Question: 

The specific vascular response to increased wall stress is not well characterized. We here set out to analyze (i) the activation of the transient receptor potential channel C3 (TRPC3), the most up-stream component of the stretch-induced signaling leading to zyxin activation known and (ii) the consequences of zyxin-mediated gene expression.

Methods: 

Primary human umbilical vein endothelial cells (EC) and aortic smooth muscle cells (aSMC) derived from wild type and zyxin-deficient mice were cultured on glass cover slips or on stretchable Flexercell membranes. A microarray analysis (n = 3; Mouse Genome 430 2.0; Affymetrix) was performed to characterize stretch-induced (10% elongation, 0.5 Hz for 6 h) gene expression. Intracellular calcium was analyzed by Fura-2 fluorescence ratio using a real time CCD camera system. Analytical methods such as real time RT-PCR or immunofluorescence were performed according to standard protocols.

Results: 

Hypothesizing that TRPC3, similar to other TRP-channels, may be activated by diacylglycerol (DAG), we exposed EC to the DAG analogue OAG (100 mmol/L). Indeed, nuclear translocation of zyxin and zyxin-induced gene expression similar to the stretch-response was observed. This effect was sensitive to the specific TRPC3-inhibitor Pyr3 (10 mmol/L). Intracellular calcium measurements revealed that OAG induces a strong Pyr3-sensitive calcium signal in EC. Similar to EC, approximately 600 gene products were stretch-regulated in wild type aSMC, 70% of these being zyxin-dependent. Pathway analysis revealed that zyxin generally seems to strengthen the contractile capacity of SMC and to suppress proliferation. In accordance, SMC migration, proliferation, contractility in response to epinephrine or endothelin-1 and the cytoskeletal organization was strongly altered in aSMC derived from zyxin-deficient mice.

Conclusion: 

The stretch-induced activation of TRPC3 may be mediated by a release of DAG. TRPC3 seems to set off stretch-induced zyxin activation via an increase of cytosolic calcium into EC. Zyxin-mediated gene expression appears to play a central role in stretch-induced phenotype changes in SMC.