Objective— Expression of human apolipoprotein (h-apo) A-IV in apoE-deficient (apoE ₀ ) mice (h-apoA-IV/E ₀ ) reduces susceptibility to atherosclerosis. Chronic infection mimicked by exposure to lipopolysaccharide (LPS) increases the size of atherosclerosis lesions in apoE ₀ mice. Thus, we used h-apoA-IV/E ₀ mice to determine whether h-apoA-IV plays a protective role after LPS administration. Methods and Results— We injected apoE ₀ , h-apoA-IV/E ₀ , and C57Bl/6 (wild-type) mice intraperitoneally with either LPS or phosphate-buffered saline (PBS) every week for 10 weeks. Atherosclerotic lesions were significantly smaller in h-apoA-IV/E ₀ mice treated with LPS than in their apoE ₀ counterparts. The titers of IgG2a and IgG2b autoantibodies to oxidized low-density lipoprotein (LDL) were higher in the LPS-group of h-apoA-IV/E ₀ mice than in apoE ₀ mice, suggesting that the Th1 response is stronger in the presence of h-apoA-IV. Lymphocytes from the blood, liver, spleen, and thymus of h-apoA-IV/E ₀ mice treated with LPS produced less IL-4, INF-γ, and TNF-α proinflammatory cytokines than their apoE ₀ counterparts. Furthermore, we demonstrated that recombinant h-apoA-IV blocks the LPS-induced stimulation of monocytes. Conclusions— The expression of h-apoA-IV in apoE ₀ mice reduces the susceptibility to atherogenesis and decreases the secretion of proinflammatory cytokines after LPS administration.

Nowadays, a wide array of procedures in mouse technology has been made available to researchers in order to establish valuable models for the study of gene function. The efficiency of gene transfer and gene targeting as methods for producing genetic changes in mice, in addition to continuous advances in molecular biology tools, has converted the mouse into the major experimental model for the study of mammalian physiology. In recent years, the emergence of site-specific recombinases as tools to engineer mammalian genomes has opened new avenues into the design of genetically modified mouse models. The original Cre and FLP recombinases have demonstrated their utility in developing conditional gene targeting, and now other analogous recombinases are also ready to be used, in the same way or in combined strategies, to achieve more sophisticated experimental schemes for addressing complex biological questions. The properties of site-specific recombinases in combination with other biotechnological tools (tet on/off system, siRNA mediated gene silencing, fluorescent proteins, et al.) make them useful instruments to induce precise mutations in specific cells or tissues in a time-controlled manner. This ability can be applied in functional genomics in several ways: from conditional and inducible gene targeting to controlled expression of transgenes and recombination-mediated cassette exchange in mouse models for the study of development or disease phenotypes. This review focuses on the use of site-specific recombinases for mouse genome manipulation. A historical perspective of site-specific recombinases is considered and a number of strategies for achieving inducible or conditional genomic manipulations are contemplated in the context of current techniques for producing genetically modified mice. Finally, several model generation approaches from recent examples in the literature are revised.

Atherosclerosis is an inflammatory disease in which oxidized low-density lipoprotein (oxLDL) plays important roles. Scavenger receptors (SR) CD36, SR-A, and LOX-1 uptake over 90% of the oxLDL leading to foam cell formation and secretion of inflammatory cytokines. To investigate whether the interindividual differences in macrophage SR gene expression could determine the inflammatory variability in response to oxLDL, we quantified the gene and protein expression of SR and inflammatory molecules from macrophages isolated from 18 volunteer subjects and incubated with oxLDL for 1, 3, 6, and 18 h. The individual gene expression profile of the studied SR at 1 h of incubation was highly variable, showing a wide fold-change range: CD36: −3.57–4.22, SR-A: −5.0–4.43, and LOX-1: −1.56–75.32. We identified subjects as high and low responders depending on whether their SR gene expression was above or below the median, showing a different inflammation response pattern. CD36 and LOX-1 gene expression correlated positively with IL-1β; SR-A correlated negatively with IL-8 and positively with PPARγ and NF-κBΙA. These results were confirmed in the same subjects 3 mo after the first sampling. Furthermore, a negative correlation existed between CD36 and SR-A at protein level after 18 h of oxLDL incubation (R = −0.926, p = 0.024). These data would suggest that the type of SR could determine the macrophage activation: more proinflammatory when associated to CD36 and LOX-1 than when associated with SR-A.

TRPV4 channels are calcium-permeable cation channels that are activated by several physicochemical stimuli. Accordingly, TRPV4 channels have been implicated in the regulation of osmosensing, mechanotransduction, thermosensation, and epithelial/endothelial barrier functions. Whether TRPV4 is also mechanistically implicated in melanoma cell proliferation is not clear. Here, we hypothesized that TRPV4 is expressed in human melanoma and that pharmacological activation interferes with cell proliferation. TRPV4 functions were studied in melanoma cell lines (A375, SK-MEL-28, MKTBR), immortalized non-cancer keratinocytes (HaCaT), and murine 3T3 fibroblasts by patch-clamp, qRT-PCR, intracellular calcium measurements, cell proliferation, and flow cytometric assays of apoptosis and cell cycle. The selective TRPV4-activator, GSK1016790A, elicited non-selective cation currents with TRPV4-typical current-voltage-relationship in all cell lines. GSK1016790A-induced currents were blocked by the TRPV4-blocker, HC067047. TRPV4 mRNA expression was demonstrated by qRT-PCR. In A375 cells, TRPV4 activation was frequently paralleled by co-activation of calcium/calmodulin-regulated KCa3.1 channels. Light microscopy showed that TRPV4-activation produced rapid cellular disarrangement, nuclear densification, and detachment of a large fraction of all melanoma cell lines and HaCaT cells. TRPV4-activation induced apoptosis and drastically inhibited A375 and HaCaT proliferation that could be partially prevented by HC067047. Our study showed that TRPV4 channels were functionally expressed in human melanoma cell lines and in human keratinocytes. Pharmacological TRPV4 activation in human melanoma cells and keratinocytes caused severe cellular disarrangement, necrosis and apoptosis. Pharmacological targeting of TRPV4 could be an alternative or adjuvant therapeutic strategy to treat melanoma progression and other proliferative skin disorders.