OBJECTIVE: To investigate the effects and underlying mechanism of ionizing radiation on the adipogenic of mesenchymal stem cells (MSCs). METHODS: Mouse MSCs were cultured in vitro and treated with 2 Gy and 6 Gy radiation with ⁶⁰Co, and the radiation dose rate was 0.98 Gy/min. Bulk RNA-seq was performed on control and irradiated MSCs. The changes of adipogenic differentiation and oxidative stress pathways of MSC were revealed by bioinformatics analysis. Oil Red O staining was used to detect the adipogenic differentiation ability of MSCs in vitro, and real-time fluorescence quantitative PCR (qPCR) was used to detect the expression differences of key regulatory factors Cebpa, Lpl and Pparg after radiation treatment. At the same time, qPCR and Western blot were used to detect the effect of inhibition of Nrf2, a key factor of antioxidant stress pathway, on the expression of key regulatory factors of adipogenesis. Moreover, the species conservation of the irradiation response of human bone marrow MSCs and mouse MSC was determined by qPCR. RESULTS: Bulk RNA-seq suggested that ionizing radiation promotes adipogenic differentiation of MSCs and up-regulation of oxidative stress-related genes and pathways. The results of Oil Red O staining and qPCR showed that ionizing radiation promoted the adipogenesis of MSCs, with high expression of Cebpa, Lpl and Pparg, as well as oxidative stress-related gene Nrf2. Nrf2 pathway inhibitors could further enhance the adipogenesis of MSCs in bone marrow after radiation. Notably, the similar regulation of oxidative pathways and enhanced adipogenesis post irradiation were observed in human bone marrow MSCs. In addition, irradiation exposure led to up-regulated mRNA expression of interleukin-6 and down-regulated mRNA expression of colony stimulating factor 2 in human bone marrow MSCs. CONCLUSION Ionizing radiation promotes adipogenesis of MSCs in mice, and oxidative stress pathway participates in this effect, blocking Nrf2 further promotes the adipogenesis of MSCs. Additionally, irradiation activates oxidative pathways and promotes adipogenic differentiation of human bone marrow MSCs.
Mesenchymal Stem Cells/cytology* ; Oxidative Stress/radiation effects* ; Animals ; Adipogenesis/radiation effects* ; Mice ; Radiation, Ionizing ; Cell Differentiation/radiation effects* ; Humans ; NF-E2-Related Factor 2/metabolism* ; PPAR gamma ; Cells, Cultured

OBJECTIVE: To establish an in vitro cell model simulating acute graft-versus-host disease (aGVHD) bone marrow microenvironment injury with the advantage of mouse serum of aGVHD model and explore the effect of serum of aGVHD mouse on the adipogenic differentiation ability of mesenchymal stem cells (MSCs). METHODS: The 6-8-week-old C57BL/6N female mice and BALB/c female mice were used as the donor and recipient mice of the aGVHD model, respectively. Bone marrow transplantation (BMT) mouse model (n=20) was established by being injected with bone marrow cells (1×10⁷ per mouse) from donor mice within 4-6 hours after receiving a lethal dose (8.0 Gy, 72.76 cGy/min) of γ ray general irradiation. A mouse model of aGVHD (n=20) was established by infusing a total of 0.4 ml of a mixture of donor mouse-derived bone marrow cells (1×10⁷ per mouse) and spleen lymphocytes (2×10⁶ per mouse). The blood was removed from the eyeballs and the mouse serum was aspirated on the 7^th day after modeling. Bone marrow-derived MSCs were isolated from 1-week-old C57BL/6N male mice and incubated with 2%, 5% and 10% BMT mouse serum and aGVHD mouse serum in the medium, respectively. The effect of serum in the two groups on the in vitro adipogenic differentiation ability of mouse MSCs was detected by Oil Red O staining. The expression levels of related proteins PPARγ and CEBPα were detected by Western blot. The expression differences of key adipogenic transcription factors including PPARγ, CEBPα, FABP4 and LPL were determined by real-time quantitative PCR (RT-qPCR). RESULTS: An in vitro cell model simulating the damage of bone marrow microenvironment in mice with aGVHD was successfully established. Oil Red O staining showed that the number of orange-red fatty droplets was significantly reduced and the adipogenic differentiation ability of MSC was impaired at aGVHD serum concentration of 10% compared with BMT serum. Western blot experiments showed that adipogenesis-related proteins PPARγ and CEBPα expressed in MSCs were down-regulated. Further RT-qPCR assay showed that the production of PPARγ, CEBPα, FABP4 and LPL, the key transcription factors for adipogenic differentiation of MSC, were significantly reduced. CONCLUSION The adipogenic differentiation capacity of MSCs is inhibited by aGVHD mouse serum.
Animals ; Mesenchymal Stem Cells/cytology* ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Adipogenesis ; Female ; Cell Differentiation ; Graft vs Host Disease/blood* ; Bone Marrow Cells/cytology* ; PPAR gamma/metabolism* ; Disease Models, Animal ; CCAAT-Enhancer-Binding Protein-alpha/metabolism*

OBJECTIVE: To prepare clinical-grade human umbilical cord-derived mesenchymal stem cells (hUC-MSC) with high expression of hematopoietic supporting factors and evaluate their stem cell characteristics. METHODS: Fetal umbilical cord tissues were collected from healthy postpartum women during full-term cesarean section. Wharton's jelly was mechanically separated and hUC-MSCs were obtained by explant culture method and enzyme digestion method in an animal serum-free culture system with addition of human platelet lysate. The phenotypic characteristics of hUC-MSCs obtained by two methods were detected by flow cytometry. The differences in proliferation ability between the two groups of hUC-MSCs were identified through CCK-8 assay and colony forming unit-fibroblast (CFU-F) assay. The differences in multilineage differentiation potential between the two groups of hUC-MSCs were identified through induction of adipogenic, osteogenic, and chondrogenic differentiation. The mRNA expression levels of hematopoietic supporting factors such as SCF, IL-3, CXCL12, VCAM1 and ANGPT1 in the two groups of hUC-MSCs were identified by real-time fluorescence quantiative PCR(RT-qPCR). RESULTS: The results of flow cytometry showed that hUC-MSCs obtained by the two methods both expressed high levels of CD73, CD90 and CD105, while lowly expressed CD31, CD45 and HLA-DR. The results of CCK-8 and CFU-F assay showed that the proliferation ability of hUC-MSCs obtained by explant culture method was better than those obtained by enzyme digestion method. The results of the triple lineage differentiation experiment showed that there was no significant difference in multilineage differentiation potential between the two grous of hUC-MSCs. The results of RT-qPCR showed that the mRNA expression levels of hematopoietic supporting factors SCF, IL-3, CXCL12, VCAM1 and ANGPT1 in hUC-MSCs obtained by explant cultrue method were higher than those obtained by enzyme digestion method. CONCLUSION Clinical-grade hUC-MSCs with high expression levels of hematopoietic supporting factors were successfully cultured in an animal serum-free culture system.
Humans ; Mesenchymal Stem Cells/metabolism* ; Umbilical Cord/cytology* ; Cell Differentiation ; Female ; Cell Proliferation ; Cells, Cultured ; Chemokine CXCL12/metabolism* ; Angiopoietin-1/metabolism* ; Vascular Cell Adhesion Molecule-1/metabolism* ; Stem Cell Factor/metabolism* ; Flow Cytometry ; Pregnancy

BACKGROUND: Quantitative flow ratio (QFR) holds significant value in guiding drug-coated balloon (DCB) treatment and enhancing outcomes. However, the predictive capability of post-angioplasty QFR for long-term clinical events in patients with de novo lesions who receive DCB treatment remains uncertain. The aim of this study was to explore the potential significance of post-angioplasty QFR measurements in predicting clinical outcomes in patients underwent DCB treatment for de novo lesions. METHODS: Patients who underwent DCB-only intervention for de novo lesions were enrolled. QFR was conducted after DCB treatment. The patients were then categorized based on post-angioplasty QFR. The primary endpoint was major adverse cardiac events (MACE), encompassing all-cause death, cardiovascular death, nonfatal myocardial infarction, stroke, and target vessel revascularization. RESULTS: A total of 553 patients with 561 lesions were included. The median follow-up period was 505 days, during which 66 (11.8%) MACEs occurred. Based on post-procedural QFR grouping, there were 259 cases in the high QFR group (QFR > 0.93) and 302 cases in the low QFR group (QFR ≤ 0.93). Kaplan-Meier analysis revealed a significantly higher cumulative incidence of MACE in the low QFR group (log-rank P = 0.004). The multivariate Cox proportional hazards model demonstrated a significant inverse correlation between QFR and the occurrence of MACEs (HR = 0.522, 95%CI: 0.289-0.942, P = 0.031). Landmark analysis indicated that high QFR had a significant reducing effect on the cumulative incidence of MACEs within 1 year (log-rank P = 0.016) and 1-5 years (log-rank P = 0.026). CONCLUSIONS In patients who underwent DCB-only treatment for de novo lesions, higher post-procedural QFR values (> 0.93) were identified as an independent protective factor against adverse prognosis.

Objective:To establish a mesenchymal stem cell(MSC)-based in vitro cell model for the evaluation of mouse bone marrow acute graft-versus-host disease(aGVHD).Methods:Female C57BL/6N mice aged 6-8 weeks were used as bone marrow and lymphocyte donors,and female BALB/c mice aged 6-8 weeks were used as aGVHD recipients.The recipient mouse received a lethal dose(8.0 Gy,72.76 cGy/min)of total body γ irradiation,and injected with donor mouse derived bone marrow cells(1× 107/mouse)in 6-8 hours post irradiation to establish a bone marrow transplantation(BMT)mouse model(n=20).In addition,the recipient mice received a lethal dose(8.0 Gy,72.76 cGy/min)of total body γ irradiation,and injected with donor mouse derived bone marrow cells(1 × 107/mouse)and spleen lymphocytes(2 × 106/mouse)in 6-8 hours post irradiation to establish a mouse aGVHD model(n=20).On the day 7 after modeling,the recipient mice were anesthetized and the blood was harvested post eyeball enucleation.The serum was collected by centrifugation.Mouse MSCs were isolated and cultured with the addition of 2％,5％,and 10％recipient serum from BMT group or aGVHD group respectively.The colony-forming unit-fibroblast(CFU-F)experiment was performed to evaluate the potential effects of serums on the self-renewal ability of MSC.The expression of CD29 and CD105 of MSC was evaluated by immunofluorescence staining.In addition,the expression of self-renewal-related genes including Oct-4,Sox-2,and Nanog in MSC was detected by real-time fluorescence quantitative PCR(RT-qPCR).Results:We successfully established an in vitro cell model that could mimic the bone marrow microenvironment damage of the mouse with aGVHD.CFU-F assay showed that,on day 7 after the culture,compared with the BMT group,MSC colony formation ability of aGVHD serum concentrations groups of 2％and 5％was significantly reduced(P＜0.05);after the culture,at day 14,compared with the BMT group,MSC colony formation ability in different aGVHD serum concentration was significantly reduced(P＜0.05).The immunofluorescence staining showed that,compared with the BMT group,the proportion of MSC surface molecules CD29+and CD 105+cells was significantly dereased in the aGVHD serum concentration group(P＜0.05),the most significant difference was at a serum concentration of 10％(P＜0.001,P＜0.01).The results of RT-qPCR detection showed that the expression of the MSC self-renewal-related genes Oct-4,Sox-2,and Nanog was decreased,the most significant difference was observed at an aGVHD serum concentration of 10％(P＜0.01,P＜0.001,P＜0.001).Conclusion:By co-culturing different concentrations of mouse aGVHD serum and mouse MSC,we found that the addition of mouse aGVHD serum at different concentrations impaired the MSC self-renewal ability,which providing a new tool for the field of aGVHD bone marrow microenvironment damage.

Entinostat plus exemestane in hormone receptor-positive (HR+) advanced breast cancer (ABC) previously showed encouraging outcomes. This multicenter phase 3 trial evaluated the efficacy and safety of entinostat plus exemestane in Chinese patients with HR + ABC that relapsed/progressed after ≥1 endocrine therapy. Patients were randomized (2:1) to oral exemestane 25 mg/day plus entinostat (n = 235) or placebo (n = 119) 5 mg/week in 28-day cycles. The primary endpoint was the independent radiographic committee (IRC)-assessed progression-free survival (PFS). The median age was 52 (range, 28-75) years and 222 (62.7%) patients were postmenopausal. CDK4/6 inhibitors and fulvestrant were previously used in 23 (6.5%) and 92 (26.0%) patients, respectively. The baseline characteristics were comparable between the entinostat and placebo groups. The median PFS was 6.32 (95% CI, 5.30-9.11) and 3.72 (95% CI, 1.91-5.49) months in the entinostat and placebo groups (HR, 0.76; 95% CI, 0.58-0.98; P = 0.046), respectively. Grade ≥3 adverse events (AEs) occurred in 154 (65.5%) patients in the entinostat group versus 23 (19.3%) in the placebo group, and the most common grade ≥3 treatment-related AEs were neutropenia [103 (43.8%)], thrombocytopenia [20 (8.5%)], and leucopenia [15 (6.4%)]. Entinostat plus exemestane significantly improved PFS compared with exemestane, with generally manageable toxicities in HR + ABC (ClinicalTrials.gov #NCT03538171).

Autophagy often occurs after cells are attacked by oxidative stress, where damaged structures are phagocytic and degraded into nutrients, thereby reducing oxidative damage, promoting the survival of cancer cells and reducing the therapeutic effect of photodynamic therapy (PDT). However, excessive activation of autophagy can promote cell apoptosis. In this paper, the photosensitizer pyropheophorbide-a (Ppa) was used to produce a large amount of reactive oxygen species (ROS) to achieve the effect of killing cancer cells. At the same time, icaritin (Ica), an autophagy inducer, was used to over-activate autophagy, which transformed the protection of cancer cells into the promotion of cancer cell apoptosis, so as to improve the effect of photodynamic therapy. In this study, the interaction force between Ica and Ppa was exploited to successfully construct a self-assembled nanomedicine IP with good stability and high drug load. The synthesis method is simple, through using the drug itself as a carrier, and the loading capacity (LA) of Ica and Ppa can be increased to 83.53% and 16.45% without introducing potential biosafety risks of nanocarriers. Compared with free Ppa, self-assembled nanomedicine IP showed superior performance in cellular uptake and reactive oxygen species production. In addition, the self-assembled nanomedicine IP can reverse the protective autophagy induced by PDT by activating the autophagy of tumor cells, and facilitate apoptosis and antitumor coordination, which significantly improves the antitumor activity of PDT.

BACKGROUND: Clinical outcomes are poor if patients with acute heart failure (AHF) are discharged with residual congestion in the presence of renal dysfunction. However, there is no single indication to reflect the combined effects of the two related pathophysiological processes. We, therefore, proposed an indicator, congestion and renal index (CRI), and examined the associations between the CRI and one-year outcomes and the incremental prognostic value of CRI compared with the established scoring systems in a multicenter prospective cohort of AHF. METHODS: We enrolled AHF patients and calculated the ratio of thoracic fluid content index divided by estimated glomerular filtration rate before discharge, as CRI. Then we examined the associations between CRI and one-year outcomes. RESULTS: A total of 944 patients were included in the analysis (mean age 63.3 ± 13.8 years, 39.3% women). Compared with patients with CRI ≤ 0.59 mL/min per kΩ, those with CRI > 0.59 mL/min per kΩ had higher risks of cardiovascular death or HF hospitalization (HR = 1.56 [1.13-2.15]) and all-cause death or all-cause hospitalization (HR = 1.33 [1.01-1.74]). CRI had an incremental prognostic value compared with the established scoring system. CONCLUSIONS In patients with AHF, CRI is independently associated with the risk of death or hospitalization within one year, and improves the risk stratification of the established risk models.

OBJECTIVE: To evaluate the effectiveness and safety of Rotarex catheter system in treating femoropopliteal artery stenosis accompanied with thrombosis. METHODS: From Jun. 2017 to Dec. 2019, the clinical data of 32 femoropopliteal artery stenosis accompanied with thrombosis cases treated with Rotarex catheter system were retrospectively analyzed. There were 23 males and 9 females aged from 50 to 89 years and the mean age was (70.7±10.3) years. Six cases had acute course of disease (≤2 weeks), 17 cases had subacute course of disease (>2 weeks, ≤3 months), and 9 cases had chronic course of disease (>3 months). Mean lesion length was (23.4±13.7) cm, mean occlusion length was (19.9±13.3) cm, and in-stent occlusion 7 cases. The superficial femoral artery (SFA) was involved in 13 cases, the popliteal artery (PA) was involved in 8 cases, and both SFA and PA were involved in the other 11 cases. All the cases were treated with Rotarex catheter system. When necessary, suction with large lumen catheter was enabled. Residual stenosis was treated with percutaneous transluminal angioplasty (PTA). Drug-coated balloon (DCB) was only used in patients with financial status, and stent was used only when it was necessary. Heparin was used for 24 h after procedures, and after that, antiplatelet agents were used. Doppler ultrasonography was taken during the followed-up. RESULTS: Technical success was 100%, and mean procedure time was (107.4±21.5) min. 8F (1F≈0.33 mm) and 6F Rotarex catheter were used in 27 and 5 cases respectively. In 27 cases, forward flow was obtained immediately after debulking with Rotarex catheter, and in the other 5 cases, suction with large lumen catheters were used. PTA was used in all 32 cases. DCB were used in 8 cases, of which 4 were used in in-stent stenosis. Twelve cases were implanted stents. There were no perioperative deaths. The only one procedure related complication was distal embolism. We took out the thrombus with guiding catheter. In all cases, mean hospital stay were (4.6±1.5) d. The ankle brachial index increased from 0.32±0.15 to 0.86±0.10 after treatment (t=-16.847, P < 0.001). The Rutherford stages decreased significantly (Z=-4.518, P < 0.001). All the patients were followed up for 6.0-36.0 months, and the median time was 16.0 months. 2 cases stopped antiplatelet agents, which resulted in acute thrombosis. Another percutaneous mechanical thrombectomy and PTA were taken in one of them. Two cases died of cardiovascular disease during the follow-up, and no amputation was observed. Target lesion restenosis occurred in 7 cases during the follow-up, and target lesion revascularization (TLR) was taken in two of them. CONCLUSION In treating femoropopliteal artery stenosis accompanied with thrombosis, Rotarex catheter can remove thrombus effectively, and that can expose underlying lesions and reduce stent use and complications rates. It is a safe and effective method.
Male ; Female ; Humans ; Middle Aged ; Aged ; Aged, 80 and over ; Femoral Artery/surgery* ; Retrospective Studies ; Constriction, Pathologic ; Platelet Aggregation Inhibitors ; Treatment Outcome ; Thrombosis ; Catheters