II. Angiogenic activity
In addition to the catalytic activity, cell binding sites which encompass residues 60-68 of the surface loop as well as asparagine-109 are necessary for angiogenesis. The variants undergoing alterations of these residues lack any angiogenic activity while the enzymatic activity remains intact. Inversely, replacing the surface loop in RNAse A (residues 59-73) with the corresponding region of ANG (residue 57-70) bestows a neovascularization activity to the RNAse A.
1) Basement membrane degradation
Amino acid residues from Lys60 to Asn68 of the ANG constitute a cell surface receptor binding site. Accordingly, a 42 kDa endothelial cell surface protein was identified as an ANG binding protein, which was later found to be a smooth muscle type alpha-actin. The ANG-actin complex dissociates from the cell surface and activates a tissue type plasminogen activator, thus accelerating degradation of the basement membrane and extracellular matrix that allows endothelial cells to penetrate or migrate through the extracellular matrix more easily, an initial step of neovascularization. Furthermore, fibulin-1, an important molecule for stabilization of the blood vessel wall, binds to ANG, suggesting that the ANG-fibulin-1 complex modulates new blood vessel formation and stabilization.
2) Signal transduction
Besides the 42 kDa ANG receptor, a 170 kDa molecule later found on the endothelial surface is responsible for signal transduction, an important process leading to cell proliferation. ANG activates several secondary message cascades such as extracellular signal related kinase 1/2 (ERK1 and ERK2), protein kinase B/Akt, and stress-associated protein kinase/c-Jun N-terminal kinase (SAPK/JNK).
3) Nuclear translocation
The nuclear mechanisms underlying the function of ANG remain elusive. Internalization could involve cell surface ANG binding to proteins as well as to other molecules such as proteoglycans, followed by endocytosis. In this event, ANG interacts directly with intracellular protein alpha-actinin-2 followed by translocation into the nucleus through the nuclear pore in a passive manner. After nuclear retention, ANG binds to carrier proteins through a sequence 29-IMRRRGL-35 (nuclear localization signal) of ANG and to the ANG-binding element of ribosomal DNA (CTCT repeats) and subsequently, stimulates ribosomal RNA transcription. Nuclear translocation is essential for cell proliferation since it is considered a third messenger and promotes gene activation and transcription events, and inhibition of the nuclear translocation of angiogenin abolishes ANG-induced angiogenesis. Interestingly, the expression of cell surface receptors responsible for internalization as well as for the nuclear translocation of ANG also depends on the cell density.

III. Roles of ANG in physiological angiogenesis
The above biological events, which are distinct from those of RNAse A, are regulated tightly by the cell density-dependent expression of ANG receptors. The discovery of the uniquely regulated expression of ANG receptors provides us with the following conceivable mechanisms for ANG related angiogenesis. In the region where neovascularization is required, ANG binds to the endothelial surface 42 kDa receptor, and the ANG-42 kDa receptor complex dissociates from the cell surface and stimulates proteolytic activity, thus facilitating the penetration of endothelial cells through the extracellular matrix. After the leading cells migrate away, the endothelial cell density in the vicinity of migrating cells might be sparse, and such cell sparsity triggers the endothelial proliferation machinery that includes signal transduction, ANG internalization, and nuclear translocation. A 170 kDa receptor is one of the receptors responsible for this orchestrated process. Once the microenvironment is filled up with the sufficient amount of endothelial cells and the vascular network is established, such cell proliferating events diminish. Therefore, the above cell density dependent biological events are intelligent mechanisms where the proliferation machinery and subsequent angiogenic switch are on when neovascularization is needed while they are off to prevent unwanted angiogenesis.