Christy Gliniak

Propranolol induces regression of hemangioma cells through HIF-1α-mediated inhibition of VEGF-A

Objective: To investigate the mechanism of propranolol on regression of infantile hemangiomas. Background: Propranolol has been found to be effective in treatment of severe hemangiomas of infancy. However, its mechanism of action is as yet unknown. Methods: Cultured proliferating and involuting hemangioma endothelial cells were treated with varying concentrations of propranolol for up to 4 days. Analysis was performed using cell viability, migration, and tubulogenesis assays, as well as quantitative RT-PCR and flow cytometry. Western blots and ELISA assays were used to assess protein expression. Results: Treatment with propranolol led to a dose dependent cytotoxic effect in hemangioma endothelial cells with decreased cell viability, migration, and tubulogenesis. This cytotoxic effect was VEGF (vascular endothelial growth factor) dependent, as demonstrated by decreased VEGF, VEGF-R1, and VEGF-R2 production. Decreased signaling through the VEGF pathway resulted in downregulation of PI3/Akt and p38/MAPK activity. Decreased VEGF activity was mediated through the hypoxia inducible factor (HIF)-1&agr; pathway but not through NF-&kgr;&bgr; signaling. Conclusions: Collectively, these data suggest that propranolol exerts its suppressive effects on hemangiomas through the HIF-1&agr;-VEGF-A angiogenesis axis, with effects mediated through the PI3/Akt and p38/MAPK pathways. These findings provide a plausible mechanism of action of propranolol on regression of infantile hemangiomas.

TGF-β1 RNA interference in mouse primary dura cell culture: Downstream effects on TGF receptors, FGF-2, and FGF-R1 mRNA levels

Background: Transforming growth factor (TGF)-&bgr;1 and fibroblast growth factor (FGF)-2 have both been shown to have significant roles in the regulation of murine calvarial suture fusion. Methods to decrease gene expression of these cytokines and their respective receptors have been established, but because of side effects, clinical applications are limited. In this study, the authors examined the effect of TGF-&bgr;1–specific small interfering RNA (siRNA) on the messenger RNA (mRNA) expression of TGF-&bgr;1, its TGF-&bgr;R1 and TGF-&bgr;R2 receptors, and FGF-2 and its R1 receptor in murine dura cells. Methods: A primary dura cell line was established from CD-1 mice. Transfection efficiency using Lipofectamine was determined using BLOCKiT. Dura cells were transfected with serial concentrations of TGF-&bgr;1 siRNA to determine the optimal dose. In subsequent experiments, cells were transfected with 16 nM TGF-&bgr;1 siRNA and harvested on posttransfection days 4, 7, 10, and 14 for RNA isolation and quantitative polymerase chain reaction. Results: Optimal inhibition of TGF-&bgr;1 mRNA expression was achieved at 16 nM siRNA. On posttransfection day 4, TGF-&bgr;1 mRNA levels were significantly decreased but returned to baseline by day 14. TGF-&bgr;R1 mRNA expression remained unaffected by transfection throughout the time course. However, TGF-&bgr;R2, FGF-2, and FGF-R1 demonstrated significant inhibition of mRNA expression on posttransfection day 4. Conclusions: These results indicate that TGF-&bgr;1 siRNA has the potential to alter the murine dura cytokines responsible for suture fusion in vitro. Manipulating underlying cranial suture biology with siRNA technology may ultimately allow control over suture fusion. This intervention may ultimately function as an effective adjunct to surgical intervention for craniosynostosis.

The contribution of endogenous and exogenous factors to male alopecia: a study of identical twins.

Background: In this study, the authors investigated the potential contribution of environmental factors and testosterone levels on androgenic alopecia in women. Methods: Ninety-eight identical female twins were recruited from 2009 to 2011. Subjects were asked to complete a comprehensive questionnaire, provide a sputum sample for testosterone analysis, and pose for standardized digital photography. Frontal, temporal, and vertex hair loss were assessed from the photographs using Adobe Photoshop. Hair loss measures were then correlated with survey responses and testosterone levels between twin pairs. Two independent, blinded observers also rated the photographs for hair thinning. Results: Factors associated with increased frontal hair loss included multiple marriages (p = 0.043); longer sleep duration (p = 0.011); higher severity of stress (p = 0.034); positive smoking history (p = 0.021); higher income (p = 0.023); absence of hat use (p = 0.017); and history of diabetes mellitus (p = 0.023), polycystic ovarian syndrome (p = 0.002), and hypertension (p = 0.001). Factors associated with increased temporal hair loss included divorce or separation (p = 0.034), multiple marriages (p = 0.040), more children (p = 0.005), longer sleep duration (p = 0.006), and history of diabetes mellitus (p = 0.008) and hypertension (p = 0.027). Lack of sun protection (p = 0.020), consuming less caffeine (p = 0.040), history of skin disease (p = 0.048), and lack of exercise (p = 0.012) were associated with increased vertex hair loss. Higher testosterone levels were associated with increased temporal and vertex hair loss patterns (p < 0.039). Increased stress, increased smoking, having more children, and having a history of hypertension and cancer were all associated with increased hair thinning (p < 0.05). Conclusion: This study implicates several environmental risk factors in the pathophysiology of female alopecia.

The role of different methods of nerve ablation in prevention of neuroma

Background: The aim of this study was to compare the incidence of neuroma formation and neuropathic pain following different techniques of nerve ablation in a rat sural nerve model. Methods: Rat sural nerve was subjected to four different techniques of ablation with standardized creation of a 1-cm gap (n = 15 in each group). These included nerve avulsion, transection and burying in muscle, transection and folding of nerve, and transection alone. Animals were killed after 3 months. Explanted nerves were sectioned and stained with Masson trichrome and S-100 stain against neural tissue. The maximal neural cross-sectional area and neural-to-connective tissue ratio was quantified. Quantitative reverse-transcriptase polymerase chain reaction (n = 5) was used to analyze relative mRNA expression of ciliary neurotrophic factor and calcitonin gene-related peptide. Results: Neural cross-sectional area was statistically increased (p < 0.05) compared with controls in folded, muscle buried, and transected specimens but decreased in avulsed specimens. The neural-to-connective tissue ratio was statistically decreased in the avulsed group. Relative mRNA expression of ciliary neurotrophic factor was lowest in muscle buried (4 percent of control) (p < 0.05) and avulsed specimens (15 percent of control) (p < 0.05) and higher in folded (52 percent of control) and transected specimens (75 percent of control). Relative mRNA expression of calcitonin gene-related peptide was highest in folded specimens (302 percent of control) (p < 0.05). Conclusions: Folding and transection lead to increased histologic evidence of neuroma formation, whereas folding leads to neuropathic pain, assayed by calcitonin gene-related peptide expression. Avulsion and muscle burying are preferable techniques for nerve ablation and inhibit nerve regeneration, evidenced by decreased ciliary neurotrophic factor expression. Avulsion offers an alternative to muscle burying when there is no muscle in the vicinity to bury the transected nerve.

Nonviral transfection of mouse calvarial organ in vitro using accell-modified siRNA

Background: Understanding the biology of cranial suture fusion and the precise role of involved molecules implicated in the process will help to identify key factors involved in regulation of suture fusion. Modulation of these key factors may serve as a tissue-engineering technique to replace the traditional surgical procedures for the correction of premature suture fusion. Modulation of gene expression by RNA interference is a widely used technique with high potential. Because there is no available report of calvarial organ transfection in vitro, the authors studied the development of a successful nonviral delivery technique of small inhibitory RNA (siRNA) to an in vitro calvarial organ culture system. Methods: In this study, 19-day-old male CD1 mice were euthanized and parallel craniotomies made through the parietal and frontal calvaria, 2 mm to either side of the sagittal suture, with care taken to preserve the underlying dura mater. Organs grown in vitro in a defined medium were transfected with transforming growth factor-&bgr;1–specific Accell-modified siRNA followed by RNA isolation and quantitative polymerase chain reaction analysis. Results: Transfection of a calvarial organ with transforming growth factor-&bgr;1–specific Accell-modified siRNA effectively knocks down the mRNA level. Conclusions: Observations from this study indicate that in an in vitro calvarial organ culture system, a specific, efficient, and durable RNA interference activity can be achieved when Accell-modified siRNA is used. In addition to bypassing the need for toxic lipid carriers, the modifications introduced in Accell-modified siRNAs make it more stable and less off-target. This technique can potentially be used for in vivo studies once the initial effect of gene-specific siRNA on in vitro suture fusion has been determined.

The Retinol-Binding Protein Receptor STRA6 Regulates Diurnal Insulin Responses

It has long been appreciated that insulin action is closely tied to circadian rhythms. However, the mechanisms that dictate diurnal insulin sensitivity in metabolic tissues are not well understood. Retinol-binding protein 4 (RBP4) has been implicated as a driver of insulin resistance in rodents and humans, and it has become an attractive drug target in type II diabetes. RBP4 is synthesized primarily in the liver where it binds retinol and transports it to tissues throughout the body. The retinol–RBP4 complex (holo-RBP) can be recognized by a cell-surface receptor known as stimulated by retinoic acid 6 (STRA6), which transports retinol into cells. Coupled to retinol transport, holo-RBP can activate STRA6-driven Janus kinase (JAK) signaling and downstream induction of signal transducer and activator of transcription (STAT) target genes. STRA6 signaling in white adipose tissue has been shown to inhibit insulin receptor responses. Here, we examined diurnal rhythmicity of the RBP4/STRA6 signaling axis and investigated whether STRA6 is necessary for diurnal variations in insulin sensitivity. We show that adipose tissue STRA6 undergoes circadian patterning driven in part by the nuclear transcription factor REV-ERBα. Furthermore, STRA6 is necessary for diurnal rhythmicity of insulin action and JAK/STAT signaling in adipose tissue. These findings establish that holo-RBP and its receptor STRA6 are potent regulators of diurnal insulin responses and suggest that the holo-RBP/STRA6 signaling axis may represent a novel therapeutic target in type II diabetes.

Abstract 95: STROMAL CELL DERIVED FACTOR (SDF)-1 WITH BMP-2 AND TGF-B1 INDUCES SITE DIRECTED CELL HOMING, OSTEOGENIC AND CHONDROGENIC DIFFERENTIATION WITHOUT THE REQUIREMENT FOR CELL SEEDING

Background: Bone defect repairs remain a major concern in reconstructive surgery. Injectable gels, as bone grafts, offer several advantages over preformed solid scaffolds, and include the capacity to  ll irregular shaped defects, the  exibility to incorporate therapeutic agents (e.g., growth factor) and/or cells, and the ease of placement by minimum invasive procedures. The aim of this study was to test an injectable gel graft made of tranglutaminase crosslinked gelatin gel / BMP-2 for bone defect repairs.

The Relative Contribution of Endogenous and Exogenous Factors to Male Alopecia: A Study of 66 Genetically Identical Males

reSultS: A total of 66 males with a mean age of 51.32 (23 – 82) were available for analysis. Genetics was the strongest predictor of midline hair loss (F 10.491, p = 0.002). A positive smoking history and increasing pack years was associated with increased midline hair loss (F 6.89 and 8.433; p = 0.011 and 0.008). In addition, increasing sun exposure and a history of dandruff was associated with increased midline hair loss (F 4.491 and 4.088, p = 0.039 and p = 0.047). Cumulative sun exposure and a history of cancer were associated with significantly more temporal hair loss. Age was a stronger predictor of coronal hair loss than genetics (F 6.409 vs. 4.033; p = 0.014 and 0.049). Again cumulative years of sun exposure and history of cancer were significant predictors of coronal hair loss. Those twins who had a history of hypertension and did not regularly exercise also had significantly more coronal hair loss (F 6.602 and 8.367; p = 0.017 and 0.005). 22 male twins (mean age 50.09) provided sputum samples for evaluation of testosterone levels. Genetics was a significant predictor of testosterone levels (F5.567); p=0.029) while age was not. However, the combination of age and genetics was a significantly better predictor of male testosterone levels than either alone (F6.019); p=0.009). Increased sputum testosterone levels were a significant predictor of decreased male temporal hair loss (F12.197); p=0.002).

48B: REGULATION OF OSTEOGENIC EXPRESSION OF PGE2 BY TGF-B1 IN MOUSE CALVARIAL CELLS

Background: Prostaglandin E2 (PGE2) plays an important role in skeletal tissue development by stimulating bone formation and resorption through the replication and differentiation of osteoblasts. Transforming Growth Factor B1 (TGF-B1) has been shown to regulate the expression of Cox2, a key enzyme in PGE2 biosynthesis, in a variety of cell types. However, to the best of our knowledge, there is no published report indicating the TGF-B1 mediated regulation of Cox-2 in calvaria. Because TGF-B1 is known to play an important role in cranial suture fusion, the present study was undertaken to study the effect of TGF-B1 on Cox-2 expression and subsequently on PGE2 expression.