Single-Cell RNA-Seq Analysis Reveals Macrophages Are Involved in the Pathogenesis of Human Sporadic Acute Type A Aortic Dissection.

Macrophages play an important role in the progression of sporadic acute type A aortic dissection (ATAAD). The aim of this study was to characterize the cellular heterogeneity of macrophages in ATAAD tissues by scRNA-seq. Ascending aortic wall tissue from six ATAAD patients and three heart transplant donors was assessed by scRNA-seq and then analyzed and validated by various bioinformatic algorithms and histopathology experiments. The results revealed that the proportion of macrophages in ATAAD tissues (24.51%) was significantly higher than that in normal tissues (13.69%). Among the six macrophage subclusters, pro-inflammatory macrophages accounted for 14.96% of macrophages in the AD group and 0.18% in the normal group. Chemokine- and inflammation-related genes (CCL2, CCL20, S100A8, and S100A9) were expressed more intensively in macrophages in ATAAD tissue than in those in normal tissue. Additionally, intercellular communication analysis and transcription factor analysis indicated the activation of inflammation and degradation of the extracellular matrix in ATAAD tissue. Finally, immunohistochemistry, immunofluorescence, and Western blot experiments confirmed the overexpression of macrophage marker genes (CD68 and CD163) and matrix metalloproteinases (MMP9 and MMP2) in ATAAD tissue. Collectively, our study provides a preliminary evaluation of the role of macrophages in ATAAD, and the results could aid in the development of therapeutic options in the future.

Prostaglandin E1 protects bone marrow-derived mesenchymal stem cells against serum deprivation-induced apoptosis.

Mesenchymal stem cells (MSCs) have become a recent focus of experimental and clinical research regarding myocardial regeneration. However, the therapeutic potential of these cells is limited by poor survival. Prostaglandin E1 (PGE1) is known to have anti­inflammatory and anti­apoptotic effects on the myocardium. The aim of the present study was to determine whether PGE1 could protect MSCs against serum deprivation (SD)­induced apoptosis. An SD model was used to induce apoptosis in MSCs in vitro. Apoptotic morphological changes were detected by Hoechst 33258 fluorescent nuclear staining; and Annexin V­fluorescein isothiocyanate/propidium iodide (PI) double staining and flow cytometry was used to quantify the rate of apoptosis. Western blot analysis was used to detect the expression levels of the apoptosis­associated proteins Bcl­2, Bax and caspase­3. The results of the present study demonstrated that SD induced apoptosis of MSCs, and that treatment with PGE1 attenuated the morphological changes characteristic of apoptosis. Annexin V/PI staining showed that the rate of apoptosis gradually increased with the duration of ischemia. Furthermore, treatment with PGE1 significantly reduced SD­induced apoptosis, decreased the protein expression levels of Bax and caspase­3, and increased the expression levels of Bcl­2. These data suggest that PGE1 is able to influence the survival of MSCs under certain conditions. These results may aid in improving the therapeutic efficacy of MSC transplantation used to treat chronic ischemic heart disease.

Inhibition of apoptosis by knockdown of caspase-3 with siRNA in rat bone marrow mesenchymal stem cells.

Transplantation of bone marrow mesenchymal stem cells is a promising new strategy for the repair of infarcted cardiac tissue. However, the majority of transplanted bone marrow mesenchymal stem cells (BMSCs) die soon after transplantation, due in part to oxidative stress in the ischemic region. Oxidative stress is known to induce apoptosis through the activation of caspase-3. The aim of this study is to determine whether small interfering RNA targeting caspase-3 can inhibit the apoptosis of rat BMSCs in vitro. Caspase-3 siRNA expression vectors were prepared and transfected into rat BMSCs in the presence of liposomes. Western blot assay and real-time polymerase chain reaction (RT-PCR) were performed to detect caspase-3 expression. A retrovirus packaging system was employed to package 293FT cells producing caspase-3 siRNA virus, which were transfected into rat BMSCs. Those stably expressing caspase-3 siRNA were screened by Western blot assay and RT-PCR to determine caspase-3 expression levels. Stable transfection of caspase-3 siRNA significantly decreased caspase-3 protein (0.26 ± 0.001 vs. 0.42 ± 0.004, P < 0.05) and mRNA expression (0.19 ± 0.002 vs. 1, P < 0.05) in BMSCs compared to non-transfected BMSCs. Cells were incubated in H2O2 to induce apoptosis, which was detected by TUNEL staining, and BMSC morphology was not altered by either transient or stable transfection of caspase-3 siRNA. H2O2-induced apoptosis of BMSCs stably transfected with caspase-3 siRNA was dramatically reduced compared to that of normal BMSCs (11.0 ± 3.2 vs. 25.8 ± 4.2, P < 0.05). Caspase-3 knockdown BMSCs are thus more resistant to apoptosis than normal BMSCs, potentially increasing their survival rates under conditions that cause oxidative stress.