Natural or artificial? Habitat-use by the bull shark, Carcharhinus leucas.

BACKGROUND: Despite accelerated global population declines due to targeted and illegal fishing pressure for many top-level shark species, the impacts of coastal habitat modification have been largely overlooked. We present the first direct comparison of the use of natural versus artificial habitats for the bull shark, Carcharhinus leucas, an IUCN 'Near-threatened' species--one of the few truly euryhaline sharks that utilises natural rivers and estuaries as nursery grounds before migrating offshore as adults. Understanding the value of alternate artificial coastal habitats to the lifecycle of the bull shark is crucial for determining the impact of coastal development on this threatened but potentially dangerous species. METHODOLOGY/FINDINGS: We used longline surveys and long-term passive acoustic tracking of neonate and juvenile bull sharks to determine the ontogenetic value of natural and artificial habitats to bull sharks associated with the Nerang River and adjoining canals on the Gold Coast, Australia. Long-term movements of tagged sharks suggested a preference for the natural river over artificial habitat (canals). Neonates and juveniles spent the majority of their time in the upper tidal reaches of the Nerang River and undertook excursions into adjoining canals. Larger bull sharks ranged further and frequented the canals closer to the river mouth. CONCLUSIONS/SIGNIFICANCE: Our work suggests with increased destruction of natural habitats, artificial coastal habitat may become increasingly important to large juvenile bull sharks with associated risk of attack on humans. In this system, neonate and juvenile bull sharks utilised the natural and artificial habitats, but the latter was not the preferred habitat of neonates. The upper reaches of tidal rivers, often under significant modification pressure, serve as nursery sites for neonates. Analogous studies are needed in similar systems elsewhere to assess the spatial and temporal generality of this research.

Matching mitochondria to metabolic needs at nodes of Ranvier.

Myelinated axons conduct nerve impulses at high speed using a unique mode of excitation, referred to as saltatory conduction, which is enabled structurally by the narrowing of the site of action potentials to a tiny gap in the axon called the node of Ranvier. With this structural specialization comes an interesting metabolic matching problem. How do mitochondria find and supply energy to these tiny nodes of Ranvier distributed sparsely along a myelinated axon? Does the intense Na(+) influx at the node, which is produced by the highest known sodium channel density in all excitable membranes, help guide where mitochondria stop? Evidence suggests that during excitation in the peripheral nervous system, Na(+) influx recruits mitochondria to the node by triggering Ca(2+) elevation and activating Na(+) pumps. Intriguingly, indirect evidence suggests that in the central nervous system, activity recruits mitochondria to the internode (myelin-covered portion of the axon). Metabolic dysfunction thus might produce spatially distinct lesions in PNS and CNS myelinated fibers. Future dissection of regional variation in mitochondrial biology in myelinated axons using live imaging will likely yield surprises about sites of vulnerability in demyelinating diseases and clues for therapeutic intervention strategy.

Age differences in speed of processing are partially mediated by differences in axonal integrity.

Advanced age is associated with declines in brain structure and in cognitive performance, but it is unclear which aspects of brain aging mediate cognitive declines. We inquired if individual differences in white matter integrity contribute to age differences in two cognitive domains with established vulnerability to aging: executive functioning and speed of processing. The participants were healthy volunteers aged 50-81, some of whom had elevated blood pressure, a known vascular risk factor. Using latent variable analyses, we examined whether age differences in regional white matter integrity mediated age-related differences in executive functions and speed of processing. Although diffusion-related latent variables showed stronger age differences than white matter volumes and white matter hyperintensity volumes, only one of them was significantly associated with cognitive performance. Smaller linear anisotropy partially mediated age-related reduction in speed of processing. The effect was significant in posterior (temporal-parietal-occipital) but not anterior (frontal) region, and appeared stronger for cognitive rather than reaction time measures of processing speed. The presence of hypertensive participants did not affect the results. We conclude that in healthy adults, deterioration of axonal integrity and ensuing breech of connectivity may underpin age-related slowing of information processing.

Using blood samples to estimate persistent organic pollutants and metals in green sea turtles (Chelonia mydas).

Persistent organic pollutants (POPs) and heavy metals have been reported in a number of green turtle (Chelonia mydas) populations worldwide. However, due to ethical considerations, these studies have generally been on tissues from deceased and stranded animals. The purpose of this study was to investigate the use of blood samples to estimate the tissue contamination of live C. mydas populations. This study analysed 125 POP compounds and eight heavy metals in the blood, liver, kidney and muscle of 16 C. mydas from the Sea World Sea Turtle Rehabilitation Program, Gold Coast, Australia. Strong correlations were observed between blood and tissue concentrations for a number of POPs and metals. Furthermore, these correlations were observed over large ranges of turtle size, sex and condition. These results indicate that blood samples are a reliable non-lethal method for predicting chemical contamination in C. mydas.

Chemical contamination of green turtle (Chelonia mydas) eggs in peninsular Malaysia: implications for conservation and public health.

BACKGROUND: Persistent organic pollutants (POPs)-such as organochlorine pesticides (OCPs), polychlorinated biphenyls (PCBs), and polybrominated diphenyl ethers (PBDEs)-and heavy metals have been reported in sea turtles at various stages of their life cycle. These chemicals can disrupt development and function of wildlife. Furthermore, in areas such as Peninsular Malaysia, where the human consumption of sea turtle eggs is prevalent, egg contamination may also have public health implications. OBJECTIVE: In the present study we investigated conservation and human health risks associated with the chemical contamination of green turtle (Chelonia mydas) eggs in Peninsular Malaysia. METHODS: Fifty-five C. mydas eggs were collected from markets in Peninsular Malaysia and analyzed for POPs and heavy metals. We conducted screening risk assessments (SRAs) and calculated the percent of acceptable daily intake (ADI) for POPs and metals to assess conservation and human health risks associated with egg contamination. RESULTS: C. mydas eggs were available in 9 of the 33 markets visited. These eggs came from seven nesting areas from as far away as Borneo Malaysia. SRAs indicated a significant risk to embryonic development associated with the observed arsenic concentrations. Furthermore, the concentrations of coplanar PCBs represented 3 300 times the ADI values set by the World Health Organization. CONCLUSIONS: The concentrations of POPs and heavy metals reported in C. mydas eggs from markets in Peninsular Malaysia pose considerable risks to sea turtle conservation and human health.

Influence of sediment metal spiking procedures on copper bioavailability and toxicity in the estuarine bivalve Indoaustriella lamprelli.

The effect of three methods for spiking sediments with Cu on the reburial behavior, mortality, and tissue Cu accumulation of a lucinid bivalve (Indoaustriella lamprelli) and the influence of the bivalve on the sediment geochemistry were investigated. Methods used to create Cu concentration gradients were direct spiking with and without pH adjustment to pH 7 and also dilution of sediment, previously spiked with Cu and adjusted to pH 7, using a low-Cu sediment (known to produce the lowest pore-water Cu concentrations). The presence of the bivalve within Cu-spiked sediment increased the flux of Cu and Mn to overlying waters at high Cu concentrations (550 microg/g). Bivalve behavioral response, metal accumulation, and mortality varied with the method by which Cu was spiked. In direct Cu-spiked sediment, the bivalves were inactive at concentrations of 550 and 1,100 microg/g, with mortality induced in sediment spiked with 1,100 microg/g (pH 6.5-7.1). Complete bivalve inactivity was observed only at 1,100 microg/g in direct Cu-spiked sediment with pH adjustment, whereas percentage reburial was reduced to 30% at 1,100 microg/g for sediment prepared by the dilution method. Relative reburial rates in the three spiked sediment types (direct << direct pH-7 < dilution) were proportional to dissolved Cu concentrations in the overlying water. Bivalve reburial, in addition to the method of Cu addition, affected tissue Cu accumulation. Inhibition of bivalve reburial decreased the amount of accumulated Cu, confounding relationships between tissue Cu and pore water, overlying water, or extractable metal fractions.

Outcome of intensive care of homozygous alpha-thalassaemia without prior intra-uterine therapy.

AIM: To review the outcome of homozygous alpha-thalassaemia without prior intra-uterine therapy treated in neonatal intensive care unit and identify the factors associated with survival. METHODS: The hospital records of all patients with homozygous alpha-thalassaemia treated in our neonatal intensive care unit in the last 15 years were reviewed. A literature search beginning in the year 1980 was done to identify homozygous alpha-thalassaemia actively treated in neonatal intensive care units. Those receiving prior intra-uterine therapy were excluded. The following information was collected: the severity of hydrops, sizes of liver and spleen, haemoglobin level, Apgar score at 5 min, ventilator settings, timing and forms of red blood cell transfusion and presence of persistent hypoxaemia. The survivors and the non-survivors were compared. RESULTS: In our centre, in the last 15 years there were six infants born with homozygous alpha-thalassaemia who did not receive intra-uterine therapy; one survived and five succumbed despite aggressive respiratory therapy. In our literature search there were more reports of survivors (10) than non-survivors (six) for these infants, suggesting a reporting bias towards selection of rare cases of survival. Apgar score of four or above occurred in seven of the eight survivors with data available in the reports, whereas this occurred in four of the 11 non-survivors (P = 0.035, Fisher Exact test). Five of the 11 survivors had abnormal neurological outcome including developmental delay and spastic quadriplegia. CONCLUSION: Without prior intra-uterine therapy, homozygous alpha-thalassaemia has grave outlook in terms of mortality and morbidity despite aggressive respiratory therapy.

Study of microfiltration behaviour of oily wastewater.

Crossflow microfiltration experiments were carried out with oily wastewater using alpha-Al(2)O(3) membranes with 0.05 microm pore size. The influence of parameters such as transmembrane pressure (TMP), crossflow velocity (CFV), oil concentration, pH and salt concentration on the microfiltration behaviors were studied based on the measurements of permeate flux, total organic carbon (TOC) removal efficiency, and size and zeta potential of the emulsion droplets. The results showed that there were different degrees of effect on the membrane separation performance by these parameters. The TOC removal efficiencies higher than 92.4% were achieved under all experimental conditions. A non-steady model of accumulation volume of permeation was developed. It was found that the calculated values were in good agreement with the experimental results. Sensitivity analysis was also conducted to identify the degree of influence of the parameters on the accumulation volume of permeation. The results indicated that the accumulation volume of permeation was significantly affected by the TMP.

Mechanism for the small-scale movement of carbon among estuarine habitats: organic matter transfer not crab movement.

In theory, carbon is highly mobile in aquatic systems. Recent evidence from carbon stable isotopes of crabs (Parasesarma erythrodactyla and Australoplax tridentata), however, shows that in subtropical Australian waters, measurable carbon movement between adjacent mangrove and saltmarsh habitats is limited to no more than a few metres. We tested whether the pattern in crab delta13C values across mangrove and saltmarsh habitats was explained by crab movement, or the movement of particulate organic matter. We estimated crab movement in a mark-recapture program using an array of pitfall traps on 13 transects (a total of 65 traps) covering an area of 600 m2 across the interface of these two habitats. Over a 19-day period, the majority of crabs (91% for P. erythrodactyla, 93% for A. tridentata) moved <2 m from the place of initial capture. Crab movement cannot, therefore, explain the patterns in delta13C values of crabs. delta13C values of detritus collected at 2-m intervals across the same habitat interface fitted a sigmoidal curve of a similar form to that fitting the delta13C values of crabs. delta13C values of detritus were 2-4 per thousand more depleted in saltmarsh (-18.5+/-0.6 per thousand), and 4-7 per thousand more depleted in mangroves (-25.9+/-0.1 per thousand) than delta13C values of crabs recorded previously in each habitat. Assimilation by crabs of very small detrital fragments or microphytobenthos, more enriched in 13C, may explain the disparity in delta13C values. Nevertheless, the pattern in delta13C values of detritus suggests that crabs obtain their carbon from up to several metres away, but without themselves foraging more then a metre or so from their burrow. Such detailed measurements of carbon movement in estuaries provide a spatially explicit understanding of the functioning of food webs in saltmarsh and mangrove habitats.

Hepatitic graft-versus-host disease after hematopoietic stem cell transplantation: clinicopathologic features and prognostic implication.

BACKGROUND: Graft-versus-host disease (GVHD) of the liver after allogeneic hematopoietic stem cell transplantation classically presents with increased bilirubin and alkaline phosphatase (ALP) levels. A hepatitic variant was recently recognized, with more than a 10-fold increase in aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. This study defines the clinicopathologic features and prognostic implications of hepatitic GVHD compared with classic liver GVHD. METHOD: A total of 38 cases of hepatitic GVHD, 68 cases of classic liver GVHD, and 13 cases of hepatitis B virus (HBV)-related hepatitis after hematopoietic stem cell transplantation were analyzed. RESULTS: Hepatitic GVHD cases showed significantly higher ALT, AST, and ALP levels compared with classic liver GVHD cases (at onset, mean ALT: 154 vs. 58 U/L, P <0.001; AST: 167 vs. 77 U/L, P <0.001; at peak, ALT: 435 vs. 112 U/L, P <0.001; AST: 587 vs. 150 U/L, P <0.001; ALP: 416 vs. 238 U/L, P =0.001), persisted longer (74 vs. 32 days, P =0.006), and showed more lobular pathologic changes in biopsy (lobular changes: 16/26 vs. 4/19, P =0.007; hepatocyte necrosis: 16/26 vs. 6/19, P =0.008; acidophil bodies: 15/26 vs. 4/19, P =0.014) but less cholestasis (4/26 vs. 8/19, P =0.045). However, cumulative doses of immunosuppressants prescribed, response, and outcome were similar. Compared with hepatitic GVHD, HBV-related hepatitis occurred later (95 vs. 184 days, P =0.049), but clinical and biochemical profiles were similar, requiring liver biopsies for their distinction. CONCLUSIONS: Hepatitic and classic liver GVHD differed biochemically and pathologically, but these differences showed no obvious impact on outcome. The distinction of hepatitic GVHD from other hepatitis is mandatory.

Fludarabine, mitoxantrone and dexamethasone in the treatment of indolent B- and T-cell lymphoid malignancies in Chinese patients.

The treatment results of indolent lymphoid malignancies in Chinese are poorly reported. The efficacy of FND (fludarabine 25 mg/m2/d, x3; mitoxantrone 10 mg/m2/d, x1; dexamethasone 20 mg/d, x5; monthly cycles, x6) in 95 Chinese patients with indolent B-cell malignancies (at diagnosis: 55, relapse/refractory disease: 40) and nine Chinese patients with T-cell large granular lymphocyte leukaemia (T-LGL leukaemia) (at diagnosis: two, refractory disease: seven) was evaluated. For B-cell malignancies, the complete response (CR), partial response (PR) and overall response (OR) rates were 50.5%, 18% and 68.5% respectively. Better results were obtained for primary versus relapse/refractory disease (CR: 60% vs. 37.5%, P = 0.03; OR: 84% vs. 47.5%, P < 0.001; median progression-free survival (PFS): 44 months vs. 22 months; 2-year PFS: 66% vs. 47%, P = 0.039; overall survival (OS): not reached vs. 32%; 2-year OS: 92% vs. 58%, P < 0.001). Responsive patients (CR/PR) had a better median PFS (44 months vs. 5 months, P < 0.001) and OS (67 months vs. 13 months, P < 0.001) than unresponsive patients. For T-LGL leukaemia, the CR and molecular-remission rates were 56% and 67% (median follow-up: 23 months). FND is an active regimen for the treatment of indolent B- and T-cell malignancies in Chinese patients, with results comparable with Western patients with similar indolent lymphomas.

HIF-1alpha-mediated up-regulation of vascular endothelial growth factor, independent of basic fibroblast growth factor, is important in the switch to the angiogenic phenotype during early tumorigenesis.

The switch to the angiogenic phenotype represents a critical checkpoint during tumor progression. The acquisition of new capillary vessels provides newly vascularized tumor nodules with a distinct biological advantage over their avascular counterparts by conferring upon them the ability to expand and develop both locally and metastatically. To identify the molecules and mechanisms underlying this rate-limiting step in successful tumorigenesis, we have developed an in vivo tumor model that reproducibly recapitulates the angiogenic switch. Using this model, we have analyzed vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and hypoxia-inducible factor-1alpha (HIF-1alpha) expression and activity in both avascular and vascular growth phases of the tumor. A significantly higher level of VEGF protein was detected in avascular tumor nodules compared with vascular nodules. As avascular tumors became vascularized, VEGF levels decreased approximately 10-fold. In contrast, bFGF levels were not elevated in avascular nodules but rather were detected at levels approximately 2 times higher in vascular nodules compared with the avascular tumor nodules. Given that VEGF is transcriptionally regulated by HIF-1alpha, immunohistochemical studies of chondrosarcoma nodules were conducted and revealed that the nuclear translocation of HIF-1alpha was detected exclusively in avascular tumor nodules. This study implicates HIF-1alpha-mediated up-regulation of VEGF but not bFGF in the switch to the angiogenic phenotype during tumorigenesis.

Effect of antiangiogenic therapy on slowly growing, poorly vascularized tumors in mice.

BACKGROUND: Angiogenesis is essential for tumor growth and progression. Therefore, inhibition of angiogenesis is being studied as a new anticancer therapy. Because cytotoxic chemotherapy is more effective on rapidly growing tumors than on slowly growing tumors, it has been assumed that antiangiogenic therapy will also be effective only on rapidly growing, highly vascularized tumors. We compared the effects of two angiogenesis inhibitors, TNP-470 and angiostatin, on slowly growing, poorly vascularized and rapidly growing, highly vascularized human tumors in mice. METHODS: Slowly growing (RT-4) and rapidly growing (MGH-U1) human bladder carcinoma cell lines were grown in severe combined immunodeficiency mice. Established tumors were treated with one of the two angiogenesis inhibitors. Tumor volumes, vascularity, and proliferation indices were determined. The in vitro effects of TNP-470 and of angiostatin on the proliferation of RT-4 and MGH-U1 cells were also investigated. All statistical tests were two-sided. RESULTS: RT-4 and MGH-U1 tumor growth was statistically significantly inhibited by both angiogenesis inhibitors (P<.001). Both inhibitors decreased the blood vessel density in both tumor types but did not alter the in vivo proliferation indices of the tumors. TNP-470, but not angiostatin, marginally decreased the in vitro proliferation of MGH-U1 cells. CONCLUSION: Slowly growing, poorly vascularized tumors in animal models respond as well as rapidly growing, highly vascularized tumors to therapy with the angiogenesis inhibitors TNP-470 and angiostatin.

Efficacy of antiangiogenic therapy with TNP-470 in superficial and invasive bladder cancer models in mice.

OBJECTIVES: To evaluate the efficacy of antiangiogenic therapy with O-(chloracetyl-carbamoyl) fumagillol (TNP-470) in a superficial and an invasive bladder cancer model in mice. The control of recurrent superficial and metastatic bladder cancer constitutes a major problem in urologic practice. Although the established therapies for these cases (immunotherapy, chemotherapy, and radiation therapy) have improved during the previous decades, further improvement and the reduction of existing side effects are needed. The inhibition of angiogenesis represents a new concept in cancer therapy. METHODS: We evaluated the in vitro effect of TNP-470 on the proliferation of bovine capillary endothelial cells (BCE), the superficial transitional cell carcinoma (TCC) cell line (KK-47), and the invasive TCC cell line (MGH-U1). To evaluate the in vivo effect of TNP-470 on the growth of advanced TCCs, both cell lines were injected subcutaneously into SCID mice. When tumors grew to a size of 100 to 200 mm(3), therapy either with TNP-470 or phosphate-buffered saline was initiated. RESULTS: TNP-470 strongly inhibited endothelial cell proliferation in vitro. The in vitro proliferation of both bladder carcinoma cell lines was also inhibited by TNP-470. However, the doses inhibitory to bladder carcinoma cells were 100-fold higher than the doses that were effective in the inhibition of endothelial cell proliferation. In vivo, TNP-470 significantly inhibited the growth of advanced KK-47 (67%) and MGH-U1 (68%) tumors in SCID mice. CONCLUSIONS: Our results indicate that antiangiogenic therapy with TNP-470 is equally effective in advanced superficial and invasive bladder carcinoma models in mice. When our results are taken together with the reports of other laboratories, TNP-470 appears to be a promising candidate as a tumor suppressor in superficial and invasive bladder cancer.

Matrix metalloproteinase-2 is required for the switch to the angiogenic phenotype in a tumor model.

Among the earliest and most important stages during tumorigenesis is the activation of the angiogenic process, an event that is termed the "switch to the angiogenic phenotype." We have developed an in vivo system that can reliably recapitulate the stages in tumor development that represent this transition. Using this model, we have harvested and studied tumor nodules that can be distinguished from each other on the basis of their degree of vascularization. Angiogenic tumor nodules were characterized by the presence of capillary vessels as determined by factor VIII immunohistochemistry, and both angiogenic and proteolytic activities in vitro. In contrast, preangiogenic nodules were devoid of microvessels and showed little angiogenic or proteolytic activity in vitro. Addition of a specific metalloproteinase inhibitor resulted in the abrogation of both angiogenic and proteolytic activities of the angiogenic nodules in vitro. Comparative substrate gel electrophoresis detected the presence of a prominent matrix metalloproteinase (MMP-2) in the angiogenic nodules when compared with the preangiogenic ones. Suppression of MMP-2 activity by antisense oligonucleotides in the vascular nodules resulted in the loss of angiogenic potential both in vitro and in vivo in the chick chorioallantoic membrane assay. Moreover, this suppression of MMP-2 activity in angiogenic nodules inhibited tumor growth in vivo by approximately 70%. These results strongly implicate the activity of MMP-2 as a requirement for the switch to the angiogenic phenotype and validate this model as a reliable and reproducible tool by which to study other cellular and biochemical factors involved in the acquisition of the angiogenic phenotype.

New role for tRNA and its fragment purified from human urinary bladder carcinoma conditioned medium: inhibition of endothelial cell growth.

The growth of endothelial cells is necessary for angiogenesis, which in turn is required for later steps of tumor progression. In an attempt to purify new modulators of endothelial cell growth from the conditioned medium of human urinary bladder carcinoma cells, we isolated a small and stable oligonucleotide containing 10 to 16 bases. This oligonucleotide inhibited the growth of endothelial cells in vitro and was identified as a fragment of transfer RNA (tRNA). When unfractionated bovine tRNA was added to the cell culture, it specifically inhibited growth of endothelial cells, but not smooth muscle cells, bovine kidney cells, 3T3 fibroblasts, and several cancer cell lines. In contrast, ribosomal RNA, total yeast RNA, and single nucleosides from tRNA hydrolysate had no effect. These results demonstrate a new role for tRNA and its fragment as a selective endothelial cell inhibitor in vitro.

Multiple forms of angiostatin induce apoptosis in endothelial cells.

Angiostatin is a circulating inhibitor of angiogenesis generated by proteolytic cleavage of plasminogen. In this study we have used recombinant human and murine angiostatins (kringles 1-4) as well as native human angiostatin (prepared by elastase digestion of plasminogen [kringles 1-3] or by plasmin autocatalysis in the presence of a free sulfhydryl donor [kringles 1-4]). We report that angiostatin reduces endothelial cell number in a 4-day proliferation assay without affecting cell cycle progression into S-phase (as determined by bromodeoxyuridine labeling). This suggested that the reduction in cell number in the proliferation assay might in part be due to cytotoxicity. This was confirmed by the observation that ethidium homodimer incorporation (a measure of plasma membrane integrity) into endothelial cells was increased by angiostatin in a manner similar to that seen with tumor necrosis factor- (TNF-) and transforming growth factor-beta1 (TGF-beta1), both of which induce apoptosis in endothelial cells. In contrast to TNF- and TGF-beta1, angiostatin did not induce cytotoxicity in human MRC-5 fibroblast, rat smooth muscle, canine MDCK epithelial, or murine B16-F10 melanoma cell lines. Angiostatin-induced apoptosis was confirmed by endothelial cell nuclear acridine orange incorporation as well as by annexin V and TUNEL staining. These in vitro findings point to endothelial cell apoptosis as a mechanism for the antiangiogenic effect of angiostatin in vivo.

Suppression of tumor growth with recombinant murine angiostatin.

Angiostatin, a 38 kDa internal fragment of plasminogen, is an antiangiogenic endothelial cell inhibitor. It regresses several primary and metastatic tumors in mice. To produce recombinant angiostatin for further structural and functional studies, the mouse angiostatin gene preceded by a sequence including a signal peptide of plasminogen was introduced into baculovirus. Recombinant murine angiostatin was purified from the culture medium of angiostatin baculovirus-infected insect cells (yield = 1 mg/liter) with a single-step of lysine-Sepharose chromatography. The angiostatin baculovirus-infected insect cells expressed and secreted a 52 kDa polypeptide that demonstrated all of the biological activities of angiostatin. A partial amino acid sequence of the NH2-terminus of the secreted protein revealed that the signal peptide was recognized and properly cleaved in insect cells. The recombinant murine angiostatin potently inhibited the proliferation of bovine capillary endothelial cells in vitro (half maximal inhibition = 50 ng/ml) and suppressed the growth of primary Lewis lung carcinoma in vivo (6 mg/kg/day, T/C = 0.08).

Endostatin: an endogenous inhibitor of angiogenesis and tumor growth.

We previously identified the angiogenesis inhibitor angiostatin. Using a similar strategy, we have identified endostatin, an angiogenesis inhibitor produced by hemangioendothelioma. Endostatin is a 20 kDa C-terminal fragment of collagen XVIII. Endostatin specifically inhibits endothelial proliferation and potently inhibits angiogenesis and tumor growth. By a novel method of sustained release, E. coli-derived endostatin was administered as a nonrefolded suspension. Primary tumors were regressed to dormant microscopic lesions. Immunohistochemistry revealed blocked angiogenesis accompanied by high proliferation balanced by apoptosis in tumor cells. There was no toxicity. Together with angiostatin data, these findings validate a strategy for identifying endogenous angiogenesis inhibitors, suggest a theme of fragments of proteins as angiogenesis inhibitors, and demonstrate dormancy therapy.

Experimental corneal neovascularisation using sucralfate and basic fibroblast growth factor.

PURPOSE: To develop a non-inflammatory model of both acute and chronic angiogenesis in the rabbit cornea using a known directly angiogenic cytokine. METHODS: Pellets made of the slow-release polymer Hydron (polyhydroxyethylmethacrylate) and containing sucralfate and/or basic fibroblast growth factor (basic-FGF) were implanted into rabbit corneas. The neovascular response to the implantation of pellets containing basic-FGF alone, sucralfate alone or a titration of basic-FGF in the presence of a constant amount of sucralfate was measured. The role of inflammation in the neovascular response was also investigated. RESULTS: The addition of sucralfate to the pellets led to the sustained release of basic-FGF resulting in a predictable and aggressive neovascular response with a low dose of basic-FGF that by itself was unable to elicit neovascularisation. At a dose of 500 ng per pellet, approximately one-third of the surface area of the cornea was vascularised within eight days of implantation. Minimal or no vascularisation occurred with the same dose of basic-FGF without sucralfate. While this dose of basic-FGF induced corneal oedema, only minimal inflammation was observed and the response was unaffected by ionising radiation. A less aggressive though still robust neovascular response with no or only minimal oedema was observed when the dose was lowered to 50 ng of basic-FGF per pellet. Some induced vessels persisted for more than three months. CONCLUSION: This is an inexpensive in vivo model of angiogenesis with the advantages of the neovascularisation being aggressive, predictable, persistent, unassociated with an obvious inflammatory response and induced by the sustained release of an agent known to have a direct stimulatory action on endothelial cells.