*2.2. Angiogenic Factors*

VEGF is a member of a family of six structurally related proteins, namely, VEGF-A, -B, -C, -D, -E (viral factor), and PDGF; these act by interacting with their relative receptors, which are differentially expressed in various cell types [41,65].The VEGF receptors are differentially implicated in angiogenesis stimulation (VEGF-A, -E/VEGFR-2-neuropilin (NRP)-1, -2), or lymphangiogenesis (VEGF-C, -D/VEGFR-2, -3) [41,66,67]. On the other hand, VEGF also acts on cell types different from vascular cells, modulating various biological activities and primarily tumor cell growth, spread, invasiveness, and drug resistance [68–70].

Our group demonstrated that, as by tumor cells, VEGF is also produced by all the cellular components of the tumor microenvironment and acts via autocrine and paracrine loops to carry out its activity [71,72]. In cancer cells and ST, VEGF expression is modulated by several pathways, including metabolic factor-induced pathways, such as hypoxia and hypoglycemia via ROS production [62,63,73]; lysophosphatidic acid (LPA), via activation of c-Jun N-terminal kinase (JNK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) [74]; PI3K/Akt signaling pathway [75]; and transcription factors, such as activator protein-1 (AP-1), NF-κB, and stimulatory protein-1 (SP-1) [76–78]. In thyroid carcinomas, VEGF over-expression has been correlated with increased growth, progression, invasiveness, spread, and metastasis of thyroid cancer cells [79–81]. A consistent increase in VEGF, VEGF-C, and angiopoietin-2 and their tyrosine kinase receptors VEGFR2, VEGFR3, and TEK receptor tyrosine kinase have been demonstrated in thyroid cancer versus normal thyroid tissues, and a strong correlation has been found between this overexpression and tumor size [50]. Moreover, the same authors showed that in the lymph node of metastatic thyroid tumors, there is an increase of VEGF-C expression and, at the same time, a reduced expression of TSP-1 near VEGF and angiopoietin-2 increased production, indicating the hematogenous metastasis capability of thyroid malignancies [50].

bFGF is an angiogenic growth factor that, by interaction with FGF receptor (highaffinity tyrosine kinase (TK) receptor) and with low-affinity heparan sulphate proteoglycans (co-receptors), induces activation, proliferation, chemotaxis, protease production, and vessel formation in endothelial cells [82,83]. In this way, it induces angiogenesis and modulates neovascularization during physiological (wound healing, inflammation) and pathological (atherosclerosis, cancer) conditions [84]. Several studies demonstrated that in thyroid tumors, the expression of bFGF and FGFR are both increased and play a role in tumor progression and angiogenesis [85–88]. As in other solid and hematologic cancers, in thyroid tumors bFGF acts as an angiogenic factor independently in the presence of other factors, such as VEGF, and directly stimulates endothelial and tumor cell growth [45].

MMPs are zinc-endopeptidases of the protease superfamily with specific proteolytic activity against a broad range of substrates located on the ECM [89,90]. MMPs are produced by thyroid tumor and microenvironment stromal cells and promote tumor growth, invasion, migration, and apoptosis inhibition. Moreover, they exert angiogenesis stimulation because the degradation of ECM causes the release of angiogenic factors stored in attachment with heparan sulphate [89,90]. The promotion of tumor growth is primarily related to MMP-2 and MMP-9 through activation of TGF-β [91]. Another growth factor, namely epidermal growth factor (EGF), is involved in promoting cell invasion and angiogenesis in thyroid carcinoma. It acts as a regulator of the production of MMP-9 through focal adhesion kinase (FAK) phosphorylation [92]. Natural inhibitors of MMPs are the tissue inhibitors of metalloproteinases (TIMPs), produced and released in the tumor microenvironment [93]. Published data from our group indicate that the MMPs' proangiogenic and pro-tumoral activities are related to the balance of MMPs and TIMPS in the microenvironment, and that the switch toward an invasive phenotype is mainly due to increased MMP production and release, and not to the reduction of TIMPs (Figure 1) [94,95].

**Figure 1.** The angiogenic microenvironment of thyroid cancer: interactions between cancer cells, stromal cells, and inflammatory cells to promote angiogenesis and tumor progression.
