Regulatory T Cells in Angiogenesis.

In contrast to the literature summarized above, Tregs have also been shown to have antiangiogenic effects especially in the context of ischemia, chronic inflammatory states and in tissues characterized by angiogenic privilege such as the cornea. 77 - 80 However, the literature on the antiangiogenic effects of Tregs and the mechanisms by which they exert this function (summarized in Table 2 ) is still scarce. To investigate the effects of Tregs on angiogenesis in the setting of acute ischemia, two models of ischemic injury have been reported. 77 , 78 Zouggari et al. used a mouse model of ischemic hindlimb by ligating the femoral artery. 77 The authors reported that in knockout mice with lower levels of endogenous Tregs, neovascularization following ischemic insult increased significantly. 77 Conversely, in mice with an increased number of endogenous Tregs, a reduction of post-ischemic neovascularization was seen. Interestingly, there was no statistical difference between the VEGF-A protein levels of the mice with down-regulated endogenous Tregs when compared to the control. 77 Bansal et al. provided insights into the mechanisms by which regulatory T cells inhibit angiogenesis following myocardial infarction. Using a mouse model of heart failure (HF), the authors demonstrated that HF Tregs, which were dysfunctional and pro-inflammatory, decrease cardiac neovascularization. This reduction is effected via a cell-contact mediated process requiring tumor necrosis factor α receptor-1 (TNFR1). 78 Moreover, their study demonstrated that HF Tregs modulate CD34 + circulating angiogenic cells via C-C chemokine ligand 5/C-C chemokine receptor 5. 78 Two studies have substantiated the role of Tregs in inhibiting angiogenesis in inappropriately persistent inflammatory states. Teige et al. described in 2009 the potential antiangiogenic effect of Tregs in association with psoriasis, an angiogenesis-dependent skin disease. 3 , 80 In a mouse model of psoriasis-like skin inflammation, they reported that administration of CD4 + CD25 + Tregs significantly diminished the VEGF-driven cutaneous inflammatory response, but the mechanisms by which this inflammatory response was dampened by Tregs was not explored in their report. Huang et al. also demonstrated the antiangiogenic effects of Tregs in chronic inflammatory states, albeit in a different animal model. 79 Using a mouse model of ovalbumin-induced airway inflammation, they showed that adoptive transfer of Tregs decreased angiogenesis by cell-contact dependent mechanisms. Specifically, their paper established that Tregs exert their antiangiogenic effects by promoting endothelial cell apoptosis via delta-like 4 notch signaling pathway, the main inhibitor of angiogenesis in the notch signaling pathway, thereby down-regulating VEGF expression. 79 In summary, these reports position Tregs as downregulators of angiogenesis in states of persistent inflammation. The concept of angiogenic privilege has been of considerable interest in the field of ophthalmology: the cornea remains one of the best in vivo models to study the molecular processes in angiogenesis. 54 Numerous studies have demonstrated that presence of neovascularization prior to corneal transplantation represents one of the most significant causes for high rates of rejection of subsequent grafts. 81 , 82 This is largely due to the fact that these new corneal blood vessels facilitate delivery of immune effector cells to the graft site. It stands to reason, then, that therapeutic strategies targeting neovascularization, principally those targeting VEGF 83 , have been shown to promote graft survival. 84 , 85 However, anti-VEGFs have also been associated with corneal toxicity, given the high expression of VEGF-Rs by corneal afferent nerves. 86 , 87 Additionally, clinical studies have also shown a substantial number of non-responders to local anti-VEGF therapy. 86 , 88 , 89 For these reasons, there exists an unmet need for novel treatments for corneal neovascularization. Data from our laboratory suggest that local administration of host naïve regulatory T cells has an antiangiogenic effect on corneal neovascularization. Indeed, using a suture-induced model of corneal neovascularization 85 , 90 , we note a robust and consistent decrease in corneal neovascularization in mice following adoptive transfer by tail vein injection (Jia Yin, ARVO Abstract 3341, 2018) and subconjunctival injection of Tregs (unpublished data). In the field of vision science, the antiangiogenic effect of Tregs has also been demonstrated in the context of retinopathy of prematurity: Deliyanti et al. demonstrated, using a mouse model of oxygen-induced retinopathy, that expansion of retinal Foxp3 + Tregs modulated microglial activation and decreases levels of neovascularization after ischemic injury. 91

The concept of distinct T lymphocytes mediating immune suppression, “suppressor T cells”, was first introduced by Gershon and Kondo in 1970. 9 Twenty-five years after the publication of Gershon and Kondo’s seminal work, Shimon Sakaguchi et al. successfully identified CD4 + CD25 + T lymphocytes and demonstrated their role in preventing auto-immunity. 7 Their work precipitated a new wave of interest in Tregs, which have, since then, been the focus of considerable research efforts in the field of immunology. Tregs are a heterogenous group of lymphocytes that act principally as mediators of immunologic self-tolerance. 10 They can differentiate naturally within the thymus, so called “naturally occurring Tregs”, and can be peripherally induced following antigen presentation. 11 They are characterized by their expression of the surface molecules CD4 and CD25 (the interleukin (IL) 2 cytokine receptor) and the transcription factor FoxP3. 12 FoxP3 is a protein that is indispensable to the normal development and function of regulatory T cells. In humans lacking functional FoxP3, a severe multi-organ autoimmune disease called IPEX syndrome (immunodysregulation, polyendocrinopathy and enteropathy, X-linked syndrome) occurs, underscoring the importance of this transcription factor. 13 , 14 The mechanisms by which Tregs suppress the immune system are complex and numerous. Briefly, Tregs regulate the activity of effector T cells and other immune cells mainly i) by immunosuppressive cytokine production (IL-10, transforming growth factor (TGF)-β, IL-35), ii) by IL-2 consumption, iii) by direct cell-to-cell interactions and cytolysis (cytotoxic effect from granzyme A and B), and iv) by modulation of dendritic cell function. 12 Beyond their critical function in preventing autoimmunity, these lymphocytes are also important promoters of allograft tolerance in transplantation 15 and maintenance of pregnancy. 16 Conversely, these same immunosuppressive and tolerogenic properties are destructive in the context of cancer. 17 Recent evidence suggests that the aforementioned mechanisms are also involved in the regulation of angiogenesis; however, this modulation is highly tissue- and context-specific and can yield either pro or antiangiogenic effects. Moreover, direct crosstalk between Tregs and vascular endothelial cells is still insufficiently understood.

Although the role of regulatory T cells in modulating angiogenesis has been the focus of this review, it bears noting that the process of angiogenesis itself has immunoregulatory function. Indeed, VEGF can directly modulate development, proliferation, migration and survival of adaptive immune cells (beyond its indirect effect through inhibiting dendritic cell function and macrophage maturation). 92 , 93 Thus the influence of VEGF signaling and angiogenesis on Treg cell function warrants further discussion and review. As previously noted in section 4.2 , numerous clinical studies have shown that VEGFR-targeting drugs both prevented tumor angiogenesis in patients with solid tumors, and reduced the number of infiltrating Tregs in the tumor microenvironment. 94 , 95 No such study, however, has demonstrated a causal relationship between infiltrating Tregs and intratumoral angiogenesis. Newly formed tumor vascular endothelial cells are known to secrete immunomodulatory factors such as TGF-β and VEGF. 93 TGF-β is a pleiotropic cytokine that, amongst its many functions, can induce phenotype conversion from CD4 + Foxp3 negative T cells to CD4 + Foxp3 + T cells. 96 In addition to its role inhibiting dendritic cell maturation in the tumor microenvironment, 97 VEGF can also directly and synergistically act on Treg cell migration and possess remarkable immunosuppressive function. 93 , 98 , 99 In addition to the change in levels of Tregs in the pathological angiogenic states as demonstrated in a variety of murine disease models (e.g. diabetic ischemia 75 and abortion 16 ), Bansal et al. demonstrated that antiangiogenic heart failure Tregs are also strikingly pro-inflammatory: these lymphocytes were found to secrete IFNγ and IL-4. 78 Signaling through VEGFR-1 and VEGFR-2 have opposing effects on lymphocyte development: pathophysiologic levels of VEGF robustly inhibit T-cell development via VEGFR-2, whereas VEGFR-1 signaling decreases this inhibition. 92 VEGFR-2, but not VEGFR-1, expression was selectively found on Foxp3 high but not Foxp3 low Treg with high immunosuppressive function in one study. 98 VEGFR-2 was expressed both on the cell surface and in the nucleus. 98 VEGFR-2 present on the cell surface can translocate into the nucleus and regulate self-transcription by activating its own promoter. 100 By contrast, another study demonstrated that VEGFR-1 activation played a critical role in angiogenesis and healing of mucosal damage via accumulation of VEGFR-1 + CXCR4 + Foxp3 + Tregs in intestinal ulcerated tissue. 101 Therefore, VEGF signaling may have important implications on Treg cell survival and function, as several studies demonstrate that it is directly implicated in Treg migration and proliferation. Studies also revealed that VEGFR-2 + Treg cells have strong inhibitory effect on CD4 + T cells. 93 VEGF was shown to directly stimulate Tregs accumulation in tumor environments. 99 In a mouse model of metastatic colon cancer, VEGF played a vital role in accumulating Treg cells into tumor tissue by signaling through VEGFR-2 or its co-receptor, neuropilin-1. 99 An increased proportion of Treg cells has also been shown in human tumor tissues 61 , 102 - 106 and high levels of tumor-infiltrating Treg cells indicated poor clinical prognosis in some cancers as discussed above. 106 , 107 In addition, VEGF has been shown to directly stimulate Tregs to synthesize and excrete VEGF in the surrounding environment via VEGFR-2 signaling. 108 VEGF-A was also shown to directly stimulate proliferation of Treg cells in a VEGFR-2-dependent manner in tumor-bearing mice and metastatic colorectal cancer patients. 99

In conclusion, regulatory T cells exert an important influence on angiogenesis and their pro or antiangiogenic function is inextricably linked to the inherent tissue characteristics and the micro-environment in which they reside. Research on the contributions of regulatory T cells in promoting angiogenesis, especially in the context of tumor pathogenesis, has gained remarkable interest; meanwhile there is emerging evidence demonstrating their antiangiogenic properties in ischemia, chronic inflammation, and ocular diseases. The precise mechanisms by which Tregs exert these opposing effects on angiogenesis remain to be fully elucidated. Further investigations dissecting the immunoregulatory function of Tregs from their effect on angiogenesis will be critical to establish the therapeutic role and safety of Tregs in treating pathologic neovascularization. As the development of cell-based therapies continues to gain tremendous popularity, Treg-based therapies may beckon a new, more targeted approach to treating patients with chronic debilitating diseases caused by pathological inflammation and vascularization.

Angiogenesis is a highly coordinated process that depends on proangiogenic and antiangiogenic factors that modulate vascular endothelial cell proliferation and migration. 1 Well-regulated angiogenesis is critical during embryonic development and continues to be essential throughout life (e.g. placentation, tissue repair and wound healing). 1 , 18 Abnormal and dysfunctional angiogenesis (i.e. angiogenesis that is dysregulated due to an imbalance between proangiogenic and antiangiogenic factors) is the basis of a variety of pathological processes including tumor growth and metastasis, chronic inflammation and several ophthalmic diseases. 18 Although the formation of new mature blood vessels is a highly coordinated process whereby numerous receptors are activated by various ligands in a step-wise fashion, the rate-limiting and most crucial step is signaled through the family of vascular endothelial growth factor (VEGF) proteins. 1 VEGF was first identified, isolated and cloned over 30 years ago. 19 The VEGF gene family includes, most notably, VEGF-A, VEGF-B, VEGF-C, VEGF-D and placental growth factor (PlGF) that bind to three VEGF receptor tyrosine kinases (VEGFR-1-3), in addition to their co-receptors, neuropilin and heparan sulfate proteoglycan. 1 By binding to different receptors, VEGF proteins exert an array of distinct biological functions. 1 For example, VEGFR-1 signaling results in inhibition of dendritic cell maturation, migration of monocytes and hematopoietic stem cell survival and recruitment; VEGFR-2 activation promotes proliferation, migration and survival of endothelial cells and increases vascular permeability; and VEGFR-3 signaling is associated with lymphangiogenesis. 1 , 20 VEGF-A, often referred to as VEGF, is the key regulator of blood vessel growth through its activation of VEGFR-2. 21 VEGF-C and VEGF-D primarily regulate lymphatic angiogenesis. 1 In addition to their role in controlling the growth and differentiation of several anatomical components of the vascular system (direct effect on vascular endothelial cells), 1 the VEGF family also has important immunomodulatory effects, which have been substantially studied in tumor pathobiology (reviewed in Yang et al.) 22 and are briefly summarized in the sections below. Emerging research in the field of vascular development has shed light on the mechanisms by which blood vessel formation is regulated during development (embryonic and fetal development), its role in maintaining physiological homeostasis and its contribution to pathology. 23 Numerous animal studies have demonstrated the importance of well-regulated angiogenesis during embryonic development. 24 Animal models of VEGF (e.g. loss of a single VEGF allele) 25 and VEGF-R knockouts 26 , 27 and VEGF-A overexpression 28 result in mouse embryonic lethality with significant defects in vascular and hematopoietic cell development. 24 Homozygous loss of the VEGFR-1 (also referred to as Flt1) results in aberrant endothelial cell-cell/matrix interaction and disorganization of blood vessels, whereas loss of the VEGFR-2 (also referred to as KDR or Flk1) results in lack of endothelial cell growth and blood vessel formation. 26 , 27 Tight regulation of angiogenesis and vascular remodeling is also essential for normal tissue repair and wound healing. Normal skin wound healing provides a basic biphasic model of rapid and profound new capillary growth and regression that is regulated by several soluble factors. 29 VEGF-A is the main proangiogenic factor that is produced in response to tissue hypoxia and contributes also to vascular permeability and wound edema. 30 , 31 Several other proangiogenic factors, such as TGF-β, fibroblast growth factor-2, and platelet derived growth factor also promote wound angiogenesis after injury thereby dramatically increasing the number of capillaries in the resolving wound. 29 During the period of vascular pruning, proangiogenic factors subside and the levels of several negative regulators of angiogenesis become elevated (e.g. Sprouty2, pigment epithelium derived factor, CXCL10) causing regression of newly formed vessel until normal (pre-injury) density of blood vessel is reached. 29 VEGF secretion is upregulated in the setting of hypoxia through the expression of hypoxia-inducible factors (HIF). 32 This family of DNA-binding transcription factors regulates the expression of myriad genes involved in tissue oxygen homeostasis (cellular metabolism, proliferation, apoptosis, and migration, etc.). 32 Their effects are far reaching with over 2% of all human genes having been shown to be directly or indirectly regulated by HIF-1 in culture of arterial endothelial cells. 33 Two distinct subunits make up the heterodimeric transcription factor HIF-1: the hypoxia responsive HIF-1α and the constitutively expressed HIF-1β subunit. 34 HIF-1α has been repeatedly proven to mediate the proangiogenic response observed following tissue ischemia. 33 , 35 , 36 In addition to their critical role as “master regulators” of angiogenesis, HIF transcription factors also play a role in the innate and adaptive immune response (summarized in a review by Palazon et al.). 37 During normal gestation, uterine angiogenesis and vascular remodeling are indispensable and regulated by two key vascular growth factor receptors, VEGFR-2 and Tie2. 38 In the placental bed, various immune cells can be found that have been reported to support implantation, in part by promoting angiogenesis. These cells are directly responsible for spiral artery remodeling by producing proangiogenic cytokines, growth factors and by phagocytizing dead cells and debris. 39 Moreover, peripherally induced placental Tregs are crucial for embryonic immune-tolerance (the tolerization against fetal antigens) and the maintenance of pregnancy in humans and mice. 40 VEGFs can also act as pro-inflammatory cytokines by increasing vascular permeability, expression of endothelial adhesion molecules, and monocyte chemo-attractants. 24 Indeed, VEGF levels have been found to be elevated in numerous chronic inflammatory disease models suggesting that it may be involved in the pathogenesis of psoriasis, 41 rheumatoid arthritis, 42 asthma, 43 and allergic eye disease. 44 Other studies have provided evidence that VEGF-A acts as a pro-inflammatory mediator involved in acute 45 and chronic allograft rejection. 46 VEGF has also been noted to be important in the molecular pathogenesis of tumor growth and metastasis. 24 Indeed, VEGF secreted by tumor and tissue stromal cells induces formation of new blood vessels that are structurally anomalous and leaky. 18 In the majority of human cancer cells, VEGF is overexpressed and correlates with tumor vascular density, invasiveness, metastasis, tumor recurrence and prognosis. 18 , 47 Though the vast majority of tissues depend on a rich vascular supply for survival, as presented above, a few tissues, namely the cornea, lens, cartilage, and heart valves, are avascular under homeostatic conditions. 48 - 50 “Angiogenic privilege” denotes the active and highly sophisticated processes involved in maintaining avascularity within these distinct tissues. 5 , 51 The process of “angiogenic privilege” is still incompletely understood, with myriad of pro and antiangiogenic factors having been shown to work in opposition to inhibit vascularization in angiogenically privileged tissues. 52 A balance must be struck between proangiogenic factors such as VEGF, fibroblast growth factor, and matrix metalloproteinases and antiangiogenic factors including pigment epithelium derived factor, angiostatin, and endostatin. 5 Over the past years, extensive evidence has shown a causal relationship between VEGF expression and several eye diseases in which increased vascular permeability and neovascularization occur. 24 In retinas and vitreous bodies of patients and experimental animals with active neovascularization from ischemic retinopathies (e.g. ischemic central retinal vein occlusion, proliferative diabetic retinopathy and retinopathy of prematurity), VEGF levels are notably elevated. 24 Moreover, numerous studies of VEGF inhibitors have confirmed that VEGF plays a central role in ischemia-induced intraocular neovascularization. 19 , 24 At the level of the cornea, a transparent tissue at the front of the eye normally devoid of any vessels in order to optimize its optical properties, a wide variety of diseases including ocular surface infection, inflammation, ischemia, or trauma (i.e. chemical or thermal injury) can also cause pathologic neovascularization from the peri-corneal limbal vascular plexus. 53 As discussed above, corneal angiogenic privilege necessitates a healthy ocular surface and the secretion of a wide array of pro and antiangiogenic factors by the corneal epithelium. 53 Interestingly, despite the presence of numerous antiangiogenic factors in the corneal epithelium, under physiologic conditions, the majority of secreted VEGF-A is mainly bound to the soluble VEGFR-1. 54 Similarly, the constitutively expressed VEGFR-3 also inhibits corneal angiogenesis, therefore suggesting that corneal angiogenic privilege is a redundant process. 55 Thus, local anti-VEGF administration is also gaining popularity in treating some corneal neovascularization etiologies. 53 , 56

Angiogenesis is defined as the formation of new blood vessels from pre-existing vasculature and is indispensable for a range of physiological processes, including normal embryonic development, gestational maintenance, skeletal growth, and wound healing. 1 However, this process is susceptible to dysregulation and can lead to development of new, aberrant blood vessels. Pathological neovascularization is the key histological hallmark of tumor malignancy and progression, 2 and is also seen in several chronic inflammatory conditions such as psoriasis 3 and rheumatoid arthritis, 4 and in ocular diseases. 5 Elucidating the mechanisms that regulate angiogenesis is therefore of great therapeutic importance. Regulatory T cells (Tregs), defined as CD4 + CD25 + forkhead box P3 (FoxP3) + T cells, are known to be the most important anti-inflammatory subset of T cells that mediate immune tolerance and tissue homeostasis. 6 , 7 In the last decade, the role of Tregs in modulating angiogenesis has become the object of substantial research interests. 8 Overall, the role of Tregs in angiogenesis is both tissue- and disease-specific ( Figure 1 ). In malignancy such as solid tumors and leukemia and in reproductive pathologies such as endometriosis and infertility, Tregs are correlated with a heightened vascular response. On the other hand, Tregs have been reported to inhibit angiogenesis in tissue ischemia, chronic inflammation, and ocular tissues. Herein, the reported mechanisms of action by which Tregs modulate angiogenesis in health and disease are reviewed.