RESUMO
BACKGROUND: Thoracic aortic dissection (TAD) is an irreversible cardiovascular disorder with high mortality and morbidity. However, the molecular mechanisms remain elusive. Thus, identifying an effective therapeutic target to prevent TAD is especially critical. The purpose of this study is to elucidate the potential mechanism of inflammation and vascular smooth muscle cell (VSMCs) phenotypic switch in ß-aminopropionitrile fumarate (BAPN)-induced TAD. METHODS: A mouse model of TAD induced by BAPN and IL-1ß -stimulated HVSMCs in vivo and in vitro models, respectively. ACE2 Knockdown mice treated with BAPN or without, and the TAD mouse model was treated with or without AAV-ACE2. Transthoracic ultrasound was conducted for assessment the maximum internal diameter of the thoracic aorta arch. RNA sequencing analysis was performed to recapitulate transcriptome profile changes. Western blot were used to detect the expression of MMP2, MMP9, ACE2, SIRT3, OPN, SM22α and other inflammatory markers. The circulating levels of ACE2 was measured by ELISA assay. Histological changes of thoracic aorta tissues were assessed by H&E, EVG and IHC analysis. RESULTS: We found that circulating levels of and the protein levels of ACE2 were increased in the TAD mouse model and in patients with TAD. For further evidence, ACE2 deficiency decelerated the formation of TAD. However, overexpression of ACE2 aggravated BAPN-induced aortic injury and VSMCs phenotypic switch via lowered SIRT3 expression and elevated inflammatory cytokine expression. CONCLUSION: ACE2 deficiency prevented the development of TAD by inhibiting inflammation and VSMCs phenotypic switch in a SIRT3-dependent manner, suggesting that the ACE2/SIRT3 signaling pathway played a pivotal role in the pathological process of TAD and might be a potential therapeutical target.
Assuntos
Enzima de Conversão de Angiotensina 2 , Aneurisma da Aorta Torácica , Dissecção Aórtica , Modelos Animais de Doenças , Inflamação , Músculo Liso Vascular , Miócitos de Músculo Liso , Sirtuína 3 , Animais , Enzima de Conversão de Angiotensina 2/metabolismo , Enzima de Conversão de Angiotensina 2/genética , Camundongos , Dissecção Aórtica/metabolismo , Dissecção Aórtica/etiologia , Dissecção Aórtica/genética , Dissecção Aórtica/patologia , Miócitos de Músculo Liso/metabolismo , Sirtuína 3/metabolismo , Sirtuína 3/genética , Sirtuína 3/deficiência , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Inflamação/metabolismo , Aneurisma da Aorta Torácica/metabolismo , Aneurisma da Aorta Torácica/etiologia , Aneurisma da Aorta Torácica/genética , Masculino , Fenótipo , Humanos , Camundongos Knockout , Aorta Torácica/metabolismo , Aorta Torácica/patologia , Aorta Torácica/efeitos dos fármacos , Aminopropionitrilo/farmacologia , Camundongos Endogâmicos C57BL , Dissecção da Aorta TorácicaRESUMO
The intima, comprising the endothelium and the subendothelial matrix, plays a crucial role in atherosclerosis pathogenesis. The mechanical stress arising from disturbed blood flow (d-flow) and the stiffening of the arterial wall contributes to endothelial dysfunction. However, the specific impacts of these physical forces on the mechanical environment of the intima remain undetermined. Here, we investigated whether inhibiting collagen crosslinking could ameliorate the detrimental effects of persistent d-flow on the mechanical properties of the intima. Partial ligation of the left carotid artery (LCA) was performed in C57BL/6J mice, inducing d-flow. The right carotid artery (RCA) served as an internal control. Carotids were collected 2 days and 2 weeks after surgery to study acute and chronic effects of d-flow on the mechanical phenotype of the intima. The chronic effects of d-flow were decoupled from the ensuing arterial wall stiffening by administration of ß-aminopropionitrile (BAPN), an inhibitor of collagen crosslinking by lysyl oxidase (LOX) enzymes. Atomic force microscopy (AFM) was used to determine stiffness of the endothelium and the denuded subendothelial matrix in en face carotid preparations. The stiffness of human aortic endothelial cells (HAEC) cultured on soft and stiff hydrogels was also determined. Acute exposure to d-flow caused a slight decrease in endothelial stiffness in male mice but had no effect on the stiffness of the subendothelial matrix in either sex. Regardless of sex, the intact endothelium was softer than the subendothelial matrix. In contrast, exposure to chronic d-flow led to a substantial increase in the endothelial and subendothelial stiffness in both sexes. The effects of chronic d-flow were largely prevented by concurrent BAPN administration. In addition, HAEC displayed reduced stiffness when cultured on soft vs. stiff hydrogels. We conclude that chronic d-flow results in marked stiffening of the arterial intima, which can be effectively prevented by inhibition of collagen crosslinking.
Assuntos
Artérias Carótidas , Camundongos Endogâmicos C57BL , Rigidez Vascular , Animais , Masculino , Rigidez Vascular/efeitos dos fármacos , Camundongos , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/patologia , Artérias Carótidas/fisiopatologia , Feminino , Túnica Íntima/patologia , Túnica Íntima/efeitos dos fármacos , Colágeno/metabolismo , Aminopropionitrilo/farmacologia , Proteína-Lisina 6-Oxidase/metabolismo , Proteína-Lisina 6-Oxidase/antagonistas & inibidores , Microscopia de Força Atômica , Humanos , Estresse Mecânico , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Endotélio Vascular/metabolismoRESUMO
There is no treatment for acute aortic dissection (AAD) targeting inflammatory cells. We aimed to identify the new therapeutic targets associated with inflammatory cells. We characterized the specific distribution of myeloid cells of both human type A AAD samples and a murine AAD model generated using angiotensin II (ANGII) and ß-aminopropionitrile (BAPN) by single-cell RNA sequencing (scRNA-seq). We also examined the effect of an anti-interleukin-1ß (IL-1ß) antibody in the murine AAD model. IL1B+ inflammatory macrophages and classical monocytes were increased in human AAD samples. Trajectory analysis demonstrated that IL1B+ inflammatory macrophages differentiated from S100A8/9/12+ classical monocytes uniquely observed in the aorta of AAD. We found increased infiltration of neutrophils and monocytes with the expression of inflammatory cytokines in the aorta and accumulation of inflammatory macrophages before the onset of macroscopic AAD in the murine AAD model. In blocking experiments using an anti-IL-1ß antibody, it improved survival of murine AAD model by preventing elastin degradation. We observed the accumulation of inflammatory macrophages expressing IL-1ß in both human AAD samples and in a murine AAD model. Anti-IL-1ß antibody could improve the mortality rate in mice, suggesting that it may be a treatment option for AAD.
Assuntos
Dissecção Aórtica , Modelos Animais de Doenças , Interleucina-1beta , Macrófagos , Dissecção Aórtica/metabolismo , Dissecção Aórtica/patologia , Interleucina-1beta/metabolismo , Animais , Humanos , Macrófagos/metabolismo , Macrófagos/imunologia , Camundongos , Masculino , Aminopropionitrilo/farmacologia , Angiotensina II/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Monócitos/metabolismo , Aorta/metabolismo , Aorta/patologia , Camundongos Endogâmicos C57BL , FemininoRESUMO
BACKGROUND: ß-aminopropionitrile (BAPN) is a pharmacological inhibitor of LOX (lysyl oxidase) and LOXLs (LOX-like proteins). Administration of BAPN promotes aortopathies, although there is a paucity of data on experimental conditions to generate pathology. The objective of this study was to define experimental parameters and determine whether equivalent or variable aortopathies were generated throughout the aortic tree during BAPN administration in mice. METHODS: BAPN was administered in drinking water for a period ranging from 1 to 12 weeks. The impacts of BAPN were first assessed with regard to BAPN dose, and mouse strain, age, and sex. BAPN-induced aortic pathological characterization was conducted using histology and immunostaining. To investigate the mechanistic basis of regional heterogeneity, the ascending and descending thoracic aortas were harvested after 1 week of BAPN administration before the appearance of overt pathology. RESULTS: BAPN-induced aortic rupture predominantly occurred or originated in the descending thoracic aorta in young C57BL/6J or N mice. No apparent differences were found between male and female mice. For mice surviving 12 weeks of BAPN administration, profound dilatation was consistently observed in the ascending region, while there were more heterogeneous changes in the descending thoracic region. Pathological features were distinct between the ascending and descending thoracic regions. Aortic pathology in the ascending region was characterized by luminal dilatation and elastic fiber disruption throughout the media. The descending thoracic region frequently had dissections with false lumen formation, collagen deposition, and remodeling of the wall surrounding the false lumen. Cells surrounding the false lumen were predominantly positive for α-SMA (α-smooth muscle actin). One week of BAPN administration compromised contractile properties in both regions equivalently, and RNA sequencing did not show obvious differences between the 2 aortic regions in smooth muscle cell markers, cell proliferation markers, and extracellular components. CONCLUSIONS: BAPN-induced pathologies show distinct, heterogeneous features within and between ascending and descending aortic regions in mice.
Assuntos
Aminopropionitrilo , Aorta Torácica , Ruptura Aórtica , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Animais , Aminopropionitrilo/toxicidade , Aminopropionitrilo/farmacologia , Aorta Torácica/patologia , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Feminino , Masculino , Ruptura Aórtica/induzido quimicamente , Ruptura Aórtica/patologia , Ruptura Aórtica/metabolismo , Ruptura Aórtica/prevenção & controle , Camundongos , Remodelação Vascular/efeitos dos fármacos , Dilatação Patológica , Músculo Liso Vascular/patologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Fatores Etários , Fatores de Tempo , Fatores Sexuais , Proliferação de Células/efeitos dos fármacos , Proteína-Lisina 6-Oxidase/metabolismoRESUMO
Inflammation assumes a pivotal role in the aortic remodeling of aortic dissection (AD). Asiatic acid (AA), a triterpene compound, is recognized for its strong anti-inflammatory properties. Yet, its effects on ß-aminopropionitrile (BAPN)-triggered AD have not been clearly established. The objective is to determine whether AA attenuates adverse aortic remodeling in BAPN-induced AD and clarify potential molecular mechanisms. In vitro studies, RAW264.7 cells pretreated with AA were challenged with lipopolysaccharide (LPS), and then the vascular smooth muscle cells (VSMCs)-macrophage coculture system was established to explore intercellular interactions. To induce AD, male C57BL/6J mice at three weeks of age were administered BAPN at a dosage of 1 g/kg/d for four weeks. To decipher the mechanism underlying the effects of AA, RNA sequencing analysis was conducted, with subsequent validation of these pathways through cellular experiments. AA exhibited significant suppression of M1 macrophage polarization. In the cell coculture system, AA facilitated the transformation of VSMCs into a contractile phenotype. In the mouse model of AD, AA strikingly prevented the BAPN-induced increases in inflammation cell infiltration and extracellular matrix degradation. Mechanistically, RNA sequencing analysis revealed a substantial upregulation of CX3CL1 expression in BAPN group but downregulation in AA-treated group. Additionally, it was observed that the upregulation of CX3CL1 negated the beneficial impact of AA on the polarization of macrophages and the phenotypic transformation of VSMCs. Crucially, our findings revealed that AA is capable of downregulating CX3CL1 expression, accomplishing this by obstructing the nuclear translocation of NF-κB p65. The findings indicate that AA holds promise as a prospective treatment for adverse aortic remodeling by suppressing the activity of NF-κB p65/CX3CL1 signaling pathway.
Assuntos
Dissecção Aórtica , Quimiocina CX3CL1 , Camundongos Endogâmicos C57BL , Triterpenos Pentacíclicos , Transdução de Sinais , Fator de Transcrição RelA , Remodelação Vascular , Animais , Camundongos , Masculino , Dissecção Aórtica/metabolismo , Dissecção Aórtica/patologia , Dissecção Aórtica/tratamento farmacológico , Triterpenos Pentacíclicos/farmacologia , Remodelação Vascular/efeitos dos fármacos , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição RelA/metabolismo , Quimiocina CX3CL1/metabolismo , Quimiocina CX3CL1/genética , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Aminopropionitrilo/farmacologia , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacosRESUMO
Two major constituents of exfoliation material, fibrillin-1 and lysyl oxidase-like 1 (encoded by FBN1 and LOXL1), are implicated in exfoliation glaucoma, yet their individual contributions to ocular phenotype are minor. To test the hypothesis that a combination of FBN1 mutation and LOXL1 deficiency exacerbates ocular phenotypes, the pan-lysyl oxidase inhibitor ß-aminopropionitrile (BAPN) was used to treat adult wild-type (WT) mice and mice heterozygous for a missense mutation in Fbn1 (Fbn1C1041G/+) for 8 weeks and their eyes were examined. Although intraocular pressure did not change and exfoliation material was not detected in the eyes, BAPN treatment worsened optic nerve and axon expansion in Fbn1C1041G/+ mice, an early sign of axonal damage in rodent models of glaucoma. Disruption of elastic fibers was detected only in Fbn1C1041G/+ mice, which increased with BAPN treatment, as shown by histologic and immunohistochemical staining of the optic nerve pia mater. Transmission electron microscopy showed that Fbn1C1041G/+ mice had fewer microfibrils, smaller elastin cores, and a lower density of elastic fibers compared with WT mice in control groups. BAPN treatment led to elastin core expansion in both WT and Fbn1C1041G/+ mice, but an increase in the density of elastic fiber was confined to Fbn1C1041G/+ mice. LOX inhibition had a stronger effect on optic nerve and elastic fiber parameters in the context of Fbn1 mutation, indicating the Marfan mouse model with LOX inhibition warrants further investigation for exfoliation glaucoma pathogenesis.
Assuntos
Aminopropionitrilo , Modelos Animais de Doenças , Fibrilina-1 , Síndrome de Marfan , Nervo Óptico , Proteína-Lisina 6-Oxidase , Animais , Camundongos , Adipocinas , Aminoácido Oxirredutases/metabolismo , Aminoácido Oxirredutases/antagonistas & inibidores , Aminoácido Oxirredutases/genética , Aminopropionitrilo/farmacologia , Tecido Elástico/patologia , Tecido Elástico/metabolismo , Tecido Elástico/ultraestrutura , Fibrilina-1/genética , Fibrilinas/metabolismo , Glaucoma/patologia , Pressão Intraocular , Síndrome de Marfan/patologia , Síndrome de Marfan/complicações , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Nervo Óptico/patologia , Nervo Óptico/ultraestrutura , Nervo Óptico/efeitos dos fármacos , Proteína-Lisina 6-Oxidase/metabolismo , Proteína-Lisina 6-Oxidase/antagonistas & inibidoresRESUMO
BACKGROUND: Thoracic aortic dissection (TAD) is one of the most fatal cardiovascular diseases. One of its important pathological characteristics is the local inflammatory response. Many studies have found that Macrophage polarization plays an extremely critical role in the inflammatory progression and tissue remodeling of TAD. Costunolide (CTD) has an improving effect on oxidative stress and inflammation in the body. However, whether it can promote the integrity of extracellular matrix in Aortic dissection and its mechanism are still unclear. METHODS: The male C57BL/6J mice were used to construct an animal model of TAD with ß-aminopropionitrile (BAPN) (100 mg/kg/day, lasting for 28 days), and then CTD (10 mg/kg or 100 mg/kg) was injected intraperitoneally for 28 days to check the survival rate, TAD incidence, aortic morphology and other indicators of the mice. Using hematoxylin-eosin (HE), Masson, Elastin van Gieson (EVG) staining, immunofluorescence (IF), and immunohistochemical staining, the study aimed to determine the therapeutic effects of CTD on an animal model with BAPN-induced TAD. To enhance the examination of the regulatory mechanism of CTD, we conducted transcriptome sequencing on arterial tissues of mice in both the BAPN group and the BAPN + CTD100 group. Next, ANG II were used to construct TAD model in vascular smooth muscle cells (VMSCs). The effects of CTD on the proliferation, migration, invasion, and apoptosis of ANG II-induced cells are to be detected. The expression of MMP2, MMP9, P65, and p-P65 in each group will be examined using Western blot. Finally, the overexpression of IκB kinaseß (IKKß) will be established in VMSCs cells to further explore the protective function of CTD. RESULTS: The result showed that CTD significantly inhibited BAPN induced mortality and TAD incidence in the animal model, improved aortic vascular morphology, promoted the integrity of extracellular matrix in TAD, reduced tissue inflammation, reduced the accumulation of M1 macrophage, promoted M2 macrophage polarization, and reduced the expression of NF-κB pathway related proteins. Mechanistically, CTD significantly weakened the proliferation, migration, invasion, and apoptosis. p-P65 protein expression of TAD cells were induced by ANG II and IKK-ß. CONCLUSION: CTD has the potential to alleviate inflammation, VSMC apoptosis, MMP2/9 levels, and enhance extracellular matrix integrity in TAD by inhibiting the NF-κB signaling pathway.
Assuntos
Dissecção Aórtica , Dissecção da Aorta Torácica , Sesquiterpenos , Masculino , Camundongos , Animais , NF-kappa B/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Aminopropionitrilo/uso terapêutico , Aminopropionitrilo/farmacologia , Camundongos Endogâmicos C57BL , Dissecção Aórtica/tratamento farmacológico , Transdução de Sinais , Inflamação/tratamento farmacológico , Modelos Animais de DoençasRESUMO
Epilepsy-associated cognitive disorder (ECD), a prevalent comorbidity in epilepsy patients, has so far uncharacterized etiological origins. Our prior work revealed that lysyl oxidase (Lox) acted as a novel contributor of ferroptosis, a recently discovered cell death mode in the regulation of brain function. However, the role of Lox-mediated ferroptosis in ECD remains unknown. ECD mouse model was established 2 months later following a single injection of kainic acid (KA) for. After chronic treatment with KA, mice were treated with different doses (30â¯mg/kg, 100â¯mg/kg and 300â¯mg/kg) of Lox inhibitor BAPN. Additionally, hippocampal-specific Lox knockout mice was also constructed and employed to validate the role of Lox in ECD. Cognitive functions were assessed using novel object recognition test (NOR) and Morris water maze test (MWM). Protein expression of phosphorylated cAMP-response element binding (CREB), a well-known molecular marker for evaluation of cognitive performance, was also detected by Western blot. The protein distribution of Lox was analyzed by immunofluorescence. In KA-induced ECD mouse model, ferroptosis process was activated according to upregulation of 4-HNE protein and a previously discovered ferroptosis in our group, namely, Lox was remarkably increased. Pharmacological inhibition of Lox by BAPN at the dose of 100â¯mg/kg significantly increased the discrimination index following NOR test and decreased escape latency as well as augmented passing times within 60â¯s following MWM test in ECD mouse model. Additionally, deficiency of Lox in hippocampus also led to pronounced improvement of deficits in ECD model. These findings indicate that the ferroptosis regulatory factor, Lox, is activated in ECD. Ablation of Lox by either pharmacological intervention or genetic manipulation ameliorates the impairment in ECD mouse model, which suggest that Lox serves as a promising therapeutic target for treating ECD in clinic.
Assuntos
Disfunção Cognitiva , Epilepsia , Humanos , Camundongos , Animais , Proteína-Lisina 6-Oxidase/genética , Proteína-Lisina 6-Oxidase/metabolismo , Aminopropionitrilo/farmacologia , Regulação da Expressão Gênica , Modelos Animais de Doenças , Disfunção Cognitiva/tratamento farmacológicoRESUMO
Acute aortic dissection (AAD) progresses rapidly and is associated with high mortality; therefore, there remains an urgent need for pharmacological agents that can protect against AAD. Herein, we examined the therapeutic effects of cannabidiol (CBD) in AAD by establishing a suitable mouse model. In addition, we performed human AAD single-cell RNA sequencing and mouse AAD bulk RNA sequencing to elucidate the potential underlying mechanism of CBD. Pathological assays and in vitro studies were performed to verify the results of the bioinformatic analysis and explore the pharmacological function of CBD. In a ß-aminopropionitrile (BAPN)-induced AAD mouse model, CBD reduced AAD-associated morbidity and mortality, alleviated abnormal enlargement of the ascending aorta and aortic arch, and suppressed macrophage infiltration and vascular smooth muscle cell (VSMC) apoptosis. Bioinformatic analysis revealed that the pro-apoptotic gene PMAIP1 was highly expressed in human and mouse AAD samples, and CBD could inhibit Pmaip1 expression in AAD mice. Using human aortic VSMCs (HAVSMCs) co-cultured with M1 macrophages, we revealed that CBD alleviated HAVSMCs mitochondrial-dependent apoptosis by suppressing the BAPN-induced overexpression of PMAIP1 in M1 macrophages. PMAIP1 potentially mediates HAVSMCs apoptosis by regulating Bax and Bcl2 expression. Accordingly, CBD reduced AAD-associated morbidity and mortality and mitigated the progression of AAD in a mouse model. The CBD-induced effects were potentially mediated by suppressing macrophage infiltration and PMAIP1 (primarily expressed in macrophages)-induced VSMC apoptosis. Our findings offer novel insights into M1 macrophages and HAVSMCs interaction during AAD progression, highlighting the potential of CBD as a therapeutic candidate for AAD treatment.
Assuntos
Dissecção Aórtica , Canabidiol , Animais , Humanos , Camundongos , Aminopropionitrilo/farmacologia , Dissecção Aórtica/tratamento farmacológico , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Canabidiol/farmacologia , Canabidiol/metabolismo , Macrófagos/metabolismo , Músculo Liso Vascular/patologiaRESUMO
OBJECTIVE: Selenium (Se) is a key part of the body's oxidation defence system. However, it is unclear whether Se affects the development of aortic aneurysm (AA). An animal experiment was conducted to clarify the role of Se in AA development. METHODS: C57BL/6N male mice were fed with a Se deficient (Se-D, < 0.05 mg/kg), Se adequate (Se-A, 0.2 mg/kg), or Se supplemented (Se-S, 1 mg/kg) diet for 8 weeks. Subsequently, an AA murine model (Se-D, n = 11; Se-A, n = 12; Se-S, n = 15) was established using angiotensin II (Ang II, 1 mg/kg/min) for four weeks plus ß-aminopropionitrile (BAPN, 1 mg/mL) for the first two weeks. Saline replaced Ang II, and BAPN was removed during the modelling process for sham mice (Se-A, n = 9). To determine whether Se deficiency promoted aortic dilation via matrix metalloproteinase-2 (MMP-2), the non-specific MMP inhibitor doxycycline (Dox, 100 mg/kg/day) was given to Se-D AA mice (n = 7) for two weeks. RESULTS: The maximum aortic diameter in Se-D AA model mice was significantly increased compared with Se-A AA model mice. MMP-2 expression and activity in the aortic media of Se-D AA model mice was significantly increased compared with Se-A AA model mice. A large number of vascular smooth muscle cells (VSMCs) were found aggregating in the media of the non-dilated aorta of Se-D AA model mice, which was completely inhibited by Dox. The percentage of VSMCs in aortic media of Se-D AA model mice was significantly higher than in Se-A AA model mice. The maximum aortic diameter and occurrence rate of AA in Se-D AA model mice with Dox were significantly reduced compared with Se-D AA model mice. CONCLUSION: Se deficiency promoted dilatation of the aorta in AA model mice by increasing expression and activity of VSMC derived MMP-2, causing abnormal aggregation and proliferation of VSMCs in aortic media.
Assuntos
Aneurisma Aórtico , Selênio , Masculino , Camundongos , Animais , Metaloproteinase 2 da Matriz/metabolismo , Músculo Liso Vascular/metabolismo , Dilatação , Selênio/farmacologia , Selênio/metabolismo , Aminopropionitrilo/farmacologia , Camundongos Endogâmicos C57BL , Aorta/metabolismo , Modelos Animais de Doenças , Miócitos de Músculo Liso/metabolismoRESUMO
Vascular inflammation is known to cause degeneration of retinal capillaries in early diabetic retinopathy (DR), a major microvascular complication of diabetes. Past studies investigating these diabetes-induced retinal vascular abnormalities have focused primarily on the role of molecular or biochemical cues. Here we show that retinal vascular inflammation and degeneration in diabetes are also mechanically regulated by the increase in retinal vascular stiffness caused by overexpression of the collagen-cross-linking enzyme lysyl oxidase (LOX). Treatment of diabetic mice with LOX inhibitor ß-aminopropionitrile (BAPN) prevented the increase in retinal capillary stiffness, vascular intracellular adhesion molecule-1 overexpression, and leukostasis. Consistent with these anti-inflammatory effects, BAPN treatment of diabetic mice blocked the upregulation of proapoptotic caspase-3 in retinal vessels, which concomitantly reduced retinal capillary degeneration, pericyte ghost formation, and the diabetes-induced loss of contrast sensitivity in these mice. Finally, our in vitro studies indicate that retinal capillary stiffening is sufficient to increase the adhesiveness and neutrophil elastase-induced death of retinal endothelial cells. By uncovering a link between LOX-dependent capillary stiffening and the development of retinal vascular and functional defects in diabetes, these findings offer a new insight into DR pathogenesis that has important translational potential.
Assuntos
Diabetes Mellitus Experimental , Retinopatia Diabética , Degeneração Retiniana , Camundongos , Animais , Células Endoteliais , Diabetes Mellitus Experimental/complicações , Aminopropionitrilo/farmacologia , Retina/patologia , Retinopatia Diabética/patologia , Inflamação/patologia , Vasos Retinianos/patologia , Camundongos Endogâmicos C57BLRESUMO
Chemotherapy based on small molecule drugs, hormones, cycline kinase inhibitors, and monoclonal antibodies has been widely used for breast cancer treatment in the clinic but with limited efficacy, due to the poor specificity and tumor microenvironment (TME)-caused diffusion barrier. Although monotherapies targeting biochemical cues or physical cues in the TME have been developed, none of them can cope with the complex TME, while mechanochemical combination therapy remains largely to be explored. Herein, a combination therapy strategy based on an extracellular matrix (ECM) modulator and TME-responsive drug for the first attempt of mechanochemically synergistic treatment of breast cancer is developed. Specifically, based on overexpressed NAD(P)H quinone oxidoreductase 1 (NQO1) in breast cancer, a TME-responsive drug (NQO1-SN38) is designed and it is combined with the inhibitor (i.e., ß-Aminopropionitrile, BAPN) for Lysyl oxidases (Lox) that contributes to the tumor stiffness, for mechanochemical therapy. It is demonstrated that NQO1 can trigger the degradation of NQO1-SN38 and release SN38, showing nearly twice tumor inhibition efficiency compared with SN38 treatment in vitro. Lox inhibition with BAPN significantly reduces collagen deposition and enhances drug penetration in tumor heterospheroids in vitro. It is further demonstrated that the mechanochemical therapy showed outstanding therapeutic efficacy in vivo, providing a promising approach for breast cancer therapy.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/patologia , Aminopropionitrilo/farmacologia , Aminopropionitrilo/uso terapêutico , Quinonas/uso terapêutico , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Microambiente TumoralRESUMO
Patients with chronic kidney disease (CKD) are at increased risk for adverse cardiovascular events. CKD is associated with increases in arterial stiffness, whereas improvements in arterial stiffness correlate with better survival. However, arterial stiffness is increased early in CKD, suggesting that there might be additional factors, unique to kidney disease, that increase arterial stiffness. Lysyl oxidase (LOX) is a key mediator of collagen cross linking and matrix remodeling. LOX is predominantly expressed in the cardiovascular system, and its upregulation has been associated with increased tissue stiffening and extracellular matrix remodeling. Thus, this study was designed to evaluate the role of increased LOX activity in inducing aortic stiffness in CKD and whether ß-aminopropionitrile (BAPN), a LOX inhibitor, could prevent aortic stiffness by reducing collagen cross linking. Eight-week-old male C57BL/6 mice were subjected to 5/6 nephrectomy (Nx) or sham surgery. Two weeks after surgery, mice were randomized to BAPN (300 mg/kg/day in water) or vehicle treatment for 4 wk. Aortic stiffness was assessed by pulse wave velocity (PWV) using Doppler ultrasound. Aortic levels of LOX were assessed by ELISA, and cross-linked total collagen levels were analyzed by mass spectrometry and Sircol assay. Nx mice showed increased PWV and aortic wall remodeling compared with control mice. Collagen cross linking was increased in parallel with the increases in total collagen in the aorta of Nx mice. In contrast, Nx mice that received BAPN treatment showed decreased cross-linked collagens and PWV compared with that received vehicle treatment. Our results indicated that LOX might be an early and key mediator of aortic stiffness in CKD.NEW & NOTEWORTHY Arterial stiffness in CKD is associated with adverse cardiovascular outcomes. However, the mechanisms underlying increased aortic stiffness in CKD are unclear. Herein, we demonstrated that 1) increased aortic stiffness in CKD is independent of hypertension and calcification and 2) LOX-mediated changes in extracellular matrix are at least in part responsible for increased aortic stiffness in CKD. Prevention of excess LOX may have therapeutic potential in alleviating increased aortic stiffness and improving cardiovascular disease in CKD.
Assuntos
Insuficiência Renal Crônica , Rigidez Vascular , Animais , Masculino , Camundongos , Aminopropionitrilo/farmacologia , Colágeno , Camundongos Endogâmicos C57BL , Proteína-Lisina 6-Oxidase , Análise de Onda de Pulso/métodos , Rigidez Vascular/fisiologiaRESUMO
Cancer immunotherapy has shown tremendous potential to train the intrinsic immune system against malignancy in the clinic. However, the extracellular matrix (ECM) in tumor microenvironment is a formidable barrier that not only restricts the penetration of therapeutic drugs but also prevents the infiltration of antitumor immune cells. We herein report a semiconducting polymer-based ECM nanoremodeler (SPNcb) to combine photodynamic antitumor activity with cancer-specific inhibition of collagen-crosslinking enzymes (lysyl oxidase (LOX) family) for activatable cancer photo-immunotherapy. SPNcb is self-assembled from an amphiphilic semiconducting polymer conjugated with a LOX inhibitor (ß-aminopropionitrile, BAPN) via a cancer biomarker (cathepsin B, CatB)-cleavable segment. BAPN can be exclusively activated to inhibit LOX activity in the presence of the tumor-overexpressed CatB, thus blocking collagen crosslinking and decreasing ECM stiffness. Such an ECM nanoremodeler synergizes immunogenic phototherapy and checkpoint blockade immunotherapy to improve the tumor infiltration of cytotoxic T cells, inhibiting tumor growth and metastasis.
Assuntos
Aminopropionitrilo , Neoplasias , Aminopropionitrilo/farmacologia , Matriz Extracelular , Colágeno , Imunoterapia , Neoplasias/patologiaRESUMO
In Duchenne muscular dystrophy (DMD), a lack of functional dystrophin leads to myofiber instability and progressive muscle damage that results in fibrosis. While fibrosis is primarily characterized by an accumulation of extracellular matrix (ECM) components, there are changes in ECM architecture during fibrosis that relate more closely to functional muscle stiffness. One of these architectural changes in dystrophic muscle is collagen cross-linking, which has been shown to increase the passive muscle stiffness in models of fibrosis including the mdx mouse, a model of DMD. We tested whether the intraperitoneal injections of beta-aminopropionitrile (BAPN), an inhibitor of the cross-linking enzyme lysyl oxidase, would reduce collagen cross-linking and passive stiffness in young and adult mdx mice compared to saline-injected controls. We found no significant differences between BAPN treated and saline treated mice in collagen cross-linking and stiffness parameters. However, we observed that while collagen cross-linking and passive stiffness scaled positively in dystrophic muscles, collagen fiber alignment scaled with passive stiffness distinctly between muscles. We also observed that the dystrophic diaphragm showed the most dramatic fibrosis in terms of collagen content, cross-linking, and stiffness. Overall, we show that while BAPN was not effective at reducing collagen cross-linking, the positive association between collagen cross-linking and stiffness in dystrophic muscles still show cross-linking as a viable target for reducing passive muscle stiffness in DMD or other fibrotic muscle conditions.
Assuntos
Distrofia Muscular de Duchenne , Proteína-Lisina 6-Oxidase , Animais , Camundongos , Aminopropionitrilo/farmacologia , Colágeno , Modelos Animais de Doenças , Fibrose , Camundongos Endogâmicos mdx , Músculo Esquelético/fisiologia , Proteína-Lisina 6-Oxidase/antagonistas & inibidoresRESUMO
Aortic dissection (AD) is a lethal aortic pathology without effective medical treatments since the underlying pathological mechanisms responsible for AD remain elusive. Matrix metalloproteinase-8 (MMP8) has been previously identified as a key player in atherosclerosis and arterial remodeling. However, the functional role of MMP8 in AD remains largely unknown. Here, we report that an increased level of MMP8 was observed in 3-aminopropionitrile fumarate (BAPN)-induced murine AD. AD incidence and aortic elastin fragmentation were markedly reduced in MMP8-knockout mice. Importantly, pharmacologic inhibition of MMP8 significantly reduced the AD incidence and aortic elastin fragmentation. We observed less inflammatory cell accumulation, a lower level of aortic inflammation, and decreased smooth muscle cell (SMC) apoptosis in MMP8-knockout mice. In line with our previous observation that MMP8 cleaves Ang I to generate Ang II, BAPN-treated MMP8-knockout mice had increased levels of Ang I, but decreased levels of Ang II and lower blood pressure. Additionally, we observed a decreased expression level of vascular cell adhesion molecule-1 (VCAM1) and a reduced level of reactive oxygen species (ROS) in MMP8-knockout aortas. Mechanistically, our data show that the Ang II/VCAM1 signal axis is responsible for MMP8-mediated inflammatory cell invasion and transendothelial migration, while MMP8-mediated SMC inflammation and apoptosis are attributed to Ang II/ROS signaling. Finally, we observed higher levels of aortic and serum MMP8 in patients with AD. We therefore provide new insights into the molecular mechanisms underlying AD and identify MMP8 as a potential therapeutic target for this life-threatening aortic disease.
Assuntos
Dissecção Aórtica , Metaloproteinase 8 da Matriz , Animais , Camundongos , Aminopropionitrilo/farmacologia , Dissecção Aórtica/sangue , Dissecção Aórtica/genética , Angiotensina II/farmacologia , Angiotensina II/metabolismo , Modelos Animais de Doenças , Elastina/metabolismo , Inflamação/genética , Metaloproteinase 8 da Matriz/sangue , Metaloproteinase 8 da Matriz/genética , Metaloproteinase 8 da Matriz/metabolismo , Camundongos Knockout , Espécies Reativas de Oxigênio/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , HumanosRESUMO
Chronic kidney disease is one of the major health burdens affecting a considerable number of people worldwide. The aberrant regulation of lysyl oxidase (LOX) family of enzymes results in establishment of dense extracellular matrix (ECM). Since, LOX enzymes need copper (Cu) for their proper catalytic activity; the present study investigated the efficacy of a copper chelator, disulfiram (DSF) in renal fibrosis. Antifibrotic activity of DSF was investigated in kidney epithelial cells stimulated by transforming growth factor-ß1 (5 ng/ml) as well as in two animal models. The renal injury was induced in animals by unilateral ureteral obstruction and folic acid administration (250 mg/kg). The DSF (3 and 10 mg/kg, every 3rd day) and standard LOX inhibitor, ß-aminopropionitrile (BAPN, 100 mg/kg, daily) administration was started on day 0 and continued till the day of sacrifice. DSF was found to be a potent LOX/LOXL2 inhibitor to reduce crosslinking of collagen fibrils leading to reduction in the collagen deposition. In addition, the DSF was demonstrated to inhibit epithelial-mesenchymal transition in the tubular cells and fibrotic kidneys. Our results suggested that DSF, being a clinically available drug could be translated to clinics for its potent antifibrotic activity due to its inhibitory effect on LOX proteins.
Assuntos
Nefropatias , Proteína-Lisina 6-Oxidase , Aminopropionitrilo/farmacologia , Animais , Colágeno/metabolismo , Cobre , Dissulfiram/farmacologia , Fibrose , Humanos , Proteína-Lisina 6-Oxidase/metabolismoRESUMO
BACKGROUND: It is necessary and useful to obtain an experimental model which steadily and rapidly induces aneurysms for investigation of the pathogenesis of cerebral aneurysm. We attempted to examine whether intraperitoneal administration of ß-aminopropionitrile fumarate (BAPN-F) with additional treatments of induced hypertension and hemodynamic stress could steadily and rapidly induce aneurysms in male rats. METHODS: Seven-week-old male Sprague-Dawley rats pretreated with ligation of left common carotid and bilateral posterior renal arteries were administrated BAPN-F intraperitoneally. Induction rate and size of aneurysms was investigated with varying dose and duration of BAPN-F administration (low dose; 400 mg/kg/week for 4 or 8 weeks and high dose; 2800 mg/kg/week for 8 or 12 weeks). RESULTS: Induction rate in the high-dose groups was significantly higher (P<0.01) than that in the low-dose groups. Making comparisons between 8 and 12 weeks of the high-dose groups, while there was no difference in induction rate (8 weeks; 85.2% vs. 12 weeks; 76.9%), aneurysmal size was larger in 12 weeks (8 weeks; 127.5 µm, vs. 12 weeks; 181.7 µm in terms of median) but lethal intrathoracic hemorrhage was increased in 12 weeks (8 weeks; 7.4% vs. 12 weeks; 30.8%). Induction rate of large aneurysm was 22.2% and 30.8% in 8 and 12 weeks of the high-dose groups, respectively. CONCLUSIONS: High-dose BAPN-F administration can cause high-frequency aneurysmal induction. Although there was the difference in size and mortality rate based on administration duration, intraperitoneal administration of 2800 mg/kg/week BAPN-F for 8 weeks would be suitable for aneurysmal induction.
Assuntos
Aminopropionitrilo , Aneurisma Intracraniano , Aminopropionitrilo/análogos & derivados , Aminopropionitrilo/farmacologia , Animais , Modelos Animais de Doenças , Aneurisma Intracraniano/induzido quimicamente , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Congenital clubfoot is a complex musculoskeletal deformity, in which a stiff, contracted tissue forms in the medial part of the foot. Fibrotic changes are associated with increased collagen deposition and lysyl oxidase (LOX)-mediated crosslinking, which impair collagen degradation and increase the tissue stiffness. First, we studied collagen deposition, as well as the expression of collagen and the amount of pyridinoline and deoxypyridinoline crosslinks in the tissue of relapsed clubfoot by immunohistochemistry, real-time PCR, and enzyme-linked immunosorbent assay (ELISA). We then isolated fibroblast-like cells from the contracted tissue to study the potential inhibition of these processes in vitro. We assessed the effects of a LOX inhibitor, ß-aminopropionitrile (BAPN), on the cells by a hydroxyproline assay, ELISA, and Second Harmonic Generation imaging. We also evaluated the cell-mediated contraction of extracellular matrix in 3D cell-populated collagen gels. For the first time, we have confirmed significantly increased crosslinking and excessive collagen type I deposition in the clubfoot-contracted tissue. We successfully reduced these processes in vitro in a dose-dependent manner with 10-40 µg/mL of BAPN, and we observed an increasing trend in the inhibition of the cell-mediated contraction of collagen gels. The in vitro inhibitory effects indicate that BAPN has good potential for the treatment of relapsed and resistant clubfeet.
Assuntos
Aminopropionitrilo/farmacologia , Pé Torto Equinovaro/tratamento farmacológico , Colágeno/química , Reagentes de Ligações Cruzadas/farmacologia , Fibroblastos/efeitos dos fármacos , Proteína-Lisina 6-Oxidase/antagonistas & inibidores , Pré-Escolar , Pé Torto Equinovaro/metabolismo , Pé Torto Equinovaro/patologia , Feminino , Humanos , MasculinoRESUMO
OBJECTIVE: Abdominal aortic aneurysm (AAA) is a significant medical problem with a high mortality rate. Nevertheless, the underlying mechanism for the progression and regression of AAA is unknown. METHODS: Experimental model of AAA was first created by porcine pancreatic elastase incubation around the infrarenal aorta of C57BL/6 mice. Then, AAA progression and regression were evaluated based on the diameter and volume of AAA. The aortas were harvested for hematoxylin-eosin staining (HE), orcein staining, sirius red staining, immunofluorescence analysis and perls' prussian blue staining at the indicated time point. Finally, ß-aminopropionitrile monofumarate (BAPN) was used to explore the underlying mechanism of the regression of AAA. RESULTS: When we extended the observation period to 100 days, we not only observed an increase in the AAA diameter and volume in the early stage, but also a decrease in the late stage. Consistent with AAA diameter and volume, the aortic thickness showed the same tendency based on HE staining. The elastin and collagen content first degraded and then regenerated, which corresponds to the early deterioration and late regression of AAA. Then, endogenous up-regulation of lysyl oxidase (LOX) was detected, accompanying the regression of AAA, as detected by an immunofluorescent assay. BAPN and LOX inhibitor considerably inhibited the regression of AAA, paralleling the degradation of elastin lamella and collagen. CONCLUSION: Taken together, we tentatively conclude that endogenous re-generation of LOX played an influential role in the regression of AAA. Therefore, regulatory factors on the generation of LOX exhibit promising therapeutic potential against AAA.