Effects of CSL525 on in vitro calcification of human vascular smooth muscle cells (hVSMCs). (A) Calcium content in cultures following calcification induction with 3.3 mM phosphate (P) and increasing concentrations of CSL525 for 21 days. (B) Calcium content in cultures following calcification induction with 3 mM P and 3 mM calcium (Ca) in the presence of increasing concentrations of CSL525 for 14 days. (C) Inhibition of calcification by CSL525 when added at the start of calcification induction. (D) Calcium content in cultures after 7 days of calcification induction with 3 mM P and 3 mM Ca, followed by CSL525 treatment initiated on day 7. (E) Inhibition of calcification by CSL525 when added after 7 days of calcification induction. Results are presented as mean ± SD. Statistical analysis: one-way ANOVA with Tukey’s post hoc test. (**) p < 0.01, (***) p < 0.001, (****) p < 0.0001.

Effect of 1 mM CSL525 in an abcc6a^cmg52/cmg52 zebrafish model of pseudoxanthoma elasticum. A total of 100 abcc6a^cmg52/cmg52 zebrafish larvae were exposed to 1 mM CSL525 at 3 days post-fertilization (dpf) for 7 days, while another group of 100 larvae was kept under the same conditions without CSL525. After 7 days of treatment (10 dpf), the fish were euthanized, fixed in 4% paraformaldehyde and stained with Alizarin Red S. (A) Representative images of the Alizarin Red S staining in abcc6^cmg52/cmg52 zebrafish untreated or treated with CSL525. (B) Survival curves during the 7 days of study. (C) Mineralized fraction area of spinal mineralization. Results are presented as mean ± SD. Statistical analysis: (A) Kaplan–Meier survival analysis. (B,C) Mann-Whitney test. (*) p < 0.05.

Effect of subcutaneous CSL525 at 15 mg/kg three times per week in a pseudoxanthoma elasticum abcc6^-/- mouse model. The abcc6^-/- mice were subcutaneously administered CSL525 at 15 mg/kg or 0.9% NaCl thrice weekly for eight weeks. A group of C57BL/6J wild-type mice, also receiving 0.9% NaCl thrice weekly, was included in the study. Each group consisted of five males and five females. All mice were maintained on a standard diet and sacrificed at 13 weeks of age, eight weeks after treatment initiation. (A) Histopathology of vibrissae calcification in the mice muzzle skin (von Kossa staining). Arrows indicate ectopic calcification. Scale bar: 0.4 mm. (B,C) Calcium (Ca) content and percentage inhibition of calcification in the muzzle skin. (D) Plasma pyrophosphate (PPi) concentration. (E) Alkaline phosphatase (ALP) activity in serum. WT, wild-type C57BL/6J mice. Results are presented as mean ± SD. Statistical analysis: one-way ANOVA with Tukey’s post hoc test. (***) p < 0.001, (****) p < 0.0001.

Effect of subcutaneous administration of CSL525 at 60 mg/kg once daily in a calcinosis cutis rat model. Calcinosis cutis was induced in 24 rats by sensitization with subcutaneous vitamin D (50,000 IU/kg on days 1, 2 and 3), followed by subcutaneous injections of FeCl₃ (0.15 mg) at each of the two ventral sites in the thorax on day 4. CSL525 at 60 mg/kg (N = 12) or 0.9% NaCl (N = 12) was administered subcutaneously on a daily basis. A group of 6 healthy rats received subcutaneous 0.9% NaCl on days 1, 2, and 3, and 0.5 mL of 0.9% NaCl at each of the two ventral thoracic sites on day 4. (A) Representative images of the skin tissue surrounding the FeCl₃ injection site. Arrows indicate ectopic calcification. Scale bar: 1 cm. (B) Calcium (Ca) content in the skin tissue surrounding the FeCl₃ injection site. (C) Calcium content in femoral arteries. (D) Inhibition of calcification in the skin and femoral arteries by CSL525. Results are presented as mean ± SD. Statistical analysis: one-way ANOVA with Tukey’s post hoc test. (**) p < 0.01, (***) p < 0.001.

Schematic representation of the protocols used in the in vivo studies. (A) Zebrafish model of pseudoxanthoma elasticum (PXE). A total of 100 abcc6a^cmg52/cmg52 zebrafish larvae were exposed to 1 mM CSL525 at 3 days post fertilization (dpf) for 7 days, while another group of 100 larvae was kept under the same conditions without CSL525. After 7 days of treatment (10 dpf), the fish were euthanized, fixed in 4% paraformaldehyde, and stained with Alizarin Red S. (B) Mouse model of PXE. The abcc6^-/- mice were subcutaneously administered CSL525 at 15 mg/kg or 0.9% NaCl thrice weekly for eight weeks. A group of C57BL/6J wild-type mice, also receiving 0.9% NaCl thrice weekly, was included in the study. Each group consisted of five males and five females. All mice were maintained on a standard diet and sacrificed at 13 weeks of age, eight weeks after treatment initiation. (C) Rat model of calcinosis cutis. Calcinosis cutis was induced in 24 rats by sensitization with subcutaneous vitamin D (50,000 IU/kg on days 1, 2 and 3), followed by subcutaneous injections of FeCl₃ (0.15 mg) at each of the two ventral thoracic sites on day 4. CSL525 at 60 mg/kg (N = 12) or 0.9% NaCl (N = 12) was administered subcutaneously on a daily basis. A group of 6 healthy rats received subcutaneous 0.9% NaCl on days 1, 2, and 3, and 0.5 mL of 0.9% NaCl at each of the two ventral thoracic sites on day 4.