MEF2B-Nox1 signaling is critical for stretch-induced phenotypic modulation of vascular smooth muscle cells.

OBJECTIVE: Blood vessel hemodynamics have profound influences on function and structure of vascular cells. One of the main mechanical forces influencing vascular smooth muscle cells (VSMC) is cyclic stretch (CS). Increased CS stimulates reactive oxygen species (ROS) production in VSMC, leading to their dedifferentiation, yet the mechanisms involved are poorly understood. This study was designed to test the hypothesis that pathological CS stimulates NADPH oxidase isoform 1 (Nox1)-derived ROS via MEF2B, leading to VSMC dysfunction via a switch from a contractile to a synthetic phenotype. APPROACH AND RESULTS: Using a newly developed isoform-specific Nox1 inhibitor and gene silencing technology, we demonstrate that a novel pathway, including MEF2B-Nox1-ROS, is upregulated under pathological stretch conditions, and this pathway promotes a VSMC phenotypic switch from a contractile to a synthetic phenotype. We observed that CS (10% at 1 Hz) mimicking systemic hypertension in humans increased Nox1 mRNA, protein levels, and enzymatic activity in a time-dependent manner, and this upregulation was mediated by MEF2B. Furthermore, we show that stretch-induced Nox1-derived ROS upregulated a specific marker for synthetic phenotype (osteopontin), whereas it downregulated classical markers for contractile phenotype (calponin1 and smoothelin B). In addition, our data demonstrated that stretch-induced Nox1 activation decreases actin fiber density and augments matrix metalloproteinase 9 activity, VSMC migration, and vectorial alignment. CONCLUSIONS: These results suggest that CS initiates a signal through MEF2B that potentiates Nox1-mediated ROS production and causes VSMC to switch to a synthetic phenotype. The data also characterize a new Nox1 inhibitor as a potential therapy for treatment of vascular dysfunction in hypertension.

In vitro evolution of an archetypal enteropathogenic Escherichia coli strain.

Enteropathogenic Escherichia coli (EPEC) is a leading cause of infantile diarrhea in developing countries. EPEC strain E2348/69 is used worldwide as a prototype to study EPEC genetics and disease. However, isolates of E2348/69 differ phenotypically, reflecting a history of in vitro selection. To identify the genomic and phenotypic changes in the prototype strain, we sequenced the genome of the nalidixic acid-resistant (Nal(r)) E2348/69 clone. We also sequenced a recent nleF mutant derived by one-step PCR mutagenesis from the Nal(r) strain. The sequencing results revealed no unintended changes between the mutant and the parent strain. However, loss of the pE2348-2 plasmid and 3 nonsynonymous mutations were found in comparison to the published streptomycin-resistant (Str(r)) E2348/69 reference genome. One mutation is a conservative amino acid substitution in ftsK. Another, in gyrA, is a mutation known to result in resistance to nalidixic acid. The third mutation converts a stop codon to a tryptophan, predicted to result in the fusion of hflD, the lysogenization regulator, to purB. The purB gene encodes an adenylosuccinate lyase involved in purine biosynthesis. The Nal(r) clone has a lower growth rate than the Str(r) isolate when cultured in minimal media, a difference which is corrected upon addition of adenine or by genetic complementation with purB. Addition of adenine or genetic complementation also restored the invasion efficiency of the Nal(r) clone. This report reconciles longstanding inconsistencies in phenotypic properties of an archetypal strain and provides both reassurance and cautions regarding intentional and unintentional evolution in vitro.