Inhibition of C5AR1 impairs osteoclast mobilization and prevents bone loss.

Age-related and chemotherapy-induced bone loss depends on cellular senescence and the cell secretory phenotype. However, the factors secreted in the senescent microenvironment that contribute to bone loss remain elusive. Here, we report a central role for the inflammatory alternative complement system in skeletal bone loss. Through transcriptomic analysis of bone samples, we identified complement factor D, a rate-limiting factor of the alternative pathway of complement, which is among the most responsive factors to chemotherapy or estrogen deficiency. We show that osteoblasts and osteocytes are major inducers of complement activation, while monocytes and osteoclasts are their primary targets. Genetic deletion of C5ar1, the receptor of the anaphylatoxin C5a, or treatment with a C5AR1 inhibitor reduced monocyte chemotaxis and osteoclast differentiation. Moreover, genetic deficiency or inhibition of C5AR1 partially prevented bone loss and osteoclastogenesis upon chemotherapy or ovariectomy. Altogether, these lines of evidence support the idea that inhibition of alternative complement pathways may have some therapeutic benefit in osteopenic disorders.

Dental Stem Cells SV40, a new cell line developed in vitro from human stem cells of the apical papilla.

AIM: To establish and fully characterize a new cell line from human stem cells of the apical papilla (SCAPs) through immortalization with an SV40 large T antigen. METHODOLOGY: Human SCAPs were isolated and transfected with an SV40 large T antigen and treated with puromycin to select the infected population. Expression of human mesenchymal surface markers CD73, CD90 and CD105 was assessed in the new cell line named Dental Stem Cells SV40 (DSCS) by flow cytometry at early and late passages. Cell contact inhibition and proliferation were also analysed. To evaluate trilineage differentiation, quantitative polymerase chain reaction and histological staining were performed. RESULTS: DSCS cell flow cytometry confirmed the expression of mesenchymal surface markers even in late passages [100% positive for CD73 and CD90 and 98.9% for CD105 at passage (P) 25]. Fewer than 0.5% were positive for haematopoietic cell markers (CD45 and CD34). DSCS cells also showed increased proliferation when compared to the primary culture after 48 h, with a doubling time of 23.46 h for DSCS cells and 40.31 h for SCAPs, and retained the capacity to grow for >45 passages (150 population doubling) and their spindle-shaped morphology. Trilineage differentiation potential was confirmed through histochemical staining and gene expression of the chondrogenic markers SOX9 and COL2A1, adipogenic markers CEBPA and LPL, and osteogenic markers COL1A1 and ALPL. CONCLUSIONS: The new cell line derived from human SCAPs has multipotency, retains its morphology and expression of mesenchymal surface markers and shows higher proliferative capacity even at late passages (P45). DSCS cells can be used for in vitro study of root development and to achieve a better understanding of the regenerative mechanisms.

The Expression of TP53-Induced Glycolysis and Apoptosis Regulator (TIGAR) Can Be Controlled by the Antioxidant Orchestrator NRF2 in Human Carcinoma Cells.

Hyperactivation of the KEAP1-NRF2 axis is a common molecular trait in carcinomas from different origin. The transcriptional program induced by NRF2 involves antioxidant and metabolic genes that render cancer cells more capable of dealing with oxidative stress. The TP53-Induced Glycolysis and Apoptosis Regulator (TIGAR) is an important regulator of glycolysis and the pentose phosphate pathway that was described as a p53 response gene, yet TIGAR expression is detected in p53-null tumors. In this study we investigated the role of NRF2 in the regulation of TIGAR in human carcinoma cell lines. Exposure of carcinoma cells to electrophilic molecules or overexpression of NRF2 significantly increased expression of TIGAR, in parallel to the known NRF2 target genes NQO1 and G6PD. The same was observed in TP53KO cells, indicating that NRF2-mediated regulation of TIGAR is p53-independent. Accordingly, downregulation of NRF2 decreased the expression of TIGAR in carcinoma cell lines from different origin. As NRF2 is essential in the bone, we used mouse primary osteoblasts to corroborate our findings. The antioxidant response elements for NRF2 binding to the promoter of human and mouse TIGAR were described. This study provides the first evidence that NRF2 controls the expression of TIGAR at the transcriptional level.

Therapeutic Applications of Rose Hips from Different Rosa Species.

Rosa species, rose hips, are widespread wild plants that have been traditionally used as medicinal compounds for the treatment of a wide variety of diseases. The therapeutic potential of these plants is based on its antioxidant effects caused by or associated with its phytochemical composition, which includes ascorbic acid, phenolic compounds and healthy fatty acids among others. Over the last few years, medicinal interest in rose hips has increased as a consequence of recent research that has studied its potential application as a treatment for several diseases including skin disorders, hepatotoxicity, renal disturbances, diarrhoea, inflammatory disorders, arthritis, diabetes, hyperlipidaemia, obesity and cancer. In this review, the role of different species of Rosa in the prevention of treatment of various disorders related to oxidative stress, is examined, focusing on new therapeutic approaches from a molecular point of view.

Colorectal Carcinoma: A General Overview and Future Perspectives in Colorectal Cancer.

Colorectal cancer (CRC) is the third most common cancer and the fourth most common cause of cancer-related death. Most cases of CRC are detected in Western countries, with its incidence increasing year by year. The probability of suffering from colorectal cancer is about 4%-5% and the risk for developing CRC is associated with personal features or habits such as age, chronic disease history and lifestyle. In this context, the gut microbiota has a relevant role, and dysbiosis situations can induce colonic carcinogenesis through a chronic inflammation mechanism. Some of the bacteria responsible for this multiphase process include Fusobacterium spp, Bacteroides fragilis and enteropathogenic Escherichia coli. CRC is caused by mutations that target oncogenes, tumour suppressor genes and genes related to DNA repair mechanisms. Depending on the origin of the mutation, colorectal carcinomas can be classified as sporadic (70%); inherited (5%) and familial (25%). The pathogenic mechanisms leading to this situation can be included in three types, namely chromosomal instability (CIN), microsatellite instability (MSI) and CpG island methylator phenotype (CIMP). Within these types of CRC, common mutations, chromosomal changes and translocations have been reported to affect important pathways (WNT, MAPK/PI3K, TGF-ß, TP53), and mutations; in particular, genes such as c-MYC, KRAS, BRAF, PIK3CA, PTEN, SMAD2 and SMAD4 can be used as predictive markers for patient outcome. In addition to gene mutations, alterations in ncRNAs, such as lncRNA or miRNA, can also contribute to different steps of the carcinogenesis process and have a predictive value when used as biomarkers. In consequence, different panels of genes and mRNA are being developed to improve prognosis and treatment selection. The choice of first-line treatment in CRC follows a multimodal approach based on tumour-related characteristics and usually comprises surgical resection followed by chemotherapy combined with monoclonal antibodies or proteins against vascular endothelial growth factor (VEGF) and epidermal growth receptor (EGFR). Besides traditional chemotherapy, alternative therapies (such as agarose tumour macrobeads, anti-inflammatory drugs, probiotics, and gold-based drugs) are currently being studied to increase treatment effectiveness and reduce side effects.

Silencing of CD47 and SIRPα by Polypurine reverse Hoogsteen hairpins to promote MCF-7 breast cancer cells death by PMA-differentiated THP-1 cells.

BACKGROUND: In the context of tumor immunology, tumor cells have been shown to overexpress CD47, an anti-phagocytic signal directed to macrophages to escape from phagocytosis by interacting with Signal Regulatory Protein α SIRPα. In the present work, we designed Polypurine reverse Hoogsteen hairpins, PPRHs, to silence the expression of CD47 in tumor cells and SIRPα in macrophages with the aim to eliminate tumor cells by macrophages in co-culture experiments. METHODS: THP-1 cells were differentiated to macrophages with PMA. The mRNA levels of differentiation markers CD14 and Mcl-1 mRNA and pro-inflammatory cytokines (IL-1ß, IL-18, IL-6, IL-8 and TNF-α) were measured by qRT-PCR. The ability of PPRHs to silence CD47 and SIRPα was evaluated at the mRNA level by qRT-PCR and at the protein level by Western Blot. Macrophages were co-cultured with tumor cells in the presence of PPRHs to silence CD47 and/or SIRPα. Cell viability was assessed by MTT assays. RESULTS: THP-1 cells differentiated to macrophages with PMA showed an increase in macrophage surface markers (CD14, Mcl-1) and pro-inflammatory cytokines (IL-1ß, IL-18, IL-6, IL-8 and TNF-α). PPRHs were able to decrease both CD47 expression in MCF-7 cell line and SIRPα expression in macrophages at the mRNA and protein levels. In the presence of PPRHs, MCF-7 cells were eliminated by macrophages in co-culture experiments, whereas they survived in the absence of PPRHs. CONCLUSIONS: Our data support the usage of PPRHs to diminish CD47/SIRPα interaction by decreasing the expression of both molecules thus resulting in an enhanced killing of MCF-7 cells by macrophages, which might translate into beneficial effects in cancer therapy. These results indicate that PPRHs could represent a new approach with immunotherapeutic applications.

TXNDC5, a newly discovered disulfide isomerase with a key role in cell physiology and pathology.

Thioredoxin domain-containing 5 (TXNDC5) is a member of the protein disulfide isomerase family, acting as a chaperone of endoplasmic reticulum under not fully characterized conditions As a result, TXNDC5 interacts with many cell proteins, contributing to their proper folding and correct formation of disulfide bonds through its thioredoxin domains. Moreover, it can also work as an electron transfer reaction, recovering the functional isoform of other protein disulfide isomerases, replacing reduced glutathione in its role. Finally, it also acts as a cellular adapter, interacting with the N-terminal domain of adiponectin receptor. As can be inferred from all these functions, TXNDC5 plays an important role in cell physiology; therefore, dysregulation of its expression is associated with oxidative stress, cell ageing and a large range of pathologies such as arthritis, cancer, diabetes, neurodegenerative diseases, vitiligo and virus infections. Its implication in all these important diseases has made TXNDC5 a susceptible biomarker or even a potential pharmacological target.

Engineering a Microphysiological Model for Regenerative Endodontic Studies.

Dental pulp infections are common buccal diseases. When this happens, endodontic treatments are needed to disinfect and prepare the root canal for subsequent procedures. However, the lack of suitable in vitro models representing the anatomy of an immature root canal hinders research on regenerative events crucial in endodontics, such as regenerative procedures. This study aimed to develop a 3D microphysiological system (MPS) to mimic an immature root canal and assess the cytotoxicity of various irrigating solutions on stem cells. Utilizing the Dental Stem Cells SV40 (DSCS) cell line derived from human apical papilla stem cells, we analyzed the effects of different irrigants, including etidronic acid. The results indicated that irrigating solutions diminished cell viability in 2D cultures and influenced cell adhesion within the microphysiological device. Notably, in our 3D studies in the MPS, 17% EDTA and 9% 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP) irrigating solutions demonstrated superior outcomes in terms of DSCS viability and adherence compared to the control. This study highlights the utility of the developed MPS for translational studies in root canal treatments and suggests comparable efficacy between 9% HEBP and 17% EDTA irrigating solutions, offering potential alternatives for clinical applications.

Differential toxicity profile of secreted and processed α-Klotho expression over mineral metabolism and bone microstructure.

The aging-protective gene α-Klotho (KL) produces two main transcripts. The full-length mRNA generates a transmembrane protein that after proteolytic ectodomain shedding can be detected in serum as processed Klotho (p-KL), and a shorter transcript which codes for a putatively secreted protein (s-KL). Both isoforms exhibit potent pleiotropic beneficial properties, although previous reports showed negative side effects on mineral homeostasis after increasing p-KL concentration exogenously. Here, we expressed independently both isoforms using gene transfer vectors, to assess s-KL effects on mineral metabolism. While mice treated with p-KL presented altered expression of several kidney ion channels, as well as altered levels of Pi and Ca2+ in blood, s-KL treated mice had levels comparable to Null-treated control mice. Besides, bone gene expression of Fgf23 showed a fourfold increase after p-KL treatment, effects not observed with the s-KL isoform. Similarly, bone microstructure parameters of p-KL-treated mice were significantly worse than in control animals, while this was not observed for s-KL, which showed an unexpected increase in trabecular thickness and cortical mineral density. As a conclusion, s-KL (but not p-KL) is a safe therapeutic strategy to exploit KL anti-aging protective effects, presenting no apparent negative effects over mineral metabolism and bone microstructure.

HERC1 deficiency causes osteopenia through transcriptional program dysregulation during bone remodeling.

Bone remodeling is a continuous process between bone-forming osteoblasts and bone-resorbing osteoclasts, with any imbalance resulting in metabolic bone disease, including osteopenia. The HERC1 gene encodes an E3 ubiquitin ligase that affects cellular processes by regulating the ubiquitination of target proteins, such as C-RAF. Of interest, an association exists between biallelic pathogenic sequence variants in the HERC1 gene and the neurodevelopmental disorder MDFPMR syndrome (macrocephaly, dysmorphic facies, and psychomotor retardation). Most pathogenic variants cause loss of HERC1 function, and the affected individuals present with features related to altered bone homeostasis. Herc1-knockout mice offer an excellent model in which to study the role of HERC1 in bone remodeling and to understand its role in disease. In this study, we show that HERC1 regulates osteoblastogenesis and osteoclastogenesis, proving that its depletion increases gene expression of osteoblastic makers during the osteogenic differentiation of mesenchymal stem cells. During this process, HERC1 deficiency increases the levels of C-RAF and of phosphorylated ERK and p38. The Herc1-knockout adult mice developed imbalanced bone homeostasis that presented as osteopenia in both sexes of the adult mice. By contrast, only young female knockout mice had osteopenia and increased number of osteoclasts, with the changes associated with reductions in testosterone and dihydrotestosterone levels. Finally, osteocytes isolated from knockout mice showed a higher expression of osteocytic genes and an increase in the Rankl/Opg ratio, indicating a relevant cell-autonomous role of HERC1 when regulating the transcriptional program of bone formation. Overall, these findings present HERC1 as a modulator of bone homeostasis and highlight potential therapeutic targets for individuals affected by pathological HERC1 variants.

Griddient: a microfluidic array to generate reconfigurable gradients on-demand for spatial biology applications.

Biological tissues are highly organized structures where spatial-temporal gradients (e.g., nutrients, hypoxia, cytokines) modulate multiple physiological and pathological processes including inflammation, tissue regeneration, embryogenesis, and cancer progression. Current in vitro technologies struggle to capture the complexity of these transient microenvironmental gradients, do not provide dynamic control over the gradient profile, are complex and poorly suited for high throughput applications. Therefore, we have designed Griddent, a user-friendly platform with the capability of generating controllable and reversible gradients in a 3D microenvironment. Our platform consists of an array of 32 microfluidic chambers connected to a 384 well-array through a diffusion port at the bottom of each reservoir well. The diffusion ports are optimized to ensure gradient stability and facilitate manual micropipette loading. This platform is compatible with molecular and functional spatial biology as well as optical and fluorescence microscopy. In this work, we have used this platform to study cancer progression.

Microphysiological model reveals the promise of memory-like natural killer cell immunotherapy for HIV± cancer.

Numerous studies are exploring the use of cell adoptive therapies to treat hematological malignancies as well as solid tumors. However, there are numerous factors that dampen the immune response, including viruses like human immunodeficiency virus. In this study, we leverage human-derived microphysiological models to reverse-engineer the HIV-immune system interaction and evaluate the potential of memory-like natural killer cells for HIV+ head and neck cancer, one of the most common tumors in patients living with human immunodeficiency virus. Here, we evaluate multiple aspects of the memory-like natural killer cell response in human-derived bioengineered environments, including immune cell extravasation, tumor penetration, tumor killing, T cell dependence, virus suppression, and compatibility with retroviral medication. Overall, these results suggest that memory-like natural killer cells are capable of operating without T cell assistance and could simultaneously destroy head and neck cancer cells as well as reduce viral latency.

Microfluidics in vascular biology research: a critical review for engineers, biologists, and clinicians.

Neovascularization, the formation of new blood vessels, has received much research attention due to its implications for physiological processes and diseases. Most studies using traditional in vitro and in vivo platforms find challenges in recapitulating key cellular and mechanical cues of the neovascularization processes. Microfluidic in vitro models have been presented as an alternative to these limitations due to their capacity to leverage microscale physics to control cell organization and integrate biochemical and mechanical cues, such as shear stress, cell-cell interactions, or nutrient gradients, making them an ideal option for recapitulating organ physiology. Much has been written about the use of microfluidics in vascular biology models from an engineering perspective. However, a review introducing the different models, components and progress for new potential adopters of these technologies was absent in the literature. Therefore, this paper aims to approach the use of microfluidic technologies in vascular biology from a perspective of biological hallmarks to be studied and written for a wide audience ranging from clinicians to engineers. Here we review applications of microfluidics in vascular biology research, starting with design considerations and fabrication techniques. After that, we review the state of the art in recapitulating angiogenesis and vasculogenesis, according to the hallmarks recapitulated and complexity of the models. Finally, we discuss emerging research areas in neovascularization, such as drug discovery, and potential future directions.

Advancing Treatment of Bone Metastases through Novel Translational Approaches Targeting the Bone Microenvironment.

Bone metastases represent a lethal condition that frequently occurs in solid tumors such as prostate, breast, lung, and renal cell carcinomas, and increase the risk of skeletal-related events (SREs) including pain, pathologic fractures, and spinal cord compression. This unique metastatic niche consists of a multicellular complex that cancer cells co-opt to engender bone remodeling, immune suppression, and stromal-mediated therapeutic resistance. This review comprehensively discusses clinical challenges of bone metastases, novel preclinical models of the bone and bone marrow microenviroment, and crucial signaling pathways active in bone homeostasis and metastatic niche. These studies establish the context to summarize the current state of investigational agents targeting BM, and approaches to improve BM-targeting therapies. Finally, we discuss opportunities to advance research in bone and bone marrow microenvironments by increasing complexity of humanized preclinical models and fostering interdisciplinary collaborations to translational research in this challenging metastatic niche.

NRF2 function in osteocytes is required for bone homeostasis and drives osteocytic gene expression.

Osteocytes, the most abundant bone cell type, are derived from osteoblasts through a process in which they are embedded in an osteoid. We previously showed that nutrient restriction promotes the osteocyte transcriptional program and is associated with increased mitochondrial biogenesis. Here, we show that increased mitochondrial biogenesis increase reactive oxygen species (ROS) levels and consequently, NRF2 activity during osteocytogenesis. NRF2 activity promotes osteocyte-specific expression of Dmp1, Mepe, and Sost in IDG-SW3 cells, primary osteocytes, and osteoblasts, and in murine models with Nfe2l2 deficiency in osteocytes or osteoblasts. Moreover, ablation of Nfe2l2 in osteocytes or osteoblasts generates osteopenia and increases osteoclast numbers with marked sexual dimorphism. Finally, treatment with dimethyl fumarate prevented the deleterious effects of ovariectomy in trabecular bone masses of mice and restored osteocytic gene expression. Altogether, we uncovered the role of NRF2 activity in osteocytes during the regulation of osteocyte gene expression and maintenance of bone homeostasis.

Interplay between BMPs and Reactive Oxygen Species in Cell Signaling and Pathology.

The integration of cell extrinsic and intrinsic signals is required to maintain appropriate cell physiology and homeostasis. Bone morphogenetic proteins (BMPs) are cytokines that belong to the transforming growth factor-ß (TGF-ß) superfamily, which play a key role in embryogenesis, organogenesis and regulation of whole-body homeostasis. BMPs interact with membrane receptors that transduce information to the nucleus through SMAD-dependent and independent pathways, including PI3K-AKT and MAPKs. Reactive oxygen species (ROS) are intracellular molecules derived from the partial reduction of oxygen. ROS are highly reactive and govern cellular processes by their capacity to regulate signaling pathways (e.g., NF-κB, MAPKs, KEAP1-NRF2 and PI3K-AKT). Emerging evidence indicates that BMPs and ROS interplay in a number of ways. BMPs stimulate ROS production by inducing NOX expression, while ROS regulate the expression of several BMPs. Moreover, BMPs and ROS influence common signaling pathways, including PI3K/AKT and MAPK. Additionally, dysregulation of BMPs and ROS occurs in several pathologies, including vascular and musculoskeletal diseases, obesity, diabetes and kidney injury. Here, we review the current knowledge on the integration between BMP and ROS signals and its potential applications in the development of new therapeutic strategies.

Inhibition of phosphatidylinositol 3-kinase α (PI3Kα) prevents heterotopic ossification.

Heterotopic ossification (HO) is the pathological formation of ectopic endochondral bone within soft tissues. HO occurs following mechanical trauma, burns, or congenitally in patients suffering from fibrodysplasia ossificans progressiva (FOP). FOP patients carry a conserved mutation in ACVR1 that becomes neomorphic for activin A responses. Here, we demonstrate the efficacy of BYL719, a PI3Kα inhibitor, in preventing HO in mice. We found that PI3Kα inhibitors reduce SMAD, AKT, and mTOR/S6K activities. Inhibition of PI3Kα also impairs skeletogenic responsiveness to BMPs and the acquired response to activin A of the Acvr1R206H allele. Further, the efficacy of PI3Kα inhibitors was evaluated in transgenic mice expressing Acvr1Q207D . Mice treated daily or intermittently with BYL719 did not show ectopic bone or cartilage formation. Furthermore, the intermittent treatment with BYL719 was not associated with any substantial side effects. Therefore, this work provides evidence supporting PI3Kα inhibition as a therapeutic strategy for HO.

ACVR1 Function in Health and Disease.

Activin A receptor type I (ACVR1) encodes for a bone morphogenetic protein type I receptor of the TGFß receptor superfamily. It is involved in a wide variety of biological processes, including bone, heart, cartilage, nervous, and reproductive system development and regulation. Moreover, ACVR1 has been extensively studied for its causal role in fibrodysplasia ossificans progressiva (FOP), a rare genetic disorder characterised by progressive heterotopic ossification. ACVR1 is linked to different pathologies, including cardiac malformations and alterations in the reproductive system. More recently, ACVR1 has been experimentally validated as a cancer driver gene in diffuse intrinsic pontine glioma (DIPG), a malignant childhood brainstem glioma, and its function is being studied in other cancer types. Here, we review ACVR1 receptor function and signalling in physiological and pathological processes and its regulation according to cell type and mutational status. Learning from different functions and alterations linked to ACVR1 is a key step in the development of interdisciplinary research towards the identification of novel treatments for these pathologies.

LPS-squalene interaction on D-galactose intestinal absorption.

The dynamic and complex interactions between enteric pathogens and the intestinal epithelium often lead to disturbances in the intestinal barrier, altered fluid, electrolyte, and nutrient transport and can produce an inflammatory response. Lipopolysaccharide (LPS) is a complex polymer forming part of the outer membrane of Gram-negative bacteria. On the other hand, squalene is a triterpene present in high levels in the extra-virgin olive oil that has beneficial effects against several diseases and it has also anti-oxidant and anti-inflammatory properties. The aim of this work was to study whether the squalene could eliminate the LPS effect on D-galactose intestinal absorption in rabbits and Caco-2 cells. The results have shown that squalene reduced the effects of LPS on sugar absorption. High LPS doses increased D-galactose uptake through via paracellular but also decreased the active sugar transport because the SGLT1 levels were diminished. However, the endotoxin effect on the paracellular way seemed to be more important than on the transcellular route. At the same time, an increased in RELM-ß expression was observed. This event could be related to inflammation and cause a decrease in SGLT1 levels. In addition, MLCK protein is also increased by LPS which could lead to an increase in sugar transport through tight junctions. At low doses, the LPS could inhibit SGLT1 intrinsic activity. Bioinformatic studies by docking confirm the interaction between LPS-squalene as well as occur through MLCK and SGLT-1 proteins.

Glucose Restriction Promotes Osteocyte Specification by Activating a PGC-1α-Dependent Transcriptional Program.

Osteocytes, the most abundant of bone cells, differentiate while they remain buried within the bone matrix. This encasement limits their access to nutrients and likely affects their differentiation, a process that remains poorly defined. Here, we show that restriction in glucose supply promotes the osteocyte transcriptional program while also being associated with increased mitochondrial DNA levels. Glucose deprivation triggered the activation of the AMPK/PGC-1 pathway. AMPK and SIRT1 activators or PGC-1α overexpression are sufficient to enhance osteocyte gene expression in IDG-SW3 cells, murine primary osteoblasts, osteocytes, and organotypic/ex vivo bone cultures. Conversely, osteoblasts and osteocytes deficient in Ppargc1a and b were refractory to the effects of glucose restriction. Finally, conditional ablation of both genes in osteoblasts and osteocytes generate osteopenia and reduce osteocytic gene expression in mice. Altogether, we uncovered a role for PGC-1 in the regulation of osteocyte gene expression.