CD248 and its cytoplasmic domain: a therapeutic target for arthritis.

OBJECTIVE: CD248 is a transmembrane glycoprotein expressed on the surface of activated perivascular and fibroblast-like cells. This study was undertaken to explore the function of CD248 and its cytoplasmic domain in arthritis. METHODS: Synovial tissue biopsy samples from healthy controls, from patients with psoriatic arthritis (PsA), and from patients with rheumatoid arthritis (RA) were stained for CD248. Transgenic mice that were CD248-deficient (CD248-knockout [CD248(KO/KO) ]) or mice with CD248 lacking the cytoplasmic domain (CD248(CyD/CyD) ) were generated. Collagen antibody-induced arthritis (CAIA) was induced in these mice and in corresponding wild-type (WT) mice as controls. Clinical signs and histologic features of arthritis were evaluated. Cytokine levels were determined by enzyme-linked immunosorbent assay, and the number of infiltrating inflammatory cells was quantified by immunohistochemistry. In vitro studies were performed with fibroblasts from CD248-transgenic mouse embryos to explain the observed effects on inflammation. RESULTS: Immunostaining of synovium from patients with PsA and patients with RA and that from mice after the induction of CAIA revealed strong CD248 expression in perivascular and fibroblast-like stromal cells. CD248(KO/KO) and CD248(CyD/CyD) mice had less severe arthritis, with lower plasma levels of proinflammatory cytokines, as compared with WT controls. Moreover, the joints of these mice had less synovial hyperplasia, reduced accumulation of inflammatory cells, and less articular cartilage and bone damage. Tumor necrosis factor α-induced monocyte adhesion to CD248(CyD/CyD) fibroblasts was impaired. CD248(CyD/CyD) fibroblasts exhibited reduced expression of hypoxia-inducible factor 1α, placental growth factor, vascular endothelial growth factor, and matrix metalloproteinase 9 activity in response to transforming growth factor ß. CONCLUSION: CD248 contributes to synovial hyperplasia and leukocyte accumulation in inflammatory arthritis, the effects of which are mediated partly via its cytoplasmic domain. CD248 is therefore a potential new target in the treatment of arthritis.

Thrombomodulin mutations in atypical hemolytic-uremic syndrome.

BACKGROUND: The hemolytic-uremic syndrome consists of the triad of microangiopathic hemolytic anemia, thrombocytopenia, and renal failure. The common form of the syndrome is triggered by infection with Shiga toxin-producing bacteria and has a favorable outcome. The less common form of the syndrome, called atypical hemolytic-uremic syndrome, accounts for about 10% of cases, and patients with this form of the syndrome have a poor prognosis. Approximately half of the patients with atypical hemolytic-uremic syndrome have mutations in genes that regulate the complement system. Genetic factors in the remaining cases are unknown. We studied the role of thrombomodulin, an endothelial glycoprotein with anticoagulant, antiinflammatory, and cytoprotective properties, in atypical hemolytic-uremic syndrome. METHODS: We sequenced the entire thrombomodulin gene (THBD) in 152 patients with atypical hemolytic-uremic syndrome and in 380 controls. Using purified proteins and cell-expression systems, we investigated whether thrombomodulin regulates the complement system, and we characterized the mechanisms. We evaluated the effects of thrombomodulin missense mutations associated with atypical hemolytic-uremic syndrome on complement activation by expressing thrombomodulin variants in cultured cells. RESULTS: Of 152 patients with atypical hemolytic-uremic syndrome, 7 unrelated patients had six different heterozygous missense THBD mutations. In vitro, thrombomodulin binds to C3b and factor H (CFH) and negatively regulates complement by accelerating factor I-mediated inactivation of C3b in the presence of cofactors, CFH or C4b binding protein. By promoting activation of the plasma procarboxypeptidase B, thrombomodulin also accelerates the inactivation of anaphylatoxins C3a and C5a. Cultured cells expressing thrombomodulin variants associated with atypical hemolytic-uremic syndrome had diminished capacity to inactivate C3b and to activate procarboxypeptidase B and were thus less protected from activated complement. CONCLUSIONS: Mutations that impair the function of thrombomodulin occur in about 5% of patients with atypical hemolytic-uremic syndrome.

Role of the 2 zebrafish survivin genes in vasculo-angiogenesis, neurogenesis, cardiogenesis and hematopoiesis.

BACKGROUND: Normal growth and development of organisms requires maintenance of a dynamic balance between systems that promote cell survival and those that induce apoptosis. The molecular mechanisms that regulate these processes remain poorly understood, and thus further in vivo study is required. Survivin is a member of the inhibitor of apoptosis protein (IAP) family, that uniquely also promotes mitosis and cell proliferation. Postnatally, survivin is hardly detected in most tissues, but is upregulated in all cancers, and as such, is a potential therapeutic target. Prenatally, survivin is also highly expressed in several tissues. Fully delineating the properties of survivin in vivo in mice has been confounded by early lethal phenotypes following survivin gene inactivation. RESULTS: To gain further insights into the properties of survivin, we used the zebrafish model. There are 2 zebrafish survivin genes (Birc5a and Birc5b) with overlapping expression patterns during early development, prominently in neural and vascular structures. Morpholino-induced depletion of Birc5a causes profound neuro-developmental, hematopoietic, cardiogenic, vasculogenic and angiogenic defects. Similar abnormalities, all less severe except for hematopoiesis, were evident with suppression of Birc5b. The phenotypes induced by morpholino knockdown of one survivin gene, were rescued by overexpression of the other, indicating that the Birc5 paralogs may compensate for each. The potent vascular endothelial growth factor (VEGF) also entirely rescues the phenotypes induced by depletion of either Birc5a and Birc5b, highlighting its multi-functional properties, as well as the power of the model in characterizing the activities of growth factors. CONCLUSION: Overall, with the zebrafish model, we identify survivin as a key regulator of neurogenesis, vasculo-angiogenesis, hematopoiesis and cardiogenesis. These properties of survivin, which are consistent with those identified in mice, indicate that its functions are highly conserved across species, and point to the value of the zebrafish model in understanding the role of this IAP in the pathogenesis of human disease, and for exploring its potential as a therapeutic target.

Protective role of the inhibitor of apoptosis protein, survivin, in toxin-induced acute renal failure.

Acute renal failure (ARF) is a major worldwide cause of morbidity and mortality, lacking specific targeted, effective therapies. Renal tubular cell apoptosis has been recognized to play a critical role in the pathogenesis of ARF, yet few studies have evaluated whether intervention in apoptotic pathways can ameliorate the deterioration in renal function associated with ARF. Using transgenic mice with diminished levels of the inhibitor of apoptosis protein, survivin, we show that survivin is required to protect the kidney from apoptosis, to suppress renal expression of p53, and to ameliorate renal dysfunction in two models of ARF. Gene delivery of survivin to wild-type mice and mice with 50% levels of survivin, prior to or at the time of induction of ARF, interferes with the deterioration of renal function and preserves the integrity of the kidneys and the renal tubular cells by inhibiting activation of apoptotic pathways in the kidneys and suppressing expression of p53. These results encourage further evaluation of survivin, its active structural domains, and other inhibitors of apoptosis proteins, for preventing and/or treating acute renal failure.

Lack of endothelial cell survivin causes embryonic defects in angiogenesis, cardiogenesis, and neural tube closure.

We explored the physiologic role of endothelial cell apoptosis during development by generating mouse embryos lacking the inhibitor of apoptosis protein (IAP) survivin in endothelium. This was accomplished by intercrossing survivin(lox/lox) mice with mice expressing cre recombinase under the control of the endothelial cell specific tie1 promoter (tie1-cre mice). Lack of endothelial cell survivin resulted in embryonic lethality. Mutant embryos had prominent and diffuse hemorrhages from embryonic day 9.5 (E9.5) and died before E13.5. Heart development was strikingly abnormal. Survivin-null endocardial lineage cells could not support normal epithelial-mesenchymal transformation (EMT), resulting in hypoplastic endocardial cushions and in utero heart failure. In addition, 30% of mutant embryos had neural tube closure defects (NTDs) that were not caused by bleeding or growth retardation, but were likely due to alterations in the release of soluble factors from endothelial cells that otherwise support neural stem cell proliferation and neurulation. Thus, regulation of endothelial cell survival, and maintenance of vascular integrity by survivin are crucial for normal embryonic angiogenesis, cardiogenesis, and neurogenesis.

The lectin-like domain of thrombomodulin confers protection from neutrophil-mediated tissue damage by suppressing adhesion molecule expression via nuclear factor kappaB and mitogen-activated protein kinase pathways.

Thrombomodulin (TM) is a vascular endothelial cell (EC) receptor that is a cofactor for thrombin-mediated activation of the anticoagulant protein C. The extracellular NH(2)-terminal domain of TM has homology to C-type lectins that are involved in immune regulation. Using transgenic mice that lack this structure (TM(LeD/LeD)), we show that the lectin-like domain of TM interferes with polymorphonuclear leukocyte (PMN) adhesion to ECs by intercellular adhesion molecule 1-dependent and -independent pathways through the suppression of extracellular signal-regulated kinase (ERK)(1/2) activation. TM(LeD/LeD) mice have reduced survival after endotoxin exposure, accumulate more PMNs in their lungs, and develop larger infarcts after myocardial ischemia/reperfusion. The recombinant lectin-like domain of TM suppresses PMN adhesion to ECs, diminishes cytokine-induced increase in nuclear factor kappaB and activation of ERK(1/2), and rescues ECs from serum starvation, findings that may explain why plasma levels of soluble TM are inversely correlated with cardiovascular disease. These data suggest that TM has antiinflammatory properties in addition to its role in coagulation and fibrinolysis.