CD13 deficiency leads to increased oxidative stress and larger atherosclerotic lesions.

BACKGROUND AND AIMS: Atherosclerosis is an inflammatory cardiovascular disorder characterized by accumulation of lipid-loaded macrophages in the intima. Prolonged accumulation leads to apoptosis of macrophages and eventually to progression of lesion development. Prevention of macrophage accumulation within the intima has been shown to reduce lesion formation. Since CD13 mediates trafficking of macrophages to sites of injury and repair, we tested the role of CD13 in atherosclerosis. METHODS: CD13+/+Ldlr-/- and CD13-/-Ldlr-/- (low density lipoprotein receptor) mice were fed basal or high fat diet (HFD) for 9, 12 and 15 weeks. Mice were euthanized and aortic roots along with innominate arteries were analyzed for atherosclerotic lesions. Cellular mechanisms were determined in vitro using CD13+/+ and CD13-/- bone marrow derived macrophages (BMDMs) incubated with highly oxidized low-density lipoprotein (oxLDL). RESULTS: At the 9 and 12 week time points, no differences were observed in the average lesion size, but at the 15 week time point, CD13-/-Ldlr-/- mice had larger lesions with exaggerated necrotic areas. CD13+/+ and CD13-/- macrophages endocytosed similar amounts of oxLDL, but CD13-/- macrophages generated higher amounts of oxidative stressors in comparison to CD13+/+ macrophages. This increased oxidative stress was due to increased nitric oxide production in oxLDL treated CD13-/- macrophages. Accumulated oxidative stress subsequently led to accelerated apoptosis and enhanced necrosis of oxLDL treated CD13-/- macrophages. CONCLUSIONS: Contrary to our prediction, CD13 deficiency led to larger atherosclerotic lesions with increased areas of necrosis. Mechanistically, CD13 deficiency led to increased nitric oxide production and consequently, greater oxidative stress.

Patient-derived heavy chain antibody targets cell surface HSP90 on breast tumors.

BACKGROUND: Monoclonal antibodies have been used to effectively treat various tumors. We previously established a unique strategy to identify tumor specific antibodies by capturing B-cell response against breast tumor antigens from patient-derived sentinel lymph nodes. Initial application of this approach led to identification of a tumor specific single domain antibody. In this paper we optimized our previous strategy by generating heavy chain antibodies (HCAbs) to overcome the deficiencies of single domain antibodies. Here we identified and characterized a heavy chain antibody (HCAb2) that targets cell surface HSP90 antigen on breast tumor cells but not normal cells. METHODS: Eight HCAbs derived from 4 breast cancer patients were generated using an in vitro expression system. HCAbs were screened against normal breast cells (MCF10A, HMEC) and tumor cell lines (MCF7, MDA-MB-231) to identify cell surface targeting and tumor specific antibodies using flow cytometry and immunofluorescence. Results observed with cell lines were validated by screening a cohort of primary human breast normal and tumor tissues using immunofluorescence. Respective antigens for two HCAbs (HCAb1 and HCAb2) were identified using immunoprecipitation followed by mass spectrometry. Finally, we generated MDA-MB-231 xenograft tumors in NOD scid gamma mice and performed in vivo tumor targeting analysis of HCAb1 and HCAb2. RESULTS: Flow cytometry screen revealed that HCAb2 selectively bound to the surface of MDA-MB-231 cells in comparison to MCF10A and MCF7 cells. HCAb2 showed punctate membrane staining on MDA-MB-231 cells and preferential binding to human breast tumor tissues in comparison to normal breast tissues. In primary breast tumor tissues, HCAb2 showed positive binding to both E-cadherin positive and negative tumor cells. We identified and validated the target antigen of HCAb2 as Heat shock protein 90 (HSP90). HCAb2 also selectively targeted MDA-MB-231 xenograft tumor cells in vivo with little targeting to mouse normal tissues. Finally, HCAb2 specifically targeted calnexin negative xenograft tumor cells. CONCLUSIONS: From our screening methodology, we identified HCAb2 as a breast tumor specific heavy chain antibody targeting cell surface HSP90. HCAb2 also targeted MDA-MB-231 tumor cells in vivo suggesting that HCAb2 could be an ideal tumor targeting antibody.

AMPKα2 Suppresses Murine Embryonic Fibroblast Transformation and Tumorigenesis.

AMP-activated kinase (AMPK) is a key metabolic sensor and stress signaling kinase. AMPK activity is known to suppress anabolic processes such as protein and lipid biosynthesis and promote energy-producing pathways including fatty acid oxidation, resulting in increased cellular energy. In addition, AMPK localizes to centrosomes during cell division, plays a role in cellular polarization, and directly targets p53, affecting apoptosis. Two distinct catalytic AMPKα isoforms exist: α1 and α2. Multiple reports indicate that both common and distinct functions exist for each of the 2 α isoforms. AMPK activation has been shown to repress tumor growth, and it has been suggested that AMPK may function as a metabolic tumor suppressor. To evaluate the potential role of each of the AMPKα isoforms in modulating cellular transformation, susceptibility to Ras-induced transformation was evaluated in normal murine embryonic fibroblasts (MEFs) obtained from genetically deleted AMPKα1- or AMPKα2-null mice. This study demonstrated that while AMPKα1 is the dominant AMPK isoform expressed in MEFs, only the AMPKα2-null MEFs displayed increased susceptibility to H-RasV12 transformation in vitro and tumorigenesis in vivo. Conversely, AMPKα1-null MEFs, which demonstrated compensation with increased expression of AMPKα2, displayed minimal transformation susceptibility, decreased cell survival, decreased cell proliferation, and increased apoptosis. Finally, this study demonstrates that AMPKα2 was selectively responsible for targeting p53, thus contributing to the suppression of transformation and tumorigenic mechanisms.