Vascular injury is one of the most common detrimental effects of cancer radiotherapy on healthy tissues. Since the efficacy of current preventive and therapeutic strategies remains limited, the exploration of new approaches to treat radiation-induced vascular injury (RIV) is on high demands. The use of mesenchymal stem cells (MSCs) to treat RIV holds great promise thanks to their well-documented function of mediating tissue regeneration after injury. Recently, we genetically modified MSCs with high mobility group box 1 (HMGB1) and demonstrated the high efficacy of these cells in treating graft atherosclerosis. The current study was to investigate the protective effect of HMGB1-modified MSCs (MSC-H) on RIV by using a rat model. Female F344 rats received an intravenous injection of male F344 MSC-H cells or vehicle control at four doses of 2 × 106 cells with a 15-day interval starting from 30 days after irradiation to the abdominal aorta. The aortas were procured for histological and biomedical analysis at 90 days after irradiation. Cell migration to irradiated aortas was traced by green fluorescent protein and sex determination region on the Y chromosome. In vitro cell migration and endothelial differentiation of MSC-H cells were analyzed by stromal-derived factor 1-induced transwell assay and RNA microarray, respectively. The contribution of extracellular HMGB1 to the bioactivity of MSC-H cells was investigated by inhibition experiments with HMGB1 antibody. MSC-H cell infusion alleviated neointimal formation, vascular inflammation, and fibrosis in irradiated aortas, which was associated with local migration and endothelial differentiation of MSC-H cells. The MSC-H cells showed high motility and potential of endothelial differentiation in vitro. Microarray analysis suggested multiple pathways like MAPK and p53 signaling were activated during endothelial differentiation. MSC-H cells highly expressed CXC chemokine receptor 4 and migrated progressively after stromal-derived factor 1 stimulation, which was blocked by the antagonist of CXC chemokine receptor 4. Finally, the migration and endothelial differentiation of MSC-H cells were inhibited by HMGB1 antibody. MSC-H cell infusion significantly attenuated RIV, which was associated with their high motility and endothelial differentiation potential. Multiple pathways that possibly contributed to the efficacy of MSC-H cells were suggested and deserved further investigation.

Background: Mesenchymal stem cells (MSC) hold great promise for treating cardiovascular disease. Recently, we genetically modified MSCs with high mobility group box 1 (HMGB1), and these cells demonstrated high mobility by efficient migrating and homing to target neointima. The possible mechanism was investigated in the current study. Methods: Rat MSCs were transfected with lentivirus containing HMGB1 cDNA to yield MSC-H cell line stably overexpressing HMGB1. The MSC-C cells which were transfected with empty lentivirus served as negative control, and the differentially expressed genes were analyzed by microarray. The cell mobility was determined by transwell migration assay. Intracellular free calcium and the expression of Cav3.2 T-type calcium channel (CACNA1H) were assayed to analyze activity of CACNA1H-mediated calcium influx. H₂S production and γ-cystathionase expression were examined to assess the activity of γ-cystathionase/H₂S signaling. The interaction of HMGB1 with γ-cystathionase in MSC-H cells was analyzed by co-immunoprecipitation. Luciferase reporter assay was performed to determine whether the promoter activity of γ-cystathionase was regulated by interaction of β-catenin and TCF/LEF binding site. Intercellular cAMP, PKA activity, phosphorylation of β-catenin, and GSK3β were investigated to reveal cAMP/PKA mediated β-catenin activation. Result: Microarray analysis revealed that differentially expressed genes were enriched in cAMP signaling and calcium signaling. CACNA1H was upregulated to increase intracellular free calcium and MSC-H cell migration. Blockage of CACNA1H by ABT-639 significantly reduced intracellular free calcium and cell migration. The γ-cystathionase/H₂S signaling was responsible for CACNA1H activation. H₂S production was increased with high expression of γ-cystathionase in MSC-H cells, which was blocked by γ-cystathionase inhibitor DL-propargylglycine. Upregulation of γ-cystathionase was not attributed to interaction with HMGB1 overexpressed in MSC-H cells although γ-cystathionase was suggested to co-immunoprecipitate with oxidized HMGB1. Bioinformatics analysis identified a conserved TCF/LEF binding site in the promoter of γ-cystathionase gene. Luciferase reporter assay confirmed that the promoter had positive response to β-catenin which was activated in MSC-H cells. Finally, cAMP/PKA was activated to phosphorylate β-catenin at Ser657 and GSK3β, enabling persisting activation of Wnt/β-catenin signaling in MSC-H cells. Conclusion: Our study revealed that modification of MSCs with HMGB1 promoted CACNA1H-mediated calcium influx via PKA/β-catenin/γ-cystathionase pathway. This was a plausible mechanism for high mobility of MSC-H cell line.

BACKGROUND Carotid atherosclerosis is a complex disease involving multiple cellular and molecular pathways. Mesenchymal stem cells (MSCs) show therapeutic potential, but their optimal targets and efficacy are still under study. MiR-126 enhances endothelial function and promotes angiogenesis by relieving vascular endothelial growth factor signaling suppression, suggesting its potential in vascular regeneration. However, its role in directing stem cell differentiation toward endothelial lineages remains unclear. We hypothesize that miR-126 may influence MSCs’ immunomodulatory and vascular reparative functions via the mitogen-activated protein kinases/extracellular signal-regulated kinase (MAPK/ERK) pathway, thereby improving carotid atherosclerosis. This study explores this mechanism to provide novel insights and support the development of miR-126-based therapeutic strategies. AIM To verify if miR-126 inhibits carotid atherosclerosis via the MAPK/ERK pathway. METHODS Rat bone marrow MSCs (product No. CP-R131, Wuhan, China) were verified by flow cytometry. The effects of miR-126 on MSCs’ proliferation, migration, apoptosis, and cytokine expression were explored using microRNA mimics and inhibitors. Fluorescence staining quantified CD31+ cells to evaluate endothelial differentiation. In vivo differentiation was assessed, and MSCs were transplanted into a rat carotid artery balloon dilatation model. Rats were randomly divided into five groups: Control, negative control mimics, miR-126 mimics, negative control inhibitor, and miR-126 inhibitor. RESULTS In vitro , MSCs treated with miR-126 mimics demonstrated enhanced proliferation, increased migration, and reduced apoptosis. These miR-126 mimics also significantly increased the secretion of vascular endothelial growth factor and basic fibroblast growth factor. Fluorescence and tissue staining indicated a higher proportion of CD31+ cells in the miR-126 mimics group. Additionally, the expression of endothelial-related genes (von Willebrand factor, endothelial nitric oxide synthase, and vascular endothelial-cadherin) was upregulated in this group. In vivo , miR-126-transfected MSCs effectively reduced neointimal thickness and promoted endothelial coverage in rats. MiR-126 stimulated MSC proliferation in a dose-dependent manner and reduced p38 and ERK1/2 phosphorylation. Conversely, miR-126 inhibition or blank controls resulted in opposing effects. CONCLUSION MiR-126 exerts significant modulatory effects on the immunoregulatory and vascular reparative functions of MSCs through the MAPK/ERK signaling pathway, promoting their differentiation into endothelial cells and thereby mitigating atherosclerosis.

Radiation vasculitis is one of the most common detrimental effects of radiotherapy for malignant tumors. This is developed at the vasculature of adjacent organs. Animal experiments have showed that transplantation of mesenchymal stem cells (MSCs) restores vascular function after irradiation. But the population of MSCs being engrafted into irradiated vessels is too low in the conventional models to make assessment of therapeutic effect difficult. This is presumably because circulating MSCs are dispersed in adjacent tissues being irradiated simultaneously. Based on the assumption, a rat model, namely, RT (radiation) plus TX (transplantation), was established to promote MSC homing by sequestering irradiated vessels. In this model, a 1.5 cm long segment of rat abdominal aorta was irradiated by 160kV X-ray at a single dose of 35Gy before being procured and grafted to the healthy counterpart. F344 inbred rats served as both donors and recipients to exclude the possibility of immune rejection. A lead shield was used to confine X-ray delivery to a 3 cm×3 cm square-shaped field covering central abdominal region. The abdominal viscera especially small bowel and colon were protected from irradiation by being pushed off the central abdominal cavity. Typical radiation-induced vasculopathy was present on the 90^thday after irradiation. The recruitment of intravenously injected MSCs to irradiated aorta was significantly improved by using the RT-plus-TX model as compared to the model with irradiation only. Generally, the RT-plus-Tx model promotes MSC recruitment to irradiated aorta by separating irradiated vascular segment from adjacent tissue. Thus, the model is preferred in the study of MSC-based therapy for radiation vasculitis when the evaluation of MSC homing is demanding.

To explore and analyze the risk factors for recurrence in patients with lower extremity arteriosclerosis obliterans (ASO) after surgical intervention and to construct and validate a nomogram prediction model. A total of 270 patients with ASO treated at our hospital were retrospectively selected as study subjects and divided into a training cohort (189 cases) and a validation cohort (81 cases) based on a 7:3 ratio. Patients in the training cohort were further divided into recurrence and non-recurrence groups based on whether they experienced recurrence within two years post-surgery. Univariate and multivariate logistic regression analyses were employed to identify independent risk factors for postoperative recurrence, which were then used to construct a predictive model and generate a nomogram. Of the 270 patients with ASO included in the study, the training cohort consisted of 189 patients, with 76 (40.21%) in the recurrence group and 113 (59.79%) in the non-recurrence group. The validation cohort consisted of 81 patients, with 32 (39.51%) in the recurrence group and 49 (60.49%) in the non-recurrence group. Univariate analysis in the training cohort revealed significant differences in age, body mass index (BMI), diabetes, hypertension, lesion location classification, use of antiplatelet drugs, triglycerides, fibrinogen (FIB), and di-dimer (D-D) (P < 0.05, respectively). Multivariate logistic regression analysis indicated that age ≥ 60 years, BMI ≥ 24 kg/m², diabetes, hypertension, discontinuation of antiplatelet therapy, FIB, and D-D were independent risk factors for recurrence after surgical intervention in patients with lower extremity ASO (OR = 2.471, 1.625, 4.568, 2.678, 5.974, 2.073 and 3.067; P < 0.05, respectively). When the training and validation cohorts were tested in the established nomogram model, the area under the curve (AUC) of the model was 0.832 (95% CI: 0.765–0.919) in the training cohort and 0.858 (95% CI: 0.745–0.964) in the validation cohort. Calibration curves indicated high consistency between the predicted and actual outcomes in both groups, suggesting good predictive accuracy of the model. Decision curve analysis showed that using this model significantly increased net clinical benefit for patients. The nomogram model constructed for predicting the risk of recurrence in patients with lower extremity ASO after surgical intervention demonstrates good predictive and discriminative abilities, offering valuable guidance for clinical screening of high-risk populations.