Robert M. Strieter, John A. Belperio, Michael P. Keane
Francis X. McCormack, Jeffrey A. Whitsett
W. Kline Bolton
Michael D. Schneider
Steven Grant, Sarah Spiegel
Joseph L. Witztum
Deficiency of the Golgi enzyme UDP-Gal:N-acetylglucosamine β-1,4-galactosyltransferase I (β4GalT I) (E.C.126.96.36.199) causes a new congenital disorder of glycosylation (CDG), designated type IId (CDG-IId), a severe neurologic disease characterized by a hydrocephalus, myopathy, and blood-clotting defects. Analysis of oligosaccharides from serum transferrin by HPLC, mass spectrometry, and lectin binding revealed the loss of sialic acid and galactose residues. In skin fibroblasts and leukocytes, galactosyltransferase activity was reduced to 5% that of controls. In fibroblasts, a truncated polypeptide was detected that was about 12 kDa smaller in size than wild-type β4GalT I and that failed to localize to the Golgi apparatus. Sequencing of the β4GalT I cDNA and gene revealed an insertion of a single nucleotide (1031-1032insC) leading to premature translation stop and loss of the C-terminal 50 amino acids of the enzyme. The patient was homozygous and his parents heterozygous for this mutation. Expression of a corresponding mutant cDNA in COS-7 cells led to the synthesis of a truncated, inactive polypeptide, which localized to the endoplasmic reticulum.
Bengt Hanßke, Christian Thiel, Torben Lübke, Martin Hasilik, Stefan Höning, Verena Peters, Peter H. Heidemann, Georg F. Hoffmann, Eric G. Berger, Kurt von Figura, Christian Körner
Increased inducible nitric oxide synthase (iNOS) expression is a component of the immune response and has been demonstrated in cardiomyocytes in septic shock, myocarditis, transplant rejection, ischemia, and dilated cardiomyopathy. To explore whether the consequences of such expression are adaptive or pathogenic, we have generated a transgenic mouse model conditionally targeting the expression of a human iNOS cDNA to myocardium. Chronic cardiac-specific upregulation of iNOS in transgenic mice led to increased production of peroxynitrite. This was associated with a mild inflammatory cell infiltrate, cardiac fibrosis, hypertrophy, and dilatation. While iNOS-overexpressing mice infrequently developed overt heart failure, they displayed a high incidence of sudden cardiac death due to bradyarrhythmia. This dramatic cardiac phenotype was rescued by specific attenuation of transgene activity. These data implicate cardiomyocyte iNOS overexpression as sufficient to cause cardiomyopathy, bradyarrhythmia, and sudden cardiac death.
Imran N. Mungrue, Robert Gros, Xiaomang You, Asif Pirani, Azar Azad, Tamas Csont, Richard Schulz, Jagdish Butany, Duncan J. Stewart, Mansoor Husain
Atherosclerosis is characterized by vascular inflammation and associated with systemic and local immune responses to oxidized LDL (oxLDL) and other antigens. Since immunization with oxLDL reduces atherosclerosis, we hypothesized that the disease might be associated with development of protective immunity. Here we show that spleen-associated immune activity protects against atherosclerosis. Splenectomy dramatically aggravated atherosclerosis in hypercholesterolemic apoE knockout (apoE°) mice. Transfer of spleen cells from atherosclerotic apoE° mice significantly reduced disease development in young apoE° mice. To identify the protective subset, donor spleen cells were divided into B and T cells by immunomagnetic separation before transfer. Protection was conferred by B cells, which reduced disease in splenectomized apoE° mice to one-fourth of that in splenectomized apoE° controls. Protection could also be demonstrated in intact, nonsplenectomized mice and was associated with an increase in antibody titers to oxLDL. Fewer CD4+ T cells were found in lesions of protected mice, suggesting a role for T-B cell cooperation. These results demonstrate that B cell–associated protective immunity develops during atherosclerosis and reduces disease progression.
Giuseppina Caligiuri, Antonino Nicoletti, Bruno Poirier, Göran K. Hansson
We have demonstrated that murine thymic dendritic cells (DCs) isolated from donor mice have the capability to home to thymi of fully allogeneic recipients after intravenous injections, where they induce T cell deletions and prolong donor-strain airway and skin graft survival. In contrast, infused splenic DCs immigrated poorly to thymi, and did not affect graft survival. These findings suggest that preferential homing may be an important mechanistic difference among subpopulations of DCs that mediate immune functions and illustrate a novel methodology that could have utility for induction of specific immunologic nonreactivity to allografts, or other disease-associated antigens.
Steven R. Duncan, Nickolas G. Capetanakis, Brian R. Lawson, Argyrios N. Theofilopoulos
To investigate the physiological role of the α1D-adrenergic receptor (α1D-AR) subtype, we created mice lacking the α1D-AR (α1D–/–) by gene targeting and characterized their cardiovascular function. In α1D–/– mice, the RT-PCR did not detect any transcript of the α1D-AR in any tissue examined, and there was no apparent upregulation of other α1-AR subtypes. Radioligand binding studies showed that α1-AR binding capacity in the aorta was lost, while that in the heart was unaltered in α1D–/– mice. Non-anesthetized α1D–/– mice maintained significantly lower basal systolic and mean arterial blood pressure conditions, relative to wild-type mice, and they showed no significant change in heart rate or in cardiac function, as assessed by echocardiogram. Besides hypotension, the pressor responses to phenylephrine and norepinephrine were decreased by 30–40% in α1D–/– mice. Furthermore, the contractile response of the aorta and the pressor response of isolated perfused mesenteric arterial beds to α1-AR stimulation were markedly reduced in α1D–/– mice. We conclude that the α1D-AR participates directly in sympathetic regulation of systemic blood pressure by vasoconstriction.
Akito Tanoue, Yoshihisa Nasa, Takaaki Koshimizu, Hitomi Shinoura, Sayuri Oshikawa, Takayuki Kawai, Sachie Sunada, Satoshi Takeo, Gozoh Tsujimoto
Tumors have been thought to initiate as avascular aggregates of malignant cells that only later induce vascularization. Recently, this classic concept of tumor angiogenesis has been challenged by the suggestion that tumor cells grow by co-opting preexisting host vessels and thus initiate as well-vascularized tumors without triggering angiogenesis. To discriminate between these two mechanisms, we have used intravital epifluorescence microscopy and multi-photon laser scanning confocal microscopy to visualize C6 microglioma vascularization and tumor cell behavior. To address the mechanisms underlying tumor initiation, we assessed the expression of VEGF, VEGF receptor-2 (VEGFR-2), and angiopoietin-2 (Ang-2), as well as endothelial cell proliferation. We show that multicellular aggregates (<< 1 mm3) initiate vascular growth by angiogenic sprouting via the simultaneous expression of VEGFR-2 and Ang-2 by host and tumor endothelium. Host blood vessels are not co-opted by tumor cells but rather are used as trails for tumor cell invasion of the host tissue. Our data further suggest that the established microvasculature of growing tumors is characterized by a continuous vascular remodeling, putatively mediated by the expression of VEGF and Ang-2. The results of this study suggest a new concept of vascular tumor initiation that may have important implications for the clinical application of antiangiogenic strategies.
Peter Vajkoczy, Mohammad Farhadi, Andreas Gaumann, Regina Heidenreich, Ralf Erber, Andreas Wunder, Jörg C. Tonn, Michael D. Menger, Georg Breier
Angiotensin II (Ang II), a potent hypertrophic stimulus, causes significant increases in TGFb1 gene expression. However, it is not known whether there is a causal relationship between increased levels of TGF-β1 and cardiac hypertrophy. Echocardiographic analysis revealed that TGF-β1–deficient mice subjected to chronic subpressor doses of Ang II had no significant change in left ventricular (LV) mass and percent fractional shortening during Ang IItreatment. In contrast, Ang II–treated wild-type mice showed a >20% increase in LV mass and impaired cardiac function. Cardiomyocyte cross-sectional area was also markedly increased in Ang II–treated wild-type mice but unchanged in Ang II–treated TGF-β1–deficient mice. No significant levels of fibrosis, mitotic growth, or cytokine infiltration were detected in Ang II–treated mice. Atrial natriuretic factor expression was ∼6-fold elevated in Ang II–treated wild-type, but not TGF-β1–deficient mice. However, the α- to β-myosin heavy chain switch did not occur in Ang II–treated mice, indicating that isoform switching is not obligatorily coupled with hypertrophy or TGF-β1. The Ang IIeffect on hypertrophy was shown not to result from stimulation of the endogenous renin-angiotensis system. These results indicate that TGF-β1 is an important mediator of the hypertrophic growth response of the heart to Ang II.
Jo El J. Schultz, Sandra A. Witt, Betty J. Glascock, Michelle L. Nieman, Peter J. Reiser, Stacey L. Nix, Thomas R. Kimball, Thomas Doetschman
The antiphospholipid syndrome (APS) is characterized by the presence of pathogenic autoantibodies against β2-glycoprotein-I (β2GPI). The factors causing production of anti-β2GPI remain unidentified, but an association with infectious agents has been reported. Recently, we identified a hexapeptide (TLRVYK) that is recognized specifically by a pathogenic anti-β2GPI mAb. In the present study we evaluated the APS-related pathogenic potential of microbial pathogens carrying sequences related to this hexapeptide. Mice immunized with a panel of microbial preparations were studied for the development of anti-β2GPI autoantibodies. IgG specific to the TLRVYK peptide were affinity purified from the immunized mice and passively infused intravenously into naive mice at day 0 of pregnancy. APS parameters were evaluated in the infused mice on day 15 of pregnancy. Following immunization, high titers of antipeptide [TLRVYK] anti-β2GPI Ab’s were observed in mice immunized with Haemophilus influenzae, Neisseria gonorrhoeae, or tetanus toxoid. The specificity of binding to the corresponding target molecules was confirmed by competition and immunoblot assays. Naive mice infused with the affinity-purified antipeptide Ab’s had significant thrombocytopenia, prolonged activated partial thromboplastin time and elevated percentage of fetal loss, similar to a control group of mice immunized with a pathogenic anti-β2GPI mAb. Our study establishes a mechanism of molecular mimicry in experimental APS, demonstrating that bacterial peptides homologous with β2GPI induce pathogenic anti-β2GPI Ab’s along with APS manifestations.
Miri Blank, Ilan Krause, Mati Fridkin, Nathan Keller, Juri Kopolovic, Iris Goldberg, Ana Tobar, Yehuda Shoenfeld
Acute intensive insulin therapy is an independent risk factor for diabetic retinopathy. Here we demonstrate that acute intensive insulin therapy markedly increases VEGF mRNA and protein levels in the retinae of diabetic rats. Retinal nuclear extracts from insulin-treated rats contain higher hypoxia-inducible factor-1α (HIF-1α) levels and demonstrate increased HIF-1α–dependent binding to hypoxia-responsive elements in the VEGF promoter. Blood-retinal barrier breakdown is markedly increased with acute intensive insulin therapy but can be reversed by treating animals with a fusion protein containing a soluble form of the VEGF receptor Flt; a control fusion protein has no such protective effect. The insulin-induced retinal HIF-1α and VEGF increases and the related blood-retinal barrier breakdown are suppressed by inhibitors of p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositol (PI) 3-kinase, but not inhibitors of p42/p44 MAPK or protein kinase C. Taken together, these findings indicate that acute intensive insulin therapy produces a transient worsening of diabetic blood-retinal barrier breakdown via an HIF-1α–mediated increase in retinal VEGF expression. Insulin-induced VEGF expression requires p38 MAPK and PI 3-kinase, whereas hyperglycemia-induced VEGF expression is HIF-1α–independent and requires PKC and p42/p44 MAPK. To our knowledge, these data are the first to identify a specific mechanism for the transient worsening of diabetic retinopathy, specifically blood-retinal barrier breakdown, that follows the institution of intensive insulin therapy.
Vassiliki Poulaki, Wenying Qin, Antonia M. Joussen, Peter Hurlbut, Stanley J. Wiegand, John Rudge, George D. Yancopoulos, Anthony P. Adamis
Nitric oxide (NO) is produced by NO synthase (NOS) in many cells and plays important roles in the neuronal, muscular, cardiovascular, and immune systems. In various disease conditions, all three types of NOS (neuronal, inducible, and endothelial) are reported to generate oxidants through unknown mechanisms. We present here the first evidence that peroxynitrite (ONOO–) releases zinc from the zinc-thiolate cluster of endothelial NOS (eNOS) and presumably forms disulfide bonds between the monomers. As a result, disruption of the otherwise SDS-resistant eNOS dimers occurs under reducing conditions. eNOS catalytic activity is exquisitely sensitive to ONOO–, which decreases NO synthesis and increases superoxide anion (O2.–) production by the enzyme. The reducing cofactor tetrahydrobiopterin is not oxidized, nor does it prevent oxidation of eNOS by the same low concentrations of OONO–. Furthermore, eNOS derived from endothelial cells exposed to elevated glucose produces more O2.–, and, like eNOS purified from diabetic LDL receptor–deficient mice, contains less zinc and fewer SDS-resistant dimers. Hence, eNOS exposure to oxidants including ONOO– causes increased enzymatic uncoupling and generation of O2.– in diabetes, contributing further to endothelial cell oxidant stress. Regulation of the zinc-thiolate center of NOS by ONOO– provides a novel mechanism for modulation of the enzyme function in disease.
Ming-Hui Zou, Chaomei Shi, Richard A. Cohen
We studied the role of protein kinase C isoform PKCδ in ceramide (Cer) formation, as well as in the mitochondrial apoptosis pathway induced by anticancer drugs in prostate cancer (PC) cells. Etoposide and paclitaxel induced Cer formation and apoptosis in PKCδ-positive LNCaP and DU145 cells but not in PKCδ-negative LN-TPA or PC-3 cells. In contrast, these drugs induced mitotic cell cycle arrest in all PC cell lines. Treatment with Rottlerin, a specific PKCδ inhibitor, significantly inhibited drug-induced Cer formation and apoptosis in LNCaP cells, as did overexpression of dominant negative–type PKCδ. Overexpression of wild-type PKCδ had an opposite effect in PC-3 cells. Notably, etoposide induced biphasic Cer formation in LNCaP cells. The early and transient Cer increase resulted from de novo Cer synthesis, while the late and sustained Cer accumulation was derived from sphingomyelin hydrolysis by neutral sphingomyelinase (nSMase). Cer, in turn, induced mitochondrial translocation of PKCδ and stimulated the activity of this kinase, promoting cytochrome c release and caspase-9 activation. Furthermore, the specific caspase-9 inhibitor LEHD-fmk significantly inhibited etoposide-induced nSMase activation, Cer accumulation, and PKCδ mitochondrial translocation. These results indicate that PKCδ plays a crucial role in activating anticancer drug–induced apoptosis signaling by amplifying the Cer-mediated mitochondrial amplification loop.
Makoto Sumitomo, Motoi Ohba, Junichi Asakuma, Takako Asano, Toshio Kuroki, Tomohiko Asano, Masamichi Hayakawa
To date, most studies have focused on the characterization of HIV-1–specific cellular immune responses in the peripheral blood (PB) of infected individuals. Much less is known about the comparative magnitude and breadth of responses in the lymphoid tissue. This study analyzed HIV-1–specific CD8+ T cell responses simultaneously in PB and lymph nodes (LNs) of persons with chronic HIV-1 infection and assessed the dynamics of these responses during antiretroviral treatment and supervised treatment interruption (STI). In untreated chronic infection, the magnitude of epitope-specific CD8+ T cell activity was significantly higher in LNs than in PB. Responses decreased in both compartments during highly active antiretroviral therapy, but this decline was more pronounced in PB. During STI, HIV-1–specific CD8+ T cell responses in PB increased significantly. Enhancement in breadth and magnitude was largely due to the expansion of pre-existing responses in the LNs, with new epitopes infrequently targeted. Taken together, these data demonstrate that HIV-1–specific CD8+ T cells are preferentially located in the LNs, with a subset of responses exclusively detectable in this compartment. Furthermore, the enhanced CD8+ T cell responses observed during STI in chronically infected individuals is largely due to expansion of pre-existing virus-specific CD8+ T cells, rather than the induction of novel responses.
Marcus Altfeld, Jan van Lunzen, Nicole Frahm, Xu G. Yu, Claus Schneider, Robert L. Eldridge, Margaret E. Feeney, Dirk Meyer-Olson, Hans-Juergen Stellbrink, Bruce D. Walker