The effect and possible clinical efficacy of in vivo inhibition of neutrophil extracellular traps by blockade of PI3K-gamma on the pathogenesis of microscopic polyangiitis.

OBJECTIVE: Neutrophil extracellular traps (NETs) are peculiar structures composed of the externalized chromatin with intracellular proteins and formed by activated neutrophils in a reactive oxygen species (ROS)-dependent manner. Aberrant NETs are considered to be autoantigens for anti-neutrophil cytoplasmic antibodies (ANCAs) underling the development of microscopic polyangiitis (MPA). However, little is known regarding the therapeutic efficacy of in vivo inhibition of NET formation (NETosis) on MPA pathogenesis. This study determines whether reducing NETosis prevents ANCA production and improves characteristic involvement. METHODS: A mouse model of MPA induced by administering a novel extract from Candida albicans was devised. By applying this method to mice lacking phosphoinositide 3-kinase gamma (PI3K-gamma), which is indispensable for ROS production in neutrophils, we investigated the levels of in vivo NETs, ANCA titers and histological damage. RESULTS: Our model exhibited accumulation of NETs in vivo, elevation of ANCA titers and characteristic pathologies mimicking human MPA, including small-vessel vasculitis and crescentic glomerulonephritis. Strikingly, these abnormalities were reduced by genetically and/or pharmacologically blocking PI3K-gamma. Moreover, a pharmacological PI3K-gamma blockade decreased the levels of human NETs. CONCLUSION: Our results suggest that in vivo inhibition of NETosis by blocking PI3K-gamma could be a promising therapeutic strategy for the pathogenesis of MPA.

Critical roles of type III phosphatidylinositol phosphate kinase in murine embryonic visceral endoderm and adult intestine.

The metabolism of membrane phosphoinositides is critical for a variety of cellular processes. Phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P(2)] controls multiple steps of the intracellular membrane trafficking system in both yeast and mammalian cells. However, other than in neuronal tissues, little is known about the physiological functions of PtdIns(3,5)P(2) in mammals. Here, we provide genetic evidence that type III phosphatidylinositol phosphate kinase (PIPKIII), which produces PtdIns(3,5)P(2), is essential for the functions of polarized epithelial cells. PIPKIII-null mouse embryos die by embryonic day 8.5 because of a failure of the visceral endoderm to supply the epiblast with maternal nutrients. Similarly, although intestine-specific PIPKIII-deficient mice are born, they fail to thrive and eventually die of malnutrition. At the mechanistic level, we show that PIPKIII regulates the trafficking of proteins to a cell's apical membrane domain. Importantly, mice with intestine-specific deletion of PIPKIII exhibit diarrhea and bloody stool, and their gut epithelial layers show inflammation and fibrosis, making our mutants an improved model for inflammatory bowel diseases. In summary, our data demonstrate that PIPKIII is required for the structural and functional integrity of two different types of polarized epithelial cells and suggest that PtdIns(3,5)P(2) metabolism is an unexpected and critical link between membrane trafficking in intestinal epithelial cells and the pathogenesis of inflammatory bowel disease.

Deletion of autophagy-related 5 (Atg5) and Pik3c3 genes in the lens causes cataract independent of programmed organelle degradation.

The lens of the eye is composed of fiber cells, which differentiate from epithelial cells and undergo programmed organelle degradation during terminal differentiation. Although autophagy, a major intracellular degradation system, is constitutively active in these cells, its physiological role has remained unclear. We have previously shown that Atg5-dependent macroautophagy is not necessary for lens organelle degradation, at least during the embryonic period. Here, we generated lens-specific Atg5 knock-out mice and showed that Atg5 is not required for lens organelle degradation at any period of life. However, deletion of Atg5 in the lens results in age-related cataract, which is accompanied by accumulation of polyubiquitinated and oxidized proteins, p62, and insoluble crystallins, suggesting a defect in intracellular quality control. We also produced lens-specific Pik3c3 knock-out mice to elucidate the possible involvement of Atg5-independent alternative autophagy, which is proposed to be dependent on Pik3c3 (also known as Vps34), in lens organelle degradation. Deletion of Pik3c3 in the lens does not affect lens organelle degradation, but it leads to congenital cataract and a defect in lens development after birth likely due to an impairment of the endocytic pathway. Taken together, these results suggest that clearance of lens organelles is independent of macroautophagy. These findings also clarify the physiological role of Atg5 and Pik3c3 in quality control and development of the lens, respectively.

Increased fatty acyl saturation of phosphatidylinositol phosphates in prostate cancer progression.

Phosphoinositides (PIPs) participate in many cellular processes, including cancer progression; however, the metabolic features of PIPs associated with prostate cancer (PCa) are unknown. We investigated PIPs profiles in PTEN-deficient prostate cancer cell lines, human prostate tissues obtained from patients with PCa and benign prostate hyperplasia (BPH) specimens using mass spectrometry. In immortalized normal human prostate PNT1B cells, PTEN deficiency increased phosphatidylinositol tris-phosphate (PIP3) and decreased phosphatidylinositol mono- and bis-phosphate (PIP1 and PIP2), consistent with PTEN's functional role as a PI(3,4,5)P3 3-phosphatase. In human prostate tissues, levels of total (sum of all acyl variants) phosphatidylinositol (PI) and PIP1 in PCa were significantly higher than in BPH, whereas PIP2 and PIP3 contents were significantly lower than in BPH. PCa patients had significantly higher proportion of PI, PIP1, and PIP2 with 0-2 double bonds in acyl chains than BPH patients. In subgroup analyses based on PCa aggressiveness, mean total levels of PI with 0-2 double bonds in acyl chains were significantly higher in patients with pathological stage T3 than in those with pathological stage T2. These data indicate that alteration of PIPs level and the saturation of acyl chains may be associated with the development and aggressiveness of prostate cancer, although it is unknown whether this alteration is causative.

Vps34 regulates myofibril proteostasis to prevent hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a common heart disease with a prevalence of 1 in 500 in the general population. Several mutations in genes encoding cardiac proteins have been found in HCM patients, but these changes do not predict occurrence or prognosis and the molecular mechanisms underlying HCM remain largely elusive. Here we show that cardiac expression of vacuolar protein sorting 34 (Vps34) is reduced in a subset of HCM patients. In a mouse model, muscle-specific loss of Vps34 led to HCM-like manifestations and sudden death. Vps34-deficient hearts exhibited abnormal histopathologies, including myofibrillar disarray and aggregates containing αB-crystallin (CryAB). These features result from a block in the ESCRT-mediated proteolysis that normally degrades K63-polyubiquitinated CryAB. CryAB deposition was also found in myocardial specimens from a subset of HCM patients whose hearts showed decreased Vps34. Our results identify disruption of the previously unknown Vps34-CryAB axis as a potentially novel etiology of HCM.

A large-scale prospective cohort study on the current status of therapeutic modalities for acute myocardial infarction in Japan: rationale and initial results of the HIJAMI Registry.

BACKGROUND: In Western countries, several multicenter collaborative studies on acute myocardial infarction (AMI) have provided much information about this disease. In Japan, on the other hand, there have been few cohort studies in which a sufficient number of Japanese patients with AMI were registered during a short period. This fact explains the absence of a database from which strategies for treating Japanese patients with AMI could be established. The purpose of this study was to build a comprehensive database on Japanese patients with AMI to elucidate their characteristics. METHODS: Between January 1999 and June 2001, we consecutively registered all patients with AMI who were admitted to 17 participating medical institutions, including The Heart Institute of Japan, Cardiology (HIJC), Tokyo Women's Medical University. A standardized case report form was used to register all the patients. RESULTS: A total of 3,021 consecutive patients was registered (2,136 men, 70.7%; 885 women, 29.3%) with a median age of 69 years [59, 77]. Among the patients, there were 851 elderly individuals (28.2%) > or = 76 years and 1102 patients with diabetes (36.5%). On index electrocardiogram, ST-elevation myocardial infarction was observed in 2,392 patients (79.2%). Within 24 hours after the onset of AMI, coronary angiography was conducted for 2,177 patients (72.1%). Primary percutaneous coronary intervention and coronary thrombolysis were conducted for 1,755 (58.1%) and 491 patients (16.3%), respectively, and percutaneous coronary intervention or coronary artery bypass grafting was additionally carried out in 303 patients. By the time of discharge, coronary angiography and coronary artery bypass grafting were performed in 2,659 (88.0%) and 137 patients (4.5%), respectively. During initial hospitalization, 285 patients died and the overall inhospital mortality rate was 9.4%. During hospitalization, cardiogenic shock and cardiac rupture were observed in 6.1% and 2.8% of the patients, respectively. The inhospital mortality rate is still high in patients with AMI with such mechanical complications and in elderly patients. CONCLUSION: In our prospective cohort, we showed that Japanese patients with AMI could be characterized as (1) having a disease severity comparable with values observed in Western populations and (2) receiving early reperfusion therapy by PCI, which was used widely and safely, but nevertheless (3) exhibiting a high inhospital mortality rate. Our data indicate that further improvements in therapy for AMI in elderly patients and for AMI with mechanical complications are essential in Japan.

INPP4B Is a PtdIns(3,4,5)P3 Phosphatase That Can Act as a Tumor Suppressor.

UNLABELLED: Inositol polyphosphate 4-phosphatase B (INPP4B) has been identified as a tumor suppressor mutated in human breast, ovary, and prostate cancers. The molecular mechanism underlying INPP4B's tumor-suppressive role is currently unknown. Here, we demonstrate that INPP4B restrains tumor development by dephosphorylating the PtdIns(3,4,5)P3 that accumulates in situations of PTEN deficiency. In vitro, INPP4B directly dephosphorylates PtdIns(3,4,5)P3. In vivo, neither inactivation of Inpp4b (Inpp4b(Δ/Δ)) nor heterozygous deletion of Pten (Pten(+/-)) in mice causes thyroid abnormalities, but a combination of these mutations induces malignant thyroid cancers with lung metastases. At the molecular level, simultaneous deletion of Inpp4b and Pten synergistically increases PtdIns(3,4,5)P3 levels and activates AKT downstream signaling proteins in thyroid cells. We propose that the PtdIns(3,4,5)P3 phosphatase activity of INPP4B can function as a "back-up" mechanism when PTEN is deficient, making INPP4B a potential novel therapeutic target for PTEN-deficient or PIK3CA-activated cancers. SIGNIFICANCE: Although INPP4B expression is reduced in several types of human cancers, our work on Inpp4B-deficient mice provides the first evidence that INPP4B is a bona fide tumor suppressor whose function is particularly important in situations of PTEN deficiency. Our biochemical data demonstrate that INPP4B directly dephosphorylates PtdIns(3,4,5)P3.

Skeletal muscle hemodynamics and endothelial function in patients after Fontan operation.

To elucidate the relation between exercise capacity and peripheral hemodynamics in patients after the Fontan operation, near-infrared spectroscopy was performed on the vastus lateralis muscle, and flow-mediated vasodilation of the brachial and posterior tibial arteries was done with high-resolution ultrasonography. In Fontan patients, diminished exercise capacity was related to a reduced blood flow supply and an attenuated post-exercise oxygen resaturation of the working skeletal muscle, which also was related to impaired endothelium-dependent vasodilation.

Prognostic significance of serum creatinine concentration for in-hospital mortality in patients with acute myocardial infarction who underwent successful primary percutaneous coronary intervention (from the Heart Institute of Japan Acute Myocardial Infarction [HIJAMI] Registry).

This study evaluated the impact of serum creatinine levels on in-hospital mortality in 1,359 consecutive patients with acute myocardial infarction (from a Japanese prospective multicenter registry) who underwent successful primary percutaneous coronary intervention (PCI). Even in the patients who underwent successful primary PCI, the in-hospital mortality of patients with mild (1.2 /=2.0 mg/dl) renal dysfunction was greater (17.1% and 34.5%, respectively) than that of patients without renal dysfunction (3.9%) (relative risk [RR] 1.72, 95% confidence interval [CI] 0.94 to 3.14, p = 0.080; and RR 4.26, 95% CI 1.48 to 12.27, p <0.0001, respectively).

Rapidly progressed secondary amyloidosis in a patient with mixed connective tissue disease.

A 39-year-old woman with mixed connective tissue disease suddenly developed repeated watery diarrhea two years after the onset of the disease. A colonic biopsy specimen revealed amyloid A protein deposition and the diagnosis of secondary amyloidosis was established. The amyloid deposition disappeared after the 8-month course of the treatment with prednisolone and azathioprine. Molecular genetic analysis showed the presence of the gamma-allele in her serum amyloid A protein 1 gene. This might be associated with the early onset and progression of secondary amyloidosis in our case, just like cases reported in rheumatoid arthritis.

Increased levels of interleukin 33 in sera and synovial fluid from patients with active rheumatoid arthritis.

OBJECTIVE: To determine levels of interleukin 33 (IL-33) in serum and synovial fluid (SF) and their clinical associations in patients with rheumatoid arthritis (RA). To evaluate the ability of activated peripheral blood mononuclear cells (PBMC) and fibroblast-like synoviocytes (FLS) from RA patients to release IL-33. METHODS: Sera were obtained from 59 patients with RA, 10 patients with infectious diseases, and 42 healthy volunteers. SF samples were obtained from 15 patients with RA and 13 with osteoarthritis. IL-33 levels were measured using a sandwich ELISA after removal of rheumatoid factor with protein A-Sepharose beads. FLS were stimulated with IL-1beta and tumor necrosis factor, and treated with or without chemical damage. PBMC were stimulated with anti-CD3/CD28 antibodies. The levels of IL-33 were measured in the culture supernatants and cell lysates by ELISA or immunoblotting. RESULTS: Serum IL-33 levels were significantly higher in RA patients, especially in the high disease activity group compared to the moderate or low activity group. IL-33 levels in SF were elevated in all 15 RA patients measured. IL-33 levels were higher in SF samples than in sera in 7 RA patients measured simultaneously. The 30-kDa IL-33 precursor was detected in the culture supernatants of damaged FLS but was not detected in those of activated PBMC and non-damaged FLS. CONCLUSION: IL-33 levels were elevated in sera and SF samples from patients with RA, and correlated with disease activity. IL-33 was produced mainly in inflamed joints; IL-33/ST2L signaling might play an important role in joint inflammation of human RA.