Potential Involvement of the South American Lungfish Intelectin-2 in Innate-Associated Immune Modulation.

Intelectins belong to a family of lectins with specific and transitory carbohydrate interaction capabilities. These interactions are related to the activity of agglutinating pathogens, as intelectins play a significant role in immunity. Despite the prominent immune defense function of intelectins, limited information about its structural characteristics and carbohydrate interaction properties is available. This study investigated an intelectin transcript identified in RNA-seq data obtained from the South American lungfish (Lepidosiren paradoxa), namely LpITLN2-B. The structural analyses predicted LpITLN2-B to be a homo-trimeric globular protein with the fibrinogen-like functional domain (FReD), exhibiting a molecular mass of 57 kDa. The quaternary structure is subdivided into three monomers, A, B, and C, and each domain comprises 11 ß-sheets: an anti-parallel ß-sheet, a ß-hairpin, and a disordered ß-sheet structure. Molecular docking demonstrates a significant interaction with disaccharides rather than monosaccharides. The preferential interaction with disaccharides highlights the potential interaction with pathogen molecules, such as LPS and Poly(I:C). The hemagglutination assay inhibited lectins activity, especially maltose and sucrose, highlighting lectin activity in L. paradoxa samples. Overall, our results show the potential relevance of LpITLN2-B in L. paradoxa immune defense against pathogens.

Light-induced shifts in opsin gene expression in the four-eyed fish Anableps anableps.

The development of the vertebrate eye is a complex process orchestrated by several conserved transcriptional and signaling regulators. Aside from partial or complete loss, examples of exceptional modifications to this intricate organ are scarce. The unique eye of the four-eyed fish Anableps anableps is composed of duplicated corneas and pupils, as well as specialized retina regions associated with simultaneous aerial and aquatic vision. In a previous transcriptomic study of the A. anableps developing eye we identified expression of twenty non-visual and eleven visual opsin genes. Here, we surveyed the expression territories of three non-visual melanopsins genes (opn4×1, opn4×2, opn4m3), one teleost multiple tissue opsin (tmt1b) and two visual opsins (lws and rh2-1) in dorsal and ventral retinas. Our data showed that asymmetry of non-visual opsin expression is only established after birth. During embryonic development, while inside pregnant females, the expression of opn4×1, opn4×2, and tmt1b spans the whole retina. In juvenile fish (post birth), the expression of opn4×1, opn4×2, opn4m3, and tmt1b genes becomes restricted to the ventral retina, which receives aerial light. Raising juvenile fish in clear water instead of the murky waters found in its natural habitat is sufficient to change gene expression territories of opn4×1, opn4×2, opn4m3, tmt1b, and rh2-1, demonstrating that different lighting conditions can shift opsin expression and potentially contribute to changes in spectral sensitivity in the four eyed fish.

The subterranean catfish Phreatobius cisternarum provides insights into visual adaptations to the phreatic environment.

Vertebrate eyes share the same general organization, though species have evolved morphological and functional adaptations to diverse environments. Cave-adapted animals are characterized by a variety of features including eye reduction, loss of body pigmentation, and enhanced non-visual sensory systems. Species that live in perpetual darkness have also evolved sensory mechanisms that are independent of light stimuli. The subterranean catfish Phreatobius cisternarum lives in the Amazonian phreatic zone and displays a diversity of morphological features that are similar to those observed in cavefish and appear to be adaptations to life in the dark. Here we combine histological and transcriptome analyses to characterize sensory adaptations of P. cisternarum to the subterranean environment. Histological analysis showed that the vestigial eyes of P. cisternarum contain a rudimentary lens. Transcriptome analysis revealed a repertoire of eleven visual and non-visual opsins and the expression of 36 genes involved in lens development and maintenance. In contrast to other cavefish species, such as Astyanax mexicanus, Phreatichthys andruzzii, Sinocyclocheilus anophthalmus and Sinocyclocheilus microphthalmus, DASPEI neuromast staining patterns did not show an increase in the number of sensory hair cells. Our work reveals unique adaptations in the visual system of P. cisternarum to underground habitats and helps to shed light into troglomorphic attributes of subterranean animals.

Salamander-like tail regeneration in the West African lungfish.

Salamanders, frog tadpoles and diverse lizards have the remarkable ability to regenerate tails. Palaeontological data suggest that this capacity is plesiomorphic, yet when the developmental and genetic architecture of tail regeneration arose is poorly understood. Here, we show morphological and molecular hallmarks of tetrapod tail regeneration in the West African lungfish Protopterus annectens, a living representative of the sister group of tetrapods. As in salamanders, lungfish tail regeneration occurs via the formation of a proliferative blastema and restores original structures, including muscle, skeleton and spinal cord. In contrast with lizards and similar to salamanders and frogs, lungfish regenerate spinal cord neurons and reconstitute dorsoventral patterning of the tail. Similar to salamander and frog tadpoles, Shh is required for lungfish tail regeneration. Through RNA-seq analysis of uninjured and regenerating tail blastema, we show that the genetic programme deployed during lungfish tail regeneration maintains extensive overlap with that of tetrapods, with the upregulation of genes and signalling pathways previously implicated in amphibian and lizard tail regeneration. Furthermore, the lungfish tail blastema showed marked upregulation of genes encoding post-transcriptional RNA processing components and transposon-derived genes. Our results show that the developmental processes and genetic programme of tetrapod tail regeneration were present at least near the base of the sarcopterygian clade and establish the lungfish as a valuable research system for regenerative biology.

Eye development in the four-eyed fish Anableps anableps: cranial and retinal adaptations to simultaneous aerial and aquatic vision.

The unique eyes of the four-eyed fish Anableps anableps have long intrigued biologists. Key features associated with the bulging eye of Anableps include the expanded frontal bone and the duplicated pupils and cornea. Furthermore, the Anableps retina expresses different photoreceptor genes in dorsal and ventral regions, potentially associated with distinct aerial and aquatic stimuli. To gain insight into the developmental basis of the Anableps unique eye, we examined neurocranium and eye ontogeny, as well as photoreceptor gene expression during larval stages. First, we described six larval stages during which duplication of eye structures occurs. Our osteological analysis of neurocranium ontogeny revealed another distinctive Anablepid feature: an ossified interorbital septum partially separating the orbital cavities. Furthermore, we identified the onset of differences in cell proliferation and cell layer density between dorsal and ventral regions of the retina. Finally, we show that differential photoreceptor gene expression in the retina initiates during development, suggesting that it is inherited and not environmentally determined. In sum, our results shed light on the ontogenetic steps leading to the highly derived Anableps eye.

Irradiation with 780 nm diode laser attenuates inflammatory cytokines but upregulates nitric oxide in lipopolysaccharide-stimulated macrophages: implications for the prevention of aneurysm progression.

BACKGROUND AND OBJECTIVES: Low level laser irradiation (LLLI) has been shown to reduce inflammation in a variety of clinical situations. We have shown that LLLI (780 nm) increases aortic smooth muscle cell proliferation and matrix protein secretion and modulates activity and expression of matrix metalloproteinases. Inflammation is a major component of arteriosclerotic diseases including aneurysm. Macrophage recruitment and secretion of pro-inflammatory cytokines and the vasodilator, nitric oxide (NO), are central to most immune responses in the arterial wall. The present study was designed to determine the effect of LLLI on cytokine gene expression and secretion as well as gene expression of inducible nitric oxide synthase (iNOS) and NO production in lipopolysaccharide (LPS)-stimulated macrophages. STUDY DESIGN/MATERIALS AND METHODS: Murine monocyte/macrophages (RAW 264.7) were irradiated with a 780 nm diode laser (2 mW/cm(2), 2.2 J/cm(2)) during stimulation with LPS (0, 0.1, and 1 microg/ml). Gene expression of chemokines, cytokines, and iNOS were assessed by RT-PCR. Secretion of interleukin (IL)-1beta and monocyte chemotactic protein (MCP)-1 and NO were assessed by ELISA and the Griess reaction, respectively. RESULTS: LLLI reduced gene expression of MCP-1, IL-1alpha, IL-10 (P<0.01), IL-1beta, and IL-6 (P<0.05) when cells were stimulated by 1 microg/ml LPS. LLLI reduced LPS-induced secretion of MCP-1 over non-irradiated cells by 17+/-5% and 13+/-5% at 12 hours (0.1 and 1 microg/ml LPS; P<0.01 and P=0.05, respectively), and reduced IL-1beta by 22+/-5% and 25+/-9% at 24 hours (0.1 and 1 microg/ml LPS, P=0.01 and P=0.06, respectively). However, LLLI increased NO secretion after 12 hours (LLLI vs. CONTROL: without LPS, 1.72+/-0.37 vs. 0.95+/-0.4 microM, P<0.05; 0.1 microg/ml LPS, 7.46+/-1.62 vs. 4.44+/-1.73 microM, P=0.06; 1 microg/ml LPS, 10.91+/-3.53 vs. 6.88+/-1.52 microM, P<0.05). CONCLUSIONS: These properties of LLLI, with its effects on smooth muscle cells reported previously, may be of profound therapeutic relevance for arterial diseases such as aneurysm where inflammatory processes and weakening of the matrix structure of the arterial wall are major pathologic components.

Low-level laser irradiation modulates matrix metalloproteinase activity and gene expression in porcine aortic smooth muscle cells.

BACKGROUND AND OBJECTIVES: The vascular extracellular matrix is maintained by a dynamic balance between matrix synthesis and degradation. This equilibrium is disrupted in arterial pathologies such as abdominal aortic aneurysm. Low-level laser irradiation (LLLI) promotes wound healing. However, its effect on smooth muscle cells (SMCs), a central player in these responses, has not been established. The current study was designed to determine the effects of LLLI on arterial SMC proliferation, inflammatory markers, and matrix proteins. STUDY DESIGN/MATERIALS AND METHODS: Porcine primary aortic SMCs were irradiated with a 780 nm laser diode (1 and 2 J/cm(2)). Trypan blue exclusion assay, immunofluorescent staining for collagen I and III, Sircol assay, gelatin zymography, and RT-PCR were used to monitor proliferation; collagen trihelix formation; collagen synthesis; matrix metalloproteinase-2 (MMP-2) activity, and gene expression of MMP-1, MMP-2, tissue inhibitor of MMP-1 (TIMP-1), TIMP-2, and IL-1-beta, respectively. RESULTS: LLLI-increased SMC proliferation by 16 and 22% (1 and 2 J/cm(2), respectively) compared to non-irradiated cells (P<0.01 and P<0.0005). Immediately after LLLI, trihelices of collagen I and III appeared as perinuclear fluorescent rings. Collagen synthesis was increased twofold (2 days after LLLI: 14.3+/-3.5 microg, non-irradiated control: 6.6+/-0.7 microg, and TGF-beta stimulated control: 7.1+/-1.2 microg, P<0.05), MMP-2 activity after LLLI was augmented (over non-irradiated control) by 66+/-18% (2 J/cm(2); P<0.05), and MMP-1 gene expression upregulated. However, TIMP-2 was upregulated, and MMP-2 gene expression downregulated. IL-1-beta gene expression was reduced. CONCLUSIONS: LLLI stimulates SMC proliferation, stimulates collagen synthesis, modulates the equilibrium between regulatory matrix remodeling enzymes, and inhibits pro-inflammatory IL-1-beta gene expression. These findings may be of therapeutic relevance for arterial diseases such as aneurysm where SMC depletion, weakened extracellular matrix, and an increase in pro-inflammatory markers are major pathologic components.

Locally delivered nanoencapsulated tyrphostin (AGL-2043) reduces neointima formation in balloon-injured rat carotid and stented porcine coronary arteries.

Local delivery of antiproliferative drugs encapsulated in biodegradable nanoparticles (NP) has shown promise as an experimental strategy for preventing restenosis development. A novel PDGFRbeta-specific tyrphostin, AGL-2043, was formulated in polylactide-based nanoparticles and was administered intraluminally to the wall of balloon-injured rat carotid and stented pig coronary arteries. The disposition and elimination kinetics within the vessel wall, as well as the antirestenotic potential of the novel drug and delivery system, were evaluated. The efficacy and the local drug elimination kinetics were affected by the size of the NP and the drug-carrier binding mode. Despite similar arterial drug levels 90 min after delivery in rats, small NP were more efficacious in comparison to large NP (90 and 160 nm, respectively). AGL-2043 selectively inhibited vascular SMC in a dose-dependent manner. The antiproliferative effect of nanoencapsulated tyrphostin was considerably higher than that of surface-adsorbed drug. In the pig model, intramural delivery of AGL-2043 resulted in reduced in-stent neointima formation in the coronary arteries over control despite similar degrees of wall injury. The results of this study suggest that locally delivered tyrphostin AGL-2043 formulated in biodegradable NP may be applicable for antirestenotic therapy independent of stent design or type of injury.

Tyrphostin AGL-2043 eluting stent reduces neointima formation in porcine coronary arteries.

OBJECTIVE: Tyrphostin AGL-2043 is a potent tricyclic quinoxaline inhibitor of PDGF beta-receptor tyrosine kinase (PTK), Kit, and Flt3. We have shown previously that selective inhibition of PDGF beta-receptor PTK by tyrphostins markedly reduces SMC proliferation and migration in vitro, reduces neointima formation in balloon-injured porcine femoral arteries, and reduces neointimal stenosis in stented porcine coronary arteries when administered intramurally within biodegradable nanoparticles. The present study was designed to determine the effect of AGL-2043 delivered from a stent-based, biodegradable polymeric coating on neointima formation in the porcine coronary artery model. METHODS AND RESULTS: Stents coated with biodegradable, polylactic/glycolic acid (PLGA) polymer, with (n=13) or without (n=11) 180 mcg AGL-2043 were implanted into the proximal LAD of 24 Sinclair mini-pigs (34+/-4 kg) to achieve a 1.1:1 stent/artery diameter ratio. The delivery of drug from stent to tissue was confirmed by high-performance liquid chromatography. After 28 days, histomorphometric analysis showed that in-stent stenosis in animals treated with AGL-2043 was reduced by 50% (51+/-21% versus 26+/-10%, p=0.001), the absolute neointimal area was reduced by 44% (2.38+/-1.04 versus 1.31+/-0.43 mm(2), p=0.004), and the absolute luminal area was increased by 57% (2.19+/-1.09 versus 3.39+/-0.59 mm(2), p=0.003). There were no significant differences between control and AGL-2043 in injury score (1.24+/-0.11 vs. 1.15+/-0.12, p=0.07) or inflammation score (1.19+/-0.35 vs. 1.07+/-0.33, p=0.41). Moreover, the difference in % in-stent stenosis between control and treated animals remained highly significant even after normalizing the % stenosis to the degree of injury (p=0.0008) or to the inflammation score (p=0.001). Mortality for this study was zero. Tissue concentration in segments 1 cm proximal and distal to the stents, were negligible or zero at 1 h, 24 h, and 4 weeks after stent implantation. CONCLUSION: Stent-based delivery of tyrphostin AGL-2043 from a biodegradable polymeric coating reduces in-stent neointimal hyperplasia in porcine coronary arteries by 50% after 28 days and preserves lumen area. Long-term studies should be the next step in testing applicability to the human interventional setting.

Metalloproteinase inhibitor attenuates neointima formation and constrictive remodeling after angioplasty in rats: augmentative effect of alpha(v)beta(3) receptor blockade.

Release of matrix metalloproteinases (MMP) from smooth muscle and foam cells following arterial injury facilitates cell migration, neointimal hyperplasia, and vessel wall remodeling. Inhibition of MMP activity using the hydroxamate, zinc-chelating mimicers of collagen, Batimastat and Marimastat, has shown efficacy in reducing constrictive vascular remodeling 6 weeks after experimental angioplasty but not intimal hyperplasia. Vitronectin receptor (alpha(v)beta(3)) blockade interferes with binding of this integrin to MMP-2 and proteolyzed collagen, thereby reducing cell invasion. This study tests the effect of MMP inhibition, with and without vitronectin receptor (alpha(v)beta(3)) blockade, on neointima formation and arterial remodeling in a long-term model (up to 212 months) of balloon injury in vivo. Male Sabra rats were treated with Batimastat (BB-94, British Biotech Pharmaceuticals Ltd., 30 mg/kg, intraperitoneally) and/or the alpha(v)beta(3) receptor inhibiting RGD peptide, G-Pen-GRGDSPCA (GIBCO BRL, 0.1 micromol), administered as a perivascular gel to the common carotid artery after balloon injury. Animals were sacrificed 3, 14, 25, and 75 days (n=21, 23, 22, and 21) after injury. Animals treated with BB-94, peptide, or both had markedly increased absolute luminal area with markedly reduced luminal cross-sectional-area narrowing by neointima and intima-to-media area ratio at all time points except for 3 days after balloon injury versus non-treated, ballooned animals. Combined treatment was significantly more effective than either one alone. Constrictive remodeling, most marked 212 months after balloon injury, was prevented at this time point in all treated animals. The pattern of reduction in luminal narrowing, neointimal formation, and constrictive remodeling across treatment groups correlated very significantly with the reduction in tissue MMP activity as determined by zymography at 3 days. Confirmation of the efficacy of this strategy in larger animals should be the next step toward testing the applicability of this novel approach to the interventional setting.