Dapagliflozin prevents ERK activation and SGLT2-dependent endoglin upregulation in a mechanically provoked cardiac injury model.

Sodium-glucose cotransporter 2 inhibitors (SGLT2i) are rapidly gaining ground in the treatment of heart failure (HF) with reduced ejection fraction (HFrEF) and acute myocardial infarction (AMI) by an unknown mechanism. Upregulation of Na+/H+ exchanger 1 (NHE1), SGLT1, and Ca2+/calmodulin-dependent protein kinase II (CaMKII) in the diseased hearts was found to be attenuated by prolonged SGLT2i treatment. Unfortunately, dapagliflozin is not well understood as to how Na+/Ca2+ homeostasis is affected in cardiomyocytes. In this study, we aimed to investigate whether mechanical stretch in cardiomyocytes upregulate SGLT2, resulted to loss of Na+/Ca2+ homeostasis via ERK and eNOS signaling. AMI (+) and AMI (-) serum levels were estimated using ELISA assays of TGFß-1 or endoglin (CD105). Human cardiomyocyte cell line AC16 was subjected to different stresses: 5% mild and 25% aggressive, at 1 Hz for 24 h. Immunofluorescence assays were used to estimate troponin I, CD105, SGLT1/2, eNOSS633, and ERK1/2T202/Y204 levels was performed for 5% (mild), and 25% elongation for 24 h. AMI (+) serum showed increased TGFß1 and CD105 compared to AMI (-) patients. In consistent, troponin I, CD105, SGLT1/2, eNOSS633 and ERK1/2T202/Y204 were upregulated after 25% of 24 h cyclic stretch. Dapagliflozin addition caused SGLT2 inhibition, which significantly decreased troponin I, CD105, SGLT1/2, eNOSS633, and ERK1/2T202/Y204 under 25% cyclic stretching. In summary, SGLT2 may have sensed mechanical stretch in a way similar to cardiac overloading as in vivo. By blocking SGLT2 in stretched cardiomyocytes, the AMI biomarkers (CD105, troponin I and P-ERK) were decreased, potentially to rescue eNOS production to maintain normal cellular function. This discovery of CD105 and SGLT2 increase in mechanically stretched cardiomyocytes suggests that SGLT2 may conceive a novel role in direct or indirect sensing of mechanical stretch, prompting the possibility of an in vitro cardiac overloaded cell model, an alternative to animal heart model.

Assessment of Tilapia Skin Collagen for Biomedical Research Applications in Comparison with Mammalian Collagen.

Collagen is an important material for biomedical research, but using mammalian tissue-derived collagen carries the risk of zoonotic disease transmission. Marine organisms, such as farmed tilapia, have emerged as a safe alternative source of collagen for biomedical research. However, the tilapia collagen products for biomedical research are rare, and their biological functions remain largely unexamined. In this study, we characterized a commercial tilapia skin collagen using SDS-PAGE and fibril formation assays and evaluated its effects on skin fibroblast adhesion, proliferation, and migration, comparing it with commercial collagen from rat tails, porcine skin, and bovine skin. The results showed that tilapia skin collagen is a type I collagen, similar to rat tail collagen, and has a faster fibril formation rate and better-promoting effects on cell migration than porcine and bovine skin collagen. We also confirmed its application in a 3D culture for kidney cells' spherical cyst formation, fibroblast-induced gel contraction, and tumor spheroid interfacial invasion. Furthermore, we demonstrated that the freeze-dried tilapia skin collagen scaffold improved wound closure in a mouse excisional wound model, similar to commercial porcine or bovine collagen wound dressings. In conclusion, tilapia skin collagen is an ideal biomaterial for biomedical research.

Microglial activation and toll-like receptor 4-Dependent regulation of angiotensin II type I receptor-mu-opioid receptor 1 heterodimerization and hypertension in fructose-fed rats.

Our previous study reported that the heterodimer of Angiotensin II Type I Receptor (AT1R) and Mu-Opioid Receptor 1 (MOR1) involves Nitric Oxide (NO) reduction which leads to elevation of blood pressure. Secondly, we showed that Toll-like Receptor 4 (TLR4) may be involved in the heterodimerization of AT1R and MOR1 in the brainstem Nucleus Tractus Solitarii (NTS), which regulates systemic blood pressure and gastric nitric oxide through the insulin pathway. Here, we investigated the role of microglial activation and TLR4 in the heterodimerization of AT1R and MOR1. Hypertensive rats were established after four weeks of fructose consumption. SBP of rats was measured using non-invasive blood pressure method. PLA technique was utilized to determine protein-protein interaction in the nucleus tractus solitarii. Results showed that the level of MOR-1 and AT1R was induced significantly in the fructose group compared with control. PLA signal potentially showed that AT1R and MOR1 were formed in the nucleus tractus solitarii after fructose consumption. Meanwhile, the innate immune cell in the CNS microglia was observed in the nucleus tractus solitarii using biomarkers and was activated. TLR4 inhibitor CLI-095, was administered to animals to suppress the neuroinflammation and microglial activation. CLI-095 treatment reduced the heterodimer formation of AT1R and MOR1 and restored nitric oxide production in the nucleus tractus solitarii. These findings imply that TLR4-primed neuroinflammation involves formation of heterodimers AT1R and MOR1 in the nucleus tractus solitarii which leads to increase in systemic blood pressure.

Mechanical Stretching-Induced Traumatic Brain Injury Is Mediated by the Formation of GSK-3ß-Tau Complex to Impair Insulin Signaling Transduction.

Traumatic brain injury confers a significant and growing public health burden. It is a major environmental risk factor for dementia. Nonetheless, the mechanism by which primary mechanical injury leads to neurodegeneration and an increased risk of dementia-related diseases is unclear. Thus, we aimed to investigate the effect of stretching on SH-SY5Y neuroblastoma cells that proliferate in vitro. These cells retain the dopamine-ß-hydroxylase activity, thus being suitable for neuromechanistic studies. SH-SY5Y cells were cultured on stretchable membranes. The culture conditions contained two groups, namely non-stretched (control) and stretched. They were subjected to cyclic stretching (6 and 24 h) and 25% elongation at 1 Hz. Following stretching at 25% and 1 Hz for 6 h, the mechanical injury changed the mitochondrial membrane potential and triggered oxidative DNA damage at 24 h. Stretching decreased the level of brain-derived neurotrophic factors and increased amyloid-ß, thus indicating neuronal stress. Moreover, the mechanical injury downregulated the insulin pathway and upregulated glycogen synthase kinase 3ß (GSK-3ß)S9/p-Tau protein levels, which caused a neuronal injury. Following 6 and 24 h of stretching, GSK-3ßS9 was directly bound to p-TauS396. In contrast, the neuronal injury was improved using GSK-3ß inhibitor TWS119, which downregulated amyloid-ß/p-Taus396 phosphorylation by enhancing ERK1/2T202/Y204 and AktS473 phosphorylation. Our findings imply that the neurons were under stress and that the inactivation of the GSK3ß could alleviate this defect.

Systems biology approach to exploring the effect of cyclic stretching on cardiac cell physiology.

Although mechanical forces are involved in pressure-overloaded cardiomyopathy, their effects on gene transcription profiles are not fully understood. Here, we used next-generation sequencing (NGS) to investigate changes in genomic profiles after cyclic mechanical stretching of human cardiomyocytes. We found that 85, 87, 32, 29, and 28 genes were differentially expressed after 1, 4, 12, 24, and 48 hours of stretching. Furthermore, 10 of the 29 genes that were up-regulated and 11 of the 28 that were down-regulated after 24 h showed the same changes after 48 h. We then examined expression of the genes that encode serpin family E member 1 (SERPINE1), DNA-binding protein inhibitor 1 (ID1), DNA-binding protein inhibitor 3 (ID3), and CCL2, a cytokine that acts as chemotactic factor in monocytes, in an RT-PCR experiment. The same changes were observed for all four genes after all cyclic stretching durations, confirming the NGS results. Taken together, these findings suggest that cyclical stretching can alter cardiac cell physiology by activating cardiac cell metabolism and impacting cholesterol biosynthesis signaling.

Vitamin D Attenuates Loss of Endothelial Biomarker Expression in Cardio-Endothelial Cells.

Vitamin D is associated with cardiovascular health through activating the vitamin D receptor that targets genes related to cardiovascular disease (CVD). The human cardiac microvascular endothelial cells (HCMECs) were used to develop mechanically and TGF-ß1-induced fibrosis models, and the rat was used as the isoproterenol (ISO)-induced fibrosis model. The rats were injected with ISO for the first five days, followed by vitamin D injection for the consecutive three weeks before being sacrificed on the fourth week. Results showed that mechanical stretching reduced endothelial cell marker CD31 and VE-cadherin protein expressions, as well as increased α-smooth muscle actin (α-SMA) and fibronectin (FN). The transforming growth factor-ß1 (TGF-ß1) reduced CD31, and increased α-SMA and FN protein expression levels. Vitamin D presence led to higher protein expression of CD31, and lower protein expressions of α-SMA and FN compared to the control in the TGF-ß1-induced fibrosis model. Additionally, protein expression of VE-cadherin was increased and fibroblast-specific protein-1 (FSP1) was decreased after vitamin D treatment in the ISO-induced fibrosis rat. In conclusion, vitamin D slightly inhibited fibrosis development in cell and animal models. Based on this study, the beneficial effect of vitamin D may be insignificant; however, further investigation of vitamin D's effect in the long-term is required in the future.

Angiotensin II inhibits DDAH1-nNOS signaling via AT1R and µOR dimerization to modulate blood pressure control in the central nervous system.

G protein-coupled receptors (GPCRs) are important drug targets. Blocking angiotensin II (Ang II) type 1 receptor signaling alleviates hypertension and improves outcomes in patients with heart failure. Changes in structure and trafficking of GPCR, and desensitization of GPCR signaling induce pathophysiological processes. We investigated whether Ang II, via induction of AT1R and µ-opioid receptor (µOR) dimerization in the nucleus tractus solitarius (NTS), leads to progressive hypertension. Ang II signaling increased µOR and adrenergic receptor α2A (α2A-AR) heterodimer levels and decreased expression of extracellular signal-regulated kinases 1/2T202/Y204, ribosomal protein S6 kinaseT359/S363, and nNOSS1416 phosphorylation. Dimethylarginine dimethylaminohydrolase 1 (DDAH1) expression was abolished in the NTS of adult spontaneously hypertensive rats (SHRs). Endomorphin-2 was overexpressed in NTS of adult SHRs compared with that in 6-week-old Wistar-Kyoto rats (WKY). Administration of µOR agonist into the NTS of WKY increased blood pressure (BP), decreased nitric oxide (NO) production, and decreased DDAH1 activity. µOR agonist significantly reduced the activity of DDAH1 and decreased neuronal NO synthase (nNOS) phosphorylation. The AT1R II inhibitor, losartan, significantly decreased BP and abolished AT1R-induced formation of AT1R and µOR, and α2A-AR and µOR, heterodimers. Losartan also significantly increased the levels of nNOSS1416 phosphorylation and DDAH1 expression. These results show that Ang II may induce expression of endomorphin-2 and abolished DDAH1 activity by enhancing the formation of AT1R and µOR heterodimers in the NTS, leading to progressive hypertension.

Dapagliflozin Prevents NOX- and SGLT2-Dependent Oxidative Stress in Lens Cells Exposed to Fructose-Induced Diabetes Mellitus.

PURPOSE: Cataracts in patients with diabetes mellitus (DM) are a major cause of blindness in developed and developing countries. This study aims to examine whether the generation of reactive oxygen species (ROS) via the increased expression of glucose transporters (GLUTs) and the receptor for advanced glycation end products (RAGE) influences the cataract development in DM. METHODS: Lens epithelial cells (LECs) were isolated during cataract surgery from patients without DM or with DM, but without diabetic retinopathy. In a rat model, fructose (10% fructose, 8 or 12 weeks) with or without dapagliflozin (1.2 mg/day, 2 weeks) treatment did induce DM, as verified by blood pressure and serum parameter measurements. Immunofluorescence stainings and immunoblottings were used to quantify the protein levels. Endogenous O2˙¯ production in the LECs was determined in vivo with dihydroethidium stainings. RESULTS: We investigated that GLUT levels in LECs differed significantly, thus leading to the direct enhancement of RAGE-associated superoxide generation in DM patients with cataracts. Superoxide production was significantly higher in LECs from rats with fructose-induced type 2 DM, whereas treatment with the sodium-glucose cotransporter 2 (SGLT2) inhibitor dapagliflozin prevented this effect in fructose-fed rats. Protein expression levels of the sodium/glucose cotransporter 2 (SGLT2), GLUT1, GLUT5, the nicotinamide adenine dinucleotide phosphate reduced form (NADPH) oxidase subunit p67-phox, NOX2/4 and RAGE were upregulated in fructose-fed animals, whereas dapagliflozin treatment reversed these effects. CONCLUSIONS: In rats with fructose-induced DM, dapagliflozin downregulates RAGE-induced NADPH oxidase expression in LECs via the inactivation of GLUTs and a reduction in ROS generation. These novel findings suggest that the SGLT2 inhibitor dapagliflozin may be a candidate for the pharmacological prevention of cataracts in patients with DM.

Closer to Nature Through Dynamic Culture Systems.

Mechanics in the human body are required for normal cell function at a molecular level. It is now clear that mechanical stimulations play significant roles in cell growth, differentiation, and migration in normal and diseased cells. Recent studies have led to the discovery that normal and cancer cells have different mechanosensing properties. Here, we discuss the application and the physiological and pathological meaning of mechanical stimulations. To reveal the optimal conditions for mimicking an in vivo microenvironment, we must, therefore, discern the mechanotransduction occurring in cells.

Mechanical Stretching Simulates Cardiac Physiology and Pathology through Mechanosensor Piezo1.

The dynamics of a living body enables organs to experience mechanical stimulation at cellular level. The human cardiomyocytes cell line provides a source for simulating heart dynamics; however, a limited understanding of the mechanical stimulation effect on them has restricted potential applications. Here, we investigated the effect of mechanical stimulation on the cardiac function-associated protein expressions in human cardiomyocytes. Human cardiomyocyte cell line AC16 was subjected to different stresses: 5% mild and 25% aggressive, at 1 Hz for 24 h. The stretched cardiomyocytes showed down-regulated Piezo1, phosphorylated-Ak transforming serine473 (P-AKTS473), and phosphorylated-glycogen synthase kinase-3 beta serine9 P-GSK3ßS9 compared to no stretch. In addition, the stretched cardiomyocytes showed increased low-density lipoprotein receptor-related protein 6 (LRP6), and phosphorylated-c-Jun N-terminal kinase threonine183/tyrosine185 (P-JNKT183/Y185). When Piezo inhibitor was added to the cells, the LRP6, and P-JNKT183/Y185 were further increased under 25%, but not 5%, suggesting that higher mechanical stress further activated the wingless integrated-(Wnt)-related signaling pathway when Piezo1 was inhibited. Supporting this idea, when Piezo1 was inhibited, the expression of phosphorylated-endothelial nitric oxide synthase serine1177 (P-eNOSS1177) and release of calcium ions were reduced under 25% compared to 5%. These studies demonstrate that cyclic mechanical stimulation affects cardiac function-associated protein expressions, and Piezo1 plays a role in the protein regulation.

Effect of Resveratrol on Reactive Oxygen Species-Induced Cognitive Impairment in Rats with Angiotensin II-Induced Early Alzheimer's Disease †.

Recent studies have indicated that several anti-hypertensive drugs may delay the development and progression of Alzheimer's disease (AD). However, the relationships among AD, hypertension, and oxidative stress remain to be elucidated. Here, we aimed to determine whether reactive oxygen species (ROS) reduction by resveratrol in the brain leads to cognitive impairment reduction in rats with angiotensin II (Ang-II)-induced early AD. Male Wistar Kyoto (WKY) rats with Ang-II-induced AD were treated with losartan or resveratrol for two weeks. Our results show decreased blood pressure, increased hippocampal brain-derived neurotrophic factor (BDNF) level, and decreased nucleus tractus solitarius (NTS) ROS production in the Ang-II groups with losartan (10 mg/kg), or resveratrol (10 mg/kg/day) treatment. Furthermore, losartan inhibition of hippocampal TauT231 phosphorylation activated AktS473 phosphorylation, and significantly abolished Ang-II-induced Aß precursors, active caspase 3, and glycogen synthase kinase 3ß (GSK-3ß)Y216 expressions. Consistently, resveratrol showed similar effects compared to losartan. Both losartan and resveratrol restored hippocampal-dependent contextual memory by NADPH oxidase 2 (NOX2) deletion and superoxide dismutase 2 (SOD2) elevation. Our results suggest that both losartan and resveratrol exert neuroprotective effects against memory impairment and hippocampal damage by oxidative stress reduction in early stage AD rat model. These novel findings indicate that resveratrol may represent a pharmacological option similar to losartan for patients with hypertension at risk of AD during old age.

Hyaluronan keeps mesenchymal stem cells quiescent and maintains the differentiation potential over time.

Hyaluronan (HA), an abundant polysaccharide found in human bodies, plays a role in the mesenchymal stem cells (MSCs) maintenance. We had previously found that HA prolonged the lifespan, and prevented the cellular aging of murine adipose-derived stromal cells. Recently, we had also summarized the potential pathways associated with HA regulation in human MSCs. In this study, we used the human placenta-derived MSCs (PDMSC) to investigate the effectiveness of HA in maintaining the PDMSC. We found that coating the culture surface coated with 30 µg cm-2 of HA (C) led to cluster growth of PDMSC, and maintained a higher number of PDMSC in quiescence compared to those grown on the normal tissue culture surface (T). PDMSC were treated for either 4 (short-term) or 19 (long-term) consecutive passages. PDMSC which were treated with HA for 19 consecutive passages had reduced cell enlargement, preserved MSCs biomarker expressions and osteogenic potential when compared to those grown only on T. The PDMSC transferred to T condition after long-term HA treatment showed preserved replicative capability compared to those on only T. The telomerase activity of the HA-treated PDMSC was also higher than that of untreated PDMSC. These data suggested a connection between HA and MSC maintenance. We suggest that HA might be regulating the distribution of cytoskeletal proteins on cell spreading in the event of quiescence to preserve MSC stemness. Maintenance of MSCs stemness delayed cellular aging, leading to the anti-aging phenotype of PDMSC.

Differential Proteomic Analysis of Human Placenta-Derived Mesenchymal Stem Cells Cultured on Normal Tissue Culture Surface and Hyaluronan-Coated Surface.

Our previous results showed that hyaluronan (HA) preserved human placenta-derived mesenchymal stem cells (PDMSC) in a slow cell cycling mode similar to quiescence, the pristine state of stem cells in vivo, and HA was found to prevent murine adipose-derived mesenchymal stem cells from senescence. Here, stable isotope labeling by amino acid in cell culture (SILAC) proteomic profiling was used to evaluate the effects of HA on aging phenomenon in stem cells, comparing (1) old and young passage PDMSC cultured on normal tissue culture surface (TCS); (2) old passage on HA-coated surface (CHA) compared to TCS; (3) old and young passage on CHA. The results indicated that senescence-associated protein transgelin (TAGLN) was upregulated in old TCS. Protein CYR61, reportedly senescence-related, was downregulated in old CHA compared to old TCS. The SIRT1-interacting Nicotinamide phosphoribosyltransferase (NAMPT) increased by 2.23-fold in old CHA compared to old TCS, and is 0.48-fold lower in old TCS compared to young TCS. Results also indicated that components of endoplasmic reticulum associated degradation (ERAD) pathway were upregulated in old CHA compared to old TCS cells, potentially for overcoming stress to maintain cell function and suppress senescence. Our data points to pathways that may be targeted by HA to maintain stem cells youth.

Molecular mechanism of extrinsic factors affecting anti-aging of stem cells.

Scientific evidence suggests that stem cells possess the anti-aging ability to self-renew and maintain differentiation potentials, and quiescent state. The objective of this review is to discuss the micro-environment where stem cells reside in vivo, the secreted factors to which stem cells are exposed, the hypoxic environment, and intracellular factors including genome stability, mitochondria integrity, epigenetic regulators, calorie restrictions, nutrients, and vitamin D. Secreted tumor growth factor-ß and fibroblast growth factor-2 are reported to play a role in stem cell quiescence. Extracellular matrices may interact with caveolin-1, the lipid raft on cell membrane to regulate quiescence. N-cadherin, the adhesive protein on niche cells provides support for stem cells. The hypoxic micro-environment turns on hypoxia-inducible factor-1 to prevent mesenchymal stem cells aging through p16 and p21 down-regulation. Mitochondria express glucosephosphate isomerase to undergo glycolysis and prevent cellular aging. Epigenetic regulators such as p300, protein inhibitors of activated Stats and H19 help maintain stem cell quiescence. In addition, calorie restriction may lead to secretion of paracrines cyclic ADP-ribose by intestinal niche cells, which help maintain intestinal stem cells. In conclusion, it is crucial to understand the anti-aging phenomena of stem cells at the molecular level so that the key to solving the aging mystery may be unlocked.

Human JC virus small tumour antigen inhibits nucleotide excision repair and sensitises cells to DNA-damaging agents.

The human JC virus (JCV) is potentially carcinogenic to humans as a Group 2B carcinogen, and it is ubiquitous in human populations. To investigate whether the small tumour (ST) antigen of the JCV contributes to genomic instability, we established cell lines stably expressing the JCV ST and examined its role in DNA repair. Results from host cell reactivation (HCR) assay revealed that the established cell lines exhibited lower nucleotide excision repair (NER) activity than the vector control cells did. The presence of γ-H2AX, a marker of DNA damage, indicated that the established cell line contained more DNA damage foci compared with vector control cells. Furthermore, the results of clonogenic analyses indicated that the JCV ST-expressing cells were more sensitive than the vector control cells to ultraviolet (UV) irradiation and cisplatin treatment. Micronuclei formation assay revealed that the JCV ST-positive cells presented more chromosomal breakages than did the JCV ST-negative cells, particularly after exposure to DNA-damaging agents. The xeroderma pigmentosum Group D protein, a DNA helicase involved in NER, was downregulated in the JCV ST-positive cells in response to UV irradiation. The effect of the protein phosphatase 2A (PP2A) inhibitor okadaic acid on NER was similar to that of the ST, which is a PP2A-binding protein. Therefore, the deactivation of the PP2A might underlie ST-mediated NER inhibition. The results of this study indicate that exposing JCV ST-positive cells to DNA-damaging agents causes genomic instability, which contributes to carcinogenesis. Our data provide further evidence on the association between the JCV ST and human cancer.

Hyaluronan regulates cell behavior: a potential niche matrix for stem cells.

Hyaluronan is a linear glycosaminoglycan that has received special attention in the last few decades due to its extraordinary physiological functions. This highly viscous polysaccharide is not only a lubricator, but also a significant regulator of cellular behaviors during embryogenesis, morphogenesis, migration, proliferation, and drug resistance in many cell types, including stem cells. Most hyaluronan functions require binding to its cellular receptors CD44, LYVE-1, HARE, layilin, and RHAMM. After binding, proteins are recruited and messages are sent to alter cellular activities. When low concentrations of hyaluronan are applied to stem cells, the proliferative activity is enhanced. However, at high concentrations, stem cells acquire a dormant state and induce a multidrug resistance phenotype. Due to the influence of hyaluronan on cells and tissue morphogenesis, with regards to cardiogenesis, chondrogenesis, osteogenesis, and neurogenesis, it is now been utilized as a biomaterial for tissue regeneration. This paper summarizes the most important and recent findings regarding the regulation of hyaluronan in cells.