The role of microenvironment in tumor angiogenesis

A wide variety of protein polypeptides are distributed in an organism. Some of these protein polypeptide factors have a role in promoting neovascularization and are known as angiogenic factors. These play an important role in regulating both normal and abnormal angiogenesis. The most important of these for tumor angiogenesis are the three peptide families of VEGF, FGF, and platelet-derived growth factor (PDGF).

VEGF is a 40–45 kD dimeric cysteine-rich protein that was discovered in 1983 and is highly conserved among mammals. It was found to increase the permeability of tumor blood vessels and promote the formation of ascites [58]. In 1989, the VEGF protein was first isolated and its role in the process of angiogenesis was identified [59, 60]. The human VEGF family has multiple members. Among them, VEGFA was identified first, and is the most specific angiogenesis-inducing factor. VEGF is commonly referred to as VEGFA. The VEGFA gene located on chromosome 6p21. 3 extends over 28 kb in length and consists of eight exons and seven introns. VEGFA mRNA undergoes alternative splicing during its maturation and generates seven spliceosomes: VEGF121, VEGF145, VEGFA162, VEGF165, VEGF183, VEGF189, and VEGF206. Each spliceosome can bind to different receptors and perform different functions [61]. VEGF regulates tumor angiogenesis by binding to its receptor (VEGFR1–3) and activates intracellular signaling pathways (Fig. 3). The VEGF receptor can be divided into three domains: extracellular VEGF-binding domain, transmembrane domain, and intracellular domain (tyrosine activation domain). VEGF binds to the extracellular domain of VEGFR to phosphorylate intracellular tyrosine residues of VEGFR, and activates intracellular signaling pathways [62]. VEGFR1 and VEGFR2 are predominantly expressed in vascular endothelial cells; however, high expression is also observed in macrophages and tumor cells [63]. VEGFR3 is highly expressed in endothelial lymphocytes [64]. It is well known that endothelial cells play a pivotal role in tumor angiogenesis; therefore, the regulation of endothelial cell signaling plays an important role in tumor angiogenesis and development. Studies have shown that the binding of VEGF to VEGFR2 can induce VEGFR2 phosphorylation at Y1175, leading to PKCγ activation, Raf-MEK-MAPK signal transduction, and proliferation of endothelial cells [65, 66]. Moreover, VEGF has also been reported to promote endothelial cell proliferation by activating MAPK signaling via growth factor receptor binding 2 and Shc [67]. In addition, VEGF-activated VEGFR2 can activate PI3K/Akt signaling via direct binding with Gab1/Gab2, and promote the proliferation of vascular endothelial cells [68]. VEGF can also modulate FAK activity by VEGFR2 activation of c-Src, shb, or RhoA, or binding with integrin proteins to alter PI3K/Akt signaling and endothelial cell proliferation [69‐72]. Furthermore, Axl plays an important role in VEGF-mediated Src/TSAd activation of PI3K/Akt [73]. The migration of endothelial cells is an important prerequisite for tumor angiogenesis. PI3K/Akt signaling is responsible for the expression of MMPs and other molecules required for invasion and metastasis of tumor cells [74]. Additionally, PI3K/Akt signaling can activate Cdc42, Rho, and Rac proteins, and promote VEGF-mediated invasion and metastasis of endothelial cells [75, 76]. Vascular permeability is required for tumor angiogenesis; Src plays an important role in VEGF-induced vascular permeability. VEGFA can activate c-Src and Yes proteins via VEGFR and phosphorylate adhesion factors such as VE-cadherin and beta-catenin in the presence of TSAd to increase vascular permeability. In addition, the phosphorylation of VE-cadherin via VEGF-induced activation of Rac can disrupt endothelial cell-cell interaction and increase the permeability of blood vessels [77]. Furthermore, activated endothelial nitric oxide synthase (eNOS) plays an important role in vascular permeability by releasing nitric oxide in blood vessels. VEGF can activate nuclear factor of activated T-cells by activating PLCγ via the PI3K/Akt signaling pathway to modulate intracellular calcium ion concentration or increase eNOS production to increase vascular permeability [78, 79]. VEGFR can also activate the P38/MAPK signaling pathway through Nck and Fyn binding, inducing alterations in the cytoskeleton and promoting tube formation in endothelial cells. In a melanoma study, VEGF was found to promote vasculogenic mimicry by activating PI3K/Akt signaling [80]. In addition, vasculogenic mimicry markers such as VE-cadherin, MMP2, and MMP9 have been shown to be modulated by VEGFA. These results suggest that VEGFA plays an important role in vasculogenic mimicry in tumor cells. The tumor microenvironment plays a key role in tumor angiogenesis as numerous cells here can secrete VEGF protein,

FGF and its receptor play an important role in cell proliferation, migration, survival, and differentiation. FGF interacts with its cofactor heparan sulfate or Klotho, and dimerizes with FGFR to exert its physiological function [81, 82]. The FGF family is divided into six subfamilies according to their sequence homology and development characteristics and are composed of 18 members in mammals. bFGF—also called FGF2—was discovered first, and plays a crucial role in tumor angiogenesis [83]. The binding of FGF to FGFR promotes autophosphorylation of FGFR, which induces a conformational change from inactive to active. Activated FGFR further activates FGFR substrate 2 and recruits PLCγ, which consequently, recruits growth factor receptor binding 2 to activate PKC, RAS/RAF/MEK/MAPK signaling and PI3K/AKT signaling. FGFR also activates the p38 MAPK and JNK signaling pathways, STAT signaling pathway, and ribosomal protein S6 kinase 2 [84, 85]. Furthermore, FGF2 activates intracellular signaling and promotes angiogenesis by interacting with the membrane-bound integrinαVβ3 [86]. FGF can modulate these signaling pathways to stimulate neovascular formation and maturation by promoting endothelial cell proliferation and ECM degradation, and altering the expression of intercellular adhesion molecules [87]. FGF2 plays an important role in tumor angiogenesis. Studies have shown that FGF2 secretion by neutrophils in the tumor microenvironment can promote angiogenesis in metastatic liver tumors [88]. Similarly, a long non-coding RNA (lncRNA), MALAT1, was found to promote angiogenesis in thyroid cancer tissues by increasing FGF2 secretion from tumor-associated macrophages [89]. Finally, FGF2 exerts a synergistic effect with PDGF-BB to increase the interaction between endothelial and mural cells, and promote tumor angiogenesis and metastasis [90]. Therefore, decreasing FGF expression in the tumor microenvironment can be an important antitumor therapeutic strategy in future.

PDGF plays an important role in embryonic development, cell growth and differentiation, and tissue repair. Several pathological conditions occur due to aberrant expression of PDGF and its receptors [91]. PDGFA expression is upregulated in several cancers. PDGFA increases tumor angiogenesis in both ovarian and hepatocellular carcinoma cells by promoting MEK/ERK signaling [92, 93]. PDGF-BB can induce proliferation, migration, and tube formation of vascular endothelial cells in addition to increasing VEGF expression [94, 95]. PDGF-BB can also facilitate peripheral migration of pericytes to surrounding tumors to promote tumor angiogenesis and vasculogenic mimicry formation [96, 97]. PDGF-D can promote tumor angiogenesis of colorectal cancer by activating Notch1/Twist1 signaling and recruiting macrophages to tumor tissues [98, 99].

The TGF-β family of peptide signaling molecules include TGFB1-B3, activins, inhibins, Nodal, bone morphogenetic proteins (BMPs), and growth differentiation factors [100]. The TGF-β family plays an important role in embryonic development and regulation of tissue homeostasis, and its aberrant expression is associated with several diseases. TGF-β plays an important role in the growth, invasion, metastasis, and immune escape of tumors. Although, the role of TGF-β in tumor angiogenesis remains controversial. While some studies have shown that TGF-β can promote tumor angiogenesis, a few other studies have revealed its inhibitory effect. TGF-β is highly expressed in several cancers; however, high TGF-β expression is negatively correlated with patient prognosis and positively correlated with tumor growth and angiogenesis [101]. In colorectal and renal cancer cells, TGF-β overexpression promotes tumor angiogenesis, and the addition of neutralizing antibodies to TGF-β1 markedly reduces tumor angiogenesis [100]. One study demonstrated that VEGF and TGFBR1 (ALK5) inhibitors can synergistically promote tumor angiogenesis by potentially blocking the downstream effectors of ALK5 such as Smad2 and Smad3 [102]. However, according to recent studies, Smad3—a tumor-promoting factor—can increase VEGF expression and promote tumor angiogenesis, and Smad2—a tumor-suppressing factor—can inhibit tumor metastasis and angiogenesis [103]. These studies also confirmed that TGF-β inhibits tumor growth in the early stages of tumorigenesis and promotes tumor growth in the advanced stages. Therefore, the potential targeting of TGF-β for tumor therapy requires further research. BMPs can also increase tumor angiogenesis. A Matrigel plug experiment revealed that BMP2 can increase angiogenesis in lung cancer cells. Moreover, BMP2 antagonists blocked the angiogenic effect of BMP2 [104, 105]. Similar results were obtained in breast cancer and melanoma cells [106, 107]. These results suggest that BMPs can either directly induce VEGF expression or recruit endothelial precursor cells to facilitate secretion of VEGF and placental growth factor (PIGF) from mesenchymal stem cells to promote tumor angiogenesis. The regulatory mechanisms of TGF-β activity in tumor angiogenesis is not well understood and requires further research.

Interferons (IFNs) are biologically active glycoproteins secreted by cells, following bacterial or viral infection. IFNs possess antiviral, antibacterial, antitumor, and immune regulatory activity, and can inhibit angiogenesis [108]. In neuroendocrine tumors, IFN-α downregulates VEGF expression by inhibiting SP1 or SP3 and reduces angiogenesis [109]. IFN-2α downregulates HIF-1α expression by inhibiting PI3K or MAPK signaling, resulting in suppression of VEGF expression and tumor angiogenesis [110]. However, some studies have showed that IFN-α can promote the formation of vasculogenic mimicry. IFN-α can increase HIF-1a expression and promote vasculogenic mimicry in the kidney, breast, ovarian, and colorectal cancer cells by activating PI3K/AKT/mTOR signaling [111]. IFN-β can effectively inhibit endothelial precursor cell-mediated tumor angiogenesis. The inhibitory effect of INF-γ on tumor angiogenesis has been extensively reported. However, recent studies have showed that IFN-γ can promote tumor angiogenesis in mesenchymal stem cells. Moreover, IFN-γ increases HIF-1α expression in MSCs, which in turn, upregulates VEGF expression and promotes tumor angiogenesis [112].

TNF was originally named owing to its ability to directly cause hemorrhagic necrosis in tumors. However, later studies found that in addition to killing tumor cells, TNF can function as an inflammatory mediator. TNF-α is produced by kinase-activated macrophages and bind to specific homotrimeric receptors on the cell membrane. TNF-α can activate caspase protease, JNK, and NF-κB signaling pathways to induce inflammation and promote cell growth, differentiation, and apoptosis. Previous studies have showed that TNF-α can inhibit tumor angiogenesis. However, recent studies have demonstrated that TNF-α exerts pro-angiogenic activity in tumors. TNF-α promotes human umbilical vein endothelial cell (HUVEC) migration and tube formation capacity by activating PI3K, p38, JNK, ERK, and NF-κb signaling pathways [113]. In prostate cancer cells, TNF-α induces VEGFA expression by activating downstream NF-κb signaling, and promotes endothelial cell angiogenesis. In contrast, miR-130b suppresses angiogenesis in prostate cancer cells by inhibiting TNF-α [114]. In choriocarcinoma cells, TNF-α can promote angiogenesis by activating the PIGF/VEGFR1 and VEGFA/VEGFR2 pathways [115]. Recent studies have suggested that TNF-α can exert both anti-angiogenic and pro-angiogenic effects in different tumor tissues, likely due to differences in its expression.

Interleukins (ILs) are a class of cytokines that play an important role in the maturation, activation, proliferation, and regulation of immune cells. In addition, they participate in various physiological and pathological processes. IL-1 is an inflammatory cytokine that is important for tumor angiogenesis. IL-1 was originally named hemopoietin-1 because of its angiogenic effect [116]. The IL-1 family cytokines bind to their receptors and activate downstream signaling pathways. Upon activation, MyD88 forms a complex with interleukin-receptor associated kinase 4 to activate downstream MAPK and IKK/NF-κb signaling pathways [117]. The secretion of IL-1α by colorectal cancer cells can increase the proliferation and tube formation capacity of HUVECs [118]. Furthermore, IL-1α exerts pro-angiogenic effects in glioma, pancreatic cancer, and prostate cancer cells by activating JNK signaling and increasing VEGF expression [119‐122]. As IL-1β and IL-1 bind to the same receptor, both can promote angiogenesis by inducing the expression of ANG-1, Tie-2, and VEGF via JNK and p38 MAPK signaling [123]. In melanoma cells, both IL-1α and IL-1β can promote tumor angiogenesis by activating NF-κB signaling pathways to induce the expression of IL-6, IL-8, intercellular adhesion molecule-1, and tissue factor [124]. Thus, IL-1 signaling promotes angiogenesis by activating JNK or p38 MAPK and NF-κB signaling, and the IL-1 receptor antagonist inhibits tumor angiogenesis by blocking IL-1 signaling [125]. In addition, several other members of the IL-1 family participate in tumor angiogenesis. IL-33 promotes colorectal cancer cell growth and liver metastasis by regulating the tumor microenvironment [126]. IL-33 can also activate endothelial cells, increase vascular permeability, and promote angiogenesis via ST2/TRAF6-Akt-eNOS signaling. Furthermore, IL-33 can phosphorylate VE-cadherin to facilitate disruption of intercellular junctions of endothelial cells and enhance vascular permeability [127]. Lastly, IL-33 can downregulate the expression of tight junction proteins such as occludins, and reduce the barrier integrity of endothelial cells [128]. In glioma cells, IL-18 facilitates VEGF-induced migration and forms a positive feedback loop wherein VEGF can upregulate IL-18 expression via ERK1/2 signaling [129]. IL-18 can promote angiogenesis via Src and JNK signaling pathways [130]. However, a few studies have demonstrated that IL-33 and IL-18 can exert anti-angiogenic effects in different tissues according to the local environment. Recent studies have showed that IL-36γ can enhance the tube formation capacity of HUVECs in a VEGF-dependent manner [131]. TGF-β can increase the ability of IL-37 to bind to the activated protein receptor-like kinase 1 receptor complex, and upregulates the expression of angiogenesis-related genes [132]. In addition, IL-37 can induce proliferation and migration of endothelial cells, increase capillary formation, and promote the survival of endothelial cells via ERK1/2 and AKT signaling [133]. IL-6 promotes angiogenesis via IL-6/STAT3/VEGFA signaling in hepatocellular carcinoma, cervical cancer, and glioma carcinoma cells [134‐136]. IL-8 can increase endothelial cell migration via PI3K/Rac1/RhoA signaling, and promote angiogenesis in prostate cancer cells by increasing MMP9 expression [137, 138]. Furthermore, IL-8 can be used as an independent prognostic factor for patients with early-stage prostate cancer [139]. Lastly, IL-8 can promote tumor angiogenesis in non-small-cell lung cancer, colorectal cancer, and glioma cells [140‐142]. IL-17 can promote tumor angiogenesis [143]. It can increase VEGF expression via activation of STAT3 signaling in non-small-cell lung cancer and glioma cells, and IL-6, IL-8, and VEGF expression via activation of STAT1 signaling in lung adenocarcinoma cells [144‐146]. Moreover, IL-17 can stimulate fatty acid β-oxidation in endothelial cells [147]. A few studies have also demonstrated that IL-22 possess pro-angiogenic activity [148]. In conclusion, ILs found in the tumor microenvironment can promote angiogenesis.