emerging evidences suggest that the angopoietin/tyrosine kinase with immunoglobulin and epidermal growth factor homology domains (Ang/Tie) signaling pathway is essential for the development of retinal and choroidal vascular. Tie receptors and their downstream pathways play a key role in modulating the vascular development, vascular stability, remodeling and angiogenesis. Angiopoietin 1 (Ang1) is a natural agonist of Tie2 receptor, which can promote vascular stability. On the other hand, angiopoietin 2 (Ang2) is an antagonist of Tie2 receptor that causes vascular instability. Currently, agents targeting the Ang/Tie signaling pathway have been used to inhibit neovascularization and vascular leakage in neovascular-AMD and DR animal models. Particularly, the AKB-9778 and Faricimab have shown promising efficacy in improving visual acuity in patients with neovascular-AMD and DR. These experimental and clinical evidences suggest that activation of Ang/Tie signaling pathway can inhibit the vascular permeability, neovascularization, thereby maintaining the normal function and structure of NVU. This review seeks to introduce the versatile functions and elucidate the modulatory mechanisms of Ang/Tie signaling pathway. Recent pharmacologic therapies targeting this pathway are also elaborated and summarized. Further translation of these findings may afford a new therapeutic strategy from bench to bedside.

Introduction

Retinal and choroidal vascular diseases are characterized by the development of neovascularization, increased vascular permeability, and inflammatory reactions at the posterior segment of eyeball [1, 2]. This pathological entity is composed of a cluster of degenerative diseases, including the neovascular age-related macular degeneration (neovascular-AMD), diabetic retinopathy (DR), retinal vein occlusion (RVO) and retinopathy of prematurity (ROP) [1,2,3]. Typically, these diseases are initiated by the development of neovascularization, which subsequently leading to the instability of vascular system. Vascular endothelial growth factor A (VEGFA) plays a significant role in regulating the neovascular formation and growth under physiological or pathological conditions [4]. In clinical practice, anti-VEGF therapy has dramatically improved the visual prognosis of these patients with neovascular-AMD, DR/diabetic retinal edema (DME), RVO and ROP [3, 5,6,7]. Nevertheless, anti-VEGF therapy has several practical limitations such as high economic burden and unwanted adverse effects [2, 8]. Some anti-VEGF drugs have short half-lives, so them require frequent administrations to control the diseases [9]. The safety of intraocular injection is controversial, and frequent administration can cause endophthalmitis, retinal detachment, thrombosis and so on [2]. These limitations have driven further researches into searching for alternative strategies to treat the aberrant angiogenesis. For instance, several therapeutics are designed to target the angopoietin/tyrosine kinase with immunoglobulin and epidermal growth factor homology domains (Ang/Tie) signaling pathway.

The Ang/Tie signaling pathway is mainly composed of the Tie receptors and angiopoietin ligands. This pathway plays a critical role in modulating the vascular development, vascular stability, vascular remodeling and angiogenesis [10,11,12,13]. Tie receptors, including Tie1 and Tie2, are mainly expressed in the endothelial cells (ECs) of blood vessels and lymphatic vessels [13]. Tie1 is considered as an orphan receptor which regulates the function of Tie2 [14]. Tie2 exerts its effects by binding angiopoietin ligands. Angiopoietin 1 (Ang1) can activate the Tie2 receptor and maintain the vascular stability [15]. On the other hand, angiopoietin 2 (Ang2) can act as a context-dependent antagonist or weak agonist of Tie2 [16]. Under inflammatory conditions, the Ang2 will switch from an agonist to an antagonist for Tie2, and the transformation is correlated with the cleavage of the Tie1 extracellular domain [14]. Besides, the expression of Ang2 is up-regulated under inflammatory conditions [17]. Another key modulator is vascular endothelial protein tyrosine phosphatase (VE-PTP), which dephosphorylates Tie2 and negatively regulates the Ang/Tie signaling pathway [18]. Preclinical evidences have shown that targeting Ang/Tie signaling can alleviate the neovascularization, restore the vascular stability and inhibit the inflammatory response in retinal and choroidal diseases [19,20,21].

In this review, we summarize recent findings of the Ang/Tie2 pathway in terms of physiological and pathophysiological conditions. In particular, some novel drug candidates (such as AKB-9778, Faricimab and ARP-1536) targeting the Ang/Tie pathway are introduced in this paper. Further refinements of these findings may shed light into the discovery of an effective medication for retinal and choroidal vascular diseases, providing accurate and personalized therapies for different patients.

Physiology of retinal and choroidal vasculature

As a highly metabolic neural tissue, the retina requires an abundant, extensive and stable blood supply to maintain the normal physiologic activity and cell survival. This need is met by the dual blood supply, including retinal vessels and choroidal circulation [2]. Inner 2/3 part of retina is nourished by the central retinal vessels, which perforate the center of optic disc. Central retinal artery gradually branches and penetrates into the inner nuclear layer (INL), forming a rich capillary network that nourishes the corresponding retinal areas. The outer 1/3 part of retina is a transparent avascular tissue, and the nutrients and oxygen required for this region are provided by the neighboring choroidal circulation. Several types of highly selective biological barriers exist in eyeball, including the blood-aqueous barrier (BAB), blood-retinal barrier (BRB) and corneal barrier [22]. These barriers provide a stable metabolic basis for retinal cells to maintain their physiological function. BRB is divided into inner blood-retinal barrier (iBRB) and outer blood-retinal barrier (oBRB) [22]. The iBRB lies on the basement membrane which is covered with processes of astrocytes and müller cells [23]. IBRB is composed of continuous capillary ECs. Tight junctions between ECs and internal vesicles are fundamental for achieving material transport and limiting permeability [2]. IBRB can regulate the transport of blood components to maintain the homeostasis of retinal cells. There are also some pericytes on the basal plate and in contact with ECs. Astrocytes, müller cells and pericytes can modulate the activity of iBRB by transmitting signals from the retinal neurons to ECs [23]. The oBRB consists of retinal pigment epithelial (RPE) cells and tight junctions between them apical sides [23, 24]. Since the oBRB has lower pericytes coverage compared with iBRB, it is able to regulate nutrients supply and eliminate metabolic waste more efficiently. Normal vascular structure is essential for maintaining the normal physiological function of retina.

Retinal neurovascular unit

Retinal NVU is a multi-cellular structure that is comprised of neurons, RPE cells, macroglia (müller cells and astrocytes), microglia, vascular ECs and mural cells (pericytes and vascular smooth muscle cells) [25]. Each component of this basic unit is closely associated with each other, and they collectively modulating the retinal energy metabolism and vasculature physiologic function. There are functional couplings among retinal neurons, which are able to transmit electrochemical signals to the brain with the support of glial cells and vascular tissue [26]. Müller cell, the major glial cell type in the NVU, is in close contact with a variety of retinal cells due to their high apical-basal structure. With their particular physiological structure, müller cells are capable of nourishing neurons, transmitting information as well as stabilizing the integrity of the BRB and neurons [27]. Astrocytes form complex networks around neuronal axons and vessels, providing mechanical and metabolic support for neurons [28]. Microglia, as the main immune-resident cell in retina, plays an essential role in immune regulation and homeostasis maintenance [29, 30]. Pericytes and vascular smooth muscle cells are located on the vascular basement membrane and cooperate with ECs to maintain vascular stability. When the structure or function of retinal NVU is disrupted, microangiopathy, oxidative stress and nerve damage will occur, giving rise to retinal degenerative diseases [31].

Mechanism of retinal and choroidal vascular diseases

Retinal and choroidal vascular diseases, mainly including neovascular-AMD, DR, RVO and ROP, have become the leading cause of blindness in older adults and children, which are characterized primarily by neovascularization. These eye diseases can be divided into two stages. Firstly, the vascular occlusion developed from the original pathological conditions will form corresponding non-perfusion area. Subsequently, the ischemic and hypoxic environment will up-regulate hypoxia inducible factor-1 (HIF-1) and various hypoxia-regulated gene products such as VEGF, Ang2 and VE-PTP, which can lead to abnormal neovascularization. Ocular neovascularization mainly includes two types depending on their occurrence site: retinal neovascularization (RNV) and choroidal neovascularization (CNV) (Fig. 1). Neovascular that grow from the retina into the vitreous cavity are called RNV. Once the pathological process of RNV is initiated, the vascular permeability will increase drastically due to the lack of tight junctions between ECs. Plasma will penetrate into the vitreous cavity and retinal tissue, thereby disrupting the balance of endogenous environment. RNV causes vitreous hemorrhage or tractional retinal detachment on account of vitreous contraction. The visual acuity of patients will be severely impaired when the retinal detachment or leaking fluid affects the macula region [32]. The RNV related diseases mainly include DR, RVO and ROP [2, 33]. CNV is defined as the process that choroidal neovascular penetrates through Bruch’s membrane into the subretinal RPE or subretinal space [34]. The RPE cells will be exposed to exudative fluid, hypoxia stimulus, oxidative stress and inflammation [1, 2]. Ultimately, CNV results in macular edema and irreversible visual impairments. The most common CNV related disease is neovascular-AMD [1].

Fig. 1

Retinal and choroidal vasculatures in the healthy eye (left), the eye with diabetic retinopathy (middle) and the eye with neovascular AMD (right). In diabetic retinopathy, the formation of retinal neovessels is accompanied by vascular leakage, which disrupts the nerve layer structure. Choroidal neovessels grow through Bruch’s membrane into the retinal pigment epithelium or subretinal space, causing destruction of the retinal pigment epithelial cells and photoreceptors. Created with BioRender.com

Age-related macular degeneration

AMD is a degenerative retinopathy that characterized by the accumulation of drusen deposits, vascular inflammation, RPE cell senescence, along with progressive photoreceptor demise. AMD causes irreversible macular degeneration in the elder population and acts as the principle cause of blindness in developed countries [35]. It is predicted that more than 288 million people worldwide will be affected by AMD by 2040 [36]. As a multifactorial disease, aging, genetic susceptibility and adverse environmental factors are related to the pathogenesis of AMD [37]. Typically, AMD is classified as the non-neovascular AMD (“dry” AMD) and neovascular-AMD (“wet” AMD) phenotypes. The incidence of neovascular-AMD is low, but its progress rate is extremely rapid. Neovascular-AMD is characterized by the CNV formation and RPE detachment. During the pathological process of neovascular-AMD, the RPE cells would be exposed to hypoxia stimulus, oxidative stress and inflammation when the CNV growing into the RPE layer. CNV results in abnormal exudation of fluid that accumulates in the macular area and leaves destructive scarring [34, 38, 39]. These disturbances eventually cause macular edema and severe visual impairments in neovascular-AMD patients. Additionally, excessive extracellular matrix (ECM) proteins can also destroy the retinal architecture and trigger neurodegeneration [40]. Notably, the VEGF expression is up-regulated under hypoxic conditions, and acts as the pathogenic factor in stimulating CNV in AMD patients [41]. Current existed treatments for AMD mainly include laser therapy, surgery and intravitreal injection of anti-VEGF drugs such as ranibizumab, pegaptanib and bevacizumab [37].

Diabetic retinopathy

DR and DME are complex complications of diabetes mellitus, and act as the leading cause of vision loss in the working age population. An increasing number of DR patients have been found to have microvascular and neurological abnormalities [42]. The damaged nerves and vessels can affect severely the retina’s ability to convert light energy into electrical signals, leading to adverse effects on vision and ultimately blindness. The loss of retinal pericytes, changes of microvascular structure and thickening of capillary basement membrane (deposition of ECM components) are recognized as typical hallmarks of DR at early stage [42]. After pericytes demise, the loss of ECs will leave behind acellular capillaries, thus causing the previously perfused retina region in a state of ischemia and hypoxia [43]. The secretion of growth factors and proinflammatory factors such as VEGF will increase under hypoxic condition [2]. The loosened EC-EC junctions of neovascularization can increase vascular permeability, resulting in the accumulation of extracellular fluid in the macula region [42]. In particular, the abnormal localization of inward rectifying potassium channel (Kir4.1) at the capillary interface of müller cell terminal foot in the retina of diabetic rat can lead to impaired iBRB and water metabolism, which will aggravate the disruption of vascular integrity [44]. Furthermore, hyperglycemia causes changes in NVU and mitigates the production of several important neurotrophic factors, such as pigment epithelial growth factor (PEGF), somatostatin, corstistatin and nerve growth factor (NGF) [45]. Current treatments for DR include the vitrectomy, laser photocoagulation and pharmacological therapies. Among them, anti-VEGF drugs are considered as the first choice for the DR treatment and have shown certain efficacy in clinical practice [6].

Retinal vein occlusion

RVO is a vascular occlusive disease that caused by the thrombus occlusion of retinal vein system [46]. Its occurrence is closely associated with the atherosclerosis, as the diseased retinal artery will compress the adjacent vein to cause RVO [47]. Generally, the RVO can be classified into three categories according to the type of vein involved. If the occlusion occurs in the central retinal vein, it is defined as the central retinal vein occlusion (CRVO), which is usually characterized by the macular oedema, accompanied by retinal hemorrhage, optic nerve oedema, cotton wool spots and capillary nonperfusion [47]. The second category is the hemi-retinal vein occlusion (HRVO), a vein embolism occurs at the main bifurcation of central retinal vein. HRVO can cause retinal hemorrhage and affect the retinal hemispheres. The last category is branch retinal vein occlusion (BRVO), which is caused by thrombosis of branch veins such as the arteriovenous junction [48]. Clinical data indicates that the prevalence of all the RVO types increases with age [49]. After embolization, the retinal vein thrombosis can cause massive capillary nonperfusion, leading to up-regulation of inflammatory cytokines such as VEGF, TNF-α and IL-1β [50]. VEGF will promote the formation of neovascular and increase capillary permeability, thereby inducing fluid leakage, and causing macula edema. Traction retinal detachment may occur in these severe cases of RVO [2, 47]. Additionally, the retinal nerve fiber layer (RNFL) and ganglion cell inner plexiform layer (GCIPL) become thinner in some BRVO patients [51]. Therapeutically, anti-VEGF drugs such as ranibizumab, bevacizumab and aflibercept are delivered into the vitreous body of RVO patients to treat the macular oedema [52].

Retinopathy of prematurity

ROP, a retinal vascular disorder that always occurs in the premature infants, has become the leading cause of visual impairment even blindness in children. This disease is caused by delayed retinal vascular development and abnormal proliferation of neovascularization after premature birth, which divides the pathogenesis of ROP into phase 1 and 2 [33]. Firstly, most premature babies who are earlier than 36 weeks or weigh less than 1.5 kg require supplemental oxygen to maintain respiratory support after birth. Hyperoxia-induced retinal vasoattenuation will lead to vasoconstriction and occlusion of developing retinal blood vessels in phase 1. In phase 2, hypoxia-induced subsequently up-regulation of VEGF promotes the abnormal formation and development of RNV, which can lead to subsequent vitreoretinal traction, retinal detachment, and finally make permanent visual loss in some severe conditions [53]. The treatment of ROP begins with timely screening of preterm infants, followed by pharmacological interventions such as anti-VEGF therapy or non-pharmacological interventions such as laser therapy, cryotherapy and vitrectomy based on the severity of ROP. With the deepening of research on the pathogenesis, the elimination of abnormal RNV has become the main therapeutic purpose. Anti-VEGF drugs have received more and more attention, but it has been reported that anti-VEGF treatment of ROP can inhibit the normal structural development and functional maturation of retinal neurons [54]. Therefore, there is still an urgent need for a safer treatment for ROP.

VEGF

VEGF is a family of homodimeric vasoactive glycoproteins that modulate angiogenesis, including VEGFA, VEGFB, VEGFC, VEGFD, VEGFE, and placental growth factor (PIGF) [55]. Among them, VEGFA, commonly known as VEGF, regulates the formation and development of blood vessels by binding to vascular endothelial growth factor receptor (VEGFR), a specific receptor expressed on ECs. Thus far, two main types of VEGFRs have been identified: VEGFR-1 (fms-like tyrosine kinase, FLT-1) and VEGFR-2 (kinase insert domain containing receptor, KDR) [56]. In particular, VEGFR-2 acts as the major mediator of VEGF-driven responses in ECs [57]. The dimer of VEGF and VEGFR-2 can activate proliferation pathways, promote DNA synthesis, and initiate ECs division [57].

The function of VEGF

Under physiological conditions, the main function of VEGF is to induce angiogenesis. It has been shown that VEGF is an endothelial cell-specific mitogen that induces angiogenesis [58]. VEGF also acts as a survival factor for ECs mediated by anti-apoptotic proteins B-cell lymphoma-2 (Bcl-2) as well as Bcl2 associated protein A1 in vivo [59]. In the past decades, the proangiogenic effect of VEGF in the pathogenesis of some blinding ocular diseases has been confirmed. Alterations in local oxygen concentration could affect the dynamic expressions of VEGF [60]. That is, under pathological conditions, retinal vascular damages or vascular obstruction can lead to retinal nonperfusion, which induces tissue hypoxia and subsequently increases the expression of HIF-1. HIF-1 enables the up-regulation of hypoxia-regulated gene products such as VEGF [60]. Eventually, the abnormal angiogenesis and blood leakage (increased vascular permeability) mediated by VEGF will cause a series of functional impairments. Accumulating evidences suggest that VEGF is expressed in the retinal tissue and intraocular aqueous of patients with ischemic diseases [61, 62]. The inhibitory effect of VEGF antagonists on NV in human eyes further confirmed the role of VEGF in promoting angiogenesis [3].

The anti-VEGF therapy and its drawback

Pioneering studies have proposed that “anti-angiogenesis” may act as a therapeutic strategy for neovascular diseases [63, 64]. The proangiogenic effect of VEGF can be blocked by the antagonistic manipulations targeting any stage of the signal transduction. Typically, therapies involve pharmacologic down-regulation of VEGF expression or inhibition of VEGF/VEGFR signaling by means of antibodies to this pathway. A cluster of experimental and clinical evidences have shown that the monoclonal antibodies of VEGF and VEGFR exert inhibitive effect on neovascularization [65,66,67]. Pegaptanib and ranibizumab are the earliest VEGF antagonists used to treat neovascular-AMD [68, 69]. Bevacizumab is a recombinant anti-VEGF monoclonal antibody that was originally developed to inhibit neovascularization in tumors, and later found a role in the treatment of CNV [70]. At present, anti-VEGF drugs have been developed into the primary therapy for neovascular diseases in ophthalmologic practice.

Although anti-VEGF therapy can alleviate the macular edema and improve the visual acuity of patients, some problems should be addressed appropriately during its application. These monoclonal and polyclonal drugs have short half-lives and require frequent injections to maintain the effective concentration, which can place a significant psychological and financial burden on patients [9]. Especially, the safety of the intraocular injections is still controversial. Adverse effects such as endophthalmitis, retinal detachment, RPE atrophy, ocular hypertension, thrombus and traumatic cataract, are proven to be associated with the intraocular injection procedures [2]. Therefore, noninvasive therapeutics or drug formulations with sustained release capabilities are in emergent need. Additionally, some patients show tolerance to the anti-VEGF therapy [2]. Notably, the growth of the CNV can nourish these damaged RPE cells within retinas in some cases, while inhibiting the neovascularization will exacerbate the death of RPE cells [71]. Therefore, reducing neovascularization with anti-VEGF therapy is not always beneficial. An alternative therapeutic strategy that stabilizing neovascularization and maintaining the homeostasis of NVU may be more prospective and effective.

Ang/Tie signaling pathway

Ang/Tie signaling pathways plays a critical role in vascular development, vascular remodeling and angiogenesis. There is a close coupling between neural tissue and vascular system in NVU, which is the basic functional unit of the retina. Therefore, maintaining the physiological structure and function of vascular ECs and pericytes is crucial for the homeostasis of NVU. Downstream pathways activated by Tie2 receptors can stabilize the endothelium and reduce the loss of pericytes, thus contributing to maintain physiological function of vascular system in NVU [10].

Composition of Ang/Tie signaling pathways

The Ang/Tie signaling pathway is mainly composed of the Tie receptors and their angiopoietin ligands. Tie is a family of receptor tyrosine kinases (RTKs) with immunoglobulin and epidermal growth factor homeodomains, including Tie1 and Tie2 [72]. Tie1 is encoded by TIE, while Tie2 is encoded by TEK. Both Tie1 and Tie2 are mainly expressed in the ECs of vascular and lymphatic vessels [13]. In addition, Tie2 is expressed in some cancer cells and hematopoietic cells such as macrophages [73, 74]. Tie1 acts as orphan receptor which does not bind directly to angiopoietin. Conversely, Tie2 exerts its effects by binding to angiopoietin ligands. Tie receptors are essential for vascular development, as the inactivation of Tie signaling is embryonic lethal, and leading to severe angiogenic defects [75]. Recent studies show that Ang /Tie signaling can serve as potential therapeutic targets for diseases with vascular dysfunction.

Angiopoietin is a family of vascular growth factors that can modulate the growth of vessels and stabilize the NVU, mainly including Ang1, Ang2, Ang3 and Ang4 [15, 16, 76]. The major ligands of the Tie2 receptor are Ang1 and Ang2, which can exert opposing effects on vascular stability and integrity. As an agonist of Tie2, Ang1 is mainly secreted by pericytes, astrocytes, mesenchymal cells and platelets [10, 77,78,79]. It acts in a paracrine manner to stabilize vessels by mediating Tie2 phosphorylation and activating the downstream signaling pathways [10, 15]. Ang2 is an environmentally dependent antagonist or weak agonist of Tie2 [16]. It is mainly produced by ECs and is stored in the intracellular Weibel-Palade bodies [80]. The Ang2 expression is up-regulated under pathological conditions such as hypoxia, inflammation, hyperglycemia and oxidative stress [17, 81, 82]. Ang2 usually acts as a competitive antagonist of Tie2, thereby disrupting the junctions between ECs and increasing the permeability of vessels [81, 82]. Ang2 also induces pericytes apoptosis via α3β1 integrin signaling in the early stage of DR [83]. However, high concentrations of Ang2 can activate the Tie2 receptor under the premise of Ang1 deficiency [16]. Moreover, like Ang2, VE-PTP is also up-regulated during hypoxia, which mainly inhibits the activation of Tie2 receptor as well as its downstream signaling pathways by mediating the dephosphorylation of Tie2 [18]. Thus far, we are still lack of in-depth understanding on the Ang3 and Ang4. Ang3 and Ang4 are two novel angiopoietins cloned from mouse and human cDNA libraries, respectively. Ang3 can be induced by hypoxia and exerts an antagonistic effect of Tie2 in human ECs [76]. While mouse Ang3 acts as an agonist of Tie2 in its own species. Ang4, the human homologue of mouse Ang3, is expressed in a specific sub-population of astrocytes at the site where venous morphogenesis occurs. Animal experiments have found that Ang4 can mediate vein-specific development and NVU remodeling [76, 84]. Currently, most of the researches focus on the Ang1 and Ang2 mediated effects on neovascularization.

Modulating mechanisms of Ang/Tie signaling pathways

The disorder of Ang/Tie signaling pathway will contribute to the occurrence of vascular pathology. Many vascular diseases, including neovascular-AMD, DR, RVO, ROP and diabetic vascular complications, are closely correlated with the abnormalities in this pathway [85, 86]. The Ang/Tie signaling pathway consists of diverse components, and each part undertakes specific function. Tie1 has long been considered an orphan receptor without natural ligand [87]. Tie1 exerts its bioactive effects by forming a heterodimer with Tie2 [88, 89]. In ECs, Tie1 is phosphorylated in a Tie2-dependent manner upon the stimulation of angiopoietin, and subsequently activates the downstream phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling [87, 88]. Activation of the downstream signaling pathway would help promote the vascular ECs survival and maintain the NVU integrity [88]. Tie1 can also regulate the expression and function of Tie2 by forming a Tie1-Tie2 heterodimer [89]. Notably, a recent study shows that Tie1 is able to regulate Tie2 responsiveness paradoxically, namely, inhibiting/maintaining Tie2 activation. On one hand, the formation of Tie1-Tie2 heterodimer restricts the recognition and binding of Tie2 to ligands, thereby inhibiting the activation of Tie2 [89]. Experiments with Tie1-deficient mice have shown that silencing Tie1 enhances the angiopoietin-induced phosphorylation of Tie2 [89]. On the other hand, it has been shown that upon Ang1 stimulation, Tie2 translocates and co-localizes with Tie1 at EC-EC junctions, preventing the rapid internalization of Tie2 [90, 91]. Tie1 can also maintain the Tie2 activation by activating PI3K/Akt [88]. These findings highlight that Tie1 is implicated in the regulation mechanism of Tie2 function. Recent experiments have pointed out that the Tie1 mediated effect on Tie2 is environmentally dependent. Tie1 inhibits the activation of Tie2 in the endothelial tip cells, but maintains the activation of Tie2 by co-localization in stalk cells [75]. Moreover, the Tie2 signaling predominates in quiescent endothelial phalanx cells due to the low expression of Tie1 [75]. Tie1 can also regulate the activity of angiopoietin. Ang1 can induce the dissociation of Tie2 from the Tie1-Tie2 dimer, which is recognized as a feature that distinguishes Ang1 from Ang2 in terms of function [89]. Tie1 is also required for Ang2 to activate Tie2, which may be attributed to the ability of Tie1 to maintain Tie2 activity. Moreover, Ang2 has been shown to switch from a weak Tie2 agonist to an antagonist of Tie2 upon the cleavage of Tie1 ectodomain during inflammation. This leads to disruption of EC-EC junctions and leakage of plasma [14]. The extracellular domains of both Tie1 and Tie2 can split and shed from the cell surface, limiting the number and availability of the Tie2 receptor [91]. The exfoliated Tie2 can also bind to extracellular ligands, which is called “ligand traps” [92, 93]. The formation of this trap regulates Tie2 signaling in a concentration-dependent manner, thereby inhibiting both Tie2 activation and Tie2 shedding [91, 92]. In addition, recent studies have shown that leukocyte cell-derived chemotaxin 2 (LECT2) is a specific functional ligand of Tie1 receptor which can exert anti-angiogenic effect both in vitro and in vivo [94]. Further molecular studies are necessary to elucidate the exact role of Tie1 in the Ang/Tie signaling pathway.

Tie2 is as a tyrosine kinase receptor of angiopoietin that plays a central role in regulating vascular homeostasis [10]. Diverse downstream signal pathways are under the control of this receptor (Fig. 2). Ang1 is a natural agonist of Tie2, mainly expressed and synthesized in pericytes and smooth muscle cells adjacent to the vascular endothelium [10, 92]. It binds to Tie2 extracellular domain on ECs in a paracrine manner [10]. This allows the Tie2 receptor to autophosphorylate and activates a series of downstream pathways. In resting vessels, Tie2 signaling is able to activate the PI3K/Akt pathway [95, 96]. Subsequently, Akt promotes the ECs survival and vascular stabilization by phosphorylating forkhead box protein O1 (FOXO1), leading to its nuclear exclusion and down-regulation of FOXO1 targeted genes such as ang2 [95,96,97]. Akt can also activate the endothelial nitric oxide synthase (eNOS) and survivin to promote vasodilation and cell survival [10, 95]. Additionally, Akt plays an anti-apoptotic role by inhibiting the mediators such as caspase-9 and Recombinant Bcl2 Associated Death Promoter (BAD) [10]. A recent study shows that Tie2 promotes the ECs migration, tube formation, sprouting and survival. The activation of extracellular regulated protein kinase (ERK) pathway also induces the proliferation and migration of ECs, thereby contributing to angiogenesis [90]. Interestingly, the activation of Akt or ERK pathways varies under different conditions. It relies on the spatial localization of Tie2, which in turn depends on the presence or absence of EC-EC junctions. In quiescent ECs, Ang1 induces the relocalization of Tie2 to cell junctions, allowing Tie2 to form a homotypic Tie2-Tie2 trans-associated complex together with VE-PTP [90]. At this stage, the Akt pathway is preferentially activated to conduct cell survival signals [90]. In motile/non-contact ECs, Tie2 is cis-associated at cell-ECM contacts upon Ang1 induction [90]. ERK is preferentially activated to induce ECs migration and proliferation. Ang1 can modulate the ECs permeability by activating Tie2. Tie2 is capable of stabilizing vessels by activating the GTPase signaling pathways such as IQ domain GTPase-activating protein 1/Ras-related C3 botulinum toxin substrate 1 (IQGAP1/RAC1) and Ras-related protein (RAP1)/RAC1 [20]. For instance, Tie2 can increase the combination of IQGAP1 and RAC1-GTP, which will keep RAC1 in an active GTP-binding state. RAC1 is able to dephosphorylate myosin light chain 2 (MLC2) and mitigates actomyosin contraction [20, 98]. The activation of RAC1-mediated pathway is instrumental for stabilizing the actin cytoskeleton and reducing ECs permeability, and maintaining the integrity of endothelial barrier [98]. The non-receptor tyr kinase Src is responsible for linking VEGF receptors to the vascular endothelial-cadherin (VEC) internalization pathway [99]. Src induces phosphorylation and internalization of VEC after it is activated by VEGF, thereby disrupting EC-EC junctions and increasing vascular permeability. The mammalian clear body (mDia), as a downstream target of the small GTPase-RhoA, can sequester Src and then prevent it from disrupting EC-EC junctions [100]. RhoA is precisely activated by Tie2 signaling to exert these beneficial effects [100]. Additionally, the activated Tie2 intracellular domain can inhibit the activation of NF-κB by interacting with A20 binding inhibitor of NF-κB activation-2 (ABIN-2), thereby mitigating the expressions of related inflammatory genes such as intracellular cell adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and E-selectin genes [21, 101].

Fig. 2

Modulating mechanisms of the Ang/Tie signaling pathway. In a healthy vessel (left), Ang1 binds the extracellular domain of Tie2 and activates Tie2 intracellular signaling pathway at EC-EC junctions. The activated PI3K/Akt signaling pathway mediates the phosphorylation of FOXO1 and transcription of ang2. Akt can also maintain cell survival by inducing eNOS and survivin, and exert anti-apoptotic effects by inhibiting caspase-9 and BAD. Activated Tie2 can inhibit the activation of NF-κB by interacting with ABIN-2, thereby inhibiting the expression of inflammatory genes. Ang1/Tie2 signaling can stabilize vessels by activating the GTPase signaling pathways. RAC1 dephosphorylates MLC2 to stabilize the actin cytoskeleton. Src, the downstream molecule of VEGF/VEGFR2, is sequestered by mDia, thereby prevent phosphorylation and internalization of VEC. In motile/non-contact ECs, Tie2 is localised at the EC-ECM and preferentially activates ERK to conduct cell migration and proliferation. Under inflammatory conditions (middle), cleavage of the Tie1 extracellular domain correlates with the switch of Ang2 from an agonist to an antagonist of Tie2. Suppression of Tie2 signaling pathway leads to cytoskeleton instability, inflammation, pericyte detachment and Ang2 positive feedback formation. At this point, VE-PTP inhibits the intracellular domain of Tie2. The meaning of the symbols involved in the figure (right). Created with BioRender.com

Ang2 is another member of the angiopoietin family that is expressed exclusively in ECs and stored in Weibel-Palade bodies [80]. In response to certain stimuli such as Ang2 itself or VEGF, Ang2 is rapidly released from the bodies and acts back into ECs in an autocrine manner [82]. In the Ang/Tie signaling pathway, Ang2 plays different roles depending on the environment. Since Ang1 and Ang2 have similar structures and affinity for Tie2, Ang2 has been previously identified as an antagonist of Tie2 that competitively inhibits Ang1 binding to Tie2 [16]. Nevertheless, some studies have shown that Ang2 can act as a Tie2 agonist under certain conditions (such as in the absence of Ang1 or in lymphatic ECs) [16, 102] Compared with Ang1, the agonistic activity of Ang2 is significantly weaker, which may be explained in structural terms. Native Ang1 exists in a highly oligomeric form, usually forming a tetramer or higher-order multimeric structure [103]. Native Ang2 exists mainly in the form of disulfide-bonded dimer [103]. It has shown that angiopoietin monomers require multimerization to form the ligands that can bind Tie [103]. Moreover, both multimerized ligand and receptor aggregation are required for Tie2 activation [103]. However, the dimeric angiopoietin is too distant from the ligand-binding site on the receptor to facilitate the formation of Tie2 dimers in the cis-orientation [103, 104]. Therefore, the Ang2, which normally exists in a dimeric form, has difficulty activating Tie2 compared with the hyperpolymerized Ang1. On one hand, when Ang2 acts as an agonist of Tie2, it can activate the PI3K/Akt pathway and inhibit the transcription of ang2 by phosphorylating FOXO1. This forms a negative feedback pathway concerning Ang2 production. On the other hand, when Ang2 acts as an antagonist of Tie2, it will inhibit the PI3K/Akt pathway and activate the FOXO1. The activation of FOXO1 will promote the generation of Ang2, thus forming a positive feedback pathway that drives ECs damage. Suppressing the remaining Tie2 intracellular signals also leads to the disruption of EC-EC junctions, instability of the actin skeleton and production of proinflammatory factors such as VEGF. Ang2 also activates Tie2 expressed on pericytes in a paracrine manner, leading to pericyte detachment, vascular structural abnormalities and vascular dysfunction [105].

Ang/Tie signaling is also regulated by VE-PTP, which is exclusively expressed in ECs and interacts with the intracellular domain of Tie2. It can catalyze the dephosphorylation of Tie2, thereby suppressing the Tie2 signaling and affecting the functional state of ECs [18]. On the other hand, inhibiting the VE-PTP can activate RAP1/RAC1, the downstream pathway of Tie2, which further contributes to stabilize blood vessels and reduce vascular permeability. Other studies have shown that VE-PTP can be recruited to EC-EC junctions by Ang1 and form complexes with VEC as binding partners [106]. Then, VE-PTP and VEC interact with each other to increase the adhesion activity of VEC, subsequently making the cell connections more tightly [106]. Conversely, the dissociation of them can lead to the phosphorylation and internalization of VEC [98]. When ECs lack VE-PTP, the adhesion function of VEC will be impaired, leading to increased vascular permeability [107]. Interestingly, inhibition of VE-PTP is independent of VEC in the presence of Tie2, and this inhibition will not induce any effect on EC-EC junctions [98]. However, in the case of Tie2 deficiency, inhibiting VE-PTP will affect VEC and increase vascular permeability [98]. VE-PTP-deficient mouse has been shown to develop embryonic lethality and impaired angiogenesis, verifying the important role of VE-PTP in the development of vasculature [108]. In essence, VE-PTP is still a negative regulator of Ang/Tie pathway. Under hypoxia, the expressions of both Ang2 and VE-PTP increase significantly, leading to pathological angiogenesis, inflammation and blood leakage [109]. Moreover, in addition to the fragmentation of Tie1 extracellular domain, the existence of VE-PTP is another factor that causes Ang2 to turn into an antagonist of Tie2 [110]. Ang2 acts as a competitive antagonist in vascular ECs in the presence of VE-PTP [102]. Thus, inhibiting VE-PTP is able to restore the agonistic activity of Ang2. In fact, VE-PTP is absent in the lymphatic endothelium, conferring Ang2 agonist properties [102]. Accordingly, the context-dependent function of Ang2 is controlled by both VE-PTP and Tie1 in vascular ECs and lymphatic ECs.

There exists a cross-talk between Ang/Tie signaling pathway and integrins. Integrin is composed of α and β subunits that linked by non-covalent bonds [111]. It serves as an intercellular adhesion molecule in multiple types of tissue. Integrin can also act as a receptor on the cell surface to recognize and bind angiopoietin [85, 111]. In ischemic retinal diseases caused by abnormal vascular degeneration, Ang1 works through a dual signaling pathway, which covers Tie2 signaling in ECs and α5β5 integrin signaling in astrocytes [85, 112]. Activated α5β5 integrin signal is able to promote fibronectin production, accumulation and radial distribution along the sprouting ECs, thereby guiding the directional migration of ECs to promote angiogenesis and vessels maturation in ischemic retinal diseases [112]. At EC-EC junctions, α5β1 integrin promotes the formation of Tie1-Tie2 complex induced by Ang1, activates Tie2 and induces phosphorylation of FOXO1 [14]. In the presence of Ang1 and extracellular fibronectin, α5β1 enables Tie2 to specifically activate ERK signaling pathway, which affects the proliferation and migration of ECs. However, the binding of Ang2 to integrin induces pericyte apoptosis, ECs migration, angiogenesis and vascular leakage [113]. Under hyperglycemia conditions, the expressions of both α3β1 integrin and Ang2 increase in the retinal tissue of DR patients [83]. The combination of Ang2 and α3β1 is able to mediate pericyte apoptosis via the p53 pathway, which does not occur in the presence of Ang2 alone [83]. Therefore, inhibiting the expression of Ang2 and α3β1 integrin or blocking the combination of them can act as a therapeutic strategy to prevent pericyte apoptosis and vascular lesions in the early stage of DR. During budding angiogenesis, Tie2 level is low in endothelial tip cells due to the inhibitory effect of Tie1. In this case, Ang2 binds to integrins and acts in a Tie2-independent manner, inducing ECs migration, vessel sprouting and angiogenesis [114]. Integrin α2β1 can reduce the adhesion between EC and ECM with the assistance of Ang2 [115]. Another study also shows that Ang2 induces the translocation and activation of α5β1 integrin in the Tie2 silenced ECs [116]. The activation of α5β1 stimulates the formation of actin stress fibril, leading to ECs instability and disruption of EC-EC junctions [116]. Integrin also interacts with Tie2 to transmit signals in the ECM through their respective extracellular domains. Integrin α5β3 is highly expressed in neovascularization, which can promote the adhesion and proliferation of ECs [117].

Ang/Tie signaling pathway can also interact with VEGF. Firstly, the Ang1 can inhibit the vascular damages caused by VEGF in angiogenic diseases. A recent experiment shows that the Ang1 has no effect on the established neovascularization, but it can inhibit the VEGF-induced neovascularization when co-expressed with VEGF [118]. In addition, Ang1-activated RhoA is able to inhibit Src-mediated signaling, which blocks VEGF-induced VEC phosphorylation and internalization, thereby protecting EC-EC junctions [100, 119]. VEGF can activate the eNOS and promote ECs to produce nitric oxide (NO) which subsequently increases cell permeability [120]. However, Ang1 was able to interrupt the signaling between VEGF and eNOS, thus alleviating blood leakage [120]. Moreover, there exists a synergistic effect and mutual promotion between Ang2 and VEGF. Co-expression of them not only accelerates neovascularization, but also further enhances vascular permeability [118, 121]. VEGF also stimulates the release of Ang2 from Weibel Palade bodies, thereby inhibiting Tie2 signaling and disrupting vascular stability [122].

Role of Ang/Tie signaling pathway in pathological activities

Neural inflammation

Inflammation is a critical pathogenetic factor of ocualr diseases, such as DR, RVO, ROP and AMD. When the retinal is exposed to pathologic insults, locally damaged RPE cells are activated to recruit choroidal dendritic cells (DCs). DCs can cause local inflammation through kinds of mechanism such as the formation of immune complexes and activation of complement system [123]. In DR, the advanced glycation end products (AGEs) and oxidative stress can activate retinal microglia and müller glia, which subsequently produce proinflammatory cytokines such as IL-6, IL-8 and TNF-α [124]. Ang/Tie2 signaling pathway plays a key role in the inflammatory response. Ang2 can competitively inhibit the binding of Ang1 to Tie2, thus suppressing ABIN-2 and promoting the NF-κB activation [21]. The resulted expression of inflammatory genes such as ICAM-1 and VCAM-1 will trigger the leukocyte recruitment [101]. Inflammation is able to induce the cleavage of the Tie1 ectodomain. This process corresponds to the switch of Ang2 from agonist to antagonist of Tie2. The Tie1 cleavage will cause vascular leakage and exacerbate the inflammation reaction [14]. Due to the stimulus from proinflammatory factors, Ang2 is promptly released from the ECs Weibel-Palade bodies, and rapidly initiates the inflammatory response [80]. Ang2 can activate the Tie2 receptor on neutrophils and enhance the adhesion of neutrophils to ECs [125]. Moreover, It has been demonstrated that Ang2 can induce β2 integrin-dependent infiltration of monocytes even in the absence of inflammatory stimuli [126]. Ang2 can also activate β1 integrins, causing endothelial instability, which may be related to inflammation [116]. TNF-α is a cytokine that contributes to the development of ocular inflammatory diseases. Ang2 enhances the activity of TNF-α and sensitizes ECs to TNF-α, which in turn modulates TNF-α-induced ICAM-1 expression and promotes ECs adhesion [17]. Multiple studies have demonstrated the potent anti-inflammatory effects of dual Ang2/VEGFA inhibition. In a retinal ischemia/reperfusion injury mouse model, dual Ang2/VEGFA inhibition can reduce macrophage/microglia accumulation around lesions [127]. The treatment also reduces clinical and histopathological scores in a mouse model of experimental autoimmune uveoretinitis [128]. Unlike Ang2, Ang1 suppresses inflammation through the activation of the PI3K/Akt pathway. AXT107 is a natural amino acid peptide that can activate Tie2 by converting Ang2 to an agonist of Tie2. AXT107 is able to inhibit ocular vascular inflammation by activating the Tie2-mediated anti-inflammatory pathway and counteracting the proinflammatory synergy between TNF and Ang2 [129]. Under inflammatory conditions, VEGF causes the dissociation of VE-PTP and VEC, promoting the extravasation of leukocytes from plasma into tissue fluid [98] On the other hand, AKB-9778, a VE-PTP inhibitor, can block leukocyte extravasation through activating the Ang/Tie signaling pathway [98].

Neovascularization

Several lines evidences suggest that ang2 and tie2 are susceptible genes for neovascular-AMD [130, 131]. The Ang2 expression in the aqueous humor of neovascular-AMD patients increases significantly, and is correlated with the severity of disease [132]. Notably, a recent study shows that Ang2 and VEGF are synergistically up-regulated, and are intensively distributed in the lesions of CNV vascularization [127]. RNV is considered as a hallmark of DR and RVO. The expression level of Ang2 is up-regulated in the vitreous of DR and RVO patients [133, 134]. In a model of ischemic retinopathy, Ang2 mRNA level is found to increase in the retinal tissue during pathological angiogenesis [135]. Studies have shown that VEGF can induce RNV, while Ang2 can sensitize retinal vessels to VEGF [136]. Under hypoxic conditions, Ang2/VEGF stimulates angiogenesis, highlighting their crucial role in the development of pathological neovascularization. Tie2 expression is evident in both the periphery and the base of the newly formed RNV and CNV. Therapeutically, RNV is inhibited in a mouse model of ROP by intravitreally injecting soluble Tie2 fusion protein (sTie-Fc), an extracellular component of Tie2 [137]. Similarly, CNV is inhibited in a mouse model of laser-induced CNV by the intramuscular injection of an adenovirus expressing the Tie2 receptor extracellular domain gene [138]. Moreover, Tie2 activation by Ang2-binding and Tie2-activating antibody (ABTAA) can reduce the VEGF-induced vascular leakage and inhibit the growth of CNV [139]. 5α-Hydroxycostic acid has a positive therapeutic effect on choroidal neovascularization in a rat model of laser-induced CNV by inhibiting the phosphorylation of VEGFR2 and Tie2 [140]. In addition, it has been reported that Ang1 inhibits the laser-induced CNV formation and vascular leakage in mice [19]. Therefore, targeting the Ang/Tie pathway affords a potential therapeutic option for neovascularization.

Vascular permeability

Fluid leakage occurs in many retinal and choroidal vascular diseases due to the increased vascular permeability. Ang/Tie signaling plays an important role in maintaining vascular integrity and controlling vascular permeability. ECs are essential for this signaling pathway to exert its protective effects. Physiologically, endothelial barriers integrity is maintained by extracellular structures such as glycocalyx, EC-EC junctions and ECM. EC-EC junctions include the adherens junctions and tight junctions. Endothelial adherens junctions contain VEC, which connects to neighboring cells through their extracellular domain. In particular, VE-PTP acts as a transmembrane binding partner of VEC. Its dissociation from VEC leads to the phosphorylation and internalization of VEC [98]. Src can be activated by VEGF to induce VEC phosphorylation and internalization, thereby increasing vascular permeability [141]. On the other hand, Ang1 can activate Tie2 and RhoA, leading to downstream mDia binding to Src. This prevents VEC phosphorylation and internalization, thus blocking VEGF-induced vascular permeability [100]. Ang1 also promotes the formation of the Tie2/VE-PTP/VEC complex through VE-PTP, ultimately preventing the increase of vascular permeability [106]. Furthermore, the interaction of Ang2 with integrins affects the vascular permeability profoundly. Combination of Ang2 and integrins can induce the Tie2-independent disruption of ECs. In an streptozotocin-induced DR mouse model, Ang2 interacts with α5β5 integrin, leading to astrocyte apoptosis and increased vascular leakage [142]. In many diseases, targeting Ang/Tie signaling is capable of reducing vascular permeability and leakage. In a transient middle cerebral artery occlusion mouse model, activation of Tie2 with VE-PTP inhibitors reduces the vascular permeability and stroke size [143]. In the human breast cancer bearing mouse model, dual inhibition of Ang2 and VEGFA effectively reduces the pathological neovascularization and enhanced pericytes coverage of blood vessels [144]. In a retinal ischemia/reperfusion injury mice model, dual Ang2/VEGFA inhibition effectively prevents retinal vascular leakage [127]. Particularly, intravitreal injection of AXT107 can alleviate Ang2-induced retinal vascular leakage in an Ang2-overexpression transgenic model [129]. Targeting Ang/Tie signaling pathway may provide a prospective approach to treating these diseases, as the levels of Ang2 is high in ischemic diseases such as DME, neovascular-AMD, and uveitis.

Fibrosis

Fibrosis refers to the excessive accumulation of ECM in various tissues that can cause organ dysfunction. Subretinal fibrosis is a consequence of the wound healing response after CNV formation [40]. This pathological process can result in irreversible central vision loss in neovascular-AMD patients. In response to hypoxia and inflammatory stimulus, proliferative diabetic retinopathy (PDR) undergoes extensive neovascularization and fibrosis after the neuroglia activation. In RVO, hypoxia induces the expression of transforming growth factor (TGF)-β1 which promotes fibroplasia [145]. Currently, the advancement to fibrosis and traction in ROP remains a crucial clinical challenge [146]. In the diabetic db/db mouse model, over-expression of Ang2 inhibits the expressions of Tie2 and VEGF in the heart, resulting in ECs apoptosis, myocardial fibrosis formation, and reduced capillary density. In addition, the fibrosis and vascular alterations could be reversed by Ang1 over-expression [147]. In another study, the histology of the αMHC∷VEGF; αMHC∷Ang2 double-transgenic mice in cardiac tissue shows abnormal angiogenesis, severe edema, and fibrosis. In contrast, all these phenotypes are improved in αMHC∷VEGF; αMHC∷Ang1; αMHC∷Ang2 triple-transgenic mice [148]. Cartilage oligomeric matrix protein-Ang1 (COMP-Ang1), a derivative of Ang1, has demonstrated efficacy in accelerating myofiber regeneration by activating N-cadherin to ameliorate ischemic muscle injury of mice [149]. Furthermore, in a 10-week-old uninephrectomized db/db mouse model, a single dose of AAVrh10.COMP-Ang1 delivered via the intracarotid artery reduces ECM accumulation in diabetic glomeruli by 61% [150]. Collectively, these findings suggest that Ang/Tie signaling is involved in modulating the vascular fibrosis. Therefore, targeting the Ang/Tie pathway provides a potential therapeutic option for vascular fibrosis in ocular diseases such as neovascular-AMD, PDR, RVO and ROP.

Neural degeneration

Neural degeneration is characterized by the progressive neuron apoptosis and glial dysfunction [151]. Not only neovascularization but also nerve damage are present in the above-mentioned eye diseases. Neural degeneration is recognized as a hallmark of AMD, DR and ROP. Multiple etiological factors are involved in the neural degeneration, such as oxidative stress, glutamate mediated excitotoxicity, and the deficiency of nutritional cytokines [45]. Recent studies show that angiopoietins exert neuroprotective and neurotrophic effects on the neural system [152]. Ang/Tie2 signaling is required for proper Purkinje cell (PC) dendritic morphogenesis and function [153]. Ang1 can protect cortical neurons from apoptotic stress by activating the PI3K/Akt pathway and inhibiting caspase-3 activation. Ang1 can also support the development of neurites and promote synapse formation in dorsal root ganglia (DRG) neurons [154].Neurons express Tie2 receptor and β1 integrin, both of which are able to activate the downstream Akt pathway and stimulate the neurogenesis in neural progenitor cells (NPCs) [154]. Ang2 is not only involved in the neurogenesis of embryonic cortical nerves, but also contributes to the NPCs proliferation and migration in adult [155]. In preclinical studies, tie2 has been proven to induce less effects on the IOP, and to exert neuroprotection on the RGCs [156, 157]. Researchers have targeted Ang/Tie pathway as a potential retinal neuroprotectant. COMP-Ang1, a more potent recombinant form of Ang1, ameliorates neurological deficits by promoting neuronal survival in central retinal artery occlusion (CRAO) [158]. Moreover, dual Ang2/VEGFA inhibition is more effective in preventing neurodegeneration than either Ang2 or VEGFA inhibition alone in a retinal ischemia/reperfusion injury model [127]. In the middle-aged rat VaD multiple microinfarction (MMI) model, intraperitoneal injection of 1 µg/kg AV-001 (Tie2 receptor agonist) significantly increases axonal density, remyelination, and neuroplasticity in the brain [159]. Therefore, these findings will encourage further investigations of the neuroprotective role of Ang/Tie pathway in retinal degenerative diseases (Fig. 3).

Fig. 3

Pathological conditions of ocular vascular diseases. (A): At the late stage of diabetic retinopathy, changes occur to the vasculature including basement membrane thickening, loss of endothelial tight junctions, loss of pericytes and abnormal neovascularization. Angiogenesis occurs in regions of low O₂, which induces VEGF production. VEGF binds to VEGFR2, triggering competition between neighboring cells as they differentiate. Therefore, these adjacent vascular endothelial cells become stalk cells and form the body of the sprouting vessel. (B): Retinal structure is intact in a healthy eye. Nevertheless, in an eye with neural degeneration, damages occur in the retinal cells. (C): In response to external stimulus, endothelial cells are damaged and converted into myofibroblasts or fibroblasts, resulting in increased ECM synthesis. Vascular fibrosis occurs when ECM accumulates excessively. Created with BioRender.com

Role of Ang/Tie pathway in other domains

Primary open-angle glaucoma (POAG) is usually caused by elevated intraocular pressure (IOP), manifested as retinal neuronal damage and retinal ganglion cells (RGCs) apoptosis. A recent study shows that the integrity of the Schlemm’s canal is compromised in either Ang1/Ang2 or Tie2-deficient adult mice, with increased IOP similar to that in POAG. The POAG phenotype in Ang1/Ang2-deficient mice is alleviated and Schlemm’s canal viability is restored after treatment with a Tie2 agonism antibody [156]. Accordingly, the Tie2 agonists may serve as a potential therapeutic option for POAG. Furthermore, the Ang/Tie signaling pathway also represents a therapeutic target for the pulmonary vascular diseases. Tie1 and Tie2 are transmembrane receptors that expressed on tumor-associated macrophages and ECs [73]. The Ang/Tie signaling pathway is able to regulate angiogenesis during tumor growth and promote capillary remodeling in tumor tissue, thereby maintaining the vascular integrity [10, 14]. Moreover, the combined application of Ang2 blocking antibody with TNF-α blockade is more effective in reducing the vascular leakage and inhibiting the inflammatory vascular remodeling after pulmonary mycoplasma infection compared with the single use of Ang2 blocking antibody [160]. In addition, inhibition of Ang2 also plays a protective role in heart transplantation. A recent study shows that the specific blockade of Ang2 can alleviate the deleterious inflammation caused by heart transplant rejection [161].

Pharmacological therapy targeting Ang/Tie signaling pathway

AKB-9778 and ARP-1536

AKB-9778 is a small molecule that can suppress the catalytic activity of VE-PTP. Pharmacological kinetic study shows that the conformation and flexibility of VE-PTP active-site will change profoundly when AKB-9778 binding to VE-PTP indicating that AKB-9778 can impede the combination of VE-PTP and Tie2 [162]. Moreover, AKB-9778 can stabilize the EC-EC junctions and vascular integrity through activating Tie2 even in the absence of VEC [98]. Currently, AKB-9778 has been used to inhibit neovascularization and vascular leakage in animal models of neovascular-AMD and DR. AKB-9778 has also shown promising efficacy in improving visual acuity in patients with DME and moderate to severe nonproliferative DR.

In mouse models of neovascular-AMD and DR, subcutaneous injection of AKB-9778 induces the phosphorylation of Tie2 and activates downstream signaling pathways. The growth of VEGF-mediated ocular neovascularization and vascular leakage in these animal models are effectively inhibited by AKB-9778 administration. In particular, AKB-9778 can inhibit the retinal neovascularization even in the presence of Ang2 [111]. In an open-label, dose-escalation phase I trial, AKB-9778 was dosed by subcutaneous injection twice daily for consecutively four weeks. There were 6 DME patients in each of the four treatment groups with different drug doses: 5 mg, 15 mg, 22.5 mg and 30 mg, respectively. Among 18 patients treated with 15 mg or more twice daily, the best corrected visual acuity (BCVA) improved by 5 or more letters from baseline in 13 patients at week 4. Moreover, 7 of these 18 patients showed improvements in the central retinal thickness (CST). There were 5 patients showed a decrease of more than 100 mm in CST and 2 patients showed a decrease of 50 to 100 mm. Systemic subcutaneous administration of AKB-9778 was well tolerated with minor adverse effects. The adverse events potentially are caused by the vasodilatory activity of AKB-9778 presented at doses of 22.5 mg or more, including reduction in blood pressure, dizziness, vasovagal events, which were transient and caused by NO. In diabetics, modest reductions in blood pressure are desirable, as most patients with DME have hypertension and this effect may reduce long-term vascular complications [163]. This also suggests that VE-PTP inhibition (AKB-9778) may be an attractive novel treatment option for the diabetes-induced endothelial dysfunction and hypertension [164].

In the phrase IIa trail (TIME-2), 144 patients with DME were randomized to three groups: subcutaneous AKB-9778 15 mg twice daily, alone or in combination with monthly 0.3 mg ranibizumab intravitreal injection, or 0.3 mg ranibizumab monotherapy. After 12 weeks, BCVA (letters) increased from baseline 6.3 ± 1.3 in the combination group, 5.7 ± 1.2 in the ranibizumab monotherapy group, and 1.5 ± 1.2 in the AKB-9778 monotherapy group. Moreover, the combination group had the most prominent reduction in CST from baseline [165]. In another phrase IIb trail (Aerpio Pharmaceuticals; https://clinicaltrials.gov Identifier: NCT03197870), 167 patients with moderate to severe nonproliferative DR were randomized to three groups: subcutaneous AKB-9778 15 mg once daily, subcutaneous AKB-9778 15 mg twice daily, or subcutaneous placebo. No significant difference was found between the AKB-9778 and placebo group in the proportion of eyes that achieved ≥ 2 steps of improvement in the DR scores. Taken together, these results suggest that the anti-VEGF drug combined with AKB-9778 is more effective in reducing DME than VEGF inhibition alone. Although subcutaneous injection can result in systemic exposure to AKB-9778, increasing the probability of side effects, AKB-9778 has shown a favorable safety profile in aformentioned clinical trials. In addition to transient hypotension and dizziness, most of those adverse effects were caused by the complications of diabetes rather than the drug. AKB-9778 administered by subcutaneous injection has the potential advantage of patient self-administration. When AKB-9778 is used in combination with intraocular injection of VEGF neutralizing protein, it theoretically can protect patients from systemic vascular complications caused by VEGF inhibition. This suggests that a longer study duration and larger trials have the potential to find more significant visual improvement results. At present, the research interest of AKB-9778 mainly focuses on the treatment of DME, and its role in AMD still should be verified by further clinical trials.

Interestingly, among the prespecified key secondary end points in the IIb study described above, subcutaneous administration of AKB-9778 reduced intraocular pressure in a dose-dependent trend. AKB-9778 was shown to reduce IOP in both mouse and rabbit models in pioneering studies. Similarly encouraging results have also been found in early clinical studies in humans: subcutaneous administration of this VE-PTP inhibitor reduced IOP in the patients with diabetic eye disease [166]. These results suggest that subcutaneous administration AKB-9778 may represent a promising therapeutic option for ocular hypertension and glaucoma. ARP-1536 is another novel monoclonal antibody with similar efficacy to AKB-9778. It can bind the extracellular domain of VE-PTP and activate the Tie2. Studies on the biological activity and efficacy of ARP-1536 are ongoing [15].

Nesvacumab/REGN910

Nesvacumab/REGN910 is a fully human IgG1 anti-Ang2 monoclonal antibody that specifically blocks the combination of Ang2 and Tie2 receptor [167]. It is administered in collaboration with the anti-VEGF agent aflibercept through intravitreal injection. Notably, it has been found that nesvacumab can prolong the antiosmotic and antiangiogenic effects of aflibercept. The phase II RUBY study compared intravitreal injection of aflibercept 2.0 mg alone, in combination with low-dose nesvacumab 3.0 mg or high-dose nesvacumab 6.0 mg in patients with DME. This study demonstrated the anatomical advantage of high-dose combination therapy. However, the absence of a significant difference in BCVA among the three groups did not demonstrate any improvement in visual acuity. Throughout the study, patients in all groups experienced one or more ocular adverse reactions in the eye, the most common case was conjunctival hemorrhage. Two serious ocular adverse events, iridocyclitis (n = 1) and retinal artery occlusion (n = 1), occurred in the high-dose combination group and they were considered associated with this study [168]. Another phase II ONYX study assigned aflibercept monotherapy, a low-dose combination of aflibercept and nesvacumab, and a high-dose group in patients with neovascular-AMD. The study showed similar vision-treatment outcomes among the three groups and no serious adverse event [169]. The limitations of this trial are the relatively small sample size and short study duration. Whether a prolonged duration of Ang2 inhibition will improve clinical outcomes requires further investigation. Collectively, the combination of nesvacumab and aflibercept fails to show efficacy in improving vision acuity in neovascular-AMD and DME patients. Considering the above results, a phase III clinical trial is not initiated.

Faricimab

Faricimab is a bispecific antibody obtained by CrossMAb. It can bind the VEGFA and Ang2 with high affinity, thereby activating the Tie2. It is 150KDa in size and its molecular structure consists of three parts: an anti-Ang2 antigen-binding fragment, an anti-VEGF antigen-binding fragment and a modified fragment crystallizable region [170]. Currently, faricimab has shown an advantage in improving visual in patients with neovascular-AMD and DME. In an animal experiment, blocking the VEGFA/Ang2 can reduce the choroidal angiogenesis in a spontaneous CNV mouse model more effectively than the anti-VEGFA or anti-Ang2 treatment alone [171]. AVENUE is a phase 2 randomized clinical trial to evaluate the safety and efficacy of different doses of faricimab and ranibizumab in patients with neovascular-AMD. The results showed that faricimab was less effective than monthly ranibizumab in improving BCVA after a 36-week follow up, but the overall visual and anatomical advantages of faricimab supported a phase 3 trial [172]. In STAIRWAY, another phase 2 randomized clinical trial for neovascular-AMD, a total of 76 patients with neovascular-AMD were randomly assigned in a 1:2:2 ratio to receive intravitreal injection of ranibizumab (0.5 mg every 4 weeks) or faricimab (6.0 mg every 12 or 16 weeks). 40 weeks after treatment, the adjusted increase from baseline in mean BCVA with faricimab every 12 weeks (+ 9.3) and faricimab every 16 weeks (+ 12.5) were comparable to that with ranibizumab every 4 weeks (+ 11.4). Moreover, initial visual and anatomical improvements were maintained after 52 weeks with faricimab (administered every 16 weeks and every 12 weeks) compared with ranibizumab (administered monthly) [173]. BOULEVARD is a phase 2 clinical trial of DME in which 168 anti-VEGF treatment-naive patients were randomized assigned to three groups. Patients received monthly intravitreal injections of 6.0 mg faricimab, 1.5 mg faricimab, or 0.3 mg ranibizumab, respectively. 61 patients previously treated with anti-VEGF were equally assigned to receive monthly intravitreal injections of either 6.0 mg faricimab or 0.3 mg ranibizumab. In this trial, patients received treatment for 20 weeks, with an observation period that continued till the 36th week. Compared with baseline, the early treatment diabetic retinopathy study (ETDRS) showed that the average improvement was 13.9 letters (6.0 mg faricimab group), 11.7 letters (1.5 mg faricimab group), and 10.3 letters (0.3 mg ranibizumabgroup), respectively. In addition, faricimab was superior to ranibizumab in improving CST, DRSS and administration frequency in both patient populations [174].

Preliminary results of LUCERNE and TENAYA, two phase 3 randomized trials of neovascular-AMD, showed that the durability of a 16-week regimen of faricimab was noninferior to that of an 8-week regimen of aflibercept in the treatment of neovascular-AMD at the 48th week. A significant reduction in central retinal thickness (CRT) was achieved in the faricimab group [175]. The LUCERNE and TENAYA trials transitioned to the treat-and-extend (T&E) personalized treatment interval (PTI) regimen in the second year (112-week), which reflected clinical practice while maintaining durable visual acuity benefits and anatomic gains in patients [176]. Furthermore, lasted results of YOSEMITE and RHINE, two phase III randomized trials of DME, showed that faricimab (intravitreal injection and T&E PTI regimen) was noninferior to aflibercept alone in improving BCVA after 2 year follow up [177, 178]. In these Phase III clinical trials, faricimab was well tolerated and had acceptable safety profiles. The adverse events in neovascular-AMD or DME patients treated with faricimab were generally consistent with anti-VEGF group. At the last endpoints of the trials, the incidence of intraocular inflammation was lower, as most intraocular inflammatory events were resolved or resolving in the end. However, the trials had some limitations. Faricimab has extended durability due to its vascular stabilization effect on the dual inhibition of Ang2 and VEGF pathways. The anti-VEGF comparison group was administered according to a fixed 8-week dosing regimen, therefore lacked a direct comparison of the durability of faricimab relative to the standard treatment group. Nevertheless, faricimab group was treated by less injection, but not influence the efficacy, indirectly reflects the durability of faricimab. In addition, neovascular-AMD and DME are chronic and progressive diseases which have long-term effects on vision, therefore continued follow-up is needed. Considering the above trials, intravitreal injection of faricimab can be used as an alternative treatment for neovascular-AMD and DME patients.

AXT107

AXT107 is a peptide derived from the type IV collagen sequence which can bind integrins α5β3 and α5β1 with high affinity. Several lines of evidences suggest that AXT107 is able to inhibit VEGF-mediated signaling pathways, promote Ang2-induced Tie2 activation, and inhibit neovascularization and vascular leakage [179]. In the presence of Ang2, AXT107 induces dissociation of α5 and β1 integrins, promotes Tie2 aggregation at EC-EC junctions, and subsequently activates the Tie2 signaling pathways. In this process, Ang2 is converted into a strong agonist of Tie2 [180]. Since the endogenous Ang1 is present at low concentrations, AXT107 will enable Ang2 to exert a similar effect on Ang1 by activating Tie2 rather than inhibiting it. AXT107 can also reduce VEGFR2 levels through a variety of mechanisms, including increased ubiquitination, internalization and degradation [181]. Furthermore, AXT107 is capable of disrupting the synergistic effect of Ang2 and TNF-α through the Tie2 signaling pathway. The TNF-α-induced vascular inflammation in ECs is alleviated by virtue of converting Ang2 into an agonist of Tie2 signaling [129]. In particular, AXT107 not only reduce experimentally induced RNV and CNV, but also reduce VEGF-induced vascular leakage in neovascular-AMD and ischemic retinopathy. A recent in vivo study shows that the AXT107 and aflibercept can mitigate the VEGF-induced vascular leakage in rabbit eyes by 86 and 69% at the first month, respectively [181]. At the second month, vascular leakage was reduced by 70% in AXT107-injected eyes, whereas there was no significant reduction in the aflibercept injected eyes. These findings suggest that AXT107 is superior to aflibercept in reducing VEGF-induced vascular leakage in neovascular-AMD. Furthermore, AXT107 in combination with aflibercept is more effective than the AXT107 alone, indicating a synergetic mechanism is implicated in the inhibitory effects. In contrast to VEGF, AXT107 can form a gel depot outside the visual axis after intraocular injection and dissipates slowly over several months, potentially prolonging the duration of action and reducing the frequency of injections. In a non-GLP (Good Laboratory Practice) safety study, no elevated intraocular pressure, intraocular media degradation, or retinal toxicity was observed in AXT107 treated rabbit eyes. Therefore, these findings highlight that AXT107 may act as a effective medication for neovascular-AMD. Currently, the phase I/IIa SHASTA trial and phase I/IIa CONGO trial for neovascular-AMD are ongoing to assess the safety, tolerability and biological activity of AXT107 [182] (Table 1).

Table 1 Summary of therapeutic agents targeting Ang/Tie signaling pathway

Other agents

ABTAA is a dual functional antibody that binds Ang2 and activates Tie2 [183]. It can block the antagonist function of Ang2 by inducing the oligomerization of Ang2 and transforming it into a strong agonist of Tie2 [183]. This differs from the traditional pharmacological mechanism which seeks to inhibit the action of Ang2. However, it also suggests that ABTAA-induced Tie2 activation and maintenance are largely dependent on Ang2 and Tie2 levels in the pathological micro-environment, which is recognized as a limitation of ABTAA. In addition, the optimal dose of ABTAA to reduce unexpected aggregation between ABTAA and Ang2, which might lead to excessive activation of Tie2, also needs to be further verified for clinical application [184]. Emerging evidences have shown that ABTAA not only induces CNV regression, but also inhibits the vascular leakage in the neovascular-AMD mouse model [145]. Moreover, ABTAA can promote healthy choriocapillary vasculature regeneration and alleviate hypoxia around CNV, which is recognized as a key step for the basic recovery of neovascular-AMD [145]. Advantageously, ABTAA also has a long half-life and the convenience of clinical production, demonstrating its potential to extend the dosing interval, thereby reducing the treatment burden for neovascular-AMD patients [145, 183]. Moreover, it has been shown that activation of Tie2 by ABTAA restores the senescent Schlemm’s canal and inhibits its degeneration [156]. Therefore, the drainage of aqueous outflow is strengthened and the IOP is reduced, highlighting that ABTAA may act as a potential therapeutic molecule for POAG [183].

AMG 386/Trebananib is a recombinant Fc-peptide fusion protein that effectively blocks the binding of Tie2 and ligands Ang1/2 [185, 186]. The drug is delivered by IV administration, which may avoid adverse events associated with invasive procedures, such as endophthalmitis, elevated intraocular pressure, subconjunctival intraocular hemorrhage, and retinal detachment. This peptibody can reduce significantly the vascular leakage, fibroplasia and angiogenesis in a cynomolgus monkey model of CNV [185]. AMG 386 also shows inhibitory effects on the pathological retinal angiogenesis of the murine model of ROP. In particular, a combination of both AMG 386 and AMG237 (a VEGFR inhibitor) yields better protective effects on the retinal angiogenesis. Based on the mechanism of action and the absence of any new safety concerns during the study, AMG386 warrants further investigation in neovascular AMD, PDR, and ROP.

Vanucizumab (RG7221/RO5520985) is an IgG1-like bispecific antibody that targets both VEGFA and Ang2 [152, 187]. In contrast to the monospecific antibodies against Ang2 or VEGF, it is more potent in inhibiting neovascularization in vivo. It can aslo reduce the microvessel density and promote blood vessel maturation [152]. Notably, vanucizumab does not exacerbate the known adverse effects of anti-VEGF therapy [187]. However, current application of this drug is still limited to anti-tumor research, and phase II clinical trial has been completed in the treatment of rectal cancer [188]. Based on its safety and promising results in the treatment of tumors, vanucizumab may have a prospective application prospect in ocular neovascular diseases.

COMP-Ang1 is an Ang1 variant with stronger agonistic activity than the native Ang1. It can not only overcome some limitations of natural molecules such as insolubility and aggregation, but also make Tie2 phosphorylation more persistent. Therefore, researchers have proposed that COMP-Ang1 may serve as a viable alternative to natural Ang1. The Tie2 phosphorylation induced by COMP-Ang1 can activate Akt pathways for a long period of time, thereby stabilizing the NVU and reduce vascular permeability effectively [189]. The half-life of the drug in the circulation of male adult mice is extremely short, and it has a strong non-specific binding force to any tissue of the body, which has great limitations in systemic administration [190]. However, intravitreal injection of the AAV-mediated gene therapy with COMP-Ang1 (AAV.COMP-Ang1) can overcome the limitations when applied to treat ocular diseases. Thus far, it has used as an anti-leakage agent for vascular diseases such as DR and neovascular-AMD. In a neovascular-AMD mice model, AAV.COMP-Ang1 was able to inhibit the CNV and effects of VEGF, thereby ameliorating neovascular-AMD. In a murine model of DR, the AAV.COMP-Ang1 could protect vascular structure, maintain the integrity of BRB, reduce the adhesion of leukocytes to ECs, and prevent neurovascular pathology [191]. However, the drug failed to alleviate retinal neurodegeneration and visual impairments in the aged DR mouse model [192]. This difference in neuroprotective effect may be caused by the different dose and timing of drug injection after the onset of the disease model, or the development of some retinal pathologies such as retinal remodeling. These pharmacological problems should be addressed before it can be applied to clinical trials (Fig. 4).

Fig. 4

Therapeutic agents targeting the Ang/Tie2 signaling pathway. (A): AKB-9778 is administered by subcutaneous injection. AXT107, nesvacumab and faricimab are administered by intravitreal injection. (B): AXT107 inhibits VEGF-mediated signaling pathways and promotes Tie2 activation. COMP-Ang1 activates Tie2 with stronger agonistic activity than the native Ang1. Both AKB-9778 and ARP-1536 inhibit the activity of VE-PTP, thus activating Tie2. (C): Nesvacumab is an anti-Ang2 monoclonal antibody. Faricimab is a bispecific antibody that can binds to VEGFA and Ang2 with high affinity. Vanucizumab is a bispecific antibody that targets both VEGFA and Ang2. AMG 386 is an antibody that effectively blocks the binding between Tie2 and ligands angiopoietin (Ang1 and Ang2). ABTAA is a dual functional antibody that binds Ang2 and activates Tie2. Created with BioRender.com

Outlook

Ang/Tie signaling pathway plays an essential role in the vascular development, NVU remodeling and angiogenesis [10, 12, 13]. Under physiological conditions, the dynamic crosstalk between the pathway components is stable, which would be helpful for maintaining the normal function of NVU. When the retina is exposed to pathological insults, the dynamic balance between pathway components is broken, leading to vascular leakage, inflammation, neovascularization, and nerve damage. Accumulating evidences suggest that the Ang/Tie signaling pathway contributes to the pathology of several types of retinal and choroidal vascular diseases, such as neovascular-AMD, DR, RVO and ROP. Targeting the Ang/Tie signaling pathway opens a new therapeutic window for many patients with ocular neovascular diseases who currently have a poor response to anti-VEGF. Currently, the specific role of neovascularization in ocular disease has not been decided yet. Some recent studies have shown that the growing CNV exerts a nutritional effect on the damaged RPE cells [71]. Therefore, it is better to stabilize NVU rather than inhibit completely neovascularization. Current research strategies mainly focus on combination therapy targeting Ang/Tie and VEGF/VEGFR signaling pathways. In preclinical and early-phase clinical studies, any single therapy has limited efficacy, but the efficacy of combination strategies is potentially additive. When anti-VEGF drugs are combined with long-acting agents targeting Ang/Tie, such as faricimab, the dosing interval can be prolonged, which may reduce the need for subsequent injections and the cost of treatment. This strategy is promising for vascular protection. Low concentration of VEGF can cause a decrease in NO level through inhibiting eNOS activity, leading to the secondary vasoconstriction and hypertension. The promotion of eNOS activity by AKB-9778 protects patients from systemic vascular complications caused by the VEGF antagonists. However, many limitations have hindered the translation of Ang/Tie targeted drugs to the clinic. The half-life of COMP-Ang1 in preclinical experimental models is very short and its neuroprotective effect is unstable. The drug has strong non-specific binding to any tissue of the body, which limits the range of safey dosage. The pharmacological effects of ABTAA and nesvacumab are limited by the pathological micro-environment, and the latter exhibits serious adverse reactions as well as unsatisfactory efficacy. Faricimab lacks a direct comparison with standard therapy in durability and had a shorter follow-up [175, 177]. The current challenge lies in conducting further studies to overcome the drug limitations and validate the clinical potential of targeting the Ang/Tie pathway for treating ocular neovascular diseases.