Matrix stiffness regulates neovascular homeostasis through autophagy in nude mice.

One of the major obstacles to the effective application of vascularized fruit is an insufficient understanding of the relationship between the microenvironment and neovascular homeostasis. The role of extracellular matrix stiffness in regulating the structural and functional stability of neovascularization has not yet been elucidated. This study explored the effects of matrix stiffness on neovascular homeostasis in nude mice. Dextran hydrogels with three different stiffnesses were separately combined with mouse bone marrow-derived endothelial progenitor cells (EPCs) and subcutaneously implanted into the backs of nude mice. After 14 days, neovascular homeostasis indicators in the different groups were measured. Cell autophagy levels were evaluated, and inhibitor assays were performed to explore the underlying mechanism. New blood vessels were generated in the three stiffnesses of the EPC-loaded dextran hydrogels 14 days after implantation. The newly formed vessels tended to have better structural stability in softer hydrogels. Endothelial function markers, such as endothelial nitric oxide synthase and E-selectin, were downregulated as the matrix stiffness increased. Furthermore, we found that cell autophagy levels decreased in stiffer matrices, and autophagy inhibition attenuated neovascular homeostasis. A soft matrix is conducive to maintaining neovascular homeostasis through autophagy in nude mice.

Substrate stiffness regulates arterial-venous differentiation of endothelial progenitor cells via the Ras/Mek pathway.

Cells sense and respond to the biophysical properties of their surrounding environment by interacting with the extracellular matrix (ECM). Therefore, the optimization of these cell-matrix interactions is critical in tissue engineering. The vascular system is adapted to specific functions in diverse tissues and organs. Appropriate arterial-venous differentiation is vital for the establishment of functional vasculature in angiogenesis. Here, we have developed a polydimethylsiloxane (PDMS)-based substrate capable of simulating the physiologically relevant stiffness of both venous (7kPa) and arterial (128kPa) tissues. This substrate was utilized to investigate the effects of changes in substrate stiffness on the differentiation of endothelial progenitor cells (EPCs). As EPCs derived from mouse bone marrow were cultured on substrates of increasing stiffness, the mRNA and protein levels of the specific arterial endothelial cell marker ephrinB2 were found to increase, while the expression of the venous marker EphB4 decreased. Further experiments were performed to identify the mechanotransduction pathway involved in this process. The results indicated that substrate stiffness regulates the arterial and venous differentiation of EPCs via the Ras/Mek pathway. This work shows that modification of substrate stiffness may represent a method for regulating arterial-venous differentiation for the fulfilment of diverse functions of the vasculature.

Substrate stiffness regulated migration and angiogenesis potential of A549 cells and HUVECs.

Tumor tissue tends to stiffen during solid tumor progression. Substrate stiffness is known to alter cell behaviors, such as proliferation and migration, during which angiogenesis is requisite. Mono- and co-culture systems of lung cancer cell line A549 and human umbilical vein endothelial cells (HUVECs), on polydimethylsiloxane substrates (PDMS) with varying stiffness, were used for investigating the effects of substrate stiffness on the migration and angiogenesis of lung cancer. The expressions of matrix metalloproteinases (MMPs) and angiogenesis-related growth factors were up-regulated with the increase of substrate stiffness, whereas that of tissue inhibitor of matrix metalloproteinase (TIMPs) were down-regulated with increasing substrate stiffness. Our data not only suggested that stiff substrate may promote the migration and angiogenesis capacities of lung cancer, but also suggested that therapeutically targeting lung tumor stiffness or response of ECs to lung tumor stiffness may help reduce migration and angiogenesis of lung tumor.

Regulating osteogenesis and adipogenesis in adipose-derived stem cells by controlling underlying substrate stiffness.

Cells reside in a complex microenvironment (niche) in which the biochemical and biophysical properties of the extracellular matrix profoundly affect cell behavior. Extracellular stiffness, one important bio-mechanical characteristic of the cell niche, is important in regulating cell proliferation, migration, and lineage specification. However, the mechanism by which mechanical signals guide osteogenic and adipogenic commitment of stem cells remains difficult to dissect. To explore this question, we generated a range of polydimethylsiloxane-based matrices with differing degrees of stiffness that mimicked the stiffness seen in natural tissues and examined adipose stem cell morphology, spreading, vinculin expression, and differentiation along the osteogenic and adipogenic pathways. Rigid matrices allowed broader cell spreading, faster growth rate and stronger expression of vinculin in adipose-derived stem cells. In the presence of inductive culture media, stiffness-dependent osteogenesis and adipogenesis of the adipose stem cells indicated that there was a combinatorial effect of biophysical and biochemical cues; no such lineage specification was observed in normal media. Osteogenic differentiation behavior showed a correlation with matrix rigidity, as well as with elevated expression of RhoA, ROCK-1/-2, and related proteins in the Wnt/ß-catenin pathway. The result provides a comprehensive understanding of how stem cells respond to the surrounding microenvironment and points to the fact that matrix stiffness is a critical element in biomaterial design and this will be an important advance in stem cell-based tissue engineering.

Notch Signaling Pathway Regulates Angiogenesis via Endothelial Cell in 3D Co-Culture Model.

This study aimed to investigate the role of Notch signaling pathway for angiogenesis in a three-dimensional (3D) collagen gel model with co-culture of adipose-derived stromal cells (ASCs) and endothelial cells (ECs). A 3D collagen gel model was established in vitro by implanting both ASCs from green fluorescent protein-labeled mouse and ECs from red fluorescent protein-labeled mouse, and the phenomena of angiogenesis with Notch signaling inducer Jagged1, inhibitor DAPT and PBS, respectively were observed by confocal laser scanning microscopy. Semi-quantitative PCR and immunofluorescent staining were conducted to detect expressions of angiogenesis-related genes and proteins. Angiogenesis in the co-culture gels was promoted by Jagged1 treatment while attenuated by DAPT treatment, compared to control group. In co-culture system of ASCs and ECs, the gene expressions of VEGFA, VEGFB, Notch1, Notch2, Hes1, Hey1, VEGFR1,and the protein expression of VEGFA, VEGFB, Notch1, Hes1, Hey1 were increased by Jagged1 treatment and decreased by DAPT treatment in ECs. And the result of VEGFR3 was the opposite. However, the same results did not appear completely in ASCs. These results revealed the VEGFA/B-Notch1/2-Hes1/Hey1- VEGFR1/3 signal axis played an important role in angiogenesis when ASCs and ECs were co-cultured in a 3D collagen gel model. J. Cell. Physiol. 232: 1548-1558, 2017. © 2016 Wiley Periodicals, Inc.

The peripheral immune cell counts and mouth ulcers: A two-sample Mendelian randomization study.

Objective: This study explored the causal association of peripheral immune cell counts with mouth ulcers (MUs) by two-sample Mendelian Randomization. Design: The counts of 12 circulating immune cell types (leukocytes, lymphocytes, monocytes, eosinophils, neutrophils, basophils, CD4+ cells, CD8+ cells, unswitched memory B cells, NK cells, B cells and a derived ratio (CD4+/CD8+)) were determined as the exposure. MUs were the outcome. The analysis was conducted mostly using the inverse-variance weighted (IVW) approach. MR Egger, weighted median, weighted mode and simple mode were used to detect the horizontal pleiotropy. Results: The IVW results for leukocytes and lymphocyte counts were OR = 0.93, 95 % CI = 0.88-0.98, p = 0.0115 and OR = 0.91, 95 % CI: 0.84-0.98, p = 0.0150, respectively. The Wald ratio result for CD4+ cell and CD8+ cell counts were OR = 0.70, 95 % CI: 0.65-0.75, p = 1.05 × 10-20 and OR = 1.25, 95 % CI: 1.19-1.31, p = 9.99 × 10-21, respectively. Conclusions: This study supports a causal effect of peripheral immune cell counts on MUs. Higher leukocyte, lymphocyte and CD4+ cell counts can protect against MUs, but higher CD8+ cell counts enhance the risk of MUs. This finding confirms host immune factors play a crucial role in the aetiology of MUs.

ATHENA: an independently validated autophagy-related epigenetic prognostic prediction model of head and neck squamous cell carcinoma.

The majority of these existing prognostic models of head and neck squamous cell carcinoma (HNSCC) have unsatisfactory prediction accuracy since they solely utilize demographic and clinical information. Leveraged by autophagy-related epigenetic biomarkers, we aim to develop a better prognostic prediction model of HNSCC incorporating CpG probes with either main effects or gene-gene interactions. Based on DNA methylation data from three independent cohorts, we applied a 3-D analysis strategy to develop An independently validated auTophagy-related epigenetic prognostic prediction model of HEad and Neck squamous cell carcinomA (ATHENA). Compared to prediction models with only demographic and clinical information, ATHENA has substantially improved discriminative ability, prediction accuracy and more clinical net benefits, and shows robustness in different subpopulations, as well as external populations. Besides, epigenetic score of ATHENA is significantly associated with tumor immune microenvironment, tumor-infiltrating immune cell abundances, immune checkpoints, somatic mutation and immunity-related drugs. Taken together these results, ATHENA has the demonstrated feasibility and utility of predicting HNSCC survival ( http://bigdata.njmu.edu.cn/ATHENA/ ).

Matrix stiffness regulates arteriovenous differentiation of endothelial progenitor cells during vasculogenesis in nude mice.

OBJECTIVES: The aim of the study was to investigate the effect of matrix stiffness on arteriovenous differentiation of endothelial progenitor cells (EPCs) during vasculogenesis in nude mice. MATERIALS AND METHODS: Dextran hydrogels of differing stiffnesses were first prepared by controlling the crosslinking reaction to generate different thioether bonds. Hydrogels with stiffnesses matching those of the arterial extracellular matrix and venous extracellular matrix were separately combined with mouse bone marrow-derived EPCs and subcutaneously implanted on either side of the backs of nude mice. After 14 days, artery-specific marker Efnb2 and vein-specific marker Ephb4 in the neovasculature were detected to determine the effect of matrix stiffness on the arteriovenous differentiation of EPCs in vivo. RESULTS: Fourteen days after the implantation of the EPC-loaded dextran hydrogels, new blood vessels were observed in both types of hydrogels. We further verified that matrix stiffness regulated the arteriovenous differentiation of EPCs during vasculogenesis via the Ras/Mek pathway. CONCLUSIONS: Matrix stiffness regulates the arteriovenous differentiation of EPCs during vasculogenesis in nude mice through the Ras/Mek pathway.

Substrate stiffness regulated migration and invasion ability of adenoid cystic carcinoma cells via RhoA/ROCK pathway.

OBJECTIVES: Human salivary adenoid cystic carcinoma (SACC) is one of the most common malignant tumours of the salivary gland and has strong migratory and invasive ability, which often lead to poor prognosis and lower survival rate. Tumour tissue tends to stiffen during solid tumour progression. This study aimed to investigate the influence of various substrate stiffness on the migration and invasion of SACC. METHODS: Salivary adenoid cystic carcinoma cell line ACC2 cells were cultured on polydimethylsiloxane substrates (PDMS) with varying stiffness for investigating the effects of substrate stiffness on the activities of MMPs and TIMPs. The underlying mechanism was also explored. RESULTS: When ACC2 cells were cultured on various stiffness of PDMS, the expressions of matrix metalloproteinases 2 (MMP2), MMP9, MMP14, RhoA, Rac1, Rho-associated protein kinase 1 (ROCK1) and ROCK2 were up-regulated with increasing substrate stiffness, whereas that of tissue inhibitor of matrix metalloproteinase 1 (TIMP1), TIMP2 and TIMP4 were down-regulated with increasing substrate stiffness. CONCLUSIONS: Our results showed that substrate stiffness regulated the activities of MMPs and TIMPs and then modulate migratory and invasive ability of ACC2 cells via RhoA/ROCK pathway. This work indicate that matrix stiffness played an important role in progression of SACC, which not only can help understand the strong invasive ability of SACC, but also suggested that therapeutically targeting matrix stiffness may help reduce migration and invasion of SACC and improve effective therapies.

Tetrahedral DNA Nanostructure Promotes Endothelial Cell Proliferation, Migration, and Angiogenesis via Notch Signaling Pathway.

The problem of tissue vascularization is one of the obstacles that currently restricts the application of tissue engineering products to the clinic. Achieving tissue vascularization and providing adequate nutrients for tissues are an urgent problem to build complex and effective tissue-engineered tissues and organs. Therefore, the aim of this study was to investigate the effect of tetrahedral DNA nanostructures (TDNs), a novel and biocompatible nanomaterial, on angiogenesis. The results showed that TDNs can enter into endothelial cells (ECs) and promote EC proliferation, migration, tube formation, and expressions of angiogenic growth factors at the concentration of 250 nmol L-1, which was accompanied by activation of the Notch signaling pathway. These results provided a theoretical basis for the further understanding and potential use of TDNs in tissue engineering vascularization.

Stiffness regulates the proliferation and osteogenic/odontogenic differentiation of human dental pulp stem cells via the WNT signalling pathway.

OBJECTIVES: Researches showed that stiffness of the extracellular matrix can affect the differentiation of many stem cells. Dental pulp stem cells (DPSCs) are a promising type of adult stem cell. However, we know little about whether and how the behaviour of DPSCs is influenced by stiffness. MATERIALS AND METHODS: We carried out a study that cultured DPSCs on tunable elasticity polydimethylsiloxane substrates to investigate the influence on morphology, proliferation, osteogenic/odontogenic differentiation and its possible mechanism. RESULTS: Soft substrates changed the cell morphology and inhibited the proliferation of DPSCs. Expression of markers related to osteogenic/odontogenic differentiation was significantly increased as the substrate stiffness increased, including ALP (alkaline phosphatase), OCN (osteocalcin), OPN (osteopontin), RUNX-2 (runt-related transcription factor-2), BMP-2 (bone morphogenetic protein-2), DSPP (dentin sialophosphoprotein) and DMP-1 (dentin matrix protein-1). Mechanical properties promote the function of DPSCs related to the Wnt signalling pathway. CONCLUSIONS: Our results showed that mechanical factors can regulate the proliferation and differentiation of DPSCs via the WNT signalling pathway. This provides theoretical basis to optimize dental or bone tissue regeneration through increasing stiffness of extracelluar matrix.

Effect of matrix stiffness on osteoblast functionalization.

OBJECTIVES: Stiffness of bone tissue differs response to its physiological or pathological status, such as osteoporosis or osteosclerosis. Consequently, the function of cells residing in bone tissue including osteoblasts (OBs), osteoclasts and osteocytes will be affected. However, to the best of our knowledge, the detailed mechanism of how extracellular matrix stiffness affects OB function remains unclear. MATERIALS AND METHODS: We conducted a study that exposed rat primary OBs to polydimethylsiloxane substrates with varied stiffness to investigate the alterations of cell morphology, osteoblastic differentiation and its potential mechanism in mechanotransduction. RESULTS: Distinctive differences of cell shapes and vinculin expression in rat osteoblasts were detected on different PDMS substrates. As representatives for OB function, expression of alkaline phosphatase, Runx2 and osteocalcin were identified and showed a decrease trend as substrates become soft, which is associated with the Rho/ROCK signalling pathway. CONCLUSIONS: Our results indicated substrate elasticity as a potent regulator in OBs functionalization, which may pave a way for further understanding of bone diseases as well as a potential therapeutic alternative in tissue regeneration.

IGF-1 promotes angiogenesis in endothelial cells/adipose-derived stem cells co-culture system with activation of PI3K/Akt signal pathway.

OBJECTIVES: The aim of this study was to investigate the role of insulin-like growth factor-1 (IGF-1) and crosstalk between endothelial cells (ECs) and adipose-derived stem cells (ASCs) in the process of angiogenesis. METHODS: A three-dimensional collagen gel used to culture mouse ASCs and mouse ECs in vitro was established. The effects of angiogenesis after exposure to IGF-1 were observed by confocal laser scanning microscopy. Western blotting and qPCR were performed to elucidate the underlying mechanisms. RESULTS: IGF-1 treatment promoted the formation of vessel-like structures and the recruitment of ASCs in the three-dimensional collagen gel. The angiogenic genes and proteins in ECs were up-regulated by IGF-1 and in co-culture. Similar changes in the genes and in the proteins were detected in ASCs after exposure to IGF-1 and co-culture. p-Akt expression levels were high in ECs and ASCs after exposure to IGF-1 and co-culture. CONCLUSIONS: IGF-1 and co-culture between cells facilitate the process of angiogenesis via the PI3-kinase/Akt signalling pathway. In ECs, IGF-1 stimulates the expression of angiogenesis-related growth factors with the activation of the PI3-kinase/Akt signalling pathway. Co-cultured ECs exposed to excess VEGF-A and other angiogenesis-related growth factors para-secreted from ASCs exhibit high expression of angiogenesis-related genes and proteins. In ASCs, IGF-1 induces the recruitment and function of ASCs by up-regulating the expression of PDGFB, MMPs and α-SMA. Crosstalk with ECs further facilitates changes in ASCs.

Curved microstructures promote osteogenesis of mesenchymal stem cells via the RhoA/ROCK pathway.

OBJECTIVES: Cells in the osteon reside in a curved space, accordingly, the curvature of the microenvironment is an important geometric feature in bone formation. However, it is not clear how curved microstructures affect cellular behaviour in bone tissue. MATERIALS AND METHODS: Rat primary bone marrow mesenchymal stem cells (BMSCs) on wavy microgrooves were exposed to PDMS substrates with various curvatures to investigate alterations in cellular morphology and osteogenic differentiation. Additionally, the expression levels of RhoA and its effectors were examined by immunofluorescence and quantitative PCR to determine the mechanisms of curvature-dependent osteogenic differentiation. RESULTS: Wavy microgrooves caused dramatic nuclear distortion and cytoskeletal remodelling. We detected a noticeable increase in the expression of osteogenic-related genes in BMSCs in wavy microgroove groups, and the maximum expression was observed in the high curvature group. Moreover, immunofluorescent staining and quantitative RT-PCR results for RhoA and its effectors showed that the RhoA/ROCK signalling pathway is associated with curvature-dependent osteogenic differentiation. CONCLUSIONS: Our results illustrated that curved microstructures could promote BMSC differentiation to the osteogenic lineage, and the osteogenic effects of higher curvature are more obvious. Wavy microstructures could also influence the RhoA/ROCK pathway. Accordingly, curved microstructures may be useful in bone tissue engineering.

The JAK/STAT3 signalling pathway regulated angiogenesis in an endothelial cell/adipose-derived stromal cell co-culture, 3D gel model.

OBJECTIVES: The aim of the study was to investigate the role of the JAK/STAT3 signalling pathway in angiogenesis. MATERIALS AND METHODS: The model established in vitro, involved a 3D collagen gel being implanted with endothelial cells (ECs) from red fluorescent protein-labelled mice, and adipose-derived stromal cells (ASCs) from green fluorescent protein-labelled mice. Phenomena of angiogenesis, after treatment by the inhibitor and the activator of JAK/STAT3 pathway respectively, were observed using confocal laser scanning microscopy. Transwell co-culture of ECs and ASCs was used to elucidate mechanisms. RESULTS: Stattic, inhibitor of JAK/STAT3 pathway, attenuated angiogenesis in the model. In contrast, angiogenesis was promoted after treatment of Olanzapine, an activator. We found that protein levels of VEGFA and cyclin D1 were regulated by the JAK/STAT3 pathway, and flow cytometry further confirmed variations in cell cycle parameters of ECs and ASCs. Genes VEGFA/B, VEGFR2, MMP-2, MMP-9, IGF-1 and b-FGF were down-regulated by Stattic in ECs, while Olanzapine significantly up-regulated mRNA levels of these genes. As for ASCs, genes VEGFA, MMP-2, MMP-9, IGF-1 and b-FGF were modulated by the JAK/STAT3 pathway. CONCLUSIONS: Angiogenesis in the 3D collagen gel was regulated by the JAK/STAT3 pathway which involved changes in vessel length, vessel diameter and sprout number. The underlying mechanism was that the JAK/STAT3 signalling pathway regulated angiogenesis by modulation of numbers of angiogenesis-related growth factors and by direct regulation of cell cycle.

Electrospun Poly(3-hydroxybutyrate-co-4-hydroxybutyrate)/Graphene Oxide Scaffold: Enhanced Properties and Promoted in Vivo Bone Repair in Rats.

Bone tissue engineering emerges as an advantageous technique to achieve tissue regeneration. Its scaffolds must present excellent biomechanical properties, where bare polymers poorly perform. Development of new biomaterials with high osteogenic capacity is urgently pursued. In this study, an electrospun poly(3-hydroxybutyrate-co-4-hydroxybutyrate)/graphene oxide (P34HB/GO) nanofibrous scaffold is successfully fabricated and characterized. The effects of GO amount on scaffold morphology, biomechanical properties, and cellular behaviors are investigated. GO reduces the fiber diameter and enhances porosity, hydrophilicity, mechanical properties, cellular performance, and osteogenic differentiation of scaffolds. P34HB/GO triumphs over P34HB in in vivo bone regeneration in critical-sized calvarial defect of rats. We believe that this study is the first to evaluate the capability of in vivo bone repair of electrospun P34HB/GO scaffold. With facile fabrication process, favorable porous structures, enhanced biomechanical properties, and fast osteogenic capability, P34HB/GO scaffold holds practical potentials for bone tissue engineering application.