Impaired angiogenesis in aminopeptidase N-null mice

Abstract

Aminopeptidase N (APN, CD13; EC 3.4.11.2) is a transmembrane metalloprotease with several functions, depending on the cell type and tissue environment. In tumor vasculature, APN is overexpressed in the endothelium and promotes angiogenesis. However, there have been no reports of in vivo inactivation of the APN gene to validate these findings. Here we evaluated, by targeted disruption of the APN gene, whether APN participates in blood vessel formation and function under normal conditions. Surprisingly, APN-null mice developed with no gross or histological abnormalities. Standard neurological, cardiovascular, metabolic, locomotor, and hematological studies revealed no alterations. Nonetheless, in oxygen-induced retinopathy experiments, APN-deficient mice had a marked and dose-dependent deficiency of the expected retinal neovascularization. Moreover, gelfoams embedded with growth factors failed to induce functional blood vessel formation in APNnull mice. These findings establish that APN-null mice develop normally without physiological alterations and can undergo physiological angiogenesis but show a severely impaired angiogenic response under pathological conditions. Finally, in addition to vascular biology research, APN-null mice may be useful reagents in other medical fields such as malignant, cardiovascular, immunological, or infectious diseases.CD13 ͉ knockout mice ͉ retinopathy ͉ vasculogenesis T he aminopeptidases are a large family of proteolytic enzymes that affect protein maturation, degradation, and regulation (1, 2). Aminopeptidase N (APN) is a membrane-bound zincdependent metalloprotease originally identified as a surface marker in myeloid cells (3,4). APN is widely distributed in many cell types, and its role in hydrolyzing unsubstituted N-terminal residues with neutral side chains varies in different locations. In the epithelium of the renal proximal tubule, APN cleaves its only known natural substrate, angiotensin (ang) III, to ang IV; in synaptic membranes, APN metabolizes enkephalins and endorphins; in the heart, it is an integral component of cardiac remodeling postmyocardial infarction (5-10); and in the respiratory system, APN is the cell surface receptor for certain human coronaviruses and potentially for the severe acute respiratory syndrome (SARS) virus (11-13). Additionally, APN functions in signal transduction, cell cycle control, and differentiation (14, 15).We have developed an in vivo system by using ligand peptides displayed on the surface of phage to study organ-and tumorspecific vascular homing; this methodology enables the identification of vascular markers (16,17). We have isolated phage displaying an asparagine-glycine-arginine (NGR)-containing peptide in a tumor-homing selection and have shown that these phage bind selectively to angiogenic blood vessels. When coupled to a cytotoxic drug (18) or fused to a proapoptotic peptide (19) or to tumor necrosis factor (20), NGR-targeted compounds were more effective and less toxic than the respective controls. The cell surface receptor fo...

Paladin (X99384) is expressed in the vasculature and shifts from endothelial to vascular smooth muscle cells during mouse development

“…However, the dynamics of their expression pattern is not known. Furthermore, when taking genes outside of the core cluster into account, there are additional vascular enriched genes expressed in both endothelial and mural cells, e.g., CD13 (aminopeptidase N; Rangel et al, 2007), and Gpr124 (Kuhnert et al, 2010). Thus, vascular genes are expressed; (i) in both endothelium and mural cells (Paladin, Gpr124), (ii) predominantly in endothelium (CD31, VE-cadherin) or (iii) preferentially in smooth muscle/pericyte (a-SMA, RGS5).…”

CD13-positive bone marrow-derived myeloid cells promote angiogenesis, tumor growth, and metastasis

“…Furthermore, by using CD13-null neonatal mice, we demonstrated an impaired angiogenic response in the oxygen-induced retinopathy-of-prematurity model, which quantifies retinal pathological neovascularization in response to hypoxia (22). We have also shown that CD13-null mice displayed dosedependent reduced tumor growth after implantation with either B16-F10 melanoma or Lewis lung carcinoma (LLC) cells, largely because of impaired angiogenesis (12).…”

“…All of the animal studies were approved through the Institutional Animal Care and Use Committee of the University of Texas M. D. Anderson Cancer Center. CD13 WT and KO BALB/c and C57BL/6 mice were generated as described (12,22). Recipient 8-to 12-wk-old CD13 WT and KO mice were whole body-irradiated in a cobalt irradiator at 12 Gy per mouse (two rounds of radiation at 6 Gy each, with a 2-h recovery interval).…”

Phase Ib study of NGR–hTNF, a selective vascular targeting agent, administered at low doses in combination with doxorubicin to patients with advanced solid tumours

“…CD13 is a membrane-bound metalloprotease, and is thought to have an important role in chemokine processing and tumour invasion, and therefore is considered an attractive target for inhibiting angiogenesis. Recently, in a CD13-null mice model it was shown that although aminopeptidase-N activity is not essential for embryonic and foetal development, including de novo blood vessel formation (i.e., vasculogenesis) and normal adult function, it is crucial for the pathological development of new blood vessels from existing blood vessels (i.e., angiogenesis) in disease (Rangel et al, 2007).…”