BackgroundNuclear factor of activated T‐cells 5 (NFAT5) has recently been described to control the phenotype of vascular smooth muscle cells (VSMCs). Although an increase in wall stress or stretch (eg, elicited by hypertension) is a prototypic determinant of VSMC activation, the impact of this biomechanical force on the activity of NFAT5 is unknown. This study intended to reveal the function of NFAT5 and to explore potential signal transduction pathways leading to its activation in stretch‐stimulated VSMCs.Methods and ResultsHuman arterial VSMCs were exposed to biomechanical stretch and subjected to immunofluorescence and protein‐biochemical analyses. Stretch promoted the translocation of NFAT5 to the nucleus within 24 hours. While the protein abundance of NFAT5 was regulated through activation of c‐Jun N‐terminal kinase under these conditions, its translocation required prior activation of palmitoyltransferases. DNA microarray and ChiP analyses identified the matrix molecule tenascin‐C as a prominent transcriptional target of NFAT5 under these conditions that stimulates migration of VSMCs. Analyses of isolated mouse femoral arteries exposed to hypertensive perfusion conditions verified that NFAT5 translocation to the nucleus is followed by an increase in tenascin‐C abundance in the vessel wall.ConclusionsCollectively, our data suggest that biomechanical stretch is sufficient to activate NFAT5 both in native and cultured VSMCs where it regulates the expression of tenascin‐C. This may contribute to an improved migratory activity of VSMCs and thus promote maladaptive vascular remodeling processes such as hypertension‐induced arterial stiffening.

Arteriogenesis—the growth of collateral arterioles—partially compensates for the progressive occlusion of large conductance arteries as it may occur as a consequence of coronary, cerebral or peripheral artery disease. Despite being clinically highly relevant, mechanisms driving this process remain elusive. In this context, our study revealed that abundance of regulator of G-protein signalling 5 (RGS5) is increased in vascular smooth muscle cells (SMCs) of remodelling collateral arterioles. RGS5 terminates G-protein-coupled signalling cascades which control contractile responses of SMCs. Consequently, overexpression of RGS5 blunted Gαq/11-mediated mobilization of intracellular calcium, thereby facilitating Gα12/13-mediated RhoA signalling which is crucial for arteriogenesis. Knockdown of RGS5 evoked opposite effects and thus strongly impaired collateral growth as evidenced by a blockade of RhoA activation, SMC proliferation and the inability of these cells to acquire an activated phenotype in RGS5-deficient mice after the onset of arteriogenesis. Collectively, these findings establish RGS5 as a novel determinant of arteriogenesis which shifts G-protein signalling from Gαq/11-mediated calcium-dependent contraction towards Gα12/13-mediated Rho kinase-dependent SMC activation.Subject Categories Vascular Biology & Angiogenesis

Cytoskeletal reorganization and migration are critical responses which enable vascular smooth muscle cells (VSMCs) cells to evade, compensate, or adapt to alterations in biomechanical stress. An increase in wall stress or biomechanical stretch as it is elicited by arterial hypertension promotes their reorganization in the vessel wall which may lead to arterial stiffening and contractile dysfunction. This adaptive remodeling process is dependent on and driven by subtle phenotype changes including those controlling the cytoskeletal architecture and motility of VSMCs. Recently, it has been reported that the transcription factor nuclear factor of activated T-cells 5 (TonEBP/NFAT5) controls critical aspects of the VSMC phenotype and is activated by biomechanical stretch. We therefore hypothesized that NFAT5 controls the expression of gene products orchestrating cytoskeletal reorganization in stretch-stimulated VSMCs. Automated immunofluorescence and Western blot analyses revealed that biomechanical stretch enhances the expression and nuclear translocation of NFAT5 in VSMCs. Subsequent in silico analyses suggested that this transcription factor binds to the promotor region of ACTBL2 encoding kappa-actin which was shown to be abundantly expressed in VSMCs upon exposure to biomechanical stretch. Furthermore, ACTBL2 expression was inhibited in these cells upon siRNA-mediated knockdown of NFAT5. Kappa-actin appeared to be aligned with stress fibers under static culture conditions, dispersed in lamellipodia and supported VSMC migration as its knockdown diminishes lateral migration of these cells. In summary, our findings delineated biomechanical stretch as a determinant of NFAT5 expression and nuclear translocation controlling the expression of the cytoskeletal protein ACTBL2. This response may orchestrate the migratory activity of VSMCs and thus promote maladaptive rearrangement of the arterial vessel wall during hypertension.

Chronic alterations in the biomechanical stimulation of vascular smooth muscle cells (VSMC) as experienced during hypertension lead to changes in VSMC phenotype and function and further enable the structural remodeling of the vessel wall. In this context, we recently reported that an increase in wall stress or biomechanical stretch is sufficient to activate nuclear factor of activated T-cells 5 (NFAT5). This transcription factor promotes the expression of gene products such as tenascin-C and κ-actin, both involved in VSMC migration. Based on these findings, we hypothesized that biomechanical stretch elicits NFAT5 mRNA expression and induces biochemical modifications of NFAT5 on the post-translational level, a prerequisite for its entry into the nucleus and transcriptional activity. To scrutinize this hypothesis, human arterial VSMC were exposed to biomechanical stretch (13%, 0.5 Hz) and subjected to detailed mRNA expression analyses. While a ~3-fold reduction in NFAT5 splice variant 1 (isoform A) mRNA expression was observed in stretch-stimulated VSMC as compared to the static controls (n=3, p<0.05), splice variant 3 (isoform C) mRNA levels were induced ~1.8-fold (n=3, p<0.05). Overexpression of corresponding Flag-tagged NFAT5 proteins in VSMC and subsequent immunofluorescence as well as biochemical analyses revealed that isoform A was primarily located in the cytoplasm of static and stretch-stimulated VSMC while isoform C was preferentially localized in the nucleus under baseline conditions and further accumulated in the nucleus upon biomechanical stimulation (n=3, p<0.05). Nuclear translocation of isoform C was amplified for phosphorylation-deficient mutants generated by exchanging serine to alanine at position 1197 even under static culture conditions while a phosphomimetic mutation at this residue (serine to glutamate) inhibited NFAT5c nuclear translocation (n=3, p<0.05). Collectively, our findings indicate that exposure of VSMC to biomechanical stretch triggers the expression of NFAT5 isoform C and controls its entry into the nucleus via phosphorylation at S-1197. Current investigations are focusing on the impact of NFAT5 on hypertensive remodeling utilizing inducible smooth muscle cell-specific NFAT5-deficient mice.