Decoy gelatin nanoparticles as a novel tool to elucidate the role of NF-kB in Kupffer cells on hepatic ischemia/reperfusion injury

Kupffer cells, the resident macrophages of the liver, play an important role in host defense and immune system. Moreover, they are also involved in several pathological conditions, like the hepatic ischemia/reperfusion injury. The transcription factor NF-kB is activated during this event. However, to date it was not possible to evaluate the consequence of this upregulation, as NF-kB possesses both detrimental and beneficial properties, regarding of the liver cell type affected. An activation in Kupffer cells is thought to lead to an increased inflammatory response. This hypothesis could not be proven in vivo, as a selective inhibition of NF-kB in Kupffer cells was not possible due to technical difficulties. In this work, solid nanoparticles made of gelatin were chosen to deliver NF-kB inhibiting decoy oligonucleotides exclusively to Kupffer cells in order to block the activation of NF-kB. Confocal scanning laser microscopy showed selective uptake of gelatin nanoparticles into the Kupffer cells without affecting hepatocytes. An increase in NF-kB binding activity during postischemic reperfusion could be diminished by the delivery of decoy oligonucleotides to the resident liver macrophages. In addition, a rise in TNF-alpha mRNA expression assessed by real time RT-PCR was also reduced, thus providing evidence for the effectiveness of this selective targeting. Thus, this work established a novel carrier for a specific Kupffer cell targeting

BIOMARKERS TO DEFINE OPTIMAL PROTEIN REQUIREMENT

2013/2014Dietary proteins are the source of the amino acids required by the body for tissue growth and maintenance. The Population Reference Intake (PRI) for proteins, as defined by the European Food Safety Authority (EFSA) for healthy adults, including the elderly, is 0.83 g/kg body weight/day. This amount is defined on the net balance of body protein (or “nitrogen balance”, given by the difference between dietary nitrogen intake and losses) equivalent to 0.66 g/kg/day plus a safety factor for interpersonal variability and differences in proteins quality of mixed diets. The PRI, however, is the minimum daily amount of protein needed to maintain the nitrogen balance and avoid a progressive loss of lean body mass in healthy people with moderate physical activity. Therefore nitrogen balance may not be adequate to define protein requirement in adults and especially in ageing characterized by loss of muscle mass and function (sarcopenia). Furthermore until recently the prevalent idea was that a protein intake above PRI had no further benefits and on the contrary could impair health. These believes are now under discussion, diets with higher protein intake have been shown beneficial in the prevention and treatment of conditions such as sarcopenia, COPD and type 2 diabetes mellitus. There is a need of more precise methods to define protein requirement. AIM. The aim of the present thesis is to investigate in human healthy volunteers new biomarkers adequate to define optimal protein intake. Recent studies have determined protein needs by measuring whole-body protein metabolism using stable labeled isotope-amino acids. METHODS. Our research group has applied two different metabolic methods based on the most widely used tracer, i.e. D5-Phe stable isotope, in two experimental bed rest campaigns (FP7 PLANHAB and INTERREG PANGaA) in healthy volunteers. BR is a suitable model to investigate physiologic adaptation to inactivity. MAIN RESUTLTS. FP7 PLANHAB. We applied the stable isotope infusion technique, to assess the effect of physical inactivity and/or hypoxic condition on whole body protein turnover as previously described in Biolo et al 2008. Chronic hypoxia has been associated with an overall reduction in protein synthesis and in total plasma and skeletal muscle protein content. During the PLANHAB study we investigated, through a crossover randomization, the net effects of 10 days normobaric hypoxia (4000 mt.), associated with either ambulatory conditions or BR, in 11 young (age 24±4 yr), healthy and normal weight male subjects maintained on eucaloric diets. Main results. Hypoxia in ambulatory conditions significantly decreased whole body protein turnover by reducing both protein synthesis (-8±2%) and protein degradation (-8±3%). Hypoxia during bed rest did not caused significant changes in protein metabolism. INTERREG PANGaA. The skeletal muscle loss in aging is caused mainly by the “anabolic resistance” i.e. the inadequate increase in the rate of protein synthesis in response to nutritional-metabolic stimuli, including exercise, protein and amino acid intake as well as insulin and insulin-like growth factor stimulation. As a consequence, the net protein balance becomes negative leading to sarcopenia. The effects of ageing on the anabolic resistance induced by inactivity are poorly investigated. During the PANGeA study we had the opportunity to perform the second documented experimental BR in in healthy elderly volunteers and the first comparing aged with young subjects. To evaluate the anabolic resistance associated with ageing and inactivity, we enrolled 7 young (23±1yr) and 8 elderly (59±1yr) normal weight individuals, in a 14-d experimental BR protocol. We replaced our previous infusion method with a new, simpler, safer and quicker technique, by which tracers are given orally instead of parenterally, the all procedure is completed in two hours, instead of 6, and only two blood draws versus 7 are sufficient. Main results. At baseline parameters of anabolic sensitivity were comparable between young and elderly individuals. The anabolic resistance significantly increased after BR in both groups (bed-rest effect p<0.01), with a statistically significant bed-rest×group interaction (p=0.01). Anabolic resistance increased significantly in elderly (18.5%±7.3%) more than in young (5.2%±9.4%) subjects. DISCUSSION. In the PLANHAB study, hypoxia in ambulatory conditions reduced by the same level both protein synthesis and catabolism, as measured by isotope infusions, suggesting an adaptive mechanism: the lower energy production and availability induced by hypoxia associated with ambulatory condition. These modifications could not have been revealed by the use of nitrogen balance method, showing the relevance of more sophisticated analysis. The direct evaluation of the muscle protein metabolism through an infusion of stable-labeled isotope tracer, considered the golden standard methodology, gave us, in the PLANHAB study, reliable results in the early protein metabolism changes during hypoxia and/or BR. This method however has the limit of being complex, onerous and invasive, therefore being unsuitable for clinical evaluation. In the PANGeA study we could confirm the presence of a reduced sensitivity to anabolic stimuli in the elderly population compared to the young men. The elderly subjects are therefore, more at risk to develop changes of protein metabolism induced by inactivity. The simpler, timesaving and less invasive method we have developed for the PANGeA study, on the other hand, could be applied to a wider ranges of experimental conditions and clinical settings.XXVII Ciclo198

Mechanisms of muscle atrophy following inactivity

2008/2009Background. Muscle atrophy is determined by specific molecular pathways controlling protein synthesis and degradation. Physical inactivity and muscle unloading are normally related to decreased muscle mass, but pathways controlling muscle atrophy during inactivity in humans are not presently clarified. Previous publications showed in animals a link between muscle atrophy and oxidative stress or inflammation. Inactivity was shown to be associated to inflammation and oxidative stress upregulation. Inflammation is controlled by several factors and polyunsaturated fatty acids (PUFA) of n-3 and n-6 series are known to play an anti-inflammatory and proinflammatory role, respectively: their relative content in cell membranes reflects the inflammatory condition at whole body level. Oxidative stress derives from reactive oxygen species (ROS) production in excess to the scavenging activity of antioxidant systems. Homocysteine is a non-proteinogenic amino acid: increased homocysteine concentration in plasma is directly linked to oxidative tissue damage. Glutathione (GSH) is an antioxidant tripeptide: upregulated GSH availability is considered a response to occurred ROS production. Glutamine, is synthesized by muscles and it is utilized by immune cells: its depletion occurs in critically ill patients. Nutrition can play a pivotal role during inactivity: dietary protein supplementation can ameliorate protein turnover while energy intake restriction was linked to enhanced muscle atrophy. Aims and experimental design. The aim of the present thesis is to investigate in human healthy volunteers: i) the impact of physical inactivity on inflammation and oxidative stress as potential mechanisms underlying muscle atrophy; ii) the impact of energy balance, and of protein supplementation during inactivity on muscle atrophy, oxidative stress and glutamine availability. Different metabolic tests were performed in five separate experimental bed rest campaigns: STBR-IP, WISE, Valdoltra Bed Rest 2006 – 2007 – 2008. Results. Eucaloric experimental bed rest at normal protein intake: decreased n-3 and increased n-6 PUFA in red blood cell membranes; increased arachidonic to eicosapentaenoic acid ratio (Valdoltra Bed Rest 2006 – 2007 – 2008); increased homocysteine plasma concentration lowering clearance by remethylation (WISE); reduced plasma glutamine concentrations lowering its de novo synthesis (STBR-IP); decreased muscle thickness and myofibres pennation angle; increased muscle protein carbonylation and GSH availability in direct correlation with atrophy progression (Valdoltra Bed Rest 2006 – 2007). High protein intake during bed rest significantly lowered homocysteine concentrations by increased clearance by transsulfuration. Calorie restriction (20% reduction) during muscle unloading failed to affect changes of glutamine kinetics and availability mediated by inactivity (STBR-IP). During bed rest, excessive fat mass gain, when matched to stable fat mass maintenance, significantly: reduced vastus lateralis muscle thickness; increased plasma leptin, myeloperoxidase and C-reactive protein levels; enhanced, in red blood cells, activity and availability of GSH (Valdoltra Bed Rest 2006 – 2007). Discussion. The experimental bed rest mediated increase in n-6 relatively to n-3 polyunsaturated fatty acids of erythrocytes membranes confirm bed rest can play a pro-inflammatory role at whole body level. Upregulated levels of homocysteine plasma concentrations after bed rest underline that immobility can induce whole body oxidative stress and cardiovascular risk. Significant correlation between changes in protein carbonylation, as marker of oxidative damage, or antioxidant GSH availability, with muscle atrophy induced by bed rest, strongly suggest that physical inactivity can induce muscle atrophy by pathways involving oxidative stress. Carbonylated proteins are, in fact, directly and rapidly degraded by the proteasome system. Moreover, dietary protein supplementation during physical inactivity potentially reduces oxidative damage lowering plasma homocysteine concentration. This evidence is confirmed by publications showing, in animals, that high protein diet can increase homocysteine transsulfuration rate. Interestingly, during inactivity, positive energy balance, when matched to fat mass maintenance, was here shown to induce greater muscle atrophy together with greater upregulation of whole body inflammation and oxidative stress. Thus, such observations further suggest that inactivity mediated atrophy can be triggered and regulated by oxidative stress and inflammation. Calorie restriction probably failed to affect glutamine kinetics due to the low extent of energy intake reduction, but glutamine metabolism can be hypothesized to be independent from energy balance during inactivity.XXII Cicl

El papel del AMPc, del estrés del retículo endoplasmático y de las adipocitoquinas en el trasplante hepático

[spa] La lesión por isquemia-reperfusión (I/R) inherente al trasplante hepático (TH), es la causa principal tanto de mal funcionamiento como del fallo primario del injerto hepático. Y si esto ocurre en hígados sanos, aun son mayores los casos de mal función o fallo primario cuando el injerto es esteatósico, ya que estos hígados toleran peor que los normales la lesión por I/R. De ahí que la esteatósis sea la causa principal del mayor número de órganos considerados no aptos para el trasplante, acentuando así la problemática de la falta de injertos hepáticos para TH. Por ello, es evidente la necesidad de desarrollar estrategias protectoras para minimizar los efectos adversos de la lesión por I/R en hígados esteatósicos. Por otro lado, el precondicionamiento (PC) isquémico basado en la inducción de breves periodos de I/R, reduce la lesión por I/R asociada a TH, pero se desconocen en gran parte sus mecanismos protectores. En la presente tesis se investigó el papel del AMP cíclico, del estrés del retículo endoplasmático y de las adipocitoquinas tales como la adiponectina, la resistina y la visfatina en la lesión por I/R en injertos hepáticos esteatósicos y no esteatósicos sometidos a TH, así como su implicación en los beneficios del PC. Además se evaluaron los mecanismos de protección de estrategias farmacológicas o quirúrgicas, como el PC capaces de modular el AMPc y la adiponectina en ambos tipos de injertos. Para el desarrollo de la presente tesis, se realizaron modelos de TH singénico y alogénico. La utilidad de estrategias dirigidas a modular AMPc en el trasplante de hígados esteatósicos no se había determinado previamente. En la presente tesis se evidencian los efectos perjudiciales del AMPc en los injertos hepáticos esteatósicos y se sugiere la utilidad clínica de estrategias que bloquean la acción del AMPc (tales como la inducción del PC o inhibidores de la adenilato ciclasa), para proteger a los injertos hepáticos esteatósicos frente a la lesión por I/R asociada al trasplante. Esta protección-mediada por el oxido nítrico-reduce el estrés oxidativo, el daño celular endotelial y las disfunciones microvasculares. Los beneficios de tales estrategias sobre el metabolismo energético en injertos esteatósicos fueron independientes del oxido nítrico. Además se demostró que el TUDCA es capaz de proteger sólo a los injertos hepáticos esteatósicos mediante un mecanismo independiente del estrés del retículo endoplasmático. Los mecanismos de protección del TUDCA cursan mediante una reducción en la expresión de PPARγ, lo cual a su vez activa la vía del TLR4, específicamente la vía del TRIF para proteger a los injertos esteatósicos frente a la lesión asociada al trasplante. Asimismo se evidencia por primera vez que la visfatina no está implicada en la vulnerabilidad de los hígados esteatósicos frente a la lesión por I/R en el trasplante hepático. Además se demostró que los injertos hepáticos esteatósicos están más predispuestos a una reducción en los niveles de adiponectina y resistina al someterlos a trasplante que los no esteatósicos. Los resultados obtenidos señalan que la adiponectina aumenta los niveles de resistina y esto a su vez favorece la activación de la vía PI3K/Akt, estableciendo entonces una posible vía de señalización. El tratamiento con adiponectina o resistina protegió a los injertos esteatósicos frente a la vulnerabilidad que presentan a la lesión inducida por I/R mientras que fue ineficaz en injertos no esteatósicos. En resumen se describen estrategias farmacológicas (inhibidores del AMPc, TUDCA, agonistas del TLR4, adiponectina y resistina) que son específicas para proteger a los injertos hepáticos esteatósicos sometidos a trasplante, y se refuerza el papel del PC como una estrategia quirúrgica promisoria que puede ejercer los mismos efectos protectores que las estrategias farmacológicas propuestas. Estos hallazgos podrían contribuir a nuevas aplicaciones del PC en la práctica clínica de los TH.[eng] In this thesis the role of cyclic AMP , the endoplasmic reticulum stress and adipocytokines such as adiponectin, resistin and visfatin injury in I/R in steatotic liver grafts and non- steatotic undergoing transplantation (TH) was investigated and their involvement in the benefits of the preconditioning (PC). Besides protection mechanisms pharmacological or surgical strategies, as the PC capable of modulating cAMP and adiponectin in both types of grafts were evaluated. The usefulness of strategies to modulate cAMP in steatotic liver transplantation has not been previously determined. In this thesis the harmful effects of cAMP in steatotic liver grafts are evident and clinical utility of strategies that block the action of cAMP (such as induction of PC or inhibitors of adenylate cyclase), is suggested to protect the grafts steatotic liver injury against I/R associated with the transplant. This protection - mediated nitric oxide reduces oxidative stress, endothelial cell damage and microvascular dysfunction. The benefits of such strategies on energy metabolism in steatotic grafts were independent of nitric oxide. In addition it was shown that TUDCA is able to protect only the steatotic liver grafts by a mechanism independent of endoplasmic reticulum stress. The mechanisms of protection by reducing TUDCA enrolled in PPARγ expression, which in turn activates TLR4 pathway, specifically TRIF pathway for steatotic grafts protect against transplant-associated lesion. Also evidenced for the first time that visfatin is not involved in the vulnerability of steatotic livers against injury by I/R in liver transplantation. It was further demonstrated that steatotic liver grafts are more prone to reduction in adiponectin and resistin when subjected to non- steatotic transplant. The results indicate that adiponectin increases resistin levels and this in turn promotes activation of the PI3K/Akt pathway, then establishing a possible signaling pathway. Treatment with adiponectin or resistin steatotic grafts protected against the vulnerability of the injury induced by I / R whereas it was ineffective in not steatotic grafts. Pharmacological strategies (cAMP inhibitors, TUDCA, TLR4 agonists, adiponectin and resistin) that are specific to protect steatotic liver grafts undergoing transplantation are described in brief, and the role of the PC is reinforced as a promising surgical strategy can exercise the same protective effects proposed that pharmacological strategies. These findings could contribute to new PC applications in clinical practice of TH. In terms of clinical application, these therapies might open new avenues for steatotic liver transplantation and improve the initial conditions of donor livers with low steatosis that are available for transplantation. Such therapies could also increase the use of numerous steatotic livers currently discarded for transplantation, thus reducing the risk of death of those patients on liver transplant waiting list