Angiotensin II–driven coronary vasculopathy and pressure-overload myocardial remodeling represent distinct vascular phenotypes

Figures

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  Figure 1. Mouse models of cardiac remodeling. Transverse aortic constriction (TAC; 28 days) induces pressure overload–mediated remodeling. AngII (1.5 µg/g/day) plus phenylephrine (50 µg/g/day) produces an HFpEF model, and high-dose AngII (HD-AngII; 3 µg/g/day for 10 or 28 days) generates a vasculopathy-predominant remodeling phenotype.

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  Figure 2. Hemodynamic assessment across cardiac remodeling models. (A-C) Retrograde left ventricular (LV) catheterization-derived systolic pressure and LV end-diastolic pressure (LVEDP) in TAC (after 28 days), AngII/PE and HD-AngII (after 28 days) groups. (A) TAC surgery produced the highest systolic and (B) diastolic blood pressure. (C) LVEDP remained comparable across all experimental conditions. (D) Quantitative assessment of neurohumoral activation by serum AngII levels at day 28. AngII concentrations were minimal in controls and significantly increased in all three stress models, with the highest concentrations in HD-AngII mice, intermediate levels in AngII/PE, and a more modest elevation in TAC. Group sizes were n≥7 per mouse model. Statistical significance was defined as: * P < 0.05; ** P < 0.01, and **** P < 0.0001. Data was analyzed using two-way ANOVA with multiple comparisons. No significant sex-related differences (males are shown in blue and females in red) in hemodynamic parameters were observed in either untreated or treated groups across all models.

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  Figure 3. Cardiomyocyte hypertrophy and death across remodeling models. (A) Low-magnification Masson’s trichrome and IF images of the whole heart provide an anatomic roadmap for the higher-magnification insets shown in subsequent panels (white arrows – area proximal to ROI; red arrows - area remote to ROI). (B) Representative wheat germ agglutinin (WGA, green), TUNEL (red), and DAPI (blue) staining of left ventricular cardiomyocytes 28 days after AngII/PE or HD-AngII infusion (regions adjacent to and remote from fibrosis/inflammation) or 28 days after TAC surgery. AngII/PE and HD-AngII hearts exhibit heterogeneous cardiomyocyte hypertrophy, with marked CM enlargement, CM necrosis in ROIs and near-normal CM size in remote zones. (C) Quantification of myocyte cross-sectional area adjacent to fibrotic regions showed increased hypertrophy in AngII/PE and HD-AngII groups at day 28; TAC demonstrates more homogeneous enlargement throughout the myocardium. (D) Heart weight-to-tibial length ratios are elevated across all treatment groups, with TAC showing the highest values. Group sizes were n≥7 per mouse model. Statistical significance was defined as: * P < 0.05; *** P < 0.001, and **** P <…

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  Figure 4. Cardiac fibrosis phenotypes across experimental models. (A) Representative Masson’s trichrome-stained myocardial sections demonstrate interstitial, perivascular, and mixed fibrosis phenotypes following 28 days of AngII/PE infusion, 28 days after TAC surgery, or 28 days of HD-AngII infusion. (B) Distribution of fibrosis phenotypes reveals perivascular predominance in the HD-AngII model and interstitial predominance in TAC. (C) Quantitative analysis of interstitial and perivascular fibrosis (ECM relative to total coronary section area) shows the greatest overall fibrosis in HD-AngII models across both compartments, with TAC exhibiting minimal perivascular involvement. Group sizes were n≥6 per each mouse model and cardiac remodeling phenotype. Statistical significance was defined as: ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Data was analyzed using two-way ANOVA with multiple comparisons.

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  Figure 5. Temporal progression of perivascular inflammation, fibrosis, and SMC-to-macrophage phenotypic transition during HD-AngII infusion. (A) Representative Masson’s trichrome staining at 10 and 28 days reveals progressive extracellular matrix deposition and cellular infiltrates in perivascular regions ((A1’–3’) inset magnifications). (B–D) Immunofluorescence for CD68 (red; perivascular macrophages/CD68+ +SMCs, yellow arrows), wheat germ agglutinin (WGA, white), smooth muscle actin (SMA, green), and DAPI (blue) shows persistent perivascular macrophage accumulation from early (day 10) through late (day 28) stages, with maturation of perivascular fibrosis over time; white double arrows define neointima (NI) and perivascular (PV) areas. (E) Quantitative analysis confirms sustained macrophage infiltration (per µm2) across both early and late phases of HD-AngII infusion. (F) Quantification reveals approximately one-third of SMCs acquire macrophage markers by day 10, with the process declining at later time points by day 28. Group sizes were n≥8 at 10 and 28 days of HD-AngII treatment. Statistical significance was defined as: ** P < 0.01, *** P < 0.001 and **** P < 0.0001. Data was analyzed…

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  Figure 6. VSMC dedifferentiation in response to AngII infusion. (A–C) Immunofluoresce staining for smooth muscle actin (SMA; green), SCA1 (red, red arrows), KLF4 (white, white arrows), and DAPI (blue) reveals SMC transition from differentiated to undifferentiated phenotype, marked by co-expression of SCA1 and KLF4. Individual channel views showing KLF4/SMA/DAPI, SCA1/KLF4/DAPI, and SCA1/KLF4/SMA/DAPI staining patterns. (D) Quantification of SCA1 + and SMA+ cells per vessel. (E) Quantification of KLF4+, SMA+ cells per vessel. Group sizes were n≥8 at 10 and 28 days of HD-AngII treatment. Statistical significance was defined as: ** P < 0.01, *** P < 0.001 and **** P < 0.0001. Data was analyzed using two-way ANOVA with multiple comparisons.

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  Figure 7. SMC proliferation and quiescence dynamics during HD-AngII infusion. (A) Bright-field immunohistochemistry for Ki67 (pink, white arrows) and SMA (brown) reveals SMC proliferation at early stages. (B–D) Immunofluorescence for Ki67 (red, red arrows) and CDKN1a (white, white arrows) shows senescent SMCs (CDKN1a+ nuclei) in controls, proliferative states (Ki67 + nuclei) at early infusion (7–10 days), and absence of proliferation at late stages. Individual channel views of CDKN1a/SMA, SMA/DAPI, Ki67/SMA, and SMA/DAPI staining. (E) Quantification show peak of proliferative state (Ki67+, SMA+ cells per vessel) at 10 days of HD-AngII infusion. (F) Quantification showing peak quiescence (CDKN1A+, SMA+ cells per vessel) of VSMC in untreated mice. Group sizes were n ≥ 8 at 10 and 28 days of HD-AngII treatment. Statistical significance was defined as: *** P < 0.001 and **** P < 0.0001. Data was analyzed using two-way ANOVA with multiple comparisons.

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  Figure 8. Vascular elastic membrane remodeling and endothelial/smooth muscle cell dynamics during HD-AngII infusion. (A) Experimental plan for SMC and EC lineage–tracing, showing SMMHC-CreER and Cdh5-CreERT2;ROSAtdTomato induction with tamoxifen at 3–5 weeks of age, followed by a 4-week washout period before HD-AngII minipump implantation. (B) Endothelial lineage tracing in AngII-induced coronary vasculopathy. Endothelial cell dynamics were assessed using Cdh5-CreERT2; ROSAtdTomato mice (EC lineage label, red) in relation to the elastic lamina (elastin, green) at early (7–10 days) and late (28 days) stages of HD-AngII infusion. (C) SMC lineage labeling (ROSA-tdTomato, red) in AngII-induced coronary vasculopathy. SMC dynamics were assessed using SMMHC-CreER; ROSAtdTomato(red), and elastin (green). (D–I) Quantitative morphometry of immunofluorescence images in HD-AngII–treated mice: (D) HD AngII infusion induced marked concentric remodeling, with a significant increase in combined tunica media plus neointima thickness compared with controls; (E) perivascular fibrosis was substantially higher in AngII treated vessels; (F) the number of SMC layers across the tunica media and neointima was…

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  Figure 9. Serum and urine desmosine levels across cardiac remodeling phenotypes (A) after 28 days of HD-AngII infusion, serum desmosine concentrations were highest in mice with a vascular phenotype and showed only a modest increase in those with an interstitial phenotype compared with untreated controls (N=9 untreated, N=28 vascular, N=13 interstitial). (B) After 28 days of HD-AngII infusion, urine desmosine concentrations were similarly elevated in treated mice irrespective of histologic subtype, with no significant differences between vascular and interstitial phenotypes (N = 8 untreated, N=12 vascular, N=8 interstitial). Statistical significance was defined as: * P < 0.05; *** P < 0.001, and **** P < 0.0001. Data was analyzed using two-way ANOVA with multiple comparisons.

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  Figure 10. AT1R distribution and pAKT activation in AngII–induced vasculopathy.(A) Representative Masson's trichrome–stained section of an untreated heart illustrating the region from which the AT1R immunofluorescence insets were obtained (yellow arrows). (B) High magnification views of regions indicated by yellow arrows in A. AT1R expression (red) in smooth muscle cells (SMA; green), endothelial cells, cardiomyocytes (ventricular and atrial), and valvular structures (WGA; white). Yellow arrows highlight regions of interest (vascular, atrioventricular valve, ventricular and atrial insets), and red arrows indicate areas with high AT1R expression. (C-E) Phosphorylated AKT (pAKT; red) in VSMCs (SMA; green) is absent in untreated hearts (C), appears by day 10 of HD-AngII infusion (D), and persists through day 28 (E) pAKT localizes predominantly to cytosolic regions adjacent to the plasma membrane rather than to nuclei (DAPI; blue).

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  Figure 11. Model of SMC phenotypic switching during AngII infusion. Smooth muscle cells undergo sequential phenotypic transitions: (1) contractile state expressing CDKN1A, (2) undifferentiated state marked by SCA1 and KLF4 expression, (3) macrophage-like state expressing CD68, and (4) hypertrophic phenotype.