Influence of the Environment on Regeneration PDF

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Warning The contents of these slides are the exclusive property of the Instructor and/or granted by third parties (textbooks’ reference for pictures) and are therefore protected by the current regulations governing the Protection of Copyright. All rights are reserved. The reproduction and/or diffusion, even partial, by any analogical and/or digital means, without the consent of the rights holder is FORBIDDEN. Any unauthorized use of the above mentioned "Contents" is under the full and exclusive responsibility of the users who will be responsible for it, according to the laws and regulations in force. It is allowed the use of the material for private and study use, however not for profit and without commercial purposes.  The influence of the environment on the regeneration process  If you follow the coloured line you can follow the possible outcomes of regeneration across different species DOI: 10.1186/s12891-020-03363-6  WHAT IS MEANT BY ENVIRONMENT? The regenerative capacity of an organism is largely determined by the properties of its cellular environment. It refers to almost any external influence on the cells involved in the regenerative process. More broadly environmental features include the hormonal and nutritional status of the individual, the immunologic environment, general physiologic conditions, such as oxygen saturation and pH, hormone and growth factors, mechanical and physical cues, extracellular matrix. Conceptually the factors based on their initial input are divided as either external cues (for example, starvation and light/dark cycle) or internal cues (for example, hormones); however, all of these inputs ultimately lead to internal responses.  Green indicates that an increase is beneficial for regeneration or repair, red indicates that an increase is detrimental to the healing process, and gray indicates a neutral or insignificant response. Gradients between colors within the same section indicate mixed results. 10.1038/s41536-021-00130-6 ROLE OF EXTERNAL CUES IN REGENERATIVE CAPACITY 1 - OXYGEN Oxygen levels and nutrient availability influence cell metabolism during regeneration. In some regenerative species, hypoxia (low oxygen levels) can trigger regenerative responses. For example, salamanders regenerate limbs in low-oxygen environments through enhanced glycolysis. In mammals, the balance between oxygen supply and demand is tightly regulated, and deviations can hinder regeneration, contributing to scarring and fibrosis. Molecular pathways governing oxygen-sensing and regeneration are coupled. The role of oxygen in mammalian wound healing In each phases of wound healing process (here referred as inflammation, new tissue formation and remodelling), oxygen plays an important role in cell proliferation, collagen production, tissue reorganization, and infection prevention. The timely progression of the wound healing, is heavily influenced by changing oxygen concentrations. 1- Initial loss of the epidermal barrier can result in a sudden influx of extracellular oxygen that it is quickly consumed by metabolically active cells or converted to ROS. 2- Therefore, immediately after injury, the wound bed can be considered a hypoxic environment. As a result, HIF-1α is stabilized in various cell types and activates the transcription of proinflammatory cytokines. In addition to mediating the inflammatory phase of healing, some of these cytokines also contribute to further HIF- 1α stabilization. Proinflammatory cytokines drives the migration of circulating neutrophils, and later monocytes to the site of injury that engulf dying cells and foreign pathogens within phagosomes. HIF-1a: sensor of O2  1) vasocostriction to reduce the efflux of blood. Produced ROS protect from infections, and acts to recruit neutrophiles 2)hipoxia 1 2 10.1016/j.biomaterials.2020.120646 3- In tissue regrowth, migration of keratinocyte is increased in hypoxia through induction of urokinase plasminogen activator and mTORC1 signaling. However, their proliferation and maturation require a sufficient supply of oxygen to allow for ATP production. like Release of the HIF-1α-target, VEGF, mediates the recruitment of endothelial cells to initiate the growth of new vasculature. HIF-1a induce theexpression of different genes. VEGF acts as a receptors to produce more vessels from the alredy existing ones This oxygen-dependent process also becomes important in later stages of the proliferative phase, which are initiated after the arrival of myofibroblasts and fibroblasts. To achieve closure, myofibroblasts generate force to contract the wound margins, while simultaneously producing collagen. 3) we have more O2 with new vessesls and production of new extracellular matrix 3 4- During the maturation or remodeling phase, oxygen is required as a co-factor for hydroxyproline (key to forming fibrillar collagens). The enzymes that are essential for collagen stability, utilize oxygen as a co-substrate, facilitating cross-linking between collagen molecules. An imbalance of oxygen during this process can lead to unstable collagen formation. In the repair of tissue that relies heavily on collagen formation and where the oxygen tension is lower than in other tissues, such as in cartilaginous tissue, the role of available oxygen may be critical. Failure to repair can open the path for degenerative diseases and chronic pain. During the final stage of healing, this acellular matrix will be remodeled through the release of matrix metalloproteins (MMPs) by ROS-stimulated macrophages, keratinocytes, endothelial cells and fibroblasts to produce the mature scar 4) we have new blood vessels, and MMPs remodel the scar 4  HIF-1α Hipoxia inducible factor HIF-1α signaling allows cells to adapt to local fluctuations in oxygen. https://doi.org/10.1016/j.kint.2017.02.035 During normoxia, HIF-1α is regulated by prolyl hydroxylation through Prolyl Hydroxylases (PHDs). This is then recognized by the von Hippel-Lindau protein (pVHL), which then marks it for degradation through a process called ubiquitination, leading to its breakdown by the proteasome. (because it is without the ubiquitin tag) In hypoxia, when the hydroxylation is suppressed or PHDs are inhibited, HIF-1α is translocated into the nucleus, where it heterodimerises with HIF1-β subunit allowing binding to hypoxia-response elements (HREs). Like VEGF or other cytokines important in the initial stages of inflammation Although local hypoxia is required to initiate the early stages of healing, its persistence can result in the formation of a chronic wound. Common causes of chronic wounds include venous insufficiency, diabetes mellitus, and hypertension, which all result in alterations to vasculature that contribute to tissue hypoxia. Fibroblasts, keratinocytes, and endothelial progenitor cells from aged patients are especially susceptible to these low oxygen conditions and show reduced migration and proliferation compared to cells derived from young adults. In diseases characterized by prolonged inflammation, severe oxygen deprivation can occur in affected tissues. For example, in inflammatory bowel disease, the intestinal epithelial cells undergo an inflammatory-induced hypoxia.  After injury resolution, the healing-promoting inflammatory microenvironments typically return to homeostasis. However, persistent stimuli can escalate acute inflammation towards a chronic state. Inflammation can paradoxically induce hypoxia (despite the increased demand for oxygen following an injury), a cycle frequently seen in ischemic injuries, where inflammation is both accompanied and triggered by hypoxia. Injury-driven hypoxia is common in ischemic injuries, blood vessel damage and thrombotic scenarios. While the development of many chronic wounds is exacerbated by the inability of cells to adapt to hypoxia, excessive upregulation of HIF-1α can also impede normal healing. This effect is seen in keloid and hypertrophic scars which are characterized by excessive ECM accumulation and fibroblast proliferation. On one hand hipoxia can help healing process, however an excess of HIF-1a can cause damges aswell  Recent studies into the use of small molecule PHD inhibitors have shown that pharmacological stabilization of HIF-1α may be used to unlock regeneration and accelerate wound healing in mammals. In many cases, the biological mechanism of this effect is not fully understood. However, the expression of various gene targets governing tissue remodeling, angiogenesis, inflammation, differentiation, and metabolism, likely play a role. https://doi.org/10.1016/j.biomaterials.2020.120646  The role of ROS in mammalian wound healing Wound healing heavily relies on reactive oxygen species (ROS), which are essential for controlling various processes, including inflammation, cell growth, angiogenesis, granulation, and the formation of extracellular matrix. ROS consist of extremely active and oxidizing compounds, which encompass the superoxide anion (O2–), hydrogen peroxide (H2O2), and hydroxyl radical (−OH). ROS can induce oxidative stress; but also redox signalling important in regeneration https://doi.org/10.1038/s41467-024-48719-x  Neutrophils, and monocytes to the site of injury engulf dying cells and foreign pathogens within phagosomes. To destroy the contents of these phagosomes, they need large amounts of molecular oxygen that is converted to ROS. These molecules, can disrupt metabolic pathways through protein oxidation and cause lethal damage to DNA. Since H2O2 can freely diffuse through lipid bilayers, a high concentration of ROS is also released into the extracellular space preventing the growth of invading microbes, and serve as an another chemoattractant and activator for inflammatory cells, and can facilitate the proliferation of fibroblasts and vascular progenitor cells in the next stage of healing.  ROS promote the proliferation and migration of fibroblasts and the synthesis and of collagen and fibronectin. In addition, ROS can stimulate angiogenesis, endothelial cell division and migration by expressing vascular endothelial growth factor (VEGF), and promote blood vessel formation. Therefore, maintaining an optimal level of reactive oxygen species (ROS) is crucial for fighting against microorganisms and ensuring the survival of cells. An increase in ROS at the wound site may lead to the formation of chronic inflammation. ROS can hinder the wound recovery process and increase the degree of tissue damage by counteracting the effects of cytokines such as vascular endothelial growth factor (VEGF) and tumor necrosis factor-α (TNF-α). DOI: 10.3389/fbioe.2023.1304835 Limiting ROS levels to the normal range has a beneficial effect on the wound healing process. Too much reactive oxygen species (ROS) can hinder the cell growth process and cause tissue damage, hindering the wound healing ROLE OF EXTERNAL CUES IN REGENERATIVE CAPACITY 2 - TEMPERATURE Temperature impacts all biological processes, notably through influence over biochemical reaction kinetics and enzyme activity affeting regenerative capacity. Typically, warmer temperatures are associated with faster regeneration rates. For example, in a variety of fish species, rates of wound healing are proportional to ambient temperature, and this increased healing speed is correlated with faster rates of cellular processes, including macrophage activity and keratocyte migration to the wound site. https://doi.org/10.1002/jez.1719 Thermal stress trigger s complex program of gene expression and biochemical adaptive responses. Biologically, the ability to survive and adapt to thermal stress appears to be a fundamental requirement of cellular life, as cell stress responses are ubiquitous among both eukaryotes and prokaryotes. Heat shock proteins (HSPs) are involved in cell stress responses and are highly conserved across evolutionary lines. Even in euthermic species, in which core temperature is tightly regulated, considerable variations in core temperature can occur during severe environmental stress, exercise, and fever. euthermic species like humans Temperature significantly influences the regenerative processes in various physiological and biochemical mechanisms. 1. Metabolic Rate and Biochemical Reactions Temperature impacts metabolic rates, which in turn affects the speed of biochemical reactions essential for regeneration. Generally, warmer temperatures lead to increased metabolic activity. 2. Cellular Processes At higher temperatures, the rates of cellular processes such as macrophage activity and keratinocyte migration to wound sites are accelerated. 3. Formation and Differentiation of Regenerative Structures Temperature affects the formation of blastema and their subsequent differentiation into specific cell types. For instance, while lower temperatures may not significantly impede blastema formation, they can drastically slow down differentiation processes necessary for effective regeneration. 5. Heat Shock Proteins (HSPs) Response Temperature changes can trigger the heat shock response, leading to the expression of heat shock proteins (HSPs). These proteins play a crucial role in the regeneration process, particularly under temperature-induced stress conditions. HSPs are synthesized in response to various stressors, including elevated temperatures, and are essential for maintaining cellular integrity and function. they protect tertiary protein structure, prevent protein aggregation (which are present in neurodegenrative diseases like Parkins) HSPs primarily function as molecular chaperones, facilitating the correct folding of newly synthesized proteins and refolding damaged proteins. This is particularly important during regeneration, as cells must produce and maintain a high turnover of proteins to support tissue repair and growth. The upregulation of HSPs in response to heat stress ensures that proteins involved in regeneration are properly folded and functional, thus enhancing the overall regenerative capacity of the organism. When organisms experience thermal stress, heat shock factors (HSFs) activate the transcription of HSP genes. This mechanism aids in protein homeostasis and enhances cell survival during regeneration by protecting against proteotoxic stress. Moreover, by modulating apoptotic pathways, HSPs can promote cell survival during the regenerative process, ensuring that sufficient cells remain to contribute to tissue repair. A detailed understanding of the complexities of the cellular response to thermal stress and the observation that adaptation to one stressor often leads to cross- protection to others have significant implications for the development of therapeutics. - In a rat model, it has been shown that chemically induced pancreatitis can be blunted by prior whole body hyperthermia. Hyperthermia conferred significant protection against subsequent arginine-induced acute pancreatitis. https://doi.org/10.1152/japplphysiol.01143.2001 Hyperthermia may promote vasodilation, increase aerobic metabolism and induce production of protective molecules such as heat shock proteins. E.g. HyMP could provide an attractive reconditioning strategy for steatotic livers. https://doi.org/10.1152/ajpgi.00101.2020  - The gene expression response to cold exposure is robust and differs in important ways from the cellular response to heat. A better understanding of the pathways that are protective during both cold exposure and rewarming can potentially enhance the benefits of hypothermic therapies that are currently used clinically, such as cryopreservation of organs to be used for transplant, and therapeutic hypothermia in the setting of traumatic brain injury. Therapeutic hypothermia provides potent and universally acknowledged benefits regarding infarct size, and cardiac during ischaemia in experimental models. However, no clinical evidence strongly supports the use of mild therapeutic hypothermia in AMI patients at present. Hypothermia as been useful in therapies for mice, but the same results hasn't been translet in humans https://doi.org/10.1016/j.acvd.2016.05.005 Mild hypothermia of 35°C promotes hNSCs to differentiate into neurons through RBM3- SOX11 signaling pathway regulates the neuronal differentiation of hNSCs. NSC = neural stem cells https://doi.org/10.1016/j.isci.2024.109435 ROLE OF EXTERNAL CUES IN REGENERATIVE CAPACITY 3 – DARK/LIGHT CYCLE Circadian rhythms affect many diverse biological processes, including regeneration. Light exposure has an important role in regulating the circadian cycle, and it consequently affects the systemic context in which regeneration occurs. Light and darkness provide external signals which, when translated by the pineal gland into day/night cycles, promote distinct hormonal and neural responses. Some of the resulting modulations in regenerative capacity may be the direct result of light fluctuations, or they may be an indirect consequence of alterations to other light/dark cycle-dependent attributes, such as activity and sleep–wake cycles. https://doi.org/10.1038/s41598-024-60014-9  Several studies in mice have found that cellular proliferation rates follow a cyclic pattern during regeneration that corresponds with circadian oscillations, with the largest spike in proliferation typically occurring in the morning. Long-term circadian rhythm dysregulation hinders the regenerative ability of skin and hair precursor cells in humans, as observed in a study comparing cultured skin biopsies of night-shift workers to diurnal workers.  In newts, increased duration of light exposure appears to have positive effects on regenerative rate, with continuous light exposure prompting the fastest regenerative rate compared with other permutations of light and darkness. This correlation is apparent even when the animals used are blind. This phenomenon is speculated to be caused by activity within the pineal gland. When stimulated by light, the pineal gland produces serotonin, which stimulates prolactin release. Serotonin-stimulated prolactin release promotes increased cell density at the blastema site, which may be due to increased cellular proliferation, decreased cell death, and/or increased blastema cell recruitment, and subsequently accelerates regeneration rate. These findings all bolster the claim that circadian perturbations have a strong impact on regenerative response, although the specific impact is variable between species and tissues. Photobiomodulation is a research field in which cells are exposed to light. It has therapeutic application in medicine and dentistry, to stimulate tissue repair processes and to reduce pain and inflammation. It is used, for example, in the treatment of nerve damage, wound healing, osteoarthritis, psoriasis. For many cell types including fibroblasts, osteoblasts and mesenchymal stem cells, the cyclic short- term exposure to red/infrared light of low intensity (laser or LED) has been shown to increase the viability and have a stimulatory effect on cellular activities such as proliferation, differentiation, and secretion of signalling factors.  In vivo studies have been shown that photobiomodulation can stimulate stem cells proliferation due to the activation of various genes. Light exposure has been shown to modulate most phases of the wound process. For example, it has been reported that red (670 nm) and green LED light (530 nm) stimulates fibroblast growth and wound healing. Light radiation (633 or 830 nm) can induce the proliferative phase in ischemic and diabetic wounds, due to the activation of fibroblast migration and proliferation, as well as the proliferation of epithelial cells.  Especially, the wavelengths around 600 and 800 nm are strongly absorbed by cytochrome c oxidase in mitochondria and it is the onset of the mechanism of PBM. Thus, cytochrome c oxidase works as a photoacceptor found at the end of the mitochondrial respiratory chain. Redox reactions in cytochrome c affect cell signaling molecules. Biochemical reactions that are induced by these molecules result in the production of intracellular ROS, ATP, NO is a signalling molecule and Nitric Oxide (NO) release at the end of the light absorption. This set of events accelerates cell division by increasing the synthesis of DNA and RNA, protein expressions, and supports the regulation of the cell cycle ROLE OF EXTERNAL CUES IN REGENERATIVE CAPACITY 4 – SEASONAL CUES Seasonal cues can also play an influential role in the regenerative response, working in tandem with related factors such as age, developmental stage, sex, and reproductive cycle. Like circadian rhythms, seasonal cues are associated with cyclic cycles. Factors related to seasonal cues may include annual fluctuations in daylight, temperature, humidity, diet, and microbiome, and they may trigger internal, hormonal changes that influence regeneration. Newts, for example, regenerate faster in the summer than in autumn or winter, even in conditions where light exposure and temperature are kept constant year-round. This regenerative difference may be owing to seasonal variations in prolactin levels; however, the overt seasonal signal remains unclear. Seasonal variations are rarely considered a contributing component to human tissue function or health, although many diseases and physiological process display annual periodicities. In this work the authors find more than 4,000 protein-coding mRNAs in white blood cells and adipose tissue to have seasonal expression profiles, with inverted patterns observed between Europe and Oceania. They also find the cellular composition of blood to vary by season, and these changes could explain the gene expression periodicity. The immune system has a profound pro-inflammatory transcriptomic profile during European winter, with increased levels of soluble IL-6 receptor and C-reactive protein, risk biomarkers for cardiovascular, psychiatric and autoimmune diseases that have peak incidences in winter. https://doi.org/10.1038/ncomms8000 EXTERNAL CUES 4 - SALINITY AND OSMOREGULATION Many highly regenerative animals live in an aqueous or moist environment. Thus, one external cue to consider in the context of regeneration and repair is environmental salinity and its effect on the osmolarity and regenerative capacity of the organism.  In zebrafish, salinity affect wound healing. A gradient in osmolarity between the environment and interstitial fluid leads to the activation of cytosolic phospholipase a2 at the wound site, which works to recruit leukocytes to the injury. Hypotonicity is required for rapid leukocyte recruitment to larval zebrafish tail fin wounds DOI: 10.1038/ncb2818  Osmoregulation has also been directly implicated in cellular responses to injury that are necessary for murine wound healing. For example, M2-activated macrophages are sensitive to osmotic conditions and have been demonstrated to use this input to guide efficient healing and repair responses in mice. When fed high-salt diets, mice show reduced M2 activation following cutaneous injury and delayed wound healing compared with controls. https://doi.org/10.1172/JCI80919.  A common consequence of osmoregulatory dysfunction in humans is hypertension, which can lead to skin ulceration and non-healing wounds of the extremities, particularly the lower legs. One important osmoregulator is the kidney, which produces a crucial protein called Renin. Renin-producing cells retain some developmental plasticity and are highly involved in the regeneration of glomeruli in the kidney. The dysfunction of these renin- In the renin-angiotensin system, some receptors have beneficial producing cells affects the regenerative ability effects, other detrimental. The final result depends on the amount and the timing of the expression of these receptors of the kidney. A general consensus exists viewing The tissue renin–angiotensin system is AT1 and AT2 on opposites ends on a implicated in all stages of epidermal wound seesaw during tissue regeneration, with healing and repair, as well as in the re- AT1 being pro-inflammatory and pro- proliferative, and AT2 counteracting activation of infarct myofibroblasts after injury. these actions to level the seesaw. https://doi.org/10.1016/j.cellsig.2018.07.011  Microenvironmental osmolarity can induce and reverse osteoarthritis- related behavior of chondrocytes via altered intracellular molecular crowding, which represents a previously unknown mechanism underlying OA pathophysiology. Decreased intracellular crowding is associated with increased sensitivity to proinflammatory triggers and decreased responsiveness to anabolic stimuli. OA-induced lowered intracellular molecular crowding could In healthy joints, condrocytes have higher intracellular crowding be renormalized via exposure to higher extracellular osmolarity such as those found in healthy joints, which reverse OA chondrocyte's sensitivity to catabolic stimuli as well as its glycolytic metabolism. https://doi.org/10.1002/advs.202306722 INTERNAL CUES 1- ENDOCRINE ENVIRONMENT Endocrine signals acting directly on receptors expressed in the tissue or via neuroendocrine pathways can affect regeneration by regulating the immune response to injury, allocation of energetic resources, or by enhancing or inhibiting proliferation and differentiation pathways involved in regeneration. https://doi.org/10.1210/en.2019-00321 The combination of extrinsic and intrinsic factors varies endocrine signals that can positively or negatively modulate local cellular processes involved in regeneration. These hormones are secreted in the periphery or the pituitary and can influence regeneration through direct and indirect pathways. Direct pathways are when the hormone acts through its receptors at the site of injury; indirect pathways are when the hormone acts on receptors in the brain or pituitary to stimulate neuroendocrine pathways that can enhance or inhibit regeneration. Endocrine signals can often be synthesized and secreted in skin or nearby tissues to also act as a paracrine factor regulating regeneration. Variation in endocrine signaling associated with life history stage or environmental conditions modulate the cellular processes involved in regeneration to either enhance or inhibit their actions, ultimately regulating the quality and tempo of regeneration. Nutritionally regulated hormones: GH (growth hormon), insulin and IGFs (Insulin Growth Factor) (this hormones have positive roles in regeneration) Growth hormones (GHs) are implicated in regenerative processes. GH stimulates IGF-I and II secretion in the brain and liver through endocrine, neuroendocrine, and paracrine factors to regulate body size and regeneration rate. Many of GH actions work through stimulating insulin, a peptide hormone secreted from beta cells in the pancreas in response to elevated glucose levels and works through the IGF1/AKT pathway to increase several growth factors, which increase cellular proliferation, cellular protection and growth. There is evidence that signals regulated by GH, fibroblast growth factor (FGF), and epithelium growth factor (EGF), are critical for increased proliferation, motility and cellular protection during the blastema formation in zebrafish.  staining of neurons and dendrits Insulin has been implicated in wound healing and regenerative events in mammalian models. Aberrant or insufficient insulin signalling, even in the absence of diabetes, has been associated with neurodegeneration in diseases characterized by dendritic pathology, notably Alzheimer’s and Parkinson’s disease, as well as glaucoma. After lowering insulin there is a reduction of dendritis, but by giving again insulin dendritis regenerates https://doi.org/10.1093/brain/awy142  Insulin growth factor (IGF) is required for zebrafish fin regeneration. The present study identifies a fundamental role of IGF signaling in mediating reciprocal communication along the epidermis- blastema axis. The picture shows zebrafish fins. The line represents the amputation site. 24 hpa = hours post amputations, here we can recognize the amputation. The purple recognize the presence of mRNA in a site (in this case of IGF) negative effects Sex steroids can influence the differentiation and behavior of several types of stem cells. Effects of both of these hormones depend on context and species Testosterone: it has shown to be immunosuppressive and decrease cellular growth factors. In two species of lizard, elevated testosterone levels (e.g., during reproductive active seasons) decrease the number of lymphocytes and immune factors such as IL-6 and TNF-α. Work in mice has also shown that elevated testosterone levels decrease the immune response and decrease growth factors such as TGF-β during wound healing. (beneficial effect) Estrogen: Estrogenic signaling enhances wound epidermal formation by stimulating collagen production in the skin, which increases elasticity. Estrogen promotes healing in various regenerative contexts, including cutaneous wound healing; skeletal muscle, bone, and cartilage regeneration; and liver regeneration. Studies in humans repeatedly demonstrate that reduced estrogen causes defects in wound healing, while exogenous estrogen may promote improved healing outcomes Estrogen has beneficial effects on inflammation, proliferation, and remodeling phases of skin wounds. The overlapping roles of several hormones may synergize to increase the ability of the endocrine system to regulate wound healing and transition quickly to blastema formation. Leptin and IGF have both been shown to enhance wound healing by reducing the time it takes to close the wound by increasing cellular proliferation, cellular growth, and motility. Insulin signaling and IGF signaling enhance blastema cell growth and protect the cells from apoptosis. Other hormones such as GCs, TH, and testosterone interact to regulate the immune response. GCs help with epidermal formation by increasing the movement of leukocytes out of the bloodstream and to the site of the wound, thus decreasing the time of the wound closure. However, in certain developmental contexts when an increase in GCs is coupled with an upregulation of TH signaling, it will result in cellular apoptosis  AEC and Blastema escrescenza Wound Healing Outgrowth Formation Cellular processes Motility, proliferation, Motility, proliferation, Motility, proliferation, apoptosis, immune dedifferentiation differentiations, pattern response formation Developmental gene Wnt, FGF-2, IL-6, IL-1α, FGF-8, FGF-10, Wnt3α, FGF-8, FGF-10, Wnt3α, networks TNFα, ROS Sall4, MMP-2, TGFB-1, 11βHSD2, SHH, Notch, FGF, PDGFAA, MSX-2, VEGF, FGF, PDGFAA, MSX- MSH, BMPs, Sox2, ProD1 2, BMPs, PEDF Endocrine signaling +GH +GH +GH +Leptin +Leptin +Leptin +/−Glucocorticoids −Glucocorticoids −Glucocorticoids +/−Thyroid hormone −Thyroid hormone −Thyroid hormone +/−Sex steroids +Prolactin +Prolactin +/−Sex steroids  INTERNAL CUES 2- GROWTH FACTORS Growth factors (GF) are small molecules capable of starting tissue healing. That includes cell replication, differentiation, migration, and other processes. Growth factors work by binding to specific cell receptors (receptor tyrosine kinases, RTKs), triggering a cascade of biological responses and influencing different responses. In regeneration, the crucial function of GFs is the establishment of correct intercellular communications, driving successful acquisition of function/phenotype by individual cells (e.g., SC differentiation) or cell division. They induce the expression of different genes RTKs binds are present in almost every cells. When the receptor is bound, the tyrosine dimerizes and induce auto- phosphorylation. https://doi.org/10.3389/fendo.2020.00384 GFs are an evolutionary established unique system that provides tissue formation in development and then via RTKs and their signaling axes supports homeostasis, cell integration, and tissue renewal. However, after damage, they may become “the cure and the cause,” as positive and negative outcomes are mediated by the same GFs depending on species or specific tissue within the organism. Signaling pathways from GF-triggered RTKs are well-conserved within the Animal Kingdom, raising the question of what has changed, altering human tissues and communication patterns and creating an inclination toward fibrosis compared to other species. A possible answer is that our epigenetic landscape is responsible for the cellular effects of RTKs. Importance in Regeneration: Cell Signaling: Growth factors act as signaling molecules, activating specific cellular pathways that lead to tissue repair and regeneration. They ensure that the right cells respond at the right time. for cell division and migration Tissue Repair: During injury, growth factors are released to orchestrate the repair process. They promote the regeneration of damaged tissues by stimulating cell division and migration, leading to the restoration of normal function. Stem Cell Activation: Certain growth factors play a pivotal role in activating stem cells, guiding their differentiation into specific cell types required for tissue regeneration. This is critical for replacing lost or damaged cells. Inflammation Regulation: Growth factors help modulate inflammatory responses, promoting healing while preventing excessive inflammation that can hinder recovery. This balance is essential for optimal tissue regeneration. Fibroblast Growth Factor (FGF): FGFs stimulates the proliferation of fibroblasts, leading to increased collagen production and tissue repair. It also plays a role in angiogenesis and neuronal development. FGFs are critical in wound healing and tissue regeneration, particularly in skin and muscle repair. Vascular Endothelial Growth Factor (VEGF): VEGF is vital for angiogenesis, the process by which new blood vessels form from pre-existing vessels. It enhances blood flow and oxygen supply to healing tissues, facilitating recovery. Increased VEGF levels are observed in wound healing, cancer progression, and various regenerative therapies, highlighting its importance in both health and disease. VEFG increases during hypoxia Angiogenesis helps cancer progression, so this factor (VEGF) is inhibited during cancer Platelet-Derived Growth Factor (PDGF): PDGF promotes the proliferation and migration of various cell types, including fibroblasts and smooth muscle cells and endothelial cells. It is crucial in the early phases of wound healing. By attracting cells to the injury site, PDGF aids in tissue formation and repair. VEGF VEGF recruits endhotelial cells, which have VEGF receptors VEGF has an internal tyrosin kinase domain Transforming Growth Factor-beta (TGF-β): TGF-β is involved in cell growth, differentiation, and immune regulation. It plays a significant role in the extracellular matrix (ECM) production, essential for tissue architecture. It helps in the transition from inflammation to tissue remodeling. While promoting tissue healing, TGF-β can also lead to fibrosis if dysregulated, emphasizing the need for balanced signaling during regeneration. Insulin-like Growth Factor (IGF): IGF supports cell growth and development by promoting anabolic processes. It enhances the effects of other growth factors, ensuring a robust regenerative response. IGF is involved in muscle repair and has been studied for its potential in treating age-related degenerative diseases. Hepatocyte Growth Factor (HGF): HGF plays important roles in regeneration, protection, and homeostasis in various cells and tissues, which includes hepatocytes, renal tubular cells, and neurons. HGF suppresses cell death and promotes the survival of cells, and this action participates in the protection of cells and tissues against injuries and pathology.  for example, cyclin are expressed during regeneration https://doi.org/10.1016/j.crneur.2022.100068 Growth factors in liver regeneration GF can be over-expressed during malignancies, this because GF leads to tudrug resistance and tumor proliferation  INTERNAL CUES: 3 - MICROBIOTA The microbiota, the set of microorganisms associated with a particular environment or host, has acquired a prominent role in the study of many physiological and developmental processes. Terms such as “microbiota” to describe the microbial taxa composition that are found within a certain environment These are the association of the microbiota with: 1) the host metabolic/digestive processes, 2) embryonic developmental processes, and 3) wound healing. From the roles ascribed to the microbiota, probably the best understood is their importance on host metabolism, which impacts their digestion and nutrition, by the assimilation of the digested food for the host physiological process. https://doi.org/10.3389/fcell.2021.768783  Many of the findings on the role of microbiota in wound healing were facilitated by studies of chronic wounds, such as diabetic foot ulcers and non- healing surgical wounds, which represent major healthcare problems. The main bacterial phyla identified in acute and chronic wounds are also found in healthy skin, however wounds are characterized by skin dysbiosis where their relative abundance differs significantly by wound type. E.g. Pseudomonas and Staphylococcus dominate in all types of chronic wounds and usually are present in acute wounds created by blunt or penetrating trauma. burns or atopic dermatitis However, higher levels of anaerobic bacteria are present in chronic wounds and are commonly associated with worse prognosis. Moreover, pathogenic microorganisms are suspected of playing a substantial role in delayed wound healing.  The microbiota that initially contaminates and colonizes wounded tissue can influence multiple cell types and phases of the wound healing process. Pathogens of the skin and soft tissue have specialized mechanisms that allow for adherence, invasion and host defense evasion. For example, commensal skin microorganisms can enhance epithelialization and barrier repair and promote lymphocyte-mediated tissue repair in mice. Probiotics and prebiotics are being studied for their ability to balance the wound microbiome, supporting healing and preventing infection 10.1038/s41579-024-01035-z  The gut microbiota has been implicated in intestinal epithelial repair. Gut microbiota enhanced epithelial wound repair in intestinal gut. Specifically, intestinal commensal bacteria have been found to regulate the proliferation, migration, and survival of host epithelial cells, as well as promote barrier function and resolution of epithelial wounds Two studies in the planaria have shown that bacteria can influence whole body regeneration. Additional model systems, mainly in vitro models comprising cell cultures, tissue explants, and organoids, have been developed to decipher the microbiota’s influence on the homeostasis and regeneration of mammalian intestines. Microbiota in gut regeneration Impairment of the integrity of the gut epithelium diminishes its ability to act as an effective gut barrier, can contribute to conditions associated to inflammation processes and can have other negative consequences. Pathogens and pathobionts have been linked with damage of the integrity of the gut epithelium, but other components of the gut microbiota and some of their metabolites can contribute to its repair and regeneration. Microbiota in gut regeneration  Microbiota in liver regeneration lower acetate reduce cell proliferation and regeneration 10.1016/j.jhep.2022.12.028 dysbiosis is associated with different types of diseas, including Parkinson