Fig 2: Effects of D2 on bone volume and its mechanical properties in a mouse model of osteogenesis imperfecta (OI). (A) Comparison of postnatal growth (left) and gross appearance (right) between wild-type (WT) and Mov13 male mice. ∗p < 0.05 (Student's t-test). (B) Bone volume of L3 vertebral bodies of WT and Mov13 female mice orally administered D2 (10 mg/kg/day) or vehicle. 3D micro-CT images (left) and BV/TV (right) of the L3 vertebrae are shown for WT and Mov13 female mice at the age of 8 weeks (after 4-week D2 administration to Mov13 mice). Data are means ± SD (n = 4 to 5). ∗p < 0.05 (Steel–Dwass test). (C) Mechanical properties of L3 vertebral bodies of WT and Mov13 female mice orally administered D2 or vehicle. Load and deformation were measured by compression tests (left). The maximum load is shown in the right panel. Data are means ± SDs (n = 11). ∗p < 0.05 (Steel–Dwass test). (D) Production of type I collagen in primary osteoblasts isolated from either WT or Mov13 mouse calvaria at P1. Cells were treated with or without 10 nM D2 for 2 days. The cellular production of type I collagen was measured by ELISA. Data are means ± SDs (n = 6). ∗p < 0.05, ∗∗p < 0.01 (Steel–Dwass test).

Fig 3: Effects of in vivo miR-199a-3p inhibition on cardiac remodeling in HCM mice. (A) Micrographs depict 5 μm paraffin-embedded heart sections from LNA-control and LNA-antimiR-199a-3p treated mice stained with Masson's Trichrome (MT). Blue staining depicts collagen deposition areas. Scale bar = 0.5 mm (B) Graph shows quantification of fibrosis in heart sections using NIH Image J Software in LNA-control (blue-filled circles) and LNA-antimiR-199a-3p (red-filled circles) treated mice hearts. Data are expressed as mean ± SEM. Statistics: Unpaired 2-tailed t-test with Welch's correction, *p-value <0.05. mRNA levels of collagens (C) Col1a1 and (D) Col3a1 were quantified by real time-quantitative PCR in left ventricular heart tissue obtained from LNA-control (n = 9, blue filled circles) or LNA-antimiR-199a-3p treated HCM mice (n = 7, red filled circles). Relative quantity of each gene was calculated using the 2-DDCT method (Tbp was used as a normalization control). Data are expressed as geometric mean ± SD. Statistics: Unpaired 2-tailed t-test with Welch's correction. P value * p < 0.05 (E) Representative immunoblots show Periostin and Gapdh protein expression (left) and densitometric analysis of Periostin normalized to Gapdh levels (LNA-control n = 9 or LNA-antimiR-199a-3p treated n = 7 HCM mice). Data are presented mean ± SEM. Statistics: Unpaired Mann-Whitney test. P value ** p = 0.005. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig 4: Macrophage–myofibroblast transition (MMT)-induced macrophages acquire fibrotic features after myocardial infarction.a Experimental protocol for inducing the MMT in cardiac macrophages (cMacs) isolated from WT mice with myocardial infarction (MI). cMacs were differentiated into inflammatory M1 macrophages with 100 ng/mL LPS and 30 ng/mL IFN-γ or into anti-inflammatory M2 macrophages with 30 ng/mL IL-4 and 30 ng/mL IL-13. Then, the cells were stimulated with the fibrosis inducer TGF-β1 (5 ng/mL) in culture media supplemented with 1% FBS. Isolated cardiac fibroblasts (cFbs) were used as a control group. b The amount of COL1A1 protein released in the culture media was measured. c Fibrotic genes were analyzed in cMacs and cFbs, as indicated. d Human heart tissues were isolated from consenting recipients with ischemic cardiomyopathy. Immunofluorescence staining revealed that periostin (POSTN)-expressing CD68(+) macrophages produced the collagen protein. White arrows, POSTN(+)CD68(+)COL1A1(+) cells. Scale bars: 20 μm. e Schematic representation showing that macrophages, particularly M2 macrophages, lead to the MMT and increased collagen production. The data are presented as the means ± SEMs. **P < 0.01 and ***P < 0.001 (one-way ANOVA with Tukey’s multiple comparisons test).

Fig 5: LDAH induces LXR-dependent transcriptional changes.A qPCR analysis of Abca1 and Abcg1 mRNA in PM isolated from LDAH-Tg (Tg, blue bars) and LDAH-KO (KO, red bars) mice treated with oxLDL (50 μg/ml) for 48 h vs. PM isolated from their respective WT controls (gray bars) (n = 3-4 independent samples). B Common genes identified in RNA-seq analysis of LDAH-KO (red bars) and LDAH-Tg (blue bars) PM vs. their WT controls at q-value < 0.05. C RNA-seq analyzes identified multiple ECM-related genes induced by LDAH. The upper row in the heat map (Tg) represents the ratio of LDAH-Tg PM vs. WT, and the lower row (KO) represents the ratio of LDAH-KO PM vs. their WT controls (n = 4). D qPCR analysis of Col1a1 and Col1a2 genes in LDAH-Tg (Tg, blue bars) and LDAH-KO (KO, red bars) vs. their respective WT control (gray bars) PM treated with oxLDL (50 μg/ml) for 48 h (n = 3–4). E WT (gray bars) and LDAH-Tg (Tg, blue bars) BMM were treated with siRNA against LXRα (LXR-si) or with non-target siRNA, followed by oxLDL (50 μg/ml) for 48 h. Expression of Nr1h3 (Lxra), Abca1, Abcg1, and Col1a1 was determined by qPCR (n = 3). (F) ELISA quantification of pro-collagen I alpha in WT (gray bar) and LDAH-Tg (Tg, blue bar) BMM treated with ox LDL (50 μg/ml) for 48 h (n = 5–6). G LDAH WT (gray bars) and KO (red bars) PM were treated with oxLDL (50 μg/mL) for 48 h and media was replaced by media with or without TO901317 (5 μM) for 6 h. TO rescued the downregulation of Abca1, Abcg1. and Col1a1 seen under LDAH deficiency (n = 4). H qPCR analysis of Scd1, Fasn and Acaca mRNA in LDAH-Tg (Tg, blue bars) and LDAH-KO (KO, red bars) PM treated with oxLDL (50 μg/ml) for 48 h vs. their respective WT controls (gray bars) (n = 3-4). In the right panel, WT PM were treated with oxLDL (50 μg/ml) for 48 h and media was replaced by media with ( + TO, purple bars with triangles) or without (-TO, gray bars with circular dots) TO901317 (5 μM) for 6 h (n = 5). Comparisons in (A), (D), (E) and (H) were performed by two-tailed unpaired t-test (normally distributed with equal variances), two-tailed Welch’s t-test (normally distributed with unequal variances), or two-tailed Mann-Whitney U (not normally distributed). Comparisons in (F) and (G) were performed by two-way ANOVA followed by Tukey’s test. *p < 0.05, **p < 0.01, ***p < 0.001. All bars represent mean ± SEM of independent samples. Source data are provided as a Source Data file.