scholarly journals 213 USE OF ACTIVE CASPASE 3 AND TUNEL ASSAYS TO ESTIMATE EMBRYONIC QUALITY IN IN VIVO SANTA INES EWE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 220 ◽  
Author(s):  
M. E. F. Oliveira ◽  
C. S. Oliveira ◽  
M. R. Lima ◽  
F. F. P. C. Barros ◽  
A. P. Perini ◽  
...  
Keyword(s):  
Breeding Season ◽  
Caspase 3 ◽  
Transition Period ◽  
Pearson Correlation ◽  
Apoptotic Cells ◽  
Nonbreeding Season ◽  
Follicular Wave ◽  
Santa Inês ◽  
Active Caspase

This study was designed to quantify the percentage of apoptotic cells using active caspase 3 and TUNEL assays, in order to estimate the quality of ovine embryos produced in vivo. For that, 60 Santa Ines ewes (n = 10 per group) were submitted to superovulation with FSH treatment started near the different follicular wave emergence of the protocols (G-1 or G-2), during breeding season, transition, and nonbreeding season. Follicular wave emergence days were defined in a previous study that evaluated the follicular dynamic in oestrus synchronization treatments (Oliveira et al. 2011 Acta Sci. Vet. 38, 361). On Day 0, all ewes received a P4 device (CIDR®) and 37.5 μg of D-cloprostenol. The P4 device was replaced by a new one on Day 7 just for G-2 in the transition period. The superovulation treatment started on Day 4, 4, and 6 of protocol for G-1 and on Day 10, 10, and 11 for G-2 in nonbreeding, transition, and breeding season, respectively. The FSH treatment consisted of eight injections of pFSH administrated twice a day in descending order (40, 30, 20, and 10 mg of pFSH). The P4 device was removed two days after beginning of FSH treatment. At these times, all ewes received another injection of 37.5 μg of D-cloprostenol and a dose of 200 IU of eCG. Ewes were mated with a fertile ram for 4 days after P4 device removal. Embryo recoveries were carried out by laparotomy, 7 days after CIDR withdrawal. Embryos were morphologically classified. Grade I to III morulas and blastocysts were considered viable. A representative sample of each treatment was fixed and stained by active caspase 3 and TUNEL assays to assess the apoptotic cells percentage. Data were analysed by GLIMMIX using SAS comparing mean values (±s.e.m.) between groups at each season (P = 0.05). Pearson correlation was estimated between active caspase 3 and TUNEL assays. No effect was detected between treatments in each season on the number of viable embryos (3.2 ± 0.8 v. 1.8 ± 0.8, 3.9 ± 1.9 v. 5.7 ± 1.4, and 3.8 ± 1.5 v. 3.4 ± 0.8 for G-1 v. G-2 in nonbreeding, transition and breeding season, respectively). The treatment G-2 increased (P < 0.05) apoptotic cells percentage in nonbreeding season group, assessed by active caspase 3 (G-1: 3.1 ± 1.6% and G-2: 12.8 ± 4.3%) and TUNEL (G-1: 1.6 ± 0.5% and G-2: 11.1 ± 3.5%) assays. The apoptotic cells percentage remained unaltered for Transition and Breeding season groups, assessed by either active caspase 3 (G-1: 6.0 ± 0.9% and 5.6 ± 1.5%; G-2: 5.6 ± 1.1% and 5.1 ± 0.5%) and by TUNEL (G-1: 7.5 ± 1.3% and 5.2 ± 1.0%; G-2: 5.0 ± 0.9% and 6.4 ± 1.1%). The Pearson correlation between active caspase 3 and TUNEL tests was r2 = 0.436 (P < 0.0001). In conclusion, the active caspase 3 and TUNEL assays presented similar results for apoptosis level assessment in Santa Ines ewes in vivo produced embryos, and both assays were considered appropriate for this purpose. The increased apoptosis levels detected in the G-2 nonbreeding season group suggest that this treatment is harmful for Santa Ines ewe embryos. Financial support was provided by FAPESP and FUNDUNESP.

scholarly journals A-Kinase-Anchoring Protein 95 Functions as a Potential Carrier for the Nuclear Translocation of Active Caspase 3 through an Enzyme-Substrate-Like Association

2005 ◽  
Vol 25 (21) ◽  
pp. 9469-9477 ◽  
Author(s):  
Shinji Kamada ◽  
Ushio Kikkawa ◽  
Yoshihide Tsujimoto ◽  
Tony Hunter
Keyword(s):  
Molecular Mechanisms ◽  
Caspase 3 ◽  
Nuclear Translocation ◽  
Morphological Changes ◽  
Central Element ◽  
Nuclear Localization Sequence ◽  
Apoptotic Cells ◽  
Enzyme Substrate ◽  
Anchoring Protein ◽  
Active Caspase

ABSTRACT Caspase-mediated proteolysis is a critical and central element of the apoptotic process, and caspase 3, one of the effector caspases, is proposed to play essential roles in the nuclear morphological changes of apoptotic cells. Although many substrates for caspase 3 localize in the nucleus and caspase 3 translocates from the cytoplasm to the nuclei after activation in apoptotic cells, the molecular mechanisms of nuclear translocation of active caspase 3 have been unclear. Recently, we suggested that a substrate-like protein(s) served as a carrier to transport caspase 3 from the cytoplasm into the nucleus. In the present study, we identified A-kinase-anchoring protein 95 (AKAP95) as a caspase 3-binding protein. Small interfering RNA-mediated depletion of AKAP95 reduced apoptotic nuclear morphological changes, suggesting that AKAP95 is involved in the process of apoptotic nuclear morphological changes. The association of AKAP95 with active caspase 3 was analogous to an enzyme-substrate interaction. Furthermore, overexpression of AKAP95 with nuclear localization sequence mutations inhibited nuclear morphological changes in apoptotic cells. These results indicate that AKAP95 is a potential carrier protein for active caspase 3 from the cytoplasm into the nuclei in apoptotic cells.

scholarly journals Targeting JAK2 Gene Rearrangements with a Novel Kinase Inhibitor in a Preclinical Model of Pediatric Acute Lymphoblastic Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1180-1180
Author(s):  
Manuel Quadri ◽  
Claudia Saitta ◽  
Sonia Palamini ◽  
Chiara Palmi ◽  
Andrea Biondi ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Pediatric Patients ◽  
Kinase Inhibitor ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
P Value ◽  
Fusion Genes ◽  
Apoptosis Induction ◽  
Active Caspase

Abstract Background. Although the Event Free Survival for Childhood Acute lymphoblastic leukemia (ALL) reaches 85%, the remaining 15% of patients relapse, and 25-40% of them die. Novel molecular targets may increase the efficacy of therapy and reduce treatment toxicity. Among B-other ALL patients, JAK/STAT pathway alterations represent about 7% of the 'Philadelphia-like' cases. JAK2 gene encodes for a non-receptor tyrosine kinase fundamental for hematopoiesis, cellular proliferation and survival. In last years, JAK2 mutations have been widely studied in leukemia and lymphoma, whereas JAK2 fusion genes are still poorly characterized. Aim. This study aims to identify JAK2 fusion genes among BCP-ALL pediatric patients and develop a target strategy in in vitro and in vivo preclinical models. Methods. A targeted capture RNA Next Generation Sequencing strategy was applied to discover JAK2 fusion genes in a large cohort of PCR-based MRD high risk (HR) BCP-ALL pediatric patients. Fusions were validated by RT-PCR and/or FISH. Primary patients' cells have been in vivo expanded in NSG mice. We performed ex vivo and in vivo drug treatments with JAK2 inhibitors; phosphoflow and apoptosis-viability assays were performed in patients' blasts in co-culture with human bone marrow stroma. Results. We identified 10 pediatric cases carrying a JAK2 fusion gene with different partners in single cases, such as ATF7IP, ZEB2, MPRIP, BCR, TLE4, GIT2 and RAB7, in addition to PAX5, which was the only recurrent in three cases. Cells were available from 3 cases, carrying PAX5-JAK2, ATF7IP-JAK2 and ZEB2-JAK2, respectively. After in vivo expansion, we demonstrated that the JAK2 signaling pathway was active at basal level, through phosphorylation on Y1007-1008 JAK2 residues inside the catalytic activation loop, compared to cases wild type for JAK2 and CRLF2 (+70%, two-tailed P value 0.0355); a positive trend was also shown compared to primary cells with P2RY8-CRLF2 rearrangements and JAK2 mutation, as positive controls (+40% two-tailed P value 0.158). The JAK2 downstream effectors pS727-STAT3 and pY694-STAT5 were also activated. We thus setup a JAK2 targeted drug treatment using CHZ868, a new class-II tyrosine kinase inhibitor (TKI) (Novartis, Basel, CH). After 30 minutes of treatment, we appreciated a mean inhibition of -62% of Y1007-1008 JAK2 residues in PAX5-JAK2, -22% in ATF7IP-JAK2 and -35% in ZEB2- JAK2. Contemporarily, we observed a decrease of pS727-STAT3 (-35-50%) and pY694-STAT5 (-15-50%). After 48h monotherapy treatment by CHZ868, we detected apoptosis induction and cell viability decrease between 20- 75% at IC50. In combination with dexamethasone, we assessed a further decrease of viability between 10 to 95%. A biological variability among the three different patients was appreciated, according to the different partner genes. Exclusively for the PAX5-JAK2 fusion, we also performed treatments with the kinase inhibitor BIBF1120/Nintedanib, targeting LCK, which is activated downstream PAX5 fusions and we observed a 20% reduction of cell viability. Importantly, combination of BIBF1120 and CHZ868 showed a synergistic effect (-45%, at IC50). Moreover, we found that ruxolitinib caused autophagy as observed by higher levels of LC3-II compared to untreated cells (+ 45%, p&lt;0.01), with consequent reduction of apoptosis induction. Indeed, active caspase 3 increased when ruxolitinib was given in combination with chloroquine, an autophagy inhibitor (+20% vs ruxolitinib alone, p&lt;0.01). CHZ868 alone or in combination with chloroquine instead does not induce autophagy as LC3-II and active caspase 3 levels are the same of untreated cells. Finally, we demonstrated the in vivo efficacy of CHZ868 in patient derived xenograft model in presence of PAX5-JAK2 fusion. After two weeks of 30mg/Kg daily treatment of CHZ868, we observed a significant reduction of leukemic CD10+/CD19+ cells both in bone marrow (p&lt;0.01, -43%), spleen (p&lt;0.001, -72%), central nervous system (-40%) and peripheral blood (p&lt;0.05, -46%), compared to vehicle mice. Further in vivo experiments are ongoing in other JAK2 fusion settings. Conclusion. CHZ868 is a promising candidate for treatment of BCP-ALL carrying JAK2 fusions, showing high efficacy and specificity, both ex vivo and in vivo. Further studies will include combination with standard chemotherapy drugs with the aim to maintain its efficacy by reducing the intensity and toxicity of chemotherapy. Disclosures Biondi: Novartis: Honoraria; Bluebird: Other: Advisory Board; Incyte: Consultancy, Other: Advisory Board; Colmmune: Honoraria; Amgen: Honoraria.

Cell Cycle Entry Potentiates Elimination of Chemotherapy-Resistant Human AML Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1422-1422
Author(s):  
Yoriko Saito ◽  
Naoyuki Uchida ◽  
Satoshi Tanaka ◽  
Mariko Tomizawa-Murasawa ◽  
Nahoko Suzuki ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Caspase 3 ◽  
T Test ◽  
Tunel Staining ◽  
Cell Cycle Entry ◽  
Cell Cycle Status ◽  
Active Caspase

Abstract Abstract 1422 Poster Board I-445 Acute myeloid leukemia (AML) is associated with poor long-term prognosis despite advances in therapeutic modalities over the past few decades. As leukemia stem cells (LSCs) capable of AML initiation may contribute to recurrent disease, LSC-targeted therapies are required to overcome disease relapse and to improve long-term patient outcomes. We previously reported that human AML CD34+CD38- cells self-renew, generate non-stem leukemic cells, and possess potential to initiate leukemia following engraftment of newborn NOD/SCID/IL2rgKO mice. In the recipient bone marrow (BM), AML LSCs were found to reside preferentially within the endosteal region and exhibited chemotherapy resistance. In addition, we observed that AML cells abutting the BM endosteum were cell cycle quiescent while AML cells in the center of the BM were cycling. Based on these findings, we hypothesized that induction of cell cycle entry in quiescent AML LSCs may increase their susceptibility to chemotherapeutic agents, leading to enhanced elimination of LSCs. To test this hypothesis, we assessed the effect of granulocyte colony-stimulating factor (G-CSF) on cell cycle status and chemotherapy susceptibility of primary human AML LSCs in vivo using the NOD/SCID/IL2rgKO xenotransplantation model. In AML-engrafted recipient mice transplanted with LSCs from seven AML patients, flow cytometric analyses demonstrated a significant reduction of quiescent LSCs following 300μg/kg G-CSF sc daily for 5 days (%G0 within hCD34+CD38- BM cells (mean+/-s.e.m): 49.2+/-2.6 (n=47) and 20.5+/-2.0 (n=36), control and G-CSF treated recipients, respectively, p<0.0001 by two-tailed t test). Direct examination of recipient BM in situ revealed cell cycle entry of human AML cells abutting the BM endosteum as evidenced by increased Ki67 expression. Next we developed an in vivo treatment model evaluating the effect of cell cycle induction on chemotherapy-responsiveness of human primary AML LSCs. Human AML-engrafted recipients received AraC alone (1g/kg ip daily for 2 days) or G-CSF followed by AraC (300μg/kg G-CSF sc daily for 5 days with 1g/kg AraC ip daily on days 4 and 5). The proportion of viable active caspase 3-negative human LSCs decreased significantly with pre-chemotherapy cell cycle induction (% active caspase 3-negative hCD34+CD38- BM cell (mean+/-s.e.m.): 82.7+/-1.3% (n=33) and 40.4+/- 3.1% (n=30), AraC alone- and G-CSF followed by AraC-treated recipients, respectively, p<0.0001 by two-tailed t test). TUNEL staining of the recipient BM showed increased apoptosis of AML cells abutting the BM endosteum in recipients receiving AraC following cell cycle induction. Limiting dilution serial transplantation of residual viable human AML cells in the BM of treated recipients showed 100-fold reduction in the frequency of LSCs capable of initiating AML in secondary recipients (BM LSC frequency: 1/560 (n=125) and 1/55,076 (n=109), AraC alone- and G-CSF then AraC-treated recipients, respectively, p=0.0001 by two-tailed t test). At 24 weeks post-transplantation, 89.4% of secondary recipients of G-CSF followed by AraC-treated mice survived compared with only 2.0% survival in secondary recipients of AraC alone-treated mice (p<0.0001, survival estimated by Kaplan-Meier method). These findings indicate that cell cycle status is a key determinant of LSC chemo-responsiveness and that therapeutic strategies promoting LSC cell cycle entry may improve outcomes in AML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Detection of Active Caspase-3 in Mouse Models of Stroke and Alzheimer’s Disease with a Novel Dual Positron Emission Tomography/Fluorescent Tracer [68Ga]Ga-TC3-OGDOTA

2019 ◽  
Vol 2019 ◽  
pp. 1-17 ◽  
Author(s):  
Valeriy G. Ostapchenko ◽  
Jonatan Snir ◽  
Mojmir Suchy ◽  
Jue Fan ◽  
M. Rebecca Cobb ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Mouse Models ◽  
Caspase 3 ◽  
Single Cells ◽  
Glucose Deprivation ◽  
Apoptotic Cells ◽  
The Brain

Apoptosis is a feature of stroke and Alzheimer’s disease (AD), yet there is no accepted method to detect or follow apoptosis in the brain in vivo. We developed a bifunctional tracer [68Ga]Ga-TC3-OGDOTA containing a cell-penetrating peptide separated from fluorescent Oregon Green and 68Ga-bound labels by the caspase-3 recognition peptide DEVD. We hypothesized that this design would allow [68Ga]Ga-TC3-OGDOTA to accumulate in apoptotic cells. In vitro, Ga-TC3-OGDOTA labeled apoptotic neurons following exposure to camptothecin, oxygen-glucose deprivation, and β-amyloid oligomers. In vivo, PET showed accumulation of [68Ga]Ga-TC3-OGDOTA in the brain of mouse models of stroke or AD. Optical clearing revealed colocalization of [68Ga]Ga-TC3-OGDOTA and cleaved caspase-3 in brain cells. In stroke, [68Ga]Ga-TC3-OGDOTA accumulated in neurons in the penumbra area, whereas in AD mice [68Ga]Ga-TC3-OGDOTA was found in single cells in the forebrain and diffusely around amyloid plaques. In summary, this bifunctional tracer is selectively associated with apoptotic cells in vitro and in vivo in brain disease models and represents a novel tool for apoptosis detection that can be used in neurodegenerative diseases.

scholarly journals Eukaryotic Translation Initiation Factor 4G Is Targeted for Proteolytic Cleavage by Caspase 3 during Inhibition of Translation in Apoptotic Cells

1998 ◽  
Vol 18 (12) ◽  
pp. 7565-7574 ◽  
Author(s):  
Wilfred E. Marissen ◽  
Richard E. Lloyd
Keyword(s):  
Translation Initiation ◽  
Caspase 3 ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Apoptotic Cells ◽  
Eukaryotic Translation ◽  
Cell Lysates ◽  
Cellular Translation

ABSTRACT Although much is known about the multiple mechanisms which induce apoptosis, comparatively little is understood concerning the execution phase of apoptosis and the mechanism(s) of cell killing. Several reports have demonstrated that cellular translation is shut off during apoptosis; however, details of the mechanism of translation inhibition are lacking. Translation initiation factor 4G (eIF4G) is a crucial protein required for binding cellular mRNA to ribosomes and is known to be cleaved as the central part of the mechanism of host translation shutoff exerted by several animal viruses. Treatment of HeLa cells with the apoptosis inducers cisplatin and etoposide resulted in cleavage of eIF4G, and the extent of its cleavage correlated with the onset and extent of observed inhibition of cellular translation. The eIF4G-specific cleavage activity could be measured in cell lysates in vitro and was inhibited by the caspase inhibitor Ac-DEVD-CHO at nanomolar concentrations. A combination of in vivo and in vitro inhibitor studies suggest the involvement of one or more caspases in the activation and execution of eIF4G cleavage. Furthermore recombinant human caspase 3 was expressed in bacteria, and when incubated with HeLa cell lysates, was shown to produce the same eIF4G cleavage products as those observed in apoptotic cells. In addition, purified caspase 3 caused cleavage of purified eIF4G, demonstrating that eIF4G could serve as a substrate for caspase 3. Taken together, these data suggest that cellular translation is specifically inhibited during apoptosis by a mechanism involving cleavage of eIF4G, an event dependent on caspase activity.

scholarly journals 317 EFFECT OF TIME OF ONSET OF FOLLICLE-STIMULATING HORMONE TREATMENT ON SUPEROVULATORY RESPONSE AND EMBRYO YIELD OF SANTA INES EWES DURING BREEDING SEASON

2013 ◽  
Vol 25 (1) ◽  
pp. 306
Author(s):  
M. E. F. Oliveira ◽  
C. C. D'Amato ◽  
C. S. Oliveira ◽  
F. F. P. C. Barros ◽  
A. P. Perini ◽  
...  
Keyword(s):  
New York ◽  
Breeding Season ◽  
Animal Health ◽  
Hormone Treatment ◽  
Corpora Lutea ◽  
Mating Period ◽  
Follicular Wave ◽  
Embryo Yield ◽  
Pfizer Animal Health ◽  
Santa Inês

This study was designed to investigate if the time of onset of FSH treatment [near the emergence of first or last follicular wave on progesterone (P4) protocol] influenced the superovulatory response and embryo yield in Santa Ines ewes during breeding season. Days of emergence of the follicular waves were defined in a previous study that evaluated the follicular dynamic in oestrus synchronization treatments (Oliveira et al. 2011 Acta Sci. Vet. 40). We observed emergence of the first and last follicular wave on 5.69 ± 0.42 and 11.25 ± 0.39 days of protocol, respectively. Twenty Santa Ines ewes were submitted to 2 superovulatory protocols according to the time that FSH treatments were initiated (G-first wave, n = 10; G-last wave, n = 10). On Day 0, all ewes received a P4 device (CIDR®; Pfizer Animal Health, New York, NY, USA) and injection of 37.5 µg of d-cloprostenol, IM. The FSH treatments started on Day 6 and Day 11 of protocol for G-first and G-last, respectively. The superovulatory regimen consisted of 8 IM injections of pFSH administrated twice daily (40, 40, 30, 30, 20, 20, 10, and 10 mg of pFSH). The P4 device was removed on Day 8 and Day 13 for G-first and G-last, respectively. At these times, all ewes received another injection of 37.5 µg of d-cloprostenol and a dose of 200 IU of eCG. During 4 days after the P4 device removal, ewes were mated by a fertile ram. Embryo collections were accomplished 7 days after CIDR withdrawal. The ovaries were evaluated by ultrasonography (3 times daily, during the mating period) and laparotomy (concomitantly with embryo collection). The superovulatory response was observed by classified by score: 0 = 4 or fewer corpora lutea (CL); 1 = between 5 and 10 CL; and 2 = 11 or more CL. Data were analysed by GLIMMIX using SAS software (SAS Institute Inc., Cary, NC, USA). All donors from G-first had superovulatory response classified as score 2, whereas 60% of ewes from G-last were classified as score 2, 20% as score 1, and 20% as score 0 (P < 0.05). There were effects between treatments (P < 0.05) for ovulation rate (G-first: 97.9 ± 1.4% v. G-last: 88.5 ± 4.4%) and number of ovulations (G-first: 17.0 ± 2.3 v. G-last: 12.5 ± 2.6). The numbers of luteinized unovulated follicles were 0.7 ± 0.5 for G-first and 1.2 ± 0.4 for G-last (P > 0.05). There was no difference between G-first and G-last (P > 0.05) in the rate of ova/embryos recovered (54.9 ± 5.7% v. 49.3 ± 8.5%), mean number of ova/embryos recovered (9.0 ± 1.4 v. 6.3 ± 1.1), number of viable embryos (3.8 ± 1.5 v. 3.4 ± 0.8), or viability rate (40.3 ± 10.8 v. 53.4 ± 12.1). In conclusion, the FSH treatment started near the emergence of the first follicular wave of progesterone protocol in Santa Ines ewes during the breeding season resulted in a higher superovulatory response than that started near the last follicular wave; however, no improvements in embryo yield were observed. Financial support: FAPESP.

Parallel single-cell analysis of active caspase-3/7 in apoptotic and non-apoptotic cells

2016 ◽  
Vol 409 (1) ◽  
pp. 269-274 ◽  
Author(s):  
Vojtěch Ledvina ◽  
Eva Janečková ◽  
Eva Matalová ◽  
Karel Klepárník
Keyword(s):  
Single Cell ◽  
Caspase 3 ◽  
Single Cell Analysis ◽  
Apoptotic Cells ◽  
Cell Analysis ◽  
Active Caspase

Quantification of Active Caspase 3 in Apoptotic Cells

2000 ◽  
Vol 284 (1) ◽  
pp. 114-124 ◽  
Author(s):  
Paul A. Saunders ◽  
Jeffrey A. Cooper ◽  
Michelle M. Roodell ◽  
Dorothy A. Schroeder ◽  
Chad J. Borchert ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Apoptotic Cells ◽  
Active Caspase

Noncanonical inhibition of caspase-3 by a nuclear microRNA confers endothelial protection by autophagy in atherosclerosis

2020 ◽  
Vol 12 (546) ◽  
pp. eaaz2294 ◽  
Author(s):  
Donato Santovito ◽  
Virginia Egea ◽  
Kiril Bidzhekov ◽  
Lucia Natarelli ◽  
André Mourão ◽  
...  
Keyword(s):  
Shear Stress ◽  
Protein Function ◽  
Caspase 3 ◽  
Control Cell ◽  
High Shear ◽  
High Shear Stress ◽  
Argonaute 2 ◽  
Endothelial Integrity ◽  
Active Caspase

MicroRNAs (miRNAs) are versatile regulators of gene expression with profound implications for human disease including atherosclerosis, but whether they can exert posttranslational functions to control cell adaptation and whether such noncanonical features harbor pathophysiological relevance is unknown. Here, we show that miR-126-5p sustains endothelial integrity in the context of high shear stress and autophagy. Bound to argonaute-2 (Ago2), miR-126-5p forms a complex with Mex3a, which occurs on the surface of autophagic vesicles and guides its transport into the nucleus. Mutational studies and biophysical measurements demonstrate that Mex3a binds to the central U- and G-rich regions of miR-126-5p with nanomolar affinity via its two K homology domains. In the nucleus, miR-126-5p dissociates from Ago2 and binds to caspase-3 in an aptamer-like fashion with its seed sequence, preventing dimerization of the caspase and inhibiting its activity to limit apoptosis. The antiapoptotic effect of miR-126-5p outside of the RNA-induced silencing complex is important for endothelial integrity under conditions of high shear stress promoting autophagy: ablation of Mex3a or ATG5 in vivo attenuates nuclear import of miR-126-5p, aggravates endothelial apoptosis, and exacerbates atherosclerosis. In human plaques, we found reduced nuclear miR-126-5p and active caspase-3 in areas of disturbed flow. The direct inhibition of caspase-3 by nuclear miR-126-5p reveals a noncanonical mechanism by which miRNAs can modulate protein function.

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