Sandra R. Severson

Cell Proliferation and Epidermal Growth Factor Signaling in Non-small Cell Lung Adenocarcinoma Cell Lines Are Dependent on Rin1

Rin1 is a Rab5 guanine nucleotide exchange factor that plays an important role in Ras-activated endocytosis and growth factor receptor trafficking in fibroblasts. In this study, we show that Rin1 is expressed at high levels in a large number of non-small cell lung adenocarcinoma cell lines, including Hop62, H650, HCC4006, HCC827, EKVX, HCC2935, and A549. Rin1 depletion from A549 cells resulted in a decrease in cell proliferation that was correlated to a decrease in epidermal growth factor receptor (EGFR) signaling. Expression of wild type Rin1 but not the Rab5 guanine nucleotide exchange factor-deficient Rin1 (Rin1Δ) complemented the Rin1 depletion effects, and overexpression of Rin1Δ had a dominant negative effect on cell proliferation. Rin1 depletion stabilized the cell surface levels of EGFR, suggesting that internalization was necessary for robust signaling in A549 cells. In support of this conclusion, introduction of either dominant negative Rab5 or dominant negative dynamin decreased A549 proliferation and EGFR signaling. These data demonstrate that proper internalization and endocytic trafficking are critical for EGFR-mediated signaling in A549 cells and suggest that up-regulation of Rin1 in A549 cell lines may contribute to their proliferative nature.

Electron microscopic and immunological evidence of nanobacterial-like structures in calcified carotid arteries, aortic aneurysms, and cardiac valves

Background: Definitive mechanisms causing vascular calcification are unknown. Experiments were designed to evaluate explanted calcified human vascular tissue for the presence of nanometer-scale objects hypothesized to be a type of bacteria associated with calcified geological specimens and human kidney stones (Folk RL; J Sed Petrology 63:990-999, 1993; Kajander EO, et al., PNAS 95:8274-9279, 1998).

Distribution and function of recombinant endothelial nitric oxide synthase in transplanted hearts

Introducing recombinant genes into donor hearts may offer a therapeutic intervention that could potentially attenuate the complications of heart transplantation, including rejection, infection and accelerated atherosclerosis. In the cardiovascular system, reduced bioactivity of endothelial nitric oxide is a feature of atherosclerosis and vascular injury. Nitric oxide is an arterial vasodilator that also inhibits proliferation of vascular smooth muscle cells and platelet aggregation. Experiments were designed to determine the distribution of adenoviral-mediated transfer of recombinant endothelial nitric oxide synthase gene (eNOS) and the effect of recombinant gene expression on the function of transplanted hearts. Adenoviral vectors for (a) bovine eNOS (AdeNOS) or (b) beta-galactosidase (AdLacZ; control) were infused into two groups (n = 12, per group) of explanted rat hearts. The transduced hearts were then implanted heterotopically into the abdomen of syngeneic recipient rats. After four days, the hearts were excised and examined for distribution and function of the recombinant genes. Polymerase chain reaction (PCR) verified the presence of the recombinant eNOS gene in eNOS-transduced but not in beta-galactosidase-transduced hearts; reverse transcriptase-PCR identified mRNA for eNOS in AdeNOS-transduced hearts. NOS activity (conversion of tritiated L-arginine to citrulline) was greater in homogenates of AdeNOS-compared to AdLacZ-transduced hearts. Positive immunoreactivity for eNOS was present in cardiomyocytes predominantly in eNOS-transduced hearts. Myocardial contractility and coronary blood flow, as determined using a Langendorff preparation, were not different between hearts transduced with AdeNOS or AdLacZ. These results suggest that, up to four days post transplantation, adenoviral-mediated transfer of eNOS into transplanted hearts is possible. However, expression of the recombinant protein did not result in measurable changes in myocardial contractility or coronary perfusion.

Endothelial function in pigs transgenic for human complement regulating factor.

Background. Expression of human complement regulating factor (hCRF) in porcine organs prevents hyperacute rejection of these organs after xenotransplantation to nonhuman primates. Experiments were designed to characterize endothelial and smooth muscle function of arteries from pigs transgenic for hCD46. Methods. Arterial blood from outbred pigs transgenic for hCD46 expression and nontransgenic animals of the same lineage was analyzed for angiotensin-converting enzyme (ACE), C-type natriuretic peptide (CNP), and nitric oxide. Aortic endothelial cells were prepared for measurement of mRNA or activity for nitric oxide synthase (NOS). Rings cut from femoral and pulmonary arteries were suspended in organ chambers for measurement of isometric tension. Results. CNP was significantly greater, ACE was similar, and nitric oxide was significantly less in plasma from transgenic compared with nontransgenic pigs. Neither mRNA nor activity of NOS differed between the groups. Endothelium-dependent relaxations to bradykinin and acetylcholine but not the calcium ionophore were shifted significantly to the left in femoral and pulmonary arteries from hCD46 transgenic pigs compared with nontransgenic pigs. The ACE-inhibitor captopril augmented relaxations similarly in both groups, but NG-monomethyl-L-arginine (L-NMMA) did not inhibit relaxations in rings from transgenic pigs. Conclusions. Data suggest that expression of hCD46 on endothelium of pigs selectively augments endothelium-dependent relaxations to bradykinin by increased release of endothelium-derived factors other than nitric oxide. There does not seem to be any change in activity of ACE or NOS with expression of the human protein. Increased relaxations to bradykinin may be beneficial in lowering vascular resistance when transgenic organs are used for xenotransplantation.

1059-13 Detection and propagation of calcified nanostructures from human aneurysms

Detection and Propagation of Calcified Nanostructures From Human Aneurysms John C. Lieske, Vivek Kumar, Gerard Farell-Baril, Shihui Yu, Jon E. Charlesworth, Ewa Rzewuska-Lech, Peter LaBreche, Sandra R. Severson, Virginia M. Miller, Mayo Clinic Rochester, Rochester, MN Background: Mechanisms leading to vascular calcification remain incompletely understood. Nanometer-sized, mineralized structures recognized by a commercially available monoclonal antibody (8D10, Nanobac OY) are present in calcified human aneurysms. These structures were not detected by TUNEL staining, suggesting they were not apoptotic bodies. The 8D10 antibody is directed against nanobacteria, a controversial, slow-growing, and calcifying microorganism. Therefore, experiments were designed to determine whether structures from aneurysms are viable, nano-sized organisrns. Methods: Aneurysms (n=3) collected as surgical waste were decalcified, sterile filtered (0.22 μm), and cultured in DMEM containing gamma-irradiated calf serum. Results: In 2 of 3 cultures micron-sized particles visible by light microscopy increased in number over 4-6 weeks. The negative culture came from an aneurysm without stainable nanoparticles. With transmission electron microscopy (EM), cultured particles showed an inner core surrounded by a shell of calcium phosphate (documented via energy dispersive microanalysis). After dissolution of the shell with EDTA, spherical structures of 50-100 nm were seen by scanning EM. These cultured particles incorporated [H]uridine at a rate 2.3 times greater than control cultures of DMEM containing serum and inorganic hydroxyapatite (HA) crystals (P<0.01). Therefore, these nanostructures appear to synthesize RNA. Particles cultured from aneurysms also stained with the 8D10 antibody, and SDS-PAGE of extracted proteins revealed multiple distinct bands, including one (Mr 47 kDa) recognized by the 8D10 antibody. The pattern of proteins extracted from inorganic HA crystals incubated with DMEM and calf serum did not contain the 47-kDa band recognized by the 8D10 antibody. Conclusion: In conclusion, these results suggest that viable nano-sized organisms are present within calcified human arterial tissue. A cause and effect relationship between the presence of these organisms and development of arterial calcification remains to be determined. This was presented at the American College of Cardiology Annual Scientific Session 2004 in New Orleans, LA on March 8, 2004

Characteristics of endothelin receptors in acutely rejecting transplanted lungs.

Experiments were designed to characterize endothelin receptors in bronchi and parenchyma of transplanted lungs during acute rejection. Third-order bronchi from autografted or allografted lungs were either cut into rings and suspended in organ chambers for the measurement of isometric force or frozen for isolation of membrane proteins. Lung parenchyma was prepared for histology or isolation of membrane protein. The grade of rejection was 2.74+/-0.17 (n= 19) in allotransplanted lungs; evidence of infection was present in 58% of the transplanted lungs. In organ chamber experiments, endothelin 1 (which stimulates endothelin A receptors) caused comparable contraction of bronchi from autotransplanted and allotransplanted rejecting lungs. Endothelin 3 (which stimulates endothelin A and B receptors) caused contractions of bronchi from autotransplanted lungs which were not different from those caused by endothelin 1. In contrast, contractions caused by endothelin 3 were reduced in bronchi from rejecting allotransplanted lungs. The magnitude of contractions caused by endothelin 3 was reduced further when infection was present with rejection. Competitive inhibition of 125I-endothelin 1 by endothelin 3 was significant for a two-site binding model in membranes prepared from all bronchi and lung parenchyma. The total number of binding sites (Bmax) was reduced significantly in bronchi and parenchyma from rejecting lungs with or without infection. The relative proportions of high-affinity and low-affinity binding sites did not change. Affinities of both high- and low-affinity receptors were not altered with rejection. These results indicate that at least two subtypes of endothelin receptors are present on canine bronchial smooth muscle and parenchyma. The number of endothelin receptors associated with bronchial contractions is reduced with rejection of lung allografts.