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, Muscle Protein Synthesis and Autophagy Relationships in Cancer. An Attempt to Understand Where Are We Going, and Why

Protein-based structures are indispensable to maintain life, so identification and removal of worn out structures achieved through proteostasis, the sum of micro and macro-autophagy (autophagy) plus ubiquitin-proteasome system, must balance renewal by new synthesis. Many of the elements controlling dynamically equilibria between protein synthesis and protein degradation have been identified and modalities of activation actively studied, still we are quite far from mastering how this balance is ruled. Failure to maintain a positive balance between protein synthesis and protein degradation would result in sarcopenia, defined as the loss of skeletal muscle mass and function, a major clinical problem frequently accompanying chronic illnesses, but peculiarly spotted in cancer and in elderly patients. Also, how cancer is fed, and how nutrition in cancer patients may affect evolution and therapy effectiveness is another field of opinions and uncertainty. On the other hand, exercise and nutrition tailored to provide adequate amounts of amino acids are widely considered a necessary strategy for prevention and treatment of protein synthetic deficits in muscles. This paper will synthetically review how different nutritional strategies and energy production may interconnect efficiently synthesis and scavenging of aged and overused protein molecules by autophagy. Finally, since energy availability rules life and death of cells and organisms, an hypothesis predicting how energy may control the ratios among protein synthesis and autophagy is proposed: in normal conditions, protein syntheses have a key role in autophagy activation by consuming large amounts of energy when forming peptidic bonds, that is adenosine tri-phosphate (ATP) is consumed to monophospahate (AMP), thus decreasing ATP to AMP ratios. Conversely, both protein syntheses and autophagy may be scarcely activated when low availability of ATP would result also in lowest concentrations of AMP. In this peculiar setting, reduced rates of both protein syntheses and autophagy would be observed, resulting in worsening of protein balance and functions

Nutrition, Nitrogen Requirements, Exercise and Chemotherapy-Induced Toxicity in Cancer Patients. A puzzle of Contrasting Truths?

Amino acids can modulate cell metabolism and control cell fate by regulating cell survival and cell death. The molecular mechanisms involved are mediated by the mTOR complexes mTORC1 and mTORC2 activity. These complexes are finely regulated and the continuous advancement of the knowledge on their composition and function is revealing that their balance may represent the condition that determines the cell fate. This is important for normal healthy cells but it is becoming clear, and it is even more important, that the balance of the mTORCs activity may also condition the cell fate of cancer cells. Here, we discuss the evidences supporting the amino acids supplementation as a cancer fighting weapon and a possible strategy to counteract the myocyte toxicity associated with chemotherapy, possibly by tipping the balance of mTORCs activity

Metabolic frailty in malnourished heart failure patients

Muscular wasting (MW) and cardiac cachexia (CC) are often present in patients with chronic heart failure (HF). Aim: To identify whether MW and CC are due to malnutrition or impairment of protein metabolism in HF patients. Material and Method: In 78 clinically stable HF patients (NYHA class II-III), aged from 32 to 89 years, we measured anthropometrical parameters and nutritional habits. In the identified 35 malnourished patients, we also measured: insulin resistance, gluconeogenetic amino acids blood concentration and nitrogen balance. Results: Seventy-five patients had eating-related symptoms. However we found significant nutritional impairment in 35 patients only. In addition, these 35 patients had: 1) significant increase of blood Alanine independently from both presence of insulin resistance or food intake reduction and 2) positive nitrogen balance. Conclusion: Food intake is not impaired in CHF patients. In spite of normal food intake, 35 of 78 patients had nutritional impairment with reduced anthropometric parameters and increased blood Alanine. These findings show alteration of proteins metabolism with proteolysis. We believe that specific physical training with nutritional supplement can be an additional therapy able to prevent protein disarrangement in CHF patients

Mammalian Target of Rapamycin: Is It Relevant to COPD Pathogenesis or Treatment?

The mammalian target of rapamycin (mTOR) signalling pathway regulates fundamental metabolic processes such as inflammation, autophagy and apoptosis, all of which influence cell fate. Recent experimental data suggest that mTOR signalling is involved in many diseases, including lung diseases, but with contrasting data. Overexpression of mTOR and its signalling proteins have been linked to lung cell senescence and development of emphysema, pulmonary hypertension and inflammation. On the other hand, mTOR inhibitors, as rapamycin and/or its derivatives, restore corticosteroid sensitivity in peripheral blood mononuclear cells from chronic obstructive pulmonary disease (COPD) patients, and overexpression of mTOR suppresses cigarette smoke-induced inflammation and emphysema, suggesting that induction of mTOR expression/activity might be useful to treat COPD. This apparent discrepancy is due to complex and heterogenic enzymatic pathway of mTOR. Translation of pre-clinical positive data on the use of mTOR inhibitors to COPD therapy needs a more in-depth knowledge of mTOR signalling

Altered vascular endothelium-dependent responsiveness in frail elderly patients recovering from {COVID}-19 pneumonia: preliminary evidence

We evaluated vascular dysfunction with the single passive leg movement test (sPLM) in 22 frail elderly patients at 84 + 31 days after hospitalization for COVID-19 pneumonia, compared to 22 age-, sex- and comorbidity-matched controls (CTRL). At rest, all COVID-19 patients were in stable clinical condition without severe comorbidities. Patients (aged 72 ± 6 years, 73% male) had moderate disability (Barthel index score 77 ± 26), hypoxemia and normocapnia at arterial blood gas analysis and mild pulmonary restriction at spirometry. Values of circulating markers of inflammation (C-reactive protein: CRP; erythrocyte sedimentation rate: ESR) and coagulation (D-dimer) were: 27.13 ± 37.52 mg/dL, 64.24 ± 32.37 mm/1 h and 1043 ± 729 ng/mL, respectively. At rest, femoral artery diameter was similar in COVID-19 and CTRL (p = 0.16). On the contrary, COVID-19 infection deeply impacted blood velocity (p = 0.001) and femoral blood flow (p < 0.0001). After sPLM, peak femoral blood flow was dramatically reduced in COVID-19 compared to CTRL (p = 0.001), as was blood flow ∆peak (p = 0.05) and the area under the curve (p < 0.0001). This altered vascular responsiveness could be one of the unknown components of long COVID-19 syndrome leading to fatigue, changes in muscle metabolism and fibers’ composition, exercise intolerance and increased cardiovascular risk. Impact of specific treatments, such as exercise training, dietary supplements or drugs, should be evaluated

Metabolic syndrome alters inflammatory and membrane markers expression in human atrium cardiomyocytes

Metabolic syndrome (MetS) is a cluster of various clinical cardiovascular risk factor and causes metabolic and structural cardiomyocytes damage. Our previous study showed that MetS increases cardiomyocytes stress chaperones (1). In this work we aimed to investigate if patients with MetS showed alteration of surface, mitochondria and inflammatory markers in atrium cardiomyocytes. Atrium samples from MetS patients with stable angina, undergoing coronary artery bypass graft surgery were used. Samples from matched age subjects without MetS and no smokers, undergoing cardiac surgery for other reasons, were used as controls. The samples, obtained before cardioplegia, were fixed and processed for Caveolin 1 (Cav1), MURC, Citrate Synthase (CS), SIRT3, SOD1, IL6-10, iNOS and eNOS by immunohistochemistry. Compared to controls, in cardiomyocytes from MetS patients decreased the expression of Cav1, MURC, SIRT3, IL10 and eNOS, whereas increased the expression of CS, SOD1, IL6, iNOS. Cardiomyocytes from MetS patients present an evident inflammatory chronic state, that alters the Cav1-MURC expression. These alterations could reduce the exchange functions of sarcolemma so impairing the contractile capacity associated with mitochondrial impairment. We speculate that these damages could induce antioxidant SOD1 over-expression to rescue the cells. All these results, first from human heart, suggest that MetS induces severe enzymatic disregulation in atrium cardiomyocytes that may predispose to cardiac surgery complications

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.

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

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