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.

Mechanisms of vasodilation in the dorsal aorta of the elephant fish, Callorhinchus milii (Chimaeriformes: Holocephali)

This study investigated vasodilator mechanisms in the dorsal aorta of the elephant fish, Callorhinchus milii, using anatomical and physiological approaches. Nitric oxide synthase could only be located in the perivascular nerve fibres and not the endothelium of the dorsal aorta, using NADPH histochemistry and immunohistochemistry. In vitro organ bath experiments demonstrated that a NO/soluble guanylyl cyclase (GC) system appeared to be absent in the vascular smooth muscle, since the NO donors SNP (10⁻⁴ mol l⁻¹) and SIN^-1 (10⁻⁵ mol l⁻¹) were without effect. Nicotine (3 × 10⁻⁴ mol l⁻¹) mediated a vasodilation that was not affected by ODQ (10⁻⁵ mol l⁻¹), _l-NNA (10⁻⁴ mol l⁻¹), indomethacin (10⁻⁵ mol l⁻¹), or removal of the endothelium. In contrast, the voltage-gated sodium channel inhibitor, tetrodotoxin (10⁻⁵ mol l⁻¹), significantly decreased the dilation induced by nicotine, suggesting that it contained a neural component. Pre-incubation of the dorsal aorta with the calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP_8–37 (10⁻⁶ mol l^−  1) also caused a significant decrease in the nicotine-induced dilation. We propose that nicotine is mediating a neurally-derived vasodilation in the dorsal aorta that is independent of NO, prostaglandins and the endothelium, and partly mediated by CGRP.

Dual mechanisms for nitric oxide control of large arteries in the estuarine crocodile crocodylus porosus

n reptiles, accumulating evidence suggests that nitric oxide (NO) induces a potent relaxation in the systemic vasculature. However, very few studies have examined the source from which NO is derived. Therefore, the present study used both anatomical and physiological approaches to establish whether NO-mediated vasodilation is via an endothelial or neural NO pathway in the large arteries of the estuarine crocodile Crocodylus porosus. Specific endothelial nitric oxide synthase (NOS) staining was observed in aortic endothelial cells following nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and endothelial NOS immunohistochemistry (IHC), suggesting that an endothelial NO pathway is involved in vascular control. This finding was supported by in vitro organ bath physiology, which demonstrated that the relaxation induced by acetylcholine (10-5 mol l-1) was abolished in the presence of the NOS inhibitor, N-omega-nitro-L-arginine (L-NNA; 10-4 mol l-1), the soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10-5 mol l-1), or when the endothelium was removed. Interestingly, evidence for a neural NO pathway was also identified in large arteries of the crocodile. Neural NOS was located in perivascular nerves of the major blood vessels following NADPH-d histochemistry and neural NOS IHC and in isolated aortic rings, L-NNA and ODQ, but not the removal of the endothelium, abolished the relaxation effect of the neural NOS agonist, nicotine (3x10-4 mol l-1). Thus, we conclude that the large arteries of C. porosus are potentially regulated by NO-derived from both endothelial and neural NOS.

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.

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.

Improved endothelial function following a 14 month resistance exercise training program in adults with type 2 diabetes

Objective
We examined the effect of a 14-month progressive resistance training (PRT) program on endothelial function in both a supervised training (Center) group and non-supervised training (Home) group of patients with type 2 diabetes. We studied 28 men and women with type 2 diabetes who participated in a 14-month PRT involving an initial 2-month supervised program and a 12-month maintenance program.

Methods
Endothelial function testing was performed through laser doppler flow responses in the skin microcirculation to iontophoresis of acetylcholine (ACh) and sodium nitroprusside (NaNP) and doses of 4, 8 and 16 mC were used. Measurements of vascular response (VR), HbA1c, weight and blood pressure were performed at 0, 2 and 14 months.

Results
VR to ACh and NaNP was significantly increased at 14 months compared with baseline in both the Center and Home groups. However, no between-group differences were observed. A significant correlation was observed between HbA1c and VR to ACh at baseline and 8 weeks using 8 mC dose of ACh. There was a strong correlation between HbA1c at baseline and VR at 14 months using all three doses of ACh (4 mC:r = −0.546, p = 0.003, 8 mC:r = −0.470, p = 0.002, 16 mC:r = −0.547, p = 0.006).

Conclusions/interpretation
Endothelial function is improved following 14 months of PRT in type 2 diabetes both in a supervised and non-supervised program. Strong correlations with HbA1c including initial HbA1c levels suggest that glycemic control may be an important factor in long-term regulation of endothelial function.

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.

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.