Vascularization of the dorsal base of the second metacarpal bone: an anatomical study using C-arm cone beam computed tomography.

BACKGROUND: Vascularized bone grafts of the hand are a promising option for treatment of hand abnormalities. Therefore, the purpose of this study was to analyze the arterial anatomy of the dorsal aspect of the second metacarpal base to further investigate this possible donor site for bone grafts. METHODS: The authors examined 16 fresh frozen cadaveric hands by using a C-arm cone beam computed tomography scanner and depicted the three-dimensional course of the second dorsal metacarpal artery and measured the diameter, length, and arc of rotation of this nutritive vessel. In addition, the authors dissected six of the hands under a dissecting microscope and, after selective injection of gelatin dye solution, the authors analyzed the vessel entrances into the bones histologically. RESULTS: In all examined hands, the second dorsal metacarpal artery was a nutritive vessel to the dorsal base of the second metacarpal. The average diameter was 1.3±0.4 mm and the average length of the vascular pedicle was approximately 3.3±0.3 cm. In 14 of 16 cases, the arc of rotation was sufficient to reach the lunate without difficulty. Histologic analysis showed an intrinsic blood supply in the donor region with a vessel diameter of approximately 58 µm. A clinical case with application in Kienböck disease is presented. CONCLUSION: Pedicled vascularized bone grafts from this area are suitable for clinical application to treat Kienböck disease if standard donor sites are unavailable.

Combined inhibition of Cdk5 and ROCK additively increase cell survival, but not the regenerative response in regenerating retinal ganglion cells.

CNS regeneration is limited by lesion-induced neuronal apoptosis and an environment inhibiting axonal elongation. Inhibition of ROCK has been previously shown to promote regeneration in retinal ganglion cells (RGC) whereas Cdk5 inhibition mainly promoted survival. Therefore, we have evaluated the effects of combined treatment with inhibitors of ROCK and Cdk5. We show that in vitro, the co-application of the Cdk5 inhibitor, Indolinone A, and the ROCK inhibitor, Y-27632, potentiated the survival-promoting effect of either substance alone. However, neurite outgrowth in vitro was promoted only by the presence of Y-27632, not by Indolinone A alone. In the ex vivo explant and the in vivo optic nerve crush model the combination of both inhibitors significantly increased neurite outgrowth at small distances, but this effect leveled off for longer neurites. In summary, the combined treatment with the Cdk5 inhibitor Indolinone A and the ROCK inhibitor Y-27632 results in a strong additive effect on neuronal survival, but is not able to increase the regenerative response beyond the effect of the ROCK inhibitor.

Stroke in an adult with HIV infection due to carotid artery stenosis successfully treated with steroids: HIV-associated arteritis?

A 33-year-old Caucasian man with a 17-year history of HIV infection developed sudden right-sided hemiplegia, with the arm more affected than the leg, and aphasia. Magnetic resonance imaging of the brain showed hemodynamic watershed stroke between the anterior and middle cerebral artery territories and an ischemic stroke within the left posterior middle cerebral artery territory. Color-coded Duplex sonography and Doppler sonography revealed hypoechogenic stenosis of the left common carotid artery, the left internal carotid artery, left external carotid artery and right internal carotid artery. An extensive diagnostic workup led us to hypothesize that HIV-associated arteritis was the cause of the stroke, and following intravenous steroid therapy, the carotid artery stenoses vanished.

BAG1 promotes axonal outgrowth and regeneration in vivo via Raf-1 and reduction of ROCK activity.

Improved survival of injured neurons and the inhibition of repulsive environmental signalling are prerequisites for functional regeneration. BAG1 (Bcl-2-associated athanogene-1) is an Hsp70/Hsc70-binding protein, which has been shown to suppress apoptosis and enhance neuronal differentiation. We investigated BAG1 as a therapeutic molecule in the lesioned visual system in vivo. Using an adeno-associated viral vector, BAG1 (AAV.BAG1) was expressed in retinal ganglion cells (RGC) and then tested in models of optic nerve axotomy and optic nerve crush. BAG1 significantly increased RGC survival as compared to adeno-associated viral vector enhanced green fluorescent protein (AAV.EGFP) treated controls and this was independently confirmed in transgenic mice over-expressing BAG1 in neurons. The numbers and lengths of regenerating axons after optic nerve crush were also significantly increased in the AAV.BAG1 group. In pRGC cultures, BAG1-over-expression resulted in a approximately 3-fold increase in neurite length and growth cone surface. Interestingly, BAG1 induced an intracellular translocation of Raf-1 and ROCK2 and ROCK activity was decreased in a Raf-1-dependent manner by BAG1-over-expression. In summary, we show that BAG1 acts in a dual role by inhibition of lesion-induced apoptosis and interaction with the inhibitory ROCK signalling cascade. BAG1 is therefore a promising molecule to be further examined as a putative therapeutic tool in neurorestorative strategies.

ROCK inhibition and CNTF interact on intrinsic signalling pathways and differentially regulate survival and regeneration in retinal ganglion cells.

Functional regeneration in the CNS is limited by lesion-induced neuronal apoptosis and an environment inhibiting axonal elongation. A principal, yet unresolved question is the interaction between these two major factors. We thus evaluated the role of pharmacological inhibition of rho kinase (ROCK), a key mediator of myelin-derived axonal growth inhibition and CNTF, a potent neurotrophic factor for retinal ganglion cells (RGC), in models of retinal ganglion cell apoptosis and neurite outgrowth/regeneration in vitro and in vivo. Here, we show for the first time that the ROCK inhibitor Y-27632 significantly enhanced survival of RGC in vitro and in vivo. In vitro, the co-application of CNTF and Y-27632 potentiated the effect of either substance alone. ROCK inhibition resulted in the activation of the intrinsic MAPK pathway, and the combination of CNTF and Y-27632 resulted in even more pronounced MAPK activation. While CNTF also induced STAT3 phosphorylation, the additional application of ROCK inhibitor surprisingly diminished the effects of CNTF on STAT3 phosphorylation. ROCK activity was also decreased in an additive manner by both substances. In vivo, both CNTF and Y-27632 enhanced regeneration of RGC into the non-permissive optic nerve crush model and additive effects were observed after combination treatment. Further evaluation using specific inhibitors delineate STAT3 as a negative regulator of neurite growth and positive regulator of cell survival, while MAPK and Akt support neurite growth. These results show that next to neurotrophic factors ROCK inhibition by Y-27632 potently supports survival of lesioned adult CNS neurons. Co-administration of CNTF and Y-27632 results in additive effects on neurite outgrowth and regeneration. The interaction of intracellular signalling pathways may, however, attenuate more pronounced synergy and has to be taken into account for future treatment strategies.

Phosducin-like protein regulates G-protein betagamma folding by interaction with tailless complex polypeptide-1alpha: dephosphorylation or splicing of PhLP turns the switch toward regulation of Gbetagamma folding.

Phosducin-like protein (PhLP) exists in two splice variants PhLP(LONG) (PhLP(L)) and PhLP(SHORT) (PhLP(S)). Whereas PhLP(L) directly inhibits Gbetagamma-stimulated signaling, the G betagamma-inhibitory mechanism of PhLP(S) is not understood. We report here that inhibition of Gbetagamma signaling in intact HEK cells by PhLP(S) was independent of direct Gbetagamma binding; however, PhLP(S) caused down-regulation of Gbeta and Ggamma proteins. The down-regulation was partially suppressed by lactacystine, indicating the involvement of proteasomal degradation. N-terminal fusion of Gbeta or Ggamma with a dye-labeling protein resulted in their stabilization against down-regulation by PhLP(S) but did not lead to a functional rescue. Moreover, in the presence of PhLP(S), stabilized Ggamma subunits did not coprecipitate with stabilized Gbeta subunits, suggesting that PhLP(S) might interfere with Gbetagamma folding. PhLP(S) and several truncated mutants of PhLP(S) interacted with the subunit tailless complex polypeptide-1alpha (TCP-1alpha) of the CCT chaperonin complex, which is involved in protein folding. Knock-down of TCP-1alpha in HEK cells by small interfering RNA also led to down-regulation of Gbetagamma. We therefore conclude that the strong inhibitory action of PhLP(S) on Gbetagamma signaling is the result of a previously unrecognized mechanism of Gbetagamma-regulation, inhibition of Gbetagamma-folding by interference with TCP-1alpha.

Regulation of phosducin-like protein by casein kinase 2 and N-terminal splicing.

Phosducin-like protein (PhLP) is a member of the phosducin family of G-protein betagamma-regulators and exists in two splice variants. The long isoform PhLP(L) and the short isoform PhLP(S) differ by the presence or absence of an 83-amino acid N terminus. In isolated biochemical assay systems, PhLP(L) is the more potent Gbetagamma-inhibitor, whereas the functional role of PhLP(S) is still unclear. We now report that in intact HEK 293 cells, PhLP(S) inhibited Gbetagamma-induced inositol phosphate generation with approximately 20-fold greater potency than PhLP(L). Radiolabeling of transfected HEK 293 cells with [(32)P] revealed that PhLP(L) is constitutively phosphorylated, whereas PhLP(S) is not. Because PhLP(L) has several consensus sites for the constitutively active kinase casein kinase 2 (CK2) in its N terminus, we tested the phosphorylation of the recombinant proteins by either HEK cell cytosol in the presence or absence of kinase inhibitors or by purified CK2. PhLP(L) was a good CK2 substrate, whereas PhLP(S) and phosducin were not. Progressive truncation and serine/threonine to alanine mutations of the PhLP(L) N terminus identified a serine/threonine cluster (Ser-18/Thr-19/Ser-20) within a small N-terminal region of PhLP(L) (amino acids 5-28) as the site in which PhLP(L) function was modified in HEK 293 cells. In native tissue, PhLP(L) also seems to be regulated by phosphorylation because phosphorylated and non-phosphorylated forms of PhLP(L) were detected in mouse brain and adrenal gland. Moreover, the alternatively spliced isoform PhLP(S) was also found in adrenal tissue. Therefore, the physiological control of G-protein regulation by PhLP seems to involve phosphorylation by CK2 and alternative splicing of the regulator.