Endoplasmic Reticulum Stress and Apoptosis Triggered by Sub-Chronic Lead Exposure in Mice Spleen: a Histopathological Study

Lead (Pb) is an environmental oncogenic metal that induces immunotoxicity and anaemia. Emerging evidence has linked Pb toxicity with endoplasmic reticulum-driven apoptosis and autophagy. Glucose-regulated protein of 78 kDa (Grp78 or binding immunoglobulin protein (BiP)), a master endoplasmic reticulum chaperone, drives macrophage activation and regulates protein folding and calcium flux in response to heavy metals. The spleen may be involved in Pb poisoning due to its crucial role in erythrocatheresis and immune response, although there are no data to support this theory. Here, we found haematic and histopathological changes in the spleen of mice exposed to medium doses of Pb acetate (200 ppm-1 mM) in drinking water for 45 days. Pb deposition was also detected in organs such as the liver, kidney, brain, bone, blood and faeces, indicating an accumulation of this metal despite relatively short exposure time. Blood Pb content (BBL) reached 21.6 μg/dL; echinocytes and poikilocytes were found in Pb smears of treated group. Inside the spleen, higher Fe(II) and Fe(III) deposits inside macrophages were observed. Grp78 immunostaining, weakly expressed in spleen cells of control mice, after Pb exposure was specifically restricted to macrophages and megakaryocytes of the marginal zone of red pulp. Furthermore, Pb exposure induced superoxide dismutase 1 (SOD1) expression, cleaved caspase-3 and p62/SQSTM1, consistent with oxidative stress, apoptosis and dysregulated autophagy in spleen compartments. We suggest that even at a middle dose, oral Pb intake induces oxidant iron deposition in the spleen and that this may trigger sustained Grp78 redistribution to cells, thus leading to oxidative and autophagy dysfunction as early local reactions to this dangerous metal

Diet enrichment with a specific essential free amino acid mixture improves healing of undressed wounds in aged rats

Chronic wounds are a major, often underestimated, health problem for the elderly. Standard wound care products are not usuallymanufactured tomeet the increased demand of nutrients by skin cells in order to regenerate new tissue and accelerate healing. This work was therefore undertaken to establish whether wound healing could be accelerated by nutritional supplementation with a specific mixture tailored to human need of essential amino acids (EAAs) without topical medication. To this end, using a skin full-thickness excisional model in aged rats,we compared the closure dynamics of undressing wounds in animals fed an EAAs-enriched diet or standard diet. We assessed the degree of fibrosis and inflammation, as well as relevant signaling molecules such as COL1A1, iNOS and TGFβ1. The results showed wound healing was accelerated in EAAs-fed rats, which was accompanied by reduced inflammation and changes in TGFβ1 and COL1A1 expression. Collectively, our findings indicate that dietary supplementation with balanced EAAs diet could serve as a strategy to accelerate wound healing without inducing fibrosis and could therefore be a simple but pivotal therapeutic approach in human also

Nutritional regulation of wound healing: the role of different amino acid mixture composition intake in wound repair. A preliminary study

The ability of the skin to repair wounds requires immediate changes in metabolism and expression and silencing of the genes controlling cell proliferation, migration, adhesion and dif- ferentiation.The energetic costs of wounds repair are enormous (1). Collagen represent around 50% of skin weight (about 20Kg), and synthesis costs are around 4 ATPs for any single amino acid (AA) inserted into the final composition of collagen (2). Moreover, the AA availability is indispensable to ensure the correct repair of the wound. Indeed, the poor state of dermal collagenous matrix is largely responsible for both the propensity to injury and slowness to heal observed in the elderly.To test whether different formulation of AA mixture could improve wound repair, we fed male rats (14 months old) with three balanced diet containing 1) 100% essential AA mixture (EAA); 2) EAA 10% plus 90% non-essential (NE) AA (deficient diet - D) and 3) EAA 40% plus NEAA 60% (casein like diet – CL) for 30 days. Then, four excisional wound (diameter 5mm) were surgically made on dorsal skin of each animal. After 3, 15 and 30 post wound days (pwd) the wound area were measured, collected and analyzed by Sirius Red and IHC methods for TGFβ1 and Collagen 1. The D diet reduced weight of the animals up to 30% in 60 days although there was an increase of >18% of spontaneous caloric introduction, whereas the EAA and CL diets increased the rat weight of 6% and 12% respectively. Three pwd, the injured area in EAA and CL diets was 92% and 98% respect to day 0 (100%); whereas it was larger (120%) in D diet. Fif- teen pwd, the wounds were completely closed with EAA diet, whereas with CL and D diets the open area were 14% and 45% respectively. Thirty pwd, all wounds were closed but large scars were observed in D diet. TGFβ1 was early and strongly expressed in D diet, whereas it was much less expressed in EAA and CL diets. Sirius-red staining showed that the collagen architecture was more organized with EAA diet and less organized with D diet. Collagen was strongly expressed 15 pwd in CL diet and less expressed in D and EAA diets, whereas 30 pwd an intense staining was observed only in EAA diet. Our data show for the first time that malnutrition, diet rich in NEAA, seriously affects metabolism, leading to rapid decay of the animals and slows down the wound closure, due to dermal collagenous matrix impairment and collagen production, and pos- sibly related to excessive TGFβ1 expression. On the contrary, EAA diet controls expression of TGFβ1 and collagen production, accelerating the wound repair and blunting scarring.

Essential amino acid mixtures drive cancer cells to apoptosis through proteasome inhibition and autophagy activation

Cancer cells require both energy and material to survive and duplicate in a competitive environment. Nutrients, such as amino acids (AAs), are not only a caloric source, as they can modulate cell metabolism and modify hormones homeostasis. Our hypothesis is that the environmental messages provided by AAs rule the dynamics of cancer cells life or death, and the alteration of the balance between essential (EAAs) and non-essential amino acids (NEAAs) (lower and higher than 50%, respectively) present in nutrients may represent a key instrument to alter environment-dependent messages thus mastering cancer cells destiny. In this study, two amino acid mixtures, one exclusively consisting of EAAs and the other consisting of 85% essential and 15% non-essential amino acids, were tested to explore their effects on the viability of both normal and cancer cell lines and to clarify the molecular mechanisms involved. Both mixtures exerted a cell-dependent anti-proliferative, cytotoxic effect involving the inhibition of proteasome activity and the consequent activation of autophagy and apoptosis. These results, besides further validating the notion of the peculiar interdependence and extensive crosstalk between the ubiquitin-proteasome system and autophagy, indicate that variation in the ratio of EAAs and NEAAs can deeply influence cancer cell survival. Consequently, customization of dietary ratios among EAAs and NEAAs by specific amino acid mixtures may therefore represent a promising anticancer strategy able to selectively induce death of cancer cells through the induction of apoptosis via both ubiquitin-proteasome system inhibition and autophagy activation

Spasmogenic Effects of the Proteasome Inhibitor Carfilzomib on Coronary Resistance, Vascular Tone and Reactivity

Background: Carfilzomib (CFZ) is a new proteasome inhibitor used for the treatment of multiple myeloma. Besides heart failure, angina and myocardial ischemia occurred following administration of CFZ, which is not contraindicated in patients with recent myocardial infarction/unstable angina excluded from the safety trials. Aimof Study: To test the effects of CFZ (10−9 to 10−7 mol/L) on vascular tone and reactivity in the isolated rabbit heart and aorta. Methods and Results: CFZ administered by bolus injection to the isolated heart increased coronary perfusion pressure (CPP) at all tested concentrations and mildly raised left ventricular pressure and heart rate, only at the highest concentration. Addition of CFZ directly into the organ bath increased the basal tone of isolated aortic strips with contraction plateau reached after 10 min. This spasmogenic effect doubled following ablation of the endothelium. Pretreatment with CFZ amplified the vasospastic action exerted by KCl, noradrenaline (NA) and angiotensin II (A) on aortic strips, and impaired vasodilation following administration of nitroglycerin (NTG) and nifedipine (NFP) on the contraction plateau induced by KCl, NA and A. Aortic strips pretreatedwith CFZ exhibited impaired relaxation, as compared to untreated strips, following administration of acetylcholine (Ach), an endothelium- dependent vasodilating agent, on the plateau of NA contraction (p b 0.05). Conclusions: CFZ increased CPP, resting vasoconstricting tone and the spasmogenic effect of different agents. Preincubation with CFZ decreased the anti-spasmogenic activity of NTG and NFP, as well as reduced by over 50% the vasodilating effect of Ach, suggesting that CFZ can impair vasodilation via an endothelium dependent mechanism. Further studies are warranted to establish its clinical safety in patients with known CAD and prior history of coronary spasm

Body Weight Loss and Tissue Wasting in Late Middle-Aged Mice on Slightly Imbalanced Essential/Non-essential Amino Acids Diet

Objective: Inadequate protein intake can impair protein balance thus leading to skeletal muscle atrophy, impaired body growth, and functional decline. Foods provide both non-essential (NEAAs) and essential amino acids (EAAs) that may convey different metabolic stimuli to specific organs and tissues. In this study, we sought to evaluate the impact of six diets, with various EAA/NEAA blends, on body composition and the risk of developing tissue wasting in late middle-aged male mice. Methods: Six groups of late middle-aged male mice were fed for 35 days with iso-nutrients, iso-caloric and iso-nitrogenous special diets containing different EAA/NEAA ratios ranging from 100%/0% to 0%/100%. One group fed with standard laboratory rodent diet (StD) served as control. Preliminarily, we verified the palatability of the diets by recording the mice preference, and by making accessible all diets simultaneously, in comparison to StD. Body weight, food and water consumption were measured every three days. Blood and urine samples, as well as heart, kidneys, liver, spleen, triceps surae, retroperitoneal WAT, and BAT were harvested and weighed. Results: Mice consuming NEAA-based diets, although showing increased food and calorie intake, suffered the most severe weight loss. Interestingly, the diet containing a EAA/NEAA-imbalance, with moderate NEAAs prevalence, was able to induce catabolic stimuli, generalized body wasting and systemic metabolic alterations comparable to those observed with diet containing NEAA alone. In addition, complete depletion of retroperitoneal white adipose tissue and a severe loss (>75%) of brown adipose tissue were observed together with muscle wasting. Conversely, EAA-containing diets induced significant decreases in body weight by reducing primarily fat reserves, but at the same time they improved the clinical parameters. On these basis we can deduce that tissue wasting was caused by altered AA quality, independent of reduced nitrogen or caloric intake. Conclusion: Our results indicate that diets containing an optimized balance of AA composition is necessary for preserving overall body energy status. These findings are particularly relevant in the context of aging and may be exploited for contrasting its negative correlates, including body wasting

Decreased expression of Klotho in cardiac atria biopsy samples from patients at higher risk of atherosclerotic cardiovascular disease

Background. Klotho proteins (α- and β) are membrane-based circulating proteins that regulate cell metabolism, as well as the lifespan modulating activity of Fibroblast Growth Factors. Recent data has shown that higher plasma circulating Klotho levels reduce cardiovascular risk, suggesting Klotho has a protective role in cardiovascular diseases. However, although so far it has been identified in various organs, it is unknown whether cardiomyocytes express Klotho and Fibroblast Growth Factors(FGFs), and whether high cardiovascular risk could affect cardiac expression of Klotho, FGFs and other molecules. Methods. We selected 20 patients with an estimated 10-year high atherosclerotic cardiovascular disease and 10 age-matched control subjects with an estimated 10-year low risk undergone cardiac surgery for reasons other than coronary artery by-pass. In myocardial biopsies, we evaluated by immuno-histochemistry whether Klotho and FGFs were expressed in cardiomyocytes, and whether higher cardiovascular risk influenced the expression of other molecules involved in endoplasmic reticulum stress, oxidative stress, inflammation and fibrosis. Results. Only cardiomyocytes of patients with a higher cardiovascular risk showed lower expression of Klotho, but higher expressions of FGFs. Furthermore, higher cardiovascular risk was associated with increased expression of oxidative and endoplasmic reticular stress, inflammation and fibrosis. Conclusions. This study showed for the first time that Klotho proteins are expressed in human cardiomyocytes and that cardiac expression of Klotho is down-regulated in higher cardiovascular risk patients, while expression of stress-related molecules were significantly increased

Klotho expression in cardiomyocytes in patients at a higher atherosclerotic cardiovascular disease risk

Klotho proteins (α- and β-Klotho) are transmembrane proteins whose extracellular domain is secreted into blood and urine by ectodomain shedding. As such they behave as circulating proteins that regulate cell metabolism, endothelial function and calcium homeostasis, as well as modulating the lifespan connected activity of Fibroblast Growth Factors (FGFs, mainly 21 and 23) and other molecules (1). Recent data have shown that highest levels of plasma circulating Klotho are associated with a lower cardiovascular risk, thereby suggesting a possible role for Klotho in cardiovascular diseases (2). However, although Klotho has been identified in various organs, including kidney, brain, adipose tissue and intestine, it is unknown whether cardiomyocytes express Klotho and FGFs and, if so, whether high cardiovascular risk can be associated with cardiac expression of Klotho, FGFs and other related molecules. We examined myocardial biopsies from 20 patients with an estimated 10-year atherosclerotic cardiovascular disease (ASCVD) risk in the range of 5% to 7.5% and 10 age-matched control subjects with an estimated 10-year ASCVD risk of <5% (3) undergoing cardiac surgery other than coronary artery by-pass. Both groups of patients were statin naive, had normal hemoglobin A1c, normal coronary arteries on left heart catheterization, and had LDL cholesterol levels between 70 and 189 mg/ DL. Using immunohistochemistry methods, we evaluated Klotho and FGFs expression in human cardiomyocytes, and whether higher ASCVD risk influenced the expression of other molecules involved in endoplasmic reticulum stress (GRP78), oxidative stress (SOD1, NFkB) and inflammation (iNOS, eNOS). Cardiomyocytes of patients with a higher ASCVD risk exhibited lower expression of Klotho, but also higher expression of FGFs, as compared to cardiomyocytes of patients with a reduced ASCVD risk. Furthermore, higher ASCVD risk was associated with significantly increased expression of GRP78, SOD1, NFkB and iNOS (all p<0.05). This study shows for the first time that Klotho proteins are inherently expressed in human cardiomyocytes and also that cardiac expression of Klotho is down regulated in higher ASCVD risk patients, while the expression of FGFs and other stress-related molecules involved in myocyte damage, such as GRP78, SOD1, NFkB and iNOS, is significantly increased. Further studies are warranted to investigate the association of klotho, FGFs and related molecules expression with cardiovascular risk