Utility of circulating serum miRNAs as biomarkers of early cartilage degeneration in animal models of post-traumatic osteoarthritis and inflammatory arthritis.

OBJECTIVE: The purpose of this study was to determine if serum microRNA (miRNA) signatures were biomarkers of early cartilage degeneration in preclinical mouse models of post-traumatic osteoarthritis (OA) and inflammatory arthritis. METHODS: Cartilage degeneration was induced in 10-12 week old male C57BL6 mice by destabilization of the medial meniscus (DMM) or intra-articular injection of methylated-bovine-serum-albumin (AIA), with sham-operated or saline-injected control animals (n = 6/treatment/time). Total serum RNA and knee joints were isolated at 1, 4 and 16 weeks post-induction. Cartilage degeneration was scored histologically. Serum miRNA expression profiling was performed using Agilent microarrays and validated by qPCR. RESULTS: DMM-operated and AIA mice had characteristic cartilage degeneration (proteoglycan loss, chondrocyte hypertrophy, structural damage), that increased significantly with time compared with controls, and with distinct temporal differences between arthritis models. However, expression profiling revealed no statistically significant dysregulation of serum miRNAs between AIA vs saline-injected or DMM vs sham-operated control mice at the critical early disease stages. The inability to detect DMM or AIA serum miRNA signatures compared with controls was not due to the insensitivity of the expression profiling approach since significant changes were observed in miRNA expression between the arthritis models and between time points. CONCLUSION: While distinct patterns of progressive cartilage degradation were induced in the arthritis models, we were unable to identify any serum miRNAs that were significantly dysregulated in early stages of disease compared with controls. This suggests circulating serum miRNAs may not be useful as cartilage biomarkers in distinguishing the early or progressive stages of arthritis cartilage degeneration.

In vitro evaluation of translating and rotating plates using a robot testing system under follower load.

BACKGROUND CONTEXT: Various modifications to standard "rigid" anterior cervical plate designs (constrained plate) have been developed that allow for some degree of axial translation and/or rotation of the plate (semi-constrained plate)-theoretically promoting proper load sharing with the graft and improved fusion rates. However, previous studies about rigid and dynamic plates have not examined the influence of simulated muscle loading. PURPOSE: The objective of this study was to compare rigid, translating, and rotating plates for single-level corpectomy procedures using a robot testing system with follower load. STUDY DESIGN: In-vitro biomechanical test. METHODS: N = 15 fresh-frozen human (C3-7) cervical specimens were biomechanically tested. The follower load was applied to the specimens at the neutral position from 0 to 100 N. Specimens were randomized into a rigid plate group, a translating plate group and a rotating plate group and then tested in flexion, extension, lateral bending and axial rotation to a pure moment target of 2.0 Nm under 100N of follower load. Range of motion, load sharing, and adjacent level effects were analyzed using a repeated measures analysis of variance (ANOVA). RESULTS: No significant differences were observed between the translating plate and the rigid plate on load sharing at neutral position and C4-6 ROM, but the translating plate was able to maintain load through the graft at a desired level during flexion. The rotating plate shared less load than rigid and translating plates in the neutral position, but cannot maintain the graft load during flexion. CONCLUSIONS: This study demonstrated that, in the presence of simulated muscle loading (follower load), the translating plate demonstrated superior performance for load sharing compared to the rigid and rotating plates.

Cartilage-specific ablation of XBP1 signaling in mouse results in a chondrodysplasia characterized by reduced chondrocyte proliferation and delayed cartilage maturation and mineralization.

OBJECTIVE: To investigate the in vivo role of the IRE1/XBP1 unfolded protein response (UPR) signaling pathway in cartilage. DESIGN: Xbp1(flox/flox).Col2a1-Cre mice (Xbp1(CartΔEx2)), in which XBP1 activity is ablated specifically from cartilage, were analyzed histomorphometrically by Alizarin red/Alcian blue skeletal preparations and X-rays to examine overall bone growth, histological stains to measure growth plate zone length, chondrocyte organization, and mineralization, and immunofluorescence for collagen II, collagen X, and IHH. Bromodeoxyuridine (BrdU) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analyses were used to measure chondrocyte proliferation and cell death, respectively. Chondrocyte cultures and microdissected growth plate zones were analyzed for expression profiling of chondrocyte proliferation or endoplasmic reticulum (ER) stress markers by Quantitative PCR (qPCR), and of Xbp1 mRNA splicing by RT-PCR to monitor IRE1 activation. RESULTS: Xbp1(CartΔEx2) displayed a chondrodysplasia involving dysregulated chondrocyte proliferation, growth plate hypertrophic zone shortening, and IRE1 hyperactivation in chondrocytes. Deposition of collagens II and X in the Xbp1(CartΔEx2) growth plate cartilage indicated that XBP1 is not required for matrix protein deposition or chondrocyte hypertrophy. Analyses of mid-gestation long bones revealed delayed ossification in Xbp1(CartΔEx2) embryos. The rate of chondrocyte cell death was not significantly altered, and only minimal alterations in the expression of key markers of chondrocyte proliferation were observed in the Xbp1(CartΔEx2) growth plate. IRE1 hyperactivation occurred in Xbp1(CartΔEx2) chondrocytes but was not sufficient to induce regulated IRE1-dependent decay (RIDD) or a classical UPR. CONCLUSION: Our work suggests roles for XBP1 in regulating chondrocyte proliferation and the timing of mineralization during endochondral ossification, findings which have implications for both skeletal development and disease.

Growth rates and energy intake of hand-reared cheetah cubs (Acinonyx jubatus) in South Africa.

Growth rate is an important factor in neonatal survival. The aim of this study was to determine growth rates in hand-reared cheetah cubs in South Africa fed a prescribed energy intake, calculated for growth in the domestic cat. Growth was then compared with previously published data from hand-reared cubs in North America and the relationship between growth and energy intake explored. Daily body weight (BW) gain, feed and energy intake data was collected from 18 hand-reared cheetah cubs up to 120 days of age. The average pre-weaning growth rate was 32 g/day, which is lower than reported in mother-reared cubs and hand-reared cubs in North American facilities. However, post-weaning growth increased to an average of 55 g/day. Growth was approximately linear prior to weaning, but over the entire age range it exhibited a sigmoidal shape with an asymptotic plateau averaging 57 kg. Energy intake associated with pre-weaning growth was 481 kJ ME/kg BW(0.75). Regression analysis described the relationship between metabolic BW, metabolisable energy (ME) intake, and hence daily weight gain. This relationship may be useful in predicting energy intake required to achieve growth rates in hand-reared cheetah cubs similar to those observed for their mother-reared counterparts.

Analysis of power law models for the creep of nucleus pulposus tissue.

Tissue engineering approaches are now being investigated for altering the course of intervertebral disc degeneration (IDD). Because the disease changes the mechanical properties of the load bearing tissues of the disc, viscoelastic tissue behavior is a key measure for comparing the efficacy of treatments. To investigate the basic viscoelastic behavior of nucleus pulposus tissue, tissue from the rabbit disc was tested in torsional creep. Both the Andrade and Nutting creep models had a good fit to the data, however, the Andrade creep model gave a much better prediction of the longer term creep. This is the first application of Andrade creep to biological tissue and results indicate that this model may be particularly well suited for characterizing the viscoelastic behavior of very soft biological tissues.

Exposure of growing and adult captive cheetahs (Acinonyx jubatus) to dietary isoflavones: twenty years later.

Dietary isoflavones are associated with oestrogenic and anti-oestrogenic effects, and have been linked to infertility in cheetahs. This study aimed to determine the isoflavone content of commercially prepared diets consumed by captive cheetahs. Sixteen international zoological facilities provided diets, and the isoflavone content of each diet was determined by acid hydrolysis and HPLC quantification. Proximate nutritional composition was also determined. Over half the diets analysed contained detectable concentrations of isoflavones, whereby total isoflavone content ranged from 1.75-183 mg/kg dry matter. The zoo-specific diets were calculated to deliver a median isoflavone dose of 0.07 mg/kg body weight (BW) and a maximum of 1.95 mg/kg BW to captive cheetahs. On a metabolic body weight basis this equates to a maximum of 4.90-5.43 mg/kg(0.75) . Some diets prepared for hand-rearing neonatal cheetahs could expose neonates to doses of up to 4.24 mg/kg BW (or 4.24-6.33 mg/kg(0.75) for cubs under 3 months of age). Only one of six zoo-specific diets was found to deliver isoflavones in doses shown to possess biological activity in other species. Therefore, on average, dietary isoflavones were not found in commercially prepared diets consumed by captive cheetahs in concentrations predicted to cause physiological changes. However, a small proportion of these diets, including hand-rearing formulas, contained elevated isoflavones concentrations which may influence cheetah fertility, behaviour or other physiological parameters.

The isoflavone content of commercially-available feline diets in New Zealand.

AIM: To identify and quantify concentrations of the isoflavones genistein, daidzein, biochanin A and formononetin in commercially- prepared feline diets sold in New Zealand. METHODS: Feline diets (n=138) were collected from supermarkets, pet stores and veterinary clinics in New Zealand. Diets were classified into five categories based on the following criteria: the presence/absence of soy, the presence/absence of non-soy plant material, and dry matter (DM) content. A high performance liquid chromatography (HPLC)-based assay was developed and validated to identify and quantify concentrations of the isoflavones genistein, daidzein, biochanin A and formononetin. RESULTS: Isoflavones were detected in all categories of diet, and at quantifiable concentrations in 104/138 (75%) of the diets tested. More dry diets (127/138; 92%) contained isoflavones at quantifiable concentrations than moist diets (83/138; 60%, p<0.001). Of the isoflavone-positive diets, moist diets that contained soy had the highest median isoflavone content (71.1 mg/kg DM and 0.018 mg/kcal metabolisable energy; ME) whilst moist meat-only diets had the lowest (3.24 mg/kg DM and 0.0004 mg/kcal ME). Isoflavone contents varied similarly between categories of diet whether evaluated on a DM or ME basis. High isoflavone contents were associated with diets containing soy and those of low cost. Biochanin A and formononetin were found at concentrations above the detection limit of the assay in 25/138 (18%) and 7/138 (5%) of the diets analysed, respectively. The estimated median daily isoflavone exposure for cats consuming feline diets sold in New Zealand ranged from 0.03 mg/kg bodyweight (BW) for moist meat-only diets to 1.47 mg/kg BW for moist diets containing soy, whilst the diet with the highest isoflavone content would provide daily exposure of 8.13 mg/kg BW. CONCLUSIONS: Isoflavones are present in commercially-prepared feline diets sold in New Zealand at concentrations that have elicited physiological responses in the reproductive, endocrine, and immune systems of other mammalian species. Since isoflavones appear to be a common constituent of feline diets, further investigation of the biological activities of these dietary compounds in cats is warranted.

The endothelin B (ETB) receptor agonist IRL 1620 is highly vasoconstrictive in two syngeneic rat tumour lines: potential for selective tumour blood flow modification.

The vascular effects of the endothelin B (ET(B)) receptor agonist IRL 1620 were investigated in the rat P22 carcinosarcoma and a range of normal tissues in BDIX rats. Tissue blood flow rate was calculated from measurements of tissue uptake of radiolabelled iodoantipyrine. A comparison of vascular effects in the P22 tumour and the HSN sarcoma growing in CBH/CBi rats was made using laser Doppler flowmetry, showing similar effects of IRL 1620, with red cell flux rapidly decreasing by 50-60% and then returning to control levels within approximately 30 min. This corresponded to similar levels but different spatial organisation of ET(B) binding sites in the two tumours, as measured by autoradiography. The decrease in tumour blood flow and an increase in vascular resistance suggest that the vascular component of ET(B) receptors in the P22 tumour is localised on contractile elements rather than on endothelial cells. ET(A) receptors were also identified. Vasoconstriction occurred uniformly throughout the P22 tumour mass, consistent with a measured homogeneous distribution of ET(B) receptors. IRL 1620 caused vasoconstriction in normal skeletal muscle, kidney and small intestine of the BDIX rat as well as in tumour, but did not affect blood flow in other tissues. These effects could be useful for limiting toxicity of certain chemotherapeutic agents. Fully functional ET(B) receptors are clearly expressed on tumour vasculature and IRL 1620 shows promise for short-term modification of tumour blood flow. Expression levels of ET(B) receptors on the tumour vasculature could be useful for predicting which tumours are likely to respond to IRL 1620.

The role of MRSA screening in joint-replacement surgery.

The aim of this prospective study was to determine the effect of screening for methicillin-resistant Staphylococcus aureus (MRSA), in patients undergoing total hip and knee replacements, on reducing hospital-acquired infections and the length of hospital stay. We included 395 patients admitted to the elective orthopaedic ward for hip and knee replacements (knee 210; hip 185) from 16 October 2000 to 15 October 2001. Group 1 included 164 admissions before 16 April 2001 when MRSA swabs were not compulsory. Group 2 included 231 admissions after 16 April 2001 when axillary, nasal and groin swabs had to be negative for MRSA. Four patients in group 1 had post-operative MRSA infection compared with none in group 2. The mean length of hospital stay decreased significantly from 10.43 days +/- SD 4.2 days in group 1 to 9.47 days +/- SD 2.6 days in group 2. There was a significant reduction in the incidence of hospital-acquired infections following the introduction of pre-admission screening.

Insulin reduces serum glycosylphosphatidylinositol phospholipase D levels in human type I diabetic patients and streptozotocin diabetic rats.

The enzyme glycosylphosphatidylinositol phospholipase D has a postulated role in the insulin-mimetic signaling pathway of glycosylphosphatidylinositol compounds. We have investigated enzyme activity in the serum of human type I diabetic patients and plasma and tissues of streptozotocin-induced diabetic rats following insulin administration. In the human diabetic patients serum enzyme activity fell by an average of 10.6% (SEM = 2.7; P = 0.008; n = 20) following administration of insulin. In addition serum enzyme activity appeared to be depleted by 27% (SEM = 8.8; P = 0.011; n = 10) compared to nondiabetic controls. In untreated diabetic rats plasma enzyme activity gradually increased 0.3-fold over a 6-week period (P < 0.001; n = 8), this increase was reversed and activity normalized when these animals were treated with insulin. Cloning of the rat glycosylphosphatidylinositol phospholipase D cDNA enabled confirmation of the liver as the principal organ of synthesis. Analysis of mRNA levels in the livers of the diabetic rats showed that gene expression was reduced in the insulin-treated animals compared to the noninsulin-treated controls by 0.7-fold (P = 0.004; n = 4). Tissue enzyme activity was also reduced in the insulin-treated rats; in skeletal muscle enzyme activity was 0.3-fold lower (P = 0.001; n = 4). Insulin therefore decreases glycosylphosphatidylinositol phospholipase D synthesis in diabetic animals resulting in decreased serum enzyme levels, suggesting a relationship between this enzyme and the function of insulin.

SOX8 expression during chick embryogenesis.

We have isolated the SOX8 gene from the chicken embryo. This gene shows a high degree of sequence homology to SOX9 and SOX10. Detailed analysis of SOX8 expression by whole-mount in situ shows a dynamic and restricted expression pattern during chick development. SOX8 is expressed in the somitic derivative, the dermomyotome, the developing heart, pancreas, enteric neurone system, limb and the neural tube. This is the first detailed expression analysis of SOX8 in any species

SOX14 is a candidate gene for limb defects associated with BPES and Möbius syndrome.

Members of the SOX gene family encode proteins with homology to the HMG box DNA-binding domain of SRY, the Y-linked testis-determining gene. SOX genes are expressed during embryogenesis and are involved in the development of a wide range of different tissues. Mutations in SRY, SOX9 and SOX10 have been shown to be responsible for XY sex reversal, campomelic dysplasia and Waardenburg-Hirschsprung disease, respectively. It is likely that mutations in other SOX genes are responsible for a variety of human genetic diseases. SOX14 has been identified from a human genomic library and the mouse and chicken sequences obtained by polymerase chain reaction amplification. The SOX14 amino acid sequence is highly conserved across these species, suggesting an important role for this protein in vertebrate development. SOX14 is expressed in the neural tube and apical ectodermal ridge of the developing chicken limb. This is the only SOX gene known to be expressed in the apical ectodermal ridge, a structure that directs outgrowth of the embryonic limb bud. Human SOX14 is localised to a 1.15-Mb yeast artificial chromosome on chromosome 3q23, close to loci for BPES (blepharophimosis, ptosis, epicanthus inversus syndrome) and Mobius syndrome. Although SOX14 maps outside these loci, its expression pattern and chromosomal localisation suggest that it is a candidate gene for the limb defects frequently associated with these syndromes.

Optical encryption of digital data.

Smart-pixel architectures, which use the cells of field-programmable gate arrays to provide electronic functionality and intraplane communication, offer a general-purpose approach to exploiting new application areas that would benefit from this kind of structure. One such area, that of the encryption of digital data, is discussed here. Some of the characteristics exhibited by encryption algorithms and ways in which these are applicable to smart-pixel technology are described. The implementation of an algorithm in current use, the SAFER K-64, and its interfacing to an electronic host are then considered in detail. It is shown that this encryption algorithm maps well onto smart-pixel technology because it involves only parallel data transfers, simple regular operations, and interconnections plus a relatively low rate of transfer to the host.

Modification of blood flow in the HSN tumour and normal tissues of the rat by the endothelin ET(B) receptor agonist, IRL 1620.

Activation of endothelin receptors on the vasculature can produce a variety of responses from potent vasoconstriction to mild vasodilation, depending on the receptor complement within the tissue. To elucidate the potential role of endothelin analogues as tumour blood flow modifiers, we have evaluated the effect of the ET(B) receptor agonist, IRL 1620 ([Suc-(Glu9, Ala(11,15))-ET-1(8-21)]) in CBH/CBi rats bearing an HSN fibrosarcoma. Tissue blood flow and vascular resistance were determined, 20 min following administration of IRL 1620 (bolus intravenous), using the uptake of radiolabelled iodoantipyrine (125I-IAP). Blood flow was unchanged in most tissues. However, at doses > or = 1.0 nmol kg(-1) IRL 1620, blood flow in the brain and heart was increased, whereas in the small intestine it was reduced. Blood flow in the skeletal muscle was reduced at 1.0 nmol kg(-1) only. Tumour blood flow was significantly reduced at 3.0 and 5.0 nmol kg(-1). Vascular resistance was unchanged in most tissues although it was increased in the skeletal muscle at 1.0 nmol kg(-1), in the kidney at 1.0 and 3.0 nmol kg(-1) and in the brain and heart, it was reduced at 5.0 nmol kg(-1) IRL 1620. Vascular resistance was significantly increased in the tumour and the small intestine at doses > or = 1 nmol kg(-1) IRL 1620. Pretreatment of rats with BQ-788, an ET(B) receptor antagonist, selectively attenuated the tumour vascular response to 3 nmol kg(-1) IRL 1620 with no changes observed in the normal tissue responses. Our results demonstrate that the HSN tumour vasculature is selectively responsive to IRL 1620 at doses > 1 nmol kg(-1) compared with the majority of normal tissues with the exception of the small intestine, and that only the tumour response is highly sensitive to BQ-788 antagonism, under the experimental dosing regime investigated. These differences may be exploitable for therapeutic benefit.

Dynorphin A(1-8): stability and implications for in vitro opioid activity.

The opioid binding profile and in vitro activity of the endogenous opioid peptide dynorphin A(1-8) have been studied. At opioid receptors in guinea-pig brain dynorphine A(1-8) was nonselective, although with some preference for the delta receptor (Ki 4.6 nM) over mu (Ki 18 nM) and kappa (Ki 40 nM) receptors. However, a high degree of metabolism was observed, with less than 10% of added dynorphin A(1-8) remaining at the end of the binding assay. In the presence of peptidase inhibitors to prevent breakdown of the N- and C-termini and the Gly3-Phe4 bond the major metabolite was [Leu5]enkephalin (representing 49% recovered material). This was reduced by inclusion of an inhibitor of endopeptidase EC 3.4.24.15. In the presence of all the peptidase inhibitors the affinity for kappa receptors (Ki 0.5 nM) relative to mu and delta receptors increased, but no selectivity of binding was observed. This lack of selectivity was confirmed using membranes from C6 glioma cells expressing rat opioid receptors. The agonist effect of dynorphin A(1-8) in the mouse vas deferens (EC50 116 nM) and guinea-pig ileum (EC50 38 nM) was mediated through the kappa receptor as evidenced by the rightward shifts afforded by the kappa-selective antagonist norbinaltorphimine. In the presence of peptidase inhibition potency was improved 2-fold in the mouse vas deferens and 20-fold in the guinea-pig ileum, but this agonist activity was mediated through delta receptors in the vas deferens and mu receptors in the ileum, as a result of the formation and stabilization of [Leu5]enkephalin. The results confirm the absence of receptor selectivity of dynorphin A(1-8) in binding assays but show that its agonist effects, at least in vitro, are mediated exclusively through the kappa opioid receptor.

Modification of tumor blood flow: current status and future directions.

Suboptimal drug distribution and hypoxia, which can contribute to treatment failure, are a direct consequence of the spatial and temporal heterogeneity in perfusion that occurs in solid tumors. Therefore, improvements in tumor blood flow have wide-ranging therapeutic importance. Paradoxically, controlled decreases in tumor blood flow can also be exploited and, if permanent, induce extensive tumor cell death on their own. We review the current knowledge of the factors controlling tumor blood flow with emphasis on the roles of the endogeneous vasodilator nitric oxide and the endogenous vasoconstrictor endothelin-1. The potential importance and application of approaches that irreversibly damage vascular function, so-called vascular targeting, are also discussed. Emphasis is given to the drug-based approaches to vascular targeting that are now entering clinical evaluation. There is no doubt that increased understanding of the processes that determine blood flow in tumors, coupled with the availability of techniques to monitor blood flow noninvasively in the clinic, will enable strategies for selectively modifying tumor blood flow to be transferred from the laboratory to the clinical setting.

The effect of oxygen and carbon dioxide on tumor cell endothelin-1 production.

Endothelin-1 (ET-1) is produced by some tumor cells, but the dependence of this production on pO2 and pCO2, conditions relevant within the tumor microenvironment, has not been described. HT29 colon adenocarcinoma cells and DU145 prostate carcinoma cells produce similar amounts of ET-1 in vitro under normal cell culture conditions of 21% O2/5% CO2 (normoxia). Exposure of HT29 cells to either 2% O2 or 0.2% O2 significantly reduced ET-1 production compared to cells in normoxia. In contrast, production of ET-1 by DU145 cells was usually unaffected by hypoxia and was even slightly increased in cells exposed to 2% O2 in HEPES-buffered EMEM (HEPES-EMEM). Exposure of cells to either 2.2% CO2 or 7.1% CO2 had no effect on the production of ET-1 by cells in bicarbonate-buffered EMEM (EMEM). However, in HEPES-EMEM, ET-1 production by both cell lines was reduced in 7.1% CO2. A slight reduction in ET-1 produced by DU145 cells was also observed in 2.2% CO2. These results illustrate that changes in ET-1 production by tumor cells in response to hypoxia and hypercapnia are tumor-dependent. It is clear that the production of ET-1 by tumor cells under normal culture conditions may not accurately reflect production within the tumor microenvironment. A greater insight into the in vivo situation, however, may be possible by modifying the cell culture conditions.

Familial Kallmann syndrome: a novel splice acceptor mutation in the KAL gene.

Kallmann syndrome is an inherited disease which is characterised by anosmia (inability to smell) and hypogonadotropic hypogonadism both of which are thought to occur as a result of a failure of correct neuronal migration. To date the only genetic lesions identified are mutations in the X-linked gene, KAL. We conducted a mutation screen of the KAL gene in a family with Kallmann syndrome. This identified a new mutation in the KAL gene which removed an acceptor site at the junction of exon 6/intron 5. Exon 6 of the KAL gene encodes the C-terminal portion of a fibronectin type III domain may be involved in axonal pathfinding. We presume that the described mutation would result in the removal of exon 6 resulting in a frame shift which terminates the protein prematurely. It has been proposed that both mental illness and vesico-ureteric reflux are associated with mutations in the KAL gene. However, results from the family presented here do not show an association between either trait and the KAL gene mutation.

Vascular response of tumour and normal tissues to endothelin-1 following antagonism of ET(A) and ET(B) receptors in anaesthetised rats.

Modification of blood flow by endothelin-1 (ET-1) was examined in the s.c. HSN fibrosarcoma and compared to normal tissues of anaesthetised CBH/CBi rats. The ET receptor subtypes involved in the response were investigated using the ET(A) and ET(B) receptor antagonists BQ-610 and BQ-788, respectively. Blood flow and vascular resistance were determined using the uptake of radiolabelled iodo-antipyrine (125I-IAP). BQ-610 or BQ-788 was infused for 30 min prior to blood flow determination. ET-1 was administered 15 min into the infusion time. BQ-610 and BQ-788 infused alone did not modify any vascular parameters. Tumour blood flow increased slightly following ET-1, contrasting with most normal tissues, in which blood flow was reduced. Vascular resistance increased in all tissues, including the tumour. Neither antagonist significantly modified the ET-1-induced changes in tumour blood flow or vascular resistance, whereas in the majority of normal tissues BQ-610 attenuated and BQ-788 potentiated the vascular resonse to ET-1. Our results show that the HSN tumour vasculature is only weakly responsive to ET- 1 and antagonism of its effects by BQ-610 and BQ-788. This contrasts with the majority of normal tissues, in which ET- 1 induces an intense vasoconstriction.

Reduced capacity of tumour blood vessels to produce endothelium-derived relaxing factor: significance for blood flow modification.

The effect of nitric oxide-dependent vasodilators on vascular resistance of tumours and normal tissue was determined with the aim of modifying tumour blood flow for therapeutic benefit. Isolated preparations of the rat P22 tumour and normal rat hindlimb were perfused ex vivo. The effects on tissue vascular resistance of administration of sodium nitroprusside (SNP) and the diazeniumdiolate (or NONO-ate) NOC-7, vasodilators which act via direct release of nitric oxide (NO), were compared with the effects of acetylcholine (ACh), a vasodilator which acts primarily via receptor stimulation of endothelial cells to release NO in the form of endothelium-derived relaxing factor (EDRF). SNP and NOC-7 effectively dilated tumour blood vessels after preconstriction with phenylephrine (PE) or potassium chloride (KCl) as indicated by a decrease in vascular resistance. SNP also effectively dilated normal rat hindlimb vessels after PE/KCl constriction. Vasodilatation in the tumour preparations was accompanied by a significant rise in nitrite levels measured in the tumour effluent. ACh induced a significant vasodilation in the normal hindlimb but an anomalous vasoconstriction in the tumour. This result suggests that tumours, unlike normal tissues are incapable of releasing NO (EDRF) in response to ACh. Capacity for EDRF production may represent a difference between tumour and normal tissue blood vessels, which could be exploited for selective pharmacological manipulation of tumour blood flow.