CD13/aminopeptidase N is a transmembrane peptidase that is induced in the vasculature of solid tumors and is a potent angiogenic regulator. Here, we demonstrate that CD13 controls endothelial cell invasion in response to the serum peptide bradykinin by facilitating signal transduction at the level of the plasma membrane. Inhibition of CD13 abrogates bradykinin B 2 receptor internalization, leading to the attenuation of downstream events such as bradykinin-induced activation of Cdc42 and filopodia formation, and thus affects endothelial cell motility.Investigation into mechanisms underlying this block led us to focus on B 2 R internalization via membrane-dependent mechanisms. Membrane disruption by depletion of cholesterol or trypsinization halts B 2 R internalization, invasion, and filopodia formation, which can be recovered with addition of cholesterol. However, this functional recovery is severely impaired in the presence of CD13 antagonists, and the distribution of membrane proteins is disordered in treated cells, suggesting a role for CD13 in plasma membrane protein organization. Finally, exogenous expression of wild-type but not mutant CD13 further alters protein distribution, suggesting peptidase activity is required for CD13's regulatory activity. Therefore, CD13 functions as a novel modulator of signal transduction and cell motility via its influence on specific plasma membrane organization, thus regulating angiogenesis. IntroductionBradykinin has long been recognized as a component of an array of potent serum factors that maintain and regulate tissue perfusion by controlling the integrity of endothelial cells. This nonapeptide is the principal effector of the kallikrein-kinin system which functions in many normal and disease-related processes including pain perception, vascular homeostasis, smooth muscle contraction, coagulation, and fibrinolysis (reviewed in Blaukat 1 and Prado et al 2 ). Recently, bradykinin and its kininogen precursor have been implicated in angiogenesis, where the ischemic environment stimulates bradykinin production. In this setting, bradykinin acts immediately as a vasodilator to increase tissue perfusion and later as a long-term angiogenic stimulator. [3][4][5][6][7][8] CD13 is a cell-surface peptidase that was originally defined as a myeloid-specific hematopoietic marker. 9 More recently, however, we have shown that while normal endothelial cells are CD13 Ϫ , neovessels in developing tumors express high levels of CD13. 10 This induction is mediated at the transcriptional level in response to angiogenic stimuli in the tumor microenvironment 11 through signals transduced via the Ras/MAPK pathway. 12 Additional data support the notion that CD13 peptidase activity is required for endothelial invasion and morphogenic phases of tumor neovessel formation. 11 CD13's role as a regulator of angiogenesis is clear: CD13 rescues angiogenesis in the presence of inhibitors of the Ras/MAPK pathway, 12 and CD13 inhibition prevents tumor growth. 10 However, the precise mechanisms mediating CD13'...

The robust and consistent expression of the CD13 cell surface marker on very early as well as differentiated myeloid hematopoietic cells has prompted numerous investigations seeking to define roles for CD13 in myeloid cells. To address the function of myeloid CD13 directly, we created a CD13 null mouse and assessed the responses of purified primary macrophages or DCs from WT and CD13 null animals in cell assays and inflammatory disease models, where CD13 has been implicated previously. We find that mice lacking CD13 develop normally with normal hematopoietic profiles except for an increase in thymic but not peripheral T cell numbers. Moreover, in in vitro assays, CD13 appears to be largely dispensable for the aspects of phagocytosis, proliferation, and antigen presentation that we tested, although we observed a slight decrease in actin-independent erythrocyte uptake. However, in agreement with our published studies, we show that lack of monocytic CD13 completely ablates anti-CD13-dependent monocyte adhesion to WT endothelial cells. In vivo assessment of four inflammatory disease models showed that lack of CD13 has little effect on disease onset or progression. Nominal alterations in gene expression levels between CD13 WT and null macrophages argue against compensatory mechanisms. Therefore, although CD13 is highly expressed on myeloid cells and is a reliable marker of the myeloid lineage of normal and leukemic cells, it is not a critical regulator of hematopoietic development, hemostasis, or myeloid cell function.

In the ischaemic heart, while compensatory mechanisms apparently relieve potential angiogenic defects, CD13 is essential for proper trafficking of the inflammatory cells necessary to prime and sustain the reparative response, thus promoting optimal post-infarction healing.

There is much evidence that pigment-epithelium-derived factor (PEDF) is a potent antiangiogenic cytokine which inhibits retinal and choroidal neovascularization by inducing apoptosis in activated vascular endothelial cells. Furthermore, the regulation of PEDF appears to be linked to the regulation of vascular endothelial growth factor (VEGF), one of the most potent inducers of intraocular neovascularization. Previous studies have established that thermal photocoagulation, the mainstay in the therapy of various neovascular diseases of the posterior segment, results in a decrease in intraocular concentrations of VEGF and other angiogenic growth factors, thereby inhibiting active retinal neovascularization. In the current study, we sought to determine whether thermal photocoagulation has the potential to regulate the expression of PEDF in human retinal pigment epithelial (RPE) cells. Cultures of RPE cells were photocoagulated with a 532-nm diode laser. Subsequently, RNA was isolated for RT-PCR, and whole-cell extracts and precipitated cell culture supernatant were subjected to Western blot analysis. According to our results, PEDF mRNA and protein are significantly upregulated after photocoagulation. Moreover, PEDF protein was found to be secreted in the cell culture medium.

The CD13/am inopeptidase N cell surface peptidase was originall y identified as a marker for tumors of the hematopoietic system. Recent evidence howe ver extends the role ofCDI3/APN to solid tumors as well where it is postulated to regulate angiogenes is and tumor metastasis.