Dedifferentiation Of Cells Research Articles

YAP1/TEAD1 upregulate platelet-derived growth factor receptor beta to promote vascular smooth muscle cell proliferation and neointima formation

We have previously demonstrated that the transcription co-factor yes-associated protein 1 (YAP1) promotes vascular smooth muscle cell (VSMC) de-differentiation. Yet, the role and underlying mechanisms of YAP1 in neointima formation in vivo remain unclear. The goal of this study was to investigate the role of VSMC-expressed YAP1 in vascular injury-induced VSMC proliferation and delineate the mechanisms underlying its action. Experiments employing gain- or loss-of-function of YAP1 demonstrated that YAP1 promotes human VSMC proliferation. Mechanistically, we identified platelet-derived growth factor receptor beta (PDGFRB) as a novel YAP1 target gene that confers the YAP1-dependent hyper-proliferative effects in VSMCs. Furthermore, we identified TEA domain transcription factor 1 (TEAD1) as a key transcription factor that mediates YAP1-dependent PDGFRβ expression. ChIP assays demonstrated that TEAD1 is enriched at a PDGFRB gene enhancer. Luciferase reporter assays further demonstrated that YAP1 and TEAD1 co-operatively activate the PDGFRB enhancer. Consistent with these observations, we found that YAP1 expression is upregulated after arterial injury and correlates with PDGFRβ expression and VSMC proliferation in vivo. Using a novel inducible SM-specific Yap1 knockout mouse model, we found that the specific deletion of Yap1 in adult VSMCs is sufficient to attenuate arterial injury-induced neointima formation, largely due to inhibited PDGFRβ expression and VSMC proliferation. Our study unravels a novel mechanism by which YAP1/TEAD1 promote VSMC proliferation via transcriptional induction of PDGFRβ, thereby enhancing PDGF-BB downstream signaling and promoting neointima formation.

Increased frequency of \u03b2 cells with abnormal NKX6.1 expression in type 2 diabetes but not in subjects with higher risk for type 2 diabetes

BackgroundNKX6.1 is a transcription factor for insulin, as well as a marker for β cell maturity. Abnormal NKX6.1 expression in β cells, such as translocation from the nucleus to cytoplasm or lost expression, has been shown as a marker for β cell dedifferentiation.MethodsWe obtained pancreatic sections from organ donors and immunofluorescence staining with NKX6.1 and insulin was performed to characterize NKX6.1 expression in subjects with or without type 2 diabetes mellitus (T2DM).ResultsOur results showed that cells with insulin expression but no nucleic NKX6.1 expression (NKX6.1Nuc-Ins+), and cells with cytoplasmic NKX6.1 expression but no insulin expression (NKX6.1cytIns−) were significantly increased in T2DM subjects and positively correlated with glycated hemoglobin (HbA1c), indicating the elevated β cell dedifferentiation with NKX6.1 inactivation in T2DM. To investigate whether β cell dedifferentiation has initiated in subjects with higher risks for T2DM, we next analyzed the association between β-cell dedifferentiation level in ND subjects with different ages, body mass index, and HbA1c. The results showed the absolute number and percentage of dedifferentiated β cells with NKX6.1 inactivation did not significantly change in subjects with advanced aging, obesity, or modest hyperglycemia, indicating that the β cell dedifferentiation might mainly occur after T2DM was diagnosed.ConclusionOur results suggested that NKX6.1 expression in β cells was changed in type 2 diabetic subjects, evidenced by significantly increased NKX6.1Nuc-Ins+ and NKX6.1cytIns− cells. This abnormality did not occur more frequently in subjects with a higher risk for T2DM, suggesting that β cell dedifferentiation might be secondary to the pathological changes in T2DM.

HaCaT‑conditioned medium supplemented with the small molecule inhibitors SB431542 and CHIR99021 and the growth factor PDGF‑AA prevents the dedifferentiation of dermal papilla cells in vitro

Hair loss, including alopecia, is a common and distressing problem for men and women, and as a result, there is considerable interest in developing treatments that can prevent or reverse hair loss. Dermal papillae closely interact with epidermal cells and play a key role during hair follicle induction and hair morphogenesis. As dermal papilla cells (DPCs) lose their hair-inducing ability in monolayer cultures in vitro, it is difficult to obtain de novo hair follicle structures following DPC transplantation in vivo. The present study aimed to explore culture conditions to maintain DPC characteristics using conditioned media (CM) from the supernatant of cultured HaCaT keratinocyte cells supplemented with other components. Initially, it was observed that during passaging of in vitro monolayer DPC cultures, the Wnt/β-catenin pathway was repressed, while the TGF-β/Smad pathway was activated, and that HaCaT cells cultivated in 1% fetal bovine serum had higher levels of expression of Wnt3a and Wnt10b compared with normal keratinocytes. Culturing of high-passage (P7) DPCs in CM from HaCaT cells (HaCaT-CM) actively stimulated cell proliferation and maintained Sox2 and Versican expression levels. Supplementation of HaCaT-CM with SB431542 (SB, a TGF-β receptor inhibitor), CHIR99021, (CHIR, a GSK3α/β inhibitor and activator of Wnt signaling) and platelet-derived growth factor (PDGF)-AA further increased the expression levels of Sox2, Versican and alkaline phosphatase (ALP) in P7 DPCs. Three-dimensional culture of P7 DPCs using hanging drop cultures in HaCaT-CM supplemented with SB, CHIR and PDGF-AA resulted in larger cell aggregates and a further significant upregulation of Sox2, ALP and Versican expression levels. Taken together, these findings demonstrated that HaCaT-CM supplemented with SB, CHIR and PDGF-AA may preserve the hair-inducing ability of high-passage DPCs and may therefore be useful in reconstructing new hair follicles in vivo.

KLF11 protects against abdominal aortic aneurysm through inhibition of endothelial cell dysfunction.

Abdominal aortic aneurysm (AAA) is a life-threatening degenerative vascular disease. Endothelial cell (EC) dysfunction is implicated in AAA. Our group recently demonstrated that Krüppel-like factor 11 (KLF11) plays an essential role in maintaining vascular homeostasis, at least partially through inhibition of EC inflammatory activation. However, the functions of endothelial KLF11 in AAA remain unknown. Here we found that endothelial KLF11 expression was reduced in the ECs from human aneurysms and was time dependently decreased in the aneurysmal endothelium from both elastase- and Pcsk9/AngII-induced AAA mouse models. KLF11 deficiency in ECs markedly aggravated AAA formation, whereas EC-selective KLF11 overexpression markedly inhibited AAA formation. Mechanistically, KLF11 not only inhibited the EC inflammatory response but also diminished MMP9 expression and activity and reduced NADPH oxidase 2–mediated production of reactive oxygen species in ECs. In addition, KLF11-deficient ECs induced smooth muscle cell dedifferentiation and apoptosis. Overall, we established endothelial KLF11 as a potentially novel factor protecting against AAA and a potential target for intervention in aortic aneurysms.

Glycyrrhizin Attenuates Carcinogenesis by Inhibiting the Inflammatory Response in a Murine Model of Colorectal Cancer.

Glycyrrhizin (GL), an important active ingredient of licorice root, which weakens the proinflammatory effects of high-mobility group box 1 (HMGB1) by blocking HMGB1 signaling. In this study, we investigated whether GL could suppress inflammation and carcinogenesis in an azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced murine model of colorectal cancer. ICR mice were divided into four groups (n = 5, each)—control group, GL group, colon cancer (CC) group, and GL-treated CC (CC + GL) group, and sacrificed after 20 weeks. Plasma levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α were measured using an enzyme-linked immunosorbent assay. The colonic tissue samples were immunohistochemically stained with DNA damage markers (8-nitroguanine and 8-oxo-7,8-dihydro-2′-deoxy-guanosine), inflammatory markers (COX-2 and HMGB1), and stem cell markers (YAP1 and SOX9). The average number of colonic tumors and the levels of IL-6 and TNF-α in the CC + GL group were significantly lower than those in the CC group. The levels of all inflammatory and cancer markers were significantly reduced in the CC + GL group. These results suggest that GL inhibits the inflammatory response by binding HMGB1, thereby inhibiting DNA damage and cancer stem cell proliferation and dedifferentiation. In conclusion, GL significantly attenuates the pathogenesis of AOM/DSS-induced colorectal cancer by inhibiting HMGB1-TLR4-NF-κB signaling.

Cilostazol is an effective causal therapy for preventing paclitaxel-induced peripheral neuropathy by suppression of Schwann cell dedifferentiation

Chemotherapy-induced peripheral neuropathy (CIPN) can lead to discontinuation of chemotherapy and is consequently a serious impediment to effective cancer treatment. Due to our limited understanding of mechanisms underlying the pathogenesis of CIPN, no causal therapy has been approved for relief of this condition. We previously demonstrated that taxanes (paclitaxel and docetaxel) induce Schwann cell dedifferentiation, characterized by increased expression of p75 and galectin-3, ultimately leading to demyelination. These changes appear to be responsible for CIPN pathogenesis. This study was designed to identify a novel candidate therapeutic for CIPN with the ability to suppress paclitaxel-induced Schwann cell dedifferentiation. Given that elevation of cyclic adenosine monophosphate (cAMP) signaling participates in Schwann cell differentiation, we performed immunocytochemical screening of phosphodiesterase (PDE) inhibitors. We found that the PDE3 inhibitor cilostazol strongly promoted differentiation of primary cultures of rat Schwann cells via a mechanism involving cAMP/exchange protein directly activated by cAMP (Epac) signaling. Co-treatment with cilostazol prevented paclitaxel-induced dedifferentiation of Schwann cell cultures and demyelination in a mixed culture of Schwann cells and dorsal root ganglia neurons. Notably, continuous oral administration of cilostazol suppressed Schwann cell dedifferentiation within the sciatic nerve and the development of mechanical hypersensitivity in a mouse model of paclitaxel-related CIPN. Importantly, cilostazol potentiated, rather than inhibited, the anti-cancer effect of paclitaxel on the human breast cancer cell line MDA-MB-231. These findings highlight the potential utility of cilostazol as a causal therapeutic that avoids the development of paclitaxel-related CIPN without compromising anti-cancer properties.

Abstract LT020: Cancer associated fibroblasts in the tumor microenvironment maintain ovarian cancer stem cells through non-canonical Wnt5a signaling

Abstract Frequent relapse and development of chemoresistance are major causes of poor survival in ovarian cancer. Cancer stem cells (CSCs) consist of a small subpopulation in the tumor that are capable of initiation and maintenance. CSCs are typically resistant to cytotoxic chemotherapy and are a potential cause of relapse and chemoresistance. Most studies on CSCs focus on cancer cells alone while CSCs exist in a complex microenvironment in tumors. This microenvironment or CSC niche provides optimal conditions to maintain their tumor initiating potential. Hence, understanding the crosstalk between CSCs and the tumor microenvironment can potentially provide effective therapeutic targets to prevent chemoresistance and relapse. Cancer associated fibroblasts (CAFs) are a major constituent of the ovarian cancer tumor microenvironment and are highly enriched in the residual tumors following chemotherapy. Therefore, we studied the role of CAFs in promoting ovarian cancer chemoresistance and disease relapse through providing an optimal microenvironment for CSCs. CAFs isolated from ovarian cancer patient tumors were used in heterotypic 3D or 2D coculture systems with high grade serous ovarian cancer cell lines or with patient-derived ovarian cancer cells to study their effect on CSCs and chemoresistance. Matched pre- and post-chemotherapy patient tumors were used to confirm our findings. CAFs significantly increased resistance to carboplatin and enriched CSCs by increasing their symmetric division as well as dedifferentiation of bulk ovarian cancer cells. Pre-coculture with CAFs increased in vivo tumor initiation capacity of the ovarian cancer cells 10-fold. The CSC-CAF crosstalk responsible for CSC induction was found to be mediated by Wnt signaling. Inhibition of Wnt secretion by CAFs could block their effect on CSCs. Wnt5a was the most highly expressed Wnt in CAFs, which was further induced by ovarian cancer cells. CRISPR knockdown of Wnt5a in CAFs or treatment with a specific Wnt5a inhibitor abrogated the induction of CSCs by CAFs. Wnt5a was found to signal through a non-canonical Wnt pathway in the CSCs involving the coreceptor ROR2, protein kinase C (PKC), and CAMP Responsive Element Binding Protein 1 (CREB1). Inhibition of each of them prevented CSC induction and functional rescue experiments confirmed the sequence of the Wnt5a-ROR2-PKC-CREB1 axis. Treatment of mouse xenografts established by co-injection of CAFs and ovarian cancer cells, with the Wnt5a inhibitor sensitized them to carboplatin, and eliminated the CSCs in the residual tumors. Our results indicate that CAF-derived Wnt5a is instrumental in ovarian cancer CSC growth and maintenance. In the long term, our studies will broaden the understanding of the mechanism of CSC maintenance by the tumor microenvironment and contribute towards the development of novel therapeutic approaches to prevent ovarian cancer chemoresistance and relapse. Citation Format: Yiming Fang, Xue Xiao, Ji Wang, Subramanyam Dasari, Kenneth P. Nephew, Dmitriy Zamarin, Anirban K. Mitra. Cancer associated fibroblasts in the tumor microenvironment maintain ovarian cancer stem cells through non-canonical Wnt5a signaling [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT020.

Down-Regulation of Long Non-Coding RNA TINCR Induces Cell Dedifferentiation and Predicts Progression in Oral Squamous Cell Carcinoma.

ObjectivesRecently long non-coding RNAs (lncRNAs) have emerged as novel gene regulators involved in tumorigenic processes, including oral squamous cell carcinoma (OSCC). Here, we identified a differentiation-related lncRNA, terminal differentiation-induced non-coding RNA (TINCR). However, its biological function and clinicopathological significance in OSCC still remain unclear.MethodsThe lncRNA expression profiles in OSCC tissues and paired adjacent non-tumor tissues (NATs) from 10 patients were detected by lncRNA microarrays. Weighted gene co-expression network analysis (WGCNA) and gene ontology (GO) enrichment were performed to identify the most significant module and module functional annotation, respectively. Potential differentiation-related lncRNAs were screened by differential expression analysis. TINCR was further confirmed in OSCC cell lines and tissues of another patient cohort by using qRT-PCR. The correlation between the TINCR expression level and clinicopathological characteristics was analyzed. The effects of TINCR on cell differentiation, migration and invasion were assessed by knockdown or knock-in in vitro and in vivo.ResultsWGCNA and GO enrichment analysis showed that one co-expression network was significantly enriched for epithelial cell differentiation, among which, TINCR was significantly downregulated. qRT-PCR analyses validated down-regulation of TINCR in tumor tissues compared with paired NATs, and its expression was closely correlated with pathological differentiation and lymph node metastasis in patients with OSCC. Patients with lower TINCR expression levels had worse survival. Cell function experiments showed that TINCR played a crucial role in epithelial differentiation. Both TINCR and epithelial differentiation-associated genes, including IVL and KRT4, were significantly upregulated during OSCC cell calcium-induced differentiation but were reduced when cell dedifferentiation occurred in tumor spheres. Overexpression of TINCR dramatically suppressed cell dedifferentiation, migration and invasion in vitro, while knockdown of TINCR had the opposite effects. Upregulation of TINCR significantly elevated the expression of terminal differentiation genes and repressed tumor growth in vivo. Moreover, TINCR significantly suppressed the activation of JAK2/STAT3 signaling in OSCC cells.ConclusionOur study suggests that TINCR functions as a tumor suppressor by inducing cell differentiation through modulating JAK2/STAT3 signaling in OSCC. TINCR may serve as a prognostic biomarker and therapeutic target for OSCC.

Delayed blockage of prostaglandin EP4 receptors can reduce dedifferentiation, epithelial-to-mesenchymal transition and fibrosis following acute kidney injury.

Dedifferentiation of tubular epithelial cells is involved in both regeneration and fibrosis following acute kidney injury (AKI). Prostaglandin E2 receptor 4 (EP4 ) antagonist can inhibit the dedifferentiation of renal tubular cells. The present study investigated whether the time of blockage of EP4 receptors, using grapiprant, could affect the tubular regeneration or interstitial fibrosis in AKI. Cisplatin was used to induce AKI in 72 C57BL/6 adult female mice. Animals were assigned to four groups; control, cisplatin-treated, cisplatin-treated with early grapiprant intervention and cisplatin-treated with late grapiprant intervention. AKI was assessed by kidney function tests and histopathology. Fibrosis was evaluated by Masson's trichrome and alpha smooth muscle actin (α-SMA) expression. Markers of dedifferentiation, CD133, and epithelial to mesenchymal transition (EMT), vimentin were assessed. Early intervention with grapiprant significantly ameliorated AKI more efficiently than late intervention. However, even late intervention was useful in reducing the overall fibrosis as demonstrated by Masson's trichrome and α-SMA expression. In both grapiprant-treated groups, a parallel reduction of dedifferentiation (CD133) and EMT (vimentin) was evident. It seems that the progressive fibrotic changes that follow AKI could still be reduced possibly by targeting dedifferentiation and/or EMT.

Optimizing Protocols for Arabidopsis Shoot and Root Protoplast Cultivation.

Procedures for the direct regeneration of entire plants from a shoot and root protoplasts of Arabidopsis thaliana have been optimized. The culture media for protoplast donor-plant cultivation and protoplast culture have been adjusted for optimal plant growth, plating efficiency, and promotion of shoot regeneration. Protocols have been established for the detection of all three steps in plant regeneration: (i) chromatin relaxation and activation of auxin biosynthesis, (ii) cell cycle progression, and (iii) conversion of cell-cycle active cells to totipotent ones. The competence for cell division was detected by DNA replication events and required high cell density and high concentrations of the auxinic compound 2,4-D. Cell cycle activity and globular structure formation, with subsequent shoot induction, were detected microscopically and by labeling with fluorescent dye Rhodamine123. The qPCR results demonstrated significantly upregulated expression of the genes responsible for nuclear reorganization, auxin responses, and auxin biosynthesis during the early stage of cell reprogramming. We further optimized cell reprogramming with this protocol by applying glutathione (GSH), which increases the sensitivity of isolated mesophyll protoplasts to cell cycle activation by auxin. The developed protocol allows us to investigate the molecular mechanism of the de-differentiation of somatic plant cells.

TAp73β Can Promote Hepatocellular Carcinoma Dedifferentiation

Simple SummaryHepatocellular carcinoma (HCC) is a highly complex and heterogeneous type of cancer. Hepatocyte dedifferentiation is one of the important steps in the development of HCC. However, its molecular mechanisms are not well known. In this study, we report that transcriptionally active TAp73 isoforms are overexpressed in HCC. We also show that TAp73β suppresses the expression of the hepatocyte markers including CYP3A4, AFP, ALB, HNF4α, while increasing the expression of several cholangiocyte markers in HCC cell lines. In conclusion, this report reveals a pro-oncogenic role for TAp73β in liver cancer.Hepatocyte dedifferentiation is a major source of hepatocellular carcinoma (HCC), but its mechanisms are unknown. We explored the p73 expression in HCC tumors and studied the effects of transcriptionally active p73β (TAp73β) in HCC cells. Expression profiles of p73 and patient clinical data were collected from the Genomic Data Commons (GDC) data portal and the TSVdb database, respectively. Global gene expression profiles were determined by pan-genomic 54K microarrays. The Gene Set Enrichment Analysis method was used to identify TAp73β-regulated gene sets. The effects of TAp73 isoforms were analyzed in monolayer cell culture, 3D-cell culture and xenograft models in zebrafish using western blot, flow cytometry, fluorescence imaging, real-time polymerase chain reaction (RT-PCR), immunohistochemistry and morphological examination. TAp73 isoforms were significantly upregulated in HCC, and high p73 expression correlated with poor patient survival. The induced expression of TAp73β caused landscape expression changes in genes involved in growth signaling, cell cycle, stress response, immunity, metabolism and development. Hep3B cells overexpressing TAp73β had lost hepatocyte lineage biomarkers including ALB, CYP3A4, AFP, HNF4α. In contrast, TAp73β upregulated genes promoting cholangiocyte lineage such as YAP, JAG1 and ZO-1, accompanied with an increase in metastatic ability. Our findings suggest that TAp73β may promote malignant dedifferentiation of HCC cells.

Dedifferentiation of Epithelial Cells Incorporates Immune Reprogramming.

Recent innovations in immunotherapies have cured cancers but remain ineffective in pancreatic cancer, which exhibits intertwined dynamics of dedifferentiation of epithelium and reprogramming of immunosuppression. Emerging evidence reveals the biological mechanisms through which dedifferentiation recapitulates immune reprogramming, providing new insights into the therapeutic potential of controlling dedifferentiation for sensitization to immunotherapies.

Circ_0004872 promotes platelet-derived growth factor-BB-induced proliferation, migration and dedifferentiation in HA-VSMCs via miR-513a-5p/TXNIP axis

The proliferation, migration and dedifferentiation of vascular smooth muscle cells (VSMCs) exert crucial roles in atherosclerosis (AS) progression. The aim of our study was to explore the influences of circular RNA 0004872 (circ_0004872) in platelet-derived growth factor-BB (PDGF-BB)-induced AS cell model and investigate the underlying mechanisms. Real-time quantitative polymerase chain reaction (RT-qPCR) was implemented for the expression detection of circ_0004872, mitogen-activated protein kinase 1 (MAPK1) messenger RNA (mRNA), microRNA-513a-5p (miR-513a-5p) and thioredoxin interacting protein (TXNIP). Cell proliferation was analyzed via Cell Counting Kit 8 (CCK8) assay. Cell migration was assessed via wound healing assay and transwell migration assay. Western blot assay was used to measure the expression of alpha smooth muscle actin (α-SMA), osteopontin (OPN), calponin and TXNIP. Dual-luciferase reporter assay and RNA-pull down assay were used for confirmation of interaction between miR-513a-5p and circ_0004872 or TXNIP. Circ_0004872 expression was elevated in PDGF-BB-induced human aortic vascular smooth muscle cells (HA-VSMCs) and carotid plaque tissues. Circ_0004872 silencing alleviated PDGF-BB-induced proliferation, migration and dedifferentiation in HA-VSMCs. MiR-513a-5p bound to circ_0004872, and circ_0004872 knockdown-induced effects in PDGF-BB-treated HA-VSMCs were largely attenuated by the silencing of miR-513a-5p. MiR-513a-5p bound to the 3′ untranslated region (3′UTR) of TXNIP, and miR-513a-5p overexpression-mediated effects were counteracted by the transfection of pcDNA-TXNIP in PDGF-BB-induced HA-VSMCs. TXNIP was modulated by circ_0004872/miR-513a-5p signaling cascade in HA-VSMCs. Circ_0004872 accelerated PDGF-BB-induced proliferation, migration and dedifferentiation in HA-VSMCs through enhancing TXNIP level via sponging miR-513a-5p.

Molecular Aspects of Regeneration Mechanisms in Holothurians.

Holothurians, or sea cucumbers, belong to the phylum Echinodermata. They show good regenerative abilities. The present review provides an analysis of available data on the molecular aspects of regeneration mechanisms in holothurians. The genes and signaling pathways activated during the asexual reproduction and the formation of the anterior and posterior parts of the body, as well as the molecular mechanisms that provide regeneration of the nervous and digestive systems, are considered here. Damage causes a strong stress response, the signs of which are recorded even at late regeneration stages. In holothurian tissues, the concentrations of reactive oxygen species and antioxidant enzymes increase. Furthermore, the cellular and humoral components of the immune system are activated. Extracellular matrix remodeling and Wnt signaling play a major role in the regeneration in holothurians. All available morphological and molecular data show that the dedifferentiation of specialized cells in the remnant of the organ and the epithelial morphogenesis constitute the basis of regeneration in holothurians. However, depending on the type of damage, the mechanisms of regeneration may differ significantly in the spatial organization of regeneration process, the involvement of different cell types, and the depth of reprogramming of their genome (dedifferentiation or transdifferentiation).

Trem-2 Promotes Emergence of Restorative Macrophages and Endothelial Cells During Recovery From Hepatic Tissue Damage.

Macrophages are pivotal in mounting liver inflammatory and tissue repair responses upon hepatic injury, showing remarkable functional plasticity. The molecular mechanisms determining macrophage transition from inflammatory to restorative phenotypes in the damaged liver remain unclear. Using mouse models of acute (APAP) and chronic (CCl4) drug-induced hepatotoxic injury we show that the immune receptor Trem-2 controls phenotypic shifts of liver macrophages and impacts endothelial cell differentiation during tissue recovery. Trem-2 gene ablation led to a delayed re-population of Kupffer cells correlating with deterred resolution of hepatic damage following acute and chronic injury. During tissue recovery, we found that macrophages transitioning to Kupffer cells expressed high levels of Trem-2. Acquisition of the transition phenotype was associated with a unique transcriptomic profile denoting strong responsiveness to oxidative stress and downmodulation of the pro-inflammatory phenotype, which was not observed in absence of Trem-2. During tissue recovery, lack of Trem-2 favored accumulation of a liver-damage associated endothelial cell population (LDECs), whose transcriptional program was compatible with endothelial de-differentiation. Accordingly, LDECs precursor potential is supported by the downregulation of surface endothelial cell markers and by striking in vitro morphological changes towards typical endothelial cells. In conclusion, we found that the dynamics of liver macrophages in response to liver injury are critically controlled by Trem-2 and this regulation is interlinked with the de-differentiation of endothelial cells and heightened liver pathology. We propose that Trem-2 promotes the transition from pro-inflammatory to tissue repair phase by driving the acquisition of restorative properties in phagocytic macrophages.

A 3D Bioprinted Material That Recapitulates the Perivascular Bone Marrow Structure for Sustained Hematopoietic and Cancer Models.

Translational medicine requires facile experimental systems to replicate the dynamic biological systems of diseases. Drug approval continues to lag, partly due to incongruencies in the research pipeline that traditionally involve 2D models, which could be improved with 3D models. The bone marrow (BM) poses challenges to harvest as an intact organ, making it difficult to study disease processes such as breast cancer (BC) survival in BM, and to effective evaluation of drug response in BM. Furthermore, it is a challenge to develop 3D BM structures due to its weak physical properties, and complex hierarchical structure and cellular landscape. To address this, we leveraged 3D bioprinting to create a BM structure with varied methylcellulose (M): alginate (A) ratios. We selected hydrogels containing 4% (w/v) M and 2% (w/v) A, which recapitulates rheological and ultrastructural features of the BM while maintaining stability in culture. This hydrogel sustained the culture of two key primary BM microenvironmental cells found at the perivascular region, mesenchymal stem cells and endothelial cells. More importantly, the scaffold showed evidence of cell autonomous dedifferentiation of BC cells to cancer stem cell properties. This scaffold could be the platform to create BM models for various diseases and also for drug screening.

Formalin-killed Propionibacterium acnes activates the aryl hydrocarbon receptor and modifies differentiation of SZ95 sebocytes in vitro

Acne vulgaris is a common pilosebaceous disease associated with Propionibacterium acnes (P. acnes). Resolution of comedones may occur in association with shrunken sebaceous glands (SGs) containing de-differentiated cells, however the role of P. acnes is unclear.To investigate the effects of P. acnes on aryl hydrocarbon receptor (AhR) activation, lipogenesis and differentiation in cultured immortalized human SZ95 sebocytes.Cultured sebocytes were incubated with formalin-killed (f-) P. acnes (f-P. acnes) at different ratios of multiplicity of infection. The mRNA levels of the AhR downstream cytochrome P450 (CYP) genes were measured by quantitative RT-PCR, nuclear translocation of AhR by western blot and immunofluorescence, lipogenesis and keratinization by gene set enrichment analysis (GSEA), lipid related analysis by Oil red O staining and Nile red staining, and sebaceous differentiation-related gene expression by western blot.f-P. acnes upregulated CYPs mRNA levels and induced translocation of AhR protein from the cytoplasm into the nucleus. GSEA revealed downregulation of lipogenesis and upregulation of keratinization. f-P. acnes inhibited linoleic acid-induced neutral lipid synthesis and expression of sebocyte markers, keratin 7 and mucin1/EMA, but increased expression of keratinocyte markers, keratin 10 and involucrin, which were abolished by AhR gene silencing. Inhibition of lipogenesis-related genes, such as sterol response element-binding protein, was also observed.f-P. acnes inhibits lipogenesis and induces terminal differentiation of sebocytes, into keratinocyte-like cells, via activation of the AhR pathway in vitro, suggesting that follicular P. acnes is not only acnegenic but also promotes acne remission through feedback regulation of sebum production.

Fatty Acids and a High-Fat Diet Induce Epithelial-Mesenchymal Transition by Activating TGFβ and β-Catenin in Liver Cells.

Nonalcoholic fatty liver disease is defined as the accumulation of excessive fat in the liver in the absence of excessive alcohol consumption or any secondary cause. Although the disease generally remains asymptomatic, chronic liver inflammation leads to fibrosis, liver cirrhosis, and even to the development of hepatocellular carcinoma (HCC). Fibrosis results from epithelial–mesenchymal transition (EMT), which leads to dedifferentiation of epithelial cells into cells with a mesenchymal-like phenotype. During EMT, epithelial cells with high expression of E-cadherin, influenced by growth factors, cytokines, and inflammatory processes, undergo morphological changes via enhanced expression of, e.g., vimentin, fibronectin, and N-cadherin. An inducer of EMT and, consequently, of fibrosis development is transforming growth factor beta (TGFβ), a pleiotropic cytokine associated with the progression of hepatocarcinogenesis. However, the understanding of the molecular events that direct the development of steatosis into steatohepatitis and liver fibrosis remains incomplete. Our study revealed that both prolonged exposure of hepatocarcinoma cells to fatty acids in vitro and high-fat diet in mice (20 weeks) result in inflammation. Prolonged treatment with fatty acids increased the levels of TGFβ, MMP9, and β-catenin, important EMT inducers. Moreover, the livers of mice fed a high-fat diet exhibited features of liver fibrosis with increased TGFβ and IL-1 levels. Increased expression of IL-1 correlated with a decrease in monocyte chemoattractant protein-induced protein 1 (MCPIP1), a negative regulator of the inflammatory response that regulates the stability of proinflammatory transcripts encoding IL-1. Our study showed that a high-fat diet induced EMT by increasing the levels of EMT-activating transcription factors, including Zeb1, Zeb2, and Snail and changed the protein profile to a profile characteristic of the mesenchymal phenotype.

Mesenchymal Stem Cell-Secreted Extracellular Vesicles Instruct Stepwise Dedifferentiation of Breast Cancer Cells into Dormancy at the Bone Marrow Perivascular Region.

In the bone marrow (BM), breast cancer cells (BCC) can survive in dormancy for decades as cancer stem cells (CSC), resurging as tertiary metastasis. The endosteal region where BCCs exist as CSCs poses a challenge to target them, mostly due to the coexistence of endogenous hematopoietic stem cells. This study addresses the early period of dormancy when BCCs enter BM at the perivascular region to begin the transition into CSCs, which we propose as the final step in dormancy. A two-step process comprises the Wnt-β-catenin pathway mediating BCC dedifferentiation into CSCs at the BM perivascular niche. At this site, BCCs responded to two types of mesenchymal stem cell (MSC)-released extracellular vesicles (EV) that may include exosomes. Early released EVs began the transition into cycling quiescence, DNA repair, and reorganization into distinct BCC subsets. After contact with breast cancer, the content of EVs changed (primed) to complete dedifferentiation into a more homogeneous population with CSC properties. BCC progenitors (Oct4alo), which are distant from CSCs in a hierarchical stratification, were sensitive to MSC EVs. Despite CSC function, Oct4alo BCCs expressed multipotent pathways similar to CSCs. Oct4alo BCCs dedifferentiated and colocalized with MSCs (murine and human BM) in vivo. Overall, these findings elucidate a mechanism of early dormancy at the BM perivascular region and provide evidence of epigenome reorganization as a potential new therapy for breast cancer. SIGNIFICANCE: These findings describe how the initial process of dormancy and dedifferentiation of breast cancer cells at the bone marrow perivascular niche requires mesenchymal stem cell-derived exosomes, indicating a potential target for therapeutic intervention.

Pancreatic β cell regeneration induced by clinical and preclinical agents.

Diabetes, one of the most common chronic diseases in the modern world, has pancreatic β cell deficiency as a major part of its pathophysiological mechanism. Pancreatic regeneration is a potential therapeutic strategy for the recovery of β cell loss. However, endocrine islets have limited regenerative capacity, especially in adult humans. Almost all hypoglycemic drugs can protect β cells by inhibiting β cell apoptosis and dedifferentiation via correction of hyperglycemia and amelioration of the consequent inflammation and oxidative stress. Several agents, including glucagon-like peptide-1 and γ-aminobutyric acid, have been shown to promote β cell proliferation, which is considered the main source of the regenerated β cells in adult rodents, but with less clarity in humans. Pancreatic progenitor cells might exist and be activated under particular circumstances. Artemisinins and γ-aminobutyric acid can induce α-to-β cell conversion, although some disputes exist. Intestinal endocrine progenitors can transdeterminate into insulin-producing cells in the gut after FoxO1 deletion, and pharmacological research into FoxO1 inhibition is ongoing. Other cells, including pancreatic acinar cells, can transdifferentiate into β cells, and clinical and preclinical strategies are currently underway. In this review, we summarize the clinical and preclinical agents used in different approaches for β cell regeneration and make some suggestions regarding future perspectives for clinical application.