Reversal of TGF-β1 stimulation of α-smooth muscle actin and extracellular matrix components by cyclic AMP in Dupuytren's - derived fibroblasts

<p>Abstract</p> <p>Background</p> <p>Myofibroblasts, a derived subset of fibroblasts especially important in scar formation and wound contraction, have been found at elevated levels in affected Dupuytren's tissues. Transformation of fibroblasts to myofibroblasts is characterized by expression of alpha- smooth muscle actin (α-SMA) and increased production of extracellular matrix (ECM) components, both events of relevance to connective tissue remodeling. We propose that increasing the activation of the cyclic AMP (cAMP)/protein kinase A signaling pathway will inhibit transforming growth factor-beta1 (TGF-β₁)-induced ECM synthesis and myofibroblast formation and may provide a means to blunt fibrosis.</p> <p>Methods</p> <p>Fibroblasts derived from areas of Dupuytren's contracture cord (DC), from adjacent and phenotypically normal palmar fascia (PF), and from palmar fascia from patients undergoing carpal tunnel release (CTR; CT) were treated with TGF-β₁(2 ng/ml) and/or forskolin (10 μM) (a known stimulator of cAMP). Total RNA and protein extracted was subjected to real time RT-PCR and Western blot analysis.</p> <p>Results</p> <p>The basal mRNA expression levels of fibronectin- extra domain A (FN1-EDA), type I (COL1A2) and type III collagen (COL3A1), and connective tissue growth factor (CTGF) were all significantly increased in DC- and in PF-derived cells compared to CT-derived fibroblasts. The TGF-β₁stimulation of α-SMA, CTGF, COL1A2 and COL3A1 was greatly inhibited by concomitant treatment with forskolin, especially in DC-derived cells. In contrast, TGF-β₁stimulation of FN1-EDA showed similar levels of reduction with the addition of forskolin in all three cell types.</p> <p>Conclusion</p> <p>In sum, increasing cAMP levels show potential to inhibit the formation of myofibroblasts and accumulation of ECM components. Molecular agents that increase cAMP may therefore prove useful in mitigating DC progression or recurrence.</p

A Device for Assessment of Hand and Wrist Coronal Plane Strength Introduction

Introduction Measurement of hand strength is critical to diagnosis and assessment of surgical outcome and rehabilitation ͓1͔. The Jamar® Dynamometer and similar devices are often used to record grip strength in clinical settings and experimental investigations ͓1-5͔ but their use is often limited because they do not address activities requiring twisting strength while gripping, such as opening and closing a jar, pill bottle or tube of toothpaste. Investigators have studied activities requiring twisting strength ͓5,6͔ and moments while grasping ͓7,8͔, but no common method or device has received acceptance in quantifying the twisting strength necessary to perform daily activities. In order to more thoroughly evaluate hand function before and after thumb surgery, a scientific device was constructed and tested for immediate use. Any scientific or clinical device suitable for the required task must fulfill several basic requirements. Design requirements dictate that the device must reproducibly record applied torques, provide end effectors of different sizes, be portable, and be usable in a clinic. A desirable feature is the ability to measure forearm rotation strength as well as the coronal plane rotational strength of the wrist ͑radial-ulnar deviation strength͒. Scientific requirements dictate that the device must reliably reproduce previously established relationships. In particular, gender, end-effect size ͓9͔, and hand dominance should produce statistically measurable differences. The current work describes ͑1͒ a device that fulfills all the design aims and ͑2͒ the application of the device in testing hypotheses that gender, end-effect size, and handedness are statistically important factors. This study also tested the hypotheses that thumb carpometacarpal arthritis adversely affects radial-ulnar strength in comparison to a normal population. Methods Description of the Instrument. A scientific device was designed and constructed ͑Fig. 1͒. A custom-fitted, flanged aluminum shaft was bolted to a torque-sensing load cell ͑17 N m maximum, model TRT50, Transducer Techniques, Inc., Temecula, CA͒ and the load cell was rigidly fixed to a stiff platform fitted with suction cups for anchoring to a table. A modified conditioning amplifier was mounted with the load cell on the platform. Disks of 8 mm thickness and diameters of 2.5, 5, 7.5, 10, and 12.5 cm were fabricated from plastic, the edges were rounded and smoothed, and each disk was rubber coated. Each disk could be firmly fixed to the load cell shaft by a simple key. Functional testing was accomplished by gripping and simultaneously twisting a disk while the device was fixed to a surface. The meter recorded the applied torque and the peak value was displayed. The device was able to test both maximal and sustained voluntary isometric strength and could record torque with radial or ulnar deviation. With the suction cups attached to a wall, supination-pronation strength was also tested, although only the radial-ulnar deviation results are presented here. Testing, Subjects, Protocol. The study was approved by the Institutional Review Board at Allegheny General Hospital and a total 77 subjects were recruited from the hospital staff and from the medical practice of the clinical investigator ͑M.E.B.͒. For the normal group of 64 subjects, only volunteers with no history of upper extremity pathology were tested with the device ͑53 righthanded, 11 left-handed; overall average age was 40.5± 12; age range 19-74; 46 females: average age 41± 12, 18 males: average age 39± 13͒. An additional 13 subjects were tested who had been diagnosed with arthritis of the thumb carpometacarpal joint ͑aver-age age 58± 8; 9 female, 4 male͒. After establishing hand dominance by determining which hand was used for writing, each subject adopted the testing position recommended by American Society of Hand Therapists in 1981 ͓1͔ and applied a torque to each of the five disks, with each hand, in both the clockwise and counterclockwise directions, for three trials; a total of 60 trials/ subject. To remove bias, tests were randomized in both the direction of applied torque and the order in which disk sizes were used. The maximum values were recorded for each trial and the results of the three trials were averaged. Nine subjects were tested 1 day later using the same general protocol. Data analysis included t tests and a three-way repeated measures ANOVA. The ages of the male and female groups were compared with a t test and found to present no statistical difference ͑p = 0.28͒. Although age may affect twisting strength, the intent of the present work was not to determine the effect of age or anthropometry on strength, but to establish the ability of the device to record expected results concerning limb dominance, gender, end-effector size, and pathology. The three-way repeated measures ANOVA was performed on the data with repeated measures groupings of dominant/nondominant, radial deviation/ulnar deviation and the five sizes of disk. Gender and subject type ͑normal-arthritic͒ were used as independent variables. A post-hoc test on disk size using Tukey&apos;s honest significant difference was planned. Reproducibility was analyzed with the additional repeated measure of day tested. / Vol. 127, NOVEMBER 2005 Copyright © 2005 by ASME Transactions of the ASME Results Figures 2 and 3 and The test-retest comparison found no statistical difference between the first day and the second day results, p = 0.930. Discussion Differences of gender, hand dominance, disk size, and pathology were all easily distinguished. Males applied greater torques than females, the dominant hand applied greater torque, and subjects diagnosed with carpometacarpal arthritis could not apply normal levels of torque. There were no differences in the testretest group due to the day of testing An effect with disk size was anticipated, but the present tests included disk sizes with diameters larger than the cylinders previously used to quantify torque applied in opening jars ͓6,9͔. Many female participants could not span the largest disk of 12.5 cm with either hand. This difference in end-effector/hand interface could be the primary reason that the average maximal torque applied by females was approximately the same for 10 and 12.5 cm disks. For these same two disks, the maximum torque applied by males increased with disk size, although at a lower rate of increase than observed with the smaller disk diameters. Larger disks, therefore, should be included in subsequent testing of males. Given that pinch strength increases with grip span ͓10͔ and that a relationship between gender and disk size exists, a relationship of applied torque with anthropometry is likely. Although anthropometric measurements were taken with the current data, the study design did not include this analysis and should include a larger population. The reduction in strength with carpometacarpal arthritis indicates that the device can be useful in diagnosis, in quantification of rehabilitation ͓11͔ and in comparison of surgical treatments ͓12͔. These broader applications could include different grip instructions to tailor the test to other tasks, such as those that have examined a variety of end-effectors ͓6͔. For example, instructions about fingertip placement could mimic turning knobs on level surfaces. A smaller disk could also be added to model turning the twist-tops on tubes. The device can readily evaluate thumb function. The protocol and device did not control or consider variation in arm or body posture and can be considered valid only for the arm at the side and the elbow at 90°. Grip strength varies with shoulder and elbow position ͓13͔ and a similar effect is expected with With this device, we have successfully quantified strength, reproduced known tests, and distinguished pathologic from normal strengths. The results specifically quantify the radial and ulnar deviation at the neutral wrist position. Future work will include clinical trials, use of additional end-effectors, analysis of the effect of age, supination-pronation testing, rehabilitation, and the effects of different pathologies

Investigating the effects of Pirfenidone on TGF-β1 stimulated non-SMAD signaling pathways in Dupuytren’s disease -derived fibroblasts

Abstract Background Dupuytren’s disease (DD) is a progressive, debilitating condition of the hand that can eventually cause contractures of the affected fingers. Transforming growth factor- β1 (TGF-β1) has been reported to play a key role in DD pathology. Increased expression of TGF-β1 has shown to be the main stimulator of myofibroblast activity and in DD contractures. Pirfenidone (PFD), a small active molecule possess the ability to inhibit TGF-β1-mediated action in various fibrotic disorders. Our recent published findings show that PFD reduced TGF-β1-mediated cellular functions implicated in DD through SMAD signaling pathways. In the present study, the effect of PFD on TGF-β1-mediated non-SMAD signaling pathways were investigated in both carpal tunnel (CT) - and DD-derived fibroblasts. Methods Fibroblasts harvested from Dupuytren’s disease (DD) and carpal tunnel (CT) tissues were cultured in the presence or absence of TGF-β1 (10 ng/ml) and/or PFD (800 μg/ml). Cell lysates were analyzed using Western blots. Equal amounts of proteins were loaded to determine the phosphorylation levels of phosphatidylinositol-3 kinase (PI3K/AKT), extracellular regulated kinases (ERK1/2), p38 mitogen-activated protein kinase and Rho family related myosin light chain (MLC). Results We show that the TGF-β1-induced phosphorylation of AKT was significantly decreased by the addition of PFD (800 μg/mL) in both CT- and DD-derived fibroblasts. Interestingly, there was no significant difference in the phosphorylation levels of both ERK and p38 on TGF-β1- induced cells in both CT-and DD-derived fibroblasts. But, PFD significantly decreased the TGF- β1-induced phosphorylation levels of ERK1/2 in both CT- and DD- cells. In contrast, PFD significantly decreased the basal and TGF- β1-induced phosphorylation levels of p38 in DD-derived fibroblasts. TGF- β1-induced phosphorylation levels of MLC was decreased by PFD in DD-derived fibroblasts. Conclusions These in-vitro results indicate for the first time that PFD has the potential to inhibit TGF-β1-induced non-SMAD signaling pathways in both CT- and DD-derived fibroblasts but pronounced statistically significant inhibition on all molecules was observed only in DD-derived fibroblasts. Our previous studies show that PFD can inhibit TGF-β1- induced SMAD signaling pathway proteins, namely p- SMAD2/SMAD3. These broad and complementary actions suggest PFD as a promising candidate to inhibit the TGF-β1- mediated molecular mechanisms leading to DD fibrosis

sj-jpg-2-han-10.1177_15589447231184894 – Supplemental material for Computed Tomography–Based Templating of Proximal Ulna Intramedullary Screw Fixation

Supplemental material, sj-jpg-2-han-10.1177_15589447231184894 for Computed Tomography–Based Templating of Proximal Ulna Intramedullary Screw Fixation by Nitin Goyal, Dominic Coutinho, Anish Ghodadra, Mark E. Baratz and Robert A. Kaufmann in HAND</p

sj-docx-1-han-10.1177_15589447231184894 – Supplemental material for Computed Tomography–Based Templating of Proximal Ulna Intramedullary Screw Fixation

Supplemental material, sj-docx-1-han-10.1177_15589447231184894 for Computed Tomography–Based Templating of Proximal Ulna Intramedullary Screw Fixation by Nitin Goyal, Dominic Coutinho, Anish Ghodadra, Mark E. Baratz and Robert A. Kaufmann in HAND</p