Kay Hopkinson

FK409 inhibits both local and remote organ damage after intestinal ischaemia

In addition to localized tissue injury, intestinal ischaemia–reperfusion (I/R) leads to remote organ damage, in particular to the lungs. Given that nitric oxide (NO) can attenuate I/R‐induced tissue injury in many situations, this study evaluated the effects of the NO donor, FK409, on leukocyte adhesion in the microcirculation of the intestinal villus and also assessed pulmonary tissue damage after intestinal I/R injury. PVG rats were subjected to 30 min intestinal ischaemia and a sub‐group of animals received the NO donor FK409 (10 mg/kg; i.v.) both 30 min prior to ischaemia and 30 min post‐reperfusion. The intestinal mucosal surface was visualized via an incision made in an exteriorized ileal segment and leukocyte adhesion in the villous microcirculation was determined by in vivo microscopy. Total and differential leukocyte counts from peripheral blood were evaluated. Lungs were removed at the end for histological assessment. Six out of ten untreated I/R animals failed to survive the 2 h reperfusion period, whereas all ten FK409‐treated animals survived. I/R induced a significant increase in villous leukocyte adhesion of untreated I/R animals (p<0.001) and this was significantly decreased by FK409 treatment (p<0.001). The total leukocyte count was significantly decreased in untreated I/R animals (p<0.001) and this primarily resulted from a reduction in circulating neutrophil numbers. This effect was not observed in FK409‐treated animals. Collapsed alveoli, thickened interstitial walls, and a dense neutrophilic infiltrate were apparent in the lungs of untreated I/R animals, whereas lung histology was normal in FK409‐treated animals. In conclusion, FK409 prevented mortality, significantly reduced villous leukocyte adhesion, maintained circulating leukocyte numbers, and prevented pulmonary tissue injury following intestinal I/R. FK409 may therefore be of value in reducing both local and remote tissue damage and improving outcome in situations where intestinal I/R injury is obligatory, such as small bowel transplantation. Copyright

Effect of Upper- and Lower-limb Exercise Training on Circulating Soluble Adhesion Molecules, hs-CRP and Stress Proteins in Patients with Intermittent Claudication

OBJECTIVES To investigate the effects of exercise training on levels of circulating biomarkers associated with the progression of atherosclerosis and risk of cardiovascular events in patients with intermittent claudication. METHODS Circulating levels of soluble adhesion molecules (sVCAM-1, sICAM-1, sE-selectin), high sensitivity C-reactive protein (hs-CRP) and stress proteins (Hsp60 and Hsp70) in patients randomised to a 24-week programme of arm- or leg-cranking exercise were compared with those in usual care controls. RESULTS Arm and leg exercise similarly improved lower-limb aerobic exercise capacity (20% vs 19%, respectively; P<0.001) and maximum walking distance (30% vs 35%, respectively; P<0.001). Improvements in training limb-specific peak oxygen consumption were attenuated for patients in the highest vs lowest quartile for circulating sVCAM-1 levels at baseline (3% vs 25% respectively, P<0.001). Although circulating hs-CRP levels tended to be lower in the arm-cranking group (-1.55 [95% CI: -1.06 to -2.26]mgl(-1)), exercise training had no effect on circulating levels of soluble adhesion molecules or stress proteins. CONCLUSIONS These findings suggest that high levels of circulating sVCAM-1 are associated with an attenuated exercise training response and that arm-cranking exercise may provide an effective stimulus for evoking systemic anti-inflammatory adaptations in patients with intermittent claudication.

Serum osteoprotegerin is increased and predicts survival in idiopathic pulmonary arterial hypertension

We previously reported that osteoprotegerin (OPG) is regulated by pathways associated with pulmonary arterial hypertension (PAH), and is present at elevated levels within pulmonary vascular lesions and sera from patients with idiopathic PAH (IPAH). Since OPG is a naturally secreted protein, we investigated the relationship between serum OPG and disease severity and outcome in patients with IPAH and animal models. OPG mRNA expression was measured in pulmonary artery smooth muscle cells (PASMC) from pulmonary arteries of patients with and without IPAH. Serum concentrations of OPG were measured in a retrospective and prospective group of patients. OPG levels were compared with phenotypic data and other putative PAH biomarkers. Prognostic significance was assessed and levels compared with healthy controls. Correlation of OPG and pulmonary vascular remodeling was also performed in rodent models of PAH. OPG mRNA was significantly increased 2-fold in PASMC isolated from explanted PAH lungs compared with control. Serum OPG concentrations were markedly elevated in IPAH compared with controls. In Cohort 1 OPG levels significantly correlated with mean right atrial pressure and cardiac index, while in Cohort 2 significant correlations existed between age-adjusted OPG levels and gas transfer. In both cohorts an OPG concentration above a ROC-derived threshold of 4728 pg/ml predicted poorer survival. In two rodent models, OPG correlated with the degree of pulmonary vascular remodeling. OPG levels are significantly elevated in patients with idiopathic PAH and are of prognostic significance. The role of OPG as a potential biomarker and therapeutic target merits further investigation.

Administration of the stress protein gp96 prolongs rat cardiac allograft survival, modifies rejection-associated inflammatory events, and induces a state of peripheral T-cell hyporesponsiveness

Abstract High-dose gp96 has been shown to inhibit experimental autoimmune disease by a mechanism that appears to involve immunoregulatory CD4+ T cells. This study tested the hypothesis that high-dose gp96 administration modifies allograft rejection and associated inflammatory events. Wistar cardiac allografts were transplanted into Lewis recipient rats and graft function was monitored daily by palpation. Intradermal administration of gp96 purified from Wistar rat livers (100 μg) at the time of transplantation and 3 days later significantly prolonged allograft survival (14 vs 8 days in phosphate-buffered saline [PBS]-treated recipients; P = 0.009). Rejected allografts from gp96-treated animals were significantly less enlarged than allografts from their PBS-treated counterparts (2.8 vs 4.3 g; P < 0.004). Gp96 was also effective when administered on days 1 and 8 (13 vs 7 days), but not if it was derived from recipient (Lewis) liver tissue or administered on days 0, 3, and 6. In parallel studies, CD3+ T cells from gp96-treated untransplanted animals secreted less interleukin (IL)-4, IL-10, and interferon (IFN)-γ after in vitro polyclonal stimulation than CD3+ T cells from PBS-treated animals. Gp96 administration might therefore influence the induction of immunity to coencountered antigenic challenges and inflammatory events by inducing what appears to be a state of peripheral T-cell hyporesponsiveness.

The stress protein gp96 is not an activator of resting rat bone marrow–derived dendritic cells, but is a costimulator and activator of CD3+ T cells

Abstract Although low doses of tumor-derived stress protein gp96 elicit protective immunity to the tumor from which it is isolated, protection is lost at high doses because of the induction of immunoregulatory CD4+ T cells. This study evaluated the influence of gp96 on resting rat bone marrow–derived dendritic cells (BMDCs) and purified CD3+ T cells. In contrast to previous reports, gp96 had no effect on adhesion and costimulatory molecule expression by BMDCs, nor did it influence interleukin (IL)-10 and IL-12 secretion or their allostimulatory capacity. Gp96 did not bind to BMDCs but dose-dependently bound to CD4+ and CD8+ T cells. At low concentrations (1 and 25 μg/mL), gp96 acted as a costimulator of CD3+ T cells, inducing proliferation and the secretion of interferon (IFN)-γ and IL-10. Gp96 also increased the proliferation of CD28-costimulated CD3+ T cells and their secretion of IFN-γ, IL-4, and IL-10. Gp96 had no effect at higher concentrations (50 and 100 μg/mL), despite the occurrence of cell surface binding at these concentrations. These findings indicate that gp96 can act as a costimulatory molecule for CD3+ T cells, and an observed increase in the IL-10:IFN-γ secretion ratio induced by gp96 suggests that it might, at appropriate concentrations, promote a regulatory T-helper 2 (Th2)–like phenotype.

Stress responses in graft and native intestine after rat heterotopic small bowel transplantation.

BACKGROUND Cardiac transplantation has been shown to induce heat shock protein expression, and reactivity to these stress proteins has been implicated in acute and chronic allograft rejection. This study assessed Hsp60 and Hsp70 expression in graft and native small intestine after rat small bowel transplantation. METHODS Heterotopic small bowel transplantation was performed between PVG donor and DA recipient rats, a subgroup of which received tacrolimus immunosuppression (1 mg x kg(-1) x day(-1)). Untransplanted and isografted (PVG-->PVG) animals served as controls. Paraffin sections of graft and native intestine on day 5 after transplantation were stained by immunohistochemistry, and heat shock protein expression was graded blindly by three observers. RESULTS Villus epithelial cell expression of Hsp60, but not Hsp70, was increased in allografts. The induction of Hsp60 in the villus epithelium was not controlled by tacrolimus. Hsp60 and Hsp70 expression was induced in the lamina propria of isografts and allografts. This response was more pronounced in allografts and was significantly reduced, but not totally abrogated, by tacrolimus. Interestingly, heat shock protein expression was also induced in the native intestine lamina propria and epithelium of allograft recipients, suggesting the induction of stress responses at sites other than the transplanted organ. CONCLUSIONS Small bowel transplantation induces a stress response in both the graft and native intestine. The early and prolonged expression of these proteins may influence the induction of anti-heat shock protein reactivity and have an adverse effect on graft outcome after small bowel transplantation.

Improvement in nutritional status reduces the clinical impact of infections in older adults

To determine the effect of a dietary intervention and micronutrient supplementation on self‐reported infections in older adults.

Differential IL-1 signaling induced by BMPR2 deficiency drives pulmonary vascular remodeling.

Bone morphogenetic protein receptor type 2 (BMPR2) mutations are present in patients with heritable and idiopathic pulmonary arterial hypertension (PAH). Circulating levels of interleukin-1 (IL-1) are raised in patients and animal models. Whether interplay between BMP and IL-1 signaling can explain the local manifestation of PAH in the lung remains unclear. Cell culture, siRNA, and mRNA microarray analysis of RNA isolated from human pulmonary artery (PASMC) and aortic (AoSMC) smooth muscle cells were used. R899X+/– BMPR2 transgenic mice fed a Western diet for six weeks were given daily injections of IL-1ß prior to assessment for PAH and tissue collection. PASMC have reduced inflammatory activation in response to IL-1ß compared with AoSMCs; however, PASMC with reduced BMPR2 demonstrated an exaggerated response. Mice treated with IL-1ß had higher white blood cell counts and significantly raised serum protein levels of IL-6 and osteoprotegerin (OPG) plasma levels recapitulating in vitro data. Phenotypically, IL-1ß treated mice demonstrated increased pulmonary vascular remodeling. IL-1ß induces an exaggerated pulmonary artery specific transcriptomic inflammatory response when BMPR2 signaling is reduced.

S85 Reduction of CD68 macrophages causes gender specific spontaneous pulmonary arterial hypertension in mice

Introduction Macrophages are proposed to play an important regulatory role in the pathogenesis of pulmonary arterial hypertension (PAH) as excessive infiltration detected around vascular lesions in patients and animal models. The exact ‘causal’ role for macrophages, and whether their presence or absence is required for the vascular remodelling seen in PAH remains unclear. Objectives Using a novel inducible macrophage depletion model (MacLow mouse) we aimed to determine the role of macrophages in pulmonary arterial remodelling associated with PAH. Methods Macrophage depletion was induced in MacLow mice by administration of doxycycline, where macrophage-specific induction of the cytotoxic diphtheria toxin A chain (DTA) is driven by the CD68 promoter. Mice were phenotyped for PAH by echocardiography, closed chest cardiac catheterization and immunohistochemistry (IHC) after 6 weeks. To investigate the origin of the effector cells, male chimeric mice were generated, and the disease stimulated by inducing macrophage ablation with doxycycline. Furthermore, to study gender-specificity of the disease phenotype, MacLow mixed gender chimeric mice were produced, and macrophage ablation induced as previous. Results Interestingly male but not female MacLow mice developed a PAH phenotype compared to controls (RVSP of 66.1 mmHg vs 24.5 mmHg, p < 0.0001, n = 5–8), associated with increased right ventricular Hypertrophy (RVH 0.264 vs 0.226, p < 0.001, n = 8) and pulmonary vascular remodelling. IHC analysis of diseased lungs demonstrated increased iNOS- |CD206+ |F4/80+ macrophages suggesting a M2-like macrophage population drive the PAH phenotype in these mice. The bone marrow transplant studies shows that bone marrow (BM) derived cells contribute in the development of the disease phenotype as wild type BM cells attenuate disease progression. Moreover, female BM transplanted into male mice alleviate but does not protect them from developing PAH. Conclusion Development of PAH in male MacLow mice suggests that macrophages play a causal role in pulmonary vascular remodelling. Results suggest that the phenotype is driven by lung resident M2- like macrophages with a contribution from bone marrow derived cells. A study to examine the probable protective effect of Oestrogen is now underway to further investigate the implication of gender difference in the incidence of PAH in this model.

P5 Pulmonary hypertension in a mouse model with reduced macrophage number (MacLow)

Introduction and Objectives Pulmonary arterial hypertension (PAH) is a devastating condition with high morbidity and poor life expectancy. Pathologically PAH is characterised by the medial thickening of the small distal pulmonary arteries. Early endothelial cell (EC) dysfunction and apoptosis, and the subsequent abnormal proliferation and migration of pulmonary artery smooth muscle cells are thought to be a major contributing factor. Macrophages are proposed to play an important role in regulating these processes and are recruited to remodelled pulmonary arteries but the exact role of macrophages, and whether they are required for this remodelling remains unclear. We have recently developed a mouse model (MacLow) where approximately 50% of macrophages are depleted and now aim to investigate whether MacLow mice would demonstrate a reduced pulmonary hypertension phenotype in response to hypoxia, when compared to non-macrophage ablated littermates. Methods Macrophage ablation was induced in CD68-rtTA-eGFP/tetDTA double transgenic mice (MacLow) where macrophage-specific (CD68) induction of the cytotoxic diphtheria toxin A chain (DTA) is achieved by administration of doxycycline containing chow diet (doxy-chow). Mice were divided by sex and then fed either regular or doxy-chow for 2 weeks prior to either 2 weeks exposure to hypoxia (10% oxygen), or room air. All mice were phenotyped for PH by echocardiography followed by closed chest cardiac catheterisation. Heart and lung tissue were harvested for morphological, immunohistochemical and biochemical analyses. Results Doxy-chow fed mice displayed the expected 50% reduction in macrophages (liver) compared to controls. MacLow mice with the induced ablation of macrophages were not protected from hypoxia induced pulmonary hypertension although females displayed a trend for higher RVSP after hypoxia (34 mm Hg vs 29 mm Hg). Interestingly male MacLow mice with induced macrophage ablation displayed a spontaneous PAH phenotype (33 mm Hg), in normoxia, that was not further increased by hypoxia. The changes in RVSP were accompanied by appropriate changes in RVH. Conclusion These data suggest that macrophages play a modulating role in pulmonary vascular remodelling but further work is required to explore the mechanisms involved in this phenotype, and to fully assess the change in macrophage number within the lungs of these mice.