Ras controls melanocyte expansion during zebrafish fin stripe regeneration.

Regenerative medicine for complex tissues like limbs will require the provision or activation of precursors for different cell types, in the correct number, and with the appropriate instructions. These strategies can be guided by what is learned from spectacular events of natural limb or fin regeneration in urodele amphibians and teleost fish. Following zebrafish fin amputation, melanocyte stripes faithfully regenerate in tandem with complex fin structures. Distinct populations of melanocyte precursors emerge and differentiate to pigment regenerating fins, yet the regulation of their proliferation and patterning is incompletely understood. Here, we found that transgenic increases in active Ras dose-dependently hyperpigmented regenerating zebrafish fins. Lineage tracing and marker analysis indicated that increases in active Ras stimulated the in situ amplification of undifferentiated melanocyte precursors expressing mitfa and kita. Active Ras also hyperpigmented early fin regenerates of kita mutants, which are normally devoid of primary regeneration melanocytes, suppressing defects in precursor function and survival. By contrast, this protocol had no noticeable impact on pigmentation by secondary regulatory melanocyte precursors in late-stage kita regenerates. Our results provide evidence that Ras activity levels control the repopulation and expansion of adult melanocyte precursors after tissue loss, enabling the recovery of patterned melanocyte stripes during zebrafish appendage regeneration.

Folate regulation of axonal regeneration in the rodent central nervous system through DNA methylation.

The folate pathway plays a crucial role in the regeneration and repair of the adult CNS after injury. Here, we have shown in rodents that such repair occurs at least in part through DNA methylation. In animals with combined spinal cord and sciatic nerve injury, folate-mediated CNS axon regeneration was found to depend on injury-related induction of the high-affinity folate receptor 1 (Folr1). The activity of folate was dependent on its activation by the enzyme dihydrofolate reductase (Dhfr) and a functional methylation cycle. The effect of folate on the regeneration of afferent spinal neurons was biphasic and dose dependent and correlated closely over its dose range with global and gene-specific DNA methylation and with expression of both the folate receptor Folr1 and the de novo DNA methyltransferases. These data implicate an epigenetic mechanism in CNS repair. Folic acid and possibly other nontoxic dietary methyl donors may therefore be useful in clinical interventions to promote brain and spinal cord healing. If indeed the benefit of folate is mediated by epigenetic mechanisms that promote endogenous axonal regeneration, this provides possible avenues for new pharmacologic approaches to treating CNS injuries.

The effects of sleep hypoxia on coagulant factors and hepatic inflammation in emphysematous rats.

OBJECTIVES: To develop a sleep hypoxia (SH) in emphysema (SHE) rat model and to explore whether SHE results in more severe hepatic inflammation than emphysema alone and whether the inflammation changes levels of coagulant/anticoagulant factors synthesized in the liver. METHODS: Seventy-five rats were put into 5 groups: SH control (SHCtrl), treated with sham smoke exposure (16 weeks) and SH exposure (12.5% O(2), 3 h/d, latter 8 weeks); emphysema control (ECtrl), smoke exposure and sham SH exposure (21% O(2)); short SHE (SHEShort), smoke exposure and short SH exposure (1.5 h/d); mild SHE (SHEMild), smoke exposure and mild SH exposure (15% O(2)); standard SHE (SHEStand), smoke exposure and SH exposure. Therefore, ECtrl, SHEShort, SHEMild and SHEStand group were among emphysematous groups. Arterial blood gas (ABG) data was obtained during preliminary tests. After exposure, hepatic inflammation (interleukin -6 [IL-6] mRNA and protein, tumor necrosis factor α [TNFα] mRNA and protein) and liver coagulant/anticoagulant factors (antithrombin [AT], fibrinogen [FIB] and Factor VIII [F VIII]) were evaluated. SPSS 11.5 software was used for statistical analysis. RESULTS: Characteristics of emphysema were obvious in emphysematous groups and ABGs reached SH criteria on hypoxia exposure. Hepatic inflammation parameters and coagulant factors are the lowest in SHCtrl and the highest in SHEStand while AT is the highest in SHCtrl and the lowest in SHEStand. Inflammatory cytokines of liver correlate well with coagulant factors positively and with AT negatively. CONCLUSIONS: When SH is combined with emphysema, hepatic inflammation and coagulability enhance each other synergistically and produce a more significant liver-derivative inflammatory and prothrombotic status.

Injectable laminin-functionalized hydrogel for nucleus pulposus regeneration.

Cell delivery to the pathological intervertebral disc (IVD) has significant therapeutic potential for enhancing IVD regeneration. The development of injectable biomaterials that retain delivered cells, promote cell survival, and maintain or promote an NP cell phenotype in vivo remains a significant challenge. Previous studies have demonstrated NP cell - laminin interactions in the nucleus pulposus (NP) region of the IVD that promote cell attachment and biosynthesis. These findings suggest that incorporating laminin ligands into carriers for cell delivery may be beneficial for promoting NP cell survival and phenotype. Here, an injectable, laminin-111 functionalized poly(ethylene glycol) (PEG-LM111) hydrogel was developed as a biomaterial carrier for cell delivery to the IVD. We evaluated the mechanical properties of the PEG-LM111 hydrogel, and its ability to retain delivered cells in the IVD space. Gelation occurred in approximately 20 min without an initiator, with dynamic shear moduli in the range of 0.9-1.4 kPa. Primary NP cell retention in cultured IVD explants was significantly higher over 14 days when cells were delivered within a PEG-LM111 carrier, as compared to cells in liquid suspension. Together, these results suggest this injectable laminin-functionalized biomaterial may be an easy to use carrier for delivering cells to the IVD.

The influence of hepatic function on prostate cancer outcomes after radical prostatectomy.

Prostate growth is dependent on circulating androgens, which can be influenced by hepatic function. Liver disease has been suggested to influence prostate cancer (CaP) incidence. However, the effect of hepatic function on CaP outcomes has not been investigated. A total of 1181 patients who underwent radical prostatectomy (RP) between 1988 and 2008 at four Veterans Affairs hospitals that comprise the Shared Equal Access Regional Cancer Hospital database and had available liver function test (LFT) data were included in the study. Independent associations of LFTs with unfavorable pathological features and biochemical recurrence were determined using logistic and Cox regression analyses. Serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) levels were elevated in 8.2 and 4.4% of patients, respectively. After controlling for CaP features, logistic regression revealed a significant association between SGOT levels and pathological Gleason sum > or =7(4+3) cancer (odds ratio=2.12; 95% confidence interval=1.11-4.05; P=0.02). Mild hepatic dysfunction was significantly associated with adverse CaP grade, but was not significantly associated with other adverse pathological features or biochemical recurrence in a cohort of men undergoing RP. The effect of moderate-to-severe liver disease on disease outcomes in CaP patients managed non-surgically remains to be investigated.

Nrg1 is an injury-induced cardiomyocyte mitogen for the endogenous heart regeneration program in zebrafish.

Heart regeneration is limited in adult mammals but occurs naturally in adult zebrafish through the activation of cardiomyocyte division. Several components of the cardiac injury microenvironment have been identified, yet no factor on its own is known to stimulate overt myocardial hyperplasia in a mature, uninjured animal. In this study, we find evidence that Neuregulin1 (Nrg1), previously shown to have mitogenic effects on mammalian cardiomyocytes, is sharply induced in perivascular cells after injury to the adult zebrafish heart. Inhibition of Erbb2, an Nrg1 co-receptor, disrupts cardiomyocyte proliferation in response to injury, whereas myocardial Nrg1 overexpression enhances this proliferation. In uninjured zebrafish, the reactivation of Nrg1 expression induces cardiomyocyte dedifferentiation, overt muscle hyperplasia, epicardial activation, increased vascularization, and causes cardiomegaly through persistent addition of wall myocardium. Our findings identify Nrg1 as a potent, induced mitogen for the endogenous adult heart regeneration program.

The DLK-1 kinase promotes mRNA stability and local translation in C. elegans synapses and axon regeneration.

Growth cone guidance and synaptic plasticity involve dynamic local changes in proteins at axons and dendrites. The Dual-Leucine zipper Kinase MAPKKK (DLK) has been previously implicated in synaptogenesis and axon outgrowth in C. elegans and other animals. Here we show that in C. elegans DLK-1 regulates not only proper synapse formation and axon morphology but also axon regeneration by influencing mRNA stability. DLK-1 kinase signals via a MAPKAP kinase, MAK-2, to stabilize the mRNA encoding CEBP-1, a bZip protein related to CCAAT/enhancer-binding proteins, via its 3'UTR. Inappropriate upregulation of cebp-1 in adult neurons disrupts synapses and axon morphology. CEBP-1 and the DLK-1 pathway are essential for axon regeneration after laser axotomy in adult neurons, and axotomy induces translation of CEBP-1 in axons. Our findings identify the DLK-1 pathway as a regulator of mRNA stability in synapse formation and maintenance and also in adult axon regeneration.