Angelica Selim

TNXB Mutations Can Cause Vesicoureteral Reflux

Primary vesicoureteral reflux (VUR) is the most common congenital anomaly of the kidney and the urinary tract, and it is a major risk factor for pyelonephritic scarring and CKD in children. Although twin studies support the heritability of VUR, specific genetic causes remain elusive. We performed a sequential genome-wide linkage study and whole-exome sequencing in a family with hereditary VUR. We obtained a significant multipoint parametric logarithm of odds score of 3.3 on chromosome 6p, and whole-exome sequencing identified a deleterious heterozygous mutation (T3257I) in the gene encoding tenascin XB (TNXB in 6p21.3). This mutation segregated with disease in the affected family as well as with a pathogenic G1331R change in another family. Fibroblast cell lines carrying the T3257I mutation exhibited a reduction in both cell motility and phosphorylated focal adhesion kinase expression, suggesting a defect in the focal adhesions that link the cell cytoplasm to the extracellular matrix. Immunohistochemical studies revealed that the human uroepithelial lining of the ureterovesical junction expresses TNXB, suggesting that TNXB may be important for generating tensile forces that close the ureterovesical junction during voiding. Taken together, these results suggest that mutations in TNXB can cause hereditary VUR.

Myofibroblasts contribute to but are not necessary for wound contraction.

Wound contraction facilitates tissue repair. The correct balance between too little contraction, which leads to non-healing wounds, and too much contraction, which leads to contractures, is important for optimal healing. Thus, understanding which cells cause wound contraction is necessary to optimize repair. Wound contraction is hypothesized to develop from myofibroblast (cells which express alpha-smooth muscle actin; ACTA2) contractility, while the role of fibroblast contractility is unknown. In this study, we utilized ACTA2 null mice to determine what role fibroblasts play in wound contraction. Human scar contractures were immunostained for ACTA2, beta-cytoplasmic actin (ACTB), and gamma-cytoplasmic actin (ACTG1). Full-thickness cutaneous wounds were created on dorsum of ACTA2+/+ mice and strain-matching ACTA2+/− and ACTA2−/− mice. Wound contraction was quantified. Tissue was harvested for histologic, immunohistochemical and protein analysis. Compared with surrounding unwounded skin, human scar tissue showed increased expression of ACTA2, ACTB, and ACTG1. ACTA2 was focally expressed in clusters. ACTB and ACTG1 were widely, highly expressed throughout scar tissue. Wound contraction was significantly retarded in ACTA2−/− mice, as compared to ACTA2+/+ controls. Control mice had increased epithelialization, cell proliferation, and neovascularization. ACTA2−/− mice had lower levels of apoptosis, and fewer total numbers of cells. Smaller amount of collagen deposition and immature collagen organization in ACTA2−/− mice demonstrate that wounds were more immature. These data demonstrate that myofibroblasts contribute to but are not necessary for wound contraction. Mechanisms by which fibroblasts promote wound contraction may include activation of contractile signaling pathways, which promote interaction between non-muscle myosin II and ACTB and ACTG1.

Dupuytren's fibroblast contractility by sphingosine-1-phosphate is mediated through non-muscle myosin II.

PURPOSE Previous studies suggest that Dupuytrens disease is caused by fibroblast and myofibroblast contractility within Dupuytrens nodules; however, the stimulus for cell contractility is unknown. Sphingosine-1-phosphate (S1P) is a serum-derived lysophospholipid mediator that enhances cell contractility by activating the S1P receptor, S1P(2). It is hypothesized that S1P stimulates Dupuytrens fibroblast contractility through S1P(2) activation of non-muscle myosin II (NMMII). This investigation examined the role of S1P and NMMII activation in Dupuytrens disease progression and suggests potential targets for treatment. METHODS We enmeshed Dupuytrens fibroblasts into fibroblast-populated collagen lattices (FPCLs) and assayed S1P-stimulated FPCL contraction in the presence of the S1P(2) receptor inhibitor JTE-013, the Rho kinase inhibitor Y-27632, the myosin light chain kinase inhibitor ML-7, and the NMMII inhibitor blebbistatin. Tissues from Dupuytrens fascia (n = 10) and normal palmar fascia (n = 10) were immunostained for NMMIIA and NMMIIB. RESULTS Sphingosine-1-phosphate stimulated FPCL contraction in a dose-dependent manner. Inhibition of S1P(2) and NMMII prevented S1P-stimulated FPCL contraction. Rho kinase and myosin light chain kinase inhibited both S1P and control FPCL contraction. Dupuytrens nodule fibroblasts robustly expressed NMMIIA and NMMIIB, compared with quiescent-appearing cords and normal palmar fascia. CONCLUSIONS Sphingosine-1-phosphate promotes Dupuytrens fibroblast contractility through S1P(2), which stimulates activation of NMMII. NMMII isoforms are ubiquitously expressed throughout Dupuytrens nodules, which suggests that nodule fibroblasts are primed to respond to S1P stimulation to cause contracture formation. S1P-promoted activation of NMMII may be a target for disease treatment.

Erratum to: angiotensin II stimulates canonical TGF-β signaling pathway through angiotensin type 1 receptor to induce granulation tissue contraction.

Hypertrophic scar contraction (HSc) is caused by granulation tissue contraction propagated by myofibroblast and fibroblast migration and contractility. Identifying the stimulants that promote migration and contractility is key to mitigating HSc. Angiotensin II (AngII) promotes migration and contractility of heart, liver, and lung fibroblasts; thus, we investigated the mechanisms of AngII in HSc. Human scar and unwounded dermis were immunostained for AngII receptors angiotensin type 1 receptor (AT1 receptor) and angiotensin type 2 receptor (AT2 receptor) and analyzed for AT1 receptor expression using Western blot. In vitro assays of fibroblast contraction and migration under AngII stimulation were conducted with AT1 receptor, AT2 receptor, p38, Jun N-terminal kinase (JNK), MEK, and activin receptor-like kinase 5 (ALK5) antagonism. Excisional wounds were created on AT1 receptor KO and wild-type (WT) mice treated with AngII ± losartan and ALK5 and JNK inhibitors SB-431542 and SP-600125, respectively. Granulation tissue contraction was quantified, and wounds were analyzed by immunohistochemistry. AT1 receptor expression was increased in scar, but not unwounded tissue. AngII induced fibroblast contraction and migration through AT1 receptor. Cell migration was inhibited by ALK5 and JNK, but not p38 or MEK blockade. In vivo experiments determined that absence of AT1 receptor and chemical AT1 receptor antagonism diminished granulation tissue contraction while AngII stimulated wound contraction. AngII granulation tissue contraction was diminished by ALK5 inhibition, but not JNK. AngII promotes granulation tissue contraction through AT1 receptor and downstream canonical transforming growth factor (TGF)-β signaling pathway, ALK5. Further understanding the pathogenesis of HSc as an integrated signaling mechanism could improve our approach to establishing effective therapeutic interventions.Key messageAT1 receptor expression is increased in scar tissue compared to unwounded tissue.AngII stimulates expression of proteins that confer cell migration and contraction.AngII stimulates fibroblast migration and contraction through AT1 receptor, ALK5, and JNK.AngII-stimulated in vivo granulation tissue contraction is AT1 receptor and ALK5 dependent.

A methodology for utilization of predictive genomic signatures in FFPE samples

BackgroundGene expression signatures developed to measure the activity of oncogenic signaling pathways have been used to dissect the heterogeneity of tumor samples and to predict sensitivity to various cancer drugs that target components of the relevant pathways, thus potentially identifying therapeutic options for subgroups of patients. To facilitate broad use, including in a clinical setting, the ability to generate data from formalin-fixed, paraffin-embedded (FFPE) tissues is essential.MethodsPatterns of pathway activity in matched fresh-frozen and FFPE xenograft tumor samples were generated using the MessageAmp Premier methodology in combination with assays using Affymetrix arrays. Results generated were compared with those obtained from fresh-frozen samples using a standard Affymetrix assay. In addition, gene expression data from patient matched fresh-frozen and FFPE melanomas were also utilized to evaluate the consistency of predictions of oncogenic signaling pathway status.ResultsSignificant correlation was observed between pathway activity predictions from paired fresh-frozen and FFPE xenograft tumor samples. In addition, significant concordance of pathway activity predictions was also observed between patient matched fresh-frozen and FFPE melanomas.ConclusionsReliable and consistent predictions of oncogenic pathway activities can be obtained from FFPE tumor tissue samples. The ability to reliably utilize FFPE patient tumor tissue samples for genomic analyses will lead to a better understanding of the biology of disease progression and, in the clinical setting, will provide tools to guide the choice of therapeutics to those most likely to be effective in treating a patients disease.

Abstract 124: Mitigation Of Hypertrophic Scar Contraction And Stiffening Via An Elastomeric Biodegradable Scaffold

1The Division of Plastic and Reconstructive Surgery, Department of Surgery, Duke University School of Medicine, Durham, NC, 2Department of Biomedical Engineering, Duke University, Durham, NC, 3Korea Institute of Science and Technology Biomaterials Research Center, Republic of Korea, 4Duke University Medical Center, Department of Pathology, Durham, NC, 5Columbia University, Department of Biomedical Engineering, New York, NY

Abstract 166: Electrospun Synthetic Scaffolds

ConClusion: Our findings indicate that the use of CD90selected ASCs may facilitate more rapid regeneration of skeletal defects. Furthermore, NOG knockdown may serve to augment bone differentiation through an increase in BMP signaling. The integration of these two strategies through magnet-assisted transfection may lead to the development of promising, temporospatially controlled treatments for clinical translation. 166 electrospun Synthetic Scaffolds: a Biomimetic approach to Prevent hypertrophic Scar contraction

Beyond Pathology: Pump-Probe Imaging of Skin Slices Provides Additional Indicators of Melanoma

Principal component analysis of images taken with a pump-probe scanning microscope resolves eumelanin and pheomelanin. Utilizing intrinsic melanin contrast in skin slices has revealed significant differences between melanoma and other lesions.