The effect of intralesional injection of bone marrow derived mesenchymal stem cells and bone marrow supernatant on collagen fibril size in a surgical model of equine superficial digital flexor tendonitis.

REASONS FOR PERFORMING STUDY: Collagen fibril size is decreased in repair tissue following tendon injury compared to normal tendon matrix in horses. Mesenchymal stem cells have been suggested to promote regeneration of tendon matrix rather than fibrotic repair following injury, although this concept remains unproven. OBJECTIVES: To explore the hypothesis that implantation of autologous mesenchymal stem cells derived from bone marrow into a surgically created central core defect in the superficial digital flexor tendon (SDFT) of horses would induce the formation of a matrix with greater ultrastructural similarities to tendon matrix than the fibrotic scar tissue formed in control defects. METHODS: Tissue was collected 16 weeks after induction of injury and 12 weeks after treatment from normal and injured regions of control and treated limbs of 6 horses and examined using transmission electron microscopy. Collagen fibril diameters were measured manually with image analysis software and surface areas calculated. Three parameters assessed for normal and injured tissue were mass average diameter (MAD), collagen fibril index (CFI) and the area dependent diameter (ADD). RESULTS: Normal regions from both treated and control limbs displayed higher MAD and CFI values, as well as a characteristic bimodal distribution in fibril size. Injured regions from both treated and control limbs displayed significantly lower MAD and CFI values, as well as a unimodal distribution in fibril size. There were no significant differences between treated and control limbs for any of the parameters assessed. CONCLUSIONS: Intralesional injection of autologous bone marrow derived mesenchymal stem cells had no measurable effect on the fibril diameter of collagen in healing tissue in the SDFT of this experimental model 16 weeks after injury. POTENTIAL RELEVANCE: Favouring matrix regeneration over fibrotic repair may not be the mechanism by which autologous mesenchymal stem cells assist healing of tendon injury.

Calmodulin-dependent cyclic nucleotide phosphodiesterase in adult and developing chick spinal cord.

We investigated the level and characteristics of "low Km" 3'-5' cyclic nucleotide phosphodiesterase (PDE) activity in adult and embryo chick spinal cord. The DEAE cellulose chromatography elution profile of Triton X-100 extracts showed a single peak of calmodulin-dependent cAMP/cGMP PDE activity. After two additional purification steps, this activity showed a five-fold activation by calmodulin (Ka = 1.5 nM) for cGMP hydrolysis, and a linear kinetic behaviour with a Km of 1.3 microM. Conversely, the activity showed a biphasic behaviour for cAMP hydrolysis, with Km values of 3.1 and 18.5 microM. The enzyme showed a Stokes radius of 4.5 nm. Western blot analysis of the purified enzyme revealed two immunoreactive bands with molecular mass of 59 and 65 kDa, respectively. Immunohistochemical staining showed motoneuron decoration both on cell soma and fibres. The developmental pattern of Ca2+-calmodulin-dependent PDE expression in spinal cord was also studied; the hydrolytic activity for both substrates has been found to increase constantly from E5 to post-hatching stages, when it appears 5.6-fold higher as compared to the early embryo levels. Furthermore, in cultured spinal cord neurons from E8 embryos, muscle extract has been shown to induce a two-fold increase of Ca2+-calmodulin-dependent cGMP activity. In conclusion, the studies reported here present three relevant findings: (1) the presence in adult and embryo chick spinal cord of PDE activities with characteristics similar to those of the mammalian PDE I enzyme; (2) its localization in the ventral horn motoneurons; (3) its regulated expression during embryogenesis that is possibly related to soluble epigenetic factors produced by the target cells.

Induction of cyclic AMP and cyclic GMP 3':5'-cyclic nucleotide phosphodiesterase activities in neuroblastoma lines under differentiating conditions.

It is now widely accepted that cyclic nucleotide phosphodiesterases (PDEs) play fundamental roles in signal transduction pathways; they show a remarkable molecular complexity, different tissue distribution and complex regulatory mechanisms. Here we report PDE isoforms expression in two dibutyryl cyclic AMP differentiated murine cell lines: the hybrid neuroblastoma-glioma 108CC15 and the parental neuroblastoma N18TG2. They differ in the ability to establish functional synapses, a feature present only in the former. Ionic exchange chromatography elution profiles of N18TG2 and 108CC15 undifferentiated cell extracts show two main peaks of activity. The first one hydrolyzes cyclic GMP and is specifically inhibited by Zaprinast, thus representing a member of the PDE5 family. The second peak hydrolyzes cyclic AMP and is significantly inhibited by rolipram, as all the PDE4 family members. The induction of differentiation by dibutyryl cyclic AMP in both clonal lines results in an increase of PDE activities only after 3 hr of treatment, suggesting that protein neosynthesis is involved. Interestingly in both clones, besides the increase in cyclic AMP hydrolyzing specific activity (3.1-fold in 108CC15 and 2.5-fold in N18TG2), we also observed an increase in cyclic GMP hydrolyzing activity (1.7-fold in 108CC15 and 4.3-fold in N18TG2). While the induction of PDE4, previously reported also in other cellular systems, could be considered as a feedback response to the higher cyclic AMP levels, this is not true for the isoform that hydrolyzes cyclic GMP. These data suggest that the induction of PDE isoforms in neuroblastoma cells could be related to the activation of neuronal differentiative pathway.

Characterization of 3':5' cyclic nucleotide phosphodiesterase activities of mouse neuroblastoma N18TG2 cells.

Characterization of 'low Km' 3':5' cyclic nucleotide phosphodiesterase activities (PDE) expressed in mouse N18TG2 neuroblastoma cells is reported. At least 3 peaks of activity were isolated by DEAE chromatography, none of which was calcium-calmodulin stimulated and cGMP stimulated or inhibited. A first peak elutes at 200 mM sodium acetate; it specifically hydrolyzes cGMP with a Km of 4.7 microM and shows sensitivity to zaprinast [M&B 22948] (1.8 microM). A second peak eluting at 410 mM sodium acetate hydrolyzes both cyclic nucleotides. A third peak, specific for cAMP hydrolysis, elutes at 580 mM sodium acetate, has a Km of 3.2 microM and is sensitive to RO 20 1724 (7.6 microM) and rolipram (2 microM). Hydrodynamic analysis showed for the first peak a Stokes radius of 5.3 nm with a sedimentation coefficient of 8.1 S, a frictional ratio (f/fo) of 1.41 and a native molecular mass of 182 kDa. The same analysis for peak 3 showed a Stokes radius of 4.1 nm with a sedimentation coefficient of 3.2 S, a frictional ratio of 1.63 and a native molecular mass of 56 kDa. The biochemical features reported for the enzyme eluting in the first peak, and its cGMP-binding activity stimulated by inhibitors of phosphodiesterase activity, demonstrate that it belongs to the PDE V subfamily; on the other hand the cAMP specific enzyme eluting in the third peak can be assigned to the 'RO 20 1724 inhibited' form. The significance of these findings is discussed in relation to the functional characteristics of the N18TG2 cell line.