Histone acetyltransferase P300 deficiency promotes ferroptosis of vascular smooth muscle cells by activating the HIF-1α/HMOX1 axis.

BACKGROUND: E1A-associated 300-kDa protein (P300), an endogenous histone acetyltransferase, contributes to modifications of the chromatin landscape of genes involved in multiple cardiovascular diseases. Ferroptosis of vascular smooth muscle cells (VSMCs) is a novel pathological mechanism of aortic dissection. However, whether P300 regulates VSMC ferroptosis remains unknown. METHODS: Cystine deprivation (CD) and imidazole ketone erastin (IKE) were used to induce VSMC ferroptosis. Two different knockdown plasmids targeting P300 and A-485 (a specific inhibitor of P300) were used to investigate the function of P300 in the ferroptosis of human aortic smooth muscle cells (HASMCs). Cell counting kit-8, lactate dehydrogenase and flow cytometry with propidium iodide staining were performed to assess the cell viability and death under the treatment of CD and IKE. BODIPY-C11 assay, immunofluorescence staining of 4-hydroxynonenal and malondialdehyde assay were conducted to detect the level of lipid peroxidation. Furthermore, co-immunoprecipitation was utilized to explore the interaction between P300 and HIF-1α, HIF-1α and P53. RESULTS: Compared with normal control, the protein level of P300 was significantly decreased in HASMCs treated with CD and IKE, which was largely nullified by the ferroptosis inhibitor ferrostatin-1 but not by the autophagy inhibitor or apoptosis inhibitor. Knockdown of P300 by short-hairpin RNA or inhibition of P300 activity by A-485 promoted CD- and IKE-induced HASMC ferroptosis, as evidenced by a reduction in cell viability and aggravation of lipid peroxidation of HASMCs. Furthermore, we found that hypoxia-inducible factor-1α (HIF-1α)/heme oxygenase 1 (HMOX1) pathway was responsible for the impacts of P300 on ferroptosis of HASMCs. The results of co-immunoprecipitation demonstrated that P300 and P53 competitively bound HIF-1α to regulate the expression of HMOX1. Under normal conditions, P300 interacted with HIF-1α to inhibit HMOX1 expression, while reduced expression of P300 induced by ferroptosis inducers would favor HIF-1α binding to P53 to trigger HMOX1 overexpression. Furthermore, the aggravated effects of P300 knockdown on HASMC ferroptosis were largely nullified by HIF-1α knockdown or the HIF-1α inhibitor BAY87-2243. CONCLUSION: Thus, our results revealed that P300 deficiency or inactivation facilitated CD- and IKE-induced VSMC ferroptosis by activating the HIF-1α/HMOX1 axis, which may contribute to the development of diseases related to VSMC ferroptosis.

A Simplified Continuous Two-stitch Suture for Bronchial Anastomosis of Left Single Lung Transplant in Dogs.

Large animal models are essential to pre-clinical trials of pulmonary transplantation and bronchial anastomosis poses a great technical challenge to the procedure. Presented here is a simplified continuous two-stitch suture technique into bronchial anastomosis during the course of left single lung transplantation in canine. Animals were divided into three groups with each group having 6 animals. Left single lung transplantation in canine was performed to assess the feasibility of using this technique for bronchial anastomosis. In the control groups, all anastomoses were done by using traditional technique. Allograft functions and hemodynamic parameters were monitored during a 3-h reperfusion period. Quality of bronchial healing and airway complications were assessed by bronchoscopic surveillance after transplantation. We successfully completed left lung transplantation in 18 dogs, and all the dogs survived the procedures. The new technique substantially simplified the procedures for bronchial anastomosis and greatly reduced the time for bronchial anastomosis (P<<0.01) and the ischemic time of the grafts (P<0.05) compared to the control group. The continuous two-stitch suture attenuated the tissue injury to allografts and led to better blood gas exchange function as compared to the control group (P<0.05). Good bronchial healing (Grade I) was observed in all the groups. A canine left single lung transplantation model is feasible by using the novel suture technique, and the new technique is as safe as the traditional method. The technique is easy to learn, particularly for less experienced operators. Simpler and time-saving, the technique has great potential to be widely employed in clinical lung transplantation.

A simplified two-stitch sleeve technique for arterial anastomosis of cervical heterotopic cardiac transplantation in mice.

Although cervical cardiac transplantation is a well recognized useful model in diverse experimental settings, its widespread use, however, has been significantly hampered by the technical challenges relevant to small vessel anastomosis. We herein introduced a simplified two-stitch sleeve technique into arterial anastomosis during the course of cervical cardiac transplantation in mice. Cervical transplantation of allogenic and syngeneic cardiac grafts was conducted to assess the feasibility of this two-stitch sleeve technique in arterial anastomosis. Venous anastomosis was completed by the one-suture end-to-end microsuture technique, while arterial anastomosis was conducted by invaginating the recipient right common carotid artery into the graft left common carotid artery along with two guiding stitches. The two-stitch sleeve technique significantly simplified the procedures for arterial anastomosis as compared with that of the traditional microsuture technique (5.5 ± 1.8 min vs. 15.7 ± 3.0 min). However, the survival time for allografts (8.0 ± 0.2 day vs. 8.0 ± 0.4 day) and the long-term patency for syngeneic grafts (> 120 days) were the same as the grafts implanted by the traditional microsuture technique. This simplified sleeve technique is easy to learn, particularly for beginners without microsuture experience, and therefore, it has the great potential for widespread use in transplant immunology.