Evelina Bertolotti

Skin wound healing in different aged Xenopus laevis

Xenopus froglets can perfectly heal skin wounds without scarring. To explore whether this capacity is maintained as development proceeds, we examined the cellular responses during the repair of skin injury in 8‐ and 15‐month‐old Xenopus laevis. The morphology and sequence of healing phases (i.e., inflammation, new tissue formation, and remodeling) were independent of age, while the timing was delayed in older frogs. At the beginning of postinjury, wound re‐epithelialization occurred in form of a thin epithelium followed by a multilayered epidermis containing cells with apoptotic patterns and keratinocytes stained by anti‐inducible nitric oxide synthase (iNOS) antibody. The inflammatory response, early activated by recruitment of blood cells immunoreactive to anti‐tumor necrosis factor (TNF)‐α, iNOS, transforming growth factor (TGF)‐β1, and matrix metalloproteinase (MMP)‐9, persisted over time. The dermis repaired by a granulation tissue with extensive angiogenesis, inflammatory cells, fibroblasts, and anti‐α‐SMA positive myofibroblasts. As the healing progressed, wounded areas displayed vascular regression, decrease in cellularity, and rearrangement of provisional matrix. The epidermis restored to a prewound morphology while granulation tissue was replaced by a fibrous tissue in a scar‐like pattern. The quantitative PCR analysis demonstrated an up‐regulated expression of Xenopus suppressor of cytokine signaling 3 (XSOCS-3) and Xenopus transforming growth factor-β2 (XTGF-β2) soon after wounding and peak levels were detected when granulation tissue was well developed with a large number of inflammatory cells. The findings indicate that X. laevis skin wound healing occurred by a combination of regeneration (in epidermis) and repair (in dermis) and, in contrast to froglet scarless wound healing, the growth to a more mature adult stage is associated with a decrease in regenerative capacity with scar‐like tissue formation. J. Morphol. 274:956–964, 2013.

Tail regenerative capacity and iNOS immunolocalization in Xenopus laevis tadpoles

The morphology and the immuno-distribution of the inducible isoform of nitric oxide synthase (iNOS) have been examined in regenerating tails from differently aged Xenopus laevis larvae. By comparing stage-50 and stage-55/56 tadpoles, various morphological aspects and immunoreactivity to anti-iNOS antibody in terms of the number and duration of positive cells have been demonstrated in the regenerating buds. Unlike in stage-50 larvae, the extent of responses to tail amputation in older larvae is more dependent on the individual tadpole and a high percentage (70%-80%) of malformed tails has been seen. The findings indicate that the decline in the efficiency of Xenopus tail regeneration is driven by differences in the inflammatory responses and in the involvement of nitric oxide. This molecule is induced and required for normal tail regeneration, whereas in excess, it is probably associated with progressive loss in the regeneration capability.

The thymus and tail regenerative capacity in Xenopus laevis tadpoles.

A morphofunctional analysis of the thymus from differently aged Xenopus laevis tadpoles during regeneration of the tail is reported. In stage 50 larvae, competent to regenerate, the appendage cut provoked thymic structural modifications that affected the medullary microenvironment cells and changes in TNF-α immunoreactivity. Mucocyte-like cells, multicellular epithelial cysts, myoid cells and cells immunoreactive to TNF-α increased in number. Increased numbers of lymphocytes were also found in regenerating areas and, at the end of regeneration, thymic structural and immunocytochemical patterns were restored to control levels. The observed cellular responses and the induction of molecules critical for thymus constitutive processes suggest a stimulation of thymic function after tail amputation. In older larvae, whose capacity to form a new complete and correctly patterned tail was reduced, thymic morphological changes were more severe and may persist throughout the regeneration process with a significant reduction in organ size. In these larvae the histological patterns and the marked thymic decrease may be related to the events occurring during regeneration, i.e. the higher inflammatory response and the reduced tail regenerative potential.

The thymus and skin wound healing in Xenopus laevis adults.

The capacity to heal wounds without scars is generally lost during the development in vertebrates. To explore the involvement of cells of the adaptive immune system in a scar-like tissue based repair, we studied the thymus in 15-month-old Xenopus after skin incisional wounding. After injury, the organ size significantly increased and marked changes in structure and TNF-α immunoreactivity were detected in the medullary microenvironment when the granulation tissue was present in the repair area. Most of the lymphocytes present in this wound connective tissue were found to be immunoreactive to specific T cell markers. Thymic mucocyte-like cells and epithelial cysts increased in number, the myoid cells acquired a faster turnover and associated in large clusters, blood vessels were dilated and corpuscles similar to mammalian Hassalls bodies were formed in medulla. A higher number of stronger medullary TNF-α immunoreactive cells, i.e., dendritic, epithelial, granular basophilic and myoid cells were also induced after wounding. With progression of healing the thymus gradually returned to histochemical patterns of controls. Our results suggest that during the scar-based skin repair of Xenopus adults the activity of the thymus may be stimulated and associated with the T lymphocyte infiltration observed into injured granulation tissue.

The spleen and skin wound healing in Xenopus adults.

In most vertebrates, the regenerative capacity to restore lost/damage tissues to original structure and functionality decreases at some time during ontogenesis. To evaluate the role of the acquired immunity in the decline of regenerative potential, we examined the cellular responses elicited in the spleen during skin repair in Xenopus adults. Modifications in the architecture were found to be induced and were remarkable 14 days postinjury when the spleen increased significantly in size. In white pulp, the periarteriolar lymphoid sheaths were associated with follicles having central light zones, morphologically similar to germinal centers. With the progress of healing, pigment‐containing cells were seen to accumulate in both white and red pulp regions. Moreover, compared to controls, the cells immunoreactive to anti‐cytokines (TNF‐α, TGF‐β1) and ‐iNOS increased from the first days after wounding. The 14th day, the positive cells formed a dense network of reticular cells in central regions of lymphoid follicles and more frequent reactive leukocytes were detected within the red pulp. A higher number of lymphoid cells immunostained with anti‐CD3ε were also observed in the perifollicular zone. The results suggest that the spleen of adult frogs is involved in skin wound healing with the expansion of lymphoid compartments. J. Morphol. 277:888–895, 2016.

Combination of cGMP analogue and drug delivery system provides functional protection in hereditary retinal degeneration

Significance Development of treatments for hereditary degeneration of the retina (RD) is hampered by the vast genetic heterogeneity of this group of diseases and by the delivery of the drug to an organ protected by the blood–retina barrier. Here, we present an approach for the treatment of different types of RD, combining an innovative drug therapy with a liposomal system that facilitates drug delivery into the retina. Using different animal models of RD we show that this pharmacological treatment preserved both the viability of cells in the retina as well as retinal function. Thus, our study provides an avenue for the development of therapies for hereditary diseases which cause blindness, an unmet medical need. Inherited retinal degeneration (RD) is a devastating and currently untreatable neurodegenerative condition that leads to loss of photoreceptor cells and blindness. The vast genetic heterogeneity of RD, the lack of “druggable” targets, and the access-limiting blood–retinal barrier (BRB) present major hurdles toward effective therapy development. Here, we address these challenges (i) by targeting cGMP (cyclic guanosine- 3′,5′-monophosphate) signaling, a disease driver common to different types of RD, and (ii) by combining inhibitory cGMP analogs with a nanosized liposomal drug delivery system designed to facilitate transport across the BRB. Based on a screen of several cGMP analogs we identified an inhibitory cGMP analog that interferes with activation of photoreceptor cell death pathways. Moreover, we found liposomal encapsulation of the analog to achieve efficient drug targeting to the neuroretina. This pharmacological treatment markedly preserved in vivo retinal function and counteracted photoreceptor degeneration in three different in vivo RD models. Taken together, we show that a defined class of compounds for RD treatment in combination with an innovative drug delivery method may enable a single type of treatment to address genetically divergent RD-type diseases.

New cGMP analogues restrain proliferation and migration of melanoma cells

Melanoma is one of the most aggressive cancers and displays high resistance to conventional chemotherapy underlining the need for new therapeutic strategies. The cGMP/PKG signaling pathway was detected in melanoma cells and shown to reduce migration, proliferation and to increase apoptosis in different cancer types. In this study, we evaluated the effects on cell viability, cell death, proliferation and migration of novel dimeric cGMP analogues in two melanoma cell lines (MNT1 and SkMel28). These new dimeric cGMP analogues, by activating PKG with limited effects on PKA, significantly reduced proliferation, migration and increased cell death. No decrease in cell viability was observed in non-tumor cells suggesting a tumor-specific effect. These effects observed in melanoma are possibly mediated by PKG2 activation based on the decreased toxic effects in tumor cell lines not expressing PKG2. Finally, PKG-associated phosphorylation of vasodilator-stimulated-phosphoprotein (VASP), linked to cell death, proliferation and migration was found increased and with a change of subcellular localization. Increased phosphorylation of RhoA induced by activation of PKG may also contribute to reduced migration ability of the SkMel28 melanoma cell line when treated with cGMP analogues. These findings suggest that the cGMP/PKG pathway can be envisaged as a therapeutic target of novel dimeric cGMP analogues for the treatment of melanoma.

New dimeric cGMP analogues reduce proliferation in three colon cancer cell lines

Activation of the cGMP-dependent protein kinase G (PKG) can inhibit growth and/or induce apoptosis in colon cancer. In this study we evaluated the effects on cell viability, cell death and proliferation of novel dimeric cGMP analogues, compared to a monomeric compound. Three colon cancer cell lines, which only express isoform 2 of PKG, were treated with these novel cGMP analogues and responded with increased PKG activity. cGMP analogues reduced cell viability in the three cell lines and this was due to a cytostatic rather than cytotoxic effect. These findings suggest that activation of PKG2 can be a therapeutic target in the treatment of colon cancer and, most importantly, that dimeric cGMP analogues can further improve the beneficial effects previously observed with monomeric cGMP analogues.