Mimicking Molecular Pathways in the Design of Smart Hydrogels for the Design of Vascularized Engineered Tissues.

Biomaterials are pivotal in supporting and guiding vascularization for therapeutic applications. To design effective, bioactive biomaterials, understanding the cellular and molecular processes involved in angiogenesis and vasculogenesis is crucial. Biomaterial platforms can replicate the interactions between cells, the ECM, and the signaling molecules that trigger blood vessel formation. Hydrogels, with their soft and hydrated properties resembling natural tissues, are widely utilized; particularly synthetic hydrogels, known for their bio-inertness and precise control over cell-material interactions, are utilized. Naturally derived and synthetic hydrogel bases are tailored with specific mechanical properties, controlled for biodegradation, and enhanced for cell adhesion, appropriate biochemical signaling, and architectural features that facilitate the assembly and tubulogenesis of vascular cells. This comprehensive review showcases the latest advancements in hydrogel materials and innovative design modifications aimed at effectively guiding and supporting vascularization processes. Furthermore, by leveraging this knowledge, researchers can advance biomaterial design, which will enable precise support and guidance of vascularization processes and ultimately enhance tissue functionality and therapeutic outcomes.

A Survey on Tubulin and Arginine Methyltransferase Families Sheds Light on P. lividus Embryo as Model System for Antiproliferative Drug Development.

Tubulins and microtubules (MTs) represent targets for taxane-based chemotherapy. To date, several lines of evidence suggest that effectiveness of compounds binding tubulin often relies on different post-translational modifications on tubulins. Among them, methylation was recently associated to drug resistance mechanisms impairing taxanes binding. The sea urchin is recognized as a research model in several fields including fertilization, embryo development and toxicology. To date, some α- and ß-tubulin genes have been identified in P. lividus, while no data are available in echinoderms for arginine methyl transferases (PRMT). To evaluate the exploiting of the sea urchin embryo in the field of antiproliferative drug development, we carried out a survey of the expressed α- and ß-tubulin gene sets, together with a comprehensive analysis of the PRMT gene family and of the methylable arginine residues in P. lividus tubulins. Because of their specificities, the sea urchin embryo may represent an interesting tool for dissecting mechanisms of tubulin targeting drug action. Therefore, results herein reported provide evidences supporting the P. lividus embryo as animal system for testing antiproliferative drugs.

Evolutionary conserved mechanisms pervade structure and transcriptional modulation of allograft inflammatory factor-1 from sea anemone Anemonia viridis.

Gene family encoding allograft inflammatory factor-1 (AIF-1) is well conserved among organisms; however, there is limited knowledge in lower organisms. In this study, the first AIF-1 homologue from cnidarians was identified and characterised in the sea anemone Anemonia viridis. The full-length cDNA of AvAIF-1 was of 913 bp with a 5' -untranslated region (UTR) of 148 bp, a 3'-UTR of 315 and an open reading frame (ORF) of 450 bp encoding a polypeptide with149 amino acid residues and predicted molecular weight of about 17 kDa. The predicted protein possesses evolutionary conserved EF hand Ca2+ binding motifs, post-transcriptional modification sites and a 3D structure which can be superimposed with human members of AIF-1 family. The AvAIF-1 transcript was constitutively expressed in all tested tissues of unchallenged sea anemone, suggesting that AvAIF-1 could serve as a general protective factor under normal physiological conditions. Moreover, we profiled the transcriptional activation of AvAIF-1 after challenges with different abiotic/biotic stresses showing induction by warming conditions, heavy metals exposure and immune stimulation. Thus, mechanisms associated to inflammation and immune challenges up-regulated AvAIF-1 mRNA levels. Our results suggest its involvement in the inflammatory processes and immune response of A. viridis.

Metallothionein Gene Family in the Sea Urchin Paracentrotus lividus: Gene Structure, Differential Expression and Phylogenetic Analysis.

Metallothioneins (MT) are small and cysteine-rich proteins that bind metal ions such as zinc, copper, cadmium, and nickel. In order to shed some light on MT gene structure and evolution, we cloned seven Paracentrotus lividus MT genes, comparing them to Echinodermata and Chordata genes. Moreover, we performed a phylogenetic analysis of 32 MTs from different classes of echinoderms and 13 MTs from the most ancient chordates, highlighting the relationships between them. Since MTs have multiple roles in the cells, we performed RT-qPCR and in situ hybridization experiments to understand better MT functions in sea urchin embryos. Results showed that the expression of MTs is regulated throughout development in a cell type-specific manner and in response to various metals. The MT7 transcript is expressed in all tissues, especially in the stomach and in the intestine of the larva, but it is less metal-responsive. In contrast, MT8 is ectodermic and rises only at relatively high metal doses. MT5 and MT6 expression is highly stimulated by metals in the mesenchyme cells. Our results suggest that the P. lividus MT family originated after the speciation events by gene duplications, evolving developmental and environmental sub-functionalization.

Gene, Protein, and in Silico Analyses of FoxO, an Evolutionary Conserved Transcription Factor in the Sea Urchin Paracentrotus lividus.

FoxO is a member of the evolutionary conserved family of transcription factors containing a Forkhead box, involved in many signaling pathways of physiological and pathological processes. In mammals, mutations or dysfunctions of the FoxO gene have been implicated in diverse diseases. FoxO homologs have been found in some invertebrates, including echinoderms. We have isolated the FoxO cDNA from the sea urchin Paracentrotus lividus (Pl-foxo) and characterized the corresponding gene and mRNA. In silico studies showed that secondary and tertiary structures of Pl-foxo protein corresponded to the vertebrate FoxO3 isoform, with highly conserved regions, especially in the DNA-binding domain. A phylogenetic analysis compared the Pl-foxo deduced protein with proteins from different animal species and confirmed its evolutionary conservation between vertebrates and invertebrates. The increased expression of Pl-foxo mRNA following the inhibition of the PI3K signaling pathway paralleled the upregulation of Pl-foxo target genes involved in apoptosis or cell-cycle arrest events (BI-1, Bax, MnSod). In silico studies comparing molecular data from sea urchins and other organisms predicted a network of Pl-foxo protein-protein interactions, as well as identified potential miRNAs involved in Pl-foxo gene regulation. Our data may provide new perspectives on the knowledge of the signaling pathways underlying sea urchin development.

Effects of cadmium exposure on sea urchin development assessed by SSH and RT-qPCR: metallothionein genes and their differential induction.

In order to study the defense strategies activated by Paracentrotus lividus embryos in response to sub-lethal doses of CdCl2, we compared the induced transcripts to that of control embryos by suppression subtractive hybridization technique. We isolated five metallothionein (MT) cDNAs and other genes related to detoxification, to signaling pathway components, to oxidative, reductive and conjugative biotransformation, to RNA maturation and protein synthesis. RT-qPCR analysis revealed that two of the five P. lividus MT (PlMT7 and PlMT8) genes appeared to be constitutively expressed and upregulated following cadmium treatment, whereas the other three genes (PlMT4, PlMT5, PlMT6) are specifically switched-on in response to cadmium treatment. Moreover, we found that this transcriptional induction is concentration dependent and that the cadmium concentration threshold for the gene activation is distinct for every gene. RT-qPCR experiments showed in fact that, among induced genes, PlMT5 gene is activated at a very low cadmium concentration (0.1 µM) whereas PlMT4 and PlMT6 are activated at intermediate doses (1-10 µM). Differently, PlMT7 and PlMT8 genes increase significantly their expression only in embryos treated with the highest dose (100 µM CdCl2). We found also that, in response to a lethal dose of cadmium (1 µM), only PlMT5 and PlMT6 mRNA levels increased further. These data suggest a hierarchical and orchestrated response of the P. lividus embryo to overcome differential environmental stressors that could interfere with a normal development.

Indicaxanthin Induces Autophagy in Intestinal Epithelial Cancer Cells by Epigenetic Mechanisms Involving DNA Methylation.

Autophagy is an evolutionarily conserved process critical in maintaining cellular homeostasis. Recently, the anticancer potential of autophagy inducers, including phytochemicals, was suggested. Indicaxanthin is a betalain pigment found in prickly pear fruit with antiproliferative and pro-apoptotic activities in colorectal cancer cells associated with epigenetic changes in selected methylation-silenced oncosuppressor genes. Here, we demonstrate that indicaxanthin induces the up-regulation of the autophagic markers LC3-II and Beclin1, and increases autophagolysosome production in Caco-2 cells. Methylomic studies showed that the indicaxanthin-induced pro-autophagic activity was associated with epigenetic changes. In addition to acting as a hypermethylating agent at the genomic level, indicaxanthin also induced significant differential methylation in 39 out of 47 autophagy-related genes, particularly those involved in the late stages of autophagy. Furthermore, in silico molecular modelling studies suggested a direct interaction of indicaxanthin with Bcl-2, which, in turn, influenced the function of Beclin1, a key autophagy regulator. External effectors, including food components, may modulate the epigenetic signature of cancer cells. This study demonstrates, for the first time, the pro-autophagic potential of indicaxanthin in human colorectal cancer cells associated with epigenetic changes and contributes to outlining its potential healthy effect in the pathophysiology of the gastrointestinal tract.

Analysis of the P. lividus sea urchin genome highlights contrasting trends of genomic and regulatory evolution in deuterostomes.

Sea urchins are emblematic models in developmental biology and display several characteristics that set them apart from other deuterostomes. To uncover the genomic cues that may underlie these specificities, we generated a chromosome-scale genome assembly for the sea urchin Paracentrotus lividus and an extensive gene expression and epigenetic profiles of its embryonic development. We found that, unlike vertebrates, sea urchins retained ancestral chromosomal linkages but underwent very fast intrachromosomal gene order mixing. We identified a burst of gene duplication in the echinoid lineage and showed that some of these expanded genes have been recruited in novel structures (water vascular system, Aristotle's lantern, and skeletogenic micromere lineage). Finally, we identified gene-regulatory modules conserved between sea urchins and chordates. Our results suggest that gene-regulatory networks controlling development can be conserved despite extensive gene order rearrangement.

In silico characterization of the neural alpha tubulin gene promoter of the sea urchin embryo Paracentrotus lividus by phylogenetic footprinting.

During Paracentrotus lividus sea urchin embryo development one alpha and one beta tubulin genes are expressed specifically in the neural cells and they are early end output of the gene regulatory network that specifies the neural commitment. In this paper we have used a comparative genomics approach to identify conserved regulatory elements in the P. lividus neural alpha tubulin gene. To this purpose, we have first isolated a genomic clone containing the entire gene plus 4.5 Kb of 5' upstream sequences. Then, we have shown by gene transfer experiments that its non-coding region drives the spatio-temporal gene expression corresponding substantially to that of the endogenous gene. In addition, we have identified by genome and EST sequence analysis the S. purpuratus alpha tubulin orthologous gene and we propose a revised annotation of some tubulin family members. Moreover, by computational techniques we delineate at least three putative regulatory regions located both in the upstream region and in the first intron containing putative binding sites for Forkhead and Nkx transcription factor families.

The Repertoire of Tissue Inhibitors of Metalloproteases: Evolution, Regulation of Extracellular Matrix Proteolysis, Engineering and Therapeutic Challenges.

Tissue inhibitors of metalloproteases (TIMPs) belong to a fascinating protein family expressed in all Metazoa. They act as regulators of the turnover of the extracellular matrix, and they are consistently involved in essential processes. Herein, we recapitulate the main activities of mammalian TIMPs (TIMP1-4) in the control of extracellular-matrix degradation and pathologies associated with aberrant proteostasis. We delineate the activity of TIMPs in the control of extracellular matrix (ECM) homeostasis and discuss the diversity of TIMPs across metazoans taking into account the emergence of the components of the ECM during evolution. Thus, the TIMP repertoire herein analysed includes the homologues from cnidarians, which are coeval with the origins of ECM components; protostomes (molluscs, arthropods and nematodes); and deuterostomes (echinoderms and vertebrates). Several questions, including the maintenance of the structure despite low sequence similarity and the strategies for TIMP engineering, shed light on the possibility to use recombinant TIMPs integrating unique features and binding selectivity for therapeutic applications in the treatment of inflammatory pathologies.

Gene Expression Changes after Parental Exposure to Metals in the Sea Urchin Affect Timing of Genetic Programme of Embryo Development.

It is widely accepted that phenotypic traits can be modulated at the epigenetic level so that some conditions can affect the progeny of exposed individuals. To assess if the exposure of adult animals could result in effects on the offspring, the Mediterranean sea urchin and its well-characterized gene regulatory networks (GRNs) was chosen as a model. Adult animals were exposed to known concentrations of zinc and cadmium (both individually and in combination) for 10 days, and the resulting embryos were followed during the development. The oxidative stress occurring in parental gonads, embryo phenotypes and mortality, and the expression level of a set of selected genes, including members of the skeletogenic and endodermal GRNs, were evaluated. Increased oxidative stress at F0, high rates of developmental aberration with impaired gastrulation, in association to deregulation of genes involved in skeletogenesis (dri, hex, sm50, p16, p19, msp130), endodermal specification (foxa, hox11/13b, wnt8) and epigenetic regulation (kat2A, hdac1, ehmt2, phf8 and UBE2a) occurred either at 24 or 48 hpf. Results strongly indicate that exposure to environmental pollutants can affect not only directly challenged animals but also their progeny (at least F1), influencing optimal timing of genetic programme of embryo development, resulting in an overall impairment of developmental success.

Aberrant gene expression profiles in Mediterranean sea urchin reproductive tissues after metal exposures.

Marine organisms are simultaneously exposed to numerous pollutants, among which metals probably represent the most abundant in marine environments. In order to evaluate the effects of metal exposure at molecular level in reproductive tissues, we profiled the sea urchin transcriptional response after non-lethal exposures using pathway-focused mRNA expression analyses. Herein, we show that exposures to relatively high concentrations of both essential and toxic metals hugely affected the gonadic expression of several genes involved in stress-response, detoxification, transcriptional and post-transcriptional regulation, without significant changes in gonadosomatic indices. Even though treatments did not result in reproductive tissues visible alterations, metal exposures negatively affected the main mechanisms of stress-response, detoxification and survival of adult P. lividus. Additionally, transcriptional changes observed in P. lividus gonads may cause altered gametogenesis and maintenance of heritable aberrant epigenetic effects. This study leads to the conclusion that exposures to metals, as usually occurs in polluted coastal areas, may affect sea urchin gametogenesis, thus supporting the hypothesis that parental exposure to environmental stressors affects the phenotype of the offspring.

Characterization of Translationally Controlled Tumour Protein from the Sea Anemone Anemonia viridis and Transcriptome Wide Identification of Cnidarian Homologues.

Gene family encoding translationally controlled tumour protein (TCTP) is defined as highly conserved among organisms; however, there is limited knowledge of non-bilateria. In this study, the first TCTP homologue from anthozoan was characterised in the Mediterranean Sea anemone, Anemonia viridis. The release of the genome sequence of Acropora digitifera, Exaiptasia pallida, Nematostella vectensis and Hydra vulgaris enabled a comprehensive study of the molecular evolution of TCTP family among cnidarians. A comparison among TCTP members from Cnidaria and Bilateria showed conserved intron exon organization, evolutionary conserved TCTP signatures and 3D protein structure. The pattern of mRNA expression profile was also defined in A. viridis. These analyses revealed a constitutive mRNA expression especially in tissues with active proliferation. Additionally, the transcriptional profile of A. viridis TCTP (AvTCTP) after challenges with different abiotic/biotic stresses showed induction by extreme temperatures, heavy metals exposure and immune stimulation. These results suggest the involvement of AvTCTP in the sea anemone defensome taking part in environmental stress and immune responses.

An Intronic cis-Regulatory Element Is Crucial for the Alpha Tubulin Pl-Tuba1a Gene Activation in the Ciliary Band and Animal Pole Neurogenic Domains during Sea Urchin Development.

In sea urchin development, structures derived from neurogenic territory control the swimming and feeding responses of the pluteus as well as the process of metamorphosis. We have previously isolated an alpha tubulin family member of Paracentrotus lividus (Pl-Tuba1a, formerly known as Pl-Talpha2) that is specifically expressed in the ciliary band and animal pole neurogenic domains of the sea urchin embryo. In order to identify cis-regulatory elements controlling its spatio-temporal expression, we conducted gene transfer experiments, transgene deletions and site specific mutagenesis. Thus, a genomic region of about 2.6 Kb of Pl-Tuba1a, containing four Interspecifically Conserved Regions (ICRs), was identified as responsible for proper gene expression. An enhancer role was ascribed to ICR1 and ICR2, while ICR3 exerted a pivotal role in basal expression, restricting Tuba1a expression to the proper territories of the embryo. Additionally, the mutation of the forkhead box consensus sequence binding site in ICR3 prevented Pl-Tuba1a expression.

Coexposure to sulfamethoxazole and cadmium impairs development and attenuates transcriptional response in sea urchin embryo.

Among sulfonamides, sulfamethoxazole represents one of the most widely employed. A considerable amount of sulfamethoxazole is introduced into the marine environment after utilization in aquaculture. The cytotoxicity of sulfamethoxazole relies mainly on arylhydroxylamine metabolites and it is associated with the production of reactive oxygen species. Cadmium represents a metal largely employed in several anthropic activities and it is toxic for all living organisms even at low concentrations. Since it is not degraded, cadmium irreversibly accumulates into cells. In order to understand the mechanisms of response to changes in the chemical environment, we investigated by light microscopy observations and RT-qPCR assays the impact of sulfamethoxazole and cadmium in P. lividus sea urchin embryos. During development, embryos were exposed to sulfamethoxazole amount comparable to that usually used in aquaculture procedures and/or sublethal levels of cadmium chloride. Impairment of development and biomarkers for inflammation, detoxification, metal scavenging and cell death were inspected. Even though treatment with sulfamethoxazole apparently did not affect development, it stimulated a remarkable molecular response to oxidative stress. Moreover, combined exposure seriously compromised development and the defense mechanisms to cadmium were blocked. This study leads to the conclusion that coexposure to sulfamethoxazole and cadmium induces neutralizing effects on sea urchin embryos. Thus, in marine areas nearby aquaculture farms, where sulfamethoxazole discharge represents an important environmental contaminant, cadmium occurrence may alter population dynamics of P. lividus.

The nucleic acid-binding protein PcCNBP is transcriptionally regulated during the immune response in red swamp crayfish Procambarus clarkii.

Gene family encoding cellular nucleic acid binding proteins (CNBP) is well conserved among vertebrates; however, there is limited knowledge in lower organisms. In this study, a CNBP homolog from the red swamp crayfish Procambarus clarkii was characterised. The full-length cDNA of PcCNBP was of 1257 bp with a 5'-untranslated region (UTR) of 63 bp and a 3'-UTR of 331 bp with a poly (A) tail, and an open-reading frame (ORF) of 864 bp encoding a polypeptide of 287 amino acids with the predicted molecular weight of about 33 kDa. The predicted protein possesses 7 tandem repeats of 14 amino acids containing the CCHC zinc finger consensus sequence, two RGG-rich single-stranded RNA-binding domain and a nuclear localization signal, strongly suggesting that PcCNBP was a homolog of vertebrate CNBP. The PcCNBP transcript was constitutively expressed in all tested tissues of unchallenged crayfish, including hepatopancreas, gill, eyestalk, haemocytes, intestine, stomach and cuticle with highest expression in haemocytes, intestine, gills and hepatopancreas. The mRNA expression of PcCNBP in haemocytes was modulated at transcriptional level by different immune challenges, suggesting its involvement in the immune response of P. clarkii during both bacteria and viruses infection.

Chromatin dynamics of the developmentally regulated P. lividus neural alpha tubulin gene.

Over 40 years ago, Allfrey and colleagues (1964) suggested that two histone modifications, namely acetylation and methylation, might regulate RNA synthesis. Nowadays it is universally accepted that activation of gene expression strictly depends on enzymatic mechanisms able to dynamically modify chromatin structure. Here, using techniques including DNaseI hypersensitive site analysis, chomatin immunoprecipitation and quantitative PCR analysis, we have analyzed the dynamics of histone post-translation modifications involved in developmentally/spatially controlled activation of the sea urchin PlTalpha2 tubulin gene. We have demonstrated that only when the PlTalpha2 core promoter chromatin is acetylated on H3K9, tri-methylated on H3K4 and not di-methylated on H3K27, RNA pol II can be enrolled. In contrast, we have shown that when chromatin is methylated both on H3K9 (me2/3) and H3K27 (me2) and mono methylated on H3K4 the promoter is not accessible to RNA pol II. Our results suggest that, during P. lividus embryogenesis, both HAT/HDAC and HMT/HDM activities, which are able to regulate accessibility of the PlTalpha2 basal promoter to RNA polymerase II, are coordinately switched-on.