RESUMEN
Atherosclerosis is associated with a haemostatic imbalance characterized by excessive activation of pro-inflammatory and pro-coagulant pathways. Non-vitamin K antagonists oral anticoagulant (NOACs) may reduce the incidence of cardiovascular events, cerebral ischemia, thromboembolic events and atherosclerosis. Chronic inflammation, vascular proliferation and the development of atherosclerosis is also influenced by 25-hydroxycholesterol (25-OHC). The aim of the study was to assess the effect of rivaroxaban and dabigatran on the messenger RNA (mRNA) expression of anti-inflammatory cytokines transforming growth factor ß (TGF-ß), interleukin (IL)-37, IL-35 as well as of pro-inflammatory cytokines IL-18 and IL-23, in endothelial cells damaged by 25-OHC. Human umbilical vascular endothelial cells (HUVECs) were treated with 25-OHC (10 µg/mL), rivaroxaban (100, 500 ng/mL), dabigatran (100, 500 ng/mL), 25-OHC + rivaroxaban, and 25-OHC + dabigatran. The mRNA expression of TGF-ß, IL-37, IL-35 subunits EBI3 and p35, IL-18, and IL-23 was analysed using real-time polymerase chain reaction (PCR). The results showed that 25-OHC decreased TGF-ß and IL-37 mRNA expression and increased EBI3, p35, IL-18, IL-23 mRNA expression in endothelial cell as compared to an untreated control (P < .05). Messenger RNA expression of TGF-ß and IL-37 significantly increased following stimulation with rivaroxaban and dabigatran as compared to an untreated control (P < .01). In HUVECs pre-treated with oxysterol, rivaroxaban and dabigatran increased mRNA expression of TGF-ß, IL-37 and decreased mRNA expression of EBI3, p35, IL-23 and IL-18 as compared to 25-OHC (P < .01). Our finding suggests that both rivaroxaban and dabigatran inhibit the inflammatory activation caused by oxysterol in vitro.
Asunto(s)
Aterosclerosis , Citocinas , Dabigatrán , Células Endoteliales de la Vena Umbilical Humana , Hidroxicolesteroles , Rivaroxabán , Administración Oral , Anticoagulantes/farmacología , Anticoagulantes/uso terapéutico , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/genética , Aterosclerosis/inmunología , Fibrilación Atrial/tratamiento farmacológico , Citocinas/genética , Citocinas/inmunología , Dabigatrán/farmacología , Dabigatrán/uso terapéutico , Células Endoteliales/efectos de los fármacos , Células Endoteliales/inmunología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/inmunología , Humanos , Hidroxicolesteroles/administración & dosificación , Hidroxicolesteroles/efectos adversos , Hidroxicolesteroles/farmacología , Interleucina-18/genética , Interleucina-18/inmunología , Interleucina-23/genética , Interleucina-23/inmunología , Oxiesteroles/administración & dosificación , Oxiesteroles/efectos adversos , Oxiesteroles/farmacología , ARN Mensajero/genética , ARN Mensajero/inmunología , Rivaroxabán/farmacología , Rivaroxabán/uso terapéutico , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/inmunologíaRESUMEN
The development of effective antiviral drugs targeting the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is urgently needed to combat the coronavirus disease 2019 (COVID-19). We have previously studied the use of semi-synthetic derivatives of oxysterols, oxidized derivatives of cholesterol as drug candidates for the inhibition of cancer, fibrosis, and bone regeneration. In this study, we screened a panel of naturally occurring and semi-synthetic oxysterols for anti-SARS-CoV-2 activity using a cell culture infection assay. We show that the natural oxysterols, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 27-hydroxycholesterol, substantially inhibited SARS-CoV-2 propagation in cultured cells. Among semi-synthetic oxysterols, Oxy210 and Oxy232 displayed more robust anti-SARS-CoV-2 activities, reducing viral replication more than 90% at 10 µM and 99% at 15 µM, respectively. When orally administered in mice, peak plasma concentrations of Oxy210 fell into a therapeutically relevant range (19 µM), based on the dose-dependent curve for antiviral activity in our cell-based assay. Mechanistic studies suggest that Oxy210 reduced replication of SARS-CoV-2 by disrupting the formation of double-membrane vesicles (DMVs); intracellular membrane compartments associated with viral replication. Our study warrants further evaluation of Oxy210 and Oxy232 as a safe and reliable oral medication, which could help protect vulnerable populations with increased risk of developing COVID-19.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Oxiesteroles/química , Oxiesteroles/farmacología , SARS-CoV-2/efectos de los fármacos , Administración Oral , Animales , Antivirales/administración & dosificación , Antivirales/farmacocinética , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Ratones , Proteínas de la Nucleocápside/efectos de los fármacos , Oxiesteroles/administración & dosificación , Oxiesteroles/farmacocinética , SARS-CoV-2/genética , Células Vero , Compartimentos de Replicación Viral/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Tratamiento Farmacológico de COVID-19RESUMEN
Bone repair requires the tightly regulated control of multiple intrinsic and extrinsic cell types and signaling pathways. One of the positive regulatory signaling pathways in membranous and endochondral bone healing is the Hedgehog (Hh) signaling family. Here, a novel therapeutic liposomal delivery vector was developed by self-assembly of an Hh-activating cholesterol analog with an emulsifier, along with the addition of Smoothened agonist (SAG) as a drug cargo, for the enhancement of Hh signaling in bone regeneration. The drug-loaded nanoparticulate agonists of Hh signaling were immobilized onto trabecular bone-mimetic apatite-coated 3D scaffolds using bioinspired polydopamine adhesives to ensure favorable microenvironments for cell growth and local therapeutic delivery. Results showed that SAG-loaded liposomes induced a significant and dose-dependent increase in Hh-mediated osteogenic differentiation, as evidenced by in vitro analysis of bone marrow stromal cells, and in vivo calvarial bone healing, as evidenced using all radiographic parameters and histomorphometric analyses. Moreover, favorable outcomes were achieved in comparison to standards of care, including collagen sponge-delivered rBMP2 or allograft bone. In summary, this study demonstrates using a nanoparticle packaged Hh small molecule as a widely applicable bone graft substitute for robust bone repair.
Asunto(s)
Regeneración Ósea/efectos de los fármacos , Ciclohexilaminas/farmacología , Proteínas Hedgehog/metabolismo , Oxiesteroles/administración & dosificación , Tiofenos/farmacología , Andamios del Tejido/química , Animales , Apatitas/química , Trasplante Óseo , Diferenciación Celular/efectos de los fármacos , Ciclohexilaminas/química , Modelos Animales de Enfermedad , Femenino , Humanos , Liposomas , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/fisiología , Ratones , Osteogénesis/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Cráneo/diagnóstico por imagen , Cráneo/lesiones , Cráneo/cirugía , Tiofenos/química , Microtomografía por Rayos XRESUMEN
Non-alcoholic fatty liver disease is strongly associated with obese and type 2 diabetes. It has been reported that an oxidized cholesterol, 7-ketocholesterol (7KC), might cause inflammatory response in macrophages and plasma 7KC concentration were higher in patients with cardiovascular diseases or diabetes. Therefore, we have decided to test whether small amount of 7KC in diet might induce hepatic steatosis and inflammation in two types of obese models. We found that addition of 0.01% 7KC either in chow diet (CD, regular chow diet with 1% cholesterol) or western type diet (WD, high fat diet with 1% cholesterol) accelerated hepatic neutral lipid accumulation by Oil Red O staining. Importantly, by lipid extraction analysis, it has been recognized that triglyceride rather than cholesterol species was significantly accumulated in CD+7KC compared to CD as well as in WD+7KC compared to WD. Immunostaining revealed that macrophages infiltration was increased in CD+7KC compared to CD, and also in WD+7KC compared to WD. These phenotypes were accompanied by inducing inflammatory response and downregulating fatty acid oxidation. Furthermore, RNA sequence analysis demonstrated that 7KC reduced expression of genes which related to autophagy process. Levels of LC3-II protein were decreased in WD+7KC compared to WD. Similarly, we have confirmed the effect of 7KC on acceleration of steatohepatitis in db/db mice model. Collectively, our study has demonstrated that small amount of dietary 7KC contributed to accelerate hepatic steatosis and inflammation in obese mice models.
Asunto(s)
Colesterol en la Dieta/administración & dosificación , Cetocolesteroles/administración & dosificación , Hígado/metabolismo , Macrófagos/metabolismo , Obesidad/metabolismo , Oxiesteroles/administración & dosificación , Animales , Peso Corporal/efectos de los fármacos , Peso Corporal/fisiología , Colesterol en la Dieta/efectos adversos , Mediadores de Inflamación/metabolismo , Cetocolesteroles/efectos adversos , Hígado/efectos de los fármacos , Hígado/patología , Macrófagos/efectos de los fármacos , Macrófagos/patología , Masculino , Ratones , Ratones Obesos , Obesidad/patología , Oxiesteroles/efectos adversosRESUMEN
PURPOSE: The aim of our study was to determine the effect of Oxy133 and rhBMP2 on fusion rates and new bone formation in a rat posterolateral fusion (PLF) model. Furthermore, we examined whether Oxy133 could inhibit the adipogenesis that is often present in rhBMP2-induced fusions. METHODS: Sixty-four male Lewis rats underwent two levels PLF (L3-L5). All animals were randomly divided into eight groups based on the test compound that they received: control (DMSO), low-dose rhBMP2 (0.5 µg), high-dose rhBMP2 (5 µg), low-dose Oxy133 (5 mg), high-dose Oxy133 (20 mg), low rhBMP2 + high Oxy133, high rhBMP2 + high Oxy133, and low rhBMP2 + low Oxy133. Fusion rates were assessed 8 weeks after surgery with manual palpation and plain radiographs. Bone parameters were measured using microCT. Histology was used to evaluate adipogenesis. RESULTS: No fusion was observed in the control group. Based on the manual palpation, 100% fusion was observed in all other groups except in the low-dose rhBMP2 group (69%). At 8 weeks based on X-rays, 100% fusion was observed in the following groups: high-dose rhBMP2, low-dose Oxy133, and low rhBMP2 + low Oxy133. In the other groups, the fusion rates were between 95 and 97%, except for the low rhBMP2 group (72%). We observed similar values in BV/TV ratio at L3-4 when Oxy133 groups were compared to rhBMP2 groups alone (44.62% in high-dose Oxy133 vs. 41.47% in high-dose rhBMP2 and 47.18% in low-dose Oxy133 vs. 54.98% in low-dose rhBMP2). Trabecular thickness was slightly lower in Oxy133 groups compared to rhBMP2 when comparing low- and high-dose groups from each group (118.44 µm for high-dose Oxy133 vs. 122.39 µm for high-dose rhBMP2 and 123.51 µm for low-dose Oxy133 vs. 135.74 µm for low-dose rhBMP2). At the same time, trabecular separation was lower in Oxy133 groups compared to rhBMP2 groups. Similar trends in bone parameters were observed at the L4-5 levels. Fusion masses with low- and high-dose Oxy133 had significantly less adipocytes than rhBMP2 groups that showed robust adipocyte formation. CONCLUSION: In our study, both low-dose and high-dose Oxy133 produced solid fusions with bone densities similar or higher than in the BMP2 groups. High-dose Oxy133 group had significantly less adipocytes than high- or low-dose rhBMP2 groups. Furthermore, high-dose Oxy133 was able to significantly inhibit high-dose BMP2-induced adipogenesis when combined together. Consistent with the previous reports, our preliminary findings suggest that Oxy133 has a significant potential as an alternative to rhBMP2 in spine fusion.