**8. Clinical impact of angiogenic inhibitors**

In contrast, DLL4 expressed in ECs stimulates Notch 3 receptors located on adjacent cells (e.g., colorectal cancer or T-cell acute lymphoblastic leukemia cells) to activate tumor progression from dormant to active phase [60]. Such findings consider Notch pathways a potential therapeutic target for the design and development of novel anti-angiogenic compounds, although the Notch cell signaling shows a mixed behavior of tumor progression and inhibition in clini-

**Figure 2.** VEGF-induced intracellular signaling in tumor angiogenesis and angiogenic inhibitors with targeted active sites: The binding of vascular endothelial growth factors (VEGF) to respective transmembrane receptors stimulates a plethora of intracellular signaling pathways which regulate nuclear transcription factors for altered gene expressions of normal cell responses including loss of tumor suppression, activation of hypoxia inducible factor (HIF-α), increased receptor tyrosine kinase activity, increased tumor cell growth, and repression of oncogene mutations. Angiogenic inhibitors to their targeted active sites are also shown with numerical circles in the figure. Only anti-angiogenic compounds approved by the US Food and Drug Administration (FDA) for the treatment of numerous solid tumors and carcinomas are depicted where circle 1 represents growth factor inhibitors (bevacizumab, aflibercept); circle 2, growth factor receptor inhibitors (sunitinib, sorafenib); circle 3, RAS inhibitors (tipifarnib, lonafarnib); circle 4, RAF inhibitors (sorafenib); circle 5, HIF-1α inhibitors (geldanamycin, chetomin, echinomycin, 2ME2); circle 6, PI3K inhibitors (wortmannin, LY294002); circle 7, AKT inhibitors (FARA-A); and circle 8, mTOR inhibitors (rapamycin and analogues). JNK = JUN N-terminal kinase; MAPK = mitogen-activated protein serine/threonine kinase, MAPKK = MAPK kinase, PDK1 = phosphoinositidedependent protein kinase-1; PLC = phospholipase C; PtdInP2 = phosphatidylinositol 4,5-bisphosphate, Ins (1,4,5) =

310 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

Transforming growth factor is a ubiquitously expressed paracrine polypeptide of approximately 25 kDa molecular weight [61]. TGF 1 to TGF 3 are three highly homologous isoforms of the polypeptide and discovered in humans and mammals [62]. TGF-β is initially synthesized as a zymogen, and after secretion, an associated peptide is proteolytically sliced to release

Active TGF-β binds to constitutively active serine/threonine kinase TGFBR2 receptors to activate TGFBR1 in a heterodimer complex which controls transcription via activation of canonical signal pathways mediated by a family of SMAD proteins (SMAD1-5) (**Figure 3b**) [64]. The

cal assays [58, 59].

inositol 1,4,5-triphosphates.

**7.3. Transforming growth factor-β (TGF-β)**

active form of the growth factor [63].

The discovery and development of angiogenic inhibitors have raised the hopes to treat a lot of tumors and carcinomas and ultimately to reduce the morbidity and mortality related to tumors and cancers [67]. Five classes of anti-angiogenic compounds have established and are still under investigation on the basis of potential antitumor drug targeted sites including proteases inhibitors (MMP synthesis inhibitors), ECs proliferation and migration inhibitors, vascular endothelial growth factor inhibitors, cell matrix protein inhibitors, and angiogenic inhibitors with unique mechanisms (**Figure 2**) [68, 69].

Although the anti-angiogenic compounds approved by the FDA show therapeutic efficacy in some categories of cancer as monotherapy, however, sufficient published data recently reveal this fact that angiogenic inhibitors are best therapeutic choices for tumors when used in combination with traditional chemotherapies [70, 71]. However, one would not expect in the first instance that angiogenic inhibitors might reduce the intratumoral delivery of cytotoxic agents (traditional chemotherapy) by decreasing perfused blood vessels with impaired blood flow and decrease drug transport in treated tumor cells [48, 72]. It would also increase tissue hypoxia and inhibit tumor cell proliferation although proliferating cells are an easy target for chemotherapy [48, 72].

To overcome such hurdles and to enhance synergistic therapeutic potential of chemo and anti-angiogenic drugs when used in combination, Kerbel proposed three mechanistic approaches in this scenario to be adopted; first, normalization of tumor microvessels by anti-angiogenic compounds [73, 74]; second, maximum tolerated dose chemotherapy during the break periods of successive courses [72, 75], and third, use of known chemotherapeutic agents having anti-angiogenic effects [72]. The additional advantages of chemotherapy while improving their anti-angiogenic effects may be grabbed by adopting "metronomic chemotherapy" which states that "the administration of chemotherapeutic agents at relatively low, minimally toxic doses on a frequent schedule of administration at regular close intervals, with no prolonged drug-free breaks [76, 77]." By such approaches, endothelial cells are directly killed, and progenitor ECs are suppressed in circulation. Furthermore, minimal use of toxic doses lowers the frequency of adverse events in treated patients [72, 76, 77]. Such treatment strategies may be adopted for a prolonged period of time with angiogenic inhibitors in the treatment of advanced solid tumors with little side effects as validated by phase II clinical trials; however, phase III clinical studies are extensively demanded in this direction [70, 71].

## **9. Conclusions**

In vivo, in vitro, and in ova assays for angiogenesis assessment are the reliable approaches in basic research and to some extent in real-world clinical practices. However, in vivo systems are difficult to perform and time consumable, and the process of quantification is much complicated than in vitro assays. Conversely, these are relatively better due to complex nature of the vascular response to the test compound. In vitro angiogenesis assays may perform in a short period and provide the accurate and reliable outcome of angiogenic processes. Mouse models based angiogenesis assays have also standardized to an improved understanding of tumor angiogenesis and lymphangiogenesis. Similarly, such models are also used to assess vasculogenesis and arteriogenesis in ischemic heart diseases, blindness, psoriasis, and arthritis. Angiogenesis assessment always plays a focal role to determine the pathogenesis and progression of certain challenging diseases in human populations in particular human cancer. An ample understanding of angiogenesis research in tumor progression, by knowing the molecular mechanisms and cellular pathways, also opens the ways to design and develop effective anti-angiogenic inhibitors. The manipulation of the human genome in a precise and predictable manner due to recently developed molecular techniques has opened new gates for the generation of more reliable models for angiogenesis studies and the testing of new therapeutic strategies.
