Conditional ablation of RGS2 in myeloid cells does not influence blood pressure and organ damage in angiotensin II ‐induced hypertension in mice

Figures

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  FIGURE 1. Model Validation of RGS2 Expression in Myeloid Cells. (a) Schematic of magnetic sorting of splenic cells from wildtype (RGS2LysM‐WT), heterozygous (RGS2LysM‐HET), and homozygous (RGS2LysM‐KO) mice. (b) Expression of CD11b in the eluted fraction (non‐myeloid cells) versus the selected fraction (myeloid cells) confirms successful enrichment of myeloid cells in the selected fraction (p < 0.0002, unpaired t‐test with Welch's correction). (c) RGS2 expression in eluted (non‐myeloid) and selected (myeloid) splenic fractions from RGS2LysM‐WT, RGS2LysM‐HET, and RGS2LysM‐KO mice, measured by RT‐qPCR and presented as fold relative to control non‐myeloid cells. Data is presented as mean ± SEM.

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  FIGURE 2. Evaluation of Pressor Response during Ang II Infusion. (a) Systolic blood pressure (SBP) measured by tail cuff plethysmography over 3 weeks of Ang II (1 μg/kg/min) infusion via osmotic minipump. Data are presented as the group mean ± SEM at each time point. (b) Mean SBP presented as a dot‐plot to reveal the distribution of individual animals at Week 3 of Ang II infusion. The error bars reflect the SEM. For statistical analysis, a 2‐way repeated‐measures ANOVA, followed by Sidak's multiple‐comparison post hoc test, was performed. Data from males (squares) and females (circles) were pooled due to the absence of sex differences (Figure S1).

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  FIGURE 3. Evaluation of Renal Injury after Ang II infusion. (a) Urinary albumin to creatinine ratio (μg/mg) in Control and RGS2LysM‐KO after 4 weeks of treatment with vehicle (veh) or Ang II. Urine albumin concentration was determined by ELISA, and urine creatinine concentration was determined using a colorimetric assay. Data were log‐transformed and expressed as μg albumin per mg of creatinine. Ang II increased urinary albumin/creatinine in both genotypes, reaching significance in Control+Ang II versus Control+Vehicle (p < 0.05, two‐way ANOVA followed by Tukey multiple comparison test). (b) Renal cortical mRNA expression levels of the Kidney Injury Molecule‐1 (Kim1), Interleukin‐1 Beta (Il1b), Tumor Necrosis Factor‐Alpha (Tnfa), Nitric Oxide Synthase 2 (Nos2) (top row), Arginase‐1 (Arg1), Mannose Receptor C‐Type 1 (Mrc1), Transforming Growth Factor Beta 1 (Tgf1b), and Collagen Type 1 Alpha 2 Chain (Col1a2) (bottom row) after 4 weeks of treatment measured by quantitative RT‐PCR and plotted as fold relative to Control+Veh. Data are shown as individual animals (males, squares; females, circles) with bar height reflecting the mean and error bars reflecting SEM. Two‐way ANOVA, followed by…

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  FIGURE 4. Evaluation of Immune Cell Infiltration in Renal Cortex after Ang II Infusion. Flow cytometric quantification of renal cortex immune cell populations in male Ang II‐treated Control and RGS2LysM‐KO mice. Total counts per tissue are presented for Gr‐1+ granulocytes, F4/80+ macrophages, CD19+ B cells (top row), CD3+ total T cells, and CD4+ helper or CD8+ cytotoxic T cell infiltrates (bottom row). All data are shown as individual male animals with bar height reflecting the mean and error bars reflecting SEM.

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  FIGURE 5. Evaluation of Vascular Function after Ang II Infusion. Vascular function was assessed using wire myography in the carotid arteries of Control and RGS2LysM‐KO treated with vehicle or Ang II (a–c). Dose response curves to (a) Acetylcholine (Ach), (b) Sodium Nitroprusside (SNP), and (c) Endothelin‐1 (ET‐1) (left) and mean percent relaxation or contraction at the specified concentration. (d) Pulse wave velocity (PWV, m/s) was measured to evaluate arterial stiffness in Ang II‐treated Control and RGS2LysM‐KO mice. Bar‐plot data are shown as individual animals (males, squares; females, circles) with bar height reflecting the mean and error bars reflecting SEM and analyzed using a repeated‐measures 2‐way ANOVA with Sidak multiple‐comparisons procedure.

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  FIGURE 6. Evaluation of Immune Cell Infiltration in Thoracic Aorta after Ang II Infusion. Flow cytometric quantification of thoracic aorta immune cell populations in male Ang II‐treated Control and RGS2LysM‐KO mice. Counts are presented for Gr‐1+ granulocytes, F4/80+ macrophages, CD19+ B cells (top row), CD3+ total T cells, and CD4+ helper or CD8+ cytotoxic T cell infiltrates (bottom row). All data are shown as individual male animals with bar height reflecting the mean and error bars reflecting SEM and analyzed using. unpaired t‐test with Welch's correction.