Background aims. Human bone marrowederived mesenchymal stromal cells (MSC) can suppress inflammat... more Background aims. Human bone marrowederived mesenchymal stromal cells (MSC) can suppress inflammation; therefore their therapeutic potential is being explored in clinical trials. Poor engraftment of infused MSC limits their therapeutic utility; this may be caused by MSC processing before infusion, in particular the method of their detachment from culture. Methods. Enzymatic methods of detaching MSC (Accutase and TrypLE) were compared with non-enzymatic methods (Cell Dissociation Buffer [CDB], ethylenediamine tetra-acetic acid and scraping) for their effect on MSC viability, chemokine receptor expression, multi-potency, immunomodulation and chemokine-dependent migration. Results. TrypLE detachment preserved MSC viability and tri-lineage potential compared with non-enzymatic methods; however, this resulted in near complete loss of surface chemokine receptor expression. Of the non-enzymatic methods, CDB detachment preserved the highest viability while retaining significant tri-lineage differentiation potential. Once re-plated, CDB-detached MSC regained their original morphology and reached confluence, unlike with the use of other non-enzymatic methods. Viability was significantly reduced with the use of ethylenediamine tetra-acetic acid and further reduced with the use of cell scraping. Addition of 1% serum during CDB detachment led to higher MSC numbers entering autophagy and increased MSC recovery after re-plating. TrypLE and CDB-detached MSC suppressed CD3 þ CD4 þ CD25 À T-cell proliferation, although TrypLE-detached MSC exhibited superior suppression at 1:20 ratio. CDB detachment retained surface chemokine receptor expression and consequently increased migration to CCL22, CXCL12 and CCL4, in contrast with TrypLE-detached MSC. Conclusions. This study demonstrates that non-enzymatic detachment of MSC with the use of CDB minimizes the negative impact on cell viability, multipotency and immunomodulation while retaining chemokine-dependent migration, which may be of importance in MSC delivery and engraftment in sites of injury.
The limited availability of organs for liver transplantation, the ultimate curative treatment for... more The limited availability of organs for liver transplantation, the ultimate curative treatment for end stage liver disease, has resulted in a growing and unmet need for alternative therapies. Mesenchymal stromal cells (MSCs) with their broad ranging anti-inflammatory and immunomodulatory properties have therefore emerged as a promising therapeutic agent in treating inflammatory liver disease. Significant strides have been made in exploring their biological activity. Clinical application of MSC has shifted the paradigm from using their regenerative potential to one which harnesses their immunomodulatory properties. Reassuringly, MSCs have been extensively investigated for over 30 years with encouraging efficacy and safety data from translational and early phase clinical studies, but questions remain about their utility. Therefore, in this review, we examine the translational and clinical studies using MSCs in various liver diseases and their impact on dampening immune-mediated liver d...
Introduction: Nrf2 transcription factor is a master regulator mediating adaptive response to cell... more Introduction: Nrf2 transcription factor is a master regulator mediating adaptive response to cellular stress. A number of genes coding for stressresponsive proteins, detoxifying and ROS scavenging enzymes are directly regulated by Nrf2 through antioxidant response element located in their promoters. Our data show that Nrf2 deficient endothelial cells (ECs) are highly dysfunctional, but intriguingly still maintain oxidative balance. We aimed to find the molecular reason of their impairment. Methods: Primary human endothelial cells transfected with siNrf2 and aortas isolated from Nrf2 KO mice, together with appropriate controls were used. Results: SDF-1-induced angiogenic response of ECs is blocked in the absence of Nrf2. This effect is evidenced, however, to be independent of direct Nrf2 transcriptional activity, what is in line with poor phosphorylation, lack of acetylation and delayed nuclear translocation of Nrf2 driven by SDF-1. We observed that Nrf2 deficient ECs present p53-dependent premature senescence, accompanied by potent S-nitrosylation of proteins, occurring concomitantly with increased activation of eNOS and suppressed expression of denitrosylating enzymes. Inhibition of p53 rescues ECs from cellular senescence, even so does not reverse detrimental effect of Nrf2 deficiency on EC function. On the other hand, lack of Nrf2 affects expression of cytoskeleton-regulating proteins, what contributes to impairment of EC migration and angiogenesis. Conclusions: A role for Nrf2 in angiogenic response of ECs is linked to its involvement in regulation of proteins responsible for actin cytoskeleton rearrangements. We suppose that S-nitrosylation of proteins in Nrf2 deficient ECs may protect them from oxidative stress, but leads to premature senescence.
Lymphocytes are the key effector cells of the adaptive immune response but are also implicated in... more Lymphocytes are the key effector cells of the adaptive immune response but are also implicated in the development of chronic inflammatory diseases such as type 2 diabetes, cardiovascular disease, inflammatory bowel disease, and allergy. In order to understand the mechanisms responsible for the global increase in such debilitating conditions it is vital to appreciate the complexity of immune cell trafficking into tissue under normal and inflamed conditions. In this chapter we introduce the mechanisms of immune cell recruitment into tissue and highlight how these processes have been targeted by new therapeutic entities based on blocking integrin or chemokine function.
BACKGROUND Non-alcoholic fatty liver disease (NAFLD) is associated with obesity, insulin resistan... more BACKGROUND Non-alcoholic fatty liver disease (NAFLD) is associated with obesity, insulin resistance and dyslipidaemia and currently is estimated to affect up to a third of all individuals in developed countries. Current standard of care for patients varies according to disease stage, but includes lifestyle interventions common insulin sensitizers, antioxidants and lipid modifiers. However, to date specific therapies have shown little histological or fibrosis stage improvement in large clinical trials, and there is still no licensed therapy for NAFLD. Given the high prevalence, limited treatment options and significant screening costs for the general population, new treatments are urgently required. AIM To assess the potential for inhibition of the amine oxidase enzyme vascular adhesion protein-1 (VAP-1) to modify hepatic lipid accumulation in NAFLD. METHODS We have used immunochemical and qPCR analysis to document expression of VAP-1 and key functional proteins and transporters across the NAFLD spectrum. We then utilised hepatocytes in culture and human precision cut liver slices in concert with selective enzyme activity inhibitors to test the effects of activating the semicarbazide-sensitive amine oxidase activity of VAP-1 on hepatic lipid uptake and triglyceride export. A murine model of NAFLD was also used to determine the consequences of VAP-1 knockout and gene expression arrays were used to quantify the effects of VAP-1 activity on key lipid modifying and proinflammatory gene expression. RESULTS We confirmed that increasing severity of NAFLD and progression to cirrhosis was associated with a significant increase in hepatocellular VAP-1 expression. Hepatocytes in vitro exposed to recombinant VAP-1 and its substrate methylamine showed increased lipid accumulation as determined by quantification of Oil Red O uptake. This was recapitulated using hydrogen peroxide, and lipid accumulation was accompanied by changes in expression of the lipid transporter molecules FABP3, FATP6, insulin receptor subunits and PPARα. Human liver tissue exposed to recombinant VAP-1 or substrates for endo/exogenous VAP-1 produced less triglyceride than untreated tissue and demonstrated an increase in steatosis. This response could be inhibited by using bromoethylamine to inhibit the SSAO activity of VAP-1, and mice deficient in VAP-1/AOC3 also demonstrated reduced steatosis on high fat diet. Exposure of human liver tissue to methylamine to activate VAP-1 resulted in increased expression of FABP2 and 4, FATP3-5, caveolin-1, VLDLR, PPARGC1 and genes associated with the inflammatory response. CONCLUSION Our data confirm that the elevations in hepatic VAP-1 expression reported in nonalcoholic steatohepatitis can contribute to steatosis, metabolic disturbance and inflammation. This suggests that targeting the semicarbazide sensitive amine oxidase capacity of VAP-1 may represent a useful adjunct to other therapeutic strategies in NAFLD.
HepG2 cells continue to be a valuable tool in early drug discovery and pharmaceutical development... more HepG2 cells continue to be a valuable tool in early drug discovery and pharmaceutical development. In the current study we develop a 3D in vitro liver model, using HepG2/C3A cells that it is predictive of human genotoxic exposure. HepG2/C3A cells cultured for 7-days in agarose-coated microplates formed spheroids which were uniform in shape and had well defined outer perimeters and no evidence of a hypoxic core. Quantitative real-time-PCR analysis showed statistically significant transcriptional upregulation of xenobiotic metabolising genes (CYP1A1, CYP1A2, UG1A1, UGT1A3, UGT1A6, EPHX, NAT2) and genes linked to liver function (ALB, CAR) in 3D cultures. In response to three model pro-genotoxicants: benzo[a]pyrene, Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-aminoanthracene (2-AA),we observed further transcriptional upregulation of xenobiotic metabolising genes (CYP1A1, CYP1A2, NAT1/2, SULT1A2, UGT1A1, UGT1A3) compared to untreated spheroids. Consistent with this, spheroids were more sensitive than 2D monolayers to compound induced single- and double- stranded DNA damage as assessed by the comet assay and γH2AX phosphorylation respectively. In contrast, levels of DNA damage induced by the direct acting mutagen 4-nitroquinolin N-oxide (4NQO) was the same in spheroids and monolayers. In support of the enhanced genotoxic response in spheroids we also observed transcriptional upregulation of genes relating to DNA-damage and cellular stress response (e.g. GADD45A and CDKN1A) in spheroids. In conclusion, HepG2/C3A 3D spheroids are a sensitive model for in vitro genotoxicity assessment with potential applications in early stage drug development.
Methods in molecular biology (Clifton, N.J.), 2017
The liver is the largest internal organ and filters around 3 pints of blood per minute. This cont... more The liver is the largest internal organ and filters around 3 pints of blood per minute. This continuous flux of blood should not be confused with rapid egress of lymphocytes through the liver; this organ has intricate corridors of specialized sinusoidal spaces, ensuring that immune cells decelerate to shear flow rates, and providing ample opportunities to interact with parenchymal cells. Migration has been intricately linked to T cell function; it is therefore important to study liver T cell biology into context within the liver microenvironment. Here we discuss the highly organized architecture of liver-resident cells (sinusoidal endothelia, Kupffer cells, stellate cells/myofibroblasts, and biliary and hepatic epithelia) and showcase basic, multicellular, and complex systems to model T cell migration through the human liver microenvironment in vitro and ex vivo.
Lymphocyte recruitment to different organs, and even alternate anatomical regions within the same... more Lymphocyte recruitment to different organs, and even alternate anatomical regions within the same organ, is differentially regulated. Key combinations of adhesion molecules and chemokines govern compartmentalization, and these can change depending upon the nature and duration of tissue injury. We are interested in understanding lymphocyte recruitment to the liver during human disease, and thus need models of the liver inflammatory milieu that are as representative as possible. Here we describe the use of precision cut liver slices as models of disease.
European Journal of Gastroenterology & Hepatology, 2001
Despite intensive medical treatment with steroids and immunosuppressants, acute colitis is still ... more Despite intensive medical treatment with steroids and immunosuppressants, acute colitis is still associated with a colectomy rate of up to 15%. Following the observation that a patient with severe steroid-resistant colitis went into remission when treated with heparin for a deep vein thrombosis, there have been a number of reports on the use of heparin in acute ulcerative colitis. Although small and uncontrolled, these studies consistently demonstrate the beneficial effects of heparin, with surprisingly few side-effects in a disease characterized by mucosal haemorrhage. The mechanisms by which heparin may ameliorate ulcerative colitis remain unclear. A simple anticoagulant effect may be responsible, but similar effects are not seen with warfarin. As a result of their intense negative charge, the glycosaminoglycans that constitute heparin have diverse biological effects. These include potent anti-inflammatory actions, in vitro and in vivo, and the potentiation of the activity of the peptide growth factors necessary for mucosal regeneration and repair. This review summarizes the clinical reports on heparin treatment for ulcerative colitis and explores the mechanisms by which this novel form of treatment may exert its effects.
In order to understand human inflammatory diseases and to develop and assess new therapeutic stra... more In order to understand human inflammatory diseases and to develop and assess new therapeutic strategies targeting leukocyte recruitment to tissue, it is necessary to study human lymphocyte interactions with endothelium. It is often not practical to carry out assays on fresh human samples and therefore cells may be cryopreserved and batched for later study. Furthermore, many forms of adoptive cell therapy use cryopreserved cells that are required to migrate to tissue after infusion in vivo. The consequences of cryopreservation on the adhesion and migration of leukocytes is not known leading us to study the effects of cryopreservation on lymphocyte phenotype, migration, and adhesion. Cryopreservation and subsequent thawing did not alter the proportion of retrieved T cell subsets. Overall levels of expression of β1 or β2 integrins were unaffected but marked changes were observed in other relevant receptors. Expression of CD69, a transmembrane protein that plays a critical role in lymphocyte egress from tissues and the chemokine receptor CXCR4, increased on thawed populations and levels of CD62L and CXCR3 were reduced on thawed cells but restored if cells were allowed to recover after thawing. These changes were associated with modulation of the ability of lymphocytes to migrate across cytokine-stimulated monolayers of endothelium toward recombinant CXCL11 and CXCL12. Thus cryopreservation and thawing of lymphocytes induces changes in their adhesive phenotype and modulates their ability to migrate across endothelial monolayers. These findings have implications for in vitro experimentation and for cell therapy in which cryopreserved cells are expected to migrate when reinfused into patients.
s / Atherosclerosis 232 (2014) e1–e9 e4 LYMPHOTOXIN-b RECEPTOR SIGNALLING LINKS STEATOHEPATITIS, ... more s / Atherosclerosis 232 (2014) e1–e9 e4 LYMPHOTOXIN-b RECEPTOR SIGNALLING LINKS STEATOHEPATITIS, HYPER-LIPIDEMIA AND ATHEROSCLEROSIS B. Apta*, M.J. Harrison, C. Benezech, S. Karim, C.F. Ware, P.F. Lalor, J.H. Caamano, G.E. Rainger. MRC Centre for Immune Regulation, University of Birmingham, Birmingham, UK * Presenting author Metabolic syndrome describes a variety of risk factors associated with the development of cardiovascular disease (CVD). Non-alcoholic fatty liver disease (NAFLD) is considered to be the hepatic manifestation of the metabolic syndrome and is strongly associated with hyper-lipidemia. Lymphotoxin-b receptor (LTbR) is a receptor from the tumour necrosis factor (TNF) family and is expressed primarily on epithelial cells and cells of the myeloid lineage, LTbR is involved in hepatic lipid storage and export, and plasma levels of the receptor have been implicated as a risk factor for CVD. We aimed to characterise the role of LTbR in steatosis, hyper-lipidemia and atherosclerosis using wild-type, LTBR-/-, ApoE -/and LTBR-/-/ ApoE -/double knockout mice on a 13-week high fat diet (HFD) as established models of NAFLD and atherosclerosis, as well as murine and human hepatic tissue and hepatocyte cell culture. Serum lipid analysis and liver/aorta histology was performed according to standard methodologies, with quantification of lipid accumulation by Oil red O staining. Hepatocyte triglyceride storage and output was measured using staining and standard quantification kits. Loss of LTbR signalling in the ApoE-/strain substantially reduced steatosis, circulating triglyceride levels and atherosclerotic plaque burden compared to ApoE-/controls when fed a HFD for 13 weeks. LTbR signalling in hepatocytes enhanced de novo lipogenesis, lipid storage and triglyceride output. Thus, LTbR signalling links fatty liver disease and hyper-lipidemia, two prominent risk factors of CVD which exacerbate atherosclerosis through the disruption of lipid homeostasis. http://dx.doi.org/10.1016/j.atherosclerosis.2013.11.008 THE FORMATION OF MONOCYTE-PLATELET AGGREGATES IN STIRRED WHOLE BLOOD IN RESPONSE TO DIFFERENT PLATELET AGONISTS C.L. Box*, C. Yates, M. Chimen, M. Harrison, G. Nash, S. Watson, G.E. Rainger. School of Clinical and Experimental Medicine, University of Birmingham, B15 2TT, UK * Presenting author Introduction: Using in vitro and in vivo models of vascular inflammation resembling aspects of the chronic inflammatory environment, we have recently described a paradigm where platelets recruited to intact endothelial monolayers form adhesive bridges between the vessel wall where they preferentially aid monocyte recruitment. It is well documented that monocyte-platelet aggregates form in circulating blood with increased incidence in patients with cardiovascular and chronic inflammatory disease. It is unknown whether heterotypic aggregation increases efficiency of monocyte recruitment during vascular inflammation, which we will investigate in the near future. The aim of the current study was to determine how efficiently monocyte-platelet aggregates could form in whole blood in response to different platelet agonists. Methods: Whole blood was treated with the platelet agonists adenosine diphosphate (30mM), thrombin receptor activating peptide (100mM), thromboxane mimetic, U46619 (10mM), rhodocytin, (100nM) or collagen related peptide (1mg/ml) and gently mixed at 37 C for 30mins. Heterotypic aggregation was assessed by flow cytometry using antibodies against monocyte markers CD14 and CD16 and against platelet CD42b. Results: After treatment with a platelet agonist there was a significant increase in heterotypic aggregate formation compared to untreated control for both CD14+CD16and CD14+CD16+monocyte subsets, the incidence of aggregate formation did not vary between the subsets. Conclusions: Monocyte-platelet aggregates form in whole blood in response to a range of platelet agonists, although the efficiency of aggregation varies in an agonist dependent manner. Both monocyte subsets form heterotypic aggregates with equal efficiency. http://dx.doi.org/10.1016/j.atherosclerosis.2013.11.009 HEME AND METFORMIN COORDINATE HUMAN AND MURINE MACROPHAGE HEME OXYGENASE 1 EXPRESSION WITH FOAM CELL RESISTANCE PARTLY VIA ADENOSINE MONOPHOSPHATE KINASE AND ACTIVATING TRANSCRIPTION FACTOR 1 (AMPK-ATF1) X. Wan , Y. Huo , M. Johns , E. Piper , J.C. Mason , D. Carling , D.O. Haskard , J.J. Boyle *. Vascular Sciences, NHLI, Imperial College London, UK; MRC CSC, Imperial College London, UK * Presenting author Rationale: Intraplaque hemorrhage (IPH) is a key driver of the progression of atherosclerotic plaques. We recently characterised Mhem as a novel macrophage phenotype that limits the atherogenicity of IPH. Mhem are directed by activating transcription factor 1 (ATF1), which is activated by phosphorylation. A better understanding of the counter-atherogenic ATF1Mhem pathway may facilitate anti-atherosclerotic therapies. Methods: We…
Background aims. Human bone marrowederived mesenchymal stromal cells (MSC) can suppress inflammat... more Background aims. Human bone marrowederived mesenchymal stromal cells (MSC) can suppress inflammation; therefore their therapeutic potential is being explored in clinical trials. Poor engraftment of infused MSC limits their therapeutic utility; this may be caused by MSC processing before infusion, in particular the method of their detachment from culture. Methods. Enzymatic methods of detaching MSC (Accutase and TrypLE) were compared with non-enzymatic methods (Cell Dissociation Buffer [CDB], ethylenediamine tetra-acetic acid and scraping) for their effect on MSC viability, chemokine receptor expression, multi-potency, immunomodulation and chemokine-dependent migration. Results. TrypLE detachment preserved MSC viability and tri-lineage potential compared with non-enzymatic methods; however, this resulted in near complete loss of surface chemokine receptor expression. Of the non-enzymatic methods, CDB detachment preserved the highest viability while retaining significant tri-lineage differentiation potential. Once re-plated, CDB-detached MSC regained their original morphology and reached confluence, unlike with the use of other non-enzymatic methods. Viability was significantly reduced with the use of ethylenediamine tetra-acetic acid and further reduced with the use of cell scraping. Addition of 1% serum during CDB detachment led to higher MSC numbers entering autophagy and increased MSC recovery after re-plating. TrypLE and CDB-detached MSC suppressed CD3 þ CD4 þ CD25 À T-cell proliferation, although TrypLE-detached MSC exhibited superior suppression at 1:20 ratio. CDB detachment retained surface chemokine receptor expression and consequently increased migration to CCL22, CXCL12 and CCL4, in contrast with TrypLE-detached MSC. Conclusions. This study demonstrates that non-enzymatic detachment of MSC with the use of CDB minimizes the negative impact on cell viability, multipotency and immunomodulation while retaining chemokine-dependent migration, which may be of importance in MSC delivery and engraftment in sites of injury.
The limited availability of organs for liver transplantation, the ultimate curative treatment for... more The limited availability of organs for liver transplantation, the ultimate curative treatment for end stage liver disease, has resulted in a growing and unmet need for alternative therapies. Mesenchymal stromal cells (MSCs) with their broad ranging anti-inflammatory and immunomodulatory properties have therefore emerged as a promising therapeutic agent in treating inflammatory liver disease. Significant strides have been made in exploring their biological activity. Clinical application of MSC has shifted the paradigm from using their regenerative potential to one which harnesses their immunomodulatory properties. Reassuringly, MSCs have been extensively investigated for over 30 years with encouraging efficacy and safety data from translational and early phase clinical studies, but questions remain about their utility. Therefore, in this review, we examine the translational and clinical studies using MSCs in various liver diseases and their impact on dampening immune-mediated liver d...
Introduction: Nrf2 transcription factor is a master regulator mediating adaptive response to cell... more Introduction: Nrf2 transcription factor is a master regulator mediating adaptive response to cellular stress. A number of genes coding for stressresponsive proteins, detoxifying and ROS scavenging enzymes are directly regulated by Nrf2 through antioxidant response element located in their promoters. Our data show that Nrf2 deficient endothelial cells (ECs) are highly dysfunctional, but intriguingly still maintain oxidative balance. We aimed to find the molecular reason of their impairment. Methods: Primary human endothelial cells transfected with siNrf2 and aortas isolated from Nrf2 KO mice, together with appropriate controls were used. Results: SDF-1-induced angiogenic response of ECs is blocked in the absence of Nrf2. This effect is evidenced, however, to be independent of direct Nrf2 transcriptional activity, what is in line with poor phosphorylation, lack of acetylation and delayed nuclear translocation of Nrf2 driven by SDF-1. We observed that Nrf2 deficient ECs present p53-dependent premature senescence, accompanied by potent S-nitrosylation of proteins, occurring concomitantly with increased activation of eNOS and suppressed expression of denitrosylating enzymes. Inhibition of p53 rescues ECs from cellular senescence, even so does not reverse detrimental effect of Nrf2 deficiency on EC function. On the other hand, lack of Nrf2 affects expression of cytoskeleton-regulating proteins, what contributes to impairment of EC migration and angiogenesis. Conclusions: A role for Nrf2 in angiogenic response of ECs is linked to its involvement in regulation of proteins responsible for actin cytoskeleton rearrangements. We suppose that S-nitrosylation of proteins in Nrf2 deficient ECs may protect them from oxidative stress, but leads to premature senescence.
Lymphocytes are the key effector cells of the adaptive immune response but are also implicated in... more Lymphocytes are the key effector cells of the adaptive immune response but are also implicated in the development of chronic inflammatory diseases such as type 2 diabetes, cardiovascular disease, inflammatory bowel disease, and allergy. In order to understand the mechanisms responsible for the global increase in such debilitating conditions it is vital to appreciate the complexity of immune cell trafficking into tissue under normal and inflamed conditions. In this chapter we introduce the mechanisms of immune cell recruitment into tissue and highlight how these processes have been targeted by new therapeutic entities based on blocking integrin or chemokine function.
BACKGROUND Non-alcoholic fatty liver disease (NAFLD) is associated with obesity, insulin resistan... more BACKGROUND Non-alcoholic fatty liver disease (NAFLD) is associated with obesity, insulin resistance and dyslipidaemia and currently is estimated to affect up to a third of all individuals in developed countries. Current standard of care for patients varies according to disease stage, but includes lifestyle interventions common insulin sensitizers, antioxidants and lipid modifiers. However, to date specific therapies have shown little histological or fibrosis stage improvement in large clinical trials, and there is still no licensed therapy for NAFLD. Given the high prevalence, limited treatment options and significant screening costs for the general population, new treatments are urgently required. AIM To assess the potential for inhibition of the amine oxidase enzyme vascular adhesion protein-1 (VAP-1) to modify hepatic lipid accumulation in NAFLD. METHODS We have used immunochemical and qPCR analysis to document expression of VAP-1 and key functional proteins and transporters across the NAFLD spectrum. We then utilised hepatocytes in culture and human precision cut liver slices in concert with selective enzyme activity inhibitors to test the effects of activating the semicarbazide-sensitive amine oxidase activity of VAP-1 on hepatic lipid uptake and triglyceride export. A murine model of NAFLD was also used to determine the consequences of VAP-1 knockout and gene expression arrays were used to quantify the effects of VAP-1 activity on key lipid modifying and proinflammatory gene expression. RESULTS We confirmed that increasing severity of NAFLD and progression to cirrhosis was associated with a significant increase in hepatocellular VAP-1 expression. Hepatocytes in vitro exposed to recombinant VAP-1 and its substrate methylamine showed increased lipid accumulation as determined by quantification of Oil Red O uptake. This was recapitulated using hydrogen peroxide, and lipid accumulation was accompanied by changes in expression of the lipid transporter molecules FABP3, FATP6, insulin receptor subunits and PPARα. Human liver tissue exposed to recombinant VAP-1 or substrates for endo/exogenous VAP-1 produced less triglyceride than untreated tissue and demonstrated an increase in steatosis. This response could be inhibited by using bromoethylamine to inhibit the SSAO activity of VAP-1, and mice deficient in VAP-1/AOC3 also demonstrated reduced steatosis on high fat diet. Exposure of human liver tissue to methylamine to activate VAP-1 resulted in increased expression of FABP2 and 4, FATP3-5, caveolin-1, VLDLR, PPARGC1 and genes associated with the inflammatory response. CONCLUSION Our data confirm that the elevations in hepatic VAP-1 expression reported in nonalcoholic steatohepatitis can contribute to steatosis, metabolic disturbance and inflammation. This suggests that targeting the semicarbazide sensitive amine oxidase capacity of VAP-1 may represent a useful adjunct to other therapeutic strategies in NAFLD.
HepG2 cells continue to be a valuable tool in early drug discovery and pharmaceutical development... more HepG2 cells continue to be a valuable tool in early drug discovery and pharmaceutical development. In the current study we develop a 3D in vitro liver model, using HepG2/C3A cells that it is predictive of human genotoxic exposure. HepG2/C3A cells cultured for 7-days in agarose-coated microplates formed spheroids which were uniform in shape and had well defined outer perimeters and no evidence of a hypoxic core. Quantitative real-time-PCR analysis showed statistically significant transcriptional upregulation of xenobiotic metabolising genes (CYP1A1, CYP1A2, UG1A1, UGT1A3, UGT1A6, EPHX, NAT2) and genes linked to liver function (ALB, CAR) in 3D cultures. In response to three model pro-genotoxicants: benzo[a]pyrene, Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-aminoanthracene (2-AA),we observed further transcriptional upregulation of xenobiotic metabolising genes (CYP1A1, CYP1A2, NAT1/2, SULT1A2, UGT1A1, UGT1A3) compared to untreated spheroids. Consistent with this, spheroids were more sensitive than 2D monolayers to compound induced single- and double- stranded DNA damage as assessed by the comet assay and γH2AX phosphorylation respectively. In contrast, levels of DNA damage induced by the direct acting mutagen 4-nitroquinolin N-oxide (4NQO) was the same in spheroids and monolayers. In support of the enhanced genotoxic response in spheroids we also observed transcriptional upregulation of genes relating to DNA-damage and cellular stress response (e.g. GADD45A and CDKN1A) in spheroids. In conclusion, HepG2/C3A 3D spheroids are a sensitive model for in vitro genotoxicity assessment with potential applications in early stage drug development.
Methods in molecular biology (Clifton, N.J.), 2017
The liver is the largest internal organ and filters around 3 pints of blood per minute. This cont... more The liver is the largest internal organ and filters around 3 pints of blood per minute. This continuous flux of blood should not be confused with rapid egress of lymphocytes through the liver; this organ has intricate corridors of specialized sinusoidal spaces, ensuring that immune cells decelerate to shear flow rates, and providing ample opportunities to interact with parenchymal cells. Migration has been intricately linked to T cell function; it is therefore important to study liver T cell biology into context within the liver microenvironment. Here we discuss the highly organized architecture of liver-resident cells (sinusoidal endothelia, Kupffer cells, stellate cells/myofibroblasts, and biliary and hepatic epithelia) and showcase basic, multicellular, and complex systems to model T cell migration through the human liver microenvironment in vitro and ex vivo.
Lymphocyte recruitment to different organs, and even alternate anatomical regions within the same... more Lymphocyte recruitment to different organs, and even alternate anatomical regions within the same organ, is differentially regulated. Key combinations of adhesion molecules and chemokines govern compartmentalization, and these can change depending upon the nature and duration of tissue injury. We are interested in understanding lymphocyte recruitment to the liver during human disease, and thus need models of the liver inflammatory milieu that are as representative as possible. Here we describe the use of precision cut liver slices as models of disease.
European Journal of Gastroenterology & Hepatology, 2001
Despite intensive medical treatment with steroids and immunosuppressants, acute colitis is still ... more Despite intensive medical treatment with steroids and immunosuppressants, acute colitis is still associated with a colectomy rate of up to 15%. Following the observation that a patient with severe steroid-resistant colitis went into remission when treated with heparin for a deep vein thrombosis, there have been a number of reports on the use of heparin in acute ulcerative colitis. Although small and uncontrolled, these studies consistently demonstrate the beneficial effects of heparin, with surprisingly few side-effects in a disease characterized by mucosal haemorrhage. The mechanisms by which heparin may ameliorate ulcerative colitis remain unclear. A simple anticoagulant effect may be responsible, but similar effects are not seen with warfarin. As a result of their intense negative charge, the glycosaminoglycans that constitute heparin have diverse biological effects. These include potent anti-inflammatory actions, in vitro and in vivo, and the potentiation of the activity of the peptide growth factors necessary for mucosal regeneration and repair. This review summarizes the clinical reports on heparin treatment for ulcerative colitis and explores the mechanisms by which this novel form of treatment may exert its effects.
In order to understand human inflammatory diseases and to develop and assess new therapeutic stra... more In order to understand human inflammatory diseases and to develop and assess new therapeutic strategies targeting leukocyte recruitment to tissue, it is necessary to study human lymphocyte interactions with endothelium. It is often not practical to carry out assays on fresh human samples and therefore cells may be cryopreserved and batched for later study. Furthermore, many forms of adoptive cell therapy use cryopreserved cells that are required to migrate to tissue after infusion in vivo. The consequences of cryopreservation on the adhesion and migration of leukocytes is not known leading us to study the effects of cryopreservation on lymphocyte phenotype, migration, and adhesion. Cryopreservation and subsequent thawing did not alter the proportion of retrieved T cell subsets. Overall levels of expression of β1 or β2 integrins were unaffected but marked changes were observed in other relevant receptors. Expression of CD69, a transmembrane protein that plays a critical role in lymphocyte egress from tissues and the chemokine receptor CXCR4, increased on thawed populations and levels of CD62L and CXCR3 were reduced on thawed cells but restored if cells were allowed to recover after thawing. These changes were associated with modulation of the ability of lymphocytes to migrate across cytokine-stimulated monolayers of endothelium toward recombinant CXCL11 and CXCL12. Thus cryopreservation and thawing of lymphocytes induces changes in their adhesive phenotype and modulates their ability to migrate across endothelial monolayers. These findings have implications for in vitro experimentation and for cell therapy in which cryopreserved cells are expected to migrate when reinfused into patients.
s / Atherosclerosis 232 (2014) e1–e9 e4 LYMPHOTOXIN-b RECEPTOR SIGNALLING LINKS STEATOHEPATITIS, ... more s / Atherosclerosis 232 (2014) e1–e9 e4 LYMPHOTOXIN-b RECEPTOR SIGNALLING LINKS STEATOHEPATITIS, HYPER-LIPIDEMIA AND ATHEROSCLEROSIS B. Apta*, M.J. Harrison, C. Benezech, S. Karim, C.F. Ware, P.F. Lalor, J.H. Caamano, G.E. Rainger. MRC Centre for Immune Regulation, University of Birmingham, Birmingham, UK * Presenting author Metabolic syndrome describes a variety of risk factors associated with the development of cardiovascular disease (CVD). Non-alcoholic fatty liver disease (NAFLD) is considered to be the hepatic manifestation of the metabolic syndrome and is strongly associated with hyper-lipidemia. Lymphotoxin-b receptor (LTbR) is a receptor from the tumour necrosis factor (TNF) family and is expressed primarily on epithelial cells and cells of the myeloid lineage, LTbR is involved in hepatic lipid storage and export, and plasma levels of the receptor have been implicated as a risk factor for CVD. We aimed to characterise the role of LTbR in steatosis, hyper-lipidemia and atherosclerosis using wild-type, LTBR-/-, ApoE -/and LTBR-/-/ ApoE -/double knockout mice on a 13-week high fat diet (HFD) as established models of NAFLD and atherosclerosis, as well as murine and human hepatic tissue and hepatocyte cell culture. Serum lipid analysis and liver/aorta histology was performed according to standard methodologies, with quantification of lipid accumulation by Oil red O staining. Hepatocyte triglyceride storage and output was measured using staining and standard quantification kits. Loss of LTbR signalling in the ApoE-/strain substantially reduced steatosis, circulating triglyceride levels and atherosclerotic plaque burden compared to ApoE-/controls when fed a HFD for 13 weeks. LTbR signalling in hepatocytes enhanced de novo lipogenesis, lipid storage and triglyceride output. Thus, LTbR signalling links fatty liver disease and hyper-lipidemia, two prominent risk factors of CVD which exacerbate atherosclerosis through the disruption of lipid homeostasis. http://dx.doi.org/10.1016/j.atherosclerosis.2013.11.008 THE FORMATION OF MONOCYTE-PLATELET AGGREGATES IN STIRRED WHOLE BLOOD IN RESPONSE TO DIFFERENT PLATELET AGONISTS C.L. Box*, C. Yates, M. Chimen, M. Harrison, G. Nash, S. Watson, G.E. Rainger. School of Clinical and Experimental Medicine, University of Birmingham, B15 2TT, UK * Presenting author Introduction: Using in vitro and in vivo models of vascular inflammation resembling aspects of the chronic inflammatory environment, we have recently described a paradigm where platelets recruited to intact endothelial monolayers form adhesive bridges between the vessel wall where they preferentially aid monocyte recruitment. It is well documented that monocyte-platelet aggregates form in circulating blood with increased incidence in patients with cardiovascular and chronic inflammatory disease. It is unknown whether heterotypic aggregation increases efficiency of monocyte recruitment during vascular inflammation, which we will investigate in the near future. The aim of the current study was to determine how efficiently monocyte-platelet aggregates could form in whole blood in response to different platelet agonists. Methods: Whole blood was treated with the platelet agonists adenosine diphosphate (30mM), thrombin receptor activating peptide (100mM), thromboxane mimetic, U46619 (10mM), rhodocytin, (100nM) or collagen related peptide (1mg/ml) and gently mixed at 37 C for 30mins. Heterotypic aggregation was assessed by flow cytometry using antibodies against monocyte markers CD14 and CD16 and against platelet CD42b. Results: After treatment with a platelet agonist there was a significant increase in heterotypic aggregate formation compared to untreated control for both CD14+CD16and CD14+CD16+monocyte subsets, the incidence of aggregate formation did not vary between the subsets. Conclusions: Monocyte-platelet aggregates form in whole blood in response to a range of platelet agonists, although the efficiency of aggregation varies in an agonist dependent manner. Both monocyte subsets form heterotypic aggregates with equal efficiency. http://dx.doi.org/10.1016/j.atherosclerosis.2013.11.009 HEME AND METFORMIN COORDINATE HUMAN AND MURINE MACROPHAGE HEME OXYGENASE 1 EXPRESSION WITH FOAM CELL RESISTANCE PARTLY VIA ADENOSINE MONOPHOSPHATE KINASE AND ACTIVATING TRANSCRIPTION FACTOR 1 (AMPK-ATF1) X. Wan , Y. Huo , M. Johns , E. Piper , J.C. Mason , D. Carling , D.O. Haskard , J.J. Boyle *. Vascular Sciences, NHLI, Imperial College London, UK; MRC CSC, Imperial College London, UK * Presenting author Rationale: Intraplaque hemorrhage (IPH) is a key driver of the progression of atherosclerotic plaques. We recently characterised Mhem as a novel macrophage phenotype that limits the atherogenicity of IPH. Mhem are directed by activating transcription factor 1 (ATF1), which is activated by phosphorylation. A better understanding of the counter-atherogenic ATF1Mhem pathway may facilitate anti-atherosclerotic therapies. Methods: We…
Uploads
Papers by Patricia Lalor