Canine Mesenchymal Stromal Cell Exosomes: State-of-the-Art Characterization, Functional Analysis and Applications in Various Diseases.

Canine mesenchymal stromal cells (MSCs) possess the capacity to differentiate into a variety of cell types and secrete a wide range of bioactive molecules in the form of soluble and membrane-bound exosomes. Extracellular vesicles/exosomes are nano-sized vesicles that carry proteins, lipids, and nucleic acids and can modulate recipient cell response in various ways. The process of exosome formation is a physiological interaction between cells. With a significant increase in basic research over the last two decades, there has been a tremendous expansion in research in MSC exosomes and their potential applications in canine disease models. The characterization of exosomes has demonstrated considerable variations in terms of source, culture conditions of MSCs, and the inclusion of fetal bovine serum or platelet lysate in the cell cultures. Furthermore, the amalgamation of exosomes with various nano-materials has become a novel approach to the fabrication of nano-exosomes. The fabrication of exosomes necessitates the elimination of extrinsic proteins, thus enhancing their potential therapeutic uses in a variety of disease models, including spinal cord injury, osteoarthritis, and inflammatory bowel disease. This review summarizes current knowledge on the characteristics, biological functions, and clinical relevance of canine MSC exosomes and their potential use in human and canine research. As discussed, exosomes have the ability to control lethal vertebrate diseases by administration directly at the injury site or through specific drug delivery mechanisms.

G Protein-Coupled Estrogen Receptor (GPER) and ERs Are Modulated in the Testis-Epididymal Complex in the Normal and Cryptorchid Dog.

There is growing evidence by the literature that the unbalance between androgens and estrogens is a relevant condition associated with a common canine reproductive disorder known as cryptorchidism. The role of estrogens in regulating testicular cell function and reproductive events is supposedly due to the wide expression of two nuclear estrogen receptors (ERs), ER-alpha and ER-beta and a trans-membrane G protein-coupled estrogen receptor (GPER) in the testis. In this study, immunohistochemistry, Western blotting and qRT-PCR were used to assess the distribution and expression of GPER in the testis-epididymal complex in the normal and cryptorchid dog. ER-alpha and ER-beta were also evaluated to better characterize the relative abundances of all three receptors. In addition, in these tissues, the expression level of two proteins as SOD1 and Nrf2 normally associated with oxidative stress was investigated to evaluate a possible relationship with ERs. Our data revealed changes in the distribution and expression of the GPER between the normal and cryptorchid dog. In particular, dogs affected by cryptorchidism showed an upregulation of GPER at level of the examined reproductive tract. Also considering the obtained result of a modulation of SOD1 and Nrf2 expression, we could hypothesize the involvement of GPER in the cryptorchid condition. Further studies are, however, necessary to characterize the role of GPER and its specific signaling mechanisms.

Novel MSC Perspectives: From Cell Regulation to Tissue Regeneration.

Three Special Issues, so far, have been dedicated to overall MSC prospective biology, from cell regulation to tissue regeneration [...].

Human Platelet-Rich Plasma Regulates Canine Mesenchymal Stem Cell Migration through Aquaporins.

Platelet products are commonly used in regenerative medicine due to their effects on the acceleration and promotion of wound healing, reduction of bleeding, synthesis of new connective tissue, and revascularization. Furthermore, a novel approach for the treatment of damaged tissues, following trauma or other pathological damages, is represented by the use of mesenchymal stem cells (MSCs). In dogs, both platelet-rich plasma (PRP) and MSCs have been suggested to be promising options for subacute skin wounds. However, the collection of canine PRP is not always feasible. In this study, we investigated the effect of human PRP (hPRP) on canine MSCs (cMSCs). We isolated cMSCs and observed that hPRP did not modify the expression levels of the primary class of major histocompatibility complex genes. However, hPRP was able to increase cMSC viability and migration by at least 1.5-fold. hPRP treatment enhanced both Aquaporin (AQP) 1 and AQP5 protein levels, and their inhibition by tetraethylammonium chloride led to a reduction of PRP-induced migration of cMSCs. In conclusion, we have provided evidence that hPRP supports cMSC survival and may promote cell migration, at least through AQP activation. Thus, hPRP may be useful in canine tissue regeneration and repair, placing as a promising tool for veterinary therapeutic approaches.

Preliminary Characterization of the Epigenetic Modulation in the Human Mesenchymal Stem Cells during Chondrogenic Process.

Regenerative medicine represents a growing hot topic in biomedical sciences, aiming at setting out novel therapeutic strategies to repair or regenerate damaged tissues and organs. For this perspective, human mesenchymal stem cells (hMSCs) play a key role in tissue regeneration, having the potential to differentiate into many cell types, including chondrocytes. Accordingly, in the last few years, researchers have focused on several in vitro strategies to optimize hMSC differentiation protocols, including those relying on epigenetic manipulations that, in turn, lead to the modulation of gene expression patterns. Therefore, in the present study, we investigated the role of the class II histone deacetylase (HDAC) inhibitor, MC1568, in the hMSCs-derived chondrogenesis. The hMSCs we used for this work were the hMSCs obtained from the amniotic fluid, given their greater differentiation capacity. Our preliminary data documented that MC1568 drove both the improvement and acceleration of hMSCs chondrogenic differentiation in vitro, since the differentiation process in MC1568-treated cells took place in about seven days, much less than that normally observed, namely 21 days. Collectively, these preliminary data might shed light on the validity of such a new differentiative protocol, in order to better assess the potential role of the epigenetic modulation in the process of the hypertrophic cartilage formation, which represents the starting point for endochondral ossification.

Neonatal infrared thermography images in the hypothermic ruminant model: Anatomical-morphological-physiological aspects and mechanisms for thermoregulation.

Hypothermia is one factor associated with mortality in newborn ruminants due to the drastic temperature change upon exposure to the extrauterine environment in the first hours after birth. Ruminants are precocial whose mechanisms for generating heat or preventing heat loss involve genetic characteristics, the degree of neurodevelopment at birth and environmental aspects. These elements combine to form a more efficient mechanism than those found in altricial species. Although the degree of neurodevelopment is an important advantage for these species, their greater mobility helps them to search for the udder and consume colostrum after birth. However, anatomical differences such as the distribution of adipose tissue or the presence of type II muscle fibers could lead to the understanding that these species use their energy resources more efficiently for heat production. The introduction of unconventional ruminant species, such as the water buffalo, has led to rethinking other characteristics like the skin thickness or the coat type that could intervene in the thermoregulation capacity of the newborn. Implementing tools to analyze species-specific characteristics that help prevent a critical decline in temperature is deemed a fundamental strategy for avoiding the adverse effects of a compromised thermoregulatory function. Although thermography is a non-invasive method to assess superficial temperature in several non-human animal species, in newborn ruminants there is limited information about its application, making it necessary to discuss the usefulness of this tool. This review aims to analyze the effects of hypothermia in newborn ruminants, their thermoregulation mechanisms that compensate for this condition, and the application of infrared thermography (IRT) to identify cases with hypothermia.

Does Orexin B-Binding Receptor 2 for Orexins Regulate Testicular and Epididymal Functions in Normal and Cryptorchid Dogs?

Orexins A (OXA) and B (OXB) and the receptors 1 (OX1R) and 2 (OX2R) for orexins are hypothalamic peptides found in several mammalian organs and participated to the control of a wide assortment of physiological and pathological functions. The distribution of OXA and OX1R has been extensively studied in the male gonad of mammals. Here, we examined the expression and localization of OXB and OX2R as well as their possible involvement in the regulation of testicular and epididymal functions, in healthy and cryptorchid dogs, employing some techniques such as immunohistochemistry, Western blotting, and real-time RT-PCR. In vitro tests were also carried out for evaluating the steroidogenic effect of OXB. OXB and OX2R were expressed in spermatocytes, spermatids, and Leydig cells in normal testis. Their localization was restricted to Sertoli and Leydig cells in cryptorchid conditions. OXB was found to be localized in all tracts of both normal and cryptorchid epididymis, whereas OX2R was found only in the caput. Because the small molecular weight of the peptides OXA and OXB, the expression of their precursor prepro-orexin (PPO), OX1R, and OX2R proteins and mRNAs were investigated by means of Western blot and real-time RT-PCR analyses, respectively, in all tested groups of. In particular, the mRNA level expression of all three genes was higher in cryptorchid dogs than in normal ones. In vitro tests demonstrated that OXB-by binding OX2R-is not involved in testicular steroidogenic processes. Therefore, the findings of this study might be the basis for further functional and molecular studies addressing the possible biochemical effects of OXB and OX2R in normal and pathological conditions of the male reproductive system.

Palmitoylethanolamide Promotes White-to-Beige Conversion and Metabolic Reprogramming of Adipocytes: Contribution of PPAR-α.

The potential role of brown and beige adipose tissue against obesity has been recognized. Browning, or beiging of white adipose tissue (WAT) is associated with the remodeling of adipocytes and the improvement of their metabolic and secretory functions. Here, palmitoylethanolamide (PEA) restore the plasticity of brown and white adipocytes impaired in mice on a high-fat diet (HFD). Young male C57Bl/6J mice were fed with control (STD) diet or HFD for 12 weeks. Ultramicronized PEA (30 mg/kg/die p.o.) was administered for an additional 7 weeks, together with HFD. PEA recovered interscapular brown fat morphology and function, increasing UCP1 positivity, noradrenergic innervation, and inducing the mRNA transcription of several specialized thermogenic genes. PEA promotes the beige-conversion of the subcutaneous WAT, increasing thermogenic markers and restoring leptin signaling and tissue hormone sensitivity. The pivotal role of lipid-sensing peroxisome proliferator-activated receptor (PPAR)-α in PEA effects was determined in mature 3T3-L1. Moreover, PEA improved mitochondrial bioenergetics in mature adipocytes measured by a Seahorse analyzer and induced metabolic machinery via AMPK phosphorylation. All these outcomes were dampened by the receptor antagonist GW6471. Finally, PEA induced adipogenic differentiation and increased AMPK phosphorylation in human adipose-derived stromal cells (ASCs) obtained from subcutaneous WAT of normal-weight patients and patients with obesity. We identify PEA and PPAR-α activation as the main mechanism by which PEA can rewire energy-storing white into energy-consuming brown-like adipocytes via multiple and converging effects that restore WAT homeostasis and metabolic flexibility.

Aquaporins Are Differentially Regulated in Canine Cryptorchid Efferent Ductules and Epididymis.

The efferent ductules and the epididymis are parts of the male reproductive system where spermatozoa mature. Specialized epithelial cells in these ducts contribute to the transport of fluids produced by spermatozoa's metabolic activity. Aquaporins (AQPs) have been demonstrated to be expressed in the spermatozoan membrane and testis epithelial cells, where they contribute to regulating spermatozoan volume and transit through environments of differing osmolality. Due to the lack of detailed literature regarding AQP expression in the canine male genital tract, the aim of this study was to investigate both the distribution and expression of AQP7, AQP8, and AQP9 in the efferent ductules and epididymal regions (caput, corpus, and cauda) from normal and cryptorchid dogs by using immunohistochemistry, Western blotting, and real-time reverse transcription polymerase chain reaction (RT-PCR). Our results show different patterns for the distribution and expression of the examined AQPs, with particular evidence of their upregulation in the caput and downregulation in the cauda region of the canine cryptorchid epididymis. These findings are associated with a modulation of Hsp70 and caspase-3 expression, suggesting the participation of AQPs in the luminal microenvironment modifications that are peculiar characteristics of this pathophysiological condition.

Differential abundances of AQP3 and AQP5 in reproductive tissues from dogs with and without cryptorchidism.

Aquaporins (AQPs) are integral transmembrane proteins facilitating transport of water and small solutes, such as glycerol and urea, between cells. In male reproductive tracts, AQPs maintain a milieu conducive for sperm formation, maturation, and storage. The aim of this study was to clarify effects of testicular and epidydimal function on male fertility by investigating localisation and abundances of AQP3 and AQP5 in testes and epididymal segments from dogs with and without unilateral cryptorchidism. Immunohistochemistry results indicated AQP3 and AQP5 have different distribution patterns in reproductive tissues of dogs with and without unilateral cryptorchidism. The AQP3, an aquaglyceroprotein, is present in different germ and Sertoli cells in testis of dogs without cryptorchidism. The AQP5 protein was not detected in germ cells but was present in Sertoli and Leydig cells and in endothelia of blood vessels. In cryptorchid dogs, AQP3 was detected in early-developing germ and Sertoli cells, and AQP5 had a distribution pattern similar to testes of dogs without cryptorchidism. In the epididymis, AQP3 and AQP5 were localised in epithelial cells of dogs with and without cryptorchidism in a cell-specific manner. The AQP3 and AQP5 protein was in larger abundance in the gonads from dogs with and without cryptorchidism. In contrast, AQP3 and AQP5 abundance increased in each segment of the cryptorchid epididymis, likely as a compensatory mechanism associated with the pathologic condition. These results indicate involvement of AQP3 and AQP5 in spermatogenesis and sperm maturation. Results from the present study indicate dogs are a useful for comparative reproductive biology studies.

Role of Aquaporins in the Physiological Functions of Mesenchymal Stem Cells.

Aquaporins (AQPs) are a family of membrane water channel proteins that control osmotically-driven water transport across cell membranes. Recent studies have focused on the assessment of fluid flux regulation in relation to the biological processes that maintain mesenchymal stem cell (MSC) physiology. In particular, AQPs seem to regulate MSC proliferation through rapid regulation of the cell volume. Furthermore, several reports have shown that AQPs play a crucial role in modulating MSC attachment to the extracellular matrix, their spread, and migration. Shedding light on how AQPs are able to regulate MSC physiological functions can increase our knowledge of their biological behaviours and improve their application in regenerative and reparative medicine.

Metabolic Plasticity of Melanoma Cells and Their Crosstalk With Tumor Microenvironment.

Cutaneous melanoma (CM) is a highly aggressive and drug resistant solid tumor, showing an impressive metabolic plasticity modulated by oncogenic activation. In particular, melanoma cells can generate adenosine triphosphate (ATP) during cancer progression by both cytosolic and mitochondrial compartments, although CM energetic request mostly relies on glycolysis. The upregulation of glycolysis is associated with constitutive activation of BRAF/MAPK signaling sustained by BRAFV600E kinase mutant. In this scenario, the growth and progression of CM are strongly affected by melanoma metabolic changes and interplay with tumor microenvironment (TME) that sustain tumor development and immune escape. Furthermore, CM metabolic plasticity can induce a metabolic adaptive response to BRAF/MEK inhibitors (BRAFi/MEKi), associated with the shift from glycolysis toward oxidative phosphorylation (OXPHOS). Therefore, in this review article we survey the metabolic alterations and plasticity of CM, its crosstalk with TME that regulates melanoma progression, drug resistance and immunosurveillance. Finally, we describe hallmarks of melanoma therapeutic strategies targeting the shift from glycolysis toward OXPHOS.

Aquaporin 1 (AQP1) Expression in Healthy Dog Tears.

Aquaporins (AQPs) are a family of thirteen membrane proteins that play an essential role in the transport of fluids across the cell plasma membrane. Recently, the expression of AQPs in different ocular tissues and their involvement in the pathophysiology of eye diseases, have garnered attention. Considering that literature on AQP expression in the lacrimal glands and their secretion is scarce, we aimed to characterise AQP1 expression in the tears of healthy dogs using two tear collection methods (Schirmer tear strips (STS) and ophthalmic sponges (OS)). Fifteen healthy dogs, free of ophthalmic diseases, were included in the study. Tear collection was performed by using STS in one eye and OS in the other. After the extraction of proteins from the tears, the expression of AQP1 was analysed by Western blotting. AQP1 was expressed as a band of 28 kDa. In addition, differences were observed in the expression of AQP1 and in the correlation between tear volume and protein concentration, in tears collected by the two different methods. Our results suggest that AQP1 has a specific role in tear secretion; further research is required to assess its particular role in the function of the ocular surface in eye physiology and pathology.

Chemical Characterisation and in Vitro Gas Production Kinetics of Eight Faba Bean Varieties.

Faba bean is an important vegetable protein source for ruminant diets. This research aimed to compare the nutritional characteristics of four commercial and four local cultivars in order to better characterise the local ones and promote their use in animal nutrition. The seeds' weight and the chemical composition, including starch and the energy, was evaluated. The in vitro fermentation characteristics were studied for 48 h using bull's rumen fluid as inoculum. All the varieties showed the values' weight corresponding to the specific botanical typology. The varieties significantly differed for protein, starch and lignin (p < 0.01) and structural carbohydrates (p < 0.05) concentration. No significant differences were observed for energy content. All the in vitro fermentation parameters resulted significantly different among the varieties. Organic matter degradability ranged between 89.9% and 85.1% and the potential gas production from 367 to 325 mL/g. The Pearson's analysis showed significant correlation between morphological characteristics, chemical data and in vitro fermentation parameters. In conclusion, this investigation confirms the possibility of using local faba bean varieties (i.e., Aquino, Castrocielo, 13#5, 4#4) in ruminant nutrition with the advantage that, being local natural resources, they are better adapted to the climate and agronomic conditions and limit environmental impact.

Platelet Function and Therapeutic Applications in Dogs: Current Status and Future Prospects.

Significant progress has been made in the functional characterization of canine platelets in the last two decades. The role of canine platelets in hemostasis includes their adhesion to the subendothelium, activation, and aggregation, leading to primary clot formation at the site of injury. Studies on canine platelet function and advancements in laboratory testing have improved the diagnosis and understanding of platelet-related disorders as well as the knowledge of the mechanisms behind these diseases. This review focuses on the most recent discoveries in canine platelet structure, function, and disorders; and discusses the efficacy of various tests in the diagnosis of platelet-related disorders. With the relatively recent discovery of angiogenetic and reparative effects of growth factors found in platelets, this review also summarizes the use of canine platelet-rich plasma (PRP) alone or in association with stem cells in regenerative therapy. The characterization of proteomic and lipidomic profiles and development of platelet gene therapy in veterinary species are areas of future study with potential for major therapeutic benefits.

Orexin A-Mediated Modulation of Reproductive Activities in Testis of Normal and Cryptorchid Dogs: Possible Model for Studying Relationships Between Energy Metabolism and Reproductive Control.

Orexin A (OxA) is a neuropeptide produced in the lateral hypothalamus that performs pleiotropic functions in different tissues, including involvement in energy homeostasis and reproductive neuroendocrine functions. The role of OxA is particularly important given the well-studied relationships between physiological mechanisms controlling energy balance and reproduction. The enzyme P450 aromatase (ARO) helps convert androgens to estrogens and has roles in steroidogenesis, spermatogenesis, and energy metabolism in several organs. The goal of this study was thus to investigate the role of OxA in ARO activity and the effects of this regulation on reproductive homeostasis in male gonads from healthy and cryptorchid dogs. The cryptorchidism is a specific condition characterized by altered reproductive and metabolic activities, the latter of which emerge from impaired glycolysis. OxA helps to stimulate testosterone (T) synthesis in the dog testis. We aimed to investigate OxA-mediated modulation of 17ß-estradiol (17ß-E) synthesis, ARO expression and metabolic indicators in testis of normal and cryptorchid dogs. Our results indicate putative effects of OxA on estrogen biosynthesis and ARO activity based on western blotting analysis and immunohistochemistry for ARO detection and in vitro tests. OxA triggered decrease in estrogen production and ARO activity inhibition; reduced ARO activity thus prevented the conversion of T to estrogens and increasing OxA-mediated synthesis of T. Furthermore, we characterized some metabolic and oxidative modulations in normal and cryptorchid dog's testis. The steroidogenic regulation by OxA and its modulation of ARO activity led us to hypothesize that OxA is a potential therapeutic target in pathological conditions associated with steroidogenic alterations and OxA possible involvement in metabolic processes in the male gonad.

Substantial Overview on Mesenchymal Stem Cell Biological and Physical Properties as an Opportunity in Translational Medicine.

Mesenchymal stem cells (MSC) have piqued worldwide interest for their extensive potential to treat a large array of clinical indications, their unique and controversial immunogenic and immune modulatory properties allowing ample discussions and debates for their possible applications. Emerging data demonstrating that the interaction of biomaterials and physical cues with MSC can guide their differentiation into specific cell lineages also provide new interesting insights for further MSC manipulation in different clinical applications. Moreover, recent discoveries of some regulatory molecules and signaling pathways in MSC niche that may regulate cell fate to distinct lineage herald breakthroughs in regenerative medicine. Although the advancement and success in the MSC field had led to an enormous increase in the amount of ongoing clinical trials, we still lack defined clinical therapeutic protocols. This review will explore the exciting opportunities offered by human and animal MSC, describing relevant biological properties of these cells in the light of the novel emerging evidence mentioned above while addressing the limitations and challenges MSC are still facing.

Urocortins in the mammalian endocrine system.

Urocortins (Ucns), peptides belonging to the corticotropin-releasing hormone (CRH) family, are classified into Ucn1, Ucn2, and Ucn3. They are involved in regulating several body functions by binding to two G protein-coupled receptors: receptor type 1 (CRHR1) and type 2 (CRHR2). In this review, we provide a historical overview of research on Ucns and their receptors in the mammalian endocrine system. Although the literature on the topic is limited, we focused our attention particularly on the main role of Ucns and their receptors in regulating the hypothalamic-pituitary-adrenal and thyroid axes, reproductive organs, pancreas, gastrointestinal tract, and other tissues characterized by "diffuse" endocrine cells in mammals. The prominent function of these peptides in health conditions led us to also hypothesize an action of Ucn agonists/antagonists in stress and in various diseases with its critical consequences on behavior and physiology. The potential role of the urocortinergic system is an intriguing topic that deserves further in-depth investigations to develop novel strategies for preventing stress-related conditions and treating endocrine diseases.

Leptin and Immunological Profile in Obesity and Its Associated Diseases in Dogs.

Growing scientific evidence has unveiled increased incidences of obesity in domestic animals and its influence on a plethora of associated disorders. Leptin, an adipokine regulating body fat mass, represents a key molecule in obesity, able to modulate immune responses and foster chronic inflammatory response in peripheral tissues. High levels of cytokines and inflammatory markers suggest an association between inflammatory state and obesity in dogs, highlighting the parallelism with humans. Canine obesity is a relevant disease always accompanied with several health conditions such as inflammation, immune-dysregulation, insulin resistance, pancreatitis, orthopaedic disorders, cardiovascular disease, and neoplasia. However, leptin involvement in many disease processes in veterinary medicine is poorly understood. Moreover, hyperleptinemia as well as leptin resistance occur with cardiac dysfunction as a consequence of altered cardiac mitochondrial metabolism in obese dogs. Similarly, leptin dysregulation seems to be involved in the pancreatitis pathophysiology. This review aims to examine literature concerning leptin and immunological status in obese dogs, in particular for the aspects related to obesity-associated diseases.

Urocortinergic system in the epididymis of the normal and cryptorchid dogs.

Cryptorchidism is associated with changes in the gonads and the spermatic duct system, which may cause infertility problems. Urocortin (UCN) is a corticotrophin-releasing hormone (CRH)-related peptide, which affects several functions of male genital organs. The aim of the present study was to investigate the expression of UCN and its receptors CRHR1 and CRHR2 using immunohistochemistry, western blotting and real-time reverse transcription polymerase chain reaction in tissues collected from the epididymis of normal and cryptorchid dogs. The lumen of the cryptic epididymal duct was found to be relatively smaller than that of the normal one, and interstitial tissue was abundant in the cryptic epididymis. In addition, only a few spermatids were observed in the lumen of the epididymal duct. Results showed that UCN, CRHR2 and CRHR1 were expressed in tissues collected from normal and cryptic epididymal ducts. Urocortin- and CRHR2-immunoreactivities (IRs) were detected in the principal cells of the caput, corpus and cauda of the normal and cryptic epididymides. CRHR1-IR was detected in vascular smooth muscles and fibromuscular cells surrounding epididymal tubules of the normal and cryptorchid dogs. Expression levels of UCN and CRHR2 mRNA were higher in cryptic epididymal ducts than that in normal epididymal ducts. These results suggest that UCN and its receptors might play a role in regulating the maturation and storage of spermatozoa. These findings indicated that the expression of these proteins could be modulated by the cryptorchidism condition.