Vascular attack by 5,6-dimethylxanthenone-4-acetic acid combined with B7.1 (CD80)-mediated immunotherapy overcomes immune resistance and leads to the eradication of large tumors and multiple tumor foci

The promise of cancer immunotherapy is that it will not only eradicate primary tumors but will generate systemic antitumor immunity capable of destroying distant metastases. A major problem that must first be surmounted relates to the immune resistance of large tumors. Here we reveal that immune resistance can be overcome by combining immunotherapy with a concerted attack on the tumor vasculature. The functionally related antitumor drugs 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and flavone acetic acid (FAA), which cause tumor vasculature collapse and tumor necrosis, were used to attack the tumor vasculature, whereas the T-cell costimulator B7.1 (CD80), which costimulates T-cell proliferation via the CD28 pathway, was used to stimulate antitumor immunity. The injection of cDNA (60–180 µg) encoding B7.1 into large EL-4 tumors (0.8 cm in diameter) established in C57BL/6 mice, followed 24 h later by i.p. administration of either DMXAA (25 mg/kg) or FAA (300 mg/kg), resulted in complete tumor eradication within 2–6 weeks. In contrast, monotherapies were ineffective. Both vascular attack and B7.1 immunotherapy led to up-regulation of heat shock protein 70 on stressed and dying tumor cells, potentially augmenting immunotherapy. Remarkably, large tumors took on the appearance of a wound that rapidly ameliorated, leaving perfectly healed skin. Combined therapy was mediated by CD8+ T cells and natural killer cells, accompanied by heightened and prolonged antitumor cytolytic activity (P < 0.001), and by a marked increase in tumor cell apoptosis. Cured animals completely rejected a challenge of 1 x 107 parental EL-4 tumor cells but not a challenge of 1 x 104 Lewis lung carcinoma cells, demonstrating that antitumor immunity was tumor specific. Adoptive transfer of 2 x 108 splenocytes from treated mice into recipients bearing established (0.8 cm in diameter) tumors resulted in rapid and complete tumor rejection within 3 weeks. Although DMXAA and B7.1 monotherapies are complicated by a narrow range of effective doses, combined therapy was less dosage dependent. Thus, a broad range of amounts of B7.1 cDNA were effective in combination with 25 mg/kg DMXAA. In contrast, DMXAA, which has a very narrow range of high active doses, was effective at a low dose (18 mg/kg) when administered with a large amount (180 µg) of B7.1 cDNA. Importantly, combinational therapy generated heightened antitumor immunity, such that gene transfer of B7.1 into one tumor, followed by systemic DMXAA treatment, led to the complete rejection of multiple untreated tumor nodules established in the opposing flank. These findings have important implications for the future direction and utility of cancer immunotherapies aimed at harnessing patients’ immune responses to their own tumors.

Small diameter Polyurethane vascular graft reinforced by elastic weft-knitted tubular fabric of polyester / spandex

Small diameter vascular grafts were fabricated from pure Polyurethane (PU) as well as PU reinforced with a tubular weft-knitted fabric. The tensile properties of the reinforced composite vascular grafts were compared with that of the tubular fabric itself and the pure PU vascular grafts. The elasticity and strength of the reinforced vascular grafts were improved compared with the tubular fabric. Strength of the reinforced vascular grafts was 5–10 times of the strength of the pure PU vascular grafts. Expanding the tubular fabric to increase the inner diameter of the reinforced vascular graft reduced the graft’s strength and initial modulus, but the difference was reduced as the PU content was increased. For grafts of the same inner diameter, increasing the PU content increased the thickness and strength of the graft wall, which led to a general increase in the strength and initial modulus of the composite vascular grafts.

Neurally-derived nitric oxide regulates vascular tone in pulmonary and cutaneous arteries of the toad, Bufo marinus

In this study, the role of nitric oxide (NO) in regulation of the pulmocutaneous vasculature of the toad, Bufo marinus was investigated. In vitro myography demonstrated the presence of a neural NO signaling mechanism in both arteries. Vasodilation induced by nicotine was inhibited by the soluble guanylyl cyclase (GC) inhibitor, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one, and the NO synthase (NOS) inhibitor, N^ω-nitro-_L-arginine (_L-NNA). Removal of the endothelium had no significant effect on the vasodilation. Furthermore, pretreatment with N⁵-(1-imino-3-butenyl)-_L-ornithine (vinyl-_L-NIO), a more specific inhibitor of neural NOS, caused a significant decrease in the nicotine-induced dilation. In the pulmonary artery only, a combination of _L-NNA and the calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP(_8-37), completely blocked the nicotine-induced dilation. In both arteries, the vasodilation was also significantly decreased by glibenclamide, an ATP-sensitive K⁺ (K⁺_ATP) channel inhibitor. Levcromakalim, a K⁺_ATP channel opener, caused a dilation that was blocked by glibenclamide in both arteries. In the pulmonary artery, NO donor-mediated dilation was significantly decreased by pretreatment with glibenclamide. The physiological data were supported by NADPH-diaphorase histochemistry and immunohistochemistry, which demonstrated NOS in perivascular nerve fibers but not the endothelium of the arteries. These results indicate that the pulmonary and cutaneous arteries of B. marinus are regulated by NO from nitrergic nerves rather than NO released from the endothelium. The nitrergic vasodilation in the arteries appears to be caused, in part, via activation of K⁺_ATP channels. Thus, NO could play an important role in determining pulmocutaneous blood flow and the magnitude of cardiac shunting.

Vascular distribution of nitric oxide synthase and vasodilation in the Australian lungfish, Neoceratodus forsteri

The presence of nitric oxide synthase (NOS) and role of nitric oxide (NO) in vascular regulation was investigated in the Australian lungfish, Neoceratodus forsteri. No evidence was found for NOS in the endothelium of large and small blood vessels following processing for NADPH-diaphorase histochemistry. However, both NADPH-diaphorase histochemistry and neural NOS immunohistochemistry demonstrated a sparse network of nitrergic nerves in the dorsal aorta, hepatic artery, and branchial arteries, but there were no nitrergic nerves in small blood vessels in tissues. In contrast, nitrergic nerves were found in non-vascular tissues of the lung, gut and kidney. Dual-wire myography was used to determine if NO signalling occurred in the branchial artery of N. forsteri. Both SNP and SIN-1 had no effect on the pre-constricted branchial artery, but the particulate guanylyl cyclase (GC) activator, C-type natriuretic peptide, always caused vasodilation. Nicotine mediated a dilation that was not inhibited by the soluble GC inhibitor, ODQ, or the NOS inhibitor, L-NNA, but was blocked by the cyclooxygenase inhibitor, indomethacin. These data suggest that NO control of the branchial artery is lacking, but that prostaglandins could be endothelial relaxing factors in the vasculature of lungfish.

The ß-amyloid peptide in Alzheimer's disease decreases adhesion of vascular muscle cells to the basement membrane

Cerebral amyloid angiopathy (CAA) is a major feature of Alzheimer's disease pathology. In CAA, degeneration of vascular smooth muscle cells (VSMCs) occurs close to regions of the basement membrane where the amyloid protein (Aβ) builds up. In this study, the possibility that Aβ disrupts adhesive interactions between VSMCs and the basement membrane was examined. VSMCs were cultured on a commercial basement membrane substrate (Matrigel). The presence of Aβ in the Matrigel decreased cell-substrate adhesion and cell viability. Full-length oligomeric Aβ was required for the effect, as N- and C-terminally truncated peptide analogues did not inhibit adhesion. Aβ that was fluorescently labelled at the N-terminus (fluo-Aβ) bound to Matrigel as well as to the basement membrane heparan sulfate proteoglycan (HSPG) perlecan and laminin. Adhesion of VSMCs to perlecan or laminin was decreased by Aβ. As perlecan influences VSMC viability through the extracellular signal-regulated kinase (ERK)1/2 signalling pathway, the effect of Aβ1–40 on ERK1/2 phosphorylation was examined. The level of phospho-ERK1/2 was decreased in cells following Aβ treatment. An inhibitor of ERK1/2 phosphorylation enhanced the effect of Aβ on cell adhesion. The studies suggest that Aβ can decrease VSMC viability by disrupting VSMC–extracellular matrix (ECM) adhesion.

Cholesterol is necessary both for the toxic effect of Abeta peptides on vascular smooth muscle cells and for Abeta binding to vascular smooth muscle cell membranes

Accumulation of beta amyloid (Aβ) in the brain is central to the pathogenesis of Alzheimer's disease. Aβ can bind to membrane lipids and this binding may have detrimental effects on cell function. In this study, surface plasmon resonance technology was used to study Aβ binding to membranes. Aβ peptides bound to synthetic lipid mixtures and to an intact plasma membrane preparation isolated from vascular smooth muscle cells. Aβ peptides were also toxic to vascular smooth muscle cells. There was a good correlation between the toxic effect of Aβ peptides and their membrane binding. 'Ageing' the Aβ peptides by incubation for 5 days increased the proportion of oligomeric species, and also increased toxicity and the amount of binding to lipids. The toxicities of various Aβ analogs correlated with their lipid binding. Significantly, binding was influenced by the concentration of cholesterol in the lipid mixture. Reduction of cholesterol in vascular smooth muscle cells not only reduced the binding of Aβ to purified plasma membrane preparations but also reduced Aβ toxicity. The results support the view that Aβ toxicity is a direct consequence of binding to lipids in the membrane. Reduction of membrane cholesterol using cholesterol-lowering drugs may be of therapeutic benefit because it reduces Aβ-membrane binding.

Variations in vascular access flows in haemodialysis can depend on needle orientation

Introduction: While using the Transonic Qc[TM] machine to assess access flow in arteriovenous fistulae (AVF), we observed that when compared to antegrade arterial needle insertion, retrograde arterial needle insertion could regularly produce lower access flow measurements. This study sought to explore this phenomenon.

Method: 23 patients entered and 20 finished the study. Patient selection criteria included: functioning AVF and an adequate AVF length for either retrograde or antegrade arterial needle insertion. After ensuring stable and similar blood pressures, 3 flow measurements were taken during the first 2 hours on the same dialysis day of 3 consecutive weeks using antegrade needle insertion then were repeated on 3 further consecutive weeks using retrograde insertion.

Results: Overall, access flows measured with retrograde insertion were significantly lower by a mean difference of 107.15 ml/min (57-484 ml/min) than the flows measured with antegrade needle placement. In 5/20, 3 recorded minimal difference and 2 had a higher access flows during retrograde insertion. No recirculation was observed during either antegrade or retrograde needle insertion. The paired t-test showed that there was significant difference between the antegrade versus retrograde mean measurements (p = 0.005).

Conclusion: Although the sample size is small and the number of measurements limited, we conclude that access flows may be greater with an antegrade arterial orientation compared to flows recorded with a retrograde orientation. The phenomenon behind this conclusion is yet to be investigated. We suggest that when using the Transonic Qc[TM] access measurement device the arterial needle should always be in the same direction for each measurement for each individual patient.

Mechanism of nitric oxide regulation of vascular tone in the mesenteric arteries of the cane toad, Bufo marinus

Nitric oxide control of large systemic blood vessels of the cane toad, Bufo marinus is provided by nitrergic nerves. However, the involvement of nitrergic nerves in the regulation of small blood vessels has yet to be determined. This study investigated the nitric oxide (NO) control of the mesenteric arteries (MA) of B. marinus. Immunohistochemistry and NADPH-diaphorase histochemistry demonstrated a dense plexus of nitrergic nerves in the MA of B. marinus. MAs (~ 500–700µm in diameter) were mounted in a myograph and placed under an initial tension equivalent to their normal diameter. MAs were pre-constricted with the thromboxane A2 mimetic, U46619, prior to the addition of putative, vasodilatory chemicals. Acetylcholine caused a vasodilation that was endothelium-independent, because removal of the endothelium had no effect on the dilation. The response to acetylcholine was blocked by the NOS inhibitor, L-NNA, demonstrating that the effect was NO-dependent. Interestingly, nicotine also caused a dilation that was not affected by removal of the endothelium, but was significantly inhibited by L-NNA and the calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP(8–37). These findings indicate that the MA of B. marinus are controlled by NO released from nitrergic nerves. In addition, a component of the response to applied nicotine appears to be mediated CGRP, which is probably released from sensory nerves.

Mucosal vascular addressin cell adhesion molecule-1 is expressed outside the endothelial lineage on fibroblasts and melanoma cells

Mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1) is predominantly expressed on high endothelial venules in inflamed tissues where it assists with leucocyte extravasation. Here we report that MAdCAM-1 has the potential to be more widely expressed outside the endothelial cell lineage than previously appreciated. Thus, MAdCAM-1 RNA transcripts and cell-surface protein were expressed by NIH 3T3 fibroblasts following activation with tumour necrosis factor-alpha (TNF-alpha), and by freshly isolated and cultured primary mouse splenic and tail fibroblasts in the absence of TNF-alpha stimulation. They were constitutively expressed by B16F10 melanoma cells, and expression was enhanced by cell activation with TNF-alpha. Mucosal vascular addressin cell adhesion molecule-1 was expressed on the apical surface of isolated cells, but became predominantly localized to cell junctions in confluent cell monolayers, suggesting it may play a role in the homotypic aggregation of cells. Tumour necrosis factor-alpha enhanced the expression of a firefly luciferase reporter directed by the MAdCAM-1 promoter in NIH 3T3 and B16F10 cells. A DNA fragment extending from nt -1727 to -673 was sufficient to confer cell-type selective expression. Mucosal vascular addressin cell adhesion molecule-1 expressed by NIH 3T3 cells was biologically active, as it supported the adhesion of TK-1 T cells in an alpha4beta7-dependent fashion. The expression of MAdCAM-1 by fibroblasts, and melanomas suggests MAdCAM-1 may play a role in regulating host responses in the periphery, leucocyte transmigration across nonendothelial boundaries, or the homotypic interactions of some malignant melanomas.

Vascular plant and small mammal communities over an elevational gradient

Plant species richness and plant and small mammal beta diversity decline over the elevational gradient in the Otway ranges. These patterns are influenced by climate, habitat and spatial structure. This highlights the need to preserve continuous habitat and understand the influence of climate, to conserve communities in the changing future.

Nitric oxide and vascular regulation in fishes and amphibians

Nitric oxide is an important molecule in the regulation of the cardiovascular system and blood pressure. This research provided new insights into the evolution of nitric oxide control of blood vessels by showing how nitric oxide signalling causes vasodilation in the circulation of amphibians and fishes.

Relationships between measures of fitness, physical activity, body composition and vascular function in children

Background : The prevalence of obesity and physical inactivity in Western countries has increased rapidly. Both are modifiable risk factors for cardiovascular disease. Atherosclerosis begins in childhood and endothelial dysfunction is its earliest detectable manifestation.

Methods : We assessed flow-mediated dilation (FMD) in 129 children (75 female; 10.3 + 0.3 yrs; 54 male; 10.4; 0.3 yrs). FMD was normalised for differences in the eliciting shear rate stimulus between subjects (SR_AUC). Fitness was assessed as peak oxygen uptake during an incremental treadmill exercise test (VO₂peak). Body composition was measured using a dual-energy X-ray absorptiometry (DEXA) scan. Physical activity (PA) was assessed using Actigraph accelerometers. The cohort was split into tertiles according to FMD% and also FMD% corrected for SR_AUC to gain insight into the determinants of vascular function.

Results : Across the cohort, significant correlations were observed between FMD%/SR_AUC and DEXA percentage fat (r = −0.23, p = 0.009) and percentage lean mass (r = 0.21, p = 0.008), and also with PA performed at moderate-to-high intensity (r = 0.363, p = 0.001). For children in the lowest FMD%/SR_AUC tertile, a stronger relationship with all PA measures was observed, particularly with high intensity PA (r = 0.572, P = 0.003). Regression analysis revealed that high intensity PA was the only predictor of impaired FMD%/SR_AUC.

Conclusions : These data suggest that traditional risk factors for CHD in adult populations impact upon vascular function in young people. Furthermore, it appears that individuals with impaired FMD may benefit from performing high intensity PA, whereas no relationships exist between FMD and lower intensities of PA or between PA and FMD in those subjects who possess preserved vascular function a priori.

Chemical coding of vascular nitrergic nerves in lower vertebrates

Nitric oxide is an important regulator of blood pressure in mammals. This study provided new information on the role of nitric-oxide releasing sympathetic nerves in vascular regulation of lower vertebrates. The research outcomes advance knowledge on the potential role of these unique nerves in the control of the lower vertebrate cardiovascular system.

Lack of relationship between sedentary behaviour and vascular function in children

Some evidence suggests that sedentary behaviour is independently associated with cardiovascular (CV) risk. Endothelial dysfunction is the earliest detectable manifestation of CVD and a strong independent predictor of CV events. No previous study has examined the relationship between sedentary behaviour and endothelial function. We assessed the basal association between conduit artery endothelial function and sedentary behaviour in children, along with the correlation between changes in sedentary behaviour and endothelial function. We studied 116 children (70$: 10.7 ± 0.3; 46#: 10.7 ± 0.3 years) on two occasions; in the summer (June) and late autumn (November). We assessed endothelial function via flowmediated dilation (FMD) using high-resolution Doppler ultrasound. Sedentary behaviour (SB) was assessed using objective uni-axial accelerometry. At baseline, there were no significant differences between girls and boys for any measured variables with the exception of total physical activity time. FMD was not associated with sedentary behaviour in either group or in the cohort as a whole. Although FMD decreased (10.0 ± 4.3–7.9 ± 3.9%, P\0.001) and SB increased (499.1 ± 103.5–559 ± 81.6 min/day, P\0.001) between the seasons, no relationship existed between changes in these variables. Our data suggest that sedentary behaviour and changes in sedentary behaviour are not associated with endothelial function in children.