In vivo CD40 Silencing by siRNA Infusion in Rodents and Evaluation by Kidney Immunostaining

Hueso, Miguel; Mallén, Adrián; Ripoll, Élia; Ramon, Laura de; Bolaños, Núria; Varela, Cristián; Guiteras, Jordi; Checa, Javier; Navarro, Estanis; Grinyó, Josep M.; Cruzado, Josep M.; Aran, Josep M.; Torrás, Joan

doi:10.21769/bioprotoc.4032

Cited by 3 publications

(3 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As previously described hearts were perfused, fixed, OCT-embedded and serially sectioned (10 µm) starting in the ascending aorta of the aortic root up to the aortic sinus 29 . The serial slides were stained with Masson’s trichrome for lesion area quantification, or with Oil-Red-O (ORO) and counterstained with hematoxylin in order to analyze aortic root neutral lipid content all together in the same anatomic location (170 µm after the appearance of the 3 rd aortic valve).…”

Section: Methodsmentioning

confidence: 99%

In vivo inhibition of miR-125b modulates monocyte trafficking through the CCR7 receptor and attenuates atherosclerosis

Mallén,

Varela,

Rotllan

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

BackgroundAtherosclerosis (ATH) is a chronic systemic inflammatory disease affecting the vessel wall, wherein regulating non-coding RNAs play a crucial role. We previously demonstrated that miR-125b is upregulated in ATH and is a main regulator of cholesterol metabolism in macrophages. Herein we hypothesized that inhibiting miR-125b may attenuate ATH.Methods and resultsIn the ApoE-/-mice model fed with a high fat diet for 14 weeks, we inhibited miR-125b using an antagomiR over a 4-week period. We observed a significant reduction in plaque size, accompanied by diminished infiltration of F4/80 macrophages and attenuation of NF-κB+activation within plaques. We explored the mechanism using a Vas-on-Chip adhesion assay using Human Aortic Endothelial Cells (HAoEC) stimulated with TNFα. We observed an impairment in the trafficking of miR-125b transfected THP-1 monocytes, accompanied by the downregulation of the CD11b/CD18 integrin and the CCR7 receptor. Furthermore, we demonstrated a direct regulation of the CCR7 receptor by miR-125b using a reporter plasmid construct (p_CCR7.WT) containing the 3’UTR region of CCR7 gene fused with a luciferase coding sequence. In addition, miR-125b transfected monocytes inhibited CCR7 cell migration induced by the CCL21 ligand but did not affect migration induced by others ligands such as MCP1. Finally, we confirmed the downregulation of CCR7 in coronary plaques in both ApoE-/-mice and patients with coronary artery disease.ConclusionsInhibiting miR-125b offers a novel therapeutic approach for ameliorating ATH that results in a reduction of macrophage content and plaque lesion size. This improvement occurs through the enhancement of monocyte trafficking via CCR7 that facilitates the exit of foam cells from the plaque.CLINICAL PERSPECTIVEWhat is New?- We found evidences of a new therapeutic approach for atherosclerosis, in which miR-125b inhibition reduces macrophage content and plaque size.- We described the molecular mechanism underlying miR-125b, which involves regulating of monocyte trafficking to plaques and the downregulation of the chemokine receptor CCR7. CCR7 plays a crucial role in facilitating the egress of macrophages and foam cells from plaques, and its downregulation contribute to progression of ATH.- The results have been validated in a cohort of patients with coronary artery disease, where CCR7 expression was reduced in plaques.What are the clinical implications?- We highlight the pivotal role of monocyte trafficking in the inflammatory mechanism of atherosclerosis. Managing miR-125b/CCR7 signaling may improve the resolution of ATH promoting the exit of foam cells from plaque.- Inhibition of miR-125b in plaque macrophages represents a novel and promising therapeutic approach for cardiovascular disease.

show abstract

Section: Methodsmentioning

confidence: 99%

In vivo inhibition of miR-125b modulates monocyte trafficking through the CCR7 receptor and attenuates atherosclerosis

Mallén,

Varela,

Rotllan

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Hearts were perfused with PBS, removed by cutting the aortic root, and further fixed in 4% paraformaldehyde in cold PBS for 12 h. These were further drained and rinsed overnight in PBS containing 20% sucrose. Hearts, aortic roots, and arches were embedded in OCT, and tissue blocks were cut into serial 10 µm thick sections from the proximal aorta, beginning at the end of the aortic sinus [49]. The composition of the plaques in the aortic root was analyzed by staining with ORO and counterstaining with haematoxylin.…”

Section: Atherosclerotic Lesions Analysismentioning

confidence: 99%

Sex Differences in Glomerular Lesions, in Atherosclerosis Progression, and in the Response to Angiotensin-Converting Enzyme Inhibitors in the ApoE−/− Mice Model

Mallén,

Rodriguez-Urquia,

Alvarez

et al. 2023

IJMS

Self Cite

View full text Add to dashboard Cite

This study analyzes sex-based differences in renal structure and the response to the Angiotensin-Converting Enzyme (ACE) inhibitor enalapril in a mouse model of atherosclerosis. Eight weeks old ApoE−/− mice received enalapril (5 mg/kg/day, subcutaneous) or PBS (control) for an additional 14 weeks. Each group consisted of six males and six females. Females exhibited elevated LDL-cholesterol levels, while males presented higher creatinine levels and proteinuria. Enalapril effectively reduced blood pressure in both groups, but proteinuria decreased significantly only in females. Plaque size analysis and assessment of kidney inflammation revealed no significant sex-based differences. However, males displayed more severe glomerular injury, with increased mesangial expansion, mesangiolysis, glomerular foam cells, and activated parietal epithelial cells (PECs). Enalapril mitigated mesangial expansion, glomerular inflammation (particularly in the female group), and hypertrophy of the PECs in males. This study demonstrates sex-based differences in the response to enalapril in a mouse model of atherosclerosis. Males exhibited more severe glomerular injury, while enalapril provided renal protection, particularly in females. These findings suggest potential sex-specific considerations for ACE inhibitor therapy in chronic kidney disease and atherosclerosis cardiovascular disease. Further research is needed to elucidate the underlying mechanism behind these observations.

show abstract

“…These were further drained and rinsed overnight in PBS containing 20% sucrose. Hearts, aortic roots and arches were embedded in OCT and tissue blocks were cut into serial 10  m-thick sections from the proximal aorta beginning at the end of the aortic sinus [38]. The composition of the plaques in the aortic root was analysed by staining with ORO and counterstaining with haematoxylin.…”

Section: Atherosclerotic Lesions Analysismentioning

confidence: 99%

Sex Differences In Glomerular Lesions, Atherosclerosis Progression, And In The Response To Angiotensin-Converting Enzyme Inhibitors In The Apoe<sup>-/-</sup> Mice Model

Mallén,

Rodriguez-Urquia,

Alvarez

et al. 2023

Preprint

View full text Add to dashboard Cite

Background: This study analyzes sex-based differences in renal structure and response to the Angiotensin-Converting Enzyme (ACE) inhibitor enalapril in a mouse model of atherosclerosis. Methods: ApoE-/- mice (8 weeks old) received enalapril (5 mg/kg/day, subcutaneous) or PBS as a control for an additional 14 weeks. Each group consisted of six males and six females. Results: Females exhibited elevated LDL-cholesterol levels, while males presented higher creatinine levels and proteinuria. Enalapril effectively reduced blood pressure in both groups, but proteinuria decreased significantly only in females. Plaque size analysis and assessment of kidney inflammation revealed no significant sex-based differences. However, males displayed more severe glomerular injury, with increased mesangial expansion, mesangiolysis, glomerular foam cells and activated parietal epithelial cells (PECs). Enalapril mitigated mesangial expansion, glomerular inflammation (particularly in females), and the hypertrophy of PECs in males. Conclusion: This study demonstrates sex-based differences in the response to enalapril in a mouse model of atherosclerosis. Males exhibited more severe glomerular injury, while enalapril provided renal protection, particularly in females. These findings suggest potential sex-specific considerations for ACE inhibitor therapy in chronic kidney disease and atherosclerosis cardiovascular disease. Further research is needed to elucidate the underlying mechanism behind these observations.

show abstract

In vivo CD40 Silencing by siRNA Infusion in Rodents and Evaluation by Kidney Immunostaining

Cited by 3 publications

References 16 publications

In vivo inhibition of miR-125b modulates monocyte trafficking through the CCR7 receptor and attenuates atherosclerosis

In vivo inhibition of miR-125b modulates monocyte trafficking through the CCR7 receptor and attenuates atherosclerosis

Sex Differences in Glomerular Lesions, in Atherosclerosis Progression, and in the Response to Angiotensin-Converting Enzyme Inhibitors in the ApoE−/− Mice Model

Sex Differences In Glomerular Lesions, Atherosclerosis Progression, And In The Response To Angiotensin-Converting Enzyme Inhibitors In The Apoe<sup>-/-</sup> Mice Model

Contact Info

Product

Resources

About