Citations

Background: One factor believed to contribute to the development of hallux valgus is an abnormality in collagen structure and makeup of the medial collateral ligament (MCL) of the first metatarsophalangeal joint (MTPJ). We hypothesized that the mechanical properties of the MCL in feet with hallux valgus are significantly different from those in normal feet and that these differences may be related to alterations in the type or distribution of collagen fibers at the interface between the MCL and the bone. Materials and Methods: Seven normal fresh-frozen cadaver feet were compared to four cadaver feet that had hallux valgus deformities. The MCL mechanical properties, structure of collagen fibers, and content proportion of type I and type III collagen were determined. Results: The load-deformation and stress-strain curves were curvilinear with three regions: laxity, toe, and linear regions. Laxity of the MCL in feet with hallux valgus was significantly larger than that of normal feet ( p = 0.022). Stiffness and tensile modulus in the toe region in feet with hallux valgus were significantly smaller than those in normal feet ( p = 0.004); however, stiffness and tensile modulus in the linear region were not significantly different. The MCL collagen fibrils in the feet with hallux valgus had a more wavy distribution than the fibrils in the normal feet. Conclusions: In general, strong staining for collagen III and to a lesser extent, collagen I was observed at the interface between the MCL and bone in the feet with hallux valgus but not in the normal feet. These results indicate that the abnormal mechanical properties of the MCL in feet with hallux valgus may be related to differences in the organization of collagen I and collagen III fibrils.

Background: It is necessary to have an understanding of ankle and hindfoot motion and stability to accurately diagnosis and treat ankle-hindfoot disorders. Methods: We devised an ankle ligament testing apparatus to more critically determine ankle stability in all planes with a constant rotational force applied (inversion, eversion, internal rotation, external rotation) throughout the range of sagittal plane motion in 13 cadaver specimens. Three-dimensional kinematics were determined with a magnetic tracking device. Results: With inversion force applied, calcanealtibial inversion was greatest in maximal plantarflexion (mean 22.1 ± 6.0 degrees) and gradually decreased with dorsiflexion, which indicated that the ankle had the most inversion instability in plantarflexion. With eversion force applied, calcaneal-tibial eversion gradually increased with increasing dorsiflexion to 12.7 ± 7.4 degrees indicating that the most eversion instability was in dorsiflexion. With internal rotation force applied, calcaneal-tibial internal rotation from plantarflexion to neutral ankle position increased. With external rotation force application, external rotation from neutral to maximal dorsiflexion increased. Conclusions: Ankle laxity was not constant but varied depending on the plantarflexion-dorsiflexion position and the direction of the applied force. The degree of ankle laxity was greater with inversion and internal rotation torque. Variation in laxity between specimens was observed, consistent with previous reports. These data indicate that the ankle is less stable in plantarflexion when inversion and internal rotation forces are applied. This may explain why the lateral ankle ligaments are most prone to injury in this position. The ankle was less stable in dorsiflexion when eversion and external rotation forces were applied. This is consistent with the observation that deltoid ligament injuries occur in the neutral to dorsiflexion position. The study demonstrates the importance of examining patients with suspected ankle ligament injuries in several ankle positions. The ankle testing device has potential application for in vivo testing of patients with suspected ankle ligament instability.

AbstractControl of knee motion in small animal models is necessary to study the effect of mechanical load on the healing process. This can be especially challenging in mice, which are being increasingly used for various orthopedic reconstruction models. We explored the feasibility of botulinum toxin (Botox; Allergan, Dublin, Ireland) paralysis and a newly designed external fixator to restrict motion of the knee in mice undergoing anterior cruciate ligament (ACL) reconstruction. Nineteen C57BL/6 mice were allocated to two groups: (1) Botox group (n = 9) and (2) external fixator group (n = 10). Mice in Botox group received two different doses of Botox: 0.25 unit (n = 3) and 0.5 unit (n = 6). Injection was performed 72 hours prior to ACL reconstruction into the quadriceps, hamstring, and calf muscles of the right hind leg. Mice in external fixator group received an external fixator following ACL reconstruction. Mice were monitored for survival, tolerance, and achievement of complete knee immobilization. All mice were meant for sacrifice on day 14 postoperatively. No perceptible change in gait was observed with 0.25 unit of Botox. All mice that received 0.5 unit of Botox had complete hind limb paralysis documented by footprint analysis 2 days after injection but failed to tolerate anesthesia and were euthanized 24 hours after operation due to their critical condition. In contrast, the external fixator was well tolerated and effectively immobilized the limb. There was a single occurrence of intraoperative technical error in the external fixator group that led to euthanasia. No mechanical failure or complication was observed. Botox paralysis was not a viable option for postoperative restriction of motion and joint loading in mice. However, external fixation was an effective method for complete knee immobilization and can be used in murine models requiring postoperative control of knee loading. This study introduces a robust research tool to allow control of postoperative joint loading in animal models such as ACL reconstruction, permitting study of the effects of mechanical load on the biologic aspects of tendon-to-bone healing.

Abstract Background: The transcription factor Krüppel-like factor 5 (KLF5) is essential for normal homeostasis, stemness, and tumorigenesis. Typically, KLF5 functions as a transcriptional activator, including in esophageal epithelia where KLF5 promotes keratinocyte proliferation and migration by activating EGFR and ILK, respectively, and prevents transformation by activating Notch1. Yet, the function of KLF5 is context dependent, and mutation of the tumor suppressor p53 switches KLF5 from a transcriptional activator to a repressor of p21Waf1/Cip1 and from pro-proliferative to anti-proliferative. Interestingly, KLF5 also inhibits p53 itself, though the mechanism of this inhibition is unknown. We hypothesized that the functional switch of KLF5 from transcriptional activator to repressor involves the recruitment of a co-repressor. Methods: Primary human esophageal keratinocytes immortalized with hTERT were stably infected with inducible lentivirus containing shRNA against KLF5 or Sin3A. p53 mRNA and protein levels were assessed by real-time quantitative PCR and Western blotting, respectively, and p53 protein stability was evaluated by pulse-chase experiments. We employed ChIP across a 4 kb region on the p53 gene, from 2 kb upstream to 2 kb downstream of the transcription start site, to identify the region of KLF5 binding on p53. Promoter activity was assessed with a series of luciferase reporters containing varying lengths of p53 and its upstream regulatory region. Protein-protein interaction was examined by co-IP. Results: KLF5 knockdown significantly increased p53 both at the mRNA and protein levels. In unstressed cells p53 protein undergoes rapid degradation such that p53 levels are typically low. Yet, with KLF5 knockdown, despite increased p53 protein levels, we saw no changes in p53 protein stability. By ChIP, we identified a region of KLF5 binding to p53 between -600 to -356 bp upstream of the transcription start site. Several other KLF family members can interact with the co-repressor Sin3A, and we found that Sin3A also complexed with KLF5 in human primary esophageal keratinocytes. Knockdown of Sin3A abolished the inhibitory effect of KLF5 on p53, resulting in elevated p53 expression, growth inhibition, and increased apoptosis. Conclusion: KLF5 transcriptionally represses p53 via the co-repressor Sin3A. Thus, KLF5 promotes cell proliferation in normal keratinocytes by both activating pro-growth signals and inhibiting the brake machinery of the cell cycle. Citation Format: Yizeng Yang, Taicheng Zhou, Zhen Zong, Gwen Lomberk, Raul Urrutia, Jonathan Katz. Regulation of p53 function by KLF5. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2087. doi:10.1158/1538-7445.AM2015-2087

Abstract Mounting evidence indicates that oncogenic Ras can modulate cell autonomous inflammatory cytokine production, although the underlying mechanism remains unclear. Here we show that squamous cell carcinoma antigen (SCCA), a member of the Serpin family of serine/cysteine protease inhibitors, is transcriptionally up-regulated by oncogenic Ras via MAPK and the ETS family transcription factor PEA3. Increased SCCA expression leads to inhibition of protein turnover, unfolded protein response (UPR), activation of NF-κB, and is essential for Ras-mediated cytokine production and tumor growth. Analysis of human colorectal and pancreatic tumor samples reveals a positive correlation between Ras mutation, enhanced SCCA expression, and IL-6 expression. These results indicate that SCCA is a Ras-responsive factor that plays an essential role in Ras-associated cytokine production and tumorigenesis. Citation Format: Joseph Catanzaro, Namratha Sheshadri, Ji-An Pan, Yu Sun, Chanjuan Shi, Jinyu Li, Scott Powers, Howard Crawford, Wei-Xing Zong. Oncogenic Ras induces inflammatory cytokine production by upregulating squamous cell carcinoma antigen (SerpinB3). [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A23. doi: 10.1158/1557-3125.RASONC14-A23

As there is evidence that ligamentous laxity is affected by the female hormones, we hypothesized that hormonal changes occurring during pregnancy could have a therapeutic role in preventing the development of a joint contracture. Knee joint contractures were created in pregnant and nonpregnant rats. After 2 wk of immobilization, the degree of contracture was measured with structural properties of the medial collateral and anterior cruciate ligaments and the pubic symphysis. Although not statistically significant, there was a general trend toward reduced contracture in pregnant compared with nonpregnant rats. Cutting the posterior capsule significantly decreased contracture for both the pregnant and nonpregnant groups, confirming the contribution of capsular structures to contracture. Ultimate loads of the medial collateral and anterior cruciate ligaments significantly decreased after immobilization compared with control, but there was no significant effect due to pregnancy. Stiffness and ultimate load of the pubic symphysis were not significantly different between pregnant and nonpregnant groups. The trend toward reduced contracture with pregnancy points toward a possible therapeutic role for female hormones in the prevention of postoperative and/or posttraumatic joint contracture.

Abstract The serine/cysteine protease inhibitor SCCA1 (SERPINB3) is upregulated in many advanced cancers with poor prognosis, but there is limited information about whether it makes functional contributions to malignancy. Here, we show that SCCA1 expression promoted oncogenic transformation and epithelial–mesenchymal transition (EMT) in mammary epithelial cells, and that SCCA1 silencing in breast cancer cells halted their proliferation. SCCA1 overexpression in neu+ mammary tumors increased the unfolded protein response (UPR), IL6 expression, and inflammatory phenotypes. Mechanistically, SCCA1 induced a prolonged nonlethal increase in the UPR that was sufficient to activate NF-κB and expression of the protumorigenic cytokine IL6. Overall, our findings established that SCCA1 contributes to tumorigenesis by promoting EMT and a UPR-dependent induction of NF-κB and IL6 autocrine signaling that promotes a protumorigenic inflammation. Cancer Res; 74(21); 6318–29. ©2014 AACR.

Eleven fresh-frozen cadaver foot specimens were tested to define changes in tarsal alignment associated with flatfoot, or pes planus, a common clinical problem. The three-dimensional position of four bones (talus, calcaneus, navicular, and first metatarsal) relative to the fixed tibia was determined with a magnetic tracking system in the intact foot and flatfoot conditions. In the flatfoot, the average metatarsal-to-talar position difference was 11.7° ± 4.4° in abduction ( P < 0.0001), 10.4° ± 3.6° in dorsiflexion ( P < 0.0001), and 10.9° ± 6.0° in eversion ( P < 0.0001) compared with the intact foot. The average calcaneal-to-talar position difference was 2.2° ± 2.6° in abduction ( P = 0.0171) and 2.7° ± 2.0° in eversion ( P = 0.0012) compared with the intact foot. The average navicular-to-talar position difference was 13.6° ± 5.0° in abduction ( P < 0.0001), 10.5° ± 6.6° in dorsiflexion ( P = 0.0004), and 8.1° ± 3.8° in eversion ( P < 0.0001) compared with normal. The average talar-to-tibial position difference was −8.2° ± 3.2° in dorsiflexion ( P < 0.001) compared with the intact foot. The average arch height difference was 6.0 ± 2.7 mm ( P < 0.0001) less than the normal condition. The deformity associated with flatfoot is complex and occurs in multiple joints and in all three planes. An understanding of the normal and pathologic (flatfoot) conditions is necessary to surgically restore appropriate alignment and function in hindfoot reconstruction operations.

A major limitation of operations commonly performed for acquired flatfoot is inadequate correction of alignment. The authors defined the efficacy of two operations, deltoid ligament reconstruction and flexor digitorum longus tendon transfer, for treatment of posterior tibial tendon dysfunction with flatfoot deformity. Twelve fresh-frozen foot specimens were used. A flatfoot deformity was created, and, to simulate the midstance phase of gait, loads were applied axially to the plantar surface of the foot and to appropriate tendons. Foot position improved substantially after deltoid ligament reconstruction but not after flexor digitorum longus tendon transfer. The average increase in arch height after deltoid ligament reconstruction was 10.3 ± 8.9 mm and after flexor digitorum longus tendon transfer, −0.6 ± 2.0 mm. Mean arch height after deltoid ligament reconstruction was 2.2 ± 1.7 mm less than intact arch height and, after flexor digitorum longus tendon transfer, 13.2 ± 9.0 mm less than intact arch height. Improvement in metatarsal-talar, calcaneal-talar, and talar-tibial positions was significantly greater after deltoid ligament reconstruction than after flexor digitorum longus tendon transfer. Although the authors do not advocate clinical use, the deltoid ligament reconstruction was more effective than flexor digitorum longus tendon transfer in restoring arch alignment in flatfoot.

We defined the mechanical behavior of the foot after an operation for treatment of the flatfoot deformity, subtalar arthrodesis, and compared results with those from flexor digitorum longus tendon transfer. Twelve fresh-frozen human foot specimens were used. Supporting elements were sectioned to create a flatfoot deformity. To simulate midstance phase of gait, loads were applied axially to the plantar foot and to five tendons. Reduction of deformity in metatarsal-talar, calcaneal-talar, and talar-tibial positions was achieved and was significantly greater after subtalar arthrodesis operation than after flexor digitorum longus transfer.