Abstract 051: The Myogenic Tone Of Resistance Arteries Is Controlled By A Molecular Pathway Driven By Tgfβ Through Emilin1 And Converging On The Regulation Of Trp Channels.

Abstract

Emilin1 (E1) is a protein of the extracellular matrix that regulates TGFβ bioavailability through proteolysis of the proTGFβ. E1 KO mice are hypertensive, with increased TGFβ activation. As E1 is expressed in blood vessels starting from embryonic life to adulthood, is still unknown whether the E1 KO phenotype results from a developmental defect or lack of a homeostatic role in the adult. To dissect this issue, we used a conditional gene targeting approach to inactivate E1 in smooth muscle cells (VSMCs) of adult mice, by the use of floxed E1 and CreERT2 [tamoxifen (TAM) inducible Cre recombinase] under the control of the smooth muscle myosin heavy chain (Smmhc) promoter. When E1fl/fl mice carrying the Smmhc-CreERT2 were given TAM blood pressure significantly increased (124±1 vs basal condition 106±1 mmHg) as well as myogenic response in resistance arteries (16.3±0.7 vs basal condition 11.4±0.1 % at 125 mmHg). In order to understand how E1 ablation in VSMCs could functionally determine the increase in myogenic tone, we used an ex vivo approach in resistance arteries, to target the molecular pathway that could regulate intracellular calcium fluxes, the main regulator of myogenic contraction. Experiments performed in isolated VSMCs from E1 KO as compared to WT arteries, suggested the involvement of a ROCE (receptor operated calcium entry) mechanism, whose main final molecular determinant is represented by the TRP channels, known to be regulated by a canonical PLC-DAG pathway, but also by a parallel one controlled by the growth factors signaling cascade. By exploring this pathway, we found that E1 regulates the activity of TRPC3 channels and thus of the myogenic response. The molecular pathway delineated by our studies started from the transactivation of EGFR through metalloproteinases and converged on the Src kinases that increased TRPC3 function and thus calcium entry. In particular, with an in vivo targeting of TRPC3 in E1 KO with an adenovirus carrying a dominant negative mutation, we demonstrated a rescue of the increased myogenic response of E1 KO resistance arteries. Overall our results suggest that this pathway in VSMC, driven by TGFβ and regulated by E1, is crucial for the myogenic tone of resistance arteries and is linked to the regulation of blood pressure.