Store-Operated Ca2+ Entry Is Up-Regulated in Tumour-Infiltrating Lymphocytes from Metastatic Colorectal Cancer Patients.

(1) Background: Store-operated Ca2+ entry (SOCE) drives the cytotoxic activity of cytotoxic T lymphocytes (CTLs) against cancer cells. However, SOCE can be enhanced in cancer cells due to an increase in the expression and/or function of its underlying molecular components, i.e., STIM1 and Orai1. Herein, we evaluated the SOCE expression and function in tumour-infiltrating lymphocytes (TILs) from metastatic colorectal cancer (mCRC) patients. (2) Methods: Functional studies were conducted in TILs expanded ex vivo from CRC liver metastases. Peripheral blood T cells from healthy donors (hPBTs) and mCRC patients (cPBTs) were used as controls. (3) Results: SOCE amplitude is enhanced in TILs compared to hPBTs and cPBTs, but the STIM1 protein is only up-regulated in TILs. Pharmacological manipulation showed that the increase in SOCE mainly depends on tonic modulation by diacylglycerol kinase, which prevents the protein kinase C-dependent inhibition of SOCE activity. The larger SOCE caused a stronger Ca2+ response to T-cell receptor stimulation by autologous mCRC cells. Reducing Ca2+ influx with BTP-2 during target cell killing significantly increases cytotoxic activity at low target:effector ratios. (4) Conclusions: SOCE is enhanced in ex vivo-expanded TILs deriving from mCRC patients but decreasing Ca2+ influx with BTP-2 increases cytotoxic activity at a low TIL density.

Stress-induced premature senescence is associated with a prolonged QT interval and recapitulates features of cardiac aging.

Rationale: Aging in the heart is a gradual process, involving continuous changes in cardiovascular cells, including cardiomyocytes (CMs), namely cellular senescence. These changes finally lead to adverse organ remodeling and resulting in heart failure. This study exploits CMs from human induced pluripotent stem cells (iCMs) as a tool to model and characterize mechanisms involved in aging. Methods and Results: Human somatic cells were reprogrammed into human induced pluripotent stem cells and subsequently differentiated in iCMs. A senescent-like phenotype (SenCMs) was induced by short exposure (3 hours) to doxorubicin (Dox) at the sub-lethal concentration of 0.2 µM. Dox treatment induced expression of cyclin-dependent kinase inhibitors p21 and p16, and increased positivity to senescence-associated beta-galactosidase when compared to untreated iCMs. SenCMs showed increased oxidative stress, alteration in mitochondrial morphology and depolarized mitochondrial membrane potential, which resulted in decreased ATP production. Functionally, when compared to iCMs, SenCMs showed, prolonged multicellular QTc and single cell APD, with increased APD variability and delayed afterdepolarizations (DADs) incidence, two well-known arrhythmogenic indexes. These effects were largely ascribable to augmented late sodium current (INaL) and reduced delayed rectifier potassium current (Ikr). Moreover sarcoplasmic reticulum (SR) Ca2+ content was reduced because of downregulated SERCA2 and increased RyR2-mediated Ca2+ leak. Electrical and intracellular Ca2+ alterations were mostly justified by increased CaMKII activity in SenCMs. Finally, SenCMs phenotype was furtherly confirmed by analyzing physiological aging in CMs isolated from old mice in comparison to young ones. Conclusions: Overall, we showed that SenCMs recapitulate the phenotype of aged primary CMs in terms of senescence markers, electrical and Ca2+ handling properties and metabolic features. Thus, Dox-induced SenCMs can be considered a novel in vitro platform to study aging mechanisms and to envision cardiac specific anti-aging approach in humans.

Liraglutide preserves CD34+ stem cells from dysfunction Induced by high glucose exposure.

BACKGROUND: Glucagon like peptide-1 receptor agonists (GLP-1RAs) have shown to reduce mortality and cardiovascular events in patients with type 2 diabetes mellitus (T2DM). Since the impairment in number and function of vasculotrophic circulating CD34+ hematopoietic stem progenitor cells (HSPCs) in T2D has been reported to increase cardiovascular (CV) risk, we hypothesized that one of the mechanisms whereby GLP-1 RAs exert CV protective effects may be related to the ability to improve CD34+ HSPC function. METHODS: In cord blood (CB)-derived CD34+ HSPC, the expression of GLP-1 receptor (GLP-1R) mRNA, receptor protein and intracellular signaling was evaluated by RT-qPCR and Western Blot respectively. CD34+ HSPCs were exposed to high glucose (HG) condition and GLP-1RA liraglutide (LIRA) was added before as well as after functional impairment. Proliferation, CXCR4/SDF-1α axis activity and intracellular ROS production of CD34+ HSPC were evaluated. RESULTS: CD34+ HSPCs express GLP-1R at transcriptional and protein level. LIRA treatment prevented and rescued HSPC proliferation, CXCR4/SDF-1α axis activity and metabolic imbalance from HG-induced impairment. LIRA stimulation promoted intracellular cAMP accumulation as well as ERK1/2 and AKT signaling activation. The selective GLP-1R antagonist exendin (9-39) abrogated LIRA-dependent ERK1/2 and AKT phosphorylation along with the related protective effects. CONCLUSION: We provided the first evidence that CD34+ HSPC express GLP-1R and that LIRA can favorably impact on cell dysfunction due to HG exposure. These findings open new perspectives on the favorable CV effects of GLP-1 RAs in T2DM patients.