MAB3242 Sigma-AldrichAnti-Smoothelin Antibody, clone R4A

Fibroblasts are important in the atrial fibrillation (AF) substrate resulting from congestive heart failure (CHF). We previously noted changes in in vivo indices of fibroblast function in a CHF dog model, but could not detect changes in isolated cells. This study assessed CHF-induced changes in the phenotype of fibroblasts freshly isolated from control versus CHF dogs, and examined effects of cell culture on these differences.Left-atrial fibroblasts were isolated from control and CHF dogs (ventricular tachypacing 240 bpm × 2 weeks). Freshly-isolated fibroblasts were compared to fibroblasts in primary culture. Extracellular-matrix (ECM) gene-expression was assessed by qPCR, protein by Western blot, fibroblast morphology with immunocytochemistry, and K(+)-current with patch-clamp. Freshly-isolated CHF fibroblasts had increased expression-levels of collagen-1 (10-fold), collagen-3 (5-fold), and fibronectin-1 (3-fold) vs. control, along with increased cell diameter (13.4 ± 0.4 µm vs control 8.4 ± 0.3 µm) and cell spreading (shape factor 0.81 ± 0.02 vs. control 0.87 ± 0.02), consistent with an activated phenotype. Freshly-isolated control fibroblasts displayed robust tetraethylammonium (TEA)-sensitive K(+)-currents that were strongly downregulated in CHF. The TEA-sensitive K(+)-current differences between control and CHF fibroblasts were attenuated after 2-day culture and eliminated after 7 days. Similarly, cell-culture eliminated the ECM protein-expression and shape differences between control and CHF fibroblasts.Freshly-isolated CHF and control atrial fibroblasts display distinct ECM-gene and morphological differences consistent with in vivo pathology. Culture for as little as 48 hours activates fibroblasts and obscures the effects of CHF. These results demonstrate potentially-important atrial-fibroblast phenotype changes in CHF and emphasize the need for caution in relating properties of cultured fibroblasts to in vivo systems.

Kruppel-like transcription factor 5 (Klf5) was detected in the developing and mature murine bladder urothelium. Herein we report a critical role of KLF5 in the formation and terminal differentiation of the urothelium. The Shh(GfpCre) transgene was used to delete the Klf5(floxed) alleles from bladder epithelial cells causing prenatal hydronephrosis, hydroureter, and vesicoureteric reflux. The bladder urothelium failed to stratify and did not express terminal differentiation markers characteristic of basal, intermediate, and umbrella cells including keratins 20, 14, and 5, and the uroplakins. The effects of Klf5 deletion were unique to the developing bladder epithelium since maturation of the epithelium comprising the bladder neck and urethra was unaffected by the lack of KLF5. mRNA analysis identified reductions in Pparγ, Grhl3, Elf3, and Ovol1expression in Klf5 deficient fetal bladders supporting their participation in a transcriptional network regulating bladder urothelial differentiation. KLF5 regulated expression of the mGrhl3 promoter in transient transfection assays. The absence of urothelial Klf5 altered epithelial-mesenchymal signaling leading to the formation of an ectopic alpha smooth muscle actin positive layer of cells subjacent to the epithelium and a thinner detrusor muscle that was not attributable to disruption of SHH signaling, a known mediator of detrusor morphogenesis. Deletion of Klf5 from the developing bladder urothelium blocked epithelial cell differentiation, impaired bladder morphogenesis and function causing hydroureter and hydronephrosis at birth.

We previously reported hedgehog (Hh) signal activation in the mucus-secreting pit cell of the stomach and in diffuse-type gastric cancer (GC). Epithelial-mesenchymal transition (EMT) is known to be involved in tumour malignancy. However, little is known about whether and how both signallings cooperatively act in diffuse-type GC. By microarray and reverse transcription-PCR, we investigated the expression of those Hh and EMT signalling molecules in pit cells and in diffuse-type GCs. How both signallings act cooperatively in those cells was also investigated by the treatment of an Hh-signal inhibitor and siRNAs of Hh and EMT transcriptional key regulator genes on a mouse primary culture and on human GC cell lines. Pit cells and diffuse-type GCs co-expressed many Hh and EMT signalling genes. Mesenchymal-related genes (WNT5A, CDH2, PDGFRB, EDNRA, ROBO1, ROR2, and MEF2C) were found to be activated by an EMT regulator, SIP1/ZFHX1B/ZEB2, which was a target of a primary transcriptional regulator GLI1 in Hh signal. Furthermore, we identified two cancer-specific Hh targets, ELK1 and MSX2, which have an essential role in GC cell growth. These findings suggest that the gastric pit cell exhibits mesenchymal-like gene expression, and that diffuse-type GC maintains expression through the Hh-EMT pathway. Our proposed extensive Hh-EMT signal pathway has the potential to an understanding of diffuse-type GC and to the development of new drugs.

OBJECT: The aim of this project was to study the perturbations of four smooth-muscle proteins and an extracellular protein, type I collagen, after subarachnoid hemorrhage (SAH) and to examine the possible preventive effects of dexamethasone. METHODS: Using a one-hemorrhage rabbit model, the authors first examined the effects of SAH on the expression of alpha-actin, h-caldesmon, vimentin, smoothelin-B, and type I collagen; second, they studied whether post-SAH systemic administration of dexamethasone (three daily injections) corrected the induced alterations. Measurements were obtained at Day 7 post-SAH. The proteins were studied by performing immunohistochemical staining and using a laser-scanning confocal microscope. Compared with control (sham-injured) arteries, the density of the media of arteries subjected to SAH was reduced for alpha-actin (-11%, p = 0.01) and h-caldesmon (-15%, p = 0.06) but increased for vimentin (+15%, p = 0.04) and smoothelin-B (+53%, p = 0.04). Among animals in which SAH was induced, arteries in those treated with dexamethasone demonstrated higher values of density for alpha-actin (+13%, p = 0.05) and h-caldesmon (+20%, p = 0.01), lower values for vimentin (-55%, p = 0.05), and nonsignificantly different values for smoothelin-B. The density of type I collagen in the adventitia decreased significantly after SAH (-45%, p = 0.01), but dexamethasone treatment had no effect on this decrease. CONCLUSIONS: The SAH-induced alterations in the density of three of four smooth-muscle proteins were prevented by dexamethasone treatment; two of these proteins--alpha-actin and h-caldesmon--are directly related to contraction. This drug may potentially be useful to prevent certain morphological and functional changes in cerebral arteries after SAH.

OBJECTIVES: The healing response to vascular injury is characterized by neointimal thickening. Proliferation and phenotypic transformation of vascular smooth muscle cells (SMCs) have been implicated in this process. We sought to investigate the role of survivin, a dual regulator of cell proliferation and apoptosis, in lesion formation after diverse forms of vascular injury. METHODS: Rabbits underwent either carotid interposition vein grafting (n = 17) or bilateral femoral balloon injury (BI; n = 29); some in the BI group were placed on a high-cholesterol diet. A subset of BI arteries were treated with local adenoviral gene delivery of a survivin dominant negative-mutant (AdT34A) versus vector or saline controls. Survivin expression in vessels was analyzed by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and by immunohistochemistry (IHC), which also included markers of SMC differentiation. Specimens of human tissue including failed lower extremity bypass grafts and carotid plaque were also examined. RESULTS: RT-PCR and IHC demonstrated increased survivin expression in all experimental models, colocalizing at early times with proliferating and alpha-actin-expressing cells but was largely absent in mature, contractile SMCs. Delivery of AdT34A after BI attenuated neointimal hyperplasia. CONCLUSION: These studies provide strong evidence supporting a role for survivin in the cellular response to vascular injury. CLINICAL RELEVANCE: The regulation of cell proliferation, death, and phenotype after vascular interventions remains incompletely understood. We investigated the role of the inhibitor of apoptosis protein survivin in diverse models of vascular injury. The results suggest that survivin is an important modulator of the generalized vascular injury response and may represent a relevant target for therapies targeting intimal hyperplasia.