Endothelial progenitor cells systemic administration alleviates multi-organ senescence by down-regulating USP7/p300 pathway in chronic obstructive pulmonary disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) has impacted approximately 390 million people worldwide and the morbidity is increasing every year. However, due to the poor treatment efficacy of COPD, exploring novel treatment has become the hotpot of study on COPD. Endothelial progenitor cells (EPCs) aging is a possible molecular way for COPD development. We aimed to explore the effector whether intravenous administration of EPCs has therapeutic effects in COPD mice. METHODS: COPD mice model was induced by cigarette smoke exposure and EPCs were injected intravenously to investigate their effects on COPD mice. At day 127, heart, liver, spleen, lung and kidney tissues of mice were harvested. The histological effects of EPCs intervention on multiple organs of COPD mice were detected by morphology assay. Quantitative real-time PCR and Western blotting were used to detect the effect of EPCs intervention on the expression of multi-organ senescence-related indicators. And we explored the effect of EPCs systematically intervening on senescence-related USP7/p300 pathway. RESULTS: Compared with COPD group, senescence-associated ß-galactosidase activity was decreased, protein and mRNA expression of p16 was down-regulated, while protein and mRNA expression of cyclin D1 and TERT were up-regulated of multiple organs, including lung, heart, liver, spleen and kidney in COPD mice after EPCs system intervention. But the morphological alterations of the tissues described above in COPD mice failed to be reversed. Mechanistically, EPCs systemic administration inhibited the expression of mRNA and protein of USP7 and p300 in multiple organs of COPD mice, exerting therapeutic effects. CONCLUSIONS: EPCs administration significantly inhibited the senescence of multiple organs in COPD mice via down-regulating USP7/p300 pathway, which presents a possibility of EPCs therapy for COPD.

Cigarette smoke extract induces the senescence of endothelial progenitor cells by upregulating p300.

INTRODUCTION: Endothelial progenitor cells (EPCs) are the main source of endothelial cells. The senescence of EPCs is involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). Cigarette smoke extract (CSE) can directly induce the dysfunction and increased expression of senescence-related markers in EPCs cultured in vitro. Histone acetyltransferase p300 is a transcriptional activator, and its changes can lead to cell senescence. The present study investigated whether CSE can induce the senescence of EPCs by upregulating p300. METHODS: EPCs were isolated from bone marrow of C57BL/6J mice by density gradient centrifugation. The p300 inhibitor C646 and agonist CTPB were used to interfere with EPCs, cell cycle and apoptosis were detected by flow cytometry, the proportion of senile cells was counted by ß-galactosidase staining, the protein expression of p300, H4K12, Cyclin D1, TERT and Ki67 were detected by western blot. RESULTS: Compared with the control group, the cell cycle of CSE group and CTPB group were blocked, the apoptosis rate and early apoptosis rate were increased, the proportion of senile cells counted by ß-galactosidase staining was increased, the expression of p300 and H4K12 protein were increased, the expression of Cyclin D1, TERT and Ki67 protein were decreased. C646 could partly alleviate the damages caused by CSE. CONCLUSIONS: CSE may promote the apoptosis and senescence of EPCs by upregulating the expression of p300 and H4K12 protein, thus preventing the transition of EPCs from G1 phase to S phase, affecting telomerase synthesis, and reducing EPCs proliferation.

Existence of multiple organ aging in animal model of emphysema induced by cigarette smoke extract.

INTRODUCTION: It is commonly considered that COPD or at least emphysema represents accelerated lung aging induced in part by oxidative damage from cigarette smoke components. However, the issue if there are any aging signs in other organs in patients with COPD or emphysema remains unclear. The aim of this study is to explore whether there is multiple organ aging in the animal model of emphysema induced by cigarette smoke extract (CSE), and to ascertain the possible mechanisms, if any. METHODS: The animal model of emphysema was induced by CSE. Histomorphological changes in lung, heart, liver, kidney and spleen tissues were measured after staining with hematoxylin and eosin (H&E). The concentrations of stem cell factor (SCF), CyclinD1 and superoxide dismutase (SOD) in serum were determined by ELISA kit. The expressions of p16 (INK4a), Sca-1, eNOS proteins and mRNA in lung, heart, liver, kidney and spleen tissues were detected by Western blotting and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), respectively. Decitabine (Dec) was applied to examine whether it could alter the changes caused by CSE. RESULTS: The histomorphology of lung tissue was significantly changed, while other organs exhibited normal structure and histomorphology. The concentrations of SCF, CyclinD1 and SOD in serum were lower in the CSE group than in the control group. The expression levels of p16(INK4a) protein and mRNA in lung, heart, liver, kidney and spleen tissues were higher in the CSE group than in the control group, while the expression levels of Sca-1 and eNOS proteins and mRNA were lower in the CSE group than in the control group, in the tissues described above. Dec could partly alleviate the damages caused by CSE and the degree of alleviation resulted by Dec varied from organ to organ. CONCLUSIONS: In addition to the aging of the lung tissue in the emphysema animal model induced by CSE, the tissues of the heart, liver, kidney and spleen were also in the progress of aging, but the sensibility and affinity of lung to CSE were higher than those of the other organs. Multiple organ aging may also exist in the animal model of emphysema induced by CSE. DEC can partly alleviate the multiple organ aging caused by CSE.

p300/Sp1-Mediated High Expression of p16 Promotes Endothelial Progenitor Cell Senescence Leading to the Occurrence of Chronic Obstructive Pulmonary Disease.

OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is a common chronic disease and develops rapidly into a grave public health problem worldwide. However, what exactly causes the occurrence of COPD remains largely unclear. Here, we are trying to explore whether the high expression of p16 mediated by p300/Sp1 can cause chronic obstructive pulmonary disease through promoting the senescence of endothelial progenitor cells (EPCs). METHODS: Peripheral blood EPCs were isolated from nonsmoking non-COPD, smoking non-COPD, and smoking COPD patients. The expressions of p16, p300, and senescence-related genes were detected by RT-PCR and Western Blot. Then, we knocked down or overexpressed Sp1 and p300 and used the ChIP assay to detect the histone H4 acetylation level in the promoter region of p16, CCK8 to detect cell proliferation, flow cytometry to detect the cell cycle, and ß-galactosidase staining to count the proportion of senescent cells. RESULTS: The high expression of p16 was found in peripheral blood EPCs of COPD patients; the cigarette smoke extract (CSE) led to the increase of p16. The high expression of p16 in EPCs promoted cell cycle arrest and apoptosis. The CSE-mediated high expression of p16 promoted cell senescence. The expression of p300 was increased in peripheral blood EPCs of COPD patients. Moreover, p300/Sp1 enhanced the histone H4 acetylation level in the promoter region of p16, thereby mediating the senescence of EPCs. And knockdown of p300/Sp1 could rescue CSE-mediated cell senescence. CONCLUSION: p300/Sp1 enhanced the histone H4 acetylation level in the p16 promoter region to mediate the senescence of EPCs.

Cigarette smoke extract affects methylation status and attenuates Sca-1 expression of mouse endothelial progenitor cell in vitro.

INTRODUCTION: Endothelial dysfunction appears in many smoking-related diseases, it is also an important pathophysiological feature. Endothelial progenitor cells (EPCs) are precursors of endothelial cells and have a crucial effect on the repair and maintenance of endothelial integrity. Sca-1 is not only common in bone marrow-derived hematopoietic stem cells (HSCs), but it is also expressed in nonhematopoietic organs by tissue-resident stem and progenitor cells. The aim of this study is to investigate the impact of cigarette smoke extract (CSE) on the function of bone marrow-derived EPCs and the expression level of Sca-1 in EPCs, and also whether the methylation of Sca-1 is involved in EPC dysfunction. METHODS: We measured EPC capacities including adhesion, secretion and proliferation, the concentration of endothelial nitric oxide synthase (eNOS) and apoptosis-inducing factor (AIF) in cell culture supernatant, and also Sca-1 expression and promoter methylation in EPCs induced by CSE. Decitabine (Dec) was applied to test whether it could alter the impact caused by CSE. RESULTS: The adhesion, proliferation and secretion ability of EPCs can be induced to be decreased by CSE in vitro, accompanied by decreased concentrations of AIF and eNOS in cell culture supernatant and decreased Sca-1 expression in EPCs. In addition, Dec could partly attenuate the impact described above. There were no significant differences in the quantitative analysis of Sca-1 promoter methylation among different groups. CONCLUSIONS: The decreased Sca-1 expression was related to EPC dysfunction induced by CSE. EPC dysfunction resulting from CSE may be related to methylation mechanism, but not the methylation of Sca-1 promoter.