Functional Activity of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes on a Mouse Renal Subcapsular Xenograft Model.

In the treatment of coronary heart disease, the most promising approach for replacing lost contractile elements involves obtaining cardiomyocytes through cardiac differentiation of pluripotent cells. The objective of this study is to develop a technology for creating a functional layer of cardiomyocytes derived from iPSCs, capable of generating rhythmic activity and synchronous contractions. To expedite the maturation of cardiomyocytes, a renal subcapsular transplantation model was employed in SCID mice. Following explantation, the formation of the cardiomyocyte contractile apparatus was assessed using fluorescence and electron microscopy, while the cytoplasmic oscillation of calcium ions was evaluated through visualization using the fluorescent calcium binding dye Fluo-8. The results demonstrate that transplanted human iPSC-derived cardiomyocyte cell layers, placed under the fibrous capsules of SCID mouse kidneys (for up to 6 weeks), initiate the development of an organized contractile apparatus and retain functional activity along with the ability to generate calcium ion oscillations even after removal from the body.

Derivation of Human Corneal Keratocytes from ReLEx SMILE Lenticules for Cell Therapy and Tissue Engineering.

Fibroblasts isolated and expanded from ReLEx SMILE lenticules can be a source of human keratocytes. Since corneal keratocytes are quiescent cells, it is difficult to expand them in vitro in suitable numbers for clinical and experimental use. In the present study, this problem was solved by isolating and growing corneal fibroblasts (CFs) with a high proliferative potential and their reversion to keratocytes in a selective serum-free medium. Fibroblasts reversed into keratocytes (rCFs) had a dendritic morphology and ultrastructural signs of activation of protein synthesis and metabolism. The cultivation of CFs in a medium with 10% FCS and their reversion into keratocytes was not accompanied by the induction of myofibroblasts. After reversion, the cells spontaneously formed spheroids and expressed keratocan and lumican markers, but not mesenchymal ones. The rCFs had low proliferative and migratory activity, and their conditioned medium contained a low level of VEGF. CF reversion was not accompanied by a change with the levels of IGF-1, TNF-alpha, SDF-1a, and sICAM-1. In the present study, it has been demonstrated that fibroblasts from ReLEx SMILE lenticules reverse into keratocytes in serum-free KGM, maintaining the morphology and functional properties of primary keratocytes. These keratocytes have a potential for tissue engineering and cell therapy of various corneal pathologies.

Trehalose Activates Hepatic and Myocardial Autophagy and Has Anti-Inflammatory Effects in db/db Diabetic Mice.

Db/db mice (carrying a mutation in the gene encoding leptin receptor) show autophagy suppression. Our aim was to evaluate the effect of autophagy inducer trehalose on liver and heart autophagy in db/db mice and to study inflammation dysregulation and the suitability of chitinases' expression levels as diabetes markers. Thirty-eight male db/db mice and C57/BL mice (control) were used. The db/db model manifested inflammation symptoms: overexpression of TNF-α in the spleen and underexpression of IL-10 in the liver and spleen (cytokine imbalance). Simultaneously, we revealed decreased expression of chitotriosidase (CHIT1) and acid mammalian chitinase (CHIA) in the liver of db/db mice. CHIA expression in db/db mice is significantly lower only in the spleen. Trehalose treatment significantly reduced blood glucose concentration and glycated hemoglobin. Treatment of db/db mice by trehalose was followed by increased autophagy induction in the heart and liver (increased autolysosomes volume density studied by morphometric electron-microscopic method). Trehalose exerted beneficial cardiac effects possibly via increased lipophagy (uptake of lipid droplets). The autophagy activation by trehalose had several positive effects on the heart and liver of db/db mice; therefore, lipophagy activation seems to be a promising therapy for diabetes.

Treatment with Autophagy Inducer Trehalose Alleviates Memory and Behavioral Impairments and Neuroinflammatory Brain Processes in db/db Mice.

Autophagy attenuation has been found in neurodegenerative diseases, aging, diabetes mellitus, and atherosclerosis. In experimental models of neurodegenerative diseases, the correction of autophagy in the brain reverses neuronal and behavioral deficits and hence seems to be a promising therapy for neuropathologies. Our aim was to study the effect of an autophagy inducer, trehalose, on brain autophagy and behavior in a genetic model of diabetes with signs of neuronal damage (db/db mice). A 2% trehalose solution was administered as drinking water during 24 days of the experiment. Expressions of markers of autophagy (LC3-II), neuroinflammation (IBA1), redox state (NOS), and neuronal density (NeuN) in the brain were assessed by immunohistochemical analysis. For behavioral phenotyping, the open field, elevated plus-maze, tail suspension, pre-pulse inhibition, and passive avoidance tests were used. Trehalose caused a slight reduction in increased blood glucose concentration, considerable autophagy activation, and a decrease in the neuroinflammatory response in the brain along with improvements of exploration, locomotor activity, anxiety, depressive-like behavior, and fear learning and memory in db/db mice. Trehalose exerted some beneficial peripheral and systemic effects and partially reversed behavioral alterations in db/db mice. Thus, trehalose as an inducer of mTOR-independent autophagy is effective at alleviating neuronal and behavioral disturbances accompanying experimental diabetes.

Hypolipidemic Effects of ß-Glucans, Mannans, and Fucoidans: Mechanism of Action and Their Prospects for Clinical Application.

The search for lipid-lowering drugs is important for clinical medicine. This review summarizes our research findings regarding the hypolipidemic activity of polysaccharides. There are several validated agents altering lipid levels which reduce the risk of atherosclerotic cardiovascular events. Nonetheless, for many people, the risk of such an event remains unacceptably high despite treatment with these agents. This situation has prompted the search for new therapies to reduce the residual cardiovascular risk. The lipid-lowering effect of ß-glucans consumed with food was demonstrated in patients with atherosclerosis. The mechanism of the protective effect of ß-glucans is poorly studied. The effects of ß-glucans are mediated by Toll-like receptors, by dectin-1, and possibly by other receptors. Nevertheless, the mechanism of the protective action of ß-glucan in lipemic mice has been studied insufficiently. This review will present up-to-date information regarding experimental hypolipidemic polysaccharide compounds that hold promise for medicine. Phagocyte-specific chitotriosidase in humans contributes to innate immune responses against chitin-containing fungi. This enzyme has been first described in patients with Gaucher disease and serves as an important diagnostic biomarker. It has been reported that, in mice, chitin particles of certain size are recognized by macrophages through Toll-like receptors, dectin-1, and to a lesser extent through mannose receptor.

SGLT2 Inhibitor Empagliflozin and DPP4 Inhibitor Linagliptin Reactivate Glomerular Autophagy in db/db Mice, a Model of Type 2 Diabetes.

Recent data have indicated the emerging role of glomerular autophagy in diabetic kidney disease. We aimed to assess the effect of the SGLT2 inhibitor empagliflozin, the DPP4 inhibitor linagliptin, and their combination, on glomerular autophagy in a model of type 2 diabetes. Eight-week-old male db/db mice were randomly assigned to treatment with empagliflozin, linagliptin, empagliflozin-linagliptin or vehicle for 8 weeks. Age-matched non-diabetic db/+ mice acted as controls. To estimate glomerular autophagy, immunohistochemistry for beclin-1 and LAMP-1 was performed. Podocyte autophagy was assessed by counting the volume density (Vv) of autophagosomes, lysosomes and autolysosomes by transmission electron microscopy. LC3B and LAMP-1, autophagy markers, and caspase-3 and Bcl-2, apoptotic markers, were evaluated in renal cortex by western blot. Vehicle-treated db/db mice had weak glomerular staining for beclin-1 and LAMP-1 and reduced Vv of autophagosomes, autolysosomes and lysosomes in podocytes. Empagliflozin and linagliptin, both as monotherapy and in combination, enhanced the areas of glomerular staining for beclin-1 and LAMP-1 and increased Vv of autophagosomes and autolysosomes in podocytes. Renal LC3B and Bcl-2 were restored in actively treated animals. LAMP-1 expression was enhanced in the empagliflozin group; caspase-3 expression decreased in the empagliflozin-linagliptin group only. Mesangial expansion, podocyte foot process effacement and urinary albumin excretion were mitigated by both agents. The data provide further explanation for the mechanism of the renoprotective effect of SGLT2 inhibitors and DPP4 inhibitors in diabetes.

Empagliflozin alleviates podocytopathy and enhances glomerular nephrin expression in db/db diabetic mice.

BACKGROUND: Modern guidelines recommend sodium-glucose cotransporter-2 (SGLT2) inhibitors as the preferred antihyperglycemic agents for patients with type 2 diabetes and chronic kidney disease. However, the mechanisms underlying the renal protective effect of SGLT2 inhibitors are not fully understood. AIM: To estimate the effect of the SGLT2 inhibitor, empagliflozin (EMPA), on the structure of podocytes and nephrin expression in glomeruli in db/db diabetic mice. METHODS: We treated 8-wk-old male db/db mice with EMPA (10 mg/kg/d) or vehicle for 8 wk. Age-matched male db/+ mice were included as non-diabetic controls. Parameters of body composition, glycemic and lipid control, and plasma concentrations of leptin, insulin and glucagon were assessed. We evaluated renal hypertrophy as kidney weight adjusted to lean mass, renal function as plasma levels of creatinine, and albuminuria as the urinary albumin-to-creatinine ratio (UACR). Renal structures were studied by light and transmission electron microscopy with a focus on mesangial volume and podocyte structure, respectively. Glomerular nephrin and transforming growth factor beta (TGF-ß) were assessed by immunohistochemistry. RESULTS: Severe obesity and hyperglycemia developed in db/db mice prior to the start of the experiment; increased plasma concentrations of fructosamine, glycated albumin, cholesterol, leptin, and insulin, and elevated UACR were detected. Mesangial expansion, glomerular basement membrane thickening, and increased area of TGF-ß staining in glomeruli were revealed in vehicle-treated mice. Podocytopathy was manifested by effacement of foot processes; nephrin-positive areas in glomeruli were reduced. EMPA decreased the levels of glucose, fructosamine and glycated albumin, UACR, kidney hypertrophy, mesangial expansion, glomerular basement membrane thickening, and glomerular TGF-ß staining, alleviated podocytopathy and restored glomerular staining of nephrin. CONCLUSION: These data indicate that EMPA attenuates podocytopathy in experimental diabetic kidney disease. The anti-albuminuric effect of EMPA could be attributed to mitigation of podocyte injury and enhancement of nephrin expression.

Glucan and Mannan-Two Peas in a Pod.

In recent decades, various polysaccharides isolated from algae, mushrooms, yeast, and higher plants have attracted serious attention in the area of nutrition and medicine. The reasons include their low toxicity, rare negative side effects, relatively low price, and broad spectrum of therapeutic actions. The two most and best-studied polysaccharides are mannan and glucan. This review focused on their biological properties.

A comparative study of the hypolipidaemic effects of a new polysaccharide, mannan Candida albicans serotype A, and atorvastatin in mice with poloxamer 407-induced hyperlipidaemia.

OBJECTIVES: We evaluated the hypolipidaemic effect of mannan Candida albicans serotype A, relative to atorvastatin, in a mouse model of hyperlipidaemia. METHODS: Mannan serotype A was investigated in vitro and in vivo to determine its effects on macrophage proliferation, nitric oxide (NO) production by cultured macrophages, serum and liver lipids, changes in liver morphology and serum chitotriosidase activity and its expression in the liver. KEY FINDINGS: Mannan serotype A stimulates the macrophage proliferation and NO production in murine peritoneal macrophages in vitro. The activity of serum chitotriosidase (an enzyme released from the activated macrophages) was found to be significantly increased in P-407-induced hyperlipidaemic mice pretreated with low-dose mannan compared with mice administered P-407 only. Mannan treatment in mice was shown to significantly increase the chitotriosidase expression in the liver of both non-hyperlipidaemic and P-407-induced hyperlipidaemic mice. Lastly, mice pretreated with mannan before the induction of hyperlipidaemia with P-407 showed a significant reduction in the serum concentration of atherogenic LDL cholesterol, total cholesterol, triglycerides and liver triglycerides. CONCLUSIONS: It is suggested that mannan serotype A, like ß-glucan, may represent another hypolipidaemic agent, which could potentially be used as an adjunctive therapy with conventional antihyperlipidaemic drugs (statins and fibrates) in humans.