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

Abstract

Within the cardiovascular system, the protein vasorin (Vasn) is predominantly expressed by vascular smooth muscle cells (VSMCs) in the coronary arteries and the aorta. Vasn knockout (Vasn^-/- ) mice die within 3 weeks of birth. In the present study, we investigated the role of vascular Vasn expression on vascular function. We used inducible Vasn knockout mice (Vasn^{CRE-ERT KO} and Vasn^{SMMHC-CRE-ERT2 KO} , in which respectively all cells or SMCs only are targeted) to analyze the consequences of total or selective Vasn loss on vascular function. Furthermore, in vivo effects were investigated in vitro using human VSMCs. The death of Vasn^{CRE-ERT KO} mice 21 days after tamoxifen injection was concomitant with decreases in blood pressure, angiotensin II levels, and vessel contractibility to phenylephrine. The Vasn^{SMMHC-CRE-ERT2 KO} mice displayed concomitant changes in vessel contractibility in response to phenylephrine and angiotensin II levels. In vitro, VASN deficiency was associated with a shift toward the SMC contractile phenotype, an increase in basal intracellular Ca²⁺ levels, and a decrease in the SMCs' ability to generate a calcium signal in response to carbachol or phenylephrine. Additionally, impaired endothelium-dependent relaxation (due to changes in nitric oxide signaling) was observed in all Vasn knockout mice models. Our present findings highlight the role played by Vasn SMC expression in the maintenance of vascular functions. The mechanistic experiments suggested that these effects are mediated by SMC phenotype switching and changes in intracellular calcium homeostasis, angiotensin II levels, and NO signaling.

Keywords: angiotensin II; artery; nitric oxide; smooth muscle cells; vascular function; vasorin.

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 anti‐Vasn antibody. Vasn protein expression was quantified and normalized against β‐ACTIN. Control mice n = 4–6, Vasn ^{CRE‐ERT KO} n = 5, Vasn ^{SMMHC‐CRE‐ERT2 KO} n = 4. *p < 0.05, **p < 0.01, ***p < 0.001 versus control, $p < 0.001 versus EC of Vasn ^{SMMHC‐CRE‐ERT2 KO} mice.

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.

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 condition). *p < 0.05, versus control mice.

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 activation in aorta samples from Vasn ^{CRE‐ERT KO} and Vasn ^{SMMHC‐CRE‐ERT2 KO} mice was assessed in Western blots and compared with extracts of aortas from control mice (n = 6, for each condition). The expression of the phosphorylated form of STAT3 (p‐STAT3) was quantified and normalized against total STAT3 (t‐STAT3). Representative immunoblots are shown above the quantification histograms. HVSMC, human vascular smooth muscle cell. *p < 0.05, **p < 0.01 versus control mice.

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.

Figure 6

Western blot analysis of relaxation signaling pathways in aortas (a–c) of Vasn ^{CRE‐ERT KO}and 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 proteins (p‐eNOS and t‐eNOS) was assessed by Western blot analysis. Activated p‐eNOS levels were quantified and normalized against those of t‐eNOS. I, II, and III indicate different independent HUVECs experiments; N and V correspond to the siNeg and siVasn conditions, respectively. Results represent the accumulated data of independent experiments (n = 4). The siNeg condition was set to 1, *p < 0.05 versus siNeg. NO production in VASN‐silenced HUVECs. NO production was assessed by measuring the fluorescence of DAF‐FM DA. Results represent the accumulated data of HUVECs independent experiments (n = 7). The Control condition was set to 1. HUVEC, human umbilical vein endothelial cell. *p < 0.05, **p < 0.01 versus Control, $p < 0.05 versus siNeg.