Abstract
Regulatory T cells (Tregs) are crucial mediators of immune homeostasis. They regulate immune response by suppressing inflammation and promoting self-tolerance. In addition to their immunoregulatory role, a growing body of evidence highlights the dynamic role of Tregs in angiogenesis, the process of forming new blood vessels. Although angiogenesis is critically important for normal tissue regeneration, it is also a hallmark of pathological processes, including malignancy and chronic inflammation. Interestingly, the role of Tregs in angiogenesis has been shown to be highly tissue- and context-specific and as a result can yield either pro- or antiangiogenic effects. For these reasons, there is considerable interest in determining the molecular underpinnings of Treg-mediated modulation of angiogenesis in different disease states. The present review summarizes the role of Tregs in angiogenesis and mechanisms by which Tregs regulate angiogenesis and discusses how these mechanisms differ in homeostatic and pathological settings.
Introduction
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+ 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 (Fig. 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. In contrast, Tregs have been reported to inhibit angiogenesis in tissue ischemia, chronic inflammation, and ocular tissues. In this article, the reported mechanisms of action by which Tregs modulate angiogenesis in health and disease are reviewed.
Role of Tregs in angiogenesis. Tregs can be pro- or antiangiogenic, depending on tissues and diseases. Tregs promote angiogenesis directly by upregulating VEGF-A and/or IL-10 levels or indirectly through their effect on other immune cells. Tregs inhibit angiogenesis directly by promoting endothelial cell apoptosis via a cell contact-mediated process through DLL4 Notch and TNFR1 signaling or indirectly by modulating CD34+-circulating angiogenic cells via CCL5/CCR5 pathway. VEC, vascular endothelial cells; DLL4, δ-like 4; TNFR1, TNF receptor 1.
Role of Tregs in angiogenesis. Tregs can be pro- or antiangiogenic, depending on tissues and diseases. Tregs promote angiogenesis directly by upregulating VEGF-A and/or IL-10 levels or indirectly through their effect on other immune cells. Tregs inhibit angiogenesis directly by promoting endothelial cell apoptosis via a cell contact-mediated process through DLL4 Notch and TNFR1 signaling or indirectly by modulating CD34+-circulating angiogenic cells via CCL5/CCR5 pathway. VEC, vascular endothelial cells; DLL4, δ-like 4; TNFR1, TNF receptor 1.
Brief overview of Tregs
The concept of distinct T lymphocytes mediating immune suppression, “suppressor T cells,” was first introduced by Gershon and Kondo (9). Twenty-five years after the publication of Gershon and Kondo’s seminal work, Sakaguchi et al. (7) successfully identified CD4+ CD25+ T lymphocytes and demonstrated their role in preventing autoimmunity. 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.
Phenotype.
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 Ag presentation (11). They are characterized by their expression of the surface molecules CD4 and CD25 (the IL-2 cytokine receptor) and the transcription factor FOXP3 (12). FOXP3, is a protein that is indispensable to the normal development and function of Tregs. In humans lacking functional FOXP3, a severe multiorgan autoimmune disease, immunodysregulation, polyendocrinopathy, and enteropathy, X-linked syndrome (IPEX) occurs, underscoring the importance of this transcription factor (13, 14).
Functions.
The mechanisms by which Tregs suppress the immune system are complex and numerous. Briefly, Tregs mainly regulate the activity of effector T cells and other immune cells in the following ways: 1) by immunosuppressive cytokine production (IL-10, TGF-β, IL-35), 2) by IL-2 consumption, 3) by direct cell-to-cell interactions and cytolysis (the cytotoxic effect from granzyme A and B), and 4) 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 cross-talk between Tregs and vascular endothelial cells is still insufficiently understood.
Angiogenesis and vascular endothelial growth factor signaling
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 because of 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).
Vascular endothelial growth factor signaling pathway.
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 that bind to three VEGF receptor tyrosine kinases (VEGFR-1–3), in addition to their coreceptors, 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 that have been substantially studied in tumor pathobiology (reviewed in Ref. 22) and are briefly summarized in the sections below.
Angiogenesis in homeostasis and diseases.
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), 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, and CXCL10) causing regression of newly formed vessel until normal (preinjury) 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 Ref. 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 and 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 Ags) and the maintenance of pregnancy in humans and mice (40).
VEGFs can also act as proinflammatory 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 proinflammatory 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).
Although 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 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 that is normally devoid of any vessels 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 pericorneal 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).
Role of Tregs in promoting angiogenesis
New vessel formation is essential for the pathogenesis of many diseases, including cancer (57), chronic inflammation (58), and endometriosis (59). The role of Tregs in promoting angiogenesis has been explored in a variety of disease states, although mainly in the context of oncology because of its powerful therapeutic potential.
The association between angiogenesis and Tregs can be categorized as either of the following: 1) related to the VEGF pathways or 2) mediated through modulation of other immune cells and their release of proangiogenic cytokines and growth factors. In this work, we outline the literature demonstrating the proangiogenic function of Tregs (summarized in Table I) with an emphasis, when applicable, on the critical underlying molecular mechanisms demonstrated in each respective study.
First Author . | Year . | Experimental Model . | Tissue . | Disease/Condition . | Reported Mechanism of Action . |
---|---|---|---|---|---|
Li et al. (66) | 2019 | Murine model and cell line | Bone marrow, liver, and spleen | ALL | CD4+CD25+Foxp3+Helios+ Tregs were shown to promote angiogenesis in vitro and in mice with ALL. This proangiogenic effect was mediated by VEGF-A/VEGFR-2 pathway. Increased expression of CCL-22 was also observed. |
Leung et al. (75) | 2018 | Murine model and human ex vivo study | Gastrocnemius muscle | Peripheral artery disease secondary to TD2 | T2D gastrocnemius muscle specimens had decreased endothelial cell density, high levels of Th1 cells and low levels of Tregs. Diabetic mouse model findings were concordant with these clinical data. Tregs were also shown to directly promote angiogenesis in diabetic states, possibly through paracrine signaling. |
D’Alessio et al. (76) | 2014 | Murine model | Lung, spleen | Lung ischemia | Treg levels increase following induction of lung ischemia. Following Treg depletion, mice undergoing lung ischemia had reduced angiogenesis. This effect was nullified with exogenous Tregs. The authors propose that Tregs modulate angiogenesis indirectly through their effect on macrophages. |
Facciabene et al. (69) | 2011 | Murine model and cell lines | Ovaries | Ovarian cancer | Tregs promote angiogenesis directly by upregulating VEGF-A levels. Increased tumoral levels of Tregs are mediated by hypoxia-induced upregulation of CCL28. |
Casares et al. (73) | 2003 | Murine model | Murine cell lines, peripheral blood | Colon cancer | Depletion of Tregs indirectly decreased angiogenesis through release of IFN-γ by CD4+ effector T cells. |
Carvalho et al. (68) | 2016 | Canine model | Mammary tumor | Malignant mammary tumors | Tumors with high levels of VEGF were associated with greater Foxp3+ Treg infiltration. High levels of Foxp3+ Tregs were also associated with more aggressive clinicopathological features. |
Li et al. (67) | 2018 | Human ex vivo study | Peripheral blood and bone marrow | Pre-B ALL | The authors reported higher levels of VEGF-A expression in the bone marrow of pre-B ALL patients and a correlation between the level of bone marrow angiogenesis and Treg expression of Helios. |
Wang et al. (72) | 2017 | Human ex vivo study | Primary human cell lines | Endometriosis | The effects of endometrial stromal cell and monocyte interactions cause high levels of CCL17 and CCL22, which recruit Tregs and may increase angiogenesis indirectly through proinflammatory cytokines, such as TGF-β1. |
Zhan et al. (64) | 2012 | Human ex vivo study | Kidney | Renal cell carcinoma | The authors report a correlation between tumor-infiltrating FOXP3+ Tregs and intratumoral microvessel density. |
Giatromanolaki et al. (65) | 2008 | Human ex vivo study | Endometrium | Stage I endometrial adenocarcinoma | The authors report a correlation between FOXP3+ Treg levels and tumor vascular density. |
Ning et al. (70) | 2012 | Human ex vivo study | Kidney, peripheral blood | Renal cell carcinoma | The authors report a positive correlation between the levels of intratumoral Tregs and VEGF expression and tumor microvessel density. |
Gupta et al. (71) | 2007 | Human ex vivo study | Breast | Ductal carcinoma in situ and invasive carcinoma | The authors report an association between FOXP3 gene expression and VEGF gene expression and intratumoral microvessel density. |
Woidacki et al. (16) | 2015 | Murine model | Placental, decidual tissues | Infertility | The authors report that adoptive transfer of Tregs in abortion-prone mice promotes macrophages and decreases sFlt-1. |
Politikos et al. (109) | 2017 | Clinical study | Peripheral blood | Hematologic malignancy | The authors report a positive correlation between levels of Treg and VEGF in adults receiving umbilical cord blood transplantations. |
First Author . | Year . | Experimental Model . | Tissue . | Disease/Condition . | Reported Mechanism of Action . |
---|---|---|---|---|---|
Li et al. (66) | 2019 | Murine model and cell line | Bone marrow, liver, and spleen | ALL | CD4+CD25+Foxp3+Helios+ Tregs were shown to promote angiogenesis in vitro and in mice with ALL. This proangiogenic effect was mediated by VEGF-A/VEGFR-2 pathway. Increased expression of CCL-22 was also observed. |
Leung et al. (75) | 2018 | Murine model and human ex vivo study | Gastrocnemius muscle | Peripheral artery disease secondary to TD2 | T2D gastrocnemius muscle specimens had decreased endothelial cell density, high levels of Th1 cells and low levels of Tregs. Diabetic mouse model findings were concordant with these clinical data. Tregs were also shown to directly promote angiogenesis in diabetic states, possibly through paracrine signaling. |
D’Alessio et al. (76) | 2014 | Murine model | Lung, spleen | Lung ischemia | Treg levels increase following induction of lung ischemia. Following Treg depletion, mice undergoing lung ischemia had reduced angiogenesis. This effect was nullified with exogenous Tregs. The authors propose that Tregs modulate angiogenesis indirectly through their effect on macrophages. |
Facciabene et al. (69) | 2011 | Murine model and cell lines | Ovaries | Ovarian cancer | Tregs promote angiogenesis directly by upregulating VEGF-A levels. Increased tumoral levels of Tregs are mediated by hypoxia-induced upregulation of CCL28. |
Casares et al. (73) | 2003 | Murine model | Murine cell lines, peripheral blood | Colon cancer | Depletion of Tregs indirectly decreased angiogenesis through release of IFN-γ by CD4+ effector T cells. |
Carvalho et al. (68) | 2016 | Canine model | Mammary tumor | Malignant mammary tumors | Tumors with high levels of VEGF were associated with greater Foxp3+ Treg infiltration. High levels of Foxp3+ Tregs were also associated with more aggressive clinicopathological features. |
Li et al. (67) | 2018 | Human ex vivo study | Peripheral blood and bone marrow | Pre-B ALL | The authors reported higher levels of VEGF-A expression in the bone marrow of pre-B ALL patients and a correlation between the level of bone marrow angiogenesis and Treg expression of Helios. |
Wang et al. (72) | 2017 | Human ex vivo study | Primary human cell lines | Endometriosis | The effects of endometrial stromal cell and monocyte interactions cause high levels of CCL17 and CCL22, which recruit Tregs and may increase angiogenesis indirectly through proinflammatory cytokines, such as TGF-β1. |
Zhan et al. (64) | 2012 | Human ex vivo study | Kidney | Renal cell carcinoma | The authors report a correlation between tumor-infiltrating FOXP3+ Tregs and intratumoral microvessel density. |
Giatromanolaki et al. (65) | 2008 | Human ex vivo study | Endometrium | Stage I endometrial adenocarcinoma | The authors report a correlation between FOXP3+ Treg levels and tumor vascular density. |
Ning et al. (70) | 2012 | Human ex vivo study | Kidney, peripheral blood | Renal cell carcinoma | The authors report a positive correlation between the levels of intratumoral Tregs and VEGF expression and tumor microvessel density. |
Gupta et al. (71) | 2007 | Human ex vivo study | Breast | Ductal carcinoma in situ and invasive carcinoma | The authors report an association between FOXP3 gene expression and VEGF gene expression and intratumoral microvessel density. |
Woidacki et al. (16) | 2015 | Murine model | Placental, decidual tissues | Infertility | The authors report that adoptive transfer of Tregs in abortion-prone mice promotes macrophages and decreases sFlt-1. |
Politikos et al. (109) | 2017 | Clinical study | Peripheral blood | Hematologic malignancy | The authors report a positive correlation between levels of Treg and VEGF in adults receiving umbilical cord blood transplantations. |
Abbreviations used in this table: pre-B ALL, precursor B cell ALL; T2D, type 2 diabetes.
Treg and VEGF signaling.
Several clinical studies in oncology suggest that the presence of Tregs provides important prognostic information for survival of cancer patients (60). In addition, evidence from clinical oncologic reports established an association between high tumor VEGF-A expression, Treg presence in tumor tissues, and less favorable clinical prognosis (61). It is worth noting, however, that most of these studies provide evidence of correlation but not causation between Treg levels and VEGF signaling. Most recently, a study by Bencsikova et al. (62) demonstrated that the initial level of circulating lymphocytes in patients with metastatic colorectal cancer predicted the clinical outcomes of first-line treatment with bevacizumab, a mAb against VEGF. Interestingly, they also reported that low levels of circulating Tregs were associated with favorable treatment outcomes. A decrease in Tregs after chemotherapy in patients with ovarian cancer was reported by Coosemans et al. (63). Similarly, an association between high Treg levels and intratumoral vessel density has been reported in the context of renal cell carcinoma (64) and endometrial adenocarcinoma (65). Published reports have also demonstrated an association between the levels of Tregs and VEGF in numerous cancer models, including acute lymphoblastic leukemia (ALL) (66), precursor B cell ALL (67), malignant mammary tumor (68), ovarian cancer (69), renal cell carcinoma (70), and breast carcinoma (71). In short, in the tumor milieu, an important correlation has been established between VEGF signaling and survival and proliferation of intratumoral Tregs.
Studies providing evidence of how Tregs directly regulate VEGF signaling are scarcer. Li et al. (66) reported that CD4+CD25+Foxp3+Helios+ Tregs promoted angiogenesis in vitro and in mice with ALL via the VEGF-A/VEGFR-2 pathway. Similarly, Facciabene et al. (69) reported that Tregs promoted angiogenesis directly by upregulating VEGF-A levels in ovarian cancer. They also provided further insights into the mechanisms by which Tregs are recruited into the cancer tissue. The authors demonstrated that the increase in intratumoral levels of Tregs are mediated by hypoxia-induced upregulation of CCL28, also known as mucosae-associated epithelial chemokine (69). Wang et al. (72) demonstrated the contribution of Tregs to angiogenesis promotion in the context of endometriosis (72). The authors noted that upregulation of the chemokines CCL17 and CCL22 secreted by endometrial stromal cells and/or monocytes had the following effects: 1) induced Treg recruitment, 2) upregulated the expression of CCR4, and 3) increased TGF-β1 secretion by Tregs. These effects in turn promoted VEGF secretion by endometrial stromal cells through the p38/ERK1/2 signaling pathways (72).
Treg- and VEGF-independent angiogenesis.
Beyond their VEGF-mediated effects, Tregs can indirectly influence angiogenesis by modulating the function of other immune cells, as has been explored and suggested in the context of malignancy and ischemia. In 2003, Casares et al. (73) demonstrated using a colon cancer mouse model that reduction of Treg levels leads to a vigorous antitumoral response and a decrease in angiogenesis. The antiangiogenic effect observed was noted to be through the release of IFN-γ by CD4+ effector T cells. The critical role of IFN-γ in tumor angiogenesis is well established and remains an important concept guiding the development of immunotherapies (74).
Leung et al. (75) have also implicated CD4+ Th1 cells and Tregs in the modulation of angiogenesis. The authors noted poor vascular density, low levels of Tregs, and high levels of Th1 cells in the muscle specimens of diabetic patients with peripheral artery disease when compared with normoglycemic control biopsy specimens. When blocking CD4+ T cells indiscriminately (i.e., Th1 cells, Tregs, etc.) in two distinct diabetic murine models, the authors report an increased endothelial cell count and improved function in ischemic tissue. In an effort to discern the direct role of Tregs in angiogenesis, the authors performed a series of coculture experiments with Tregs and endothelial cells and demonstrated that Tregs or their paracrine factors (e.g., IL-10 and amphiregulin) directly promoted tube formation (75).
D’Alessio et al. (76) postulated that the proangiogenic effect of Tregs they observed in a lung ischemia mouse model could be attributed to the effect of Tregs on macrophages and the cytokines they released. They noted that Treg levels increased following induction of lung ischemia, and Treg depletion led to reduced lung angiogenesis and a decrease in the number of macrophages. They did not identify any significant phenotypical or functional changes in the macrophages after Treg depletion (76).
The role of Tregs in angiogenesis has also been explored in the context of infertility. A recent study by Woidacki et al. (16) showed that adoptive transfer of Tregs in abortion-prone mice promoted normal pregnancy development. The authors demonstrated that abortion-prone mice, which have impaired placentation and insufficient spiral artery remodeling, exhibit significantly elevated levels of soluble VEGFR-1 (16). Soluble VEGFR-1 can function as a “decoy” receptor, inhibiting angiogenesis by binding VEGF and preventing its mitogenic function on vascular endothelial cells (1). The adoptive transfer of Tregs and the subsequent upregulation of uterine macrophages at the feto–maternal interface were associated with downregulated levels of soluble VEGFR-1 (16).
In short, the aforementioned studies have linked Tregs’ angiogenic function to their well-established immunoregulatory role. As is the case with most of the literature on the angiogenic function of Tregs, these reports do not demonstrate a strong causal relationship and instead rely on observed correlations. Further research will be necessary to dissect the precise mechanisms by which Tregs augment angiogenesis.
Role of Tregs in inhibiting 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 II) is still scarce.
First Author . | Year . | Experimental Model . | Tissue . | Disease/Condition . | Reported Mechanism of Action . |
---|---|---|---|---|---|
Bansal et al. (78) | 2019 | Murine model | Peripheral blood, heart, spleen, and lymph nodes | HF | HF Tregs inhibit cardiac neovascularization via a cell contact-mediated process and TNFR1. HF Tregs modulate CD34+-circulating angiogenic cells via CCL5/CCR5. |
Deliyanti et al. (92) | 2017 | Murine model | Retina | Oxygen-induced retinopathy | Expansion of retinal Foxp3+ Tregs modulates microglial activation and decreases the levels of neovascularization after ischemic injury. |
Huang et al. (79) | 2009 | Murine model | Lung and trachea | OVA-induced airway inflammation | Tregs inhibit angiogenesis by promoting endothelial cell apoptosis via DLL4-Notch signaling and downregulating VEGF expression. This antiangiogenic effect was demonstrated to be cell contact-dependent. |
Zouggari et al. (77) | 2009 | Murine model | Calf muscle | Ischemic hindlimb | In mice with fewer endogenous Tregs, there were increased levels of neovascularization after ischemic injury. In mice with more endogenous Tregs, a reduction of postischemic neovascularization was seen. Exogenous administration of Tregs from CD28 knockout mice decreased neovascularization in Rag1−/− mice following ischemic injury. |
Teige et al. (80) | 2009 | Murine model | Ear skin | 12-O-Tetradecanoylphorbol-13-acetate–induced psoriasis | Administration of CD4+ CD25+ Tregs significantly reduced VEGF-driven inflammation of the skin. |
First Author . | Year . | Experimental Model . | Tissue . | Disease/Condition . | Reported Mechanism of Action . |
---|---|---|---|---|---|
Bansal et al. (78) | 2019 | Murine model | Peripheral blood, heart, spleen, and lymph nodes | HF | HF Tregs inhibit cardiac neovascularization via a cell contact-mediated process and TNFR1. HF Tregs modulate CD34+-circulating angiogenic cells via CCL5/CCR5. |
Deliyanti et al. (92) | 2017 | Murine model | Retina | Oxygen-induced retinopathy | Expansion of retinal Foxp3+ Tregs modulates microglial activation and decreases the levels of neovascularization after ischemic injury. |
Huang et al. (79) | 2009 | Murine model | Lung and trachea | OVA-induced airway inflammation | Tregs inhibit angiogenesis by promoting endothelial cell apoptosis via DLL4-Notch signaling and downregulating VEGF expression. This antiangiogenic effect was demonstrated to be cell contact-dependent. |
Zouggari et al. (77) | 2009 | Murine model | Calf muscle | Ischemic hindlimb | In mice with fewer endogenous Tregs, there were increased levels of neovascularization after ischemic injury. In mice with more endogenous Tregs, a reduction of postischemic neovascularization was seen. Exogenous administration of Tregs from CD28 knockout mice decreased neovascularization in Rag1−/− mice following ischemic injury. |
Teige et al. (80) | 2009 | Murine model | Ear skin | 12-O-Tetradecanoylphorbol-13-acetate–induced psoriasis | Administration of CD4+ CD25+ Tregs significantly reduced VEGF-driven inflammation of the skin. |
Tissue ischemia.
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. (77) used a mouse model of ischemic hindlimb by ligating the femoral artery. The authors reported that in knockout mice with lower levels of endogenous Tregs, neovascularization following ischemic insult increased significantly (77). Conversely, in mice with more endogenous Tregs, a reduction of postischemic neovascularization was seen. Interestingly, there was no statistical difference between the VEGF-A protein levels of the mice with downregulated endogenous Tregs when compared with the control (77). Bansal et al. (78) provided insights into the mechanisms by which Tregs inhibit angiogenesis following myocardial infarction. Using a mouse model of heart failure (HF), the authors demonstrated that HF Tregs, which were dysfunctional and proinflammatory, decrease cardiac neovascularization. This reduction is effected via a cell contact-mediated process requiring TNF-αR-1 (TNFR1). Moreover, their study demonstrated that HF Tregs modulate CD34+-circulating angiogenic cells via CCL5/CCR5 (78).
Chronic inflammation.
Two studies have substantiated the role of Tregs in inhibiting angiogenesis in inappropriately persistent inflammatory states. In 2009, Teige et al. (80) described the potential antiangiogenic effect of Tregs in association with psoriasis, an angiogenesis-dependent skin disease (3). 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. (79) also demonstrated the antiangiogenic effects of Tregs in chronic inflammatory states albeit in a different animal model. Using a mouse model of OVA-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 the δ-like 4 Notch signaling pathway, the main inhibitor of angiogenesis in the Notch signaling pathway, thereby downregulating VEGF expression (79). In summary, these reports position Tregs as downregulators of angiogenesis in states of persistent inflammation.
Ocular diseases.
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 the 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 because of 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 nonresponders 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 naive Tregs 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 (91) and subconjunctival injection of Tregs (Z. Luznik, S. Anchouche, J. Yin, and R. Dana, 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. (92) demonstrated, using a mouse model of oxygen-induced retinopathy, that expansion of retinal Foxp3+ Tregs modulated microglial activation and decreased levels of neovascularization after ischemic injury.
Effects of VEGF signaling and angiogenesis on Treg function
Although the role of Tregs 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) (93, 94). Thus the influence of VEGF signaling and angiogenesis on Treg function warrants further discussion and review.
Altered Treg function in pathological angiogenesis.
As previously noted in Treg- and VEGF-independent angiogenesis, 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 (95, 96). 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 (94). TGF-β is a pleiotropic cytokine that, among its many functions, can induce phenotype conversion from CD4+ Foxp3− T cells to CD4+ Foxp3+ T cells (97). In addition to its role in inhibiting dendritic cell maturation in the tumor microenvironment (98), VEGF can also directly and synergistically act on Treg migration and possess remarkable immunosuppressive function (94, 99, 100).
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. (78) demonstrated that antiangiogenic HF Tregs are also strikingly proinflammatory; these lymphocytes were found to secrete IFN-γ and IL-4.
Direct effect of VEGF signaling on Treg function.
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 (93). VEGFR-2 but not VEGFR-1 expression was selectively found on FOXP3high but not FOXP3low Tregs with high immunosuppressive function in one study (99). VEGFR-2 was expressed both on the cell surface and in the nucleus (99). VEGFR-2 present on the cell surface can translocate into the nucleus and regulate self-transcription by activating its own promoter (101). 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 (102). Therefore, VEGF signaling may have important implications on Treg survival and function, as several studies demonstrate that it is directly implicated in Treg migration and proliferation. Studies also revealed that VEGFR-2+ Tregs have a strong inhibitory effect on CD4+ T cells (94).
VEGF was shown to directly stimulate Tregs accumulation in tumor environments (100). In a mouse model of metastatic colon cancer, VEGF played a vital role in accumulating Tregs into tumor tissue by signaling through VEGFR-2 or its coreceptor, neuropilin-1 (100). An increased proportion of Tregs has also been shown in human tumor tissues (61, 103–107), and high levels of tumor-infiltrating Tregs indicated poor clinical prognosis in some cancers, as discussed above (107, 108). In addition, VEGF has been shown to directly stimulate Tregs to synthesize and excrete VEGF in the surrounding environment via VEGFR-2 signaling (69). VEGF-A was also shown to directly stimulate proliferation of Tregs in a VEGFR-2–dependent manner in tumor-bearing mice and metastatic colorectal cancer patients (100).
Conclusions
In conclusion, Tregs exert an important influence on angiogenesis, and their pro- or antiangiogenic function is inextricably linked to the inherent tissue characteristics and the microenvironment in which they reside. Research on the contributions of Tregs 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.
Footnotes
This work was supported by the National Institutes of Health (5R01EY012963 to R.D. and 5K12EY016335 to R.D., principal investigator, and J.Y., trainee). The funding source had no direct involvement in study design, in the collection, analysis and interpretation of data, in the writing of the report, or in the decision to submit the article for publication.
References
Disclosures
R.D. is a consultant for Dompe, Kala, Aldeyra, Novaliq, and Santen. The other authors have no financial conflicts of interest.