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BACKGROUND: The prevalence of vascular calcification (VC) in chronic kidney disease (CKD) patients remains substantial, but currently, there are no effective pharmaceutical therapies available. BRCA1/BRCA2-containing complex subunit 36 (BRCC36) has been implicated in osteoblast osteogenic conversion; however, its specific role in VC remains to be fully elucidated. The aim of this study was to investigate the role and underlying mechanisms of BRCC36 in VC. METHODS: The association between BRCC36 expression and VC was examined in radial arteries from patients with CKD, high-adenine-induced CKD mice, and vascular smooth muscle cells (VSMCs). Western blotting, real-time polymerase chain reaction, immunofluorescence, and immunohistochemistry were used to analyse gene expression. Gain- and loss-of-function experiments were performed to comprehensively investigate the effects of BRCC36 on VC. Coimmunoprecipitation and TOPFlash luciferase assays were utilized to further investigate the regulatory effects of BRCC36 on the Wnt/ß-catenin pathway. RESULTS: BRCC36 expression was downregulated in human calcified radial arteries, calcified aortas from CKD mice, and calcified VSMCs. VSMC-specific BRCC36 overexpression alleviated calcium deposition in the vasculature, whereas BRCC36 depletion aggravated VC progression. Furthermore, BRCC36 inhibited the osteogenic differentiation of VSMCs in vitro. Rescue experiments revealed that BRCC36 exerts the protective effects on VC partly by regulating the Wnt/ß-catenin signalling pathway. Mechanistically, BRCC36 inhibited the Wnt/ß-catenin pathway by decreasing the K63-linked ubiquitination of ß-catenin. Additionally, pioglitazone attenuated VC partly through upregulating BRCC36 expression. CONCLUSIONS: Our research results emphasize the critical role of the BRCC36-ß-catenin axis in VC, suggesting that BRCC36 or ß-catenin may be promising therapeutic targets to prevent the progression of VC in CKD patients.
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Ratones Endogámicos C57BL , Insuficiencia Renal Crónica , Ubiquitinación , Calcificación Vascular , Vía de Señalización Wnt , beta Catenina , Calcificación Vascular/metabolismo , Calcificación Vascular/patología , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patología , Insuficiencia Renal Crónica/complicaciones , Animales , beta Catenina/metabolismo , Humanos , Masculino , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Ratones , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Osteogénesis , Persona de Mediana Edad , Diferenciación CelularRESUMEN
OBJECTIVE: This study aimed to investigate the effect of the soluble Klotho (sKlotho)/Wnt/ß-catenin signaling pathway on vascular calcification in rat models of chronic kidney disease (CKD) and the intervention effect of Shenyuan granules. METHODS: Rats with 5/6 nephrectomy and high phosphorus feeding were used to establish the vascular calcification model. The rats were given gradient doses of Shenyuan granules aqueous solution and calcitriol solution by gavage for 8 weeks, which were divided into experimental group and positive control group. RESULTS: The 5/6 nephrectomy combined with high phosphorus feeding induced thoracic aortic calcification in rats. Shenyuan granules intervention increased the serum sKlotho level, inhibited the mRNA and protein expression of Wnt1, ß-catenin, and Runx2 in the thoracic aorta, and alleviated thoracic aortic media calcification in rats. CONCLUSION: Shenyuan granules may partially regulate the Wnt/ß-catenin signaling pathway via serum sKl to interfere with the expression of Runx2, thereby improving vascular calcification in CKD.
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Medicamentos Herbarios Chinos , Glucuronidasa , Proteínas Klotho , Insuficiencia Renal Crónica , Calcificación Vascular , Vía de Señalización Wnt , beta Catenina , Animales , Masculino , Ratas , Aorta Torácica/metabolismo , Aorta Torácica/patología , beta Catenina/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Modelos Animales de Enfermedad , Medicamentos Herbarios Chinos/farmacología , Medicamentos Herbarios Chinos/uso terapéutico , Glucuronidasa/metabolismo , Glucuronidasa/genética , Proteínas Klotho/metabolismo , Nefrectomía , Ratas Sprague-Dawley , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/complicaciones , Calcificación Vascular/metabolismo , Calcificación Vascular/etiología , Calcificación Vascular/patología , Vía de Señalización Wnt/efectos de los fármacos , Proteína Wnt1/metabolismo , Proteína Wnt1/genéticaRESUMEN
The present study aimed to explore the effect of swimming exercise on vascular calcification in type 2 diabetic rats and its related molecular mechanism. Male Sprague Dawley (SD) rats were randomly divided into normal control (NC), diabetes control (DC) and diabetes+exercise (DE) groups. The DC and DE groups were intraperitoneally injected with streptozotocin (STZ) and fed with high-fat diet to establish type 2 diabetes mellitus model. The NC and DC groups did not exercise, and the DE group performed swimming exercise for 8 weeks. ELISA was used to detect the serum glycated hemoglobin A1c (HbA1c) level. The aortas of rats were taken as sample. Assay kits were used to detect vascular calcium content and alkaline phosphatase (ALP) activity. Von Kossa staining was used to detect calcium deposition. qRT-PCR was used detect the expression of microRNA-145 (miR-145). Western blot was used to detect the protein expression levels of smooth muscle contraction markers, calcification marker and related proteins. The results showed that, compared with the NC group, the blood glucose, serum HbA1c level, vascular calcium content and ALP activity in the DC group were significantly increased, the protein expression levels of smooth muscle contraction markers smooth muscle protein 22α (SM22α) and α-smooth muscle actin (α-SMA) were significantly down-regulated, and the protein expression level of calcification marker osteopontin (OPN) was significantly up-regulated; Compared with the DC group, the serum HbA1c level, vascular calcium content and ALP activity in the DE group were significantly decreased, the protein expression levels of SM22α and α-SMA were significantly up-regulated, and the protein expression level of OPN was significantly down-regulated; Compared with the NC group, the expression of miR-145-5p in the DC group was significantly down-regulated, and the protein expression levels of transforming growth factor-ß (TGF-ß), SMAD2, ERK1/2 and p-ERK1/2 were significantly up-regulated; Compared with the DC group, the expression of miR-145-5p was significantly up-regulated in the DE group, while the expressions of TGF-ß, ERK1/2 and p-ERK1/2 were significantly down-regulated. These results suggest that miR-145/TGF-ß signaling is involved in the improving effects of 8-week swimming exercise on glucose metabolism disorder, vascular smooth muscle cell phenotype switching and vascular calcification in type 2 diabetes mellitus.
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Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , MicroARNs , Condicionamiento Físico Animal , Ratas Sprague-Dawley , Natación , Calcificación Vascular , Animales , Masculino , Ratas , MicroARNs/metabolismo , MicroARNs/genética , Calcificación Vascular/metabolismo , Calcificación Vascular/etiología , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Experimental/metabolismo , Natación/fisiología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
BACKGROUND: Vascular calcification is a common vascular lesion associated with high morbidity and mortality from cardiovascular events. Antibiotics can disrupt the gut microbiota (GM) and have been shown to exacerbate or attenuate several human diseases. However, whether antibiotic-induced GM disruption affects vascular calcification remains unclear. METHODS: Antibiotic cocktail (ABX) treatment was utilized to test the potential effects of antibiotics on vascular calcification. The effects of antibiotics on GM and serum short-chain fatty acids (SCFAs) in vascular calcification mice were analyzed using 16 S rRNA gene sequencing and targeted metabolomics, respectively. Further, the effects of acetate, propionate and butyrate on vascular calcification were evaluated. Finally, the potential mechanism by which acetate inhibits osteogenic transformation of VSMCs was explored by proteomics. RESULTS: ABX and vancomycin exacerbated vascular calcification. 16 S rRNA gene sequencing and targeted metabolomics analyses showed that ABX and vancomycin treatments resulted in decreased abundance of Bacteroidetes in the fecal microbiota of the mice and decreased serum levels of SCFAs. In addition, supplementation with acetate was found to reduce calcium salt deposition in the aorta of mice and inhibit osteogenic transformation in VSMCs. Finally, using proteomics, we found that the inhibition of osteogenic transformation of VSMCs by acetate may be related to glutathione metabolism and ubiquitin-mediated proteolysis. After adding the glutathione inhibitor Buthionine sulfoximine (BSO) and the ubiquitination inhibitor MG132, we found that the inhibitory effect of acetate on VSMC osteogenic differentiation was weakened by the intervention of BSO, but MG132 had no effect. CONCLUSION: ABX exacerbates vascular calcification, possibly by depleting the abundance of Bacteroidetes and SCFAs in the intestine. Supplementation with acetate has the potential to alleviate vascular calcification, which may be an important target for future treatment of vascular calcification.
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Acetatos , Antibacterianos , Ácidos Grasos Volátiles , Microbioma Gastrointestinal , Calcificación Vascular , Animales , Microbioma Gastrointestinal/efectos de los fármacos , Calcificación Vascular/metabolismo , Calcificación Vascular/etiología , Calcificación Vascular/tratamiento farmacológico , Ratones , Ácidos Grasos Volátiles/metabolismo , Acetatos/farmacología , Antibacterianos/efectos adversos , Antibacterianos/farmacología , Masculino , Osteogénesis/efectos de los fármacos , ARN Ribosómico 16S/genética , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Vancomicina/efectos adversos , Vancomicina/farmacología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/efectos de los fármacosRESUMEN
Hyperphosphatemic familial tumoral calcinosis (HFTC) is a rare disorder caused by deficient FGF23 signaling and resultant ectopic calcification. Here, we systematically characterized and quantified macro- and micro-calcification in a HFTC cohort using CT and 18F-sodium fluoride PET/CT (18F-NaF PET/CT). Fourier-transform infrared (FTIR) spectroscopy was performed on 4 phenotypically different calcifications from a patient with HFTC, showing the dominant component to be hydroxyapatite. Eleven patients with HFTC were studied with CT and/or 18F-NaF PET/CT. Qualitative review was done to describe the spectrum of imaging findings on both modalities. CT-based measures of volume (eg, total calcific burden and lesion volume) and density (Hounsfield units) were quantified and compared to PET-based measures of mineralization activity (eg, mean standardized uptake values-SUVs). Microcalcification scores were calculated for the vasculature of 6 patients using 18F-NaF PET/CT and visualized on a standardized vascular atlas. Ectopic calcifications were present in 82% of patients, predominantly near joints and the distal extremities. Considerable heterogeneity was observed in total calcific burden per patient (823.0 ± 670.1 cm3, n = 9) and lesion volume (282.5 ± 414.8 cm3, n = 27). The largest lesions were found at the hips and shoulders. 18F-NaF PET offered the ability to differentiate active vs quiescent calcifications. Calcifications were also noted in multiple anatomic locations, including brain parenchyma (50%). Vascular calcification was seen in the abdominal aorta, carotid, and coronaries in 50%, 73%, and 50%, respectively. 18F-NaF-avid, but CT-negative calcification was seen in a 17-year-old patient, implicating early onset vascular calcification. This first systematic assessment of calcifications in a cohort of patients with HFTC has identified the early onset, prevalence, and extent of calcification. It supports 18F-NaF PET/CT as a clinical tool for distinguishing between active and inactive calcification, informing disease progression, and quantification of ectopic and vascular disease burden.
Hyperphosphatemic familial tumoral calcinosis (HFTC) is a rare disorder in which patients develop sometimes large debilitating calcifications of soft tissues and blood vessels. It is caused by deficient fibroblast growth factor-23 that leads to high phosphate levels, which contributes to the calcifications. The calcifications and manifestations of this disorder have not been well characterized. We determined the mineral composition of the calcifications to be hydroxyapatite. Capitalizing on the fact fluoride can be integrated into hydroxyapatite, we used radiolabeled sodium fluoride PET/CT scans (18F-NaF PET/CT) to characterize and quantify the calcifications in 11 patients. Eighty-two percent of the patients had calcifications, with the largest located at the hips and shoulders. Micro-calcifications were found in the blood vessels of most patients, including children. The technique also enabled us to differentiate between active vs stable calcifications. This first systematic assessment of calcifications in patients with HFTC showed the utility of 18F-NaF PET/CT as a tool to identify and quantify calcifications, as well as distinguish between active and stable calcifications. This approach will inform disease progression and may prove useful for measuring response to treatment.
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Calcinosis , Factor-23 de Crecimiento de Fibroblastos , Hiperfosfatemia , Tomografía Computarizada por Tomografía de Emisión de Positrones , Calcificación Vascular , Humanos , Calcinosis/diagnóstico por imagen , Calcinosis/patología , Calcinosis/genética , Femenino , Masculino , Hiperfosfatemia/diagnóstico por imagen , Hiperfosfatemia/patología , Hiperfosfatemia/complicaciones , Hiperfosfatemia/genética , Adulto , Calcificación Vascular/diagnóstico por imagen , Calcificación Vascular/patología , Calcificación Vascular/metabolismo , Persona de Mediana Edad , Adolescente , Niño , Imagen Molecular/métodos , Hiperostosis Cortical Congénita/diagnóstico por imagen , Hiperostosis Cortical Congénita/genética , Hiperostosis Cortical Congénita/patología , Hiperostosis Cortical Congénita/complicaciones , Hiperostosis Cortical Congénita/metabolismo , Fluoruro de Sodio , Adulto JovenRESUMEN
BACKGROUND: Hyponatremia, frequently observed in patients with chronic kidney disease, is associated with increased cardiovascular morbidity and mortality. Hyponatremia or low osmolality induces oxidative stress and cell death, both of which accelerate vascular calcification (VC), a critical phenotype in patients with chronic kidney disease. Whether hyponatremia or low osmolality plays a role in the pathogenesis of VC is unknown. METHODS: Human vascular smooth muscle cells (VSMCs) and mouse aortic rings were cultured in various osmotic conditions and calcifying medium supplemented with high calcium and phosphate. The effects of low osmolality on phenotypic change and oxidative stress in the cultured VSMCs were examined. Microarray analysis was conducted to determine the main signaling pathway of osmolality-related VC. The transcellular sodium and calcium ions flux across the VSMCs were visualized by live imaging. Furthermore, the effect of osmolality on calciprotein particles (CPPs) was investigated. Associations between arterial intimal calcification and hyponatremia or low osmolality were examined by a cross-sectional study using human autopsy specimens obtained in the Hisayama Study. RESULTS: Low osmolality exacerbated calcification of the ECM (extracellular matrix) of cultured VSMCs and mouse aortic rings. Oxidative stress and osteogenic differentiation of VSMCs were identified as the underlying mechanisms responsible for low osmolality-induced VC. Microarray analysis showed that low osmolality activated the Rac1 (Ras-related C3 botulinum toxin substrate 1)-Akt (protein kinase B) pathway and reduced NCX1 (Na-Ca exchanger 1) expression. Live imaging showed synchronic calcium ion efflux and sodium ion influx via NCX1 when extracellular sodium ion concentrations were increased. An NCX1 inhibitor promoted calcifying media-induced VC by reducing calcium ion efflux. Furthermore, low osmolality accelerated the generation and maturation steps of CPPs. The cross-sectional study of human autopsy specimens showed that hyponatremia and low osmolality were associated with a greater area of arterial intimal calcification. CONCLUSIONS: Hyponatremia and low osmolality promote VC through multiple cellular processes, including the Rac1-Akt pathway activation.
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Hiponatremia , Músculo Liso Vascular , Miocitos del Músculo Liso , Estrés Oxidativo , Calcificación Vascular , Animales , Humanos , Hiponatremia/metabolismo , Calcificación Vascular/metabolismo , Calcificación Vascular/patología , Concentración Osmolar , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Masculino , Células Cultivadas , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Ratones Endogámicos C57BL , Femenino , Transducción de Señal , Intercambiador de Sodio-Calcio/metabolismo , Anciano , Estudios Transversales , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratones , Osteogénesis , Persona de Mediana Edad , Modelos Animales de Enfermedad , Calcio/metabolismo , Fenotipo , Proteína de Unión al GTP rac1RESUMEN
BACKGROUND: Abdominal aortic aneurysms expand over time and increase the risk of fatal ruptures. To predict expansion, the isolated assessment of 18F-fluorodeoxyglucose (FDG) and sodium fluoride (NaF) uptake or calcification volume in aneurysms has been investigated with variability in results. We systematically evaluated whether 18F-FDG and 18F-NaF uptake was predictive of abdominal aortic aneurysm expansion. METHODS: Seventy-four male Sprague-Dawley rat abdominal aortic aneurysm models were imaged using positron emission tomography-computed tomography with 18F-FDG and 18F-NaF at 1, 2, 4, 6, and 8 weeks after CaCl2 or saline stimulation. In the 1-week cohort (n=25), the correlation between 18F-FDG or 18F-NaF uptake and pathological markers was investigated. In the time course cohort (n=49), animals received either atorvastatin, losartan, aldactone, or risedronate to assess the effect of these drugs, and the relationship between aortic size and sequential 18F-FDG and 18F-NaF uptake or calcification volume was examined. RESULTS: In the 1-week cohort, the maximum standard unit value of 18F-FDG and 18F-NaF uptake correlated with CD68- (r=0.82; P=0.001) and von Kossa staining-positive areas (r=0.89; P<0.001), respectively. In the time course cohort, 18F-FDG and 18F-NaF uptake changed in a time-dependent manner and drugs attenuated this uptake. Specifically, 18F-FDG showed high uptake at weeks 1 and 2, whereas a high 18F-NaF uptake was noted throughout the study period. Atorvastatin and risedronate showed a decreased and increased aortic size, respectively. The final aortic area correlated well with 18F-FDG and 18F-NaF uptake and calcification volume, especially at 1 and 2 weeks (18F-NaF [1 week]: r=0.61, 18F-FDG [2 weeks]: r=0.51, calcification volume [1 week]: r=0.59; P<0.001). Multiple linear regression analysis showed that the combination of these factors predicted the final aortic size, with 18F-NaF uptake at 1 week being the strongest predictor. CONCLUSIONS: The uptake of 18F-NaF and 18F-FDG and the calcification volume at appropriate times correlated with the development of abdominal aortic aneurysms, with 18F-NaF uptake being the strongest predictor.
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Aorta Abdominal , Aneurisma de la Aorta Abdominal , Modelos Animales de Enfermedad , Fluorodesoxiglucosa F18 , Tomografía Computarizada por Tomografía de Emisión de Positrones , Radiofármacos , Ratas Sprague-Dawley , Fluoruro de Sodio , Calcificación Vascular , Animales , Masculino , Fluorodesoxiglucosa F18/farmacocinética , Aneurisma de la Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/diagnóstico por imagen , Aneurisma de la Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/inducido químicamente , Aorta Abdominal/diagnóstico por imagen , Aorta Abdominal/metabolismo , Aorta Abdominal/patología , Aorta Abdominal/efectos de los fármacos , Calcificación Vascular/diagnóstico por imagen , Calcificación Vascular/metabolismo , Calcificación Vascular/patología , Valor Predictivo de las Pruebas , Factores de Tiempo , Radioisótopos de Flúor , Progresión de la Enfermedad , RatasRESUMEN
Vascular calcification is frequently seen in patients with chronic kidney disease (CKD), and significantly increases cardiovascular mortality and morbidity. Sirt7, a NAD+-dependent histone deacetylases, plays a crucial role in cardiovascular disease. However, the role of Sirt7 in vascular calcification remains largely unknown. Using in vitro and in vivo models of vascular calcification, this study showed that Sirt7 expression was significantly reduced in calcified arteries from mice administered with high dose of vitamin D3 (vD3). We found that knockdown or inhibition of Sirt7 promoted vascular smooth muscle cell (VSMC), aortic ring and vascular calcification in mice, whereas overexpression of Sirt7 had opposite effects. Intriguingly, this protective effect of Sirt7 on vascular calcification is dependent on its deacetylase activity. Unexpectedly, Sirt7 did not alter the osteogenic transition of VSMCs. However, our RNA-seq and subsequent studies demonstrated that knockdown of Sirt7 in VSMCs resulted in increased intracellular reactive oxygen species (ROS) accumulation, and induced an Nrf-2 mediated oxidative stress response. Treatment with the ROS inhibitor N-acetylcysteine (NAC) significantly attenuated the inhibitory effect of Sirt7 on VSMC calcification. Furthermore, we found that knockdown of Sirt7 delayed cell cycle progression and accelerated cellular senescence of VSMCs. Taken together, our results indicate that Sirt7 regulates vascular calcification at least in part through modulation of ROS and cellular senescence of VSMCs. Sirt7 may be a potential therapeutic target for vascular calcification.
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Senescencia Celular , Músculo Liso Vascular , Miocitos del Músculo Liso , Estrés Oxidativo , Especies Reactivas de Oxígeno , Sirtuinas , Calcificación Vascular , Animales , Calcificación Vascular/patología , Calcificación Vascular/metabolismo , Calcificación Vascular/genética , Especies Reactivas de Oxígeno/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Ratones , Sirtuinas/metabolismo , Sirtuinas/genética , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Humanos , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/genética , Masculino , Colecalciferol/farmacología , Insuficiencia Renal Crónica/patología , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/genética , Ratones Endogámicos C57BL , Células CultivadasAsunto(s)
Antraquinonas , Aterosclerosis , Calcificación Vascular , Humanos , Calcificación Vascular/metabolismo , Calcificación Vascular/patología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Nitrato de Plata , Insuficiencia Renal Crónica/metabolismo , Coloración y Etiquetado/métodos , Aneurisma de la Aorta/metabolismo , Aneurisma de la Aorta/patología , Calcio/metabolismo , Tinción con Nitrato de PlataRESUMEN
BACKGROUND: Cellular senescence, macrophages infiltration, and vascular smooth muscle cells (VSMCs) osteogenic transdifferentiation participate in the pathophysiology of vascular calcification in chronic kidney disease (CKD). Senescent macrophages are involved in the regulation of inflammation in pathological diseases. In addition, senescent cells spread senescence to neighboring cells via Interferon-induced transmembrane protein3 (IFITM3). However, the role of senescent macrophages and IFITM3 in VSMCs calcification remains unexplored. AIMS: To explore the hypothesis that senescent macrophages contribute to the calcification and senescence of VSMCs via IFITM3. METHODS: Here, the macrophage senescence model was established using Lipopolysaccharides (LPS). The VSMCs were subjected to supernatants from macrophages (MCFS) or LPS-induced macrophages (LPS-MCFS) in the presence or absence of calcifying media (CM). Senescence-associated ß-galactosidase (SA-ß-gal), Alizarin red (AR), immunofluorescent staining, and western blot were used to identify cell senescence and calcification. RESULTS: The expression of IFITM3 was significantly increased in LPS-induced macrophages and the supernatants. The VSMCs transdifferentiated into osteogenic phenotype, expressing higher osteogenic differentiation markers (RUNX2) and lower VSMCs constructive makers (SM22α) when cultured with senescent macrophages supernatants. Also, senescence markers (p16 and p21) in VSMCs were significantly increased by senescent macrophages supernatants treated. However, IFITM3 knockdown inhibited this process. CONCLUSIONS: Our study showed that LPS-induced senescence of macrophages accelerated the calcification of VSMCs via IFITM3. These data provide a new perspective linking VC and aging, which may provide clues for diagnosing and treating accelerated vascular aging in patients with CKD.
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Senescencia Celular , Lipopolisacáridos , Macrófagos , Proteínas de la Membrana , Músculo Liso Vascular , Proteínas de Unión al ARN , Calcificación Vascular , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Lipopolisacáridos/farmacología , Calcificación Vascular/patología , Calcificación Vascular/metabolismo , Macrófagos/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas de Unión al ARN/metabolismo , Humanos , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patología , Células Cultivadas , Animales , Osteogénesis , Transdiferenciación CelularAsunto(s)
Técnicas de Cocultivo , Células Endoteliales , Músculo Liso Vascular , Miocitos del Músculo Liso , Calcificación Vascular , Humanos , Miocitos del Músculo Liso/patología , Miocitos del Músculo Liso/metabolismo , Calcificación Vascular/patología , Calcificación Vascular/fisiopatología , Calcificación Vascular/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiopatología , Músculo Liso Vascular/patología , Animales , Células Endoteliales/metabolismo , Células Endoteliales/patología , Células CultivadasRESUMEN
Vascular calcification, which is a major complication of diabetes mellitus, is an independent risk factor for cardiovascular disease. Osteogenic differentiation of vascular smooth muscle cells (VSMCs) is one of the key mechanisms underlying vascular calcification. Emerging evidence suggests that macrophage-derived extracellular vesicles (EVs) may be involved in calcification within atherosclerotic plaques in patients with diabetes mellitus. However, the role of macrophage-derived EVs in the progression of vascular calcification is largely unknown. In this study, we investigated whether macrophage-derived EVs contribute to the osteogenic differentiation of VSMCs under high glucose conditions. We isolated EVs that were secreted by murine peritoneal macrophages under normal glucose (EVs-NG) or high glucose (EVs-HG) conditions. miRNA array analysis in EVs from murine macrophages showed that miR-17-5p was significantly increased in EVs-HG compared with EVs-NG. Prediction analysis with miRbase identified transforming growth factor ß receptor type II (TGF-ß RII) as a potential target of miR-17-5p. EVs-HG as well as miR-17-5p overexpression with lipid nanoparticles inhibited the gene expression of Runx2, and TGF-ß RII. Furthermore, we demonstrated that VSMCs transfected with miR-17-5p mimic inhibited calcium deposition. Our findings reveal a novel role of macrophage-derived EVs in the negative regulation of osteogenic differentiation in VSMCs under high glucose conditions.
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Diferenciación Celular , Vesículas Extracelulares , Glucosa , MicroARNs , Músculo Liso Vascular , Miocitos del Músculo Liso , Osteogénesis , Transducción de Señal , Factor de Crecimiento Transformador beta , MicroARNs/genética , MicroARNs/metabolismo , Animales , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/citología , Glucosa/farmacología , Glucosa/metabolismo , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Factor de Crecimiento Transformador beta/metabolismo , Ratones , Miocitos del Músculo Liso/metabolismo , Vesículas Extracelulares/metabolismo , Calcificación Vascular/metabolismo , Calcificación Vascular/genética , Calcificación Vascular/patología , Receptor Tipo II de Factor de Crecimiento Transformador beta/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta/genética , Masculino , Ratones Endogámicos C57BL , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genéticaRESUMEN
To investigate the mechanism of curcumin (CUR) on vascular calcification (VC), we screen for common targets of CUR and atherosclerosis and verify the targets genes in vivo and in vitro experiments. The common targets of CUR and AS were screened and obtained using different databases. These target genes were analyzed by GO and KEGG pathway enrichment analysis. PPI network analysis was performed and to analyze the key targets. A rat VC model was constructed and CUR was fed for three weeks. The changes of vascular structure and calcium salt deposition were observed in H&E and Von Kossa staining. Further, the expression of these target proteins was detected in the primary VSMCs of VC. The 31 common targets were obtained. GO functional enrichment analysis obtained 1284 terms and KEGG pathway enriched 66 pathways. The key genes were identified in the cytoHubba plugin. The molecular docking analysis showed that CUR bound strongly to EGFR, STAT3 and BCL2. The animal experiments showed the deposition calcium salt reduced by the CUR administration. These proteins BMP2, RUNX2, EGFR, STAT3 and BAX expression were upregulated in VC group and CUR attenuated the upregulated expression. The signal protein Akt and p65 expression increased in VC group and decreased in CUR group. We identified some common target genes of CUR and AS and identified these key genes. The anti-VC effect of CUR was associated with the inhibition of upregulation of EGFR, STAT3 and RUNX2 expression in VSMCs.
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Subunidad alfa 1 del Factor de Unión al Sitio Principal , Curcumina , Simulación del Acoplamiento Molecular , Factor de Transcripción STAT3 , Calcificación Vascular , Animales , Curcumina/farmacología , Calcificación Vascular/metabolismo , Calcificación Vascular/tratamiento farmacológico , Calcificación Vascular/patología , Ratas , Factor de Transcripción STAT3/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Receptores ErbB/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Proteína Morfogenética Ósea 2/metabolismo , Proteína Morfogenética Ósea 2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Masculino , Transducción de Señal/efectos de los fármacos , Proteína X Asociada a bcl-2/metabolismo , Proteína X Asociada a bcl-2/genética , Mapas de Interacción de Proteínas/efectos de los fármacos , Ratas Sprague-Dawley , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Modelos Animales de EnfermedadAsunto(s)
Músculo Liso Vascular , Miocitos del Músculo Liso , Transducción de Señal , Calcificación Vascular , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Miocitos del Músculo Liso/efectos de los fármacos , Animales , Calcificación Vascular/metabolismo , Calcificación Vascular/patología , Humanos , Fosfatos/metabolismo , Quinasas Janus/metabolismo , Factores de Transcripción STAT/metabolismo , Inflamación/metabolismo , Inflamación/patologíaRESUMEN
Vascular calcification (VC) is a cardiovascular disease characterized by calcium salt deposition in vascular smooth muscle cells (VSMCs). Standard in vitro models used in VC investigations are based on VSMC monocultures under static conditions. Although these platforms are easy to use, the absence of interactions between different cell types and dynamic conditions makes these models insufficient to study key aspects of vascular pathophysiology. The present study aimed to develop a dynamic endothelial cell-VSMC co-culture that better mimics the in vivo vascular microenvironment. A double-flow bioreactor supported cellular interactions and reproduced the blood flow dynamic. VSMC calcification was stimulated with a DMEM high glucose calcification medium supplemented with 1.9 mM NaH2PO4/Na2HPO4 (1:1) for 7 days. Calcification, cell viability, inflammatory mediators, and molecular markers (SIRT-1, TGFß1) related to VSMC differentiation were evaluated. Our dynamic model was able to reproduce VSMC calcification and inflammation and evidenced differences in the modulation of effectors involved in the VSMC calcified phenotype compared with standard monocultures, highlighting the importance of the microenvironment in controlling cell behavior. Hence, our platform represents an advanced system to investigate the pathophysiologic mechanisms underlying VC, providing information not available with the standard cell monoculture.
Asunto(s)
Diferenciación Celular , Técnicas de Cocultivo , Músculo Liso Vascular , Miocitos del Músculo Liso , Calcificación Vascular , Humanos , Calcificación Vascular/metabolismo , Calcificación Vascular/patología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Células Cultivadas , Supervivencia Celular , Factor de Crecimiento Transformador beta1/metabolismo , Sirtuina 1/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/patología , Reactores BiológicosRESUMEN
The Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 (ENPP1) ectoenzyme regulates vascular intimal proliferation and mineralization of bone and soft tissues. ENPP1 variants cause Generalized Arterial Calcification of Infancy (GACI), a rare genetic disorder characterized by ectopic calcification, intimal proliferation, and stenosis of large- and medium-sized arteries. ENPP1 hydrolyzes extracellular ATP to pyrophosphate (PPi) and AMP. AMP is the precursor of adenosine, which has been implicated in the control of neointimal formation. Herein, we demonstrate that an ENPP1-Fc recombinant therapeutic inhibits proliferation of vascular smooth muscle cells (VSMCs) in vitro and in vivo. Addition of ENPP1 and ATP to cultured VSMCs generated AMP, which was metabolized to adenosine. It also significantly decreased cell proliferation. AMP or adenosine alone inhibited VSMC growth. Inhibition of ecto-5'-nucleotidase CD73 decreased adenosine accumulation and suppressed the anti-proliferative effects of ENPP1/ATP. Addition of AMP increased cAMP synthesis and phosphorylation of VASP at Ser157. This AMP-mediated cAMP increase was abrogated by CD73 inhibitors or by A2aR and A2bR antagonists. Ligation of the carotid artery promoted neointimal hyperplasia in wild-type mice, which was exacerbated in ENPP1-deficient ttw/ttw mice. Prophylactic or therapeutic treatments with ENPP1 significantly reduced intimal hyperplasia not only in ttw/ttw but also in wild-type mice. These findings provide the first insight into the mechanism of the anti-proliferative effect of ENPP1 and broaden its potential therapeutic applications beyond enzyme replacement therapy.
Asunto(s)
5'-Nucleotidasa , Adenosina , Proliferación Celular , Músculo Liso Vascular , Miocitos del Músculo Liso , Hidrolasas Diéster Fosfóricas , Pirofosfatasas , Transducción de Señal , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Pirofosfatasas/metabolismo , Pirofosfatasas/genética , 5'-Nucleotidasa/metabolismo , 5'-Nucleotidasa/genética , Animales , Proliferación Celular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Adenosina/metabolismo , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Miocitos del Músculo Liso/efectos de los fármacos , Ratones , Humanos , Adenosina Monofosfato/metabolismo , Ratones Endogámicos C57BL , AMP Cíclico/metabolismo , Masculino , Calcificación Vascular/metabolismo , Calcificación Vascular/patología , Calcificación Vascular/genéticaRESUMEN
Coronary artery calcification (CAC) is a hallmark event in the pathogenesis of cardiovascular disease, involving the phenotypic transformation of vascular smooth muscle cells (VSMC) towards an osteogenic state. Despite this understanding, the molecular mechanisms governing the VSMC osteogenic switch remain incompletely elucidated. Here, we sought to examine the potential role of circular RNA (circRNA) in the context of CAC. Through transcriptome analysis of circRNA-seq, we identified circTOP1 as a potential candidate circRNA in individuals with CAC. Furthermore, we observed that overexpression of circTOP1 exacerbated vascular calcification in a CAC model. Subsequent pull-down assays revealed an interaction between circTOP1 and PTBP1, a putative target gene of circTOP1 in the context of CAC. In both in vivo and in vitro experiments, we observed heightened expression of circTOP1 and PTBP1 in the CAC model, and noted that reducing circTOP1 expression effectively reduced calcium salt deposits and mineralized nodules in model mice. Additionally, in vitro experiments demonstrated that overexpression of PTBP1 reversed the weakening of signaling caused by silencing circTOP1, thereby exacerbating the osteogenic transition and calcification of VSMC. Collectively, our findings suggested that circTOP1 promotes CAC by modulating PTBP1 expression to mediate VSMC transdifferentiation.
Asunto(s)
Ribonucleoproteínas Nucleares Heterogéneas , Músculo Liso Vascular , Miocitos del Músculo Liso , Proteína de Unión al Tracto de Polipirimidina , ARN Circular , Calcificación Vascular , Animales , Humanos , Masculino , Ratones , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/patología , Enfermedad de la Arteria Coronaria/metabolismo , Vasos Coronarios/patología , Vasos Coronarios/metabolismo , Progresión de la Enfermedad , Regulación de la Expresión Génica/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ratones Endogámicos C57BL , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Osteogénesis/genética , Proteína de Unión al Tracto de Polipirimidina/genética , Proteína de Unión al Tracto de Polipirimidina/metabolismo , ARN Circular/genética , ARN Circular/metabolismo , Calcificación Vascular/genética , Calcificación Vascular/patología , Calcificación Vascular/metabolismoRESUMEN
Vascular calcification is a prevalent hallmark of cardiovascular risk in elderly and diabetic individuals. Senescent vascular smooth muscle cells (VSMCs) participate in calcification; however, the associated underlying mechanisms remain unknown. Aberrant activation of the cytosolic DNA sensing adaptor stimulator of interferon gene 1 (STING1) caused by cytosolic DNA, particularly that leaked from damaged mitochondria, is a catalyst for aging-related diseases. Although oleoylethanolamide (OEA) is an endogenous bioactive lipid mediator with lipid overload-associated vasoprotective effects, its benefit in diabetic vascular calcification remains uncharacterized. This study focused on the role of STING1 in mitochondrial dysfunction-mediated calcification and premature VMSC senescence in diabetes and the effects of OEA on these pathological processes. In diabetic in vivo rat/mouse aorta calcification models and an in vitro VSMC calcification model induced by Nε-carboxymethyl-lysine (CML), senescence levels, STING1 signaling activation, and mitochondrial damage markers were significantly augmented; however, these alterations were markedly alleviated by OEA, partially in a nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent manner, and similar anti-calcification and senescence effects were observed in STING1-knockout mice and STING1-knockdown VSMCs. Mechanistically, mitochondrial DNA (mtDNA) damage was aggravated by CML in a reactive oxygen species-dependent manner, followed by mtDNA leakage into the cytosol, contributing to VSMC senescence-associated calcification via STING1 pathway activation. OEA treatment significantly attenuated the aforementioned cytotoxic effects of CML by enhancing cellular antioxidant capacity through the maintenance of Nrf2 translocation to the nucleus. Collectively, targeting STING1, a newly defined VSMC senescence regulator, contributes to anti-vascular calcification effects.
Asunto(s)
Senescencia Celular , ADN Mitocondrial , Endocannabinoides , Proteínas de la Membrana , Músculo Liso Vascular , Factor 2 Relacionado con NF-E2 , Ácidos Oléicos , Estrés Oxidativo , Calcificación Vascular , Animales , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Músculo Liso Vascular/efectos de los fármacos , Ratones , Senescencia Celular/efectos de los fármacos , Ratas , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Calcificación Vascular/patología , Calcificación Vascular/metabolismo , Calcificación Vascular/tratamiento farmacológico , Calcificación Vascular/genética , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/genética , Ácidos Oléicos/farmacología , Estrés Oxidativo/efectos de los fármacos , Masculino , Endocannabinoides/metabolismo , Endocannabinoides/farmacología , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Miocitos del Músculo Liso/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Transducción de Señal/efectos de los fármacos , Humanos , Ratones Noqueados , Ratones Endogámicos C57BLRESUMEN
Zinc (Zn) is a normal trace element in mineralizing tissues, but it is unclear whether it is primarily bound to the mineral phase or to organic molecules involved in the mineralization process, or both. Tissue-nonspecific alkaline phosphatase (TNAP) is a Zn metalloenzyme with two Zn ions bound to the M1 and M2 catalytic sites that functions to control the phosphate/pyrophosphate ratio during biomineralization. Here, we studied aortas from Tagln-Cre +/-; HprtALP/Y TNAP overexpressor (TNAP-OE) mice that develop severe calcification. Zn histochemistry was performed using the sulfide-silver staining method in combination with a Zn partial extraction procedure to localize mineral-bound (mineral Zn) and TNAP-bound Zn (tenacious Zn), since soluble Zn (loose Zn) is extracted during fixation of the specimens. Two synthetic bone mineral composites with different Zn content, bone ash, and rat epiphyseal growth plate cartilage were used as controls for Zn staining. In order to correlate the distribution of mineral and tenacious Zn with the presence of mineral deposits, the aortas were examined histologically in unstained and stained thin sections using various light microscopy techniques. Our results show that 14 and 30 dpn, TNAP is concentrated in the calcifying matrix and loses Zn as Ca2+ progressively displaces Zn2+ at the M1 and M2 metal sites. Thus, in addition to its catalytic role TNAP has an additional function at calcifying sites as a Ca-binding protein.
Asunto(s)
Fosfatasa Alcalina , Aorta , Zinc , Animales , Fosfatasa Alcalina/metabolismo , Zinc/metabolismo , Ratones , Aorta/patología , Aorta/metabolismo , Calcificación Vascular/metabolismo , Calcificación Vascular/patología , Calcinosis/metabolismo , Calcinosis/patología , Ratas , Calcio/metabolismoRESUMEN
While it is known that calcium phosphate (CaP) minerals deposit in elastin-rich medial layers of arteries during medial calcification, their nucleation and growth sites are still debated. Neutral carbonyl groups and carboxylate groups are possible candidates. Also, while it is known that elastin degradation leads to calcification, it is unclear whether this is due to formation of new carboxylate groups or elastin fragmentation. In this work, we disentangle effects of carboxylate groups and particle size on elastin calcification; in doing so, we shed light on CaP mineralization sites on elastin. We find carboxylate groups accelerate calcification only in early stages; they mainly function as Ca2+ ion chelation sites but not calcification sites. Their presence promotes formation (likely on Ca2+ ions adsorbed on nearby carbonyl groups) of CaP minerals with high calcium-to-phosphate ratio as intermediate phases. Larger elastin particles calcify slower but reach similar amounts of CaP minerals in late stages; they promote direct formation of hydroxyapatite and CaP minerals with low calcium-to-phosphate ratio as intermediate phases. This work provides new perspectives on how carboxylate groups and elastin particle size influence calcification; these parameters can be tuned to study the mechanism of medial calcification and design drugs to inhibit the process.