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1.
Arterioscler Thromb Vasc Biol ; 44(7): 1555-1569, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38779856

RESUMEN

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.


Asunto(s)
Aminopropionitrilo , Aorta Torácica , Rotura de la Aorta , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Animales , Aminopropionitrilo/toxicidad , Aminopropionitrilo/farmacología , Aorta Torácica/patología , Aorta Torácica/efectos de los fármacos , Aorta Torácica/metabolismo , Femenino , Masculino , Rotura de la Aorta/inducido químicamente , Rotura de la Aorta/patología , Rotura de la Aorta/metabolismo , Rotura de la Aorta/prevención & control , Ratones , Remodelación Vascular/efectos de los fármacos , Dilatación Patológica , Músculo Liso Vascular/patología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Factores de Edad , Factores de Tiempo , Factores Sexuales , Proliferación Celular/efectos de los fármacos , Proteína-Lisina 6-Oxidasa/metabolismo
2.
Arterioscler Thromb Vasc Biol ; 43(8): 1524-1532, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-37345525

RESUMEN

BACKGROUND: Angiotensinogen (AGT) is an essential component in the renin-angiotensin system. AGT has highly conserved sequences in the loop and ß-sheet regions among species; however, their functions have not been studied. METHODS: Adeno-associated viral vector (AAV) serotype 2/8 encoding mouse AGT with mutations of conserved sequences in the loop (AAV.loop-Mut), ß-sheet (AAV.ßsheet-Mut), or both regions (AAV.loop/ßsheet-Mut) was injected into male hepatocyte-specific AGT-deficient (hepAGT-/-) mice in an LDL (low-density lipoprotein) receptor-deficient background. AAV containing mouse wild-type AGT (AAV.mAGT) or a null vector (AAV.null) were used as controls. Two weeks after AAV administration, all mice were fed a western diet for 12 weeks. To determine how AGT secretion is regulated in hepatocytes, AAVs containing the above mutations were transducted into HepG2 cells. RESULTS: In hepAGT-/- mice infected with AAV.loop-Mut or ßsheet-Mut, plasma AGT concentrations, systolic blood pressure, and atherosclerosis were comparable to those in AAV.mAGT-infected mice. Interestingly, plasma AGT concentrations, systolic blood pressure, and atherosclerotic lesion size in hepAGT-/- mice infected with AAV.loop/ßsheet-Mut were not different from mice infected with AAV.null. In contrast, hepatic Agt mRNA abundance was elevated to a comparable magnitude as AAV.mAGT-infected mice. Immunostaining showed that AGT protein was accumulated in hepatocytes of mice infected with AAV.loop/ßsheet-Mut or HepG2 cells transducted with AAV.loop/ßsheet-Mut. Accumulated AGT was not located in the endoplasmic reticulum. CONCLUSIONS: The conserved sequences in either the loop or ß-sheet region individually have no effect on AGT regulation, but the conserved sequences in both regions synergistically contribute to the secretion of AGT from hepatocytes.


Asunto(s)
Angiotensinógeno , Animales , Ratones , Angiotensinógeno/sangre , Angiotensinógeno/química , Angiotensinógeno/genética , Angiotensinógeno/metabolismo , Secuencia Conservada , Secuencia de Aminoácidos , Masculino , Femenino , Hepatocitos/metabolismo , Conformación Proteica en Lámina beta , Aterosclerosis/metabolismo , Aterosclerosis/patología , Retículo Endoplásmico/metabolismo , Glicosilación , Hígado/citología , Hígado/metabolismo , Sistema Renina-Angiotensina
3.
Circulation ; 145(13): 987-1001, 2022 03 29.
Artículo en Inglés | MEDLINE | ID: mdl-35143327

RESUMEN

BACKGROUND: The ascending aorta is a common location for aneurysm and dissection. This aortic region is populated by a mosaic of medial and adventitial cells that are embryonically derived from either the second heart field (SHF) or the cardiac neural crest. SHF-derived cells populate areas that coincide with the spatial specificity of thoracic aortopathies. The purpose of this study was to determine whether and how SHF-derived cells contribute to ascending aortopathies. METHODS: Ascending aortic pathologies were examined in patients with sporadic thoracic aortopathies and angiotensin II (AngII)-infused mice. Ascending aortas without overt pathology from AngII-infused mice were subjected to mass spectrometry-assisted proteomics and molecular features of SHF-derived cells were determined by single-cell transcriptomic analyses. Genetic deletion of either Lrp1 (low-density lipoprotein receptor-related protein 1) or Tgfbr2 (transforming growth factor-ß receptor type 2) in SHF-derived cells was conducted to examine the effect of SHF-derived cells on vascular integrity. RESULTS: Pathologies in human ascending aortic aneurysmal tissues were predominant in outer medial layers and adventitia. This gradient was mimicked in mouse aortas after AngII infusion that was coincident with the distribution of SHF-derived cells. Proteomics indicated that brief AngII infusion before overt pathology occurred evoked downregulation of smooth muscle cell proteins and differential expression of extracellular matrix proteins, including several LRP1 ligands. LRP1 deletion in SHF-derived cells augmented AngII-induced ascending aortic aneurysm and rupture. Single-cell transcriptomic analysis revealed that brief AngII infusion decreased Lrp1 and Tgfbr2 mRNA abundance in SHF-derived cells and induced a unique fibroblast population with low abundance of Tgfbr2 mRNA. SHF-specific Tgfbr2 deletion led to embryonic lethality at E12.5 with dilatation of the outflow tract and retroperitoneal hemorrhage. Integration of proteomic and single-cell transcriptomics results identified PAI1 (plasminogen activator inhibitor 1) as the most increased protein in SHF-derived smooth muscle cells and fibroblasts during AngII infusion. Immunostaining revealed a transmural gradient of PAI1 in both ascending aortas of AngII-infused mice and human ascending aneurysmal aortas that mimicked the gradient of medial and adventitial pathologies. CONCLUSIONS: SHF-derived cells exert a critical role in maintaining vascular integrity through LRP1 and transforming growth factor-ß signaling associated with increases of aortic PAI1.


Asunto(s)
Angiotensina II , Proteómica , Angiotensina II/farmacología , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero , Receptor Tipo II de Factor de Crecimiento Transformador beta/genética , Factores de Crecimiento Transformadores
4.
Arterioscler Thromb Vasc Biol ; 42(10): 1254-1261, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-36004642

RESUMEN

BACKGROUND: Cross-linking of lysine residues in elastic and collagen fibers is a vital process in aortic development. Inhibition of lysyl oxidase by BAPN (ß-aminopropionitrile) leads to thoracic aortopathies in mice. Although the renin-angiotensin system contributes to several types of thoracic aortopathies, it remains unclear whether inhibition of the renin-angiotensin system protects against aortopathy caused by the impairment of elastic fiber/collagen crosslinking. METHODS: BAPN (0.5% wt/vol) was started in drinking water to induce aortopathies in male C57BL/6J mice at 4 weeks of age for 4 weeks. Five approaches were used to investigate the impact of the renin-angiotensin system. Bulk RNA sequencing was performed to explore potential molecular mechanisms of BAPN-induced thoracic aortopathies. RESULTS: Losartan increased plasma renin concentrations significantly, compared with vehicle-infused mice, indicating effective angiotensin II type 1 receptor inhibition. However, losartan did not suppress BAPN-induced aortic rupture and dilatation. Since losartan is a surmountable inhibitor of the renin-angiotensin system, irbesartan, an insurmountable inhibitor, was also tested. Although increased plasma renin concentrations indicated effective inhibition, irbesartan did not ameliorate aortic rupture and dilatation in BAPN-administered mice. Thus, BAPN-induced thoracic aortopathies were refractory to angiotensin II type 1 receptor blockade. Next, we inhibited angiotensin II production by pharmacological or genetic depletion of AGT (angiotensinogen), the unique precursor of angiotensin II. However, neither suppressed BAPN-induced thoracic aortic rupture and dilatation. Aortic RNA sequencing revealed molecular changes during BAPN administration that were distinct from other types of aortopathies in which angiotensin II type 1 receptor inhibition protects against aneurysm formation. CONCLUSIONS: Inhibition of either angiotensin II action or production of the renin-angiotensin system does not attenuate BAPN-induced thoracic aortopathies in mice.


Asunto(s)
Aneurisma de la Aorta Torácica , Rotura de la Aorta , Sistema Renina-Angiotensina , Aminopropionitrilo/efectos adversos , Angiotensina II , Angiotensinógeno , Animales , Aneurisma de la Aorta Torácica/inducido químicamente , Aneurisma de la Aorta Torácica/genética , Aneurisma de la Aorta Torácica/prevención & control , Rotura de la Aorta/inducido químicamente , Dilatación Patológica , Modelos Animales de Enfermedad , Irbesartán/farmacología , Losartán , Lisina , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína-Lisina 6-Oxidasa/genética , Receptor de Angiotensina Tipo 1/genética , Renina/genética
6.
Arterioscler Thromb Vasc Biol ; 41(10): 2538-2550, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34407634

RESUMEN

Objective: A cardinal feature of Marfan syndrome is thoracic aortic aneurysm. The contribution of the renin-angiotensin system via AT1aR (Ang II [angiotensin II] receptor type 1a) to thoracic aortic aneurysm progression remains controversial because the beneficial effects of angiotensin receptor blockers have been ascribed to off-target effects. This study used genetic and pharmacological modes of attenuating angiotensin receptor and ligand, respectively, to determine their roles on thoracic aortic aneurysm in mice with fibrillin-1 haploinsufficiency (Fbn1C1041G/+). Approach and Results: Thoracic aortic aneurysm in Fbn1C1041G/+ mice was found to be strikingly sexual dimorphic. Males displayed aortic dilation over 12 months while aortic dilation in Fbn1C1041G/+ females did not differ significantly from wild-type mice. To determine the role of AT1aR, Fbn1C1041G/+ mice that were either +/+ or -/- for AT1aR were generated. AT1aR deletion reduced expansion of ascending aorta and aortic root diameter from 1 to 12 months of age in males. Medial thickening and elastin fragmentation were attenuated. An antisense oligonucleotide against angiotensinogen was administered to male Fbn1C1041G/+ mice to determine the effects of Ang II depletion. Antisense oligonucleotide against angiotensinogen administration attenuated dilation of the ascending aorta and aortic root and reduced extracellular remodeling. Aortic transcriptome analyses identified potential targets by which inhibition of the renin-angiotensin system reduced aortic dilation in Fbn1C1041G/+ mice. Conclusions: Deletion of AT1aR or inhibition of Ang II production exerted similar effects in attenuating pathologies in the proximal thoracic aorta of male Fbn1C1041G/+ mice. Inhibition of the renin-angiotensin system attenuated dysregulation of genes within the aorta related to pathology of Fbn1C1041G/+ mice.


Asunto(s)
Angiotensinógeno/metabolismo , Aorta Torácica/metabolismo , Aneurisma de la Aorta Torácica/prevención & control , Fibrilina-1/genética , Eliminación de Gen , Síndrome de Marfan/genética , Receptor de Angiotensina Tipo 1/genética , Sistema Renina-Angiotensina , Angiotensinógeno/genética , Animales , Aorta Torácica/patología , Aneurisma de la Aorta Torácica/genética , Aneurisma de la Aorta Torácica/metabolismo , Aneurisma de la Aorta Torácica/patología , Modelos Animales de Enfermedad , Femenino , Fibrilina-1/metabolismo , Predisposición Genética a la Enfermedad , Haploinsuficiencia , Masculino , Síndrome de Marfan/metabolismo , Síndrome de Marfan/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Fenotipo , Receptor de Angiotensina Tipo 1/deficiencia , Sistema Renina-Angiotensina/genética , Caracteres Sexuales , Factores Sexuales , Transcriptoma
7.
Arterioscler Thromb Vasc Biol ; 40(9): 2108-2113, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32640904

RESUMEN

OBJECTIVE: Renin cleavage of angiotensinogen has species specificity. As the residues at positions 11 and 12 are different between human angiotensinogen and mouse angiotensinogen, we determined whether these 2 residues in angiotensinogen affect renin cleavage and angiotensin II-mediated blood pressure regulation and atherosclerosis using an adenoassociated viral approach for manipulating angiotensinogen in vivo. Approach and Results: Hepatocyte-specific angiotensinogen deficient (hepAGT-/-) mice in an LDL receptor-deficient background were infected with adenoassociated virals containing a null insert, human angiotensinogen, or mouse angiotensinogen expressing the same residues of the human protein at positions 11 and 12 (mouse angiotensinogen [L11V;Y12I]). Expression of human angiotensinogen in hepAGT-/- mice led to high plasma human angiotensinogen concentrations without changes in plasma endogenous mouse angiotensinogen, plasma renin concentrations, blood pressure, or atherosclerosis. This is consistent with human angiotensinogen not being cleaved by mouse renin. To determine whether the residues at positions 11 and 12 in human angiotensinogen lead to the inability of mouse renin to cleave human angiotensinogen, hepAGT-/- mice were injected with adenoassociated viral vector encoding mouse angiotensinogen (L11V;Y12I). Expression of mouse angiotensinogen (L11V;Y12I) in hepAGT-/- mice resulted in increased plasma mouse angiotensinogen concentrations, reduced renin concentrations, and increased renal AngII concentrations that were comparable to their concentrations in hepAGT+/+ mice. This mouse angiotensinogen variant increased blood pressure and atherosclerosis in hepAGT-/- mice to the magnitude of hepAGT+/+ mice. CONCLUSIONS: Replacement of L11 and Y12 to V11 and I12, respectively, in mouse angiotensinogen does not affect renin cleavage, blood pressure, and atherosclerosis in LDL receptor-deficient mice.


Asunto(s)
Angiotensina II/metabolismo , Angiotensinógeno/metabolismo , Aterosclerosis/metabolismo , Presión Sanguínea , Hepatocitos/metabolismo , Hipertensión/metabolismo , Renina/metabolismo , Sustitución de Aminoácidos , Angiotensinógeno/deficiencia , Angiotensinógeno/genética , Animales , Aterosclerosis/genética , Aterosclerosis/patología , Modelos Animales de Enfermedad , Femenino , Humanos , Hipertensión/genética , Hipertensión/fisiopatología , Masculino , Ratones Noqueados , Placa Aterosclerótica , Receptores de LDL/genética , Receptores de LDL/metabolismo , Especificidad de la Especie
8.
Arterioscler Thromb Vasc Biol ; 39(2): 150-155, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30567480

RESUMEN

Objective- AGT (Angiotensinogen) is the unique precursor of the renin-angiotensin system that is sequentially cleaved by renin and ACE (angiotensin-converting enzyme) to produce Ang II (angiotensin II). In this study, we determined how these renin-angiotensin components interact with megalin in kidney to promote atherosclerosis. Approach and Results- AGT, renin, ACE, and megalin were present in the renal proximal convoluted tubules of wild-type mice. Hepatocyte-specific AGT deficiency abolished AGT protein accumulation in proximal tubules and diminished Ang II concentrations in kidney, while renin was increased. Megalin was most abundant in kidney and exclusively present on the apical side of proximal tubules. Inhibition of megalin by antisense oligonucleotides (ASOs) led to ablation of AGT and renin proteins in proximal tubules, while leading to striking increases of urine AGT and renin concentrations, and 70% reduction of renal Ang II concentrations. However, plasma Ang II concentrations were unaffected. To determine whether AGT and megalin interaction contributes to atherosclerosis, we used both male and female low-density lipoprotein receptor-/- mice fed a saturated fat-enriched diet and administered vehicles (PBS or control ASO) or megalin ASO. Inhibition of megalin did not affect plasma cholesterol concentrations, but profoundly reduced atherosclerotic lesion size in both male and female mice. Conclusions- These results reveal a regulatory role of megalin in the intrarenal renin-angiotensin homeostasis and atherogenesis, positing renal Ang II to be an important contributor to atherosclerosis that is mediated through AGT and megalin interactions.


Asunto(s)
Angiotensinógeno/fisiología , Aterosclerosis/etiología , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/fisiología , Angiotensina II/biosíntesis , Animales , Femenino , Hipercolesterolemia/complicaciones , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/antagonistas & inhibidores , Masculino , Ratones , Ratones Endogámicos C57BL , Oligonucleótidos Antisentido/farmacología , Sistema Renina-Angiotensina/fisiología
9.
Circ J ; 84(5): 825-829, 2020 04 24.
Artículo en Inglés | MEDLINE | ID: mdl-32238693

RESUMEN

BACKGROUND: Angiotensin (Ang)I is cleaved by angiotensin-converting enzyme (ACE) to generate AngII. The purpose of this study was to determine the roles of ACE in endothelial and smooth muscle cells in aortic aneurysms.Methods and Results:AngI infusion led to thoracic and abdominal aortic aneurysms in low-density lipoprotein receptor-deficient mice, which were ablated by ACE inhibition. Endothelial or smooth muscle cell-specific ACE deletion resulted in reduction of AngI-induced thoracic, but not abdominal, aortic dilatation. CONCLUSIONS: AngI infusion causes thoracic and abdominal aortic aneurysms in mice. ACE in aortic resident cells has differential effects on AngI-induced thoracic and abdominal aortic aneurysms.


Asunto(s)
Angiotensina I , Aorta Abdominal/enzimología , Aorta Torácica/enzimología , Aneurisma de la Aorta Abdominal/enzimología , Aneurisma de la Aorta Torácica/enzimología , Peptidil-Dipeptidasa A/metabolismo , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Aorta Abdominal/efectos de los fármacos , Aorta Abdominal/patología , Aorta Torácica/efectos de los fármacos , Aorta Torácica/patología , Aneurisma de la Aorta Abdominal/inducido químicamente , Aneurisma de la Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/prevención & control , Aneurisma de la Aorta Torácica/inducido químicamente , Aneurisma de la Aorta Torácica/patología , Aneurisma de la Aorta Torácica/prevención & control , Dilatación Patológica , Modelos Animales de Enfermedad , Células Endoteliales/enzimología , Células Endoteliales/patología , Ratones Noqueados , Miocitos del Músculo Liso/enzimología , Miocitos del Músculo Liso/patología , Peptidil-Dipeptidasa A/deficiencia , Peptidil-Dipeptidasa A/genética , Receptores de LDL/deficiencia , Receptores de LDL/genética
10.
Arterioscler Thromb Vasc Biol ; 36(6): 1085-9, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-27055902

RESUMEN

OBJECTIVE: Angiotensin-converting enzyme (ACE) is present in many cell types of atherosclerotic lesions. This study determined whether ACE activity in endothelial and smooth muscle cells (SMCs), 2 major resident cell types of the aorta, contributes to hypercholesterolemia-induced atherosclerosis. APPROACH AND RESULTS: All study mice were in low-density lipoprotein receptor(-/-) background. To determine the contribution of ACE on endothelial cells to atherosclerosis, female ACE floxed mice were bred to male Tie2-Cre transgenic mice. Endothelial cell-specific deletion of ACE significantly decreased serum ACE activity, but had no effect on systolic blood pressure and atherosclerosis. Because ACE protein is present on SMCs, the most abundant cell type of the aorta, we then determined whether ACE on SMCs contributes to atherosclerosis. ACE was depleted from SMCs by breeding female ACE floxed mice with male SM22-Cre transgenic mice. SMC-specific deficiency of ACE did not affect ACE activity in serum, but ablated its presence and activity in the aortic media. Although SMC-specific deficiency of ACE had no effect on systolic blood pressure, it significantly attenuated hypercholesterolemia-induced atherosclerosis in both male and female mice. CONCLUSIONS: These studies provide direct evidence that ACE derived from endothelial cells does not play a critical role in atherosclerosis. Rather, SMC-derived ACE contributes to atherosclerosis, independent of circulating ACE activity and blood pressure.


Asunto(s)
Enfermedades de la Aorta/enzimología , Aterosclerosis/enzimología , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/enzimología , Peptidil-Dipeptidasa A/metabolismo , Animales , Aorta Torácica/enzimología , Aorta Torácica/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Enfermedades de la Aorta/prevención & control , Aterosclerosis/genética , Aterosclerosis/patología , Aterosclerosis/prevención & control , Presión Sanguínea , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Células Endoteliales/enzimología , Células Endoteliales/patología , Femenino , Predisposición Genética a la Enfermedad , Hipercolesterolemia/enzimología , Hipercolesterolemia/genética , Masculino , Ratones Noqueados , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Peptidil-Dipeptidasa A/deficiencia , Peptidil-Dipeptidasa A/genética , Fenotipo , Placa Aterosclerótica , Receptores de LDL/deficiencia , Receptores de LDL/genética
11.
Arterioscler Thromb Vasc Biol ; 36(9): 1753-7, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27470509

RESUMEN

OBJECTIVE: Gain-of-function mutations of PCSK9 (proprotein convertase subtilisin/kexin type 9) lead to hypercholesterolemia. This study was to determine whether infection of normocholesterolemic mice with an adeno-associated viral (AAV) vector expressing a gain-of-function mutation of mouse PCSK9 increased angiotensin II (AngII)-induced abdominal aortic aneurysms. APPROACH AND RESULTS: In an initial study, male C57BL/6 mice were injected intraperitoneally with either an empty vector or PCSK9 gain-of-function mutation (D377Y). AAV at 3 doses and fed a saturated fat-enriched diet for 6 weeks. Two weeks after AAV injection, mice were infused with AngII for 4 weeks. Plasma PCSK9 concentrations were increased dose dependently in mice injected with AAV containing PCSK9D377Y mutation and positively associated with elevations of plasma cholesterol concentrations. Infection with intermediate and high doses of PCSK9D377Y.AAV led to equivalent increases of maximal width of abdominal aortas in C57BL/6 mice infused with AngII. Therefore, the intermediate dose was used in subsequent experiments. We then determined effects of PCSK9D377Y.AAV infection on 5 normolipidemic mouse strains, demonstrating that C57BL/6 mice were the most susceptible to this AAV infection. PCSK9D377Y.AAV infected male C57BL/6 mice were also compared with age-matched male low-density lipoprotein receptor(-/-) mice. Although plasma cholesterol concentrations were lower in mice infected with PCSK9D377Y.AAV, these mice had equivalent abdominal aortic aneurysmal formation, compared to low-density lipoprotein receptor(-/-) mice. In a separate study, reduced plasma PCSK9 concentrations by PCSK9 antisense oligonucleotides in male low-density lipoprotein receptor(-/-) mice did not influence AngII-induced abdominal aortic aneurysms. CONCLUSION: AAV-mediated infection with a mouse PCSK9 gain-of-function mutation is a rapid, easy, and efficient approach for inducing hypercholesterolemia and promoting abdominal aortic aneurysms in C57BL/6 mice infused with AngII.


Asunto(s)
Angiotensina II , Aneurisma de la Aorta Abdominal/inducido químicamente , Hipercolesterolemia/genética , Mutación , Proproteína Convertasa 9/genética , Animales , Aneurisma de la Aorta Abdominal/sangre , Aneurisma de la Aorta Abdominal/enzimología , Aneurisma de la Aorta Abdominal/genética , Biomarcadores/sangre , Colesterol/sangre , Dependovirus/genética , Modelos Animales de Enfermedad , Técnicas de Transferencia de Gen , Predisposición Genética a la Enfermedad , Vectores Genéticos , Hipercolesterolemia/sangre , Hipercolesterolemia/complicaciones , Hipercolesterolemia/enzimología , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Proproteína Convertasa 9/sangre , Receptores de LDL/deficiencia , Receptores de LDL/genética , Especificidad de la Especie , Factores de Tiempo
12.
Arterioscler Thromb Vasc Biol ; 36(5): 835-45, 2016 05.
Artículo en Inglés | MEDLINE | ID: mdl-26966280

RESUMEN

OBJECTIVE: Angiotensin II (AngII) infusion profoundly increases activity of calpains, calcium-dependent neutral cysteine proteases, in mice. Pharmacological inhibition of calpains attenuates AngII-induced aortic medial macrophage accumulation, atherosclerosis, and abdominal aortic aneurysm in mice. However, the precise functional contribution of leukocyte-derived calpains in AngII-induced vascular pathologies has not been determined. The purpose of this study was to determine whether calpains expressed in bone marrow (BM)-derived cells contribute to AngII-induced atherosclerosis and aortic aneurysms in hypercholesterolemic mice. APPROACH AND RESULTS: To study whether leukocyte calpains contributed to AngII-induced aortic pathologies, irradiated male low-density lipoprotein receptor(-/-) mice were repopulated with BM-derived cells that were either wild-type or overexpressed calpastatin, the endogenous inhibitor of calpains. Mice were fed a fat-enriched diet and infused with AngII (1000 ng/kg per minute) for 4 weeks. Overexpression of calpastatin in BM-derived cells significantly attenuated AngII-induced atherosclerotic lesion formation in aortic arches, but had no effect on aneurysm formation. Using either BM-derived cells from calpain-1-deficient mice or mice with leukocyte-specific calpain-2 deficiency generated using cre-loxP recombination technology, further studies demonstrated that independent deficiency of either calpain-1 or -2 in leukocytes modestly attenuated AngII-induced atherosclerosis. Calpastatin overexpression significantly attenuated AngII-induced inflammatory responses in macrophages and spleen. Furthermore, calpain inhibition suppressed migration and adhesion of macrophages to endothelial cells in vitro. Calpain inhibition also significantly decreased hypercholesterolemia-induced atherosclerosis in the absence of AngII. CONCLUSIONS: The present study demonstrates a pivotal role for BM-derived calpains in mediating AngII-induced atherosclerosis by influencing macrophage function.


Asunto(s)
Angiotensina II , Aneurisma de la Aorta Abdominal/prevención & control , Aterosclerosis/prevención & control , Calpaína/deficiencia , Inflamación/prevención & control , Leucocitos/enzimología , Animales , Aneurisma de la Aorta Abdominal/inducido químicamente , Aneurisma de la Aorta Abdominal/enzimología , Aneurisma de la Aorta Abdominal/genética , Aterosclerosis/inducido químicamente , Aterosclerosis/enzimología , Aterosclerosis/genética , Trasplante de Médula Ósea , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Calpaína/genética , Calpaína/metabolismo , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Inhibidores de Cisteína Proteinasa/farmacología , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Predisposición Genética a la Enfermedad , Inflamación/inducido químicamente , Inflamación/enzimología , Inflamación/genética , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Fenotipo , Receptores de LDL/deficiencia , Receptores de LDL/genética , Irradiación Corporal Total
13.
Arterioscler Thromb Vasc Biol ; 36(2): 256-65, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26681751

RESUMEN

OBJECTIVE: This study determined whether angiotensinogen (AGT) has angiotensin II-independent effects using multiple genetic and pharmacological manipulations. APPROACH AND RESULTS: All study mice were in low-density lipoprotein receptor -/- background and fed a saturated fat-enriched diet. In mice with floxed alleles and a neomycin cassette in intron 2 of the AGT gene (hypoAGT mice), plasma AGT concentrations were >90% lower compared with their wild-type littermates. HypoAGT mice had lower systolic blood pressure, less atherosclerosis, and diminished body weight gain and liver steatosis. Low plasma AGT concentrations and all phenotypes were recapitulated in mice with hepatocyte-specific deficiency of AGT or pharmacological inhibition of AGT by antisense oligonucleotide administration. In contrast, inhibition of AGT cleavage by a renin inhibitor, aliskiren, failed to alter body weight gain and liver steatosis in low-density lipoprotein receptor -/- mice. In mice with established adiposity, administration of AGT antisense oligonucleotide versus aliskiren led to equivalent reductions of systolic blood pressure and atherosclerosis. AGT antisense oligonucleotide administration ceased body weight gain and further reduced body weight, whereas aliskiren did not affect body weight gain during continuous saturated fat-enriched diet feeding. Structural comparisons of AGT proteins in zebrafish, mouse, rat, and human revealed 4 highly conserved sequences within the des(angiotensin I)AGT domain. des(angiotensin I)AGT, through adeno-associated viral infection in hepatocyte-specific AGT-deficient mice, increased body weight gain and liver steatosis, but did not affect atherosclerosis. CONCLUSIONS: AGT contributes to body weight gain and liver steatosis through functions of the des(angiotensin I)AGT domain, which are independent of angiotensin II production.


Asunto(s)
Angiotensina II/metabolismo , Angiotensinógeno/metabolismo , Aterosclerosis/metabolismo , Hígado Graso/metabolismo , Hepatocitos/metabolismo , Hipertensión/metabolismo , Hígado/metabolismo , Amidas/farmacología , Secuencia de Aminoácidos , Angiotensinógeno/deficiencia , Angiotensinógeno/genética , Animales , Aterosclerosis/genética , Aterosclerosis/patología , Aterosclerosis/prevención & control , Presión Sanguínea , Secuencia Conservada , Dependovirus/genética , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Hígado Graso/genética , Hígado Graso/patología , Hígado Graso/prevención & control , Fumaratos/farmacología , Vectores Genéticos , Genotipo , Hepatocitos/patología , Hipertensión/genética , Hipertensión/fisiopatología , Hipertensión/prevención & control , Hígado/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Moleculares , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Fenotipo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Receptores de LDL/deficiencia , Receptores de LDL/genética , Renina/antagonistas & inhibidores , Renina/metabolismo , Transducción de Señal , Factores de Tiempo , Transducción Genética , Aumento de Peso
14.
Circ J ; 81(6): 888-890, 2017 May 25.
Artículo en Inglés | MEDLINE | ID: mdl-28420827

RESUMEN

BACKGROUND: This study determined whether relaxin or matrix metalloproteinase (MMP)-9 influences angiotensin II (AngII)-induced abdominal aortic aneurysms (AAA).Methods and Results:Male C57BL/6 or apolipoprotein E-/-mice were infused with AngII with or without relaxin. Relaxin did not influence AngII-induced AAA in either mouse strain. Infusion of AngII reduced, but relaxin increased, MMP-9 mRNA in macrophages. We then determined the effects of MMP-9 deficiency on AAA in apolipoprotein E-/-mice. MMP-9 deficiency led to AAA formation in the absence of AngII, and augmented AngII-induced aortic rupture and AAA incidence. CONCLUSIONS: MMP-9 deficiency augmented AngII-induced AAA.


Asunto(s)
Angiotensina II/efectos adversos , Aneurisma de la Aorta Abdominal/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Relaxina/biosíntesis , Angiotensina II/farmacología , Animales , Aneurisma de la Aorta Abdominal/inducido químicamente , Aneurisma de la Aorta Abdominal/genética , Aneurisma de la Aorta Abdominal/patología , Apolipoproteínas E/deficiencia , Metaloproteinasa 9 de la Matriz/genética , Ratones , Ratones Noqueados , Relaxina/genética
15.
J Biol Chem ; 290(18): 11547-56, 2015 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-25784555

RESUMEN

Lipid accumulation in liver and skeletal muscle contributes to co-morbidities associated with diabetes and obesity. We made a transgenic mouse in which the adiponectin (Adipoq) promoter drives expression of lipoprotein lipase (LPL) in adipocytes to potentially increase adipose tissue lipid storage. These mice (Adipoq-LPL) have improved glucose and insulin tolerance as well as increased energy expenditure when challenged with a high fat diet (HFD). To identify the mechanism(s) involved, we determined whether the Adipoq-LPL mice diverted dietary lipid to adipose tissue to reduce peripheral lipotoxicity, but we found no evidence for this. Instead, characterization of the adipose tissue of the male mice after HFD challenge revealed that the mRNA levels of peroxisome proliferator-activated receptor-γ (PPARγ) and a number of PPARγ-regulated genes were higher in the epididymal fat pads of Adipoq-LPL mice than control mice. This included adiponectin, whose mRNA levels were increased, leading to increased adiponectin serum levels in the Adipoq-LPL mice. In many respects, the adipose phenotype of these animals resembles thiazolidinedione treatment except for one important difference, the Adipoq-LPL mice did not gain more fat mass on HFD than control mice and did not have increased expression of genes in adipose such as glycerol kinase, which are induced by high affinity PPAR agonists. Rather, there was selective induction of PPARγ-regulated genes such as adiponectin in the adipose of the Adipoq-LPL mice, suggesting that increasing adipose tissue LPL improves glucose metabolism in diet-induced obesity by improving the adipose tissue phenotype. Adipoq-LPL mice also have increased energy expenditure.


Asunto(s)
Adipocitos/metabolismo , Dieta Alta en Grasa/efectos adversos , Glucosa/metabolismo , Lipoproteína Lipasa/genética , Lipoproteína Lipasa/metabolismo , Obesidad/metabolismo , Obesidad/patología , Adipocitos/efectos de los fármacos , Animales , Femenino , Humanos , Resistencia a la Insulina , Masculino , Ratones , Ratones Transgénicos , Obesidad/enzimología , Obesidad/genética , Fenotipo , Tiazolidinedionas/farmacología
18.
Arterioscler Thromb Vasc Biol ; 35(9): 1995-2002, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26160957

RESUMEN

OBJECTIVE: Angiotensin II (Ang II) infusion causes aortic medial thickening via stimulation of angiotensin II type 1a (AT1a) receptors. The purpose of this study was to determine the cellular loci of AT1a receptors that mediate this Ang II-induced aortic pathology. APPROACH AND RESULTS: Saline or Ang II was infused into AT1a receptor floxed mice expressing Cre under control of cell-specific promoters. Initially, AT1a receptors were depleted in aortic smooth muscle cell and endothelium by expressing Cre under control of SM22 and Tie2 promoters, respectively. Deletion of AT1a receptors in either cell type had no effect on Ang II-induced medial thickening. To determine whether this effect was related to neural stimulation, AT1a receptors were depleted using an enolase 2-driven Cre. Depletion of AT1a receptors in neural cells attenuated Ang II-induced medial thickening of the ascending, but not descending aorta. Lineage tracking studies, using ROSA26-LacZ, demonstrated that enolase 2 was also expressed in adventitial cells adjacent to the region of attenuated thickening. To determine whether adventitial fibroblasts contributed to this attenuation, AT1a receptors in fibroblasts were depleted using S100A4 driven Cre. Similar to enolase 2-Cre, Ang II-induced medial thickening was attenuated in the ascending, but not the descending aorta. Lineage tracking demonstrated an increase of S100A4-LacZ positive cells in the media of the ascending region during Ang II infusion. CONCLUSIONS: AT1a receptor depletion in fibroblasts attenuates Ang II-induced medial hyperplasia in the ascending aorta.


Asunto(s)
Aorta Torácica/efectos de los fármacos , Aterosclerosis/genética , ADN/genética , Fibroblastos/metabolismo , Regulación de la Expresión Génica , ARN Mensajero/genética , Receptor de Angiotensina Tipo 1/genética , Angiotensina II/toxicidad , Animales , Aorta Torácica/metabolismo , Aorta Torácica/patología , Aterosclerosis/metabolismo , Aterosclerosis/prevención & control , Modelos Animales de Enfermedad , Fibroblastos/patología , Genotipo , Hiperplasia/tratamiento farmacológico , Hiperplasia/genética , Hiperplasia/patología , Infusiones Intravenosas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor de Angiotensina Tipo 1/administración & dosificación , Receptor de Angiotensina Tipo 1/biosíntesis , Túnica Media/efectos de los fármacos , Túnica Media/metabolismo , Túnica Media/patología
19.
Arterioscler Thromb Vasc Biol ; 35(8): 1826-34, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26044581

RESUMEN

OBJECTIVE: Dyslipidemia is implicated in abdominal aortic aneurysms (AAAs) in humans and angiotensin (Ang) II-infused mice. This study determined effects of major lipoprotein classes on AngII-induced AAAs using multiple mouse strains with dietary and pharmacological manipulations. APPROACH AND RESULTS: Western diet had minor effects on plasma cholesterol concentrations and the low incidence of AngII-induced AAAs in C57BL/6J mice. Low incidence of AAAs in this strain was not attributed to protection from high-density lipoprotein, because apolipoprotein (apo) AI deficiency did not increase AngII-induced AAAs. ApoAI deletion also failed to alter AAA occurrence in hypercholesterolemic mice. Low-density lipoprotein receptor-/- mice fed normal diet had low incidence of AngII-induced AAAs. Western diet feeding of this strain provoked pronounced hypercholesterolemia because of increased apoB-containing lipoproteins with attendant increases of atherosclerosis in both sexes, but AAAs only in male mice. ApoE-deficient mice fed normal diet were modestly hypercholesterolemic, whereas this strain fed Western diet was severely hypercholesterolemic because of increased apoB-containing lipoprotein concentrations. The latter augmented atherosclerosis, but did not change the high incidence of AAAs in this strain. To determine whether reductions in apoB-containing lipoproteins influenced AngII-induced AAAs, ezetimibe was administered at a dose that partially reduced plasma cholesterol concentrations to ApoE-deficient mice fed Western diet. This decreased atherosclerosis, but not AAAs. This ezetimibe dose in ApoE-deficient mice fed normal diet significantly decreased plasma apoB-containing lipoprotein concentrations and reduced AngII-induced AAAs. CONCLUSIONS: ApoB-containing lipoproteins contribute to augmentation of AngII-induced AAA in male mice. However, unlike atherosclerosis, AAA occurrence was not correlated with increases in plasma apoB-containing lipoprotein concentrations.


Asunto(s)
Angiotensina II , Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/inducido químicamente , Apolipoproteína A-I/sangre , Apolipoproteínas B/sangre , Hipercolesterolemia/complicaciones , Animales , Anticolesterolemiantes/farmacología , Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/sangre , Aneurisma de la Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/prevención & control , Apolipoproteína A-I/deficiencia , Apolipoproteína A-I/genética , Apolipoproteína B-100 , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Azetidinas/farmacología , Dieta Occidental , Modelos Animales de Enfermedad , Ezetimiba , Femenino , Hipercolesterolemia/sangre , Hipercolesterolemia/tratamiento farmacológico , Hipercolesterolemia/genética , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de LDL/deficiencia , Receptores de LDL/genética , Factores Sexuales
20.
Arterioscler Thromb Vasc Biol ; 35(1): 155-62, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25395615

RESUMEN

OBJECTIVE: Low-density lipoprotein receptor-related protein 1 (LRP1), a multifunctional protein involved in endocytosis and cell signaling pathways, leads to several vascular pathologies when deleted in vascular smooth muscle cells (SMCs). The purpose of this study was to determine whether LRP1 deletion in SMCs influenced angiotensin II-induced arterial pathologies. APPROACH AND RESULTS: LRP1 protein abundance was equivalent in selected arterial regions, but SMC-specific LRP1 depletion had no effect on abdominal and ascending aortic diameters in young mice. To determine the effects of LRP1 deficiency on angiotensin II vascular responses, SMC-specific LRP1 (smLRP1(+/+)) and smLRP1-deficient (smLRP1(-/-)) mice were infused with saline, angiotensin II, or norepinephrine. Several smLRP(-/-) mice died of superior mesenteric arterial (SMA) rupture during angiotensin II infusion. In surviving mice, angiotensin II profoundly augmented SMA dilation in smLRP1(-/-) mice. SMA dilation was blood pressure dependent as demonstrated by a similar response during norepinephrine infusion. SMA dilation was also associated with profound macrophage accumulation, but minimal elastin fragmentation. Angiotensin II infusion led to no significant differences in abdominal aorta diameters between smLRP1(+/+) and smLRP1(-/-) mice. In contrast, ascending aortic dilation was exacerbated markedly in angiotensin II-infused smLRP1(-/-) mice, but norepinephrine had no significant effect on either aortic region. Ascending aortas of smLRP1(-/-) mice infused with angiotensin II had minimal macrophage accumulation but significantly increased elastin fragmentation and mRNA abundance of several LRP1 ligands including MMP-2 (matrix metalloproteinase-2) and uPA (urokinase plasminogen activator). CONCLUSIONS: smLRP1 deficiency had no effect on angiotensin II-induced abdominal aortic aneurysm formation. Conversely, angiotensin II infusion in smLRP1(-/-) mice exacerbated SMA and ascending aorta dilation. Dilation in these 2 regions had differential association with blood pressure and divergent pathological characteristics.


Asunto(s)
Aneurisma/metabolismo , Angiotensina II , Aneurisma de la Aorta/metabolismo , Eliminación de Gen , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Receptores de LDL/deficiencia , Proteínas Supresoras de Tumor/deficiencia , Aneurisma/inducido químicamente , Aneurisma/genética , Aneurisma/patología , Aneurisma/fisiopatología , Animales , Aorta Abdominal/metabolismo , Aorta Abdominal/patología , Aneurisma de la Aorta/inducido químicamente , Aneurisma de la Aorta/genética , Aneurisma de la Aorta/patología , Aneurisma de la Aorta/fisiopatología , Aneurisma de la Aorta Abdominal/inducido químicamente , Aneurisma de la Aorta Abdominal/genética , Aneurisma de la Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/patología , Presión Arterial , Células Cultivadas , Dilatación Patológica , Modelos Animales de Enfermedad , Elastina/metabolismo , Ligandos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Macrófagos/metabolismo , Masculino , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Arteria Mesentérica Superior/metabolismo , Arteria Mesentérica Superior/patología , Ratones Noqueados , Músculo Liso Vascular/patología , Músculo Liso Vascular/fisiopatología , Miocitos del Músculo Liso/patología , Norepinefrina , ARN Mensajero/metabolismo , Receptores de LDL/genética , Proteínas Supresoras de Tumor/genética , Activador de Plasminógeno de Tipo Uroquinasa/genética , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo
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