Micro/nano topological modification of TiO2 nanotubes activates Thy-1 signaling to control osteogenic differentiation of stem cells.

Micro/nano topological modification is critical for improving the in vivo behaviors of bone implants, regulating multiple cellular functions. Titania (TiO2) nanotubes show the capacity of promoting osteoblast-related cell differentiation and induce effective osseointegration, serving as a model material for studying the effects of micro/nano-topological modifications on cells. However, the intracellular signaling pathways by which TiO2 nanotubes regulate the osteogenic differentiation of stem cells are not fully defined. Thy-1 (CD90), a cell surface glycoprotein anchored by glycosylphosphatidylinositol, has been considered a key molecule in osteoblast differentiation in recent years. Nevertheless, whether the micro/nano topology of the implant surface leads to changes in Thy-1 is unknown, as well as whether these changes promote osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Here, TiO2 nanotubes of various diameters were prepared by adjusting the anodizing voltage. qPCR and immunoblot were carried out to assess the mechanism by which TiO2 nanotubes regulate Thy-1. The results revealed Ti plates harboring TiO2 nanotubes ∼100-nm diameter (TNT-100) markedly upregulated Thy-1. Subsequently, upregulated Thy-1 promoted the activation of Fyn/RhoA/MLC Ⅱ/F-actin axis, which enhanced the nuclear translocation of YAP. After Thy-1 knockdown by siRNA, the Fyn/RhoA/MLC Ⅱ/F-actin axis was significantly inhibited and TiO2 nanotubes showed decreased effects on osteogenic differentiation. Therefore, Thy-1 upregulation might be a major mechanism by which micro/nano-topological modification of TiO2 nanotubes promotes osteogenic differentiation in BMSCs. This study provides novel insights into the molecular mechanism of TiO2 nanotubes, which may help design improved bone implants for clinical application.

Thy-1 knockdown promotes the osteogenic differentiation of GMSCs via the Wnt/ß-catenin pathway.

Gingival mesenchymal stem cells (GMSCs) are newly developed seed cells for tissue engineering owing to their easy isolation, abundance and high growth rates. Thy-1 is an important regulatory molecule in the differentiation of mesenchymal stem cells (MSCs). In this study, we investigated the function of Thy-1 in the osteogenic differentiation of GMSCs by reducing the expression of Thy-1 using a lentivirus. The results demonstrated that Thy-1 knockdown promoted the osteogenic differentiation of GMSCs in vitro. Validation by RNA-seq revealed an obvious decrease in Vcam1 and Sox9 gene expression with Thy-1 knockdown. Kyoto Encyclopedia of Genes and Genomes pathway analysis suggested that the differentially expressed genes were enriched in the Wnt signalling pathway. We further demonstrated that Thy-1 knockdown promoted osteogenic differentiation of GMSCs by activating the Wnt/ß-catenin signalling pathway. Therefore, Thy-1 has a key regulatory role in the differentiation of GMSCs and maybe a core molecule connecting transcription factors related to the differentiation of MSCs. Our study also highlighted the potential of Thy-1 to modify MSCs, which may help improve their use in tissue engineering.

CD44, CD90 and CD96 expression in immune thrombocytopenia purpura (ITP) patients.

Studying the expression of hematopoietic stem cell markers from different sources might be useful in understanding stem cell biology in different niche conditions. The study aimed to assess the difference in cell surface markers (CD44, CD90, CD96) on hematopoietic stem cells in three different niche conditions; umbilical cord blood (UCB), normal bone marrow (NBM) and bone marrow samples from idiopathic (immune) thrombocytopenic purpura (IBM). This study was conducted on 300 cases divided into three study groups; 100 umbilical cord blood units collected from mothers undergoing cesarian section in gynecology and obstetrics department, 100 bone marrow samples from idiopathic (immune) thrombocytopenic purpura patients collected from university children hospital and 100 normal bone marrow samples with no evidence of disease in bone marrow tissue. CD44 was significantly elevated in UCB and NBM groups compared to IBM group (<0.001). There was also a significant elevation of CD90 and CD96 in IBM group compared to NBM group and UCB (<0.001). CD90 and CD96 play a role in the pathogenesis of ITP disorder and could be applied as a targeted therapy to improve the outcome of this disease.

The GPI-Anchored Protein Thy-1/CD90 Promotes Wound Healing upon Injury to the Skin by Enhancing Skin Perfusion.

Wound healing is a highly regulated multi-step process that involves a plethora of signals. Blood perfusion is crucial in wound healing and abnormalities in the formation of new blood vessels define the outcome of the wound healing process. Thy-1 has been implicated in angiogenesis and silencing of the Thy-1 gene retards the wound healing process. However, the role of Thy-1 in blood perfusion during wound closure remains unclear. We proposed that Thy-1 regulates vascular perfusion, affecting the healing rate in mouse skin. We analyzed the time of recovery, blood perfusion using Laser Speckle Contrast Imaging, and tissue morphology from images acquired with a Nanozoomer tissue scanner. The latter was assessed in a tissue sample taken with a biopsy punch on several days during the wound healing process. Results obtained with the Thy-1 knockout (Thy-1-/-) mice were compared with control mice. Thy-1-/- mice showed at day seven, a delayed re-epithelialization, increased micro- to macro-circulation ratio, and lower blood perfusion in the wound area. In addition, skin morphology displayed a flatter epidermis, fewer ridges, and almost no stratum granulosum or corneum, while the dermis was thicker, showing more fibroblasts and fewer lymphocytes. Our results suggest a critical role for Thy-1 in wound healing, particularly in vascular dynamics.

Thy-1 plays a pathogenic role and is a potential biomarker for skin fibrosis in scleroderma.

Thy-1 (CD90) is a well-known marker of fibroblasts implicated in organ fibrosis, but its contribution to skin fibrosis remains unknown. We examined Thy-1 expression in scleroderma skin and its potential role as a biomarker and pathogenic factor in animal models of skin fibrosis. Skin from patients with systemic sclerosis demonstrated markedly elevated Thy-1 expression compared with controls, colocalized with fibroblast activator protein in the deep dermis, and correlated with the severity of skin involvement (modified Rodnan skin score). Serial imaging of skin from Thy-1 yellow fluorescent protein reporter mice by IVIS showed an increase in Thy-1 expression that correlated with onset and progression of fibrosis. In contrast to lung fibrosis, Thy-1-KO mice had attenuated skin fibrosis in both bleomycin and tight skin-1 murine models. Moreover, Thy-1 regulated key pathogenic pathways involved in fibrosis, including inflammation, myofibroblast differentiation, apoptosis, and multiple additional canonical fibrotic pathways. Therefore, although Thy-1 deficiency leads to exacerbated lung fibrosis, in skin it is protective. Moreover, Thy-1 may serve as a longitudinal marker to assess skin fibrosis.

Neurovascular dynamics of repeated cortical spreading depolarizations after acute brain injury.

Cortical spreading depolarizations (CSDs) are increasingly suspected to play an exacerbating role in a range of acute brain injuries, including stroke, possibly through their interactions with cortical blood flow. We use simultaneous wide-field imaging of neural activity and hemodynamics in Thy1-GCaMP6f mice to explore the neurovascular dynamics of CSDs during and following Rose Bengal-mediated photothrombosis. CSDs are observed in all mice as slow-moving waves of GCaMP fluorescence extending far beyond the photothrombotic area. Initial CSDs are accompanied by profound vasoconstriction and leave residual oligemia and ischemia in their wake. Later, CSDs evoke variable responses, from constriction to biphasic to vasodilation. However, CSD-evoked vasoconstriction is found to be more likely during rapid, high-amplitude CSDs in regions with stronger oligemia and ischemia, which, in turn, worsens after each repeated CSD. This feedback loop may explain the variable but potentially devastating effects of CSDs in the context of acute brain injury.

Combining CUBIC Optical Clearing and Thy1-YFP-16 Mice to Observe Morphological Axon Changes During Wallerian Degeneration.

OBJECTIVE: Wallerian degeneration is a pathological process closely related to peripheral nerve regeneration following injury, and includes the disintegration and phagocytosis of peripheral nervous system cells. Traditionally, morphological changes are observed by performing immunofluorescence staining after sectioning, which results in the loss of some histological information. The purpose of this study was to explore a new, nondestructive, and systematic method for observing axonal histological changes during Wallerian degeneration. METHODS: Thirty male Thy1-YFP-16 mice (SPF grade, 6 weeks old, 20±5 g) were randomly selected and divided into clear, unobstructed brain imaging cocktails and computational analysis (CUBIC) optical clearing (n=15) and traditional method groups (n=15). Five mice in each group were sacrificed at 1st, 3rd, and 5th day following a crush operation. The histological axon changes were observed by CUBIC light optical clearing treatment, direct tissue section imaging, and HE staining. RESULTS: The results revealed that, compared with traditional imaging methods, there was no physical damage to the samples, which allowed for three-dimensional and deep-seated tissue imaging through CUBIC. Local image information could be nicely obtained by direct fluorescence imaging and HE staining, but it was difficult to obtain image information of the entire sample. At the same time, the image information obtained by fluorescence imaging and HE staining was partially lost. CONCLUSION: The combining of CUBIC and Thy1-YFP transgenic mice allowed for a clear and comprehensive observation of histological changes of axons in Wallerian degeneration.

Photostimulation activates fast-spiking interneurons and pyramidal cells in the entorhinal cortex of Thy1-ChR2-YFP line 18 mice.

The application of optogenetics in animals has provided new insights into both fundamental neuroscience and diseases of the nervous system. This is primarily due to the fact that optogenetics allows selectively activating or inhibiting particular types of neurons. One of the first transgenic mouse lines developed for the optogenetic experiment was Thy1-ChR2-YFP. Thy1 is an immunoglobulin superfamily member expressing in projection neurons, so it was assumed that channelrhodopsin-2 (ChR2) would be primarily expressed in projection neurons. However, the specificity of ChR2 expression under promoter Thy1 in different lines has to be clarified yet. Therefore, we aimed to determine the cell specificity of ChR2 expression in the entorhinal cortex of Thy1-ChR2-YFP line 18 mice. We have found that both pyramidal cells and fast-spiking interneurons in deep layers of the entorhinal cortex depolarized and fired in response to 470-nm photostimulation. To exclude the effect of synaptic activation of interneurons by pyramidal cells, we used a selective antagonist of AMPA receptors. Under these conditions, inhibitory postsynaptic currents decreased but did not disappear completely. Furthermore, gabazine inhibited these postsynaptic currents entirely, thus confirming the direct activation of interneurons by light. These data demonstrate that ChR2 is expressed in both pyramidal neurons and fast-spiking interneurons of the entorhinal cortex in Thy1-ChR2-YFP mice.

NGF Expression and Elevation in Hip Osteoarthritis Patients with Pain and Central Sensitization.

Osteoarthritis (OA) is a chronic degenerative musculoskeletal disease that causes articular cartilage degeneration and chronic pain. Research into OA animal models suggests that elevated NGF levels in the synovium contribute to pain and central sensitization (CS). However, it is unclear whether synovial NGF contributes to CS in patients with OA. We investigated the association between synovial NGF expression and clinical assessments of pain and CS in hip OA (hOA) patients. We also aimed to identify which cells in the synovium of hOA patients express NGF. Sixty-six patients who received total hip replacement and a diagnosis of hOA were enrolled. We measured NGF mRNA expression in synovial samples obtained from 50 patients using qPCR and analyzed the correlation of NGF expression with the CS inventory (CSI) score and Japanese Orthopaedic Association (JOA) score, a clinical scoring system for OA. To identify the synovial cells expressing NGF, we analyzed NGF mRNA expression in CD14+ and CD14- cells, which represent macrophage-rich and fibroblast-rich fractions, respectively, extracted from 8 patients. To further identify which macrophage subtypes express NGF, we examined NGF mRNA expression in CD14high and CD14low cells sorted from 8 patients. Synovial NGF mRNA expression was negatively correlated with JOA score but positively correlated with CSI score (JOA pain, r = -0.337, P = 0.017; CSI score, r = 0.358, P = 0.011). Significantly greater levels of NGF were observed in CD14- cells compared to CD14+ cells (P = 0.036) and in CD14high cells compared to CD14low cells (P = 0.008). In conclusion, synovial NGF expression is correlated with the degree of pain and CS in hOA patients. NGF is predominantly expressed in synovial fibroblasts. Further, CD14high synovial macrophages expressed higher levels of NGF. Our results may provide a novel NGF-targeted therapeutic strategy for hOA pain.

Immunotherapy-based targeting of MSLN+ activated portal fibroblasts is a strategy for treatment of cholestatic liver fibrosis.

We investigated the role of mesothelin (Msln) and thymocyte differentiation antigen 1 (Thy1) in the activation of fibroblasts across multiple organs and demonstrated that Msln-/- mice are protected from cholestatic fibrosis caused by Mdr2 (multidrug resistance gene 2) deficiency, bleomycin-induced lung fibrosis, and UUO (unilateral urinary obstruction)-induced kidney fibrosis. On the contrary, Thy1-/- mice are more susceptible to fibrosis, suggesting that a Msln-Thy1 signaling complex is critical for tissue fibroblast activation. A similar mechanism was observed in human activated portal fibroblasts (aPFs). Targeting of human MSLN+ aPFs with two anti-MSLN immunotoxins killed fibroblasts engineered to express human mesothelin and reduced collagen deposition in livers of bile duct ligation (BDL)-injured mice. We provide evidence that antimesothelin-based therapy may be a strategy for treatment of parenchymal organ fibrosis.

Visualization of early oligomeric α-synuclein pathology and its impact on the dopaminergic system in the (Thy-1)-h[A30P]α-syn transgenic mouse model.

Aggregation of alpha-synuclein (α-syn) into Lewy bodies and Lewy neurites is a pathological hallmark in the Parkinson´s disease (PD) brain. The formation of α-syn oligomers is believed to be an early pathogenic event and the A30P mutation in the gene encoding α-syn, causing familial PD, has been shown to cause an accelerated oligomerization. Due to the problem of preserving antigen conformation on tissue surfaces, α-syn oligomers are difficult to detect ex vivo using conventional immunohistochemistry with oligomer-selective antibodies. Herein, we have instead employed the previously reported α-syn oligomer proximity ligation assay (ASO-PLA), along with a wide variety of biochemical assays, to discern the pathological progression of α-syn oligomers and their impact on the dopaminergic system in male and female (Thy-1)-h[A30P]α-syn transgenic (A30P-tg) mice. Our results reveal a previously undetected abundance of α-syn oligomers in midbrain of young mice, whereas phosphorylated (pS129) and proteinase k-resistant α-syn species were observed to a larger extent in aged mice. Although we did not detect loss of dopaminergic neurons in A30P-tg mice, a dysregulation in the monoaminergic system was recorded in older mice. Taken together, ASO-PLA should be a useful method for the detection of early changes in α-syn aggregation on brain tissue, from experimental mouse models in addition to post mortem PD cases.

Sublytic C5b-9 triggers glomerular mesangial cell proliferation via enhancing FGF1 and PDGFα gene transcription mediated by GCN5-dependent SOX9 acetylation in rat Thy-1 nephritis.

Rat Thy-1 nephritis (Thy-1N) is an animal model of human mesangioproliferative glomerulonephritis (MsPGN), accompanied by glomerular mesangial cell (GMC) proliferation and extracellular matrix (ECM) deposition. Although sublytic C5b-9 formed on GMC membrane could induce cell proliferation, the mechanism is still unclear. In this study, we first demonstrated that the level of SRY related HMG-BOX gene 9 (SOX9), general control nonderepressible 5 (GCN5), fibroblast growth factor 1 (FGF1) and platelet-derived growth factor α (PDGFα) was all elevated both in the renal tissues of Thy-1N rats (in vivo) and in the GMCs (in vitro) with sublytic C5b-9 stimulation. Then, we not only discovered that sublytic C5b-9 caused GMC proliferation through increasing SOX9, GCN5, FGF1 and PDGFα expression, but also proved that SOX9 and GCN5 formed a complex and combined with FGF1 and PDGFα promoters, leading to FGF1 and PDGFα gene transcription. More importantly, GCN5 could mediate SOX9 acetylation at lysine 62 (K62) to enhance SOX9 binding to FGF1 or PDGFα promoter and promote FGF1 or PDGFα synthesis and GMC proliferation. Besides, the experiments in vivo also showed that FGF1 and PDGFα expression, GMC proliferation and urinary protein secretion in Thy-1N rats were greatly reduced by silencing renal SOX9, GCN5, FGF1 or PDGFα gene. Furthermore, the renal tissues of MsPGN patients also exhibited positive expression of these genes mentioned above. Collectively, our findings indicate that GCN5, SOX9 and FGF1/PDGFα can form an axis and play an essential role in sublytic C5b-9-triggered GMC proliferation, which might provide a novel insight into the pathogenesis of Thy-1N and MsPGN.

Bone marrow stromal cells (BMSCs CD45- /CD44+ /CD73+ /CD90+ ) isolated from osteoporotic mice SAM/P6 as a novel model for osteoporosis investigation.

Available therapies aimed at treating age-related osteoporosis are still insufficient. Therefore, designing reliable in vitro model for the analysis of molecular mechanisms underlying senile osteoporosis is highly required. We have isolated and characterized progenitor cells isolated from bone marrow (BMSCs) of osteoporotic mice strain SAM/P6 (BMSCSAM/P6 ). The cytophysiology of BMSCSAM/P6 was for the first time compared with BMSCs isolated from healthy BALB/c mice (BMSCBALB/c ). Characterization of the cells included evaluation of their multipotency, morphology and determination of specific phenotype. Viability of BMSCs cultures was determined in reference to apoptosis profile, metabolic activity, oxidative stress, mitochondrial membrane potential and caspase activation. Additionally, expression of relevant biomarkers was determined with RT-qPCR. Obtained results indicated that BMSCSAM/P6 and BMSCBALB/c show the typical phenotype of mesenchymal stromal cells (CD44+, CD73+, CD90+) and do not express CD45. Further, BMSCSAM/P6 were characterized by deteriorated multipotency, decreased metabolic activity and increased apoptosis occurrence, accompanied by elevated oxidative stress and mitochondria depolarisation. The transcriptome analyses showed that BMSCSAM/P6 are distinguished by lowered expression of molecules crucial for proper osteogenesis, including Coll-1, Opg and Opn. However, the expression of Trap, DANCR1 and miR-124-3p was significantly up-regulated. Obtained results show that BMSCSAM/P6 present features of progenitor cells with disturbed metabolism and could serve as appropriate model for in vitro investigation of age-dependent osteoporosis.

Glucocorticoids and Androgens Protect From Gastric Metaplasia by Suppressing Group 2 Innate Lymphoid Cell Activation.

BACKGROUND & AIMS: The immune compartment is critical for maintaining tissue homeostasis. A weak immune response increases susceptibility to infection, but immune hyperactivation causes tissue damage, and chronic inflammation may lead to cancer development. In the stomach, inflammation damages the gastric glands and drives the development of potentially preneoplastic metaplasia. Glucocorticoids are potent anti-inflammatory steroid hormones that are required to suppress gastric inflammation and metaplasia. However, these hormones function differently in males and females. Here, we investigate the impact of sex on the regulation of gastric inflammation. METHODS: Endogenous glucocorticoids and male sex hormones were removed from mice using adrenalectomy and castration, respectively. Mice were treated with 5α-dihydrotestosterone (DHT) to test the effects of androgens on regulating gastric inflammation. Single-cell RNA sequencing of gastric leukocytes was used to identify the leukocyte populations that were the direct targets of androgen signaling. Type 2 innate lymphoid cells (ILC2s) were depleted by treatment with CD90.2 antibodies. RESULTS: We show that adrenalectomized female mice develop spontaneous gastric inflammation and spasmolytic polypeptide-expressing metaplasia (SPEM) but that the stomachs of adrenalectomized male mice remain quantitatively normal. Simultaneous depletion of glucocorticoids and sex hormones abolished the male-protective effects and triggered spontaneous pathogenic gastric inflammation and SPEM. Treatment of female mice with DHT prevented gastric inflammation and SPEM development when administered concurrent with adrenalectomy and also reversed the pathology when administered after disease onset. Single-cell RNAseq of gastric leukocytes revealed that ILC2s expressed abundant levels of both the glucocorticoid receptor (Gr) and androgen receptor (Ar). We demonstrated that DHT treatment potently suppressed the expression of the proinflammatory cytokines Il13 and Csf2 by ILC2s. Moreover, ILC2 depletion protected the stomach from SPEM development. CONCLUSIONS: Here, we report a novel mechanism by which glucocorticoids and androgens exert overlapping effects to regulate gastric inflammation. Androgen signaling within ILC2s prevents their pathogenic activation by suppressing the transcription of proinflammatory cytokines. This work revealed a critical role for sex hormones in regulating gastric inflammation and metaplasia.

THY-Tau22 mouse model accumulates more tauopathy at late stage of the disease in response to microglia deactivation through TREM2 deficiency.

The role played by microglia has taken the center of the stage in the etiology of Alzheimer's disease (AD). Several genome-wide association studies carried out on large cohorts of patients have indeed revealed a large number of genetic susceptibility factors corresponding to genes involved in neuroinflammation and expressed specifically by microglia in the brain. Among these genes TREM2, a cell surface receptor expressed by microglia, arouses strong interest because its R47H variant confers a risk of developing AD comparable to the ε4 allele of the APOE gene. Since this discovery, a growing number of studies have therefore examined the role played by TREM2 in the evolution of amyloid plaques and neurofibrillary tangles, the two brain lesions characteristic of AD. Many studies report conflicting results, reflecting the complex nature of microglial activation in AD. Here, we investigated the impact of TREM2 deficiency in the THY-Tau22 transgenic line, a well-characterized model of tauopathy. Our study reports an increase in the severity of tauopathy lesions in mice deficient in TREM2 occurring at an advanced stage of the pathology. This exacerbation of pathology was associated with a reduction in microglial activation indicated by typical morphological features and altered expression of specific markers. However, it was not accompanied by any further changes in memory performance. Our longitudinal study confirms that a defect in microglial TREM2 signaling leads to an increase in neuronal tauopathy occurring only at late stages of the disease.

Tumor-Specific T Cells Exacerbate Mortality and Immune Dysregulation during Sepsis.

Sepsis induces significant immune dysregulation characterized by lymphocyte apoptosis and alterations in the cytokine milieu. Because cancer patients exhibit a 10-fold greater risk of developing sepsis compared with the general population, we aimed to understand how pre-existing malignancy alters sepsis-induced immune dysregulation. To address this question, we assessed the impact of tumor-specific CD8+ T cells on the immune response in a mouse model of cecal ligation and puncture (CLP)-induced sepsis. Tumor-bearing animals containing Thy1.1+ tumor-specific CD8+ T cells were subjected to CLP, and groups of animals received anti-Thy1.1 mAb to deplete tumor-specific CD8+ T cells or isotype control. Results indicated that depleting tumor-specific T cells significantly improved mortality from sepsis. The presence of tumor-specific CD8+ T cells resulted in increased expression of the 2B4 coinhibitory receptor and increased apoptosis of endogenous CD8+ T cells. Moreover, tumor-specific T cells were not reduced in number in the tumors during sepsis but did exhibit impaired IFN-γ production in the tumor, tumor draining lymph node, and spleen 24 h after CLP. Our research provides novel insight into the mechanisms by which pre-existing malignancy contributes to increased mortality during sepsis.

Glutamine switches vascular smooth muscle cells to synthetic phenotype through inhibiting miR-143 expression and upregulating THY1 expression.

AIMS: Vascular smooth muscle cells (VSMCs) are involved in the pathogenesis of many human cardiovascular diseases. They modulate their phenotype from "contractile" to "synthetic" in response to changes in local environmental cues. How glutamine regulates the differentiation of VSMCs and the underlying mechanisms remain largely unknown. MAIN METHODS: Here, we explored the effects of various doses of glutamine (0 mM, 1 mM, 2 mM, and 4 mM) on the proliferation, migration, and phenotypic switch of human VSMCs in vitro. Glutamine dose-dependently enhanced VSMC proliferation, and markedly increased VSMC migration. KEY FINDINGS: Notably, glutamine promoted the phenotypic switch of VSMCs towards a synthetic phenotype, as evidenced by significantly decreased expression of contractile markers myosin heavy chain 11 (MYH11) and calponin while increased expression of synthetic markers collagen I and vimentin. Importantly, these changes upon glutamine treatments were attenuated after additional treatments with glutamine metabolism inhibitor BPTES. Additionally, glutamine downregulated miR-143 expression, and miR-143 inactivation alone resulted in enhanced proliferation, migration, and promoted the synthetic phenotype of VSMCs. Moreover, Thy-1 cell surface antigen (THY1) was validated as a downstream target of miR-143, and THY1 expression was upregulated by glutamine in VSMCs. Furthermore, either miR-143 overexpression or THY1 silencing abolished the effect of glutamine on proliferation, migration, and phenotypic switch of VSMCs, supporting a novel glutamine-miR-143-THY1 pathway in modulating VSMC functions. SIGNIFICANCE: This study demonstrated a novel mechanism of glutamine in modulation of VSMC phenotypic switch by targeting miR-143 and THY1, and provides significant insight on targeted therapy of patients with cardiovascular diseases.

Biological characteristics and metabolic profile of canine mesenchymal stem cells isolated from adipose tissue and umbilical cord matrix.

Despite the increasing demand of cellular therapies for dogs, little is known on the differences between adult and fetal adnexa canine mesenchymal stem cells (MSCs), and data on their metabolic features are lacking. The present study aimed at comparing the characteristics of canine adipose tissue (AT) and umbilical cord matrix (UC) MSCs. Moreover, for the first time in the dog, the cellular bioenergetics were investigated by evaluating the two main metabolic pathways (oxidative phosphorylation and glycolysis) of ATP production. Frozen-thawed samples were used for this study. No differences in mean cell proliferation were found (P>0.05). However, while AT-MSCs showed a progressive increase in doubling time over passages, UC-MSCs showed an initial post freezing-thawing latency. No differences in migration, spheroid formation ability, and differentiation potential were found (P>0.05). RT-PCR analysis confirmed the expression of CD90 and CD44, the lack of CD14 and weak expression of CD34, mostly by AT-MSCs. DLA-DRA1 and DLA-DQA1 were weakly expressed only at passage 0 by UC-MSCs, while they were expressed at different passages for AT-MSCs. There was no difference (P>0.05) in total ATP production between cell cultures, but the ratio between the "mitochondrial ATP Production Rate" and the "glycolytic ATP Production Rate" was higher (P<0.05) in AT- than in UC-MSCs. However, in both MSCs types the mitochondrial respiration was the main pathway of ATP production. Mitochondrial respiration and ATP turnover in UC-MSCs were higher (P<0.05) than in AT-MSCs, but both had a 100% coupling efficiency. These features and the possibility of increasing the oxygen consumption by a spare respiratory capacity of four (AT-MSCSs) and two (UC-MSCs) order of magnitude greater than basal respiration, can be taken as indicative of the cell propensity to differentiate. The findings may efficiently contribute to select the most appropriate MSCs, culture and experimental conditions for transplantation experiments in mesenchymal stem cell therapy for companion animals.

Age-related increase of alpha-synuclein oligomers is associated with motor disturbances in L61 transgenic mice.

The pathogenesis of Parkinson's disease involves fibrillization and deposition of alpha-synuclein (α-syn) into Lewy bodies. Accumulating evidence suggests that α-syn oligomers are particularly neurotoxic. Transgenic (tg) mice overexpressing wild-type human α-syn under the Thy-1 promoter (L61) reproduce many Parkinson's disease features, but the pathogenetic relevance of α-syn oligomers in this mouse model has not been studied in detail. Here, we report an age progressive increase of α-syn oligomers in the brain of L61 tg mice. Interestingly, more profound motor symptoms were observed in animals with higher levels of membrane-bound oligomers. As this tg model is X-linked, we also performed subset analyses, indicating that both sexes display a similar age-related increase in α-syn oligomers. However, compared with females, males featured increased brain levels of oligomers from an earlier age, in addition to a more severe behavioral phenotype with hyperactivity and thigmotaxis in the open field test. Taken together, our data indicate that α-syn oligomers are central to the development of brain pathology and behavioral deficits in the L61 tg α-syn mouse model.

Small molecule inhibitors and culture conditions enhance therapeutic cell and EV potency via activation of beta-catenin and suppression of THY1.

Primary cell therapy continues to face significant hurdles to therapeutic translation including the inherent variations that exist from donor to donor, batch to batch, and scale-up driven modifications to the manufacturing process. Cardiosphere-derived cells (CDCs) are stromal/progenitor cells with clinically demonstrated tissue reparative capabilities. Mechanistic investigations have identified canonical Wnt/ß-catenin signaling as a therapeutic potency marker, and THY1 (CD90) expression as inversely correlated with potency. Here we demonstrate that the cardiosphere formation process increases ß-catenin levels and enriches for therapeutic miR content in the extracellular vesicles of these cells, namely miR-146a and miR-22. We further find that loss of potency is correlated with impaired cardiosphere formation. Finally, our data show that small GSK3ß inhibitors including CHIR, and BIO and "pro-canonical Wnt" culturing conditions can rescue ß-catenin signaling and reduce CD90 expression. These findings identify strategies that could be used to maintain CDC potency and therapeutic consistency.