Functional mechanism underlying cyclooxygenase-2 expression in rat small intestine following administration of indomethacin: Relation to intestinal hypermotility

Background and Aim:  We recently reported that cyclooxygenase (COX)-2 is upregulated in the rat small intestine after administration of indomethacin, and this may be the key to non-steroidal anti-inflammatory drug (NSAID)-induced intestinal damage. The present study investigated the mechanism for COX-2 expression induced in the rat small intestine by indomethacin, in relation with ulcerogenic processes.
Methods:  Animals were given indomethacin or SC-560 p.o., and the intestinal mucosa was examined 24 h later.
Results:  Indomethacin caused hemorrhagic lesions in the small intestine, accompanied with an increase in intestinal motility, bacterial invasion and inducible nitric oxide synthase (iNOS) activity, as well as the expression of COX-2 mRNA in the mucosa. Although SC-560 did not cause any damage, this agent caused intestinal hypermotility, the bacterial invasion and the upregulation of COX-2 expression. The mucosal PGE₂ content was decreased by SC-560 at 3 h but recovered 12 h later, and this recovery of PGE₂ was attenuated by both atropine and ampicillin, in addition to rofecoxib. The intestinal hypermotility response to indomethacin was prevented by both 16,16-dimethyl PGE₂ and atropine, but not ampicillin. Yet all these agents inhibited not only the bacterial invasion but also the expression of COX-2 and iNOS activity in the intestinal mucosa following indomethacin treatment, resulting in the prevention of intestinal lesions.
Conclusion:  These results suggest that COX-2 expression in the intestinal mucosa following the administration of indomethacin is associated with intestinal hypermotility and bacterial invasion. The intestinal hypermotility caused by COX-1 inhibition may be a key to COX-2 expression after administration of NSAIDs and their intestinal ulcerogenic properties.     

HtrA2 is up-regulated in the rat testis after experimental cryptorchidism

AIM: The aim of the present study was to elucidate the role of high temperature requirement A2 (HtrA2) in germ cell loss in the heat-stressed testis. METHODS: We examined the expression of HtrA2, caspase-9 activity and proteolytic activity of HtrA2 in the rat testis, and their in vivo responses to experimental cryptorchid treatment. RESULTS: Northern analysis revealed the expression of HtrA2 mRNA peaked at days 1 and 7 after cryptorchid treatment. While expression of HtrA2 mRNA was seen in the spermatogonium, spermatocytes and some spermatids in normal adult rat testis, experimental cryptorchidism treatment resulted in a marked increase in its signal intensity in spermatocytes and some spermatids, and the layers of spermatogonium and early primary spermatocytes became negative at days 1 and 7 after the treatment. However, the spermatogonium, Sertoli cells and interstitial cells appeared to have strong intensities at days 14, 28 and 56 after the treatment. Western analysis revealed the expression of HtrA2 protein peaked at day 2 coinciding with the increase of positive spermatogonium, the appearance of protein-positive interstitial cells, and day 28 coinciding with the reappearance of protein-positive interstitial cells. Caspase-9 activity peaked at day 2 and HtrA2 proteolytic activity peaked at day 28. Consequently, the first peak of HtrA2 mRNA expression was followed by the peak of caspase-9 activity and the second peak was followed by the peak of proteolytic activity; however, the second peak of mRNA expression had considerable chronological difference from that of the protein. CONCLUSION: These findings suggest the probabilities that the heat stress results in germ cell death by a caspase-independent manner with the elevation of HtrA2 proteolytic activity, as well as a caspase-dependent manner with the elevation of caspase-9 activity.     

Relationship between cellular ATP content and cellular functions of primary cultured rat hepatocytes in hypoxia

The importance of oxygen in maintaining the functional integrity of hepatocytes has been well established in a variety of experimental models, such as in vivo , perfused liver and isolated hepatocytes. However, one of the shortcomings of these systems is their short life span. Therefore, we have examined the effects of long-term hypoxia on cellular adenine nucleotide content and cellular functions, such as albumin production, urea production and DNA synthesis, in adult rat hepatocytes in primary culture. Hepatocytes were cultured at a density of 11 × 10⁴ and 5 × 10⁴ cells/0.18 mL per cm² for the study of albumin and urea production and DNA synthesis, respectively, at various oxygen tensions (20, 12, 8 and 5%) for 24 h. Cellular ATP content in cultured hepatocytes in hypoxia gradually declined, corresponding to the decrease in oxygen tension, and the cellular ATP level at 5% oxygen was approximately 20% of that at 20% oxygen. Albumin production also decreased in parallel with the decrease in cellular ATP content in cultured hepatocytes in hypoxia. However, even when cellular ATP content gradually declined corresponding with the decrease in oxygen tension in cultured hepatocytes in hypoxia, such as at 8 or 5% oxygen, urea production remained at a high level; in contrast, DNA synthesis was completely suppressed. These results suggest that the cellular ATP content decreases in cultured hepatocytes during long-term hypoxia in relation to oxygen tension and that the relationship between decreased ATP levels and liver function in cultured hepatocytes during hypoxia differs for albumin production, urea production and DNA synthesis.     

Functional thrombomodulin expression on epithelial skin tumours as a differentiation marker for suprabasal keratinocytes

Summary In normal human skin, immtmoreactive thrombomodulin (TM) is expressed in a strict differentiation related pattern. solely in suprabasal spinous layer keratinocytes. To evaluate the polential application of TM as a differentiation marker for keratinocyte-derived skin tumours, we have studied immunohistopathological, biochemical and functional TM activities in various skin tumours. Immunoreactive, full sized and enzymatically active TM was expressed in keratinocyte-derived skin tumours (squamous cell carcinoma, seborrhoeic keratosis and partly Bowen's disease), as well as normal epidermal keratinoeytes and endothelial cells. However, no TM was detected in basal cell carcinotnas, senile keratosis or non-squanious epithelial tumours such as malignant melanoma, naevus pigmentosus and Paget's disease. Interestingly, decreased expression was observed in verruca vulgaris. These findings suggested that differentiation-dependent TM expression was restricted to epithelial skin tumours and undetectable on neural crest derived tumours. TM is a differentiation marker for spinous layer keratinocytes and is a useful tool in histopathological study of epithelial tumours.     

Functional analysis of titin/connectin N2-B mutations found in cardiomyopathy

Hypertrophic cardiomyopathy and dilated cardiomyopathy are two major clinical phenotypes of “idiopathic” cardiomyopathy. Recent molecular genetic analyses have now revealed that “idiopathic” cardiomyopathy is caused by mutations in genes for sarcomere components. We have recently reported several mutations in titin/connectin gene found in patients with hypertrophic cardiomyopathy or dilated cardiomyopathy. A hypertrophic cardiomyopathy-associated titin/connectin mutation (Arg740Leu) was found to increase the binding to actinin, while other dilated cardiomyopathy-associated titin/connectin mutations (Ala743Val and Val54Met) decreased the binding to actinin and Tcap/telethonin, respectively. We also reported several other mutations in the N2-B region of titin/connectin found in hypertrophic cardiomyopathy and dilated cardiomyopathy. Since the N2-B region expresses only in the heart, it was speculated that functional alterations due to the mutations cause cardiomyopathies. In this study, we investigated the functional changes caused by the N2-B region mutations by using yeast-two-hybrid assays. It was revealed that a hypertrophic cardiomyopathy-associated mutation (Ser3799Tyr) increased the binding to FHL2 protein, whereas a dilated cardiomyopathy-associated mutation (Gln4053ter) decreased the binding. In addition, another TTN mutation (Arg25618Gln) at the is2 region was found in familial DCM. Because FHL2 protein is known to tether metabolic enzymes to N2-B and is2 regions of titin/connectin, these observations suggest that altered recruitment of metabolic enzymes to the sarcomere may play a role in the pathogenesis of cardiomyopathies.     

Combined Repeat Dose and Reproductive/Developmental Toxicity Screening Test (OECD): Familiarization Using Cyclophosphamide

Combined Repeat Dose and Reproductive/Developmental ToxicityScreening Test (OECD): Familiarization Using Cyclophosphamide.TANAKA, S., KAWASHIMA, K., NAITO, K., USAMI, M., NAKADATE, M.,IMAIDA, K., TAKAHASHI, M., HAYASHI, Y., KUROKAWA, Y., AND TOBE,M. (1992A, Y., AND TOBE, M. (1992). Fundam. Appl. Toxicol. 18,89–95. A familiarization study was conducted on the "Combined RepeatDose and Reproductive/Developmental Toxicity Screening Test(ReproTox)" proposed by the OECD. Cyclophosphamide (CP) at dosesof 6.7, 4.5, 3, 2, and 0 mg/kg body wt was given daily by gavageto groups of 12 male and 12 female Sprague-Dawley rats. As aresult, anemia and leukopenia were evident in treated males.The absolute and relative thymus and spleen weights were decreasedin treated rats. Histopathologically, atrophy of the thymus,spleen, and bone marrow was observed. With respect to the reproductive/developmentaltoxicity, dose-dependent increases in postimplantation lossof fetuses and postnatal death were found in dams given CP.The body weight of pups treated with CP was significantly loweredin a dose-related manner. Thus the results demonstrated mostof the known toxicological properties of CP, except the adverseeffects on spermatogenesis and fertility. Therefore ReproToxcan be considered as a useful screening test for assessing repeatdose and reproductive/developmental toxicity of existing chemicalsof high production volume.     

Correlation between deletion patterns of SMN and NAIP genes and the clinical features of spinal muscular atrophy in Japanese patients

We conducted molecular analysis of two candidate genes for spinal muscular atrophy (SMA), the survival motor neuron gene (SMN) and the neuronal apoptosis inhibitory protein gene (NAIP), in 16 Japanese patients with SMA and compared the phenotypic features of SMA in these patients with the corresponding genotypes. Exons 7 and/or 8 of SMN were homozygously deleted in 11 SMA type I (Werdnig-Hoffmann disease) patients, two SMA type II patients and one SMA type III patient. Exons 5 and 6 of NAIP were homozygously deleted in six SMA type I patients. No patient had a deletion in NAIP without a deletion in SMN. Mechanical ventilation was required during the first 7 months of life in the SMA type I patients who had a deletion in both SMN and NAIP. Ventilatory support was initiated within 2 years after birth in patients who had a deletion in SMN but not in NAIP. We detected homozygous deletion of exon 5 of NAIP in the unaffected mothers of two SMA type I patients. In these families, the patients exhibited a deletion in both SMN and NAIP. The parents and unaffected siblings of these patients did not have a deletion in SMN. The present findings support the hypothesis that SMN deletion plays an important role in the development of SMA and suggest that combined deletion of both SMN and NAIP may be relevant for determining the disease severity.     

Regulatory mechanisms of C4b‐binding protein (C4BP)α and β expression in rat hepatocytes by lipopolysaccharide and interleukin‐6

Summary. Background: C4b‐binding protein (C4BP), a multimeric protein structurally composed of α chains (C4BPα) and a β chain (C4BPβ), regulates the anticoagulant activity of protein S (PS). Patients with sepsis have increased levels of plasma C4BP, which appears to be induced by interleukin (IL)‐6. However, it is not fully understood how lipopolysaccharide (LPS) and IL‐6 affect the plasma C4BP antigen level and C4BPα and C4BPβ expression in hepatocytes. Objectives: To assess the effect of LPS and IL‐6 on plasma C4BP, PS–C4BP complex levels, PS activity, and C4BP expression by rat liver in vivo and on C4BP expression by isolated rat hepatocytes in vitro. Results: Plasma C4BP antigen level transiently decreased from 2 to 12 h after LPS (2 mg kg^?1) injection, and then it abruptly increased up to 24 h after LPS injection. Plasma C4BP antigen level increased until 8 h after IL‐6 (10 μg kg^?1) injection, and then gradually decreased up to 24 h after IL‐6 injection. LPS significantly decreased the protein and mRNA expression of both C4BPα and C4BPβ in rat hepatocytes, and this effect was inhibited by NFκB and MEK/ERK inhibitors. IL‐6 mediated increase in C4BPβ expression in rat hepatocytes, which leads to increased plasma PS–C4BP complex level and to decreased plasma PS activity, was inhibited by inhibition of STAT‐3. Conclusion: LPS decreases both C4BPα and C4BPβ expression via the NFκB and MEK/ERK pathways, whereas IL‐6 specifically increases C4BPβ expression via the STAT‐3 pathway, causing an increase in plasma PS–C4BP complex, and thus decreasing the anticoagulant activity of PS.     

Successful Coil Embolization of a Ruptured Basilar Artery Aneurysm in a Child with Leukemia: A Case Report

Ruptured intracranial aneurysms are rare in the pediatric population compared to adults. This has incited considerable discussion on how to treat children with this condition. Here, we report a child with a ruptured saccular basilar artery aneurysm that was successfully treated with coil embolization. A 12-year-old boy with acute lymphoblastic leukemia and accompanying abdominal candidiasis after chemotherapy suddenly complained of a severe headache and suffered consciousness disturbance moments later. Computed tomography scans and cerebral angiography demonstrated acute hydrocephalus and subarachnoid hemorrhage caused by saccular basilar artery aneurysm rupture. External ventricular drainage was performed immediately. Because the patient was in severe condition and did not show remarkable signs of central nervous system infection in cerebrospinal fluid studies, we applied endovascular treatment for the ruptured saccular basilar artery aneurysm, which was successfully occluded with coils. The patient recovered without new neurological deficits after ventriculoperitoneal shunting. Recent reports indicate that both endovascular and microsurgical techniques can be used to effectively treat ruptured cerebral aneurysms in pediatric patients. A minimally invasive endovascular treatment was effective in the present case, but long-term follow-up will be necessary to confirm the efficiency of endovascular treatment for children with ruptured saccular basilar artery aneurysms.     

Vascular Endothelial Growth Factor–Bound Stents: Application of In Situ Capture Technology of Circulating Endothelial Progenitor Cells in Porcine Coronary Model

Objectives

We evaluated the in vivo performance of a newly devised vascular endothelial growth factor (VEGF)‐bound stent in a porcine coronary model.

Background

An anti‐CD34 antibody‐bound stent, which captures endothelial progenitor cells (EPCs) to accelerate tissue formation, did not reduce intimal hyperplasia. By targeting the VEGF receptor, which is expressed on endothelial‐lineage cells, we developed VEGF‐bound stents that may enable selective capture of EPCs followed by rapid endothelialization.

Methods

Metallic stents were first coated with poly‐(ethylene‐co‐vinyl alcohol), and then chemically bound with either VEGF or anti‐CD34 antibody. These stents were placed in porcine coronary arteries for up to 14 days. Stent surface was evaluated by immunohistochemistry and by scanning electron microscope (SEM).

Results

After 2‐day stenting with VEGF‐bound stents, small populations of KDR (VEGF receptor‐2)‐positive cells adhered to the stent struts. After 7‐ and 14‐day stenting, struts were fully covered with newly regenerated tissue. SEM images showed that the uniform tissue formed on struts was morphologically similar to native endothelium and was continuously connected with adjacent native endothelium. On the other hand, for the anti‐CD34 antibody‐bound stents, stent struts were rapidly covered by newly generated tissue that consisted of multicellular aggregates.

Conclusions

Compared with anti‐CD34 antibody‐bound stents, VEGF‐bound stents provide highly selective capture of EPCs, followed by rapid formation of intact endothelium tissue at an early period of stenting. These results suggest that VEGF‐bound stents could represent a promising therapeutic option for cardiovascular stenting, although further long‐term follow‐up experiment with double‐blinded fashion is needed prior to clinical application. (J Interven Cardiol 2014;27:63–72)