Figure 3.
Feasibility of endothelial Vwf inhibition in vivo in mice using siVwf encapsulated in 7C1 oligomeric LNPs. (A) The nonselective siVwf was encapsulated in 7C1 LNPs and injected at a dose of 0.5 to 1.5 siRNA mg/kg in male (squares) and female (inverse triangles) B6 (blue symbols) and 129S (red symbols) mice. Lung Vwf mRNA (left 2 panels) and plasma VWF (right 2 panels) levels were determined and normalized to the scrambled siControl. (B) Images of immunofluorescent staining of 2 representative areas of the lungs of siControl- (left) and siVwf-treated (right) B6 mice. Images include nuclei staining (Hoechst) in blue and VWF in green. Scale bar is representative for 100 μm. (C) Representative VWF multimeric pattern of B6 mice treated with siVwf or siControl. Analysis was performed on platelet-free plasma, and NMP was used as a reference for normal multimer patterns. Samples loaded on the gel were diluted to a final sample concentration of 0.05 U/mL, except for ∗, where the sample concentration was accidentally 0.10 U/mL. At the bottom of this panel, the plasma VWF levels are indicated for the individual mice that were used for this VWF multimeric pattern analysis. (D) Duration of siRNA–mediated endothelial Vwf inhibition in B6 mice treated with siVwf (1.5 mg/kg) and at 72 hours, 96 hours, and 10 days after injection sacrificed for analysis of lung Vwf mRNA (left panel) and plasma VWF protein (right panel). n = 6 mice per group; however, 1 siControl-treated animal was removed because of a failed siRNA injection, incidentally a blood sample was removed because of unwanted clotting activation (as established visually). Values are presented as median with range. P ≥ 0.05, denotes not significant (ns); ∗P ≤ 0.05; ∗∗P ≤ 0.01.

Feasibility of endothelial Vwf inhibition in vivo in mice using siVwf encapsulated in 7C1 oligomeric LNPs. (A) The nonselective siVwf was encapsulated in 7C1 LNPs and injected at a dose of 0.5 to 1.5 siRNA mg/kg in male (squares) and female (inverse triangles) B6 (blue symbols) and 129S (red symbols) mice. Lung Vwf mRNA (left 2 panels) and plasma VWF (right 2 panels) levels were determined and normalized to the scrambled siControl. (B) Images of immunofluorescent staining of 2 representative areas of the lungs of siControl- (left) and siVwf-treated (right) B6 mice. Images include nuclei staining (Hoechst) in blue and VWF in green. Scale bar is representative for 100 μm. (C) Representative VWF multimeric pattern of B6 mice treated with siVwf or siControl. Analysis was performed on platelet-free plasma, and NMP was used as a reference for normal multimer patterns. Samples loaded on the gel were diluted to a final sample concentration of 0.05 U/mL, except for ∗, where the sample concentration was accidentally 0.10 U/mL. At the bottom of this panel, the plasma VWF levels are indicated for the individual mice that were used for this VWF multimeric pattern analysis. (D) Duration of siRNA–mediated endothelial Vwf inhibition in B6 mice treated with siVwf (1.5 mg/kg) and at 72 hours, 96 hours, and 10 days after injection sacrificed for analysis of lung Vwf mRNA (left panel) and plasma VWF protein (right panel). n = 6 mice per group; however, 1 siControl-treated animal was removed because of a failed siRNA injection, incidentally a blood sample was removed because of unwanted clotting activation (as established visually). Values are presented as median with range. P ≥ 0.05, denotes not significant (ns); ∗P ≤ 0.05; ∗∗P ≤ 0.01.

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