• Antimycotic Ciclopirox Olamine in the Diabetic Environment Promotes Angiogenesis and Enhances Wound Healing

      Ko, Sae Hee; Nauta, Allison; Morrison, Shane D.; Zhou, Hongyan; Zimmermann, Andrew; Gurtner, Geoffrey C.; Ding, Sheng; Longaker, Michael T.; McNeil, Paul L.; Department of Cellular Biology and Anatomy; et al. (2011-11-18)
      Diabetic wounds remain a major medical challenge with often disappointing outcomes despite the best available care. An impaired response to tissue hypoxia and insufficient angiogenesis are major factors responsible for poor healing in diabetic wounds. Here we show that the antimycotic drug ciclopirox olamine (CPX) can induce therapeutic angiogenesis in diabetic wounds. Treatment with CPX in vitro led to upregulation of multiple angiogenic genes and increased availability of HIF-1α. Using an excisional wound splinting model in diabetic mice, we showed that serial topical treatment with CPX enhanced wound healing compared to vehicle control treatment, with significantly accelerated wound closure, increased angiogenesis, and increased dermal cellularity. These findings offer a promising new topical pharmacologic therapy for the treatment of diabetic wounds.
    • A Cost-Effective Transparency-Based Digital Imaging for Efficient and Accurate Wound Area Measurement

      Li, Pei-Nan; Li, Hong; Wu, Mo-Li; Wang, Shou-Yu; Kong, Qing-You; Zhang, Zhen; Sun, Yuan; Liu, Jia; Lv, De-Cheng; McNeil, Paul L.; et al. (2012-05-30)
      Wound measurement is an objective and direct way to trace the course of wound healing and to evaluate therapeutic efficacy. Nevertheless, the accuracy and efficiency of the current measurement methods need to be improved. Taking the advantages of reliability of transparency tracing and the accuracy of computer-aided digital imaging, a transparency-based digital imaging approach is established, by which data from 340 wound tracing were collected from 6 experimental groups (8 rats/group) at 8 experimental time points (Day 1, 3, 5, 7, 10, 12, 14 and 16) and orderly archived onto a transparency model sheet. This sheet was scanned and its image was saved in JPG form. Since a set of standard area units from 1 mm2 to 1 cm2 was integrated into the sheet, the tracing areas in JPG image were measured directly, using the â Magnetic lasso toolâ in Adobe Photoshop program. The pixel values/PVs of individual outlined regions were obtained and recorded in an average speed of 27 second/region. All PV data were saved in an excel form and their corresponding areas were calculated simultaneously by the formula of Y (PV of the outlined region)/X (PV of standard area unit) Ã Z (area of standard unit). It took a researcher less than 3 hours to finish area calculation of 340 regions. In contrast, over 3 hours were expended by three skillful researchers to accomplish the above work with traditional transparency-based method. Moreover, unlike the results obtained traditionally, little variation was found among the data calculated by different persons and the standard area units in different sizes and shapes. Given its accurate, reproductive and efficient properties, this transparency-based digital imaging approach would be of significant values in basic wound healing research and clinical practice.
    • The Effects of Mechanical Stress on the Growth, Differentiation, and Paracrine Factor Production of Cardiac Stem Cells

      Kurazumi, Hiroshi; Kubo, Masayuki; Ohshima, Mako; Yamamoto, Yumi; Takemoto, Yoshihiro; Suzuki, Ryo; Ikenaga, Shigeru; Mikamo, Akihito; Udo, Koichi; Hamano, Kimikazu; et al. (2011-12-28)
      Stem cell therapies have been clinically employed to repair the injured heart, and cardiac stem cells are thought to be one of the most potent stem cell candidates. The beating heart is characterized by dynamic mechanical stresses, which may have a significant impact on stem cell therapy. The purpose of this study is to investigate how mechanical stress affects the growth and differentiation of cardiac stem cells and their release of paracrine factors. In this study, human cardiac stem cells were seeded in a silicon chamber and mechanical stress was then induced by cyclic stretch stimulation (60 cycles/min with 120% elongation). Cells grown in non-stretched silicon chambers were used as controls. Our result revealed that mechanical stretching significantly reduced the total number of surviving cells, decreased Ki-67-positive cells, and increased TUNEL-positive cells in the stretched group 24 hrs after stretching, as compared to the control group. Interestingly, mechanical stretching significantly increased the release of the inflammatory cytokines IL-6 and IL-1β as well as the angiogenic growth factors VEGF and bFGF from the cells in 12 hrs. Furthermore, mechanical stretching significantly reduced the percentage of c-kit-positive stem cells, but increased the expressions of cardiac troponin-I and smooth muscle actin in cells 3 days after stretching. Using a traditional stretching model, we demonstrated that mechanical stress suppressed the growth and proliferation of cardiac stem cells, enhanced their release of inflammatory cytokines and angiogenic factors, and improved their myogenic differentiation. The development of this in vitro approach may help elucidate the complex mechanisms of stem cell therapy for heart failure.
    • Ex Vivo Stretch Reveals Altered Mechanical Properties of Isolated Dystrophin-Deficient Hearts

      Barnabei, Matthew S.; Metzger, Joseph M.; Department of Cellular Biology and Anatomy (2012-03-9)
      Duchenne muscular dystrophy (DMD) is a progressive and fatal disease of muscle wasting caused by loss of the cytoskeletal protein dystrophin. In the heart, DMD results in progressive cardiomyopathy and dilation of the left ventricle through mechanisms that are not fully understood. Previous reports have shown that loss of dystrophin causes sarcolemmal instability and reduced mechanical compliance of isolated cardiac myocytes. To expand upon these findings, here we have subjected the left ventricles of dystrophin-deficient mdx hearts to mechanical stretch. Unexpectedly, isolated mdx hearts showed increased left ventricular (LV) compliance compared to controls during stretch as LV volume was increased above normal end diastolic volume. During LV chamber distention, sarcomere lengths increased similarly in mdx and WT hearts despite greater excursions in volume of mdx hearts. This suggests that the mechanical properties of the intact heart cannot be modeled as a simple extrapolation of findings in single cardiac myocytes. To explain these findings, a model is proposed in which disruption of the dystrophin-glycoprotein complex perturbs cell-extracellular matrix contacts and promotes the apparent slippage of myocytes past each other during LV distension. In comparison, similar increases in LV compliance were obtained in isolated hearts from b-sarcoglycan-null and laminin-a2 mutant mice, but not in dysferlin-null mice, suggesting that increased whole-organ compliance in mdx mice is a specific effect of disrupted cell-extracellular matrix contacts and not a general consequence of cardiomyopathy via membrane defect processes. Collectively, these findings suggest a novel and cell-death independent mechanism for the progressive pathological LV dilation that occurs in DMD.
    • Genetic Ablation of CD68 Results in Mice with Increased Bone and Dysfunctional Osteoclasts

      Ashley, Jason W.; Shi, Zhenqi; Zhao, Haibo; Li, Xingsheng; Kesterson, Robert A.; Feng, Xu; McNeil, Paul L.; Department of Cellular Biology and Anatomy; College of Graduate Studies (2011-10-3)
      CD68 is a member of the lysosome associated membrane protein (LAMP) family that is restricted in its expression to cells of the monocyte/macrophage lineage. This lineage restriction includes osteoclasts, and, while previous studies of CD68 in macrophages and dendritic cells have proposed roles in lipid metabolism, phagocytosis, and antigen presentation, the expression and function of CD68 in osteoclasts have not been explored. In this study, we investigated the expression and localization of CD68 in macrophages and osteoclasts in response to the monocyte/macrophage-colony stimulating factor (M-CSF) and the receptor activator of NF-κB ligand (RANKL). We found that M-CSF stimulates CD68 expression and RANKL alters the apparent molecular weight of CD68 as measured by Western immunoblotting. In addition, we explored the significance of CD68 expression in osteoclasts by generating mice that lack expression of CD68. These mice have increased trabecular bone, and in vitro assessment of CD68â /â osteoclasts revealed that, in the absence of CD68, osteoclasts demonstrate an accumulation of intracellular vesicle-like structures, and do not efficiently resorb bone. These findings demonstrate a role for CD68 in the function of osteoclasts, and future studies will determine the mechanistic nature of the defects seen in CD68â /â osteoclasts.
    • In vivo MRI Characterization of Progressive Cardiac Dysfunction in the mdx Mouse Model of Muscular Dystrophy

      Stuckey, Daniel J.; Carr, Carolyn A.; Camelliti, Patrizia; Tyler, Damian J.; Davies, Kay E.; Clarke, Kieran; McNeil, Paul L.; Department of Cellular Biology and Anatomy; College of Graduate Studies (2012-01-3)
      Aims: The mdx mouse has proven to be useful in understanding the cardiomyopathy that frequently occurs in muscular dystrophy patients. Here we employed a comprehensive array of clinically relevant in vivo MRI techniques to identify early markers of cardiac dysfunction and follow disease progression in the hearts of mdx mice.
    • Label-Free 3D Visualization of Cellular and Tissue Structures in Intact Muscle with Second and Third Harmonic Generation Microscopy

      Rehberg, Markus; Krombach, Fritz; Pohl, Ulrich; Dietzel, Steffen; Department of Cellular Biology and Anatomy; College of Graduate Studies (2011-11-28)
      Second and Third Harmonic Generation (SHG and THG) microscopy is based on optical effects which are induced by specific inherent physical properties of a specimen. As a multi-photon laser scanning approach which is not based on fluorescence it combines the advantages of a label-free technique with restriction of signal generation to the focal plane, thus allowing high resolution 3D reconstruction of image volumes without out-of-focus background several hundred micrometers deep into the tissue. While in mammalian soft tissues SHG is mostly restricted to collagen fibers and striated muscle myosin, THG is induced at a large variety of structures, since it is generated at interfaces such as refraction index changes within the focal volume of the excitation laser. Besides, colorants such as hemoglobin can cause resonance enhancement, leading to intense THG signals. We applied SHG and THG microscopy to murine (Mus musculus) muscles, an established model system for physiological research, to investigate their potential for label-free tissue imaging. In addition to collagen fibers and muscle fiber substructure, THG allowed us to visualize blood vessel walls and erythrocytes as well as white blood cells adhering to vessel walls, residing in or moving through the extravascular tissue. Moreover peripheral nerve fibers could be clearly identified. Structure down to the nuclear chromatin distribution was visualized in 3D and with more detail than obtainable by bright field microscopy. To our knowledge, most of these objects have not been visualized previously by THG or any label-free 3D approach. THG allows label-free microscopy with inherent optical sectioning and therefore may offer similar improvements compared to bright field microscopy as does confocal laser scanning microscopy compared to conventional fluorescence microscopy.
    • Lack of Correlation between Outcomes of Membrane Repair Assay and Correction of Dystrophic Changes in Experimental Therapeutic Strategy in Dysferlinopathy

      Lostal, William; Bartoli, Marc; Roudaut, Carinne; Bourg, Nathalie; Krahn, Martin; Pryadkina, Marina; Borel, Perrine; Suel, Laurence; Roche, Joseph A.; Stockholm, Daniel; et al. (2012-05-29)
      Mutations in the dysferlin gene are the cause of Limb-girdle Muscular Dystrophy type 2B and Miyoshi Myopathy. The dysferlin protein has been implicated in sarcolemmal resealing, leading to the idea that the pathophysiology of dysferlin deficiencies is due to a deficit in membrane repair. Here, we show using two different approaches that fullfiling membrane repair as asseyed by laser wounding assay is not sufficient for alleviating the dysferlin deficient pathology. First, we generated a transgenic mouse overexpressing myoferlin to test the hypothesis that myoferlin, which is homologous to dysferlin, can compensate for the absence of dysferlin. The myoferlin overexpressors show no skeletal muscle abnormalities, and crossing them with a dysferlin-deficient model rescues the membrane fusion defect present in dysferlin-deficient mice in vitro. However, myoferlin overexpression does not correct muscle histology in vivo. Second, we report that AAV-mediated transfer of a minidysferlin, previously shown to correct the membrane repair deficit in vitro, also fails to improve muscle histology. Furthermore, neither myoferlin nor the minidysferlin prevented myofiber degeneration following eccentric exercise. Our data suggest that the pathogenicity of dysferlin deficiency is not solely related to impairment in sarcolemmal repair and highlight the care needed in selecting assays to assess potential therapies for dysferlinopathies.
    • Modulation of Syndecan-1 Shedding after Hemorrhagic Shock and Resuscitation

      Haywood-Watson, Ricky J.; Holcomb, John B.; Gonzalez, Ernest A.; Peng, Zhanglong; Pati, Shibani; Park, Pyong Woo; Wang, WeiWei; Zaske, Ana Maria; Menge, Tyler; Kozar, Rosemary A.; et al. (2011-08-19)
      The early use of fresh frozen plasma as a resuscitative agent after hemorrhagic shock has been associated with improved survival, but the mechanism of protection is unknown. Hemorrhagic shock causes endothelial cell dysfunction and we hypothesized that fresh frozen plasma would restore endothelial integrity and reduce syndecan-1 shedding after hemorrhagic shock. A prospective, observational study in severely injured patients in hemorrhagic shock demonstrated significantly elevated levels of syndecan-1 (554±93 ng/ml) after injury, which decreased with resuscitation (187±36 ng/ml) but was elevated compared to normal donors (27±1 ng/ml). Three pro-inflammatory cytokines, interferon-γ, fractalkine, and interleukin-1β, negatively correlated while one anti-inflammatory cytokine, IL-10, positively correlated with shed syndecan-1. These cytokines all play an important role in maintaining endothelial integrity. An in vitro model of endothelial injury then specifically examined endothelial permeability after treatment with fresh frozen plasma orlactated Ringers. Shock or endothelial injury disrupted junctional integrity and increased permeability, which was improved with fresh frozen plasma, but not lactated Ringers. Changes in endothelial cell permeability correlated with syndecan-1 shedding. These data suggest that plasma based resuscitation preserved endothelial syndecan-1 and maintained endothelial integrity, and may help to explain the protective effects of fresh frozen plasma after hemorrhagic shock.