Spatio-Temporal Changes of Extracellular Matrix (ECM) Stiffness in the Development of the Leech Hirudo verbana

The invertebrate leech Hirudo verbana represents a powerful experimental animal model for improving the knowledge about the functional interaction between the extracellular matrix (ECM) and cells within the tissue microenvironment (TME), and the key role played by ECM stiffness during development and growth. Indeed, the medicinal leech is characterized by a simple anatomical organization reproducing many aspects of the basic biological processes of vertebrates and in which a rapid spatiotemporal development is well established and easily assessed. Our results show that ECM structural organization, as well as the amount of fibrillar and non-fibrillar collagen are deeply different from hatching leeches to adult ones. In addition, the changes in ECM remodelling occurring during the different leech developmental stages, leads to a gradient of stiffness regulating both the path of migratory cells and their fates. The ability of cells to perceive and respond to changes in ECM composition and mechanics strictly depend on nuclear or cytoplasmic expression of Yes-Associated Protein 1 (YAP1), a key mediator converting mechanical signals into transcriptional outputs, expression, and activation

Functional amyloid formation in LPS activated cells from invertebrates to vertebrates

LPS stimulation provokes serious cellular stress with an increase of cytoplasmic reactive oxygen species (ROS). We have investigated, among the different cellular defenses, amyloidogenesis as common physiological response to attenuate oxidative stress. Optical and electron microscopic observations of the following LPS activated cell lines [insect (larval hemocytes, IPLB-LdFB and Drosophila Schneider\u2019s S2 cells); mouse (NIH3T3 embryonic fibroblasts); Human (Human Umbilical Vein Endothelial Cells (HUVEC), neutrophils, and mesenchymal stem cells] reveal that, all are characterized by irregular profiles, cytoplasmic empty vacuoles or by cisternae containing fibrillar material. The compartmentalized fibrillar material shows staining properties typical of amyloid fibrils. LPS activation leads to ROS generation, resulting in pH acidification. Stimulated cells show pink cytoplasm in May-Gr\ufcnwald Giemsa differential staining, giving a gross indication of a lower intracellular pH. Moreover the activation of amyloidogenesis is also linked with an extensive production of ACTH and \u3b1-MSH in all cultured cell types. We suggest that amyloidogenesis is a common, physiological cellular response to weak ROS, starting when other anti-stress cellular systems failed to restore homeostasis. The morphological evidence and/or functional characterization of synthesized amyloid fibrils could be an early indicator of oxidative stress that may lead to a general inflammatory process

Hirudo verbana as an alternative model to dissect the relationship between innate immunity and regeneration

Given the key role of innate immunity in both defense against pathogens and tissue regeneration, innovative studies are becoming crucial to provide further information on how both processes are linked together and to clarify how immune cells perform the coordinated regulation of the aforementioned processes. The present review is mainly focused on two proteins that have been recently found to carry out critical functions in innate immune system regulation, i. e. the Allograft inflammatory factor-1 (AIF-1) and RNASET2, a protein belonging to the T2 ribonuclease family. Their crucial role in both the activation and modulation of the inflammatory response and in the remodeling of connective tissue during grafts and wound repair have been thoroughly investigated in the medicinal leech and will pave the way for novel therapeutic approaches to control immune and systemic responses to disease, injury, and bacterial infection, based on the functionalities of these biomolecules

HvRNASET2 Regulate Connective Tissue and Collagen I Remodeling During Wound Healing Process

Several studies have recently demonstrated that the correct regeneration of damaged tissues and the maintaining of homeostasis after wounds or injuries are tightly connected to different biological events, involving immune response, fibroplasia, and angiogenetic processes, in both vertebrates and invertebrates. In this context, our previous data demonstrated that the Hirudo verbana recombinant protein rHvRNASET2 not only plays a pivotal role in innate immune modulation, but is also able to activate resident fibroblasts leading to new collagen production, both in vivo and in vitro. Indeed, when injected in the leech body wall, which represents a consolidated invertebrate model for studying both immune response and tissue regeneration, HvRNASET2 induces macrophages recruitment, fibroplasia, and synthesis of new collagen. Based on this evidence, we evaluate the role of HvRNASET2 on muscle tissue regeneration and extracellular matrix (ECM) remodeling in rHvRNASET2-injected wounded leeches, compared to PBS-injected wounded leeches used as control. The results presented here not only confirms our previous evidence, reporting that HvRNASET2 leads to an increased collagen production, but also shows that an overexpression of this protein might influence the correct progress of muscle tissue regeneration. Moreover, due to its inhibitory effect on vasculogenesis and angiogenesis, HvRNASET2 apparently interfere with the recruitment of the myoendothelial vessel-associated precursor cells that in turn are responsible for muscle regeneration during wound healing repair

In silico characterization of the leech Helobdella robusta\u2019s matrisome

Leeches (Phylum: Annelida) have been extensively studied to elucidate sophisticated evolutionary-conserved biological processes that are similar to those of mammals. Recently, the growing interest on extracellular matrix (ECM), owing to its multifaceted roles in physiological and pathological processes, has led the scientific community towards the definition of the matrisome as the list of all the structural ECM components, of those proteins affiliated to them, and of all secreted factors that the ECM is usually impregnated with. This annotated list is an invaluable tool to get a better insight on the role of a substantial component of every process and could help in the design and analysis of data coming from both classical and \u201c-omics\u201d experiments. Despite the human matrisome as well as those of several animal models have been already annotated, no specific information regarding Annelids and leeches in particular is available. In this work, using a combination of in silico evolutionary conservation, gene onthology, and structural analyses we conduct a first annotation of the Helobdella robusta\u2019s matrisome

Morpho-functional characterization of the microenvironment in muscle cell development of Hirudo verbana

Muscle regeneration is a process of great interest to the scientific community because is involved in a big number of disorders that are still without a cure. To study the mechanisms behind this process, the most used animal models have always been rodents, for their closeness to humans. Nonetheless, the rising ethical awareness has encouraged a reduction in the use of vertebrates in the research, but at the same time the complex puzzle in which the different factors interact for muscle regeneration has not been clarified, so the contribution of the in vivo experiments is still fundamental. The leech is an inexpensive and easily manageable animal model and represents an invaluable and necessary alternative for these studies. In fact, in a simple body organization it is able to elicit complex processes (wound-healing, angiogenesis, immune response, and muscle regeneration) characterized by the same phases described for vertebrates. For all these reasons, taking also in consideration that regeneration recapitulates some of the processes employed during development and that no satellite cells were identified in leech, we aimed to study the structure and composition of the microenvironment where myogenic precursors mature during the body wall growth of the juvenile leech. In this regard, we performed morphological analyses of the adult and of the juvenile of Hirudo verbana, focusing on the extracellular matrix (ECM) organization and on the cell\u2019s level of differentiation. Moreover, we also reconstructed one of the main pathways by which mechanical stimuli are integrated and transduced into transcriptional activity: the Hippo signaling pathway. Our data suggest that ECM has a pivotal role in controlling myocytes\u2019 proliferation, migration and differentiation thanks to the control exerted by Yap1. Moreover, many cellular types, which concur in this development as stem-like cells of different lineages, were identified in these processes. These preliminary data confirmed that juvenile leech can be a good model for understanding how muscle fibres can growth and differentiate in relation to the 21 stiffness of ECM and to its conformations, to other mechanical, and chemical stimuli and to the interactions with different cell types

In vivo isolation and characterization of telocytes using supplemented biomatrices

The leeches (Hirudo verbana) have proven to be a good model for deciphering basic biological processes for two main reasons: first of all, they have a reduced dimension and, despite a relative anatomical simplicity, share with vertebrates the complexity of immunological mechanisms and wound-healing processes; secondly, in animal kingdom there is a remarkable evolutionary conservation of biological responses, cell types, cellular mechanisms, and molecules. One of the most phylogenetically conserved system, from lower invertebrates to man, is the innate immune system that, strictly interacting with neuro-endocrine one, guarantee a powerful protection to organisms. In invertebrates, in addition to a plethora of cytokines, a wide range of immunocytes such as macrophages, NK cells, granulocytes and a new type of cells, the telocytes, are involved. Leech telocytes (TCs) are stromal cells engaged in surveillance and protection spread in various tissues and strategically localized among resident cells, nearby the capillaries and the nerve endings. These cells, contacting via gap junctions, are organized in an extensive three-dimensional network, and show cell-cell contacts with other cell types and interactions with collagen bundles of connective tissue. The interactions among these and the other cells is obtained in two ways: physically, by direct cell\u2013cell contacts and, chemically, via the release of microvesicles and exosomes, which can transport a variety of soluble factors involved in the regulation of different physiological processes. Leech-TCs originate from circulating precursor cells and, once activated in response to chemical or physical stimuli, are able to change their morphology and behaviour, moving towards the injured area to participate in repair/regenerative processes. To better characterize leech-TCs we have isolated and cultured these cells. The injection of an appropriate combination of Matrigel biopolymer, supplemented with selected factors in the leech H. verbana, has allowed to recruit these cells. While few migrating cells are present in control Matrigel specimens lacking factors, an increased number of cells in relation to the time elapsed from the injection of the supplemented biomatrix, colonize Matrigel specimens containing: Monocyte Chemoattractant Protein-1 (MCP-1/ CCL2) or IL-8 or Ribonuclease T2 (RNASET2)

Telocytes in invertebrates

Vertebrate telocytes (TCs) are stromal cells described in humans and rodens cavitary and non cavitary organs such as heart, gut, gallblader, uterus, lung, pancreas, mammary gland, skeletal muscles. Telocytes, present in many types of tissues, are strategically located among specific resident cells, close to the capillaries and to the nerve endings. TCs are always characterized by a small cell body, containing the nucleus and very thin, long tubular processes named telopodes (TPs) that are considered the ultrastructural peculiar trait of these cells. Each telopode, long and convoluted is moniliform showing thin tubes (podomers) alternated with dilations (podoms). TCs may contact with each other and with virtually all type of cells forming a close network. The cross talk among TCs and the other cells can be direct via gap junctions, and/or indirect via the release of extracellular vesicles (multivescicular bodies, exosomes). TCs are hypothesized as an extensive intercellular information transmission system able to modulate homeostasis, stem cell activity, and other functions working as a primitive nervous system at the cellular level. Our study shows the existence of these type of cells in invertebrate body wall and we firstly identified telocytes in medicinal leech body. An ultrastructural portrait and immunophenotypic characteristics as well as the possible role played by invertebrate telocytes has been proposed