Biological Targets for Dupuytren Disease.

ABSTRACT: Dupuytren disease is a connective tissue disorder occurring on the palm, causing flexion contractures of fingers. There is a pressing need for therapeutic interventions that can slow, stop, or even incrementally reverse the progression of the disease. Numerous in vitro studies have shed light on cellular and molecular agents that contribute to contractures. This article comprehensively reviews various growth factors that can be targeted to prevent and limit the progression and recurrence of Dupuytren contracture (DC). Fibroblasts are the major cell population that has been reported for the contractures in DC, and they are also known to exacerbate the cytokine production. Limiting the fibroblast function by targeting the growth factor production will be of great benefit in treating DC. This review will focus on the studies that have shown to limit the exaggerated function of fibroblasts by reducing the expression of profibrotic growth factors by using antagonizing agents.

Contribution of Organic Anion-Transporting Polypeptides 1A/1B to Doxorubicin Uptake and Clearance.

The organic anion-transporting polypeptides represent an important family of drug uptake transporters that mediate the cellular uptake of a broad range of substrates including numerous drugs. Doxorubicin is a highly efficacious and well-established anthracycline chemotherapeutic agent commonly used in the treatment of a wide range of cancers. Although doxorubicin is a known substrate for efflux transporters such as P-glycoprotein (P-gp; MDR1, ABCB1), significantly less is known regarding its interactions with drug uptake transporters. Here, we investigated the role of organic anion transporting polypeptide (OATP) transporters to the disposition of doxorubicin. A recombinant vaccinia-based method for expressing uptake transporters in HeLa cells revealed that OATP1A2, but not OATP1B1 or OATP1B3, and the rat ortholog Oatp1a4 were capable of significant doxorubicin uptake. Interestingly, transwell assays using Madin-Darby canine kidney II cell line cells stably expressing specific uptake and/or efflux transporters revealed that OATP1B1, OATP1B3, and OATP1A2, either alone or in combination with MDR1, significantly transported doxorubicin. An assessment of polymorphisms in SLCO1A2 revealed that four variants were associated with significantly impaired doxorubicin transport in vitro. In vivo doxorubicin disposition studies revealed that doxorubicin plasma area under the curve was significantly higher (1.7-fold) in Slco1a/1b-/- versus wild-type mice. The liver-to-plasma ratio of doxorubicin was significantly decreased (2.3-fold) in Slco1a/1b2-/- mice and clearance was reduced by 40% compared with wild-type mice, suggesting Oatp1b transporters are important for doxorubicin hepatic uptake. In conclusion, we demonstrate important roles for OATP1A/1B in transporter-mediated uptake and disposition of doxorubicin.

Interindividual and interethnic variability in drug disposition: polymorphisms in organic anion transporting polypeptide 1B1 (OATP1B1; SLCO1B1).

OATP1B1 (SLCO1B1) is predominantly expressed at the basolateral membrane of hepatocytes and is critically important for the hepatic uptake and clearance of numerous drug substrates and endogenous compounds. In general, the organic anion transporting polypeptides (OATP; SLCO) represent a superfamily of uptake transporters that mediate the sodium-independent transport of a diverse range of amphipathic organic compounds including bile salts, steroid conjugates, thyroid hormones, anionic peptides, numerous drugs and other xenobiotic substances. OATP1B1 is highly polymorphic and a number of relevant and ethnically dependent polymorphisms have been identified and functionally characterized. In particular, the SLCO1B1 521T>C and 388A>G polymorphisms are commonly occurring variants in ethnically diverse populations and numerous in vitro and clinical studies have evaluated the consequences of these variants to interindividual differences in drug disposition and response. OATP1B1 is particularly important for the disposition of HMG-CoA reductase inhibitors, or statins, as it is known to efficiently transport most statins to their site of action within hepatocytes. Many studies have focused on the consequences of OATP1B1 variants to statin disposition in vitro and in vivo and would suggest that genetic variability in SLCO1B1 has important implications for statin pharmacokinetics, risk for statin-induced myopathy, and modulation of statin treatment response. This review describes what is currently known regarding SLCO1B1 genotype, OATP1B1 protein expression and interindividual and interethnic consequences to drug disposition, with particular focus on statin pharmacokinetics and implications for drug response and toxicity.

Agarose disk electroporation method for ex vivo retinal tissue cultured at the air-liquid interface reveals electrical stimulus-induced cell cycle reentry in retinal cells.

It is advantageous to culture the ex vivo murine retina along with many other tissue types at the air-liquid interface. However, gene delivery to these cultures can be challenging. Electroporation is a fast and robust method of gene delivery, but typically requires submergence in a liquid buffer to allow electric current flow. We have developed a submergence-free electroporation technique using an agarose disk that allows for efficient gene delivery to the ex vivo murine retina. This method advances our ability to use ex vivo retinal tissue for genetic studies and can easily be adapted for any tissue cultured at an air-liquid interface. We found an increased ability to transfected Muller glia at 14 days ex vivo and an increase in BrdU incorporation in Muller glia following electrical stimulation. Use of this method has revealed valuable insights on the state of ex vivo retinal tissues and the effects of electrical stimulation on retinal cells.

Effects of doxycycline on mesenchymal stem cell chondrogenesis and cartilage repair.

OBJECTIVE: Strategies to improve cartilage repair tissue quality after bone marrow cell-based procedures may reduce later development of osteoarthritis. Doxycycline is inexpensive, well-tolerated, and has been shown to reduce matrix-metalloproteinases (MMPs) and osteoarthritis progression. This study tests the hypotheses that doxycycline reduces MMP, enhances chondrogenesis of human bone marrow-derived mesenchymal stem cells (hMSC), and improves in vivo cartilage repair. DESIGN: Ninety hMSC pellets were cultured in chondrogenic media with either 0-, 1- or 2-µg/mL doxycycline. Pellets were evaluated with stereomicroscopy, proteoglycan assay, qRT-PCR, and histology. Osteochondral defects (OCDs) were created in the trochlear grooves of 24-Sprague-Dawley rats treated with/without oral doxycycline. Rats were sacrificed at 12-weeks and repair tissues were examined grossly and histologically. RESULTS: hMSC pellets with 1-µg/mL (P = 0.014) and 2-µg/mL (P = 0.002) doxycycline had larger areas than pellets without doxycycline. hMSC pellets with 2-µg/mL doxycycline showed reduced mmp-13 mRNA (P = 0.010) and protein at 21-days. Proteoglycan, DNA contents, and mRNA expressions of chondrogenic genes were similar (P > 0.05). For the in vivo study, while the histological scores were similar between the two groups (P = 0.116), the gross scores of the OCD repair tissues in doxycycline-treated rats were higher at 12-weeks (P = 0.017), reflective of improved repair quality. The doxycycline-treated repairs also showed lower MMP-13 protein (P = 0.029). CONCLUSIONS: This study shows that doxycycline improves hMSC chondrogenesis and decreases MMP-13 in pellet cultures and within rat OCDs. Doxycycline exerted no negative effect on multiple measures of chondrogenesis and cartilage repair. These data support potential use of doxycycline to improve cartilage repair to delay the onset of osteoarthritis.

Graphene-based microfluidic perforated microelectrode arrays for retinal electrophysiological studies.

Perforated microelectrode arrays (pMEAs) have become essential tools for ex vivo retinal electrophysiological studies. pMEAs increase the nutrient supply to the explant and alleviate the accentuated curvature of the retina, allowing for long-term culture and intimate contacts between the retina and electrodes for electrophysiological measurements. However, commercial pMEAs are not compatible with in situ high-resolution optical imaging and lack the capability of controlling the local microenvironment, which are highly desirable features for relating function to anatomy and probing physiological and pathological mechanisms in retina. Here we report on microfluidic pMEAs (µpMEAs) that combine transparent graphene electrodes and the capability of locally delivering chemical stimulation. We demonstrate the potential of µpMEAs by measuring the electrical response of ganglion cells to locally delivered high K+ stimulation under controlled microenvironments. Importantly, the capability for high-resolution confocal imaging of the retina tissue on top of the graphene electrodes allows for further analyses of the electrical signal source. The new capabilities provided by µpMEAs could allow for retinal electrophysiology assays to address key questions in retinal circuitry studies.

Localization of the Median Nerve and Flexor Pollicis Longus at the Carpal Tunnel Inlet in Patients With and Without Carpal Tunnel Syndrome Using Ultrasound.

BACKGROUND: Current illustrations of the carpal tunnel vary greatly. The relative positions of the components such as the median nerve and flexor pollicis longus (FPL) tendon seem often arbitrarily chosen. The purpose of this study was to determine the locations of the median nerve and FPL in the carpal tunnel using ultrasound (US) and to determine whether the position of the median nerve changes in carpal tunnel syndrome (CTS). METHOD: Patients with and without CTS underwent US examination of the wrist. A 4 × 10 grid was fitted to each saved cross-sectional image. The center points of the median nerve and FPL were identified, and their horizontal and vertical coordinates were recorded. RESULTS: The median nerve was identified in 115 wrists (average x = 0.70, y = 0.82), and FPL was identified in 90 wrists (average x = 0.86, y = 0.59). A scatter plot was created by stacking all US images to demonstrate the average positions of the median nerve and FPL. There were 97 wrists without CTS (No CTS) and 17 wrists with CTS. There was a significant difference in the vertical position of the median nerve between No CTS and CTS wrists (P = .0006). CONCLUSIONS: The locations of the median nerve and FPL within the carpal tunnel were determined using US of 115 wrists, and a heat map was created to illustrate these locations. The median nerve was found to be more superficial in the setting of CTS.

The intestine-specific transcription factor Cdx2 inhibits beta-catenin/TCF transcriptional activity by disrupting the beta-catenin-TCF protein complex.

Cdx2 is an intestine-specific transcription factor known to regulate proliferation and differentiation. We have reported previously that Cdx2 limits the proliferation of human colon cancer cells by inhibiting the transcriptional activity of the beta-catenin-T-cell factor (TCF) bipartite complex. Herein we further elucidate this mechanism. Studies with a classic Cdx2 target gene and a canonical Wnt/beta-catenin/TCF reporter suggest that Cdx2 regulates these promoters by distinctly different processes. Specifically, inhibition of beta-catenin/TCF activity by Cdx2 does not require Cdx2 transcriptional activity. Instead, Cdx2 binds beta-catenin and disrupts its interaction with the DNA-binding TCF factors, thereby silencing beta-catenin/TCF target gene expression. Using Cdx2 mutants, we map the Cdx2 domains required for the inhibition of beta-catenin/TCF activity. We identify a subdomain in the N-terminus that is highly conserved and when mutated significantly reduces Cdx2 inhibition of beta-catenin/TCF transcriptional activity. Mutation of this subdomain also abrogates Cdx2's anti-proliferative effects in colon cancer cells. In summary, we conclude that Cdx2 binds beta-catenin and disrupts the beta-catenin-TCF complex. Considering the pivotal role of beta-catenin/TCF activity in driving proliferation of normal intestinal epithelial and colon cancer cells, our findings suggest a novel mechanism for Cdx2-mediated regulation of Wnt/beta-catenin signaling and cell proliferation.

Accuracy and Reliability of Visual Inspection and Smartphone Applications for Measuring Finger Range of Motion.

Measurement of finger range of motion is critical in clinical settings, especially for outcome analysis, clinical decision making, and rehabilitation/disability assessment. Although goniometer measurement is clinically considered the gold standard, its accuracy compared with the true radiographic measurements of the joint angles remains questionable. The authors compared 3 smartphone applications and visual inspection measurements of the finger joints with the radiographic measurements and determined interrater reliability for these measurement tools. A finger was held in place using an aluminum-alloy splint, and a fluoroscopic image was acquired by a mini C-arm. An independent observer measured each joint flexion angle of the fluoroscopic image using a universal handheld goniometer, and this was used as the reference. Finger joint flexion angles were then independently measured by 3 observers using 3 different smartphone applications. In addition, visual inspection was used to estimate the flexion angles of finger joints. The results of this study suggest that all 3 smartphone measurement tools, as well as visual inspection, agree and correlate well with the reference fluoroscopic image measurement. Average differences between the fluoroscopic image measurements with the measured angles using the tools studied ranged from 9.4° to 12.2°. The mean correlation coefficients for each smartphone application exceeded 0.7. Overall interrater reliabilities were similar, with the interclass correlation coefficient being greater than 0.9 for all of the measurement tools. These data suggest that new smartphone applications hold promise for providing accurate and reliable measures of range of motion. [Orthopedics. 2018; 41(2):e217-e221.].

Proximal Phalanx Fracture Management.

BACKGROUND: The goal of proximal phalangeal fracture management is to allow for fracture healing to occur in acceptable alignment while maintaining gliding motion of the extensor and flexor tendons. METHODS: We reviewed the most current literature on various treatment methods of proximal phalanx fractures, focusing on the indications and outcomes of nonoperative as well as operative interventions. RESULTS: Stable fractures can be successfully treated nonoperatively, whereas unstable injuries benefit from surgery. Regardless of the surgical intervention employed, the overriding goal is to restore anatomy and impart enough stability to allow for early motion. The surgical dissection contributes to soft tissue scarring and should be minimized. CONCLUSIONS: Clinical success is achieved when acceptable fracture alignment and stability occur in the setting of unobstructed tendon gliding and early active range of motion.

Contribution of hepatic organic anion-transporting polypeptides to docetaxel uptake and clearance.

The antimicrotubular agent docetaxel is a widely used chemotherapeutic drug for the treatment of multiple solid tumors and is predominantly dependent on hepatic disposition. In this study, we evaluated drug uptake transporters capable of transporting radiolabeled docetaxel. By screening an array of drug uptake transporters in HeLa cells using a recombinant vaccinia-based method, five organic anion-transporting polypeptides (OATP) capable of docetaxel uptake were identified: OATP1A2, OATP1B1, OATP1B3, OATP1C1, and Oatp1b2. Kinetic analysis of docetaxel transport revealed similar kinetic parameters among hepatic OATP1B/1b transporters. An assessment of polymorphisms (SNPs) in SLCO1B1 and SLCO1B3 revealed that a number of OATP1B1 and OATP1B3 variants were associated with impaired docetaxel transport. A Transwell-based vectorial transport assay using MDCKII stable cells showed that docetaxel was transported significantly into the apical compartment of double-transfected (MDCKII-OATP1B1/MDR1 and MDCKII-OATP1B3/MDR1) cells compared with single-transfected (MDCKII-OATP1B1 and MDCKII-OATP1B3) cells (P < 0.05) or control (MDCKII-Co) cells (P < 0.001). In vivo docetaxel transport studies in Slco1b2(-/-) mice showed approximately >5.5-fold higher plasma concentrations (P < 0.01) and approximately 3-fold decreased liver-to-plasma ratio (P < 0.05) of docetaxel compared with wild-type (WT) mice. The plasma clearance of docetaxel in Slco1b2(-/-) mice was 83% lower than WT mice (P < 0.05). In conclusion, this study demonstrates the important roles of OATP1B transporters to the hepatic disposition and clearance of docetaxel, and supporting roles of these transporters for docetaxel pharmacokinetics.

Persistence, localization, and external control of transgene expression after single injection of adeno-associated virus into injured joints.

A single intra-articular injection of adeno-associated virus (AAV) results in stable and controllable transgene expression in normal rat knees. Because undamaged joints are unlikely to require treatment, the study of AAV delivery in joint injury models is crucial to potential therapeutic applications. This study tests the hypotheses that persistent and controllable AAV-transgene expression are (1) highly localized to the cartilage when AAV is injected postinjury and (2) localized to the intra-articular soft tissues when AAV is injected preinjury. Two AAV injection time points, postinjury and preinjury, were investigated in osteochondral defect and anterior cruciate ligament transection models of joint injury. Rats injected with AAV tetracycline response element (TRE)-luciferase received oral doxycycline for 7 days. Luciferase expression was evaluated longitudinally for 6 months. Transgene expression was persistent and controllable with oral doxycycline for 6 months in all groups. However, the location of transgene expression was different: postinjury AAV-injected knees had luciferase expression highly localized to the cartilage, while preinjury AAV-injected knees had more widespread signal from intra-articular soft tissues. The differential transgene localization between preinjury and postinjury injection can be used to optimize treatment strategies. Highly localized postinjury injection appears advantageous for treatments targeting repair cells. The more generalized and controllable reservoir of transgene expression following AAV injection before anterior cruciate ligament transection (ACLT) suggests an intriguing concept for prophylactic delivery of joint protective factors to individuals at high risk for early osteoarthritis (OA). Successful external control of intra-articular transgene expression provides an added margin of safety for these potential clinical applications.

Clinical and Basic Science of Cartilage Injury and Arthritis in the Football (Soccer) Athlete.

Joint injuries are very common in the athletic population, especially professional soccer players, with an incidence of 10 to 35.5 injuries per 1000 hours. Most soccer-related joint injuries occur in the lower extremities, with 16% to 46% occurring in the knee and 17% to 40% occurring in the ankle. Because of the limited healing capacity of cartilage and other intra-articular soft tissue structures, such as anterior cruciate ligament (ACL) and meniscus, joint injuries often lead to the development of early disabling osteoarthritis. Osteoarthritis in soccer players is 5 to 12 times more frequent than in the general population and diagnosed 4 to 5 years earlier. It remains a major cause of disability from this sport. This review focuses on the epidemiology of soccer-related joint injuries and subsequent development of osteoarthritis in the hip, knee, and ankle joints. As well, two different pathways for pathogenesis are described: (1) primary osteoarthritis via direct trauma to the articular cartilage and (2) secondary osteoarthritis that occurs indirectly through injury to the soft tissue structures that subsequently result in articular cartilage degeneration and loss.

Release of bioactive adeno-associated virus from fibrin scaffolds: effects of fibrin glue concentrations.

Fibrin glue (FG) is used in a variety of clinical applications and in the laboratory for localized and sustained release of factors potentially important for tissue engineering. However, the effect of different fibrinogen concentrations on FG scaffold delivery of bioactive adeno-associated viruses (AAVs) has not been established. This study was performed to test the hypothesis that FG concentration alters AAV release profiles, which affect AAV bioavailability. Gene transfer efficiency of AAV-GFP released from FG was measured using HEK-293 cells. Bioactivity of AAV transforming growth factor-beta1 (TGF-ß(1)) released from FG was assessed using the mink lung cell assay, and by measuring induction of cartilage-specific gene expression in human mesenchymal stem cells (hMSCs). Nondiluted FG had longer clotting times, smaller pore sizes, thicker fibers, and slower dissolution rate, resulting in reduced release of AAV. AAV release and gene transfer efficiency was higher with 25% and 50% FG than with the 75% and 100% FG. AAV-TGF-ß(1) released from dilute-FG transduced hMSCs, resulting in higher concentrations of bioactive TGF-ß(1) and greater upregulation of cartilage-specific gene expression compared with hMSC from undiluted FG. This study, showing improved release, transduction efficiency, and chondrogenic effect on hMSC of bioactive AAV-TGF-ß(1) released from diluted FG, provides information important to optimization of this clinically available scaffold for therapeutic gene delivery, both in cartilage regeneration and for other tissue engineering applications.