Senolytics prevent caveolar CaV 3.2-RyR axis malfunction in old vascular smooth muscle.

Aging is a major risk factor for cardiovascular diseases. Our previous studies demonstrate that aging impairs the caveolar T-type CaV 3.2-RyR axis for extracellular Ca2+ influx to trigger Ca2+ sparks in vascular smooth muscle cells (VSMCs). We hypothesize that the administration of senolytics, which can selectively clear senescent cells, could preserve the caveolar CaV 3.2-RyR axis in aging VSMCs. In this study, 10-month-old mice were administered the senolytics cocktail consisting of dasatinib (5 mg/kg) and quercetin (50 mg/kg) or vehicle bi-weekly for 4 months. Using VSMCs from mouse mesenteric arteries, we found that Ca2+ sparks were diminished after caveolae disruption by methyl-ß-cyclodextrin (10 mM) in cells from D + Q treated but not vehicle-treated 14-month-old mice. D + Q treatment promoted the expression of CaV 3.2 in 14-month-old mesenteric arteries. Structural analysis using electron tomography and immunofluorescence staining revealed the remodeling of caveolae and co-localization of CaV 3.2-Cav-1 in D + Q treatment aged mesenteric arteries. In keeping with theoretical observations, Cav 3.2 channel inhibition by Ni2+ (50 µM) suppressed Ca2+ in VSMCs from the D + Q group, with no effect observed in vehicle-treated arteries. Our study provides evidence that age-related caveolar CaV 3.2-RyR axis malfunction can be alleviated by pharmaceutical intervention targeting cellular senescence. Our findings support the potential of senolytics for ameliorating age-associated cardiovascular disease.

Establishment and Characterization of hTERT Immortalized Hutchinson-Gilford Progeria Fibroblast Cell Lines.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging syndrome caused by a dominant mutation in the LMNA gene. Previous research has shown that the ectopic expression of the catalytic subunit of telomerase (hTERT) can elongate the telomeres of the patients' fibroblasts. Here, we established five immortalized HGP fibroblast cell lines using retroviral infection with the catalytic subunit of hTERT. Immortalization enhanced the proliferative life span by at least 50 population doublings (PDs). The number of cells with typical senescence signs was reduced by 63 + 17%. Furthermore, the growth increase and phenotype improvement occurred with a lag phase of 50-100 days and was not dependent on the degree of telomere elongation. The initial telomeric stabilization after hTERT infection and relatively low amounts of hTERT mRNA were sufficient for the phenotype improvement but the retroviral infection procedure was associated with transient cell stress. Our data have implications for therapeutic strategies in HGP and other premature aging syndromes.

MiR-149 Aggravates Pyroptosis in Myocardial Ischemia-Reperfusion Damage via Silencing FoxO3.

BACKGROUND MicroRNAs (miRNAs), which modulate the expression of their target genes, are commonly involved in stimulating and adjusting of many processes that result in cardiovascular diseases, contain cardiac ischemia/reperfusion (I/R) damage. However, the expression and role of miR-149 in pyroptosis mediated myocardial I/R damage remains unclear. MATERIAL AND METHODS Real-time polymerase chain reaction was performed to measure the miR-149 and FoxO3 expression in I/R stimulated H9C2 cells. The cell proliferation, pyroptosis-related inflammatory genes in I/R-treated H9C2 cells transfected miR-149 mimics or miR-149 inhibitor were both explored. We predicted and confirmed miR-149 targets by using bioinformatics analyses and luciferase reporter assay. In addition, the potential relationship between miR-149 and FoxO3 in pyroptosis from I/R treated H9C2 cells was analyzed. RESULTS Our results showed that miR-149 was upregulated, while FoxO3 was downregulated in I/R stimulated H9C2 cells. Over-expression of miR-149 inhibited cell viability and promote pyroptosis, however, down-expression of miR-149 had an opposite effect in I/R treated H9C2 cells. Furthermore, miR-149 could negatively regulate FoxO3 expression by binding 3'UTR, whereas silencing of FoxO3 attenuated the effect of miR-149-mimics on cell proliferation and pyroptosis in I/R treated H9C2 cells. CONCLUSIONS Our study found that miR-149 played a critical role in pyroptosis during cardiac I/R injury, and thus, might provide a novel therapeutic target.

[Predictive value of glycosylated serum protein combined with glycemic variability on secondary persistent inflammatory immunosuppressed catabolic syndrome prediction in elderly septic patients].

OBJECTIVE: To investigate the predictive value of glycosylated serum protein (GSP) combined with glycemic variability (GV) in persistent inflammation immunosuppressive catabolic syndrome (PICS) in elderly septic patients. METHODS: A retrospective study was conducted. The septic patients aged ≥ 60 years old with 28 days hospitalized duration admitted to geriatric intensive care unit (ICU) of Guangzhou General Hospital of Guangzhou Military Command from January 2014 to December 2017 were enrolled. The patients were divided into two groups according to whether PICS occurred within 14 days after ICU admission according to the PICS diagnostic criteria. General patients' data including gender, age, underlying disease, site of infection, the length of ICU stay were collected, acute physiology and chronic health evaluation II (APACHE II), GSP at 1 day after hospitalization and lymphocyte count (LYM), C-reactive protein (CRP), albumin (ALB), prealbumin (PA) levels at 1 day and 14 days were recorded. The levels of blood glucose on the 1st day and 14th day were observed, the GV was calculated. Data were cross-validated using the random forest method. Receiver operating characteristic (ROC) curve was plotted to evaluate the predictive value of glycemic parameters for PICS. RESULTS: A total of 315 elderly septic patients were included. Patients with malignant tumors, severe autoimmune diseases, and immunosuppressive therapy or dead within 28 days of hospitalization were excluded. A total of 132 patients were enrolled in the analysis, including 45 in the PICS group and 87 in the non-PICS group. The length of ICU stay in the PICS group was significantly longer than that in the non-PICS group [days: 35.0 (22.0, 49.5) vs. 8.0 (5.0, 23.0), P < 0.01]. No significant difference in the baseline data of gender, age, underlying disease, infection site or APACHE II score between the two groups was found. (1) Parameters for PICS diagnosis: with the prolongation of ICU stay, LYM and PA in the non-PICS group were increased and those in the PICS group were decreased, and CRP and ALB levels were decreased in both groups. LYM, ALB and PA levels in the PICS group were significantly lower than those in the non-PICS group at 14 days after ICU admission [LYM (×109/L): 0.6 (0.5, 0.7) vs. 1.1 (0.9, 1.6), ALB (g/L): 25.4±2.7 vs. 29.9±4.3, PA (g/L): 0.08 (0.05, 0.14) vs. 0.11 (0.10, 0.21), all P < 0.01], and CRP level was significantly higher than that in the non-PICS group (mg/L: 87.5±56.3 vs. 49.2±49.1, P < 0.01). (2) Glycemic parameters: the GSP level of the PICS group at 1 day after ICU admission was significantly lower than that of the non-PICS group (mmol/L: 2.3±0.6 vs. 2.7±0.6, P < 0.01), but there was no statistically significant difference in the level of blood glucose or GV at 1 day and 14 days after ICU admission as compared with the non-PICS group [blood glucose (mmol/L): 10.0±3.3 vs. 9.4±3.3 at 1 day, 10.8±3.6 vs. 10.4±3.5 at 14 days; GV: (24.2±1.4)% vs. (23.7±1.2)% at 1 day, (24.8±7.8)% vs. (24.7±7.7)% at 14 days, all P > 0.05]. (3) ROC curve analysis: 1-day GSP as well as 1-day and 14-day GV had certain predictive value for PICS secondary to sepsis in the elderly. The predictive value of 1-day GSP combined with 14-day GV was the highest, its area under ROC curve (AUC) was 0.637, with a sensitivity of 95.8% and a specificity of 25.0%, while the positive likelihood ratio was 1.278, the negative likelihood ratio was 0.167, the positive predictive value was 71.9%, and the negative predictive value was 75.0%. CONCLUSIONS: GSP combined with GV could effectively predict secondary PICS in elderly septic patients.

[Progress in circulating microRNAs as biomarkers of sepsis].

OBJECTIVE: Sepsis is as life-threatening organ failure caused by a dysregulated host response to infection, with urgent onset, complex mechanism and high mortality. Its early diagnosis and treatment is particularly important. However, the sensitivity and specificity of current laboratory markers for the diagnosis of sepsis is not ideal. MicroRNAs represent a novel group of small RNA molecules that do not encode for proteins. MicroRNAs are synthesized in the cytoplasm and transferred to circulation primarily by micro-vesicles, and they are stable in circulation and can be rapidly detected. The researches regarding circulating miRNA as biomarkers of sepsis were collected to analyze the characteristics of differential expression of miRNAs including miR-150, miR-133a, miR-122, miR-223, miR-4772, miR-297 and miR-574-5p etc. in the pathogenesis of sepsis, which suggest that they can be used as biomarkers for the diagnosis and prognosis of sepsis.

Cell-penetrating peptide TAT-mediated delivery of acidic FGF to retina and protection against ischemia-reperfusion injury in rats.

The development of non-invasive ocular drug delivery systems is of practical importance in the treatment of retinal disease. In this study, we evaluated the efficacy of transactivator of transcription protein transduction domain (TAT-PTD, TAT(49-57)) as a vehicle to deliver acidic FGF (aFGF) to retina in rats. TAT-conjugated aFGF-His (TAT-aFGF-His) exhibited efficient penetration into the retina following topical administration to the ocular surface. Immunochemical staining with anti-His revealed that TAT-aFGF-His proteins were readily found in the retina (mainly in the ganglion cell layer) at 30 min. and remained detectable for at least 8 hrs after administration. In contrast, His(+) proteins were undetectable in the retina after topical administration of aFGF-His, indicating that aFGF-His cannot penetrate the ocular barrier. Furthermore, TAT-aFGF-His, but not aFGF-His, mediated significant protection against retinal ischemia-reperfusion (IR) injury. After IR injury, retina from TAT-aFGF-His-treated rats showed better-maintained inner retinal layer structure, reduced apoptosis of retinal ganglion cells and improved retinal function compared to those treated with aFGF-His or PBS. These results indicate that conjugation of TAT to aFGF-His can markedly improve the ability of aFGF-His to penetrate the ocular barrier without impairing its biological function. Thus, TAT(49-57) provides a potential vehicle for efficient drug delivery in the treatment of retinal disease.