The magnitude of shear stimulus has been shown to determine the level of growth factor expression in cell culture. However, little is known regarding what effect shear level has on specific arterial wall remodeling events in vivo. We have hypothesized that the rate of luminal diameter change and specific remodeling events within the arterial wall layers are dependent on shear level. Selective ligations were made to alter the number of microvascular perfusion units of mesenteric arteries within the same animal to approximately 50%, 200%, and 400% of control. Arterial blood flow and wall shear rate were correlated with the degree of alteration in perfusion units. Luminal diameters were decreased in 50% arteries by day 2 and increased approximately 17% and 33% respectively, in 200% and 400% arteries at day 7. The rate of diameter change was greatest in 50% and 400% arteries. Wall areas (medial +37%; intimal +18% at day 2) and cell densities (intimal +26%; adventitial +44% at day 2) were altered only in the 400% arteries. A positive correlation existed by day 2 between endothelial staining for endothelial nitric oxide synthase and shear level. The results demonstrate that shear level influences the rate of luminal expansion, specific remodeling events within each wall layer, and the degree of endothelial gene expression. A greater understanding of how shear level influences specific remodeling events within each wall layer should aid in the development of targeted therapies to manipulate the remodeling process in health and disease.

BackgroundThe origins of neointimal smooth muscle cells that arise following vascular injury remains controversial. Studies have suggested that these cells may arise from previously differentiated medial vascular smooth muscle cells, resident stem cells or blood born progenitors. In the current study we examined the contribution of the previously differentiated vascular smooth muscle cells to the neointima that forms following carotid artery ligation.MethodsWe utilized transgenic mice harboring a cre recombinase-dependent reporter gene (mTmG). These mice express membrane targeted tandem dimer Tomato (mTomato) prior to cre-mediated excision and membrane targeted EGFP (mEGFP) following excision. The mTmG mice were crossed with transgenic mice expressing either smooth muscle myosin heavy chain (Myh11) or smooth muscle α-actin (Acta2) driven tamoxifen regulated cre recombinase. Following treatment of adult mice with tamoxifen these mice express mEGFP exclusively in differentiated smooth muscle cells. Subsequently vascular injury was induced in the mice by carotid artery ligation and the contribution of mEGFP positive cells to the neointima determined.ResultsAnalysis of the cellular composition of the neointima that forms following injury revealed that mEGFP positive cells derived from either Mhy11 or Acta2 tagged medial vascular smooth muscle cells contribute to the majority of neointima formation (79 ± 17% and 81 ± 12%, respectively).ConclusionThese data demonstrate that the majority of the neointima that forms following carotid ligation is derived from previously differentiated medial vascular smooth muscle cells.

Cyclic nucleotides can relax smooth muscle without a change in [Ca 2؉ ]i, a phenomenon termed Ca 2؉ desensitization, contributing to vasodilation, gastrointestinal motility, and airway resistance. The physiological importance of telokin, a 17-kDa smooth musclespecific protein and target for cyclic nucleotide-induced Ca 2؉ desensitization, was determined in telokin null mice bred to a congenic background. Telokin null ileal smooth muscle homogenates compared to wild type exhibited an Ϸ30% decrease in myosin light-chain phosphatase (MLCP) activity, which was reflected in a significant leftward shift (up to 2-fold at pCa 6.3) of the Ca 2؉ force relationship accompanied by an increase in myosin light-chain phosphorylation. No difference in the Ca 2؉ force relationship occurred in telokin WT and knockout (KO) aortas, presumably reflecting the normally Ϸ5-fold lower telokin content in aorta vs. ileum smooth muscle. Ca 2؉ desensitization of contractile force by 8-Br-cGMP was attenuated by 50% in telokin KO intestinal smooth muscle. The rate of force relaxation reflecting MLCP activity, in the presence of 50 M 8-Br-cGMP, was also significantly slowed in telokin KO vs. WT ileum and was rescued by recombinant telokin. Normal thick filaments in telokin KO smooth muscles indicate that telokin is not required for filament formation or stability. Results indicate that a primary role of telokin is to modulate force through increasing MLCP activity and that this effect is further potentiated through phosphorylation by cGMP in telokin-rich smooth tissues. S mooth muscle (SM) myosin II ATPase activity and associated contraction is activated by actin only when Ser-19 of the myosin regulatory light chain (RLC 20 ) is phosphorylated. The extent of RLC 20 phosphorylation is a reflection of the balance between Ca 2ϩ calmodulin-dependent myosin light-chain kinase (MLCK) and myosin light-chain phosphatase (MLCP) activities. Although the major mechanism for initiating contraction is the rise in [Ca 2ϩ ] i , force can be further increased or decreased through signaling pathways that modulate MLCK and͞or MLCP activities giving rise to processes termed Ca 2ϩ sensitization or -desensitization (reviewed in ref. 1). Ca 2ϩ sensitization due to inhibition of MLCP activity is mediated by an agonist G protein-coupled, Ca 2ϩ -independent process that activates RhoA͞Rho-kinase, phosphorylates the myosin regulatory subunit of MLCP (MYPT1), and leads to increased force (1). On the other hand, cyclic nucleotideactivated kinases, in addition to decreasing cytosolic Ca 2ϩ , make a significant contribution to dephosphorylation of RLC 20 and relaxation through Ca 2ϩ -desensitization processes (2-5) and can reverse G protein-coupled Ca 2ϩ sensitization (2, 3). Interaction between the leucine zipper motifs of protein kinase G and MYPT1 leads to direct stimulation of MLCP (6). A role for this interaction in Ca 2ϩ desensitization is supported by the observation that chicken gizzard MYPT1 lacking the leucine zipper motifs is resistant to 8-BrcGMP-induced depho...

Serum response factor (SRF) is a key regulator of smooth muscle differentiation, proliferation, and migration. Myocardin-related transcription factor A (MRTFA) is a co-activator of SRF that can induce expression of SRF-dependent, smooth musclespecific genes and actin/Rho-dependent genes, but not MAPKregulated growth response genes. How MRTFA and SRF discriminate between these sets of target genes is still unclear. We hypothesized that SWI/SNF ATP-dependent chromatin remodeling complexes, containing Brahma-related gene 1 (Brg1) or Brahma (Brm), may play a role in this process. Results from Western blotting and qRT-PCR analysis demonstrated that dominant negative Brg1 blocked the ability of MRTFA to induce expression of smooth muscle-specific genes, but not actin/Rhodependent early response genes, in fibroblasts. In addition, dominant negative Brg1 attenuated expression of smooth muscle-specific genes in primary cultures of smooth muscle cells. MRTFA overexpression did not induce expression of smooth muscle-specific genes in SW13 cells, which lack endogenous Brg1 or Brm. Reintroduction of Brg1 or Brm into SW13 cells restored their responsiveness to MRTFA. Immunoprecipitation assays revealed that Brg1, SRF, and MRTFA form a complex in vivo, and Brg1 directly binds MRTFA, but not SRF, in vitro. Results from chromatin immunoprecipitation assays demonstrated that dominant negative Brg1 significantly attenuated the ability of MRTFA to increase SRF binding to the promoters of smooth muscle-specific genes, but not early response genes. Together these data suggest that Brg1/Brm containing SWI/ SNF complexes play a critical role in regulating expression of SRF/MRTFA-dependent smooth muscle-specific genes but not SRF/MRTFA-dependent early response genes.There are many clinical diseases, such as atherosclerosis, hypertension and asthma that involve abnormal differentiation of smooth muscle cells. An important pathological process that occurs in these diseases is the disruption of the balance between differentiation and proliferation of smooth muscle cells (1-4). Serum response factor (SRF) 4 has been shown to play an essential role in regulating smooth muscle differentiation, proliferation, and migration through its interaction with various accessory proteins (5). Smooth muscle-specific genes, such as SM ␣-actin, SM MHC, 130-kDa MLCK, SM22␣, and telokin, are activated by SRF-myocardin, SRF/MRTFA, SRF/GATA6/ CRP2, or SRF/Nkx complexes (6 -21). The immediate early growth factor responsive genes, such as c-fos and egr1 are regulated by SRF/ELK-1 (ets) complexes (22-24). The later early response genes, such as SRF itself and vinculin, that are actin/ Rho-dependent, are regulated by SRF-MRTFA complexes (16,25,26). Myocardin-related transcription factor A (MRTFA, or Mkl1, MAL, BSAC) is a unique co-activator of SRF in that it is involved in the regulation of multiple SRF-dependent gene families (reviewed in Ref. 27). MRTFA has been reported to induce SRF-dependent, smooth muscle-specific genes such as telokin, SM22␣, and SM ␣-act...