Claudia Romano

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 (FGFs). 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 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.

Protein-Amino Acid Metabolism Disarrangements: The Hidden Enemy of Chronic Age-Related Conditions

Proteins are macro-molecules crucial for cell life, which are made up of amino acids (AAs). In healthy people, protein synthesis and degradation are well balanced. However, in the presence of hypercatabolic stimulation (i.e., inflammation), protein breakdown increases as the resulting AAs are consumed for metabolic proposes. Indeed, AAs are biochemical totipotent molecules which, when deaminated, can be transformed into energy, lipids, carbohydrates, and/or biochemical intermediates of fundamental cycles, such as the Krebs’ cycle. The biochemical consequence of hyper-catabolism is protein disarrangement, clinically evident with signs such as sarcopenia, hypalbuminemia, anaemia, infection, and altered fluid compartmentation, etc. Hypercatabolic protein disarrangement (HPD) is often underestimated by clinicians, despite correlating with increased mortality, hospitalization, and morbidity quite independent of the primary disease. Simple, cheap, repeatable measurements can be used to identify HPD. Therefore, identification and treatment of proteins’ metabolic impairment with appropriate measurements and therapy is a clinical strategy that could improve the prognosis of patients with acute/chronic hypercatabolic inflammatory disease. Here, we describe the metabolism of protein and AAs in hypercatabolic syndrome, illustrating the clinical impact of protein disarrangement. We also illustrate simple, cheap, repeatable, and worldwide available measurements to identify these conditions. Finally, we provide scientific evidence for HPD nutritional treatment.

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

Abstract Chronic wounds are a major, often underestimated, health problem for the elderly. Standard wound care products are not usually manufactured to meet 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 TGF&bgr;1. The results showed wound healing was accelerated in EAAs‐fed rats, which was accompanied by reduced inflammation and changes in TGF&bgr;1 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. HighlightsThe nutritional status is an important factor in promoting wound healing.Topical dressing usually does not provide nutrients to the wound.Nutrition with stoichiometric mixture of essential amino acid could promote healing without dressing.Nutrition with essential amino acid reduces inflammation, fibrosis and healing time.

Metabolic syndrome and chronic simvastatin therapy enhanced human cardiomyocyte stress before and after ischemia-reperfusion in cardio-pulmonary bypass patients.

Metabolic syndrome (MetS) is a set of metabolic alterations including high levels of low-density lipoprotein (LDL), which increase the risk of cardiomyopathy often leading to surgery. Despite inducing myopathy, statins are widely used to lower LDL. Cardiopulmonary bypass (Cpb) causes oxidative stress and metabolic injury, altering mitochondrial expression (Grp75) and endoplasmic reticulum (Grp78) chaperones, apoptotic enzymes (Bcl2 family) and increasing cardiomyocyte lipid/lipofuscin storage. We believe that cardiomyocytes from patients with MetS may be more sensitive to surgical stress, in particular after simvastatin therapy (MetS+Stat). The study group included ten patients with MetS, ten patients with Mets+Stat and ten healthy subjects. Myocardial biopsies were obtained both before and after-Cpb. Grp75, Grp78, Bax, Bcl2, lipids, lipofuscin and fibrosis were evaluated by immuno/histochemistry. MetS cardiomyocytes had higher Grp75, Bax, fibrosis and lipofuscin. MetS+Stat had lower Grp75 and higher Grp78 expressions, high Bax, fewer fibrosis and higher lipofuscin content. Cpb did not vary the fibrosis and lipids/lipofuscin content, although it influenced the chaperones and Bax expression in all groups. These changes were more profound in patients with MetS and even more so in patients with MetS+Stat. The results suggest that MetS and MetS+Stat cardiomyocytes were more highly stressed after-Cpb. Interestingly, simvastatin caused high stress even before-Cpb.

Aging Skin: Nourishing from the Inside Out, Effects of Good Versus Poor Nitrogen Intake on Skin Health and Healing

Skin is the outermost defense organ which protects us from the environment, constituting around 8 % of an adult’s body weight. Healthy skin contains one-eighth of the body’s total proteins. The balance of turnover and synthesis of skin proteins is primarily dependent on the availability of sufficient nitrogen-containing substrates, namely, amino acids, essential for protein metabolism in any other tissue and body organs. The turnover of skin proteins has been shown to be rapid, and the mobilization of amino acids at the expense of skin proteins is relevant in experimental models of protein malnutrition. As a result, alterations in nutritional status should be suspected, diagnosed, and eventually treated for any skin lesions. Protein malnutrition has a dramatic prevalence in patients aged >70 or more, independent of the reason for hospitalization. The quality of nutrition and content of essential amino acids are strictly connected to skin health and integrity of its protein components. Collagen fiber deposition is highly and rapidly influenced by alterations in the essential to nonessential amino acid ratios. The most relevant nutritional factor of skin health is the prevalence of essential amino acids.

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.

Is the Response of Tumours Dependent on Dietary Input of Some Amino Acids or on the Ratios Among Essential and Non-Essential Amino Acids? All That Glisters is Not Gold

Production of energy is a main task of cancer cells metabolism, since costs of duplicating are enormous. Although energy is derived in cells by dismantling carbon to carbon bonds of any macronutrient, cancer nutritional needs for energetic purposes have been studied primarily as dependent on glycolysis. Since the end of the last century, awareness of dependence of cancer metabolism on amino acids not only for protein syntheses but also for matching energy needs has grown. The roles of specific amino acids, like glutamine, glycine and serine have been explored in different experimental conditions and reviewed. Moreover, there are epidemiological evidences that some amino acids used as supplement for therapeutic reasons (the branched chain ones) may reduce incidence of liver cancer, and some molecular mechanism has been proposed as functional to their protective action. On the contrary, metabolic signature of some pathology connected with increased risk of cancer, like prolonged hyperinsulinemia in insulin resistant patients, is signed by plasma elevated levels of the same branched chain amino acids, posing puzzling questions to clinicians. Most recently, peculiar formulations of amino acids, deeply different if compared to amino acids compositions normally present in foods, have shown the power to master epigenetics slowing growth or driving cancer cells to apoptotic death, while being even beneficial for normal cells and for animals health and life span. In this review, we will analyze and try to disentangle some of the many knots dealing with complexities of amino acids biology and linked to cancer metabolism.

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 3 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.

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.

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.