Vasorin plays a critical role in vascular smooth muscle cells and arterial functions

Figures

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  Figure 1. Vasn expression in ECs, SMCs, aorta, and organs of Vasn CRE‐ERT KO and Vasn SMMHC‐CRE‐ERT2 KO mice. (a) Sections of aorta from a Vasn Venus transgenic mouse. Immunofluorescence of ECs (CD31; red), aortic elastic fibers (green) and Venus+ cells (pink). Nuclei were counterstained with Hoechst reagent (blue). The red arrow indicates Venus‐positive ECs, and the yellow arrow indicates Venus‐positive SMCs. In Vasn CRE‐ERT KO mice (b) and Vasn SMMHC‐CRE‐ERT2 KO mice (c), total RNA from thoracic aortas, heart, kidney, and lung were measured and compared with control mice. Vasn protein expression in aortas was assessed in Western blots and compared with extracts from control mice. Representative immunoblots show the inhibition of Vasn protein synthesis. Quantification of Vasn expression in ECs and SMCs using Vasn immunostaining, scored from 0 (absence of immunostaining) to 4 (greatest possible immunostaining). In all qPCR experiments, Gapdh was used as the internal reference gene. For Vasn immunostaining, five thoracic aorta sections were averaged for each animal. EC, endothelial cell; SMC, smooth muscle cell. Negative control (Negative) corresponds to immunostaining without the primary…

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  Figure 2. Effect of Vasn deletion on arterial blood pressure, heart rate, and Ang II levels in Vasn CRE‐ERT KO and Vasn SMMHC‐CRE‐ERT2 KO mice. Systolic, diastolic, and mean arterial blood pressures and heart rate 21 days after tamoxifen injection in (a) 8‐week‐old Vasn CRE‐ERT KO (b) 8‐week‐old Vasn SMMHC‐CRE‐ERT2 KO mice and (d) 1‐year‐old (Aged) Vasn CRE‐ERT KO mice. The results correspond to cumulative data from 13 to 16 control mice, 17 Vasn CRE‐ERT KO mice, 16 Vasn SMMHC‐CRE‐ERT2 KO mice, and 4 Aged Vasn CRE‐ERT KO mice. *p < 0.05, **p < 0.01, ***p < 0.0005, ****p < 0.0001 versus controls. (c) Ang II levels in control, Vasn CRE‐ERT KO and Vasn SMMHC‐CRE‐ERT2 KO mice after 14 days (D14) and 21 days (D21). Control mice n = 12, Vasn CRE‐ERT KO D14 n = 6, Vasn CRE‐ERT KO D21 n = 10, Vasn SMMHC‐CRE‐ERT2 KO D14 n = 6, Vasn SMMHC‐CRE‐ERT2 KO D21 n = 10. *p < 0.05, **p < 0.005 versus control mice. $$$$p < 0.0001 versus Vasn CRE‐ERT KO D14. £££p < 0.0005 versus Vasn SMMHC‐CRE‐ERT2 KO D21.

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  Figure 3. Impact of Vasn deletion on vascular function in Vasn CRE‐ERT KO and Vasn SMMHC‐CRE‐ERT2 KO mice, and correlation between Ang II levels and contraction in Vasn SMMHC‐CRE‐ERT2 KO mice. (a) Contraction obtained with phenylephrine and relaxation obtained with acetylcholine in Vasn CRE‐ERT KO mouse vessels 14 days (Vasn CRE‐ERT KO D14, n = 8) and 21 days (Vasn CRE‐ERT KO D21, n = 8) after Vasn KO. (b) Contraction obtained with phenylephrine and relaxation obtained with acetylcholine in Vasn SMMHC‐CRE‐ERT2 KO mouse vessels 14 days (Vasn SMMHC‐CRE‐ERT2 KO D14, n = 7) and 21 days (Vasn SMMHC‐CRE‐ERT2 KO D21, n = 4) after Vasn KO. Control mice n = 7. *p < 0.05, **p < 0.005, ***p < 0.0005 versus control mice; $p < 0.01, $$$p < 0.0001 versus D14 group. (c) Correlation between the contraction in response to phenylephrine and Ang II levels in Vasn SMMHC‐CRE‐ERT2 KO mice at 14 days and 21 days. Control mice n = 12, Vasn SMMHC‐CRE‐ERT2 KO D14 n = 6, Vasn SMMHC‐CRE‐ERT2 KO D21 n = 10. (d) p‐MYPT1 protein expression levels in aortas from Vasn CRE‐ERT KO and Vasn SMMHC‐CRE‐ERT2 KO mice were assessed using Western blot analysis and compared with extracts from control mice (n = 6, for each…

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  Figure 4. Influence of Vasn silencing on SMC phenotypic marker expression and STAT3 activation in aortic samples from Vasn CRE‐ERT KO and Vasn SMMHC‐CRE‐ERT2 KO mice. (a) mRNA expression in silenced (siVasn‐transfected) HVSMCs versus control cells treated with a scrambled siRNA (siNeg), representative Western blots of VASN protein expression, and quantification of VASN protein expression in siRNA‐transfected HVSMCs from six donors (n = 6); I, II and III are different donors; N and V correspond to the siNeg and siVasn conditions, respectively. (b) mRNA expression of collagen 8 (Coll8), collagen 1 (Coll1), and osteopontin (OPN) was analyzed using qPCR in siVasn HVSMCs and compared with control cells (siNeg). (c) mRNA expression of calponin (CNN1), α‐smooth muscle actin (α‐SMA), and smooth muscle myosin heavy chains (SMMHCs) was analyzed using qPCR in siVasn HVSMCs and compared with control cells (siNeg). For all mRNA expression analyses, the results correspond to cumulative data from two independent reverse transcriptions of total RNA from six donors (n = 12). The value in the siNeg condition was set to 1. *p < 0.05, **p < 0.01, ***p < 0.0001 versus siNeg. (d) STAT3 protein expression and…

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  Figure 5. Effect of VASN silencing on calcium homeostasis in HVSMCs. (a) Typical frequency responses obtained to carbachol or phenylephrine stimulation in silenced (siVasn‐transfected cells) and control (scrambled siRNA, siNeg) cells (n = 45 cells). (b) Magnitude of Ca2+ responses and the basal Ca2+ concentration in siVasn‐transfected cells (n = 245) and siNeg control cells (n = 250). (c) Recordings of the extracellular application of Mn2+ (quenching the Fura‐2 fluorescent probe) and analysis of the corresponding slopes (siNeg, n = 32; siVasn, n = 24) in transfected HVSMCs. (d) Typical recordings of basal entry of Ca2+ and quantification in siNeg‐transfected HVSMCs (n = 19) and siVasn transfected HVSMCs (n = 21). HVSMC, human vascular smooth muscle cell. *p < 0.05, ***p < 0.001 versus siNeg.

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  Figure 6. Western blot analysis of relaxation signaling pathways in aortas (a–c) of Vasn CRE‐ERT KOand Vasn SMMHC‐CRE‐ERT2 KO mice, and (d,e) HUVECs. Protein expression in aortas from Vasn CRE‐ERT KO and Vasn SMMHC‐CRE‐ERT2 KO mice was assessed using Western blots of selected markers and compared with extracts from control mice (n = 6 for each condition). The expression of the phosphorylated forms of AKT and eNOS proteins (p‐AKT and p‐eNOS) was quantified and normalized against the total protein forms (t‐AKT and t‐eNOS). Representative immunoblots are shown above each quantification histogram. (a) p‐AKT expression in Vasn CRE‐ERT KO and Vasn SMMHC‐CRE‐ERT2 KO mice. (b) p‐eNOS expression in Vasn CRE‐ERT KO and Vasn SMMHC‐CRE‐ERT2 KO KO mice. *p < 0.05, **p < 0.01 versus control mice. (c) ADMA levels in the different mice groups (n = 15–16). (d) VASN mRNA expression and VASN protein expression in HUVECs through passages (P). Results represent the accumulated data of independent qPCR experiments (n = 5) and the accumulated data of independent Western blot experiments (n = 3). The P2 condition was set to 1, *p < 0.05 versus P2. (e) The expression of the phosphorylated and total forms of eNOS…