Propofol inhibits myocardial injury induced by microvesicles derived from hypoxia-reoxygenated endothelial cells via lncCCT4-2/CCT4 signaling.

BACKGROUND: Ischemia-reperfusion (IR) induces increased release of extracellular vesicles in the heart and exacerbates myocardial IR injury. We have previously shown that propofol attenuates hypoxia/reoxygenation (HR)-induced injury in human umbilical vein endothelial cells (HUVECs) and that microvesicles derived from propofol-treated HUVECs inhibit oxidative stress in endothelial cells. However, the role of microvesicles derived from propofol post-treated HUVECs ((HR + P)-EMVs) in IR-injured cardiomyocytes is unclear. In this study, we aimed to investigate the role of (HR + P)-EMVs in cardiac IR injury compared to microvesicles derived from hypoxic/reoxygenated HUVECs (HR-EMVs) and to elucidate the underlying mechanisms. METHODS: Hypoxia/reoxygenation (HR) models of HUVECs and AC16 cells and a mouse cardiac IR model were established. Microvesicles from HR-injured HUVECs, DMSO post-treated HUVECs and propofol post-treated HUVECs were extracted by ultra-high speed centrifugation, respectively. The above EMVs were co-cultured with HR-injured AC16 cells or injected intracardially into IR mice. Flow cytometry and immunofluorescence were used to determine the levels of oxidative stress and apoptosis in cardiomyocytes. Apoptosis related proteins were detected by Western blot. Echocardiography for cardiac function and Evans blue-TTC staining for myocardial infarct size. Expression of lncCCT4-2 in EMVs and AC16 cells was analysed by whole transcriptome sequencing of EMVs and RT-qPCR. The molecular mechanism of inhibition of myocardial injury by (HR + P)-EMVs was elucidated by lentiviral knockdown of lncCCT4-2, plasmid overexpression or knockdown of CCT4, and actinomycin D assay. RESULTS: In vitro and in vivo experiments confirmed that HR-EMVs exacerbated oxidative stress and apoptosis in IR-injured cardiomyocytes, leading to increased infarct size and worsened cardiac function. Notably, (HR + P)-EMVs induced significantly less oxidative stress and apoptosis in IR-injured cardiomyocytes compared to HR-EMVs. Mechanistically, RNA sequencing of EMVs and RT-qPCR showed that lncCCT4-2 was significantly upregulated in (HR + P)-EMVs and cardiomyocytes co-cultured with (HR + P)-EMVs. Reduction of lncCCT4-2 in (HR + P)-EMVs enhanced oxidative stress and apoptosis in IR-injured cardiomyocytes. Furthermore, the anti-apoptotic activity of lncCCT4-2 from (HR + P)-EMVs was achieved by increasing the stability of CCT4 mRNA and promoting the expression of CCT4 protein in cardiomyocytes. CONCLUSIONS: Our study showed that (HR + P)-EMVs uptake by IR-injured cardiomyocytes upregulated lncCCT4-2 in cardiomyocytes and promoted CCT4 expression, thereby inhibiting HR-EMVs induced oxidative stress and apoptosis.

Propofol inhibits myocardial injury induced by microvesicles derived from hypoxiareoxygenated endothelial cells via lncCCT4-2/CCT4 signaling

BACKGROUND: Ischemia-reperfusion (IR) induces increased release of extracellular vesicles in the heart and exacerbates myocardial IR injury. We have previously shown that propofol attenuates hypoxia/reoxygenation (HR)-induced injury in human umbilical vein endothelial cells (HUVECs) and that microvesicles derived from propofol-treated HUVECs inhibit oxidative stress in endothelial cells. However, the role of microvesicles derived from propofol post-treated HUVECs ((HR + P)-EMVs) in IR-injured cardiomyocytes is unclear. In this study, we aimed to investigate the role of (HR + P)-EMVs in cardiac IR injury compared to microvesicles derived from hypoxic/reoxygenated HUVECs (HR-EMVs) and to elucidate the underlying mechanisms. METHODS: Hypoxia/reoxygenation (HR) models of HUVECs and AC16 cells and a mouse cardiac IR model were established. Microvesicles from HR-injured HUVECs, DMSO post-treated HUVECs and propofol post-treated HUVECs were extracted by ultra-high speed centrifugation, respectively. The above EMVs were co-cultured with HR-injured AC16 cells or injected intracardially into IR mice. Flow cytometry and immunofluorescence were used to determine the levels of oxidative stress and apoptosis in cardiomyocytes. Apoptosis related proteins were detected by Western blot. Echocardiography for cardiac function and Evans blue-TTC staining for myocardial infarct size. Expression of lncCCT4-2 in EMVs and AC16 cells was analysed by whole transcriptome sequencing of EMVs and RT-qPCR. The molecular mechanism of inhibition of myocardial injury by (HR + P)-EMVs was elucidated by lentiviral knockdown of lncCCT4-2, plasmid overexpression or knockdown of CCT4, and actinomycin D assay. RESULTS: In vitro and in vivo experiments confirmed that HR-EMVs exacerbated oxidative stress and apoptosis in IR-injured cardiomyocytes, leading to increased infarct size and worsened cardiac function. Notably, (HR + P)-EMVs induced significantly less oxidative stress and apoptosis in IR-injured cardiomyocytes compared to HR-EMVs. Mechanistically, RNA sequencing of EMVs and RT-qPCR showed that lncCCT4-2 was significantly upregulated in (HR + P)-EMVs and cardiomyocytes co-cultured with (HR + P)-EMVs. Reduction of lncCCT4-2 in (HR + P)-EMVs enhanced oxidative stress and apoptosis in IR-injured cardiomyocytes. Furthermore, the anti-apoptotic activity of lncCCT4-2 from (HR + P)-EMVs was achieved by increasing the stability of CCT4 mRNA and promoting the expression of CCT4 protein in cardiomyocytes. CONCLUSIONS: Our study showed that (HR + P)-EMVs uptake by IR-injured cardiomyocytes upregulated lncCCT4-2 in cardiomyocytes and promoted CCT4 expression, thereby inhibiting HR-EMVs induced oxidative stress and apoptosis. Highlights Microvesicles from hypoxic/reoxygenated HUVECs (HR-EMVs) exacerbated oxidative stress and apoptosis in IR-injured cardiomyocytes. Microvesicles from propofol post-treated HUVECs ((HR + P)-EMVs) induced diminished oxidative stress and apoptosis in IR-injured cardiomyocytes compared with microvesicles from hypoxic/reoxygenated HUVECs (HR-EMVs). lncCCT4-2 was significantly highly expressed in (HR + P)-EMVs and cardiomyocytes co-cultured with (HR + P)-EMVs, and reduction of lncCCT4-2 in (HR + P)-EMVs enhanced oxidative stress and apoptosis in IR-injured cardiomyocytes. lncCCT4-2 inhibited HR-EMVs induced oxidative stress and apoptosis in HR-injured AC16 cells by increasing the stability of CCT4 mRNA and promoting the expression of CCT4 protein in AC16 cells.

Identification and characterization of a common B-cell epitope on EIAV capsid proteins.

The equine infectious anemia virus (EIAV) capsid protein (p26) is one of the major immunogenic proteins during EIAV infection and is widely used for the detection of EIAV antibodies in horses. However, few reports have described the use of EIAV-specific monoclonal antibodies (MAbs) in etiological and immunological detection. Previously, we developed an antigen capture enzyme-linked immunosorbent assay (AC-ELISA) for the quantification of the EIAV p26 protein level. However, the epitopes recognized by the MAbs were not identified, and the utilization of the MAbs needs to be evaluated. In this study, we characterized two monoclonal antibodies (9H8 and 1G11 MAbs) against EIAV p26. Two B-cell epitopes are located in amino acid residues, 73NLDKIAEE81 (HE) and 199KNAMRHLRPEDTLEEKMYAC218 (GE) for the 9H8 and 1G11 MAbs, respectively. The 1G11 epitope (GE) varied among viruses isolated worldwide but can be recognized by anti-EIAV sera from different regions, including China, the USA, and Argentina. Meanwhile, 1G11 MAb could react with the mutants of almost all the EIAV strains. Furthermore, we found that the histidine at position 204 (H204), leucine at position 205 (L205), and aspartic acid at position 209 (D209) of EIAV p26 individually played pivotal roles in binding with the 1G11 MAb. Our results revealed that the GE peptide might be a common B-cell binding epitope of EIAV antibodies. This is also the first report to identify a broad-spectrum monoclonal antibody (1G11) against p26 of EIAV. These findings may provide a useful basis for the development of new diagnostic assays for EIAV.