MABC950 Sigma-AldrichAnti-Aminopeptidase N/CD13 Antibody, clone SL13

CD13 is a multifunctional cell surface molecule that regulates inflammatory and angiogenic mechanisms in vitro, but its contribution to these processes in vivo or potential roles in stem cell biology remains unexplored. We investigated the impact of loss of CD13 on a model of ischemic skeletal muscle injury that involves angiogenesis, inflammation, and stem cell mobilization. Consistent with its role as an inflammatory adhesion molecule, lack of CD13 altered myeloid trafficking in the injured muscle, resulting in cytokine profiles skewed toward a prohealing environment. Despite this healing-favorable context, CD13(KO) animals showed significantly impaired limb perfusion with increased necrosis, fibrosis, and lipid accumulation. Capillary density was correspondingly decreased, implicating CD13 in skeletal muscle angiogenesis. The number of CD45-/Sca1-/α7-integrin+/β1-integrin+ satellite cells was markedly diminished in injured CD13(KO) muscles and adhesion of isolated CD13(KO) satellite cells was impaired while their differentiation was accelerated. Bone marrow transplantation studies showed contributions from both host and donor cells to wound healing. Importantly, CD13 was coexpressed with Pax7 on isolated muscle-resident satellite cells. Finally, phosphorylated-focal adhesion kinase and ERK levels were reduced in injured CD13(KO) muscles, consistent with CD13 regulating satellite cell adhesion, potentially contributing to the maintenance and renewal of the satellite stem cell pool and facilitating skeletal muscle regeneration.

To determine the role of CD13 as an adhesion molecule in trafficking of inflammatory cells to the site of injury in vivo and its function in wound healing following myocardial infarction induced by permanent coronary artery occlusion.Seven days post-permanent ligation, hearts from CD13 knockout (CD13(KO)) mice showed significant reductions in cardiac function, suggesting impaired healing in the absence of CD13. Mechanistically, CD13(KO) infarcts showed an increase in small, endothelial-lined luminal structures, but no increase in perfusion, arguing against an angiogenic defect in the absence of CD13. Cardiac myocytes of CD13(KO) mice showed normal basal contractile function, eliminating myocyte dysfunction as a mechanism of adverse remodelling. Conversely, immunohistochemical and flow cytometric analysis of CD13(KO) infarcts demonstrated a dramatic 65% reduction in infiltrating haematopoietic cells, including monocytes, macrophages, dendritic, and T cells, suggesting a critical role for CD13 adhesion in inflammatory trafficking. Accordingly, CD13(KO) infarcts also contained fewer myofibroblasts, consistent with attenuation of fibroblast differentiation resulting from the reduced inflammation, leading to adverse remodelling.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.

CD13 is a large cell surface peptidase expressed on the monocytes and activated endothelial cells that is important for homing to and resolving the damaged tissue at sites of injury. We showed previously that cross-linking of human monocytic CD13 with activating Abs induces strong adhesion to endothelial cells in a tyrosine kinase- and microtubule-dependent manner. In the current study, we examined the molecular mechanisms underlying these observations in vitro and in vivo. We found that cross-linking of CD13 on U937 monocytic cells induced phosphorylation of a number of proteins, including Src, FAK, and ERK, and inhibition of these abrogated CD13-dependent adhesion. We found that CD13 itself was phosphorylated in a Src-dependent manner, which was an unexpected finding because its 7-aa cytoplasmic tail was assumed to be inert. Furthermore, CD13 was constitutively associated with the scaffolding protein IQGAP1, and CD13 cross-linking induced complex formation with the actin-binding protein α-actinin, linking membrane-bound CD13 to the cytoskeleton, further supporting CD13 as an inflammatory adhesion molecule. Mechanistically, mutation of the conserved CD13 cytoplasmic tyrosine to phenylalanine abrogated adhesion; Src, FAK, and ERK phosphorylation; and cytoskeletal alterations upon Ab cross-linking. Finally, CD13 was phosphorylated in isolated murine inflammatory peritoneal exudate cells, and adoptive transfer of monocytic cell lines engineered to express the mutant CD13 were severely impaired in their ability to migrate into the inflamed peritoneum, confirming that CD13 phosphorylation is relevant to inflammatory cell trafficking in vivo. Therefore, this study identifies CD13 as a novel, direct activator of intracellular signaling pathways in pathophysiological conditions.

Human cytomegalovirus (HCMV) infects cells by a series of processes including attachment, penetration via fusion of the envelope with the plasma membrane, and transport of the viral DNA to the nucleus. The details of the early events of HCMV infection are poorly understood. We have recently reported that CD13, human aminopeptidase N, a metalloprotease, is present on blood cells susceptible in vitro to HCMV infection (C. Söderberg, S. Larsson, S. Bergstedt-Lindqvist, and E. Möller, J. Virol. 67:3166-3175, 1993). Here we report that human CD13 is involved in HCMV infection. Antibodies directed against human CD13 not only inhibit infection but also block binding of HCMV virions to susceptible cells. Compounds known to inhibit aminopeptidase activity block HCMV infection. HCMV-resistant murine fibroblasts have heightened susceptibility to HCMV infection after transfection with complementary DNA encoding human CD13. A significant increase in binding of HCMV was observed in the CD13-expressing transfectants compared with neomycin-resistant control mouse cells. However, murine fibroblasts transfected with mutant CD13, lacking a portion of the aminopeptidase active site, remained susceptible to HCMV infection. Thus, human CD13 appears to mediate HCMV infection by a process that increases binding, but its enzymatic domain is not necessary for infection.