Monocyte-to-M0/M1 macrophage differentiation with unclear molecular mechanisms is a pivotal cellular event in many cardiovascular diseases including atherosclerosis.Long non-coding RNAs(lncRNAs)are a group of protein ...Monocyte-to-M0/M1 macrophage differentiation with unclear molecular mechanisms is a pivotal cellular event in many cardiovascular diseases including atherosclerosis.Long non-coding RNAs(lncRNAs)are a group of protein expression regulators;however,the roles of monocyte-lncRNAs in macrophage differentiation and its related vascular diseases are still unclear.The study aims to investigate whether the novel leukocyte-specific lncRNA Morrbid could regulate macrophage differentiation and atherogenesis.We identified that Morrbid was increased in monocytes and arterial walls from atherosclerotic mouse and from patients with atherosclerosis.In cultured monocytes,Morrbid expression was markedly increased during monocyte to M0 macrophage differentiation with an additional increase during M0 macrophage-to-M1 macrophage differentiation.The differentiation stimuli-induced monocyte-macrophage differentiation and the macrophage activity were inhibited by Morrbid knockdown.Moreover,overexpression of Morrbid alone was sufficient to elicit the monocyte-macrophage differentiation.The role of Morrbid in monocyte-macrophage differentiation was also identified in vivo in atherosclerotic mice and was verified in Morrbid knockout mice.We identified that PI3-kinase/Akt was involved in the up-regulation of Morrbid expression,whereas s100a10 was involved in Morrbid-mediated effect on macrophage differentiation.To provide a proof of concept of Morrbid in pathogenesis of monocyte/macrophage-related vascular disease,we applied an acute atherosclerosis model in mice.The results revealed that overexpression of Morrbid enhanced but monocyte/macrophage-specific Morrbid knockout inhibited the monocytes/macrophages recruitment and atherosclerotic lesion formation in mice.The results suggest that Morrbid is a novel biomarker and a modulator of monocyte-macrophage phenotypes,which is involved in atherogenesis.展开更多
Midkine is a heparin-binding growth factor, which plays important roles in the regulation of cell growth and differentiation. The non-tagged recombinant human midkine （rhMK） is therefore required to facilitate its f...Midkine is a heparin-binding growth factor, which plays important roles in the regulation of cell growth and differentiation. The non-tagged recombinant human midkine （rhMK） is therefore required to facilitate its functional studies of this important growth factor. In the present work, rhMK was expressed in Escherichia coli （E. coli） BL21 （DE3）. The expression of midkine was efficiently induced by isopropyl-13-D-thiogalactopyranoside （IPTG）. After sonication, midkine was recovered in an insoluble form, and was dissolved in guanidine hydrochloride buffer. Renaturation of the denatured protein was carried out in the defined protein refolding buffer, and the refolded protein was purified using S-Sepharose ion-exchange chromatography. The final preparation of the rhMK was greater than 98% pure as measured by sodium dodecylsulfate-polyacrylamid gel electrophoresis （SDS-PAGE） and reverse phase high performance liquid chromatography （RP-HPLC）. The purified rhMK enhanced the proliferation of NIH3T3 cells.展开更多
基金supported by the National Natural Science Foundation of China(Grant Nos.82030007,81670398,and 91639102)the Central Government Guides Local Science and Technology Development Project(Grant No.2022ZYD0057)+2 种基金the Sichuan Science and Technology Program(Grant Nos.2022YFS0578 and 2022YFS0614)the Start-up Research Fund of Southwest Medical University(Grant No.00040155)supported in part by the Start-up fund from the University of Alabama at Birmingham.
文摘Monocyte-to-M0/M1 macrophage differentiation with unclear molecular mechanisms is a pivotal cellular event in many cardiovascular diseases including atherosclerosis.Long non-coding RNAs(lncRNAs)are a group of protein expression regulators;however,the roles of monocyte-lncRNAs in macrophage differentiation and its related vascular diseases are still unclear.The study aims to investigate whether the novel leukocyte-specific lncRNA Morrbid could regulate macrophage differentiation and atherogenesis.We identified that Morrbid was increased in monocytes and arterial walls from atherosclerotic mouse and from patients with atherosclerosis.In cultured monocytes,Morrbid expression was markedly increased during monocyte to M0 macrophage differentiation with an additional increase during M0 macrophage-to-M1 macrophage differentiation.The differentiation stimuli-induced monocyte-macrophage differentiation and the macrophage activity were inhibited by Morrbid knockdown.Moreover,overexpression of Morrbid alone was sufficient to elicit the monocyte-macrophage differentiation.The role of Morrbid in monocyte-macrophage differentiation was also identified in vivo in atherosclerotic mice and was verified in Morrbid knockout mice.We identified that PI3-kinase/Akt was involved in the up-regulation of Morrbid expression,whereas s100a10 was involved in Morrbid-mediated effect on macrophage differentiation.To provide a proof of concept of Morrbid in pathogenesis of monocyte/macrophage-related vascular disease,we applied an acute atherosclerosis model in mice.The results revealed that overexpression of Morrbid enhanced but monocyte/macrophage-specific Morrbid knockout inhibited the monocytes/macrophages recruitment and atherosclerotic lesion formation in mice.The results suggest that Morrbid is a novel biomarker and a modulator of monocyte-macrophage phenotypes,which is involved in atherogenesis.
基金Project supported in part by the Hi-Tech Research and Development Program(863) of China(No.2007AA02Z149)the Science and Technology Commissionof Shanghai Municipality(No.075407071),China
文摘Midkine is a heparin-binding growth factor, which plays important roles in the regulation of cell growth and differentiation. The non-tagged recombinant human midkine （rhMK） is therefore required to facilitate its functional studies of this important growth factor. In the present work, rhMK was expressed in Escherichia coli （E. coli） BL21 （DE3）. The expression of midkine was efficiently induced by isopropyl-13-D-thiogalactopyranoside （IPTG）. After sonication, midkine was recovered in an insoluble form, and was dissolved in guanidine hydrochloride buffer. Renaturation of the denatured protein was carried out in the defined protein refolding buffer, and the refolded protein was purified using S-Sepharose ion-exchange chromatography. The final preparation of the rhMK was greater than 98% pure as measured by sodium dodecylsulfate-polyacrylamid gel electrophoresis （SDS-PAGE） and reverse phase high performance liquid chromatography （RP-HPLC）. The purified rhMK enhanced the proliferation of NIH3T3 cells.
