Vivien E. Prise

Blood vessel maturation and response to vascular-disrupting therapy in single vascular endothelial growth factor-A isoform-producing tumors

Tubulin-binding vascular-disrupting agents (VDA) are currently in clinical trials for cancer therapy but the factors that influence tumor susceptibility to these agents are poorly understood. We evaluated the consequences of modifying tumor vascular morphology and function on vascular and therapeutic response to combretastatin-A4 3-O-phosphate (CA-4-P), which was chosen as a model VDA. Mouse fibrosarcoma cell lines that are capable of expressing all vascular endothelial growth factor (VEGF) isoforms (control) or only single isoforms of VEGF (VEGF120, VEGF164, or VEGF188) were developed under endogenous VEGF promoter control. Once tumors were established, VEGF isoform expression did not affect growth or blood flow rate. However, VEGF188 was uniquely associated with tumor vascular maturity, resistance to hemorrhage, and resistance to CA-4-P. Pericyte staining was much greater in VEGF188 and control tumors than in VEGF120 and VEGF164 tumors. Vascular volume was highest in VEGF120 and control tumors (CD31 staining) but total vascular length was highest in VEGF188 tumors, reflecting very narrow vessels forming complex vascular networks. I.v. administered 40 kDa FITC-dextran leaked slowly from the vasculature of VEGF188 tumors compared with VEGF120 tumors. Intravital microscopy measurements of vascular length and RBC velocity showed that CA-4-P produced significantly more vascular damage in VEGF120 and VEGF164 tumors than in VEGF188 and control tumors. Importantly, this translated into a similar differential in therapeutic response, as determined by tumor growth delay. Results imply differences in signaling pathways between VEGF isoforms and suggest that VEGF isoforms might be useful in vascular-disrupting cancer therapy to predict tumor susceptibility to VDAs.

Nitric oxide in biological fluids: analysis of nitrite and nitrate by high-performance ion chromatography

The analysis of nitric oxide-derived nitrite and nitrate ions in biological fluids represents a proven strategy for determining nitric oxide participation in a diverse range of physiological and pathophysiological processes in vivo. In this article we describe a versatile method for the simultaneous measurement of NO2- and NO3- anions in both plasma and isolated tumour models based on anion-exchange chromatography with spectrophotometric detection (214 nm). This method compares well with the capillary electrophoresis technique, exhibiting an equivalent sensitivity for NO2-/NO3- anions and short run-times, i.e. not greater than 4 min. Comparisons are also made with two alternative but less satisfactory methods which employ ion-exchange or reversed-phase ion-pair chromatography with conductimetric as well as spectrophotometric detection. Technical problems associated with each method, particularly those arising from nitrate contamination, have been addressed.

Estimation of Apparent Tumor Vascular Permeability from Multiphoton Fluorescence Microscopic Images of P22 Rat Sarcomas In Vivo

Objective: To develop an image processing‐based method to quantify the rate of extravasation of fluorescent contrast agents from tumor microvessels, and to investigate the effect of the tumor vascular disrupting agent combretastatin A‐4‐P (CA‐4‐P) on apparent tumor vascular permeability to 40 kDa fluorescein isothiocyanate (FITC) labeled dextran.

The comparative effects of the NOS inhibitor, nω-nitro-l-arginine, and the haemoxygenase inhibitor, zinc protoporphyrin IX, on tumour blood flow

PURPOSE To determine the relative effects of inhibiting nitric oxide synthase (NOS) and haemoxygenase (HO) on blood flow to the rat P22 carcinosarcoma. METHODS AND MATERIALS HO is the enzyme responsible for in vivo production of carbon monoxide (CO). The vascular effects of zinc protoporphyrin IX (ZnPP), a competitive inhibitor of HO, were compared with those of copper protoporphyrin IX (CuPP), a poor inhibitor of HO, in isolated ex vivo perfusions of the P22 tumour and in intact tumour-bearing rats. In ex vivo perfusions, tumour vascular resistance was calculated from measurements of perfusion pressure at a known flow rate. In intact animals, blood flow to tumour and normal tissues was calculated using a radiotracer uptake method. The effects of ZnPP were compared with those of the NOS inhibitor, N(omega)-nitro-L-arginine (L-NNA), and the combination of the two drugs. RESULTS HO activity in the P22 tumour was reduced by 50% following administration of either ZnPP or CuPP directly to ex vivo perfused tumours, suggesting an indirect effect on the enzyme. Enzyme inhibition was not associated with any significant vasoactive effect. Neither ZnPP nor CuPP, at a dose of 45 micromol x kg(-1) administered i.p., inhibited tumour HO in vivo. However, they did significantly decrease tumour blood flow to 60-70% of control, with similar effects in skin and brain. Skeletal muscle blood flow was increased to 150% of control. L-NNA decreased both tumour and skeletal muscle blood flow to around 40% of control. These differences suggest that the nonspecific effects of ZnPP and CuPP were not mediated by NOS inhibition. The combination of ZnPP and L-NNA improved the selective reduction in tumour blood flow achieved with either agent alone. CONCLUSION This suggests that the HO/CO pathway does not play a major vasodilatory role in this tumour. However, ZnPP and CuPP could be useful for inducing a relatively selective decrease in tumour blood flow via mechanisms unrelated to HO inhibition, especially when combined with NOS inhibition.

Effect of endothelin-1 and sarafotoxin S6c on blood flow in a rat tumor.

Summary: The modification of tumor blood flow resulting from administration of endothelin-1 (ET-1) and sarafotoxin S6c (SX6c) was examined in female CBH rats. Blood flow in subcutaneous HSN tumors and normal tissues was measured by tissue uptake of 125I-labeled io-doantipyrine ([125I]IAP). A 75% increase in tumor blood flow was observed after 1 nM/kg ET-1, contrasting with flow in normal tissues, which was unaffected or reduced. The exception to this was the brain, in which blood flow was increased by 30%, resulting from a rise in mean arterial blood pressure (MABP) and the absence of vasoconstriction. Paradoxically, a significant drop in the tumor vascular resistance was observed after 1 nM/kg ET-1, whereas in all other tissues the vascular resistance was significantly increased. Vascular responses to SX6c differed from those observed with ET-1. At 1 nM/kg SX6c, blood flow in the tumor was increased to 175% of the control as a result of the increase in MABP, which was similar to ET-1. However, unlike ET-1, there was no associated vasodilatation. Vascular resistance was increased in all normal tissues with 1 nM/kg SX6c, corresponding to decreases in blood flow in the contralateral skin, skeletal muscle, and small intestine. This study therefore demonstrates that the vascular responses to ET-1 and SX6c are unique in the HSN tumor compared to normal tissues. This atypical response of the tumor vasculature may therefore be exploitable to improve the delivery of blood-borne anti-cancer agents in therapy.

Nitric Oxide Synthase Inhibition Enhances the Tumor Vascular-Damaging Effects of Combretastatin A-4 3-O-Phosphate at Clinically Relevant Doses

Purpose: The therapeutic potential of combining the prototype tumor vascular-disrupting agent combretastatin A-4 3-O-phosphate (CA-4-P) with systemic nitric oxide synthase (NOS) inhibition was investigated preclinically. Experimental Design: Vascular response (uptake of 125I-labeled iodoantipyrine; laser Doppler flowmetry) and tumor response (histologic necrosis; cytotoxicity and growth delay) were determined. Results: Inducible NOS selective inhibitors had no effect on blood flow in the P22 rat sarcoma. In contrast, the non–isoform-specific NOS inhibitor Nω-nitro- l-arginine (l-NNA; 1 and 10 mg/kg i.v. or chronic 0.1 or 0.3 mg/mL in drinking water) decreased the P22 blood flow rate selectively down to 36% of control at 1 hour but did not induce tumor necrosis at 24 hours. CA-4-P, at clinically relevant doses, decreased the P22 blood flow rate down to 6% of control at 1 hour for 3 mg/kg but with no necrosis induction. However, l-NNA administration enhanced both CA-4-P–induced tumor vascular resistance at 1 hour (chronic l-NNA administration) and necrosis at 24 hours, with 45% or 80% necrosis for 3 and 10 mg/kg CA-4-P, respectively. Bolus l-NNA given 3 hours after CA-4-P was the most effective cytotoxic schedule in the CaNT mouse mammary carcinoma, implicating a particular enhancement by l-NNA of the downstream consequences of CA-4-P treatment. Repeated dosing of l-NNA with CA-4-P produced enhanced growth delay over either treatment alone in P22, CaNT, and spontaneous T138 mouse mammary tumors, which represented a true therapeutic enhancement. Conclusions: The combination of NOS inhibition with CA-4-P is a promising approach for targeting tumor vasculature, with relevance for similar vascular-disrupting agents in development.

Changes in tumour morphology with alterations in oxygen availability: further evidence for oxygen as a limiting substrate

The ability of cancer cells to survive at a distance from blood vessels should be dependent on the local supply of nutrients to each vessel. The corded growth of tumour cells around blood vessels within regions of necrosis in the RH carcinoma in the mouse allows the limit to which cells can be supported by individual vessels to be observed. The thickness of individual tumour cords was measured in conventionally stained tumour sections using a scanning technique to determine the distance between the blood vessel wall and the most distant viable cell adjacent to necrosis. Cord radius was found to vary with the oxygen supply conditions. Control animals had a mean radius of 105 +/- 2 microns while animals that had breathed 10% oxygen had significantly narrower cords (93 +/- 3 microns after 48 h) and animals breathing 100% oxygen had significantly wider cords (117 +/- 3 microns after 24 h). Mice made anaemic (mean hct. 28%) by phlebotomy and plasma transfusion had cord radii that were not significantly different from controls at any time up to 48 h. We conclude that this relatively slow growing mouse tumour is capable of rapid morphological adaptation (less than 3 h) to changes in nutrient availability and that oxygen is probably the limiting substrate.

The Influence of Nitric Oxide on Tumour Vascular Tone

Acetylcholine and sodium nitroprusside, which vasodilate via release of NO by endothelium-dependent and endothelium-independent mechanisms respectively, had little effect on tumour vascular resistance when administered to tissue-isolated tumours perfused in their normal state. However, under phenylephrine-induced vasoconstriction, sodium nitroprusside induced vasodilation whilst acetylcholine induced a small vasoconstriction. Phenylephrine itself induced an oscillatory change in tumour perfusion pressure. The nitric oxide synthase (NOS) inhibitor N omega-nitro-L-arginine (L-NNA) caused a dose-dependent increase in vascular resistance in ex vivo perfused tumours which was greater than that in normal perfused hindlimbs. Systemic administration of L-NNA caused a 50% decrease in tumour blood flow which was a larger effect than in any of the normal tissues studied except spleen and skeletal muscle. Modification of NOS activity in tumours is a promising means for selective tumour blood flow modification. Investigation of endothelium-dependent versus endothelium-independent methods for modifying tumour blood flow may provide methods for further selectivity.

Lipoprotein-associated phospholipase A2 (Lp-PLA2) as a therapeutic target to prevent retinal vasopermeability during diabetes

Significance Breakdown of the blood–retinal barrier (BRB) is central to diabetic macular edema (DME). Here, we demonstrate that lipoprotein-associated phospholipase A2 (Lp-PLA2) plays a critical role in diabetes-related retinal vasopermeability, a response that is mediated by lysophosphatidylcholine (LPC). Because neutralization of VEGF is the current gold-standard treatment for DME, we assessed suboptimal systemic treatment of an Lp-PLA2 inhibitor alongside a suboptimal intravitreal injection with a rat-specific VEGF antibody and demonstrated that protection against diabetes-mediated retinal vasopermeability was additive. We have also shown a coalescence of the LPC and VEGF pathways in retinal vascular endothelium via a common VEGF receptor 2-mediated mechanism. Alongside currently administered anti-VEGF agents, Lp-PLA2 may be a useful therapeutic target for DME. Lipoprotein-associated phospholipase A2 (Lp-PLA2) hydrolyses oxidized low-density lipoproteins into proinflammatory products, which can have detrimental effects on vascular function. As a specific inhibitor of Lp-PLA2, darapladib has been shown to be protective against atherogenesis and vascular leakage in diabetic and hypercholesterolemic animal models. This study has investigated whether Lp-PLA2 and its major enzymatic product, lysophosphatidylcholine (LPC), are involved in blood–retinal barrier (BRB) damage during diabetic retinopathy. We assessed BRB protection in diabetic rats through use of species-specific analogs of darapladib. Systemic Lp-PLA2 inhibition using SB-435495 at 10 mg/kg (i.p.) effectively suppressed BRB breakdown in streptozotocin-diabetic Brown Norway rats. This inhibitory effect was comparable to intravitreal VEGF neutralization, and the protection against BRB dysfunction was additive when both targets were inhibited simultaneously. Mechanistic studies in primary brain and retinal microvascular endothelial cells, as well as occluded rat pial microvessels, showed that luminal but not abluminal LPC potently induced permeability, and that this required signaling by the VEGF receptor 2 (VEGFR2). Taken together, this study demonstrates that Lp-PLA2 inhibition can effectively prevent diabetes-mediated BRB dysfunction and that LPC impacts on the retinal vascular endothelium to induce vasopermeability via VEGFR2. Thus, Lp-PLA2 may be a useful therapeutic target for patients with diabetic macular edema (DME), perhaps in combination with currently administered anti-VEGF agents.

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 (ETB) 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 ETB 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 ETB receptors in the P22 tumour is localised on contractile elements rather than on endothelial cells. ETA receptors were also identified. Vasoconstriction occurred uniformly throughout the P22 tumour mass, consistent with a measured homogeneous distribution of ETB 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 ETB receptors are clearly expressed on tumour vasculature and IRL 1620 shows promise for short-term modification of tumour blood flow. Expression levels of ETB receptors on the tumour vasculature could be useful for predicting which tumours are likely to respond to IRL 1620.