The controls of microvascular survival.

The regulation of microvascular survival impacts both developmental remodeling of the vasculature, and various microvascular pathologies. In pathological settings of vascular insufficiency, molecular targets to affect stabilization of neovascularization are needed. Conversely, an important part of anti-tumor angiogenesis is the de-stabilization of the tumor vasculature. In the study of vascular remodeling, one difficult challenge is to understand the molecular controls that allow regression of one entire vessel segment and not another. This phenomenon requires coordination of the survival signaling pathways to successfully impact vascular structure. This review describes the known mechanisms and molecules involved in microvascular and endothelial cell survival. In particular the mechanisms of molecular signaling for survival in vitro are discussed in light of what is known about microvascular survival in vivo. Possible ways to bring these data together to explain the complex regulation of vessel survival are discussed.

The antiangiogenic agent linomide inhibits the growth rate of von Hippel-Lindau paraganglioma xenografts to mice.

The aim of this study was to ascertain the potential usefulness of the antiangiogenic compound linomide for treatment of von Hippel-Lindau (VHL)-related tumors. Paraganglioma tissue fragments obtained at surgery from a VHL type 2a patient were transplanted s.c. to male BALB/c nu/nu (nude) mice: (a) 2-3-mm fragments for "prevention" experiments; and (b) 2-3-mm fragments allowed to grow to 1 cm for "intervention" studies. Both groups received either 0.5 mg/ml linomide in drinking water or acidified water and were followed until tumor diameter reached 3 cm or for 4 weeks. In both the prevention and intervention experiments, a significant diminution of tumor size and weight was observed in the drug-treated animals. In vivo nuclear magnetic resonance analysis of tumor blood flow in linomide-treated animals showed localization of blood vessels almost exclusively to the periphery of the poorly vascularized tumors with a significant reduction of both vascular functionality and vasodilation. Histological examination of tumors from linomide-treated animals revealed marked avascularity. Treated animals also displayed a 2.4-fold reduction of tumor vascular endothelial growth factor mRNA levels. Taken together, our data indicate that in VHL disease, therapy directed at inhibition of constitutively expressed VEGF induction of angiogenesis by VHL tumors may constitute an effective medical treatment.

In vivo prediction of vascular susceptibility to vascular susceptibility endothelial growth factor withdrawal: magnetic resonance imaging of C6 rat glioma in nude mice.

One of the hallmarks of tumor neovasculature is the prevalence of immature vessels manifested by the low degree of recruitment of vascular mural cells such as pericytes and smooth muscle cells. This difference in the architecture of the vascular bed provides an important therapeutic window for inflicting tumor-selective vascular damage. Here we demonstrate the application of gradient echo magnetic resonance imaging (MRI) for noninvasive in vivo mapping of vascular maturation, manifested by the ability of mature vessels to dilate in response to elevated levels of CO2. Histological alpha-actin staining showed a match between dilating vessels detected by MRI and vessels coated with smooth muscle cells. Switchable, vascular endothelial growth factor (VEGF)-overexpressing tumors (C6-pTET-VEGF rat glioma s.c. tumors in nude mice) displayed high vascular function and significant vascular damage upon VEGF withdrawal. However, damage was restricted to nondilating vessels, whereas mature dilating tumor vessels were resistant to VEGF withdrawal. Thus, MRI provides in vivo visualization of vascular maturity and prognosis of vascular obliteration induced by VEGF withdrawal.

Selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal.

Features that distinguish tumor vasculatures from normal blood vessels are sought to enable the destruction of preformed tumor vessels. We show that blood vessels in both a xenografted tumor and primary human tumors contain a sizable fraction of immature blood vessels that have not yet recruited periendothelial cells. These immature vessels are selectively obliterated as a consequence of vascular endothelial growth factor (VEGF) withdrawal. In a xenografted glioma, the selective vulnerability of immature vessels to VEGF loss was demonstrated by downregulating VEGF transgene expression using a tetracycline-regulated expression system. In human prostate cancer, the constitutive production of VEGF by the glandular epithelium was suppressed as a consequence of androgen-ablation therapy. VEGF loss led, in turn, to selective apoptosis of endothelial cells in vessels devoid of periendothelial cells. These results suggest that the unique dependence on VEGF of blood vessels lacking periendothelial cells can be exploited to reduce an existing tumor vasculature.

A plasticity window for blood vessel remodelling is defined by pericyte coverage of the preformed endothelial network and is regulated by PDGF-B and VEGF.

Little is known about how the initial endothelial plexus is remodelled into a mature and functioning vascular network. Studying postnatal remodelling of the retina vasculature, we show that a critical step in vascular maturation, namely pericyte recruitment, proceeds by outmigration of cells positive for (alpha)-smooth muscle actin from arterioles and that coverage of primary and smaller branches lags many days behind formation of the endothelial plexus. The transient existence of a pericyte-free endothelial plexus coincides temporally and spatially with the process of hyperoxia-induced vascular pruning, which is a mechanism for fine tuning of vascular density according to available oxygen. Acquisition of a pericyte coating marks the end of this plasticity window. To substantiate that association with pericytes stabilizes the vasculature, endothelial-pericyte associations were disrupted by intraocular injection of PDGF-BB. Ectopic PDGF-BB caused the detachment of PDGF-beta receptor-positive pericytes from newly coated vessels, presumably through interference with endogenous cues, but had no effect on mature vessels. Disruption of endothelial-pericyte associations resulted in excessive regression of vascular loops and abnormal remodelling. Conversely, intraocular injection of VEGF accelerated pericyte coverage of the preformed endothelial plexus, thereby revealing a novel function of this pleiotropic angiogenic growth factor. These findings also provide a cellular basis for clinical observations that vascular regression in premature neonates subjected to oxygen therapy [i.e. in retinopathy of prematurity] drops precipitously upon maturation of retina vessels and a mechanistic explanation to our previous findings that VEGF can rescue immature vessels from hyperoxia-induced regression.

Conditional switching of vascular endothelial growth factor (VEGF) expression in tumors: induction of endothelial cell shedding and regression of hemangioblastoma-like vessels by VEGF withdrawal.

We have recently shown that VEGF functions as a survival factor for newly formed vessels during developmental neovascularization, but is not required for maintenance of mature vessels. Reasoning that expanding tumors contain a significant fraction of newly formed and remodeling vessels, we examined whether abrupt withdrawal of VEGF will result in regression of preformed tumor vessels. Using a tetracycline-regulated VEGF expression system in xenografted C6 glioma cells, we showed that shutting off VEGF production leads to detachment of endothelial cells from the walls of preformed vessels and their subsequent death by apoptosis. Vascular collapse then leads to hemorrhages and extensive tumor necrosis. These results suggest that enforced withdrawal of vascular survival factors can be applied to target preformed tumor vasculature in established tumors. The system was also used to examine phenotypes resulting from over-expression of VEGF. When expression of the transfected VEGF cDNA was continuously "on," tumors became hyper-vascularized with abnormally large vessels, presumably arising from excessive fusions. Tumors were significantly less necrotic, suggesting that necrosis in these tumors is the result of insufficient angiogenesis.

Fusion of the EWS1 and WT1 genes as a result of the t(11;22)(p13;q12) translocation in desmoplastic small round cell tumors.

The isolation and molecular analysis of genes which cause and/or predispose to Wilms' tumor have yielded fascinating insights into the role of tissue-specific gene regulation in both development and disease processes. Analysis of the WT1 transcription factor has clearly established its role in Wilms' tumorigenesis and a broader role in both urogenital organogenesis and mesenchymal cell differentiation events. Clearly, loss of function mutations in WT1 is correlated with aberrant function as a regulator of gene expression, ultimately resulting in neoplastic transformation in the developing kidney. A question we have pursued is whether alterations of WT1 structure and/or function can be associated with other types of malignancies, possibly reflecting WT1's broader role in mesenchymal differentiation. To this end, we have analyzed a rare solid tumor designated Intra-Abdominal Desmoplastic Small Round Cell Sarcoma (IADSRCT) which often displays a recurrent chromosomal translocation t(11;22)(p13;q12) involving the WT1 genomic locus. We have shown that the EWS1 gene fron chromosome 22q12 is fused to the WT1 gene in IADSRCT and that a fusion protein is produced which functions as a potent activator of transcription. Our results suggest that WT1 has sustained a gain-of function alteration as a results of this fusion and that the fusion gene functions as a dominant oncogene in this disease. Thus, the WT1 locus may be the target for both gain- and loss-of-function mutations resulting in different disease outcomes. A summary of our ongoing analysis of the EWS-WT1 fusion gene is presented.

Genetic mapping and embryonic expression of a novel, maternally transcribed gene Mem3.

To study the molecular function of genes expressed during preimplantation development, we isolated a novel maternal transcript SSEC (Stage Specific Embryonic cDNA)-26 from a partial subtraction library of mouse unfertilized eggs and preimplantation embryos. The SSEC-26 transcript is abundant in the unfertilized egg and also actively transcribed from the newly formed zygotic genome. On the basis of its expression in eggs and embryos, this new mouse gene is named Mem (maternal-embryonic) 3. The genomic locus of Mem3 has been mapped to Chromosome (Chr) 8 near the D8Mit78 marker and the glutaryl CoA dehydrogenase (Gcdh) locus. The deduced amino acid sequence of MEM3 resembles that of the yeast VPS (Vacuolar Protein Sorting) 35 in two separate domains. A cDNA sequence of the potential human homolog of Mem3 has been assembled with partial clones from the EST database and assigned to human Chr 16.

The IGF-I receptor gene promoter is a molecular target for the Ewing's sarcoma-Wilms' tumor 1 fusion protein.

Desmoplastic small round cell tumor (DSRCT) is an abdominal malignancy in children which is characterized by a recurrent chromosomal translocation, t(11;22)(p13;q12). This rearrangement results in the fusion of the ubiquitously expressed EWS1 gene to the Wilms' tumor suppressor (WT1) gene. The chimeric protein contains the N-terminal domain of EWS1 fused to the DNA-binding domain of WT1, including zinc fingers 2-4. Because WT1 has been shown previously to bind and repress the insulin-like growth factor I (IGF-I-R) promoter, we investigated whether this promoter is, in addition, a target for the aberrant EWS/WT1 transcription factor. EWS/WT1 activated the IGF-I-R promoter approximately 340%, whereas a fusion protein containing a three-amino acid insert (KTS) between zinc fingers 3 and 4 had no effect. On the other hand, expression vectors encoding either WT1 or EWS1 reduced the activity of the promoter to 46 and 58% of control values, respectively. Results of gel shift assays indicate that the binding affinity of EWS/WT1 to a fragment of the 5'-flanking region of the receptor promoter was higher than the affinity of WT1 itself. Consistent with the results of functional assays, the binding of EWS/WT1(+KTS) was significantly reduced. Due to the central role of the IGF-I-R in tumorigenesis, activation of the receptor promoter by EWS/WT1 may constitute a potential mechanism for the etiology and/or progression of DSRCT.

Intra-abdominal desmoplastic small round-cell tumor: expansion of the pathologic profile.

This report describes an intra-abdominal desmoplastic small round-cell tumor in a 29-year-old man that significantly differed from the classically described appearances of this unique tumor. It showed extensive papillary areas, no necrosis, and very little desmoplasia. The latter was limited, paucicellular, and present in areas separate from the papillary structures. Also, areas of back-to-back, single-cell infiltration, which mimicked lobular breast carcinoma, were present. These epithelial features suggested the diagnosis of adenocarcinoma or peculiar mesothelioma. But, the immunohistochemical features (tumor cells positive for keratin, desmin, and vimentin) were more consistent with an intra-abdominal desmoplastic small round-cell tumor. The diagnosis became clear after application of reverse transcriptase-polymerase chain reaction techniques to formalin-fixed, paraffin-embedded tissue, which showed the presence of a 100-base pair product containing the fusion junction of Ewing's sarcoma-1 exon 7 to Wilms' tumor-1 exon 8. This feature is considered unique to intra-abdominal desmoplastic small round-cell tumors. This case illustrates the less common histologic findings that can be found in intra-abdominal desmoplastic small round-cell tumor. This deviation from the classic histologic findings may be an expression of an uncommon morphologic variant and/or partially produced by the effects of prior chemotherapy. In either event, only by illustrating the various histologic appearances of intra-abdominal desmoplastic small round-cell tumor are the chances increased for the accurate diagnosis of this aggressive neoplasm with a poor prognosis.

Novel oncogenic mutations in the WT1 Wilms' tumor suppressor gene: a t(11;22) fuses the Ewing's sarcoma gene, EWS1, to WT1 in desmoplastic small round cell tumor.

These studies suggest that the WT1 tumor suppressor gene, originally identified as a recessive oncogene in Wilms' tumors, is capable of sustaining a gain-of-function mutation which results in its contribution to a completely different disease entity: desmoplastic small round cell tumor. Two independent biochemical functions of WT1, DNA-binding activity and mode of transcriptional regulation, are altered as a consequence of the chromosomal translocation and fusion with EWS. The fusion of EWS and WT1 genes in DSRCT thus provides a unique paradigm for a means by which different alterations of transcription factor function can lead to diverse oncogenic processes.

The effect of dietary protein on nitrogen and sulfur metabolism in portacaval-shunted rats.

The effect of varying the amount of protein in the diet on postoperative recovery, plasma ammonia, urinary orotic acid and metabolism of sulfur-containing amino acids was examined in rats with portacaval shunts (PCS). Food intake and weight gain were lower in both PCS and control rats fed a low (6%) casein diet unsupplemented with methionine compared with rats fed an adequate (18%) casein diet. PCS rats fed 60% casein ate slightly less and took longer to recover their preoperative body weight compared to 60% controls. Shunted rats were consistently hyperammonemic and orotic aciduric compared to controls. Increasing protein in the diet elevated plasma ammonia and urinary orotic acid in all rats to levels above those of the rats fed 18% casein, but the effect was greater in rats with PCS. After i.p. injection of L-[35S]methionine or L-[35S]cysteine, urinary 35S and [35S]sulfate excretion increased and [35S]taurine and total taurine excretion decreased in all rats fed 60% casein. These changes are consistent with our observation that hepatic activities of cysteine dioxygenase and cysteine sulfinate:alpha-ketoglutarate aminotransferase increased and that of cysteine sulfinate decarboxylase decreased in rats fed the high protein diet. The effect of dietary treatment on both urinary taurine excretion and decarboxylase activity was greater in PCS rats than in controls. Although PCS rats fed a high protein diet may have a decreased taurine-synthesizing capability compared to controls, their ability to oxidize a methionine or cysteine load to sulfate is not compromised by feeding either an 18 or 60% casein diet.

Methionine metabolism after portacaval shunt in the rat.

The effect of portacaval shunt (PCS) on methionine metabolism in the rat was investigated. Male Sprague-Dawley rats were subjected to PCS and maintained on an 18% casein diet. Growth curves of operated rats were similar to controls. PCS rats excreted more urinary 35SO4 and less [35S]taurine than controls after intraperitoneal injection of 0.3 mmol/100 g [35S]methionine or [35S]cysteine. Total urinary taurine excretion was similar in PCS and control rats after a methionine or cysteine load; however, under basal conditions PCS rats had higher urinary taurine levels than controls, indicating that PCS may cause the taurine pool to be expanded. Hepatic methionine, S-adenosylmethionine, and cysteine pools were significantly decreased in PCS rats, while S-adenosylhomocysteine levels were unchanged. Relative rates of transsulfuration in PCS and control rats were studied by following the decrease in the 3H-to-35S ratio in liver protein after injection of [methyl-3H]methionine and [35S]methionine, and no difference in flux of 35S from [35S]methionine to [35S]cysteine was found. Similarly, total hepatic activities of methionine adenosyltransferase, cystathionine synthase, and cystathionine gamma-lyase were unchanged in PCS rats. These results indicate that altered methionine metabolism in PCS rats is not explained by changes in conversion of methionine to cysteine via the transsulfuration pathway.

2-benzazepines. 5. Synthesis of pyrimido[5,4-d][2]benzazepines and their evaluation as anxiolytic agents.

A series of 5H-pyrimido[5,4-d][2]benzazepines has been synthesized, starting from the corresponding 2-benzazepin-5-ones, and evaluated as potential anxiolytic agents. Selected compounds from this series show a pharmacological profile of action different than that of diazepam. They are more potent than diazepam in the anti-pentylenetetrazole test and in the [3H]diazepam binding assay, yet show less activity in the inclined screen test. A pharmacological data profile is given for 9-chloro-7-(2-chlorophenyl)-5H-pyrimido[5,4-d] [2]benzazepine (7c). The structure-activity relationships of these potential anxiolytic agents are discussed.

2-Benzazepines. 2. Thiazolo[5,4-d][2]benzazepines.

As part of a program in the area of annelated 2-benzazepines, several thiazolo[5,4-d))[2]benzazepines were prepared. Treatment of the bromo ketones 7-9 with various thio amides gave the thiazoles 10-15, which when treated with methylamine gave the title compounds. The preliminary pharmacology of these compounds showed that they had central nervous system activity similar to the 1,4-benzodiazepines, such as diazepam. The thiazolo[5,4-d][2]benzazepines were also found to bind to the benzodiazepine-receptor complex, indicating that their pharmacological actions are probably related to the 1,4-benzodiazepines.

Effect of portacaval shunt on sulfur amino acid metabolism in rats.

Male rats were subjected to portacaval shunts (PCS). Postoperative growth curves were similar between PCS and sham-operated control animals, with complete recovery of preoperative body weight occurring within 7 days. PCS rats had 30% smaller livers and 39% larger spleens at autopsy. Total liver levels of reduced glutathione were decreased by 38% in PCS rats. Sulfur amino acid metabolism was studied 6 wk after PCS by injection of L-[methyl-14C]- or L-[1-14C]methionine with 14CO2 collection or by similarly administering L-[35S]methionine or L-[35S] cysteine and measuring 24-h urinary 35SO4, [35S]taurine, and total 35S. There were no significant differences in 14CO2 production over an 8-h period between PCS and sham rats. PCS rats excreted 31% more 35SO4 and 25% more total 35S when injected with 0.9 mmol of [35S]methionine and excreted 38% less [35S]taurine than controls when injected with [35S]-cysteine. These results indicate altered sulfur amino acid metabolism in PCS rats, an animal model of portal-systemic shunting.