Angiogenesis as a novel component of inflammatory bowel disease pathogenesis

BACKGROUND & AIMS: Angiogenesis is a critical component of neoplastic and chronic inflammatory disorders, but whether angiogenesis also occurs in inflammatory bowel disease (IBD) has yet to be established. We assessed mucosal vascularization, expression of endothelial alphaVbeta3 integrin, angiogenic factors, and their bioactivity in Crohn's disease (CD) and ulcerative colitis (UC) mucosa. METHODS: Mucosal endothelium was immunostained for CD31 and factor VIII and quantified by digital morphometry. alphaVbeta3 expression was studied in vivo by confocal microscopy and in vitro by flow cytometric analysis of human intestinal microvascular endothelial cells (HIMECs). Vascular endothelial growth factor (VEGF), interleukin (IL)-8, and bFGF levels were measured in mucosal extracts and cells and angiogenic bioactivity shown by induction of HIMEC migration and the corneal and chorioallantoic membrane angiogenesis assays. RESULTS: Microvessel density was increased in IBD mucosa. Endothelial alphaVbeta3 was strongly expressed in IBD but only sporadically in normal mucosa and was up-regulated in HIMECs by VEGF, tumor necrosis factor alpha, and bFGF. IBD mucosal extracts induced a significantly higher degree of HIMEC migration than control mucosa, and this response was mostly dependent on IL-8 and less on basic fibroblast growth factor or vascular endothelial growth factor. Compared with normal mucosa, IBD mucosal extracts induced a potent angiogenic response in both the corneal and chorioallantoic membrane assays. CONCLUSIONS: These results provide morphological, phenotypic and functional evidence of potent angiogenic activity in both CD and UC mucosa, indicating that the local microvasculature undergoes an intense process of inflammation-dependent angiogenesis. Thus, angiogenesis appears to be an integral component of IBD pathogenesis, providing the practical and conceptual framework for anti-angiogenic therapies in IBD.     

A Mechanistic Proof-of-concept Clinical Trial With JX-594, a Targeted Multi-mechanistic Oncolytic Poxvirus,in Patients With Metastatic Melanoma

JX-594 is a targeted and granulocyte macrophage-colony stimulating factor (GM-CSF)-expressing oncolytic poxvirus designed to selectively replicate in and destroy cancer cells through viral oncolysis and tumor-specific immunity. In order to study the mechanisms-of-action (MOA) of JX-594 in humans, a mechanistic proof-of-concept clinical trial was performed at a low dose equivalent to ≤10% of the maximum-tolerated dose (MTD) in other clinical trials. Ten patients with previously treated stage IV melanoma were enrolled. Tumors were injected weekly for up to nine total treatments. Blood samples and tumor biopsies were analyzed for evidence of transgene activity, virus replication, and immune stimulation. The β-galactosidase (β-gal) transgene was expressed in all patients as evidenced by antibody induction. Six patients had significant induction of GM-CSF-responsive white blood cell (WBC) subsets such as neutrophils (25–300% increase). JX-594 replication and subsequent shedding into blood was detectable in five patients after cycles 1–9. Tumor biopsies demonstrated JX-594 replication, perivascular lymphocytic infiltration, and diffuse tumor necrosis. Mild flu-like symptoms were the most common adverse events. In sum, JX-594 replication, oncolysis, and expression of both transgenes were demonstrated; replication was still evident after multiple cycles. These findings have implications for further clinical development of JX-594 and other transgene-armed oncolytic viruses.     

Reactions of purines-containing butenolides with L-cysteine or N-acetyl-L-cysteine as model biological nucleophiles: a potent mechanism-based inhibitor of ribonucleotide reductase caused apoptosis in breast carcinoma MCF7 cells

Thiols are the most reactive nucleophilic reagents among the biological models investigated. The reactivity of butenolides 1a-c, 2-4, and 6-8 toward L-cysteine, a model biological nucleophile, was studied spectrophotometrically. The rates of the reactions were measured and correlated with antitumour activity of these molecules. N-Acetylcysteine addition product 5, resulting from the treatment of butenolide 4 with glutathione precursor, N-acetyl-L-cysteine, was isolated. Unlike purine-containing gamma-(Z)-ethylidene-2,3-dimethoxybutenolides 1a-c, 4, 6, and 7, adduct 5 and butenolides 10-12 did not exhibit inhibitory activity against murine leukemias (L1210 and P388), breast carcinoma (MCF7), and human T-lymphoblasts (Molt4/C8 and CEM/0) cell lines. As such, the biological activity of purine-containing butenolides can be attributed to their adenine moiety as a recognition site as well as their reactivity towards the cysteine residues of functional proteins forming covalent bond via reverse Michael type addition. Adenine-containing phosphonothioanhydride derivative 8 was also synthesised. Its reaction with N-acetyl-L-cysteine produced N,S-diacetylcysteine and thiophosphonate 9. Compound 9 did not exhibit anticancer activity; yet its precursor 8 displayed the most pronounced inhibition on all the examined malignant tumour cell lines. In the presence of L-cysteine, cytotoxicity of 4 and 8 was decreased, whereas glutathione addition more influenced on the cytotoxicity of 8. It was found that adenine-containing phosphonothioanhydride 8 functions as a significant irreversible inactivator of the Escherichia coli ribonucleoside diphosphate reductase. After treatment of MCF7 cells with compound 8, fluorescence microscopy demonstrated the presence of nucleus shrinkage or segmentation. This apoptotic morphology, however, was not pronounced in the presence of glutathione or dithiotheritol.     

Viscocanalostomy and deep sclerectomy for the surgical treatment of glaucoma: a longterm follow-up

PURPOSE: To study the outcome of viscocanalostomy (VC) and deep sclerectomy (DS) for the surgical management of medically uncontrolled glaucoma. PATIENTS AND METHODS: A non-randomized, prospective study of all consecutive non-penetrating glaucoma filtering procedures was carried out in two centres. In the first centre, one surgeon (MSW) performed VC in 105 eyes (27 VC and 78 phaco VC). In the second centre, one surgeon (PKW) performed DS in 87 eyes (52 DS and 35 phaco DS). RESULTS: The mean follow-up was 36 months (range 9-60 months). At final follow-up the complete success rate (intraocular pressure < or = 21 Hg without medication) was 92.6% for VC eyes, 96% for phaco VC eyes, 77% for DS eyes and 94% for phaco DS eyes. Kaplan-Meier survival analysis for complete success showed no significant difference between DS and VC nor between phaco VC and phaco DS (p > 0.05). By 36 months postoperatively, mean IOP was 16.8 mmHg (SD 3) in VC eyes, 16.6 mmHg (SD 3.1) in phaco VC eyes, 16.7 mmHg (SD 5.7) in DS eyes and 15 mmHg (SD 3.2) in phaco DS eyes. Postoperative Nd:YAG laser goniopuncture was necessary in 10 eyes in the DS group. Large or cystic drainage blebs occurred only in the DS eyes. CONCLUSIONS: Viscocanalostomy and DS are effective and safe methods of achieving sustained IOP reduction in glaucomatous eyes and both techniques can be successfully combined with cataract extraction.     

cIAP2 Is an Independent Signaling and Survival Factor during Mammary Lactational Involution and Tumorigenesis

Cellular inhibitor of apoptosis proteins-1 and -2 (cIAP1/2) are integral to regulation of apoptosis and signaling by the tumor necrosis factor (TNF) and related family of receptors. The expression of cIAP2 in tissues is typically low and considered functionally redundant with cIAP1, however cIAP2 can be activated by a variety of cellular stresses. Members of the TNFR family and their ligands have essential roles in mammary gland biology. We have found that cIAP2^?/? virgin mammary glands have reduced ductal branching and delayed lobuloalveogenesis in early pregnancy. Post-lactational involution involves two phases where the first phase is reversible and is mediated, in part, by TNFR family ligands. In cIAP2?/? mice mammary glands appeared engorged at mid-lactation accompanied by enhanced autophagic flux and decreased cIAP1 protein expression. Severely stretched myoepithelium was associated with BIM-EL expression and other indicators of anoikis. Within 24 h after forced or natural weaning, cIAP2^?/? glands had nearly completed involution. The TNF-related weak inducer of apoptosis (Tweak) which results in degradation of cIAP1 through its receptor, Fn14, began to increase in late lactation and was significantly increased in cIAP2^?/? relative to WT mice by 12 h post weaning accompanied by decreased cIAP1 protein expression. Carcinogen/progesterone-induced mammary tumorigenesis was significantly delayed in cIAP2^?/? mice and tumors contained high numbers of apoptotic cells. We conclude that cIAP2 has a critical role in the mammary gland wherein it prevents rapid involution induced by milk stasis-induced stress associated with Tweak activation and contributes to the survival of mammary tumor cells.     

Fetal genotype for specific inherited thrombophilias is not associated with severe preeclampsia

OBJECTIVE: Little is known about the association between fetal thrombophilias and severe preeclampsia. The objective of this study was to examine the association between fetal genotype for factor V Leiden, prothrombin, and methylene tetrahydrofolate reductase (MTHFR) mutations and severe preeclampsia. METHODS: Patients with severe preeclampsia or HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome admitted to Georgetown University Hospital were retrospectively identified. Controls were patients with uncomplicated, term deliveries. Fetal DNA was extracted from placental specimens and amplified by polymerase chain reaction (PCR) with locus-specific primers. The presence of polymorphisms was determined by enzymatic digestion with specific enzymes, and analyzed by polyacrylamide gels. Statistical analysis used Student t test for continuous variables and Fisher exact test for categorical data. RESULTS: Patients with preeclampsia (n = 27) and controls (n = 17) were similar for maternal age, but, as expected, they were significantly different for gestational age at delivery, birth weight, Apgar scores at 5 minutes, rate of preterm delivery less than 37 weeks, and fetal growth restriction (all P <.05). DNA extraction was successful in 25 of 27 cases from the severe preeclampsia group and 14 of 17 controls. None of the placentas analyzed in the preeclamptic or control group revealed mutations in the factor V Leiden or prothrombin genes. There was no significant difference in the rate of fetuses heterozygous for MTHFR in the preeclampsia versus control group (48% vs 43%, P >.05). CONCLUSION: In our study, fetal genotype for specific inherited thrombophilias does not appear to be associated with severe preeclampsia.