Chondroitin Sulfate N-acetylgalactosaminyltransferase-2 Impacts Foam Cell Formation and Atherosclerosis by Altering Macrophage Glycosaminoglycan Chain.

OBJECTIVE: Chondroitin sulfate proteoglycans are the primary constituents of the macrophage glycosaminoglycan and extracellular microenvironment. To examine their potential role in atherogenesis, we investigated the biological importance of one of the chondroitin sulfate glycosaminoglycan biosynthesis gene, ChGn-2 (chondroitin sulfate N-acetylgalactosaminyltransferase-2), in macrophage foam cell formation. Approach and Results: ChGn-2-deficient mice showed decreased and shortened glycosaminoglycans. ChGn-2-/-/LDLr-/- (low-density lipoprotein receptor) mice generated less atherosclerotic plaque after being fed with Western diet despite exhibiting a metabolic phenotype similar to that of the ChGn-2+/+/LDLr-/- littermates. We demonstrated that in macrophages, ChGn-2 expression was upregulated in the presence of oxLDL (oxidized LDL), and glycosaminoglycan was substantially increased. Foam cell formation was significantly altered by ChGn-2 in both mouse peritoneal macrophages and the RAW264.7 macrophage cell line. Mechanistically, ChGn-2 enhanced oxLDL binding on the cell surface, and as a consequence, CD36-an important macrophage membrane scavenger receptor-was differentially regulated. CONCLUSIONS: ChGn-2 alteration on macrophages conceivably influences LDL accumulation and subsequently accelerates plaque formation. These results collectively suggest that ChGn-2 is a novel therapeutic target amenable to clinical translation in the future. Graphic Abstract: A graphic abstract is available for this article.

Family with sequence similarity 13, member A modulates adipocyte insulin signaling and preserves systemic metabolic homeostasis.

Adipose tissue dysfunction is causally implicated in the impaired metabolic homeostasis associated with obesity; however, detailed mechanisms underlying dysregulated adipocyte functions in obesity remain to be elucidated. Here we searched for genes that provide a previously unknown mechanism in adipocyte metabolic functions and identified family with sequence similarity 13, member A (Fam13a) as a factor that modifies insulin signal cascade in adipocytes. Fam13a was highly expressed in adipose tissue, predominantly in mature adipocytes, and its expression was substantially reduced in adipose tissues of obese compared with lean mice. We revealed that Fam13a accentuated insulin signaling by recruiting protein phosphatase 2A with insulin receptor substrate 1 (IRS1), leading to protection of IRS1 from proteasomal degradation. We further demonstrated that genetic loss of Fam13a exacerbated obesity-related metabolic disorders, while targeted activation of Fam13a in adipocytes ameliorated it in association with altered adipose tissue insulin sensitivity in mice. Our data unveiled a previously unknown mechanism in the regulation of adipocyte insulin signaling by Fam13a and identified its significant role in systemic metabolic homeostasis, shedding light on Fam13a as a pharmacotherapeutic target to treat obesity-related metabolic disorders.

Chondroitin sulfate N-acetylgalactosaminyltransferase-2 deletion alleviates lipoprotein retention in early atherosclerosis and attenuates aortic smooth muscle cell migration.

Glycosaminoglycans (GAGs) play an integral role in low-density lipoprotein (LDL) retention in the vascular intimal layer and have emerged as attractive therapeutic targets for atherosclerosis. GAG biosynthesis involves the cooperation of numerous enzymes. Chondroitin sulfate N-acetylgalactosaminyltransferase-2 (ChGn-2) is a vital Golgi transferase that participates in enzymatic elongation of GAGs. Here, we investigated the effects of ChGn-2 gene deletion on the development of atherosclerosis. Partial carotid artery ligation was performed on ChGn-2-/-/LDLr-/- and ChGn-2+/+/LDLr-/- mice to induce diffuse intimal thickening (DIT). Aortic smooth muscle cells (ASMCs) were isolated to investigate cellular LDL binding and migration. Histological analysis of human coronary artery sections revealed that ChGn-2 was expressed in early and advanced atherosclerotic lesions. Deletion of the ChGn-2 gene significantly reduced LDL retention in the DIT mouse model. Furthermore, LDL binding, visualized using rhodamine-labeled LDLs, was dramatically reduced. Interestingly, a functional assay of ASMCs prepared from ChGn-2-/- mice displayed abrogation of platelet-derived growth factor (PDGF)-mediated migration via reduced PDGF receptor phosphorylation. Taken together, these findings indicate that ChGn-2 is functionally involved in the progression of atherosclerosis both in its early and advanced stages. Therefore, ChGn-2 may serve as a plausible target to treat atherosclerotic-related diseases in the future.

Neuregulin-4 is an angiogenic factor that is critically involved in the maintenance of adipose tissue vasculature.

Adipose tissue (AT) contains well-developed vascular networks. Pathological AT expansion often accompany the reduction in AT blood vessels, which further exacerbates adipocyte dysfunction due to hypoxia; however, it remains unclear whether AT vascular rarefaction is simply secondary to adipocyte hypertrophy, or if there is an actively regulated pathway that mediates impaired AT angiogenesis in obesity. We searched for growth factors whose expression in AT is down-regulated in obesity; accordingly, we identified neuregulin-4 (Nrg4), a member of the EGF family of proteins. Nrg4 is highly and preferentially expressed in healthy adipocytes, while its expression was substantially reduced in obesity. Nrg4 activated endothelial angiogenic functions and angiogenesis both in vitro and in vivo. Genetic loss of Nrg4 caused reduction in brown and white AT blood vessels, and induced overweight even while consuming normal chow. Conditional knockout of Nrg4 in brown adipocytes caused blood vessel reduction in brown but not in white AT, and was sufficient to induce obese phenotype. Our data demonstrated that Nrg4 plays a critical role in maintaining AT vasculature and its metabolic functions. Considering the substantial reduction of Nrg4 in obesity, disruption of Nrg4-mediated angiogenesis could be an active mechanism for the obesity-associated vascular rarefaction in AT, and thus Nrg4 is an attracting pharmacotherapeutic target in the prevention and/or treatment of obesity-related metabolic disorders.

Systemic inhibition of Janus kinase induces browning of white adipose tissue and ameliorates obesity-related metabolic disorders.

Browning of white adipose tissue is a promising strategy to tackle obesity. Recently, Janus kinase (JAK) inhibition was shown to induce white-to-brown metabolic conversion of adipocytes in vitro; however effects of JAK inhibition on browning and systemic metabolic health in vivo remain to be elucidated. Here, we report that systemic administration of JAK inhibitor (JAKi) ameliorated obesity-related metabolic disorders. Administration of JAKi in mice fed a high-fat diet increased UCP-1 and PRDM16 expression in white adipose tissue, indicating the browning of white adipocyte. Food intake was increased in JAKi-treated mice, while the body weight and adiposity was similar between the JAKi- and vehicle-treated mice. In consistent with the browning, thermogenic capacity was enhanced in mice treated with JAKi. Chronic inflammation in white adipose tissue was not ameliorated by JAKi-treatment. Nevertheless, insulin sensitivity was well preserved in JAKi-treated mice comparing with that in vehicle-treated mice. Serum levels of triglyceride and free fatty acid were significantly reduced by JAKi-treatment, which is accompanied by ameliorated hepatosteatosis. Our data demonstrate that systemic administration of JAKi has beneficial effects in preserving metabolic health, and thus inhibition of JAK signaling has therapeutic potential for the treatment of obesity and its-related metabolic disorders.

Acid-suppressing Drugs and a Low 1 Level of Antithrombin as Risk Factors for L-Asparaginase-associated Pancreatitis: A Case-control Study in the Japan Association of Childhood Leukemia Study (JACLS).

L-Asparaginase has significantly improved outcome for children with acute lymphoblastic leukemia and has become an essential component of multiagent chemotherapy. However, there are many adverse events due to L-asparaginase, including acute pancreatitis. The pathology of L-asparaginase-associated pancreatitis (AAP) remains unclear. We compared patients who developed AAP (n=29) and random matched controls (n=36) who had been enrolled in the Japan Association of Childhood Leukemia Study of the ALL-02 protocol. AAP and control patients were matched for age, sex, treatment, and protocol risk. We examined correlations between AAP development and clinical symptoms, laboratory data, and concomitant medication. Abdominal pain and nausea were common presenting symptoms for AAP. There was an increased risk of AAP in patients using gastric acid-suppressing agents and antithrombin (AT) supplementation. Mean fibrinogen and AT levels before the onset of AAP were lower in AAP patients than in controls. Decreased AT and fibrinogen levels resulting from the strong suppression of protein synthesis by L-asparaginase were predictive signs for AAP. Our epidemiological approach should prove clinically useful for the diagnosis the AAP as early as possible.

Long-term Results of the Risk-adapted Treatment for Childhood B-Cell Acute Lymphoblastic Leukemia: Report From the Japan Association of Childhood Leukemia Study ALL-97 Trial.

PURPOSE: This study was conducted as the first clinical trial by Japan Association of Childhood Leukemia Study to improve the outcome of B-cell acute lymphoblastic leukemia and explore a less toxic reinduction block. PATIENTS AND METHODS: From 1997 to 2002, 563 patients with B-cell acute lymphoblastic leukemia aged 1 to 15 years were enrolled. The patients were assigned into 4 risk groups (standard, intermediate, high, or extremely high risk) and treated with regimens intensified according to the risk. Two randomized trials were conducted to compare 2 regimens with and without a 3-week reinduction therapy in the standard-risk group, and to compare the efficacy of pirarubicin with daunorubicin in the intermediate-risk and high-risk groups. Prophylactic cranial irradiation was restricted in patients with high or extremely high risk. RESULTS: The event-free survival (EFS) rate at 10 years for all patients was 77.0%. Those in the standard-risk to extremely high-risk groups were 79.3%, 72.5%, 71.7%, and 66.3%, respectively. The 15-week induction/consolidation not followed by reinduction produced 76.4% of the EFS at 10 years comparable with the regimen with reinduction therapy. Pirarubicin at 25 mg/m administered 11 times throughout the treatment produced the EFS comparable with daunorubicin at 30 mg/m. CONCLUSION: The trial produced high survival rates in NCI-HR patients, although the outcomes in NCI-SR patients were not satisfactory possibly due to less intensive central nervous system-directed therapy.

Acute and late toxicities of pirarubicin in the treatment of childhood acute lymphoblastic leukemia: results from a clinical trial by the Japan Association of Childhood Leukemia Study.

BACKGROUND: Anthracyclines are used to treat childhood acute lymphoblastic leukemia (ALL). Even when administered at low doses, these agents are reported to cause progressive cardiac dysfunction. We conducted a clinical trial comparing the toxicities of two anthracyclines, pirarubicin (THP) and daunorubicin (DNR), in the treatment of childhood ALL. The results from our study that relate to acute and late toxicities are reported here. METHODS: 276 children with B-ALL were enrolled in the trial from April 1997 to March 2002 and were randomly assigned to receive a regimen including either THP (25 mg/m2 × 11) or DNR (30 mg/m2 × 11). Acute toxicity was prospectively assessed based on the National Cancer Institute Common Toxicity Criteria. Acute hematological toxicity was also examined via some parameters. Patients with event-free survival of >5 years were retrospectively surveyed for cardiac function at 5 and 10 years and at the most recent assessment more than 10 years from the onset of ALL. RESULTS: Acute hematological toxicity in the early phase was more significant in the THP arm. Based on ultrasound cardiography, cardiac function was impaired in both groups during the follow-up period, but there was no significant difference between the groups except for a greater decline in fractional shortening on ultrasound cardiography in the DNR arm. CONCLUSIONS: While acute hematological toxicity was more significant in the THP arm, THP also appeared to be less cardiotoxic. However, the evaluation of late cardiotoxicity was limited because only a few subjects were followed beyond 10 years after ALL onset. Considering that the THP regimen produced an EFS rate comparable with that of the DNR regimen, the efficacy and toxicity of THP at reduced doses should be studied in order to identify potentially safer regimens.

Identification of novel kinase fusion transcripts in paediatric B cell precursor acute lymphoblastic leukaemia with IKZF1 deletion.

Activating tyrosine kinase mutations or cytokine receptor signalling alterations have attracted attention as therapeutic targets for high-risk paediatric acute lymphoblastic leukaemia (ALL). We identified two novel kinase fusions, OFD1-JAK2 and NCOR1-LYN, in paediatric ALL patients with IKZF1 deletion, by mRNA sequencing. The patient with CSF2RA-CRLF2 also harboured IGH-EPOR. All these patients had high-risk features, such as high initial white blood cell counts and initial poor response to prednisolone. The functional analysis of these novel fusions is on-going to determine whether these genetic alterations can be targeted by drugs.

Targeted activation of endothelin-1 exacerbates hypoxia-induced pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a fatal disease that eventually results in right heart failure and death. Current pharmacologic therapies for PAH are limited, and there are no drugs that could completely cure PAH. Enhanced activity of endothelin system has been implicated in PAH severity and endothelin receptor antagonists have been used clinically to treat PAH. However, there is limited experimental evidence on the direct role of enhanced endothelin system activity in PAH. Here, we investigated the correlation between endothelin-1 (ET-1) and PAH using ET-1 transgenic (ETTG) mice. Exposure to chronic hypoxia increased right ventricular pressure and pulmonary arterial wall thickness in ETTG mice compared to those in wild type mice. Of note, ETTG mice exhibited modest but significant increase in right ventricular pressure and vessel wall thickness relative to wild type mice even under normoxic conditions. To induce severe PAH, we administered SU5416, a vascular endothelial growth factor receptor inhibitor, combined with exposure to chronic hypoxia. Treatment with SU5416 modestly aggravated hypoxia-induced pulmonary hypertension, right ventricular hypertrophy, and pulmonary arterial vessel wall thickening in ETTG mice in association with increased interleukin-6 expression in blood vessels. However, there was no sign of obliterative endothelial cell proliferation and plexiform lesion formation in the lungs. These results demonstrated that enhanced endothelin system activity could be a causative factor in the development of PAH and provided rationale for the inhibition of endothelin system to treat PAH.

An overall characterization of pediatric acute lymphoblastic leukemia with CRLF2 overexpression.

For an overall characterization of pediatric B-cell precursor acute lymphoblastic leukemia (BCPALL) with CRLF2 overexpression (OE), we conducted genetic analysis of CRLF2 in 167 pediatric BCPALL patients. CRLF2 OE was detected in 30 (18%) of 167 patients, the P2RY8-CRLF2 fusion was identified in only 3 (1.8%) of 167 patients, all of which demonstrated CRLF2 OE. Moreover, CRLF2 gain was identified in 18 (11%) of 167 patients. Messenger RNA sequencing revealed a novel fusion transcript, CSF2RA-CRLF2, in a case with CRLF2 OE, suggesting that this fusion is associated with CRLF2 OE. In survival analysis, no significant differences in 5-year event-free survival (EFS) and overall survival were observed between patients with and without CRLF2 OE (70.7 vs. 75.4%, log rank P = 0.68 and 96.4 vs. 82.1%, log rank P = 0.11, respectively). However, a significant difference in 5-year EFS between CRLF2 OE patients with and without IKZF1 deletion was observed (44.4 vs. 83.1%, log rank P = 0.02). In multivariate analysis, only IKZF1 deletion was a significant predictor of inferior OS (hazard ratio: 2.427, P = 0.04).These findings suggest that CRLF2 OE is not an independent prognostic factor in pediatric BCPALL.

Endothelin-converting enzyme-1 gene ablation attenuates pulmonary fibrosis via CGRP-cAMP/EPAC1 pathway.

Endothelin-1 (ET-1) has been shown to be involved in human pulmonary fibrosis. However, recent clinical trials targeting the ET-1 pathway with ET-1 receptor antagonists failed to achieve beneficial outcomes. Another strategy opposing the actions of ET-1 involves the inhibition of endothelin-converting enzyme-1 (ECE-1). We hypothesize that ECE-1 inhibition exerts beneficial effects on pulmonary fibrosis. Pulmonary fibrosis was induced by instilling bleomycin intratracheally into ECE-1 heterozygous knockout mice (ECE-1(+/-)) and their wild-type control mice (ECE-1(+/+)). Lung inflammation and fibrosis were assessed on Days 7, 14, and 28 after bleomycin instillation. The activity of ECE-1 and the concentrations of its related peptides, ET-1, bradykinin, atrial natriuretic peptide (ANP), and calcitonin gene-related peptide (CGRP), were determined. ECE-1(+/-) mice demonstrated less lung inflammation and limited fibrosis compared with control mice. ECE-1 activity was half-reduced in ECE-1(+/-) mice, and this activity also altered ET-1 and CGRP concentrations, but not concentrations of bradykinin and ANP. ET-1 concentrations were found to be lower in ECE-1(+/-) mice after the development of fibrosis, in contrast to the unaltered concentrations during inflammation. Reduced ECE-1 activity resulted in higher CGRP concentrations, which altered the pathological functionality of the lung, indicating the activation of the CGRP pathway involving cyclic adenosine monophosphate (cAMP)/exchange protein directly activated by cAMP and cAMP/protein kinase A in ECE-1(+/-) mice. Bleomycin instillation on Day 14 induced the accumulation of M2 macrophages expressing CGRP receptors in ECE-1(+/-) mice. Our results emphasize that the in vivo ECE-1-mediated degradation of CGRP promotes the transition from lung inflammation to fibrosis. Further, our study identified M2 macrophages as the target cells of CGRP action during this transition.

Expression of multidrug resistance 1 gene in B-cell lymphomas: association with follicular dendritic cells.

AIMS: Multidrug resistance (MDR) in B-cell lymphomas still constitutes a major obstacle to the effectiveness of chemotherapy even in the anti-CD20 antibody therapy era. The aim of this study was to investigate the expression of MDR-associated molecules in reactive lymphadenopathy (RL), follicular lymphoma (FL), and diffuse large B-cell lymphoma (DLBCL). METHODS AND RESULTS: The expression of mRNA for ABC-transporter family genes was determined by real-time RT-PCR in lymph nodes from RL, FL, and DLBCL cases. MDR1 exhibited significantly stronger expression in RL, FL, and DLBCL than Raji B-cell lymphoma cells. RL and FL showed significantly higher expression than DLBCL. Immunohistochemically, MDR1 positive cells were localized in the germinal centers of RL and center of the nodular lesions of FL showing associations with CD21 positive follicular dendritic cells (FDCs). Raji cells were co-cultured with FDC sarcoma-derived cells and the expression of MDR1 and drug resistance were analyzed. The co-culture of Raji cells with FDCs induced strong expression of MDR1 and introduced resistance to doxorubicin-induced apoptosis. CONCLUSIONS: These results suggest that FDCs induce MDR1 expression in reactive as well as neoplastic B-cells. Inhibition of the interaction of FDCs with B-cells may provide a novel strategy for treating the chemotherapy resistant fraction.

ET-1 deletion from endothelial cells protects the kidney during the extension phase of ischemia/reperfusion injury.

BACKGROUND: The prognosis of patients after acute kidney injury (AKI) is poor and treatment is limited. AKI is mainly caused by renal ischemia/reperfusion injury (IRI). During the extension phase of IRI, endothelial damage may participate in ischemia and inflammation. Endothelin-1 (ET-1) which is mostly secreted by endothelial cells is an important actor of IRI, particularly through its strong vasoconstrictive properties. We aimed to analyze the specific role of ET-1 from the endothelial cells in AKI. METHODS: We used mice lacking ET-1 in the vascular endothelial cells (VEETKO). We induced IRI in VEETKO mice and wild type controls by clamping both kidneys for 30min. Sham operated mice were used as controls. Mice were sacrificed one day after IRI in order to investigate the extension phase of IRI. Kidney function was assessed based on serum creatinine concentration. Levels of expression of ET-1, its receptor ET(A), protein kinase C, eNOS, E-Cadherin and inflammation markers were evaluated by real time PCR or western blot. Tubular injury was scored on periodic acid Schiff stained kidney preparations. Lumen and wall area of small intrarenal arteries were measured on kidney slices stained for alpha smooth muscle cell actin. Oxidative stress, macrophage infiltration and cell proliferation was evaluated on slices stained for 8-hydroxy-2'-deoxyguanosine, F4/80 and PCNA, respectively. RESULTS: IRI induced kidney failure and increased ET-1 and ET(A) receptor expression. This was accompanied by tubular injury, wall thickening and reduction of lumen area/wall area ratio of small renal arteries, increased oxidative stress and inflammation. These parameters were attenuated in VEETKO mice. CONCLUSION: Our results suggest that suppression of ET-1 from the endothelial cells attenuates IRI kidney injury. Blocking ET-1 effects may represent a therapeutic strategy in the management of AKI.

JACLS ALL-02 SR protocol reduced-intensity chemotherapy produces excellent outcomes in patients with low-risk childhood acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. As overall cure rates of childhood ALL have improved, reduction of overall treatment intensity while still ensuring excellent outcomes is imperative for low-risk patients. We report the outcomes of patients treated following the standard-risk protocol from the prospective Japan Association of Childhood Leukemia Study (JACLS) ALL-02 study, which was conducted between 2002 and 2008 for patients with newly diagnosed ALL aged 1-18 years. Of 1138 patients with B-cell precursor ALL, 388 (34.1%) were allocated to this protocol. Excellent outcomes were achieved despite the overall treatment intensity being lower than that of most contemporary protocols: 4 years event-free survival (EFS) was 92.3% and 4 years overall survival 98.2%. Patients with high hyperdiploidy (HHD) involving triple trisomy (trisomy of chromosomes 4, 10, and 17) or ETV6-RUNX1 had even better outcomes (4 years EFS 97.6% and 100%, respectively). Unique characteristics of this protocol include a selection of low-risk patients with a low initial WBC count and good early treatment response and reduction of cumulative doses of chemotherapeutic agents while maintaining dose density. In Japan, we are currently investigating the feasibility of this protocol while incorporating minimal residual disease into the patient stratification strategy.

Endothelial cell-derived endothelin-1 promotes cardiac fibrosis in diabetic hearts through stimulation of endothelial-to-mesenchymal transition.

BACKGROUND: Persistently high plasma endothelin-1 (ET-1) levels in diabetic patients have been associated with the development of cardiac fibrosis, which results from the deposition of extracellular matrix and fibroblast recruitment from an as-yet unknown source. The underlying mechanism, however, remains elusive. Here, we hypothesize that ET-1 might contribute to the accumulation of cardiac fibroblasts through an endothelial-to-mesenchymal transition in diabetic hearts. METHODS AND RESULTS: We induced diabetes mellitus in vascular endothelial cell-specific ET-1 knockout [ET-1(f/f);Tie2-Cre (+)] mice and their wild-type littermates using the toxin streptozotocin. Gene expression and histological and functional parameters were examined at 8, 24, and 36 weeks after the induction of diabetes mellitus. Diabetes mellitus increased cardiac ET-1 expression in wild-type mice, leading to mitochondrial disruption and myofibril disarray through the generation of superoxide. Diabetic mice also showed impairment of cardiac microvascularization and a decrease in cardiac vascular endothelial growth factor expression. ET-1 further promotes cardiac fibrosis and heart failure through the accumulation of fibroblasts via endothelial-to-mesenchymal transition. All of these features were abolished in ET-1(f/f);Tie2-Cre (+) hearts. Targeted ET-1 gene silencing by small interfering RNA in cultured human endothelial cells ameliorated high glucose-induced phenotypic transition and acquisition of a fibroblast marker through the inhibition of transforming growth factor-beta signaling activation and preservation of the endothelial cell-to-cell contact regulator VE-cadherin. CONCLUSIONS: These results provide new insights suggesting that diabetes mellitus-induced cardiac fibrosis is associated with the emergence of fibroblasts from endothelial cells and that this endothelial-to-mesenchymal transition process is stimulated by ET-1. Targeting endothelial cell-derived ET-1 might be beneficial in the prevention of diabetic cardiomyopathy.

Correlation of C4ST-1 and ChGn-2 expression with chondroitin sulfate chain elongation in atherosclerosis.

Subendothelial retention of lipoproteins by proteoglycans (PGs) is the initiating event in atherosclerosis. The elongation of chondroitin sulfate (CS) chains is associated with increased low-density lipoprotein (LDL) binding and progression of atherosclerosis. Recently, it has been shown that 2 Golgi enzymes, chondroitin 4-O-sulfotransferase-1 (C4ST-1) and chondroitin N-acetylgalactosaminyltransferase-2 (ChGn-2), play a critical role in CS chain elongation. However, the roles of C4ST-1 and ChGn-2 during the progression of atherosclerosis are not known. The aim of this study was to analyze the expression of C4ST-1 and ChGn-2 in atherosclerotic lesions in vivo and determine whether their expression correlated with CS chain elongation. Low-density lipoprotein receptor knockout (LDLr KO) mice were fed a western diet for 2, 4, and 8weeks to stimulate development of atherosclerosis. The binding of LDL and CS PG in this mouse model was confirmed by chondroitinase ABC (ChABC) digestion and apolipoprotein B (apo B) staining. Gel filtration analysis revealed that the CS chains began to elongate as early as 2weeks after beginning a western diet and continued as the atherosclerosis progressed. Furthermore, quantitative real-time polymerase chain reaction (qRT-PCR) showed that the mRNA levels of C4ST-1 and ChGn-2 increased after 8weeks of this diet. In contrast, the mRNA levels of their homologs, C4ST-2 and ChGn-1, were unchanged. In addition, immunohistochemical analysis demonstrated that the expression of C4ST-1 and ChGn-2 appeared to have similar site-specific patterns and coincided with biglycan expression at the aortic root. Our results suggested that C4ST-1 and ChGn-2 may be involved in the elongation of CS chains in the arterial wall during the progression of atherosclerosis. Therefore, modulating their expression and activity might be a novel therapeutic strategy for atherosclerosis.

Outcome of childhood acute lymphoblastic leukemia with induction failure treated by the Japan Association of Childhood Leukemia study (JACLS) ALL F-protocol.

BACKGROUND: Children with acute lymphoblastic leukemia (ALL) who fail to achieve complete remission (CR) after induction therapy (induction failure: IF) have a poor prognosis; however, there have been few prospective studies in patients with IF. PATIENTS AND METHODS: Between April 1997 and March 2005, 27 of 1,237 leukemic patients (2.2%) failed to achieve CR after four- or five-drug induction therapy. Twenty-three of these patients entered the F-protocol study, which mainly consisted of acute-myeloid-leukemia-oriented chemotherapy followed by scheduled hematopoietic cell transplantation (HCT). RESULTS: Seventeen (73.9%) of the 23 patients responded to re-induction chemotherapy with CR. Of note, 15 (93.8%) of 16 patients with Philadelphia-chromosome-negative (non-Ph(+)) ALL achieved CR; in contrast, only 2 (28.6%) of 7 Ph(+) patients achieved CR. Fourteen (82.4%) of 17 patients remained in CR (CCR) until their scheduled HCT, 12 of the 14 with CCR underwent HCT as scheduled, and 6 patients remain in first CR after a median of 78 months (range, 49-107 months). The 5-year overall survival (OS) rates of 16 patients with non-Ph(+) and 7 patients with Ph(+) were 43.8 +/- 12.4% and 14.3 +/- 13.2%, respectively (P = 0.012). The 5-year OS rate of the 17 patients who obtained CR by re-induction therapy and the 6 who did not were 47.1 +/- 12.1% and 0%, respectively (P < 0.001). CONCLUSION: Acute-myeloid-leukemia-oriented chemotherapy followed by scheduled HCT is a promising treatment strategy for non-Ph(+) ALL patients with IF.