Xiaofang Guo, Maja Okuka, Brittney Short, Asli Ozmen, Nihan Semerci Gunay, Jake Rymer, Burak Un, Ozlem Guzeloglu-Kayisli, Thomas J. Rutherford, Umit Kayisli, Matthew L. Anderson
Found in as many as 80% of women, uterine leiomyomas are a frequent cause of abnormal uterine bleeding, pelvic pain, and infertility. Despite their significant clinical impact, the mechanisms responsible for driving leiomyoma growth remain poorly understood. After obtaining IRB permission, expression of ecto-5′-nucleotidase (NT5E, CD73) was assessed in matched specimens of myometrium and leiomyoma by real-time qPCR, Western blot, and immunohistochemistry (IHC). Adenosine concentrations were measured by enzyme-linked assay. Primary cultures were used to assess the impact of adenosine and/or adenosine receptor agonists on proliferation, apoptosis, and patterns of intracellular signaling in vitro. When compared to matched specimens of healthy myometrium, uterine leiomyomas were characterized by reduced CD73 expression. Largely limited to thin-walled vascular structures and the pseudocapsule of leiomyomas despite diffuse myometrial distribution. Restricted intra-tumoral CD73 expression was accompanied by decreased levels of intra-tumoral adenosine. In vitro, incubation of primary leiomyoma cultures with adenosine or its hydrolysis-resistant analog 2-chloro-adenosine (2-CL-AD) inhibited proliferation, induced apoptosis, and reduced proportion of myocytes in S- and G2-M phases of the cell cycle. Decreased proliferation was accompanied by reduced expression of phospho-Akt, phospho-Cdk2-Tyr15, and phospho-Histone H3. Enforced expression of the A2B adenosine receptor (ADORA2B) and ADORA2B-selective agonists similarly suppressed proliferation and inhibited Akt phosphorylation. Collectively, these observations broadly implicate CD73 and reduced extracellular concentrations of adenosine as key regulators of leiomyoma growth and potentially identify novel strategies for clinically managing these common tumors.
{"title":"Spatially restricted ecto-5′-nucleotidase expression promotes the growth of uterine leiomyomas by modulating Akt activity","authors":"Xiaofang Guo, Maja Okuka, Brittney Short, Asli Ozmen, Nihan Semerci Gunay, Jake Rymer, Burak Un, Ozlem Guzeloglu-Kayisli, Thomas J. Rutherford, Umit Kayisli, Matthew L. Anderson","doi":"10.1096/fj.202401432R","DOIUrl":"10.1096/fj.202401432R","url":null,"abstract":"<p>Found in as many as 80% of women, uterine leiomyomas are a frequent cause of abnormal uterine bleeding, pelvic pain, and infertility. Despite their significant clinical impact, the mechanisms responsible for driving leiomyoma growth remain poorly understood. After obtaining IRB permission, expression of ecto-5′-nucleotidase (<i>NT5E,</i> CD73<i>)</i> was assessed in matched specimens of myometrium and leiomyoma by real-time qPCR, Western blot, and immunohistochemistry (IHC). Adenosine concentrations were measured by enzyme-linked assay. Primary cultures were used to assess the impact of adenosine and/or adenosine receptor agonists on proliferation, apoptosis, and patterns of intracellular signaling in vitro. When compared to matched specimens of healthy myometrium, uterine leiomyomas were characterized by reduced CD73 expression. Largely limited to thin-walled vascular structures and the pseudocapsule of leiomyomas despite diffuse myometrial distribution. Restricted intra-tumoral CD73 expression was accompanied by decreased levels of intra-tumoral adenosine. In vitro, incubation of primary leiomyoma cultures with adenosine or its hydrolysis-resistant analog 2-chloro-adenosine (2-CL-AD) inhibited proliferation, induced apoptosis, and reduced proportion of myocytes in S- and G2-M phases of the cell cycle. Decreased proliferation was accompanied by reduced expression of phospho-Akt, phospho-Cdk2-Tyr15, and phospho-Histone H3. Enforced expression of the A2B adenosine receptor (ADORA2B) and ADORA2B-selective agonists similarly suppressed proliferation and inhibited Akt phosphorylation. Collectively, these observations broadly implicate CD73 and reduced extracellular concentrations of adenosine as key regulators of leiomyoma growth and potentially identify novel strategies for clinically managing these common tumors.</p>","PeriodicalId":50455,"journal":{"name":"FASEB Journal","volume":null,"pages":null},"PeriodicalIF":4.4,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142362448","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
As renal progenitor cells, parietal epithelial cells (PECs) have demonstrated multilineage differentiation potential in response to kidney injury. However, the function of exosomes derived from PECs has not been extensively explored. Immunofluorescent staining of Claudin-1 was used to identify primary PECs isolated from mouse glomeruli. Transmission electron microscopy, nanoparticle tracking analysis, and western blotting were used to characterize the properties of PECs-derived exosomes (PEC-Exo). The therapeutic role of PEC-Exo in tubulointerstitial fibrosis was investigated in the unilateral ureteral obstruction (UUO) mouse model and TGF-β1-stimulated HK-2 cells. High-throughput miRNA sequencing was employed to profile PEC-Exo miRNAs. One of the most enriched miRNAs in PEC-Exo was knocked down by transfecting miRNA inhibitor, and then we investigated whether this candidate miRNA was involved in PEC-Exo-mediated tubular repair. The primary PECs expressed Claudin-1, PEC-Exo was homing to obstructed kidney, and TGF-β1 induced HK-2 cells. PEC-Exo significantly alleviated renal inflammation and ameliorated tubular fibrosis both in vivo and in vitro. Mechanistically, let-7b-5p, highly enriched in PEC-Exo, downregulated the protein levels of transforming growth factor beta receptor 1(TGFβR1) and AT-Rich Interaction Domain 3A(ARID3a) in tubular epithelial cells (TECs), leading to the inhibition of p21 and p27 to restoring cell cycle. Furthermore, administration of let-7b-5p agomir mitigated renal fibrosis in vivo. Our findings demonstrated that PEC-derived exosomes significantly repressed the expression of TGFβR1 and ARID3a by delivering let-7b-5p, thereby alleviating renal fibrosis. This study provides novel insights into the role of PEC-Exo in the repair of kidney injury and new ideas for renal fibrosis intervention.
{"title":"Exosomal let-7b-5p deriving from parietal epithelial cells attenuate renal fibrosis through suppression of TGFβR1 and ARID3a in obstructive kidney disease","authors":"Ahui Song, Minzhou Wang, Kewei Xie, Jiayue Lu, Bingru Zhao, Wangshu Wu, Cheng Qian, Wenkai Hong, Leyi Gu","doi":"10.1096/fj.202400802RR","DOIUrl":"10.1096/fj.202400802RR","url":null,"abstract":"<p>As renal progenitor cells, parietal epithelial cells (PECs) have demonstrated multilineage differentiation potential in response to kidney injury. However, the function of exosomes derived from PECs has not been extensively explored. Immunofluorescent staining of Claudin-1 was used to identify primary PECs isolated from mouse glomeruli. Transmission electron microscopy, nanoparticle tracking analysis, and western blotting were used to characterize the properties of PECs-derived exosomes (PEC-Exo). The therapeutic role of PEC-Exo in tubulointerstitial fibrosis was investigated in the unilateral ureteral obstruction (UUO) mouse model and TGF-β1-stimulated HK-2 cells. High-throughput miRNA sequencing was employed to profile PEC-Exo miRNAs. One of the most enriched miRNAs in PEC-Exo was knocked down by transfecting miRNA inhibitor, and then we investigated whether this candidate miRNA was involved in PEC-Exo-mediated tubular repair. The primary PECs expressed Claudin-1, PEC-Exo was homing to obstructed kidney, and TGF-β1 induced HK-2 cells. PEC-Exo significantly alleviated renal inflammation and ameliorated tubular fibrosis both in vivo and in vitro. Mechanistically, let-7b-5p, highly enriched in PEC-Exo, downregulated the protein levels of transforming growth factor beta receptor 1(TGFβR1) and AT-Rich Interaction Domain 3A(ARID3a) in tubular epithelial cells (TECs), leading to the inhibition of p21 and p27 to restoring cell cycle. Furthermore, administration of let-7b-5p agomir mitigated renal fibrosis in vivo. Our findings demonstrated that PEC-derived exosomes significantly repressed the expression of TGFβR1 and ARID3a by delivering let-7b-5p, thereby alleviating renal fibrosis. This study provides novel insights into the role of PEC-Exo in the repair of kidney injury and new ideas for renal fibrosis intervention.</p>","PeriodicalId":50455,"journal":{"name":"FASEB Journal","volume":null,"pages":null},"PeriodicalIF":4.4,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142331631","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Nasal obstruction leads to a hypoxia condition throughout the entire body. In this study, the unilateral nasal obstruction (UNO) mouse model was established by blocking the left nostril of mice. The aim of this study was to investigate the effects of UNO-induced hypoxia on mandibular condyle in juvenile (3-week-old), adolescent (6-week-old) and adult (12-week-old) male C57BL/6J mice from the perspective of H-type angiogenesis coupling osteogenesis. Firstly, UNO exerted a significant inhibitory effect on weight gain in mice of all ages. However, only in adolescent mice did UNO have an obvious detrimental effect on femoral bone mass accrual. Subsequently, micro-computed tomography (CT) analysis of mandibular condylar bone mass revealed that UNO significantly retarded condylar head volume gain but increased condylar head trabecular number (Tb.N) in juvenile and adolescent mice. Furthermore, UNO promoted the ratio of proliferative layer to cartilage layer in condylar cartilage and facilitated the chondrocyte-to-osteoblast transformation in juvenile and adolescent mice. Moreover, although UNO enhanced the positive expression of hypoxia-inducible factor (HIF)-1α in the condylar subchondral bone of mice in all ages, an increase in H-type vessels and Osterix+ cells was only detected in juvenile and adolescent mice. In summary, on the one hand, in terms of condylar morphology, UNO has a negative effect on condylar growth, hindering the increase in condylar head volume in juvenile and adolescent mice. However, on the other hand, in terms of condylar microstructure, UNO has a positive effect on condylar osteogenesis, promoting the increase of condylar Tb.N, chondrocyte-to-osteoblast transformation, HIF-1α expression, H-type angiogenesis and Osterix+ cells in juvenile and adolescent mice. Although the changes in condylar morphology and microstructure caused by UNO have not yet been fully elucidated, these findings improve our current understanding of the effects of UNO on condylar bone homeostasis.
鼻阻塞会导致全身缺氧。本研究通过阻塞小鼠的左鼻孔建立了单侧鼻阻塞(UNO)小鼠模型。本研究的目的是从H型血管生成耦联成骨的角度研究UNO诱导的缺氧对幼年(3周龄)、青少年(6周龄)和成年(12周龄)雄性C57BL/6J小鼠下颌骨髁状突的影响。首先,UNO 对各年龄段小鼠的体重增加都有显著的抑制作用。然而,只有在青春期小鼠中,UNO 才对股骨骨量的增加有明显的不利影响。随后,对下颌骨髁状突骨质进行的显微计算机断层扫描(CT)分析表明,在幼年和青春期小鼠中,UNO明显延缓了髁状突头体积的增加,但增加了髁状突头骨小梁数量(Tb.N)。此外,UNO 还能提高髁状突软骨中增殖层与软骨层的比例,促进幼年和青春期小鼠软骨细胞向成骨细胞的转化。此外,虽然 UNO 增强了各年龄段小鼠髁突软骨下骨中缺氧诱导因子(HIF)-1α 的阳性表达,但只有在幼年和青春期小鼠中才能检测到 H 型血管和 Osterix+ 细胞的增加。总之,一方面,在髁状突形态方面,UNO 对髁状突的生长有负面影响,阻碍了幼年和青春期小鼠髁状突头体积的增加。但另一方面,在髁状突微结构方面,UNO 对髁状突的成骨具有积极作用,可促进幼年和青春期小鼠髁状突 Tb.N、软骨细胞向成骨细胞转化、HIF-1α 表达、H 型血管生成和 Osterix+ 细胞的增加。尽管 UNO 引起的髁状突形态和微观结构的变化尚未完全阐明,但这些发现提高了我们目前对 UNO 对髁状突骨稳态影响的认识。
{"title":"Effects of unilateral nasal obstruction on mandibular condyle in mice of different ages: An exploration based on H-type angiogenesis coupling osteogenesis","authors":"Yun Hu, Hegang Li","doi":"10.1096/fj.202401273R","DOIUrl":"10.1096/fj.202401273R","url":null,"abstract":"<p>Nasal obstruction leads to a hypoxia condition throughout the entire body. In this study, the unilateral nasal obstruction (UNO) mouse model was established by blocking the left nostril of mice. The aim of this study was to investigate the effects of UNO-induced hypoxia on mandibular condyle in juvenile (3-week-old), adolescent (6-week-old) and adult (12-week-old) male C57BL/6J mice from the perspective of H-type angiogenesis coupling osteogenesis. Firstly, UNO exerted a significant inhibitory effect on weight gain in mice of all ages. However, only in adolescent mice did UNO have an obvious detrimental effect on femoral bone mass accrual. Subsequently, micro-computed tomography (CT) analysis of mandibular condylar bone mass revealed that UNO significantly retarded condylar head volume gain but increased condylar head trabecular number (Tb.N) in juvenile and adolescent mice. Furthermore, UNO promoted the ratio of proliferative layer to cartilage layer in condylar cartilage and facilitated the chondrocyte-to-osteoblast transformation in juvenile and adolescent mice. Moreover, although UNO enhanced the positive expression of hypoxia-inducible factor (HIF)-1α in the condylar subchondral bone of mice in all ages, an increase in H-type vessels and Osterix<sup>+</sup> cells was only detected in juvenile and adolescent mice. In summary, on the one hand, in terms of condylar morphology, UNO has a negative effect on condylar growth, hindering the increase in condylar head volume in juvenile and adolescent mice. However, on the other hand, in terms of condylar microstructure, UNO has a positive effect on condylar osteogenesis, promoting the increase of condylar Tb.N, chondrocyte-to-osteoblast transformation, HIF-1α expression, H-type angiogenesis and Osterix<sup>+</sup> cells in juvenile and adolescent mice. Although the changes in condylar morphology and microstructure caused by UNO have not yet been fully elucidated, these findings improve our current understanding of the effects of UNO on condylar bone homeostasis.</p>","PeriodicalId":50455,"journal":{"name":"FASEB Journal","volume":null,"pages":null},"PeriodicalIF":4.4,"publicationDate":"2024-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142331630","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Shu Zhang, Jingxian Zhu, Siyu Jin, Wei Sun, Wei Ji, Zhi Chen
The jawbone periosteum, the easily accessible tissue responding to bone repair, has been overlooked in the recent development of cell therapy for jawbone defect reconstruction. Therefore, this study aimed to elucidate the in vitro and in vivo biological characteristics of jawbone periosteum-derived cells (jb-PDCs). For this purpose, we harvested the jb-PDCs from 8-week-old C57BL/6 mice. The in vitro cultured jb-PDCs (passages 1 and 3) contained skeletal stem/progenitor cells and exhibited clonogenicity and tri-lineage differentiation capacity. When implanted in vivo, the jb-PDCs (passage 3) showed evident ectopic bone formation after 4-week subcutaneous implantation, and active contribution to repair the critical-size jawbone defects in mice. Molecular profiling suggested that R-spondin 3 was strongly associated with the superior in vitro and in vivo osteogenic potentials of jb-PDCs. Overall, our study highlights the significance of comprehending the biological characteristics of the jawbone periosteum, which could pave the way for innovative cell-based therapies for the reconstruction of jawbone defects.
{"title":"Jawbone periosteum-derived cells with high osteogenic potential controlled by R-spondin 3","authors":"Shu Zhang, Jingxian Zhu, Siyu Jin, Wei Sun, Wei Ji, Zhi Chen","doi":"10.1096/fj.202400988RR","DOIUrl":"10.1096/fj.202400988RR","url":null,"abstract":"<p>The jawbone periosteum, the easily accessible tissue responding to bone repair, has been overlooked in the recent development of cell therapy for jawbone defect reconstruction. Therefore, this study aimed to elucidate the in vitro and in vivo biological characteristics of jawbone periosteum-derived cells (jb-PDCs). For this purpose, we harvested the jb-PDCs from 8-week-old C57BL/6 mice. The in vitro cultured jb-PDCs (passages 1 and 3) contained skeletal stem/progenitor cells and exhibited clonogenicity and tri-lineage differentiation capacity. When implanted in vivo, the jb-PDCs (passage 3) showed evident ectopic bone formation after 4-week subcutaneous implantation, and active contribution to repair the critical-size jawbone defects in mice. Molecular profiling suggested that <i>R-spondin 3</i> was strongly associated with the superior in vitro and in vivo osteogenic potentials of jb-PDCs. Overall, our study highlights the significance of comprehending the biological characteristics of the jawbone periosteum, which could pave the way for innovative cell-based therapies for the reconstruction of jawbone defects.</p>","PeriodicalId":50455,"journal":{"name":"FASEB Journal","volume":null,"pages":null},"PeriodicalIF":4.4,"publicationDate":"2024-09-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142331632","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Diabetes osteoporosis (DOP) is a chronic metabolic bone disease. This study aimed to identify potential biomarkers of DOP and explore their underlying mechanisms through bioinformatics methods and experimental verification. Bioinformatics methods were used to identify differentially expressed genes (DEGs) for DOP based on GEO data and the GeneCards database. GO and KEGG enrichment analyses were used to search the key pathways. The STRING website was used to construct a protein–protein interaction (PPI) network and identify key genes. Then, 50 mg/mL glucose was used to interveneosteoblasts (OBs).CCK-8 and Alizarin Red staining were used to investigate the proliferation and differentiation changes in OBs. Flowcytometry was used to investigate apoptosis. The membrane protein chip, WB, and RT-PCR were used to verify the expression of key targets or pathways about DOP. Forty-two common genes were screened between DOP-related targets and DEGs. GO and KEGG enrichment analysis showed that DOP was mainly associated with cytokine-cytokine receptor interactions, and apoptosis. PPI network analysis showed that TNF, IL1A, IL6, IL1B, IL2RA, Fas ligand (FASLG), and Fas cell surface death receptor (FAS) were key up-regulated genes in the occurrence of DOP. The experiment results show that 50 mg/mL glucose significantly inhibited OBs proliferation but presented an increase in apoptosis. Membrane protein chip, WB, and RT-PCR-verified a significantly active in the expression of TNF/FASLG/FAS pathway. High glucose activated the TNF-α/FAS/FASLG pathway and induced the inflammatory microenvironment and apoptosis, then impaired osteogenic differentiation of OBs. These may be an important mechanism for the occurrence and development of DOP.
{"title":"Understanding the relationship between inflammation, apoptosis, and diabetes osteoporosis: A bioinformatics approach and experimental verification","authors":"Jun Quan Li, Bo Li, Zhang Qing Fei, Shan Shan Lei","doi":"10.1096/fj.202401452R","DOIUrl":"10.1096/fj.202401452R","url":null,"abstract":"<p>Diabetes osteoporosis (DOP) is a chronic metabolic bone disease. This study aimed to identify potential biomarkers of DOP and explore their underlying mechanisms through bioinformatics methods and experimental verification. Bioinformatics methods were used to identify differentially expressed genes (DEGs) for DOP based on GEO data and the GeneCards database. GO and KEGG enrichment analyses were used to search the key pathways. The STRING website was used to construct a protein–protein interaction (PPI) network and identify key genes. Then, 50 mg/mL glucose was used to interveneosteoblasts (OBs).CCK-8 and Alizarin Red staining were used to investigate the proliferation and differentiation changes in OBs. Flowcytometry was used to investigate apoptosis. The membrane protein chip, WB, and RT-PCR were used to verify the expression of key targets or pathways about DOP. Forty-two common genes were screened between DOP-related targets and DEGs. GO and KEGG enrichment analysis showed that DOP was mainly associated with cytokine-cytokine receptor interactions, and apoptosis. PPI network analysis showed that TNF, IL1A, IL6, IL1B, IL2RA, Fas ligand (FASLG), and Fas cell surface death receptor (FAS) were key up-regulated genes in the occurrence of DOP. The experiment results show that 50 mg/mL glucose significantly inhibited OBs proliferation but presented an increase in apoptosis. Membrane protein chip, WB, and RT-PCR-verified a significantly active in the expression of TNF/FASLG/FAS pathway. High glucose activated the TNF-α/FAS/FASLG pathway and induced the inflammatory microenvironment and apoptosis, then impaired osteogenic differentiation of OBs. These may be an important mechanism for the occurrence and development of DOP.</p>","PeriodicalId":50455,"journal":{"name":"FASEB Journal","volume":null,"pages":null},"PeriodicalIF":4.4,"publicationDate":"2024-09-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142331633","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Sunny Hoi-Sang Yeung, Ralph Hon-Sun Lee, Gerald Wai-Yeung Cheng, Iris Wai-Ting Ma, Julia Kofler, Candice Kent, Fulin Ma, Karl Herrup, Myriam Fornage, Ken Arai, Kai-Hei Tse
White matter hyperintensity (WMH) is strongly correlated with age-related dementia and hypertension, but its pathogenesis remains obscure. Genome-wide association studies identified TRIM47 at the 17q25 locus as a top genetic risk factor for WMH formation. TRIM family is a class of E3 ubiquitin ligase with pivotal functions in autophagy, which is critical for brain endothelial cell (ECs) remodeling during hypertension. We hypothesize that TRIM47 regulates autophagy and its loss-of-function disturbs cerebrovasculature. Based on transcriptomics and immunohistochemistry, TRIM47 is found highly expressed by brain ECs in human and mouse, and its transcription is upregulated by artificially induced autophagy while downregulated in hypertension-like conditions. Using in silico simulation, immunocytochemistry and super-resolution microscopy, we predicted a highly conserved binding site between TRIM47 and the LIR (LC3-interacting region) motif of LC3B. Importantly, pharmacological autophagy induction increased Trim47 expression on mouse ECs (b.End3) culture, while silencing Trim47 significantly increased autophagy with ULK1 phosphorylation induction, transcription, and vacuole formation. Together, we demonstrate that TRIM47 is an endogenous inhibitor of autophagy in brain ECs, and such TRIM47-mediated regulation connects genetic and physiological risk factors for WMH formation but warrants further investigation.
{"title":"White matter hyperintensity genetic risk factor TRIM47 regulates autophagy in brain endothelial cells","authors":"Sunny Hoi-Sang Yeung, Ralph Hon-Sun Lee, Gerald Wai-Yeung Cheng, Iris Wai-Ting Ma, Julia Kofler, Candice Kent, Fulin Ma, Karl Herrup, Myriam Fornage, Ken Arai, Kai-Hei Tse","doi":"10.1096/fj.202400689RR","DOIUrl":"10.1096/fj.202400689RR","url":null,"abstract":"<p>White matter hyperintensity (WMH) is strongly correlated with age-related dementia and hypertension, but its pathogenesis remains obscure. Genome-wide association studies identified TRIM47 at the 17q25 locus as a top genetic risk factor for WMH formation. TRIM family is a class of E3 ubiquitin ligase with pivotal functions in autophagy, which is critical for brain endothelial cell (ECs) remodeling during hypertension. We hypothesize that TRIM47 regulates autophagy and its loss-of-function disturbs cerebrovasculature. Based on transcriptomics and immunohistochemistry, TRIM47 is found highly expressed by brain ECs in human and mouse, and its transcription is upregulated by artificially induced autophagy while downregulated in hypertension-like conditions. Using in silico simulation, immunocytochemistry and super-resolution microscopy, we predicted a highly conserved binding site between TRIM47 and the LIR (LC3-interacting region) motif of LC3B. Importantly, pharmacological autophagy induction increased Trim47 expression on mouse ECs (b.End3) culture, while silencing <i>Trim47</i> significantly increased autophagy with ULK1 phosphorylation induction, transcription, and vacuole formation. Together, we demonstrate that TRIM47 is an endogenous inhibitor of autophagy in brain ECs, and such TRIM47-mediated regulation connects genetic and physiological risk factors for WMH formation but warrants further investigation.</p>","PeriodicalId":50455,"journal":{"name":"FASEB Journal","volume":null,"pages":null},"PeriodicalIF":4.4,"publicationDate":"2024-09-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1096/fj.202400689RR","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142331634","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The inability to efficiently metabolize homocysteine (Hcy) due to nutritional and genetic deficiencies, leads to hyperhomocysteinemia (HHcy) and endothelial dysfunction, a hallmark of atherosclerosis which underpins cardiovascular disease (CVD). PHF8 is a histone demethylase that demethylates H4K20me1, which affects the mammalian target of rapamycin (mTOR) signaling and autophagy, processes that play important roles in CVD. PHF8 is regulated by microRNA (miR) such as miR-22-3p and miR-1229-3p. Biochemically, HHcy is characterized by elevated levels of Hcy, Hcy-thiolactone and N-Hcy-protein. Here, we examined the effects of these metabolites on miR-22-3p, miR-1229-3p, and their target PHF8, as well as on the downstream consequences of these effects on H4K20me1, mTOR-, and autophagy-related proteins and mRNAs expression in human umbilical vein endothelial cells (HUVEC). We found that treatments with N-Hcy-protein, Hcy-thiolactone, or Hcy upregulated miR-22-3p and miR-1229-3p, attenuated PHF8 expression, upregulated H4K20me1, mTOR, and phospho-mTOR. Autophagy-related proteins (BECN1, ATG5, ATG7, lipidated LC3-II, and LC3-II/LC3-I ratio) were significantly downregulated by at least one of these metabolites. We also found similar changes in the expression of miR-22-3p, Phf8, mTOR- and autophagy-related proteins/mRNAs in vivo in hearts of Cbs−/− mice, which show severe HHcy and endothelial dysfunction. Treatments with inhibitors of miR-22-3p or miR-1229-3p abrogated the effects of Hcy-thiolactone, N-Hcy-protein, and Hcy on miR expression and on PHF8, H4K20me1, mTOR-, and autophagy-related proteins/mRNAs in HUVEC. Taken together, these findings show that Hcy metabolites upregulate miR-22-3p and miR-1229-3p expression, which then dysregulate the PHF8/H4K20me1/mTOR/autophagy pathway, important for vascular homeostasis.
{"title":"Homocysteine metabolites impair the PHF8/H4K20me1/mTOR/autophagy pathway by upregulating the expression of histone demethylase PHF8-targeting microRNAs in human vascular endothelial cells and mice","authors":"Łukasz Witucki, Hieronim Jakubowski","doi":"10.1096/fj.202302116R","DOIUrl":"https://doi.org/10.1096/fj.202302116R","url":null,"abstract":"<p>The inability to efficiently metabolize homocysteine (Hcy) due to nutritional and genetic deficiencies, leads to hyperhomocysteinemia (HHcy) and endothelial dysfunction, a hallmark of atherosclerosis which underpins cardiovascular disease (CVD). PHF8 is a histone demethylase that demethylates H4K20me1, which affects the mammalian target of rapamycin (mTOR) signaling and autophagy, processes that play important roles in CVD. PHF8 is regulated by microRNA (miR) such as miR-22-3p and miR-1229-3p. Biochemically, HHcy is characterized by elevated levels of Hcy, Hcy-thiolactone and <i>N</i>-Hcy-protein. Here, we examined the effects of these metabolites on miR-22-3p, miR-1229-3p, and their target PHF8, as well as on the downstream consequences of these effects on H4K20me1, mTOR-, and autophagy-related proteins and mRNAs expression in human umbilical vein endothelial cells (HUVEC). We found that treatments with <i>N</i>-Hcy-protein, Hcy-thiolactone, or Hcy upregulated miR-22-3p and miR-1229-3p, attenuated PHF8 expression, upregulated H4K20me1, mTOR, and phospho-mTOR. Autophagy-related proteins (BECN1, ATG5, ATG7, lipidated LC3-II, and LC3-II/LC3-I ratio) were significantly downregulated by at least one of these metabolites. We also found similar changes in the expression of miR-22-3p, Phf8, mTOR- and autophagy-related proteins/mRNAs in vivo in hearts of <i>Cbs</i><sup>−/−</sup> mice, which show severe HHcy and endothelial dysfunction. Treatments with inhibitors of miR-22-3p or miR-1229-3p abrogated the effects of Hcy-thiolactone, <i>N</i>-Hcy-protein, and Hcy on miR expression and on PHF8, H4K20me1, mTOR-, and autophagy-related proteins/mRNAs in HUVEC. Taken together, these findings show that Hcy metabolites upregulate miR-22-3p and miR-1229-3p expression, which then dysregulate the PHF8/H4K20me1/mTOR/autophagy pathway, important for vascular homeostasis.</p>","PeriodicalId":50455,"journal":{"name":"FASEB Journal","volume":null,"pages":null},"PeriodicalIF":4.4,"publicationDate":"2024-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1096/fj.202302116R","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142324529","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Paolo Bellotti, Zachary Ladd, Victoria Leroy, Gang Su, Shiven Sharma, Joseph B. Hartman, Jonathan Krebs, Chelsea Viscardi, Robert Maile, Lyle L. Moldawer, Phillip A. Efron, Ashish K. Sharma, Gilbert R. Upchurch Jr.
Abdominal aortic aneurysm (AAA) formation is a chronic vascular pathology characterized by inflammation, leukocyte infiltration, and vascular remodeling. The aim of this study was to delineate the protective role of Resolvin D2 (RvD2), a bioactive isoform of specialized pro-resolving lipid mediators, via G-protein-coupled receptor 18 (GPR18) receptor signaling in attenuating AAAs. Importantly, RvD2 and GPR18 levels were significantly decreased in aortic tissue of AAA patients compared with controls. Furthermore, using an established murine model of AAA in C57BL/6 (WT) mice, we observed that treatment with RvD2 significantly attenuated aortic diameter, pro-inflammatory cytokine production, immune cell infiltration (neutrophils and macrophages), elastic fiber disruption, and increased smooth muscle cell α-actin expression as well as increased TGF-β2 and IL-10 expressions compared to untreated mice. Moreover, the RvD2-mediated protection from vascular remodeling and AAA formation was blocked when mice were previously treated with siRNA for GPR18 signifying the importance of RvD2/GPR18 signaling in vascular inflammation. Mechanistically, RvD2-mediated protection significantly enhanced infiltration and activation of monocytic myeloid-derived suppressor cells (M-MDSCs) by increasing TGF-β2 and IL-10 secretions in a GPR18-dependent manner to attenuate aortic inflammation and vascular remodeling. Collectively, this study demonstrates that RvD2 treatment induces an expansion of myeloid-lineage committed progenitors, such as M-MDSCs, activates GPR18-dependent signaling to enhance TGF-β2 and IL-10 secretion, and mitigates SMC activation that contributes to resolution of aortic inflammation and remodeling during AAA formation.
{"title":"Resolvin D2/GPR18 signaling enhances monocytic myeloid-derived suppressor cell function to mitigate abdominal aortic aneurysm formation","authors":"Paolo Bellotti, Zachary Ladd, Victoria Leroy, Gang Su, Shiven Sharma, Joseph B. Hartman, Jonathan Krebs, Chelsea Viscardi, Robert Maile, Lyle L. Moldawer, Phillip A. Efron, Ashish K. Sharma, Gilbert R. Upchurch Jr.","doi":"10.1096/fj.202400414RRR","DOIUrl":"https://doi.org/10.1096/fj.202400414RRR","url":null,"abstract":"<p>Abdominal aortic aneurysm (AAA) formation is a chronic vascular pathology characterized by inflammation, leukocyte infiltration, and vascular remodeling. The aim of this study was to delineate the protective role of Resolvin D2 (RvD2), a bioactive isoform of specialized pro-resolving lipid mediators, via G-protein-coupled receptor 18 (GPR18) receptor signaling in attenuating AAAs. Importantly, RvD2 and GPR18 levels were significantly decreased in aortic tissue of AAA patients compared with controls. Furthermore, using an established murine model of AAA in C57BL/6 (WT) mice, we observed that treatment with RvD2 significantly attenuated aortic diameter, pro-inflammatory cytokine production, immune cell infiltration (neutrophils and macrophages), elastic fiber disruption, and increased smooth muscle cell α-actin expression as well as increased TGF-β2 and IL-10 expressions compared to untreated mice. Moreover, the RvD2-mediated protection from vascular remodeling and AAA formation was blocked when mice were previously treated with siRNA for GPR18 signifying the importance of RvD2/GPR18 signaling in vascular inflammation. Mechanistically, RvD2-mediated protection significantly enhanced infiltration and activation of monocytic myeloid-derived suppressor cells (M-MDSCs) by increasing TGF-β2 and IL-10 secretions in a GPR18-dependent manner to attenuate aortic inflammation and vascular remodeling. Collectively, this study demonstrates that RvD2 treatment induces an expansion of myeloid-lineage committed progenitors, such as M-MDSCs, activates GPR18-dependent signaling to enhance TGF-β2 and IL-10 secretion, and mitigates SMC activation that contributes to resolution of aortic inflammation and remodeling during AAA formation.</p>","PeriodicalId":50455,"journal":{"name":"FASEB Journal","volume":null,"pages":null},"PeriodicalIF":4.4,"publicationDate":"2024-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142320608","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Uric acid (UA) is the end product of purine metabolism. In recent years, UA has been found to be associated with the prognosis of clinical cancer patients. However, the intricate mechanisms by which UA affects the development and prognosis of tumor patients has not been well elucidated. In this study, we explored the role of UA in breast cancer, scrutinizing its impact on breast cancer cell function by treating two types of breast cancer cell lines with UA. The role of UA in the cell cycle and proliferation of tumors and the underlying mechanisms were further investigated. We found that the antioxidant effect of UA facilitated the scavenging of reactive oxygen species (ROS) in breast cancer, thereby reducing aryl hydrocarbon receptor (AhR) expression and affecting the breast cancer cell cycle, driving the proliferation of breast cancer cells through the AhR/p27Kip1/cyclin E1 pathway. Moreover, in breast cancer patients, the expression of AhR and its downstream genes may be closely associated with cancer progression in patients. Therefore, an increase in UA could promote the proliferation of breast cancer cells through the AhR/p27Kip1/cyclin E1 pathway axis.
尿酸(UA)是嘌呤代谢的最终产物。近年来,人们发现尿酸与临床癌症患者的预后有关。然而,尿酸影响肿瘤患者病情发展和预后的复杂机制尚未得到很好的阐明。在这项研究中,我们探讨了 UA 在乳腺癌中的作用,通过用 UA 处理两种乳腺癌细胞系,仔细研究其对乳腺癌细胞功能的影响。我们还进一步研究了 UA 在肿瘤细胞周期和增殖中的作用及其内在机制。我们发现,UA的抗氧化作用有利于清除乳腺癌细胞中的活性氧(ROS),从而降低芳香烃受体(AhR)的表达,影响乳腺癌细胞周期,通过AhR/p27Kip1/细胞周期蛋白E1途径驱动乳腺癌细胞增殖。此外,在乳腺癌患者中,AhR 及其下游基因的表达可能与患者的癌症进展密切相关。因此,UA 的增加可通过 AhR/p27Kip1/cyclin E1 通路轴促进乳腺癌细胞的增殖。
{"title":"UA influences the progression of breast cancer via the AhR/p27Kip1/cyclin E pathway","authors":"Zhiying Wang, Yuanqi Zhang, Shengchao Huang, Zhihong Liao, Mingzhang Huang, Wei Lei, Xiaorong Shui","doi":"10.1096/fj.202400938R","DOIUrl":"https://doi.org/10.1096/fj.202400938R","url":null,"abstract":"<p>Uric acid (UA) is the end product of purine metabolism. In recent years, UA has been found to be associated with the prognosis of clinical cancer patients. However, the intricate mechanisms by which UA affects the development and prognosis of tumor patients has not been well elucidated. In this study, we explored the role of UA in breast cancer, scrutinizing its impact on breast cancer cell function by treating two types of breast cancer cell lines with UA. The role of UA in the cell cycle and proliferation of tumors and the underlying mechanisms were further investigated. We found that the antioxidant effect of UA facilitated the scavenging of reactive oxygen species (ROS) in breast cancer, thereby reducing aryl hydrocarbon receptor (AhR) expression and affecting the breast cancer cell cycle, driving the proliferation of breast cancer cells through the AhR/p27<sup>Kip1</sup>/cyclin E1 pathway. Moreover, in breast cancer patients, the expression of AhR and its downstream genes may be closely associated with cancer progression in patients. Therefore, an increase in UA could promote the proliferation of breast cancer cells through the AhR/p27<sup>Kip1</sup>/cyclin E1 pathway axis.</p>","PeriodicalId":50455,"journal":{"name":"FASEB Journal","volume":null,"pages":null},"PeriodicalIF":4.4,"publicationDate":"2024-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1096/fj.202400938R","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142320607","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Michael Chen, Krit Suwannaphoom, Yas Sanaiha, Yuan Luo, Peyman Benharash, Michael C. Fishbein, René R. Sevag Packard
Coronary plaque rupture remains the prominent mechanism of myocardial infarction. Accurate identification of rupture-prone plaque may improve clinical management. This study assessed the discriminatory performance of electrochemical impedance spectroscopy (EIS) in human cardiac explants to detect high-risk atherosclerotic features that portend rupture risk. In this single-center, prospective study, n = 26 cardiac explants were collected for EIS interrogation of the three major coronary arteries. Vessels in which advancement of the EIS catheter without iatrogenic plaque disruption was rendered impossible were not assessed. N = 61 vessels underwent EIS measurement and histological analyses. Plaques were dichotomized according to previously established high rupture-risk parameter thresholds. Diagnostic performance was determined via receiver operating characteristic areas-under-the-curve (AUC). Necrotic cores were identified in n = 19 vessels (median area 1.53 mm2) with a median fibrous cap thickness of 62 μm. Impedance was significantly greater in plaques with necrotic core area ≥1.75 mm2 versus <1.75 mm2 (19.8 ± 4.4 kΩ vs. 7.2 ± 1.0 kΩ, p = .019), fibrous cap thickness ≤65 μm versus >65 μm (19.1 ± 3.5 kΩ vs. 6.5 ± 0.9 kΩ, p = .004), and ≥20 macrophages per 0.3 mm-diameter high-power field (HPF) versus <20 macrophages per HPF (19.8 ± 4.1 kΩ vs. 10.2 ± 0.9 kΩ, p = .002). Impedance identified necrotic core area ≥1.75 mm2, fibrous cap thickness ≤65 μm, and ≥20 macrophages per HPF with AUCs of 0.889 (95% CI: 0.716–1.000) (p = .013), 0.852 (0.646–1.000) (p = .025), and 0.835 (0.577–1.000) (p = .028), respectively. Further, phase delay discriminated severe stenosis (≥70%) with an AUC of 0.767 (0.573–0.962) (p = .035). EIS discriminates high-risk atherosclerotic features that portend plaque rupture in human coronary artery disease and may serve as a complementary modality for angiography-guided atherosclerosis evaluation.
{"title":"Electrochemical impedance spectroscopy unmasks high-risk atherosclerotic features in human coronary artery disease","authors":"Michael Chen, Krit Suwannaphoom, Yas Sanaiha, Yuan Luo, Peyman Benharash, Michael C. Fishbein, René R. Sevag Packard","doi":"10.1096/fj.202401200R","DOIUrl":"10.1096/fj.202401200R","url":null,"abstract":"<p>Coronary plaque rupture remains the prominent mechanism of myocardial infarction. Accurate identification of rupture-prone plaque may improve clinical management. This study assessed the discriminatory performance of electrochemical impedance spectroscopy (EIS) in human cardiac explants to detect high-risk atherosclerotic features that portend rupture risk. In this single-center, prospective study, <i>n</i> = 26 cardiac explants were collected for EIS interrogation of the three major coronary arteries. Vessels in which advancement of the EIS catheter without iatrogenic plaque disruption was rendered impossible were not assessed. <i>N</i> = 61 vessels underwent EIS measurement and histological analyses. Plaques were dichotomized according to previously established high rupture-risk parameter thresholds. Diagnostic performance was determined via receiver operating characteristic areas-under-the-curve (AUC). Necrotic cores were identified in <i>n</i> = 19 vessels (median area 1.53 mm<sup>2</sup>) with a median fibrous cap thickness of 62 μm. Impedance was significantly greater in plaques with necrotic core area ≥1.75 mm<sup>2</sup> versus <1.75 mm<sup>2</sup> (19.8 ± 4.4 kΩ vs. 7.2 ± 1.0 kΩ, <i>p</i> = .019), fibrous cap thickness ≤65 μm versus >65 μm (19.1 ± 3.5 kΩ vs. 6.5 ± 0.9 kΩ, <i>p</i> = .004), and ≥20 macrophages per 0.3 mm-diameter high-power field (HPF) versus <20 macrophages per HPF (19.8 ± 4.1 kΩ vs. 10.2 ± 0.9 kΩ, <i>p</i> = .002). Impedance identified necrotic core area ≥1.75 mm<sup>2</sup>, fibrous cap thickness ≤65 μm, and ≥20 macrophages per HPF with AUCs of 0.889 (95% CI: 0.716–1.000) (<i>p</i> = .013), 0.852 (0.646–1.000) (<i>p</i> = .025), and 0.835 (0.577–1.000) (<i>p</i> = .028), respectively. Further, phase delay discriminated severe stenosis (≥70%) with an AUC of 0.767 (0.573–0.962) (<i>p</i> = .035). EIS discriminates high-risk atherosclerotic features that portend plaque rupture in human coronary artery disease and may serve as a complementary modality for angiography-guided atherosclerosis evaluation.</p>","PeriodicalId":50455,"journal":{"name":"FASEB Journal","volume":null,"pages":null},"PeriodicalIF":4.4,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142309016","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}