**2. TGF-β signaling pathways**

terminus is latency-associated protein (LAP), also referred to as the prodomain. Regardless of this processing by furin, the mature and LAP domains remain associated by noncovalent bonds to form the small latent complex (SLC). This complex subsequently covalently interacts with a second gene product, the latent TGF-β binding protein (LTBP), and is incorporated into a larger latent complex (LLC) that associates with the extracellular matrix (ECM) [6]. Threedimensional crystal structure of porcine latent TGF-β1 shows a conformation that resembles a ring-like shape [7]. Two domains were defined in the structure: (i) an arm domain that contains an integrin-binding Arg-Gly-Asp (RGD) peptide motif and (ii) a "straitjacket" domain where the mature TGF-β is encased. At the opposite end of the arm domain, LTBP binds the

After secretion, the LLC complex interacts with various ECM proteins, such as fibronectin and fibrillin, and is maintained in an inactivated form [8]. TGF-β is activated by different mechanisms, including interactions with integrins, alterations in pH and extracellular proteases. αν integrin, which forms heterodimers with five different β integrin subunits (β1, β3, β5, β6 and β8), that bind to LAP-TGF-β1 and LAP-TGFβ-3 [9, 10]. However, only ανβ6 and ανβ8 have been shown to activate the latent TGF-β complex [11]. Activation by both ανβ6 and ανβ8 integrins requires the RGD motif in LAP. Activation by ανB6 requires an intact cytoplasmic domain [12, 13] and the presence of other ECM proteins [14]. Activation by ανβ8, however,

**Figure 1.** TGF-β processing and activation. **(a)**TGF-β precursor undergoes proteolysis at its N-terminus (black arrow head) which results in the removal of its signal peptide. **(b)**In a second proteolytic cleavage event by furin (blue arrow head), the precursor is separated into a large LAP or prodomain (gray) and the mature TGF-β (red) and **(c)**Schematic view of the closed ring structure (left) and unfastened straitjacket (right) conformation corresponding to the inactive

prodomain forming the "ring head" [7] (**Figure 1**).

4 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

LAP-TGF-β and mature TGF-β, respectively.

Signaling is regulated by three major receptors: TGF-β receptor type I (TβRI), type II (TβRII) and type III (TβRIII). In general, TGF-β binds TβRIII, which facilitates its delivery to TβRII, a constitutively active kinase, leading to the subsequent phosphorylation and activation of TβRI. In humans, there are seven TβRIs, also known as activin receptor-like kinases (ALK), and five TβRIIs [5]. In most cells, ALK-5 forms a heterodimer with TβRII bound to TGF-β, which activates the ALK-5 kinase domain via phosphorylation of its GS domain. This receptor activation propagates intracellular signaling through 'canonical' effector proteins mothers against decapentaplegic homolog 2/SMAD family member 2 **(**Smad2) and Smad3, which are transcription factors. Once phosphorylated, these Smad proteins form a complex with Smad4 leading to nuclear translocation and initiation of genes transcription. In most normal cells, TGF-β-mediated activation of Smads leads to inhibition of cell growth. More specifically, the Smad2/3-4 complex partners with foxhead box O (FOXO) factors to activate p21Cip1 (*CDKN1A*), which inhibits cyclin-dependent kinase 1(CDK1), resulting in cell cycle arrest. Similarly, TGF-β can also activate p15Inkab (*CDKN2B*), the CDK4 inhibitor, through the SMAD2-3/4-FOXO1 axis (**Figure 2**) [5].

**Figure 2.** TGF-β canonical pathway. **(a)**In normal cells and early stages of cancer TGF-β promotes cell cycle arrest. Repressors of the pathway are shown in red. Blue dots represent protein phosphorylation and **(b)**in endothelial cells, an alternative pathway promotes cell proliferation.

Likewise, TGF-β acts as a cytostatic factor by decreasing c-Myc expression and downregulating the inhibitor of DNA-binding protein (ID) 1 and ID3 transcription factors. ID1 and ID3 are involved in differentiation, cell cycle progression and self-renewal of stem cells [21, 22]. TGF-β elicits c- Flk-1myc repression by promoting SMAD3 binding to a repressing Smadbinding element (RSBE) at the c-myc promoter [23]. c-Myc can be recruited to the promoters of *CDKN1A* and *CDKN2B* by the Myc-interacting zinc-finger (MIZ-1). This blocks CDK expression and results in apoptosis [24]. Additionally, in endothelial cells (ECs), TGF-β can target a second receptor type 1, ALK-1, which signals through Smad1/5/8 and stimulates angiogenic factors, such as interleukin 1 receptor-like 1 and ID1 (**Figure 2**) [25].

Several proteins are known to antagonize canonical TGF-β signaling. For example, (i) PI3K activates AKT which phosphorylates the SMADs-FOXO complex and inhibits its translocation to the nucleus [21], (ii) foxhead box G1 (FOXG1) inhibits the SMADs-FOXO complex [21], (iii) SMAD7 can trigger TβRI for proteosomal degradation by recruiting SMAD-specific E3 ubiquitin protein ligase (SMURF1) and SMURF2 [26], (iv) SMAD6 blocks SMAD1 through SMAD4 binding, (v) Erk proteins phosphorylate SMADs and inhibit their nuclear translocation, (vi) BAMBI, a pseudoreceptor, dimerizes with TβRI leading to its inactivation, (vii) FKBP12 binds to TβRI and impedes its phosphorylation, activation and signaling [27] and (viii) protein arginine N-methyltransferase 1 (PRMT1) methylates SMAD6 and allows BMP signaling through SMADs1/5 [28–30].
