RESUMEN
Single-cell technologies such as flow cytometry and single-cell RNA sequencing have allowed for comprehensive characterization of the kidney cellulome. However, there is a disparity in the various protocols for preparing kidney single-cell suspensions. We aimed to address this limitation by characterizing kidney cellular heterogeneity using three previously published single-cell preparation protocols. Single-cell suspensions were prepared from male and female C57BL/6 kidneys using the following kidney tissue dissociation protocols: a scRNAseq protocol (P1), a multi-tissue digestion kit from Miltenyi Biotec (P2), and a protocol established in our laboratory (P3). Following dissociation, flow cytometry was used to identify known major cell types including leukocytes (myeloid and lymphoid), vascular cells (smooth muscle and endothelial), nephron epithelial cells (intercalating, principal, proximal, and distal tubule cells), podocytes, and fibroblasts. Of the protocols tested, P2 yielded significantly less leukocytes and type B intercalating cells compared with the other techniques. P1 and P3 produced similar yields for most cell types; however, endothelial and myeloid-derived cells were significantly enriched using P1. Significant sex differences were detected in only two cell types: granulocytes (increased in males) and smooth muscle cells (increased in females). Future single-cell studies that aim to enrich specific kidney cell types may benefit from this comparative analysis.NEW & NOTEWORTHY This study is the first to evaluate published single-cell suspension preparation protocols and their ability to produce high-quality cellular yields from the mouse kidney. Three single-cell digestion protocols were compared and each produced significant differences in kidney cellular heterogeneity. These findings highlight the importance of the digestion protocol when using single-cell technologies. This study may help future single-cell science research by guiding researchers to choose protocols that enrich certain cell types of interest.
Asunto(s)
Riñón , Ratones Endogámicos C57BL , Análisis de la Célula Individual , Animales , Análisis de la Célula Individual/métodos , Femenino , Masculino , Ratones , Riñón/metabolismo , Riñón/citología , Citometría de Flujo/métodos , Células Endoteliales/metabolismo , Células Endoteliales/citología , Células Epiteliales/metabolismo , Células Epiteliales/citologíaRESUMEN
Influenza A virus (IAV) infection during pregnancy causes severe maternal and perinatal complications, despite a lack of vertical transmission of IAV across the placenta. Here, we demonstrate a significant alteration in the maternal vascular landscape that underpins the maternal and downstream fetal pathology to IAV infection in mice. In IAV infection of nonpregnant mice, the local lung inflammatory response was contained to the lungs and was self-resolving, whereas in pregnant mice, virus dissemination to major maternal blood vessels, including the aorta, resulted in a peripheral "vascular storm," with elevated proinflammatory and antiviral mediators and the influx of Ly6Clow and Ly6Chigh monocytes, plus neutrophils and T cells. This vascular storm was associated with elevated levels of the adhesion molecules ICAM and VCAM and the pattern-recognition receptors TLR7 and TLR9 in the vascular wall, resulting in profound vascular dysfunction. The sequalae of this IAV-driven vascular storm included placental growth retardation and intrauterine growth restriction, evidence of placental and fetal brain hypoxia, and increased circulating cell free fetal DNA and soluble Flt1. In contrast, IAV infection in nonpregnant mice caused no obvious alterations in endothelial function or vascular inflammation. Therefore, IAV infection during pregnancy drives a significant systemic vascular alteration in pregnant dams, which likely suppresses critical blood flow to the placenta and fetus. This study in mice provides a fundamental mechanistic insight and a paradigm into how an immune response to a respiratory virus, such as IAV, is likely to specifically drive maternal and fetal pathologies during pregnancy.
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Inmunidad Adaptativa/genética , Inmunidad Innata/genética , Inflamación/genética , Virus de la Influenza A/genética , Animales , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patología , Femenino , Feto/inmunología , Humanos , Inflamación/inmunología , Inflamación/patología , Inflamación/virología , Virus de la Influenza A/patogenicidad , Gripe Humana/genética , Gripe Humana/inmunología , Gripe Humana/virología , Glicoproteínas de Membrana/genética , Ratones , Monocitos/metabolismo , Monocitos/patología , Placenta/irrigación sanguínea , Placenta/inmunología , Placenta/virología , Embarazo , Linfocitos T/inmunología , Linfocitos T/virología , Receptor Toll-Like 7/genética , Receptor Toll-Like 9/genéticaRESUMEN
BACKGROUND: Cardiac fibrosis is a key antecedent to many types of cardiac dysfunction including heart failure. Physiological factors leading to cardiac fibrosis have been recognized for decades. However, the specific cellular and molecular mediators that drive cardiac fibrosis, and the relative effect of disparate cell populations on cardiac fibrosis, remain unclear. METHODS: We developed a novel cardiac single-cell transcriptomic strategy to characterize the cardiac cellulome, the network of cells that forms the heart. This method was used to profile the cardiac cellular ecosystem in response to 2 weeks of continuous administration of angiotensin II, a profibrotic stimulus that drives pathological cardiac remodeling. RESULTS: Our analysis provides a comprehensive map of the cardiac cellular landscape uncovering multiple cell populations that contribute to pathological remodeling of the extracellular matrix of the heart. Two phenotypically distinct fibroblast populations, Fibroblast-Cilp and Fibroblast-Thbs4, emerged after induction of tissue stress to promote fibrosis in the absence of smooth muscle actin-expressing myofibroblasts, a key profibrotic cell population. After angiotensin II treatment, Fibroblast-Cilp develops as the most abundant fibroblast subpopulation and the predominant fibrogenic cell type. Mapping intercellular communication networks within the heart, we identified key intercellular trophic relationships and shifts in cellular communication after angiotensin II treatment that promote the development of a profibrotic cellular microenvironment. Furthermore, the cellular responses to angiotensin II and the relative abundance of fibrogenic cells were sexually dimorphic. CONCLUSIONS: These results offer a valuable resource for exploring the cardiac cellular landscape in health and after chronic cardiovascular stress. These data provide insights into the cellular and molecular mechanisms that promote pathological remodeling of the mammalian heart, highlighting early transcriptional changes that precede chronic cardiac fibrosis.
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Cardiomegalia/metabolismo , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Miocardio/metabolismo , Análisis de la Célula Individual , Estrés Fisiológico , Animales , Cardiomegalia/patología , Fibroblastos/patología , Fibrosis , Ratones , Miocardio/patología , Pirofosfatasas/metabolismo , Trombospondinas/metabolismoRESUMEN
BACKGROUND: High blood pressure (BP) continues to be a major, poorly controlled but modifiable risk factor for cardiovascular death. Among key Western lifestyle factors, a diet poor in fiber is associated with prevalence of high BP. The impact of lack of prebiotic fiber and the associated mechanisms that lead to higher BP are unknown. Here we show that lack of prebiotic dietary fiber leads to the development of a hypertensinogenic gut microbiota, hypertension and its complications, and demonstrate a role for G-protein coupled-receptors (GPCRs) that sense gut metabolites. METHODS: One hundred seventy-nine mice including C57BL/6J, gnotobiotic C57BL/6J, and knockout strains for GPR41, GPR43, GPR109A, and GPR43/109A were included. C57BL/6J mice were implanted with minipumps containing saline or a slow-pressor dose of angiotensin II (0.25 mg·kg-1·d-1). Mice were fed diets lacking prebiotic fiber with or without addition of gut metabolites called short-chain fatty acids ([SCFA)] produced during fermentation of prebiotic fiber in the large intestine), or high prebiotic fiber diets. Cardiac histology and function, BP, sodium and potassium excretion, gut microbiome, flow cytometry, catecholamines and methylation-wide changes were determined. RESULTS: Lack of prebiotic fiber predisposed mice to hypertension in the presence of a mild hypertensive stimulus, with resultant pathological cardiac remodeling. Transfer of a hypertensinogenic microbiota to gnotobiotic mice recapitulated the prebiotic-deprived hypertensive phenotype, including cardiac manifestations. Reintroduction of SCFAs to fiber-depleted mice had protective effects on the development of hypertension, cardiac hypertrophy, and fibrosis. The cardioprotective effect of SCFAs were mediated via the cognate SCFA receptors GPR43/GPR109A, and modulated L-3,4-dihydroxyphenylalanine levels and the abundance of T regulatory cells regulated by DNA methylation. CONCLUSIONS: The detrimental effects of low fiber Westernized diets may underlie hypertension, through deficient SCFA production and GPR43/109A signaling. Maintaining a healthy, SCFA-producing microbiota is important for cardiovascular health.
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Fibras de la Dieta/deficiencia , Ácidos Grasos Volátiles/metabolismo , Microbioma Gastrointestinal , Hipertensión , Mucosa Intestinal , Prebióticos , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Animales , Hipertensión/genética , Hipertensión/metabolismo , Hipertensión/microbiología , Hipertensión/patología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Masculino , Ratones , Ratones Noqueados , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Despite its well-known antithrombotic properties, the effect of aspirin on blood pressure (BP) and hypertension pathology is unclear. The hugely varying doses used clinically have contributed to this confusion, with high-dose aspirin still commonly used due to concerns about the efficacy of low-dose aspirin. Because prostaglandins have been shown to both promote and inhibit T-cell activation, we also explored the immunomodulatory properties of aspirin in hypertension. Although the common preclinical high dose of 100 mg/kg/d improved vascular dysfunction and cardiac hypertrophy, this effect was accompanied by indices of elevated adaptive immunity, renal T-cell infiltration, renal fibrosis, and BP elevation in stroke-prone spontaneously hypertensive rats and in angiotensin II-induced hypertensive mice. The cardioprotective effects of aspirin were conserved with a lower dose (10 mg/kg/d) while circumventing heightened adaptive immunity and elevated BP. We also show that low-dose aspirin improves renal fibrosis. Differential inhibition of the COX-2 isoform may underlie the disparate effects of the 2 doses. Our results demonstrate the efficacy of low-dose aspirin in treating a vast array of cardiovascular parameters and suggest modulation of adaptive immunity as a novel mechanism underlying adverse cardiovascular profiles associated with COX-2 inhibitors. Clinical studies should identify the dose of aspirin that achieves maximal cardioprotection with a new awareness that higher doses of aspirin could trigger undesired autoimmunity in hypertensive individuals. This work also warrants an evaluation of high-dose aspirin and COX-2 inhibitor therapy in sufferers of inflammatory conditions who are already at increased risk for cardiovascular disease.-Khan, S. I., Shihata, W. A., Andrews, K. L., Lee, M. K. S., Moore, X.-L., Jefferis, A.-M., Vinh, A., Gaspari, T., Dragoljevic, D., Jennings, G. L., Murphy, A. J., Chin-Dusting, J. P. F. Effects of high- and low-dose aspirin on adaptive immunity and hypertension in the stroke-prone spontaneously hypertensive rat.
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Inmunidad Adaptativa/efectos de los fármacos , Aspirina/farmacología , Hipertensión/tratamiento farmacológico , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/inmunología , Angiotensina II/farmacología , Animales , Aspirina/administración & dosificación , Aspirina/uso terapéutico , Biomarcadores/sangre , Presión Sanguínea/efectos de los fármacos , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/fisiopatología , Cardiomegalia/tratamiento farmacológico , Ciclooxigenasa 1/genética , Ciclooxigenasa 2/genética , Citocinas/sangre , Susceptibilidad a Enfermedades , Relación Dosis-Respuesta a Droga , Epoprostenol/biosíntesis , Hipertensión/inducido químicamente , Riñón/efectos de los fármacos , Riñón/enzimología , Riñón/patología , Ratones , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas SHR , Reacción en Cadena en Tiempo Real de la Polimerasa , Sístole , Linfocitos T/inmunología , Tromboxanos/sangreRESUMEN
The essential role of the Y chromosome in male sex determination has largely overshadowed the possibility that it may exert other biologic roles. Here, we show that Y-chromosome lineage is a strong determinant of perivascular and renal T-cell infiltration in the stroke-prone spontaneously hypertensive rat, which, in turn, may influence vascular function and blood pressure (BP). We also show, for the first time to our knowledge, that augmented perivascular T-cell levels can directly instigate vascular dysfunction, and that the production of reactive oxygen species that stimulate cyclo-oxygenase underlies this. We thus provide strong evidence for the consideration of Y-chromosome lineage in the diagnosis and treatment of male hypertension, and point to the modulation of cardiovascular organ T-cell infiltration as a possible mechanism that underpins Y- chromosome regulation of BP.-Khan, S. I., Andrews, K. L., Jackson, K. L., Memon, B., Jefferis, A.-M., Lee, M. K. S., Diep, H., Wei, Z., Drummond, G. R., Head, G. A., Jennings, G. L., Murphy, A. J., Vinh, A., Sampson, A. K., Chin-Dusting, J. P. F. Y-chromosome lineage determines cardiovascular organ T-cell infiltration in the stroke-prone spontaneously hypertensive rat.
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Presión Sanguínea , Hipertensión/metabolismo , Hipertensión/fisiopatología , Linfocitos T/metabolismo , Cromosoma Y/metabolismo , Animales , Hipertensión/genética , Masculino , Ratas , Ratas Endogámicas SHR , Ratas Transgénicas , Linfocitos T/patología , Cromosoma Y/genéticaRESUMEN
Initial studies suggest that increased age is associated with worse outcomes after traumatic brain injury (TBI), though the pathophysiological mechanisms responsible for this remain unclear. Immunosenescence (i.e., dysregulation of the immune system due to aging) may play a significant role in influencing TBI outcomes. This study therefore examined neurological outcomes and immune response in young-adult (i.e., 10â¯weeks old) compared to middle-aged (i.e., 1â¯year old) rats following a TBI (i.e., fluid percussion) or sham-injury. Rats were euthanized at either 24â¯h or one-week post-injury to analyze immune cell populations in the brain and periphery via flow cytometry, as well as telomere length (i.e., a biomarker of neurological health). Behavioral testing, as well as volumetric and diffusion-weighted MRI, were also performed in the one-week recovery rats to assess for functional deficits and brain damage. Middle-aged rats had worse sensorimotor deficits and shorter telomeres after TBI compared to young rats. Both aging and TBI independently worsened cognitive function and cortical volume. These changes occurred in the presence of fewer total leukocytes, fewer infiltrating myeloid cells, and fewer microglia in the brains of middle-aged TBI rats compared to young rats. These findings indicate that middle-aged rats have worse sensorimotor deficits and shorter telomeres after TBI than young rats, and this may be related to an altered neuroimmune response. Although further studies are required, these findings have important implications for understanding the pathophysiology and optimal treatment strategies in TBI patients across the life span.
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Lesiones Traumáticas del Encéfalo/inmunología , Neuroinmunomodulación/inmunología , Recuperación de la Función/inmunología , Factores de Edad , Animales , Encéfalo/fisiopatología , Lesiones Traumáticas del Encéfalo/metabolismo , Cognición/fisiología , Trastornos del Conocimiento/complicaciones , Modelos Animales de Enfermedad , Masculino , Microglía/inmunología , Microglía/metabolismo , Ratas , Ratas Wistar , Recuperación de la Función/fisiología , Homeostasis del Telómero/inmunología , Resultado del TratamientoRESUMEN
Macrophages, which accumulate in tissues during inflammation, may be polarized toward pro-inflammatory (M1) or tissue reparative (M2) phenotypes. The balance between these phenotypes can have a substantial influence on the outcome of inflammatory diseases such as atherosclerosis. Improved biomarkers of M1 and M2 macrophages would be beneficial for research, diagnosis, and monitoring the effects of trial therapeutics in such diseases. To identify novel biomarkers, we have characterized the global proteomes of THP-1 macrophages polarized to M1 and M2 states in comparison with unpolarized (M0) macrophages. M1 polarization resulted in increased expression of numerous pro-inflammatory proteins including the products of 31 genes under the transcriptional control of interferon regulatory factor 1 (IRF-1). In contrast, M2 polarization identified proteins regulated by components of the transcription factor AP-1. Among the most highly upregulated proteins under M1 conditions were the three interferon-induced proteins with tetratricopeptide repeats (IFITs: IFIT1, IFIT2, and IFIT3), which function in antiviral defense. Moreover, IFIT1, IFIT2, and IFIT3 mRNA were strongly upregulated in M1 polarized human primary macrophages and IFIT1 was also expressed in a subset of macrophages in aortic sinus and brachiocephalic artery sections from atherosclerotic ApoE-/- mice. On the basis of these results, we propose that IFITs may serve as useful markers of atherosclerosis and potentially other inflammatory diseases.
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Factor 1 Regulador del Interferón/genética , Macrófagos/inmunología , Proteínas/análisis , Proteómica/métodos , Repeticiones de Tetratricopéptidos , Animales , Aterosclerosis/diagnóstico , Aterosclerosis/patología , Biomarcadores/análisis , Humanos , Inflamación/diagnóstico , Inflamación/patología , Macrófagos/química , Ratones , Ratones Noqueados , Proteínas/genética , Células THP-1 , Regulación hacia Arriba/genéticaRESUMEN
BACKGROUND AND PURPOSE: Human amnion epithelial cells (hAECs) are nonimmunogenic, nontumorigenic, anti-inflammatory cells normally discarded with placental tissue. We reasoned that their profile of biological features, wide availability, and the lack of ethical barriers to their use could make these cells useful as a therapy in ischemic stroke. METHODS: We tested the efficacy of acute (1.5 hours) or delayed (1-3 days) poststroke intravenous injection of hAECs in 4 established animal models of cerebral ischemia. Animals included young (7-14 weeks) and aged mice (20-22 months) of both sexes, as well as adult marmosets of either sex. RESULTS: We found that hAECs administered 1.5 hours after stroke in mice migrated to the ischemic brain via a CXC chemokine receptor type 4-dependent mechanism and reduced brain inflammation, infarct development, and functional deficits. Furthermore, if hAECs administration was delayed until 1 or 3 days poststroke, long-term functional recovery was still augmented in young and aged mice of both sexes. We also showed proof-of-principle evidence in marmosets that acute intravenous injection of hAECs prevented infarct development from day 1 to day 10 after stroke. CONCLUSIONS: Systemic poststroke administration of hAECs elicits marked neuroprotection and facilitates mechanisms of repair and recovery.
Asunto(s)
Amnios/trasplante , Células Epiteliales/trasplante , Neuroprotección , Accidente Cerebrovascular/terapia , Animales , Callithrix , Modelos Animales de Enfermedad , Femenino , Xenoinjertos , Humanos , Masculino , Ratones , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/patologíaRESUMEN
Recent studies have emphasized the role of perivascular inflammation in cardiovascular disease. We studied mechanisms of perivascular leukocyte infiltration in angiotensin II (Ang II)-induced hypertension and their links to vascular dysfunction. Chronic Ang II infusion in mice increased immune cell content of T cells (255 ± 130 to 1664 ± 349 cells/mg; P < 0.01), M1 and M2 macrophages, and dendritic cells in perivascular adipose tissue. In particular, the content of T lymphocytes bearing CC chemokine receptor (CCR) 1, CCR3, and CCR5 receptors for RANTES chemokine was increased by Ang II (CCR1, 15.6 ± 1.5% vs. 31 ± 5%; P < 0.01). Hypertension was associated with an increase in perivascular adipose tissue expression of the chemokine RANTES (relative quantification, 1.2 ± 0.2 vs. 3.5 ± 1.1; P < 0.05), which induced T-cell chemotaxis and vascular accumulation of T cells expressing the chemokine receptors CCR1, CCR3, and CCR5. Mechanistically, RANTES(-/-) knockout protected against vascular leukocyte, and in particular T lymphocyte infiltration (26 ± 5% in wild type Ang II vs. 15 ± 4% in RANTES(-/-)), which was associated with protection from endothelial dysfunction induced by Ang II. This effect was linked with diminished infiltration of IFN-γ-producing CD8(+) and double-negative CD3(+)CD4(-)CD8(-) T cells in perivascular space and reduced vascular oxidative stress while FoxP3(+) T-regulatory cells were unaltered. IFN-γ ex vivo caused significant endothelial dysfunction, which was reduced by superoxide anion scavenging. In a human cohort, a significant inverse correlation was observed between circulating RANTES levels as a biomarker and vascular function measured as flow-mediated dilatation (R = -0.3, P < 0.01) or endothelial injury marker von Willebrand factor (R = +0.3; P < 0.01). Thus, chemokine RANTES is important in the regulation of vascular dysfunction through modulation of perivascular inflammation.-Mikolajczyk, T. P., Nosalski, R., Szczepaniak, P., Budzyn, K., Osmenda, G., Skiba, D., Sagan, A., Wu, J., Vinh, A., Marvar, P. J., Guzik, B., Podolec, J., Drummond, G., Lob, H. E., Harrison, D. G., Guzik, T. J. Role of chemokine RANTES in the regulation of perivascular inflammation, T-cell accumulation, and vascular dysfunction in hypertension.
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Quimiocina CCL5/metabolismo , Hipertensión/metabolismo , Linfocitos T/fisiología , Vasculitis/metabolismo , Angiotensina II/farmacología , Animales , Quimiocina CCL5/genética , Femenino , Regulación de la Expresión Génica/fisiología , Humanos , Interferón gamma/genética , Interferón gamma/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Transducción de SeñalRESUMEN
α-Tocopheryl phosphate (TP) is a naturally occurring form of vitamin E found in the body. In the present study we compared the ability of an α-TP mixture (TPM) against a standard vitamin E supplement, α-tocopherol acetate (TA) on the development of atherosclerotic lesions in ApoE-deficient mice. Mice were maintained on either a normal chow diet for 24 weeks (Normal Diet), vs a group in which the final 8 weeks of the 24-week period mice were placed on a high fat (21%), high cholesterol (0.15%) challenge diet (HFHC), to exacerbate atherosclerotic lesion development.. The difference in these two control groups established the extent of the diet-induced atherosclerotic lesion development. Mice in the various treatment groups received either TA (300 mg/kg chow) or TPM (6.7-200 mg/kg chow) for 24 weeks, with TPM treatment resulting in dose-dependent significant reductions in atherosclerotic lesion formation and plasma levels of pro-inflammatory cytokines. TA-treated mice, with the tocopherol equivalent TPM dose (200 mg/kg chow), showed no significant reduction in plasma lipid levels or evidence for aortic lesion regression. At this TPM equivalent TA dose, a 44% reduction in aortic lesion formation was observed. In addition, these TPM treated mice, also showed a marked reduction in aortic superoxide formation and decreased circulating plasma levels of known pro-inflammatory markers IL-6, MCP-1, IL-1ß, IFN-γ and TNF-α. These findings indicate that TPM treatment slows progression of atherosclerotic lesions in ApoE-deficient mice with this effect potentially involving reduced oxidative stress and decreased inflammation.
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Antiinflamatorios/farmacología , Antioxidantes/farmacología , Aorta/efectos de los fármacos , Enfermedades de la Aorta/prevención & control , Aterosclerosis/prevención & control , Placa Aterosclerótica , alfa-Tocoferol/análogos & derivados , Animales , Aorta/metabolismo , Aorta/patología , Enfermedades de la Aorta/sangre , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Aterosclerosis/sangre , Aterosclerosis/genética , Aterosclerosis/patología , Citocinas/sangre , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Mediadores de Inflamación/sangre , Masculino , Ratones Noqueados para ApoE , Estrés Oxidativo/efectos de los fármacos , Superóxidos/metabolismo , alfa-Tocoferol/farmacologíaRESUMEN
Macrophages accumulate in blood vessels during hypertension. However, their contribution to vessel remodeling is unknown. In the present study, we examined the polarization state of macrophages (M1/M2) in aortas of mice during hypertension and investigated whether antagonism of chemokine receptors involved in macrophage accumulation reduces vessel remodeling and blood pressure (BP). Mice treated with ANG II (0.7 mg·kg(-1)·day(-1), 14 days) had elevated systolic BP (158 ± 3 mmHg) compared with saline-treated animals (122 ± 3 mmHg). Flow cytometry revealed that ANG II infusion increased numbers of CD45(+)CD11b(+)Ly6C(hi) monocytes and CD45(+)CD11b(+)F4/80(+) macrophages by 10- and 2-fold, respectively. The majority of macrophages were positive for the M2 marker CD206 but negative for the M1 marker inducible nitric oxide synthase. Expression of other M2 genes (arginase-1, Fc receptor-like S scavenger receptor, and receptor-1) was elevated in aortas from ANG II-treated mice, whereas M1 genes [TNF and chemokine (C-X-C motif) ligand 2] were unaltered. A PCR array to identify chemokine receptor targets for intervention revealed chemokine (C-C motif) receptor 2 (CCR2) to be upregulated in aortas from ANG II-treated mice, while flow cytometry identified Ly6C(hi) monocytes as the main CCR2-expressing cell type. Intervention with a CCR2 antagonist (INCB3344; 30 mg·kg(-1)·day(-1)), 7 days after the commencement of ANG II infusion, reduced aortic macrophage numbers. INCB334 also reduced aortic collagen deposition, elastin loss, and BP in ANG II-treated mice. Thus, ANG II-dependent hypertension in mice is associated with Ly6C(hi) monocyte and M2 macrophage accumulation in the aorta. Inhibition of macrophage accumulation with a CCR2 antagonist prevents ANG II-induced vessel fibrosis and elevated BP, highlighting this as a promising approach for the future treatment of vessel remodeling/stiffening in hypertension.
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Aorta/patología , Presión Sanguínea , Elastina/metabolismo , Hipertensión/patología , Macrófagos/metabolismo , Angiotensina II/toxicidad , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos Ly/genética , Antígenos Ly/metabolismo , Aorta/efectos de los fármacos , Aorta/metabolismo , Arginasa/genética , Arginasa/metabolismo , Colágeno/genética , Colágeno/metabolismo , Elastina/genética , Fibrosis/metabolismo , Fibrosis/patología , Hipertensión/etiología , Hipertensión/metabolismo , Macrófagos/clasificación , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Receptores CCR2/genética , Receptores CCR2/metabolismoRESUMEN
Introduction: Marginal zone and follicular B cells are known to contribute to the development of angiotensin II-induced hypertension in mice, but the effector function(s) mediating this effect (e.g., antigen presentation, antibody secretion and/or cytokine production) are unknown. B cell differentiation into antibody secreting cells (ASCs) requires the transcription factor Blimp-1. Here, we studied mice with a Blimp-1 deficiency in follicular B cells to evaluate whether antibody secretion underlies the pro-hypertensive action of B cells. Methods: 10- to 14-week-old male follicular B cell Blimp-1 knockout (FoB-Blimp-1-KO) and floxed control mice were subcutaneously infused with angiotensin II (0.7â mg/kg/d) or vehicle (0.1% acetic acid in saline) for 28 days. BP was measured by tail-cuff plethysmography or radiotelemetry. Pulse wave velocity was measured by ultrasound. Aortic collagen was quantified by Masson's trichrome staining. Cell types and serum antibodies were quantified by flow cytometry and a bead-based multiplex assay, respectively. Results: In control mice, angiotensin II modestly increased serum IgG3 levels and markedly increased BP, cardiac hypertrophy, aortic stiffening and fibrosis. FoB-Blimp-1-KO mice exhibited impaired IgG1, IgG2a and IgG3 production despite having comparable numbers of B cells and ASCs to control mice. Nevertheless, FoB-Blimp-1-KO mice still developed hypertension, cardiac hypertrophy, aortic stiffening and fibrosis following angiotensin II infusion. Conclusions: Inhibition of follicular B cell differentiation into ASCs did not protect against angiotensin II-induced hypertension or vascular compliance. Follicular B cell functions independent of their differentiation into ASCs and ability to produce high-affinity antibodies, or other B cell subtypes, are likely to be involved in angiotensin II-induced hypertension.
RESUMEN
Aortic diseases such as atherosclerosis, aortic aneurysms, and aortic stiffening are significant complications that can have significant impact on end-stage cardiovascular disease. With limited pharmacological therapeutic strategies that target the structural changes in the aorta, surgical intervention remains the only option for some patients with these diseases. Although there have been significant contributions to our understanding of the cellular architecture of the diseased aorta, particularly in the context of atherosclerosis, furthering our insight into the cellular drivers of disease is required. The major cell types of the aorta are well defined; however, the advent of single-cell RNA sequencing provides unrivaled insights into the cellular heterogeneity of each aortic cell type and the inferred biological processes associated with each cell in health and disease. This review discusses previous concepts that have now been enhanced with recent advances made by single-cell RNA sequencing with a focus on aortic cellular heterogeneity.
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Enfermedades de la Aorta , Aterosclerosis , Humanos , ARN , Aorta/metabolismo , Enfermedades de la Aorta/genética , Perfilación de la Expresión Génica , Aterosclerosis/genética , Aterosclerosis/metabolismoRESUMEN
Metabolic syndrome (MetS) is a cluster of metabolic abnormalities affecting ~25% of adults and is linked to chronic diseases such as cardiovascular disease, cancer, and neurodegenerative diseases. Oxidative stress and inflammation are key drivers of MetS. Hesperidin, a citrus bioflavonoid, has demonstrated antioxidant and anti-inflammatory properties; however, its effects on MetS are not fully established. We aimed to determine the optimal dose of hesperidin required to improve oxidative stress, systemic inflammation, and glycemic control in a novel mouse model of MetS. Male 5-week-old C57BL/6 mice were fed a high-fat, high-salt, high-sugar diet (HFSS; 42% kcal fat content in food and drinking water with 0.9% saline and 10% high fructose corn syrup) for 16 weeks. After 6 weeks of HFSS, mice were randomly allocated to either the placebo group or low- (70 mg/kg/day), mid- (140 mg/kg/day), or high-dose (280 mg/kg/day) hesperidin supplementation for 12 weeks. The HFSS diet induced significant metabolic disturbances. HFSS + placebo mice gained almost twice the weight of control mice (p < 0.0001). Fasting blood glucose (FBG) increased by 40% (p < 0.0001), plasma insulin by 100% (p < 0.05), and HOMA-IR by 150% (p < 0.0004), indicating insulin resistance. Hesperidin supplementation reduced plasma insulin by 40% at 140 mg/kg/day (p < 0.0001) and 50% at 280 mg/kg/day (p < 0.005). HOMA-IR decreased by 45% at both doses (p < 0.0001). Plasma hesperidin levels significantly increased in all hesperidin groups (p < 0.0001). Oxidative stress, measured by 8-OHdG, was increased by 40% in HFSS diet mice (p < 0.001) and reduced by 20% with all hesperidin doses (p < 0.005). In conclusion, hesperidin supplementation reduced insulin resistance and oxidative stress in HFSS-fed mice, demonstrating its dose-dependent therapeutic potential in MetS.
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Citrus , Suplementos Dietéticos , Modelos Animales de Enfermedad , Hesperidina , Resistencia a la Insulina , Síndrome Metabólico , Ratones Endogámicos C57BL , Estrés Oxidativo , Animales , Hesperidina/farmacología , Estrés Oxidativo/efectos de los fármacos , Síndrome Metabólico/tratamiento farmacológico , Síndrome Metabólico/metabolismo , Masculino , Ratones , Citrus/química , Relación Dosis-Respuesta a Droga , Glucemia/metabolismo , Glucemia/efectos de los fármacos , Dieta Alta en Grasa/efectos adversos , Antioxidantes/farmacologíaRESUMEN
INTRODUCTION: Animal models are regularly used to test the role of the gut microbiome in hypertension. Small-scale pre-clinical studies have investigated changes to the gut microbiome in the angiotensin II hypertensive model. However, the gut microbiome is influenced by internal and external experimental factors which are not regularly considered in the study design. Once these factors are accounted for, it is unclear if microbiome signatures are reproduceable. We aimed to determine the influence of angiotensin II treatment on the gut microbiome using a large and diverse cohort of mice and to quantify the magnitude by which other factors contribute to microbiome variations. METHODS AND RESULTS: We conducted a retrospective study to establish a diverse mouse cohort resembling large human studies. We sequenced the V4 region of the 16S rRNA gene from 538 samples across the gastrointestinal tract of 303 male and female C57BL/6J mice randomised into sham or angiotensin II treatment from different genotypes, diets, animal facilities, and age groups. Analysing over 17 million sequencing reads, we observed that angiotensin II treatment influenced α-diversity (P = 0.0137) and ß-diversity (i.e., composition of the microbiome, P < 0.001). Bacterial abundance analysis revealed patterns consistent with a reduction in short-chain fatty acid-producers, microbial metabolites that lower blood pressure. Furthermore, animal facility, genotype, diet, age, sex, intestinal sampling site, and sequencing batch had significant effects on both α- and ß-diversity (all P < 0.001). Sampling site (6.8%) and diet (6%) had the largest impact on the microbiome, while angiotensin II and sex had the smallest effect (each 0.4%). CONCLUSIONS: Our large-scale data confirmed findings from small-scale studies that angiotensin II impacted the gut microbiome. However, this effect was modest relative to most of the other factors studied. Accounting for these factors in future pre-clinical hypertensive studies will increase the likelihood that microbiome findings are replicable and translatable.
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Vascular inflammation and fibrosis are hallmarks of hypertension and contribute to the development of cardiovascular disease and cognitive impairment. However, current anti-hypertensive drugs do not treat the underlying tissue damage, such as inflammation-associated fibrosis. Human amnion epithelial cells have several properties amenable for treating vascular pathology. This study tested the effect of amnion epithelial cells on vascular pathology and cognitive impairment during hypertension. Male C57Bl6 mice (8-12 weeks) were administered vehicle (saline; n = 58) or angiotensin II (0.7 mg/kg/d, n = 56) subcutaneously for 14 d. After surgery, a subset of mice were injected with 106 amnion epithelial cells intravenously. Angiotensin II infusion increased systolic blood pressure, aortic pulse wave velocity, accumulation of aortic leukocytes, and aortic mRNA expression of collagen subtypes compared to vehicle-infused mice (n = 9-11, P < 0.05). Administration of amnion epithelial cells attenuated these effects of angiotensin II (P < 0.05). Angiotensin II-induced cognitive impairment was prevented by amnion epithelial cell therapy (n = 7-9, P < 0.05). In the brain, amnion epithelial cells modulated some of the inflammatory genes that angiotensin II promoted differential expression of (n = 6, p-adjusted < 0.05). These findings suggest that amnion epithelial cells could be explored as a potential therapy to inhibit vascular pathology and cognitive impairment during hypertension.
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Disfunción Cognitiva , Hipertensión , Humanos , Animales , Masculino , Ratones , Amnios , Angiotensina II , Análisis de la Onda del Pulso , Ratones Endogámicos C57BL , Hipertensión/terapia , Disfunción Cognitiva/etiología , Disfunción Cognitiva/terapia , Células Epiteliales , Inflamación , FibrosisRESUMEN
Introduction: Depletion of mature B cells affords protection against experimental hypertension. However, whether B cell-mediated hypertension is dependent on differentiation into antibody-secreting cells (ASCs) remains unclear. Using the proteasome inhibitor, bortezomib, the present study tested the effect of ASC reduction on angiotensin II-induced hypertension. Methods: Male C57BL6/J mice were infused with angiotensin II (0.7â mg/kg/day; s.c.) for 28 days via osmotic minipump to induce hypertension. Normotensive control mice received saline infusion. Bortezomib (750â µg/kg) or vehicle (0.1% DMSO) was administered (i.v.) 3 days prior to minipump implantation, and twice weekly thereafter. Systolic blood pressure was measured weekly using tail-cuff plethysmography. Spleen and bone marrow B1 (CD19+B220-), B2 (B220+CD19+) and ASCs (CD138hiSca-1+Blimp-1+) were enumerated by flow cytometry. Serum immunoglobulins were quantified using a bead-based immunoassay. Results: Bortezomib treatment reduced splenic ASCs by â¼68% and â¼64% compared to vehicle treatment in normotensive (2.00 ± 0.30 vs. 0.64 ± 0.15 × 105 cells; n = 10-11) and hypertensive mice (0.52 ± 0.11 vs. 0.14 ± 0.02 × 105 cells; n = 9-11), respectively. Bone marrow ASCs were also reduced by bortezomib in both normotensive (4.75 ± 1.53 vs. 1.71 ± 0.41 × 103 cells; n = 9-11) and hypertensive mice (4.12 ± 0.82 vs. 0.89 ± 0.18 × 103 cells; n = 9-11). Consistent with ASC reductions, bortezomib reduced serum IgM and IgG2a in all mice. Despite these reductions in ASCs and antibody levels, bortezomib did not affect angiotensin II-induced hypertension over 28 days (vehicle: 182 ± 4â mmHg vs. bortezomib: 177 ± 7â mmHg; n = 9-11). Conclusion: Reductions in ASCs and circulating IgG2a and IgM did not ameliorate experimental hypertension, suggesting other immunoglobulin isotypes or B cell effector functions may promote angiotensin II-induced hypertension.
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The association between constipation and cardiovascular risk is unclear. This population-level matched cohort study compared the association of constipation with hypertension and incident cardiovascular events in 541,172 hospitalized patients aged ≥ 60 years. For each constipation admission, one exact age-matched non-constipated admission was randomly selected from all hospitalizations within 2 weeks to form the comparison cohort. The association of constipation with hypertension and cardiovascular events (myocardial infarction, angina, stroke and transient ischemic attack) were analysed using a series of binary logistic regressions adjusting for age, sex, cardiovascular risk factors, gastrointestinal disorders and sociological factors. Patients with constipation had a higher multivariate-adjusted risk for hypertension (odds ratio [OR], 1.96; 95% confidence interval [CI] 1.94-1.99; P < 0.001). Compared to patients with neither constipation nor hypertension, there was a higher multivariate-adjusted risk for cardiovascular events in patients with constipation alone (OR, 1.58; 95% CI 1.55-1.61; P < 0.001) or hypertension alone (OR, 6.12; 95% CI 5.99-6.26; P < 0.001). In patients with both constipation and hypertension, the risk for all cardiovascular events appeared to be additive (OR, 6.53; 95% CI 6.40-6.66; P < 0.001). In conclusion, among hospital patients aged 60 years or older, constipation is linked to an increased risk of hypertension and cardiovascular events. These findings suggest that interventions to address constipation may reduce cardiovascular risk in elderly patients.
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Enfermedades Cardiovasculares , Hipertensión , Accidente Cerebrovascular , Anciano , Humanos , Australia/epidemiología , Enfermedades Cardiovasculares/epidemiología , Enfermedades Cardiovasculares/etiología , Estudios de Cohortes , Estreñimiento/complicaciones , Estreñimiento/epidemiología , Hipertensión/complicaciones , Hipertensión/epidemiología , Pacientes Internos , Factores de Riesgo , Accidente Cerebrovascular/complicacionesRESUMEN
Obesity is a major global health concern, with prevalence rates rapidly rising due to increased availability of highly processed foods rich in fats and/or sugars and technological advances promoting more sedentary behaviour. There is increasing evidence to suggest that obesity predisposes individuals to developing cognitive impairment and dementia. However, the relationship between the brain and the peripheral metabolic state is complex, and many of the underlying mechanisms of cognitive impairment in obesity are yet to be fully elucidated. To better understand the links between obesity and dementia, further work is required to determine pathological changes occurring in the brain during obesity. In this mini-review, we discuss the role of two pathological features of obesity (the gut-brain axis and systemic inflammation) and their potential contribution to dementia.