Matrix stiffness changes affect astrocyte phenotype in an in vitro injury model

Simple SummaryYAP1 is an oncogene that can be activated by matrix stiffness, as it can act as a mechanotransducer. So far, only in vitro studies regarding YAP1 activation and matrix stiffness are present. We confirmed the activation of YAP1 in breast cancer using human breast cancer tissue and immunohistochemistry. Tumor stiffness was quantified by shear-wave elastography. Nuclear localization of YAP1 showed correlation with tumor stiffness in hormone-receptor positive (HR+) breast cancer. Also, tumors with non-collagen-type stroma showed an association between YAP1 expression and tumor stiffness. YAP1 expression, along with tumor stiffness, may serve as a prognostic candidate in HR+ breast cancer.(1) Background: Yes-associated protein 1 (YAP1) is an oncogene activated under the dysregulated Hippo pathway. YAP1 is also a mechanotransducer that is activated by matrix stiffness. So far, there are no in vivo studies on YAP1 expression related to stiffness. We aimed to investigate the association between YAP1 activation and tumor stiffness in human breast cancer samples, using immunohistochemistry and shear-wave elastography (SWE). (2) Methods: We included 488 patients with treatment-naïve breast cancer. Tumor stiffness was measured and the mean, maximal, and minimal elasticity values and elasticity ratios were recorded. Nuclear YAP1 expression was evaluated by immunohistochemistry and tumor-infiltrating lymphocytes (TILs); tumor-stroma ratio (TSR) and stroma type of tumors were also evaluated. (3) Results: Tumor stiffness was higher in tumors with YAP1 positivity, low TILs, and high TSR and was correlated with nuclear YAP1 expression; this correlation was observed in hormone receptor positive (HR+) tumors, as well as in tumors with non-collagen-type stroma. (4) Conclusions: We confirmed the correlation between nuclear YAP1 expression and tumor stiffness, and nuclear YAP1 expression was deemed a prognostic candidate in HR+ tumors combined with SWE-measured tumor stiffness.

Interplay of cell adhesion matrix stiffness and cell type for non-viral gene delivery

Reactive astrocytes and therapeutic potential in focal ischemic stroke

To detect the expression of telomerase in glial scar and its correlation with glial scar. There were 120 Sprague Dawley rats were randomly divided into non-interference group of telomerase, interference group of telomerase and control group. Non-interference group and interference group were for spinal cord injury, which adopted Allen's Weight Dropping to make molding; control group was for sham operation to open the vertebral plate and expose spinal marrow, in which spinal cord injury would not be caused. The expression of telomerase and glial fibrillary acidic profein (GFAP) was detected by PCR-ELISA and Western blot, and the formation of glial scar was observed by immunofluorescence on the 1st, 3rd, 5th, 7th, 14th, 28th, 42 th and 56th day after the spinal injury, and analyzed its relativity. The expression of telomerase in non-interference group was (0.180 ± 0.004 - 1.217 ± 0.072), which was significantly higher than those in interference group (0.028 ± 0.007 - 0.092 ± 0.004, χ(2) = 28.753 - 37.518, P < 0.05) and control group (0.072 ± 0.007 - 0.075 ± 0.004, χ(2) = 18.618 - 41.093, P < 0.05) at all the time, with statistical significance. The expression of GFAP in non-interference group was (1.98 ± 0.15 - 19.40 ± 0.55) which was significantly higher than those in interference group (1.10 ± 0.13 - 16.64 ± 1.02, χ(2) = 14.538 - 37.366, P < 0.05) and control group (0.44 ± 0.05 - 0.48 ± 0.04, χ(2) = 16.733 - 34.041, P < 0.05) at all the time, with statistical significance. The expression of GFAP showed a linear correlation with that of telomerase in non-interference group, and with statistical differences (r = 0.755, P < 0.01). The expression of telomerase in interference group and control group were always negative. Glial scar observed by immunofluorescence in non-interference group was heavier than that in interference group, and control group showed no formation of glial scar. Telomerase shows a dynamic expression in glial scar and has positive correlational linear relationship with GFAP which shows the formation of glial scar. And the telomerase may be an important factor in promoting the formation of glial scar.

Glia scar is a pathological marker in late phase of brain ischemia disease, which constitutes a major physical biochemical barrier to impede axonal regrowth. Astrocytes are known to be critically involved in the formation of glial scar. However, their response to ischemia and their role in neuroprotection after central nervous system (CNS) injury are not completely clear. Recently, we have demonstrated for the first time that Ski was up-regulated in reactive astrocytes after spinal cord injury in vivo and in vitro, which indicates Ski may be a new molecule that control astrocytes biologic properties after CNS injury. However, its role in the process of reactive astrogliosis after cerebral ischemia and its definite mechanism still remains unknown. This study is to elucidate the role of Ski in reactive astrocytes induced by oxygen-glucose deprivation/reoxygenation (OGD/R) model in vitro. The expression of Ski was proved to be up-regulated in OGD/R model. Meanwhile, Up-regulation of Ski was accompanied with high ratio of EdU (+) cells and up-expression of related proteins including GFAP, PCNA, CDK4, and CyclinD1, which demonstrated the distinct activation and proliferation of astrocytes after stimulation by OGD/R. Astrocytes were transfected with Ski-specific siRNA to knockdown Ski expression and subsequently attenuated OGD-induced astrocyte proliferation. Our results also showed that Ski down-regulation could suppress the activity of the Ras-Raf-ERK1/2 signaling pathway. Together, knockdown of Ski can effectively inhibit the proliferation of reactive astrogliosis via suppressing the Ras-Raf-ERK1/2 pathway. These findings indicated that maybe Ski is a promising therapeutic target for cerebral ischemic injury.

During the recovery phase of ischemic stroke, one of the major barriers for the spontaneous neuronal axon regeneration is the formation of astrogliosis and glial scar, and targeting astrogliosis becomes a therapeutic strategy for ischemic stroke. However, the mechanism regulating the process of scar components after ischemia still remains poorly understood. The aim of this study was to observe the role of RIP1 kinase (RIP1K), the key regulator of necroptosis (programmed necrosis) in the brain functional recovery after ischemic stroke and in the ischemic stroke-induced astrogliosis and glial scar formation in both in vitro and in vivo glial scar models. The glial scar formation model in vitro or in vivo was established by using primary cultured astrocyte subjected to 6 hours of oxygen-glucose deprivation (OGD) following 12 hours or 24 hours reperfusion, or by 90 min of transient middle cerebral artery occlusion (tMCAO) and reperfusion in rats. Western blotting analysis and immunohistochemical assay showed that knockdown of RIP1K by lentivirally-delivered shRNAs against RIP1K (shRNA RIP1K) could decrease several protein levels of glial scar markers such as glial fibillary acidic protein (GFAP), neurocan and phosphacan both in in vitro and in vivo glial scar models. Furthermore, western blotting analysis showed that knockdown of RIP1K reduced the protein levels of VEGF-D receptor 3 in in vitro glial scar models. In addition, knockdown of RIP1K also notably reduced the shrinking volume and ameliorated the behavioral symptoms in the recovery phase of rats after tMCAO. And immunocytochemistry assay demonstrated that RIP1K knockdown promoted the neuronal axonal generation in a neuron and astrocyte co-culture system. Our data indicates that RIP1K might play an important role in the formation of glial scar after ischemic stroke via promoting the function of VEGF-D receptor 3 in astrocytes.

Endothelial mechanobiology is a key consideration in the progression of vascular dysfunction, including atherosclerosis. However mechanistic connections between the clinically associated physical stimuli, vessel stiffness and shear stress, and how they interact to modulate plaque progression remain incompletely characterized. Vessel-chip systems are excellent candidates for modeling vascular mechanobiology as they may be engineered from the ground up, guided by the mechanical parameters present in human arteries and veins, to recapitulate key features of the vasculature. Here, we report extensive validation of a vessel-chip model of endothelial yes-associated protein (YAP) mechanobiology, a protein sensitive to both matrix stiffness and shearing forces and, importantly, implicated in atherosclerotic progression. Our model captures the established endothelial mechanoresponse, with endothelial alignment, elongation, reduction of adhesion molecules, and YAP cytoplasmic retention under high laminar shear. Conversely, we observed disturbed morphology, inflammation, and nuclear partitioning under low, high, and high oscillatory shear. Examining targets of YAP transcriptional co-activation, connective tissue growth factor (CTGF) is strongly downregulated by high laminar shear, whereas it is strongly upregulated by low shear or oscillatory flow. Ankyrin repeat domain 1 (ANKRD1) is only upregulated by high oscillatory shear. Verteporfin inhibition of YAP reduced the expression of CTGF but did not affect ANKRD1. Lastly, substrate stiffness modulated the endothelial shear mechanoresponse. Under high shear, softer substrates showed the lowest nuclear localization of YAP whereas stiffer substrates increased nuclear localization. Low shear strongly increased nuclear localization of YAP across stiffnesses. Together, we have validated a model of endothelial mechanobiology and describe a clinically relevant biological connection between matrix stiffness, shear stress, and endothelial activation via YAP mechanobiology.

Reactive astrocytes play an essential role in determining the tissue response to ischaemia. Formation of a glial scar can block the neuronal outgrowth that is required for restoration of damaged tissue. Therefore, regulation of astrocyte activation is important; however, the mediator of this process has not been fully elucidated. Apoptosis signal-regulating kinase 1 (ASK1) is an early responder to oxidative stress, and plays a pivotal role in the intracellular signalling pathway of apoptosis, inflammation, and differentiation. To confirm whether ASK1 mediates astrocyte activation and leads to glial scar formation after cerebral ischaemia, we conducted in vivo and in vitro experiments. C57BL/6 mice were subjected to occlusion of the middle cerebral artery, and astrocyte cultures were exposed to oxygen-glucose deprivation. After silencing of ASK1 , astrocyte-associated genes were downregulated, as seen with the use of microarrays. The glial fibrillary acidic protein (GFAP) level was decreased, and correlated with the reduction in the ASK1 level. In astrocytes, reduction in the ASK1 level decreased the activity of the p38 pathway, and the levels of transcription factors for GFAP and GFAP transcripts after hypoxia. In the chronic phase, ASK1 depletion reduced glial scar formation and conserved neuronal structure, which may lead to better functional recovery. These data suggest that ASK1 may be an important mediator of ischaemia-induced astrocyte activation and scar formation, and could provide a potential therapeutic target for treatment after ischaemic stroke.

Objective To investigate the mechanism of matrix stiffness promoting epithelial-mesenchymal transformation (EMT) and vasculogenic mimicry (VM) of glioma cells by yes-related proteins (YAP). Methods Human glioma U87 cell lines were cultured under different matrix stiffness conditions. Matrix stiffness 0.2, 16.0 and 64.0 kPa represented normal brain, glioma and extreme stiffness, respectively. CCK-assay was used to examine the proliferation of U87 cell line.The localization of YAP cells was detected by immunofluorescence staining. The expression of YAP targeting genes including CTGF and CRY61 were detected by real-time quantitative PCR. The expression and phosphorylation expression of YAP, proliferation signal including Akt and ERK1/2 were detected by Western blot. Immunofluorescence and Western blot were used to detect the expression of EMT-related proteins including Vimentin, E-cadherin and Twist. VM formation was detected in soft and stiff substrates under 3D culture. U87 cells were transfected with shYAP lentivirus, and then the expression of EMT proteins and VM formation were examined. Results With the increase of matrix stiffness, the proliferation activity of glioma cells and phosphorylation expression of Akt and ERK1/2 were increased (all P<0.05), the expression of phosphorylated Yap was decreased (P<0.05), while the expression levels of CTGF and CRY61 were increased (both P<0.05) as the nuclear translocation of YAP was increased. The results of immunofluorescence staining and Western blot showed that with the increase of matrix stiffness, the EMT component expression levels of Vimentin and Twist were increased, the epithelial marker E-cadherin was decreased (all P<0.05). Compared with that under soft matrix 3D culture, the formation of VM tubular structure in U87 cells was increased in stiff matrix (P<0.05). Interference with Yap expression inhibited the enhancement of the proliferation of glioma cells, the expression of Vimentin and Twist, and VM formation induced by matrix stiffness (all P<0.05). Conclusions Matrix stiffness promotes the proliferation of glioma cells, EMT and VM formation through YAP. Targeted matrix stiffness and YAP can be used as new strategies for glioma treatment. Key words: Glioma; Matrix stiffness; Yes-associated protein; Epithelial-mesenchymal transition; Vasculogenic mimicry

Tendinopathy is characterised by pathological changes in tendon matrix composition, architecture, and stiffness, alterations in tendon resident cell characteristics, and fibrosis, with inflammation also emerging as an important factor in tendinopathy progression. The sequence of pathological changes in tendinopathy and the cellular effects of the deteriorating matrix are largely unknown. This study investigated the effects of substrate stiffness on tendon-derived cells (TDCs) and THP-1 macrophages using PDMS substrates representing physiological tendon stiffness (1.88 MPa), a stiff gel (3.17 MPa) and a soft gel (0.61 MPa). Human TDCs were cultured on the different gel substrates and on tissue culture plastic. Cell growth was determined by alamarBlue™ assay, cell morphology was analysed in f-actin labelled cells, and phenotypic markers were analysed by real-time PCR. We found that in comparison to TDCs growing on gels with physiological stiffness, cell growth increased on soft gels at 48 h (23%, p = 0.003). Cell morphology was similar on all three gels. SCX expression was slightly reduced on the soft gels (1.4-fold lower, p = 0.026) and COL1A1 expression increased on the stiff gels (2.2-fold, p = 0.041). Culturing THP-1 macrophages on soft gels induced increased expression of IL1B (2-fold, p = 0.018), and IL8 expression was inhibited on the stiffer gels (1.9-fold, p = 0.012). We also found that culturing TDCs on plastic increased cell growth, altered cell morphology, and inhibited the expression of SCX, SOX9, MMP3, and COL3. We conclude that TDCs and macrophages respond to changes in matrix stiffness. The magnitude of responses measured in TDCs were minor on the range of substrate stiffness tested by the gels. Changes in THP-1 macrophages suggested a more inflammatory phenotype on substrates with non-physiological stiffness. Although cell response to subtle variations in matrix stiffness was moderate, it is possible that these alterations may contribute to the onset and progression of tendinopathy.

Matrix stiffness is an essential physical microenvironment in solid cancer. However, its influence on cancer stemness still remains elusive. Colorectal cancer (CRC) cell line HCT-116 was cultured in the matrix with various stiffness. The siYAP was applied to detect the changes of stemness markers. The cancer stemness markers, Yes-associated protein (YAP), Lamin A/C and downstream protein molecules, and their activation were measured after the treatment with anti-β1-integrin and FAK inhibitors. In CRC tissue samples, collagen deposition and the expression of α-SMA and CD133 were detected. The study found that the expression level of stemness markers and Lamin A/C increased as the matrix stiffness raised and was regulated by YAP activation in CRC stem cells. Inhibition of β1-integrin and FAK activation in a high stiffness cell culture medium significantly decreased the activation of YAP, PI3K, and AKT. Collagen was highly deposited in the CRC invasive tumor front (ITF), and the expression of CD133 was higher in ITF compared with normal tissue and the tumor cells. Moreover, the expression level of α-SMA was positively correlated with CD133 expression level. Together, our results suggest that activation of YAP in CRC plays an important role in the promotion of cancer stem cell properties by extracellular matrix stiffness in CRC.

BackgroundCarcinoma associated fibroblasts (CAFs or myofibroblasts) are activated fibroblasts which participate in breast tumor growth, angiogenesis, invasion, metastasis and therapy resistance. As such, recent efforts have been directed toward understanding the factors responsible for activation of the phenotype. In this study, we have investigated how changes in the mechanical stiffness of a 3D hydrogel alter the behavior and myofibroblast-like properties of human mammary fibroblasts (HMFs).ResultsHere, we utilized microbial transglutaminase (mTG) to mechanically tune the stiffness of gelatin hydrogels and used rheology to show that increasing concentrations mTG resulted in hydrogels with greater elastic moduli (G’). Upon encapsulation of HMFs in 200 (compliant), 300 (moderate) and 1100 Pa (stiff) mTG hydrogels, it was found that the HMFs remained viable and proliferated over the 7 day culture period. Specifically, rates of proliferation were greatest for HMFs in moderate hydrogels. Regarding morphology, HMFs in compliant and moderate hydrogels exhibited a spindle-like morphology while HMFs in stiff hydrogels exhibited a rounded morphology with several large cellular protrusions. Quantification of cell morphology revealed that HMFs cultured in all mTG hydrogels overall assumed a more elongated phenotype over time in culture; however, few significant differences in morphology were observed between HMFs in each of the hydrogel conditions. To determine whether matrix stiffness upregulated expression of ECM and myofibroblast markers, western blot was performed on HMFs in compliant, moderate and stiff hydrogels. It was found that ECM and myofibroblast proteins varied in expression during both the culture period and according to matrix stiffness with no clear correlation between matrix stiffness and a myofibroblast phenotype. Finally, TGF-β levels were quantified in the conditioned media from HMFs in compliant, moderate and stiff hydrogels. TGF-β was significantly greater for HMFs encapsulated in stiff hydrogels.ConclusionsOverall, these results show that while HMFs are viable and proliferate in mTG hydrogels, increasing matrix stiffness of mTG gelatin hydrogels doesn’t support a robust myofibroblast phenotype from HMFs. These results have important implications for further understanding how modulating 3D matrix stiffness affects fibroblast morphology and activation into a myofibroblast phenotype.

Injury to the central nervous system (CNS) such as spinal cord results in multifaceted cellular responses, including the glial scars, a structural formation of reactive glia around an area of severe tissue damage. However, the functional heterogeneity within the cells in glial scars remains unclear. In this study, we found Aldh1L1 lineage cells exhibited reactive astrocyte distribution, culminating in glial scar formation via proliferation and differentiation after SCI. Interestingly, the Aldh1L1+ glial scars were distinct from the GFAP+ glial scars, encircled an area closer to the injury center and harbored scattered Aldh1L1 lineage cells within the central zone. At day 7 after SCI, Aldh1L1 lineage cells reached peak activation; some of them exhibited an injury responsive intermediate state with neural stem cell-like properties, and a significant portion of the Aldh1L1+ glial scar arose from the differentiation and migration of central canal ependymal cells, not derived from invading meningeal cell lineages or pial astroglia. Some of the Aldh1L1+ cells in glial scar exhibited expression of oligodendrocyte progenitor cell markers. Mechanistically, YAP was highly expressed and activated in Aldh1L1+ cells after SCI, and YAP deletion in Aldh1L1+ cells inhibited glial scar formation and differentiation into astrocytes from Aldh1L1+ ependymal cells, exacerbated neuronal loss, and inhibited the functional recovery of mice after SCI. These results suggest that Aldh1L1 lineage cells contribute to the functional heterogeneity within the cells in glial scars after SCI and uncover the pivotal role of YAP signaling in the formation of glial scars derived from Aldh1L1 lineage cells.

Astrocytes are specialized and most numerous glial cell type in the central nervous system and play important roles in physiology. Astrocytes are also critically involved in many neural disorders including focal ischemic stroke, a leading cause of brain injury and human death. One of the prominent pathological features of focal ischemic stroke is reactive astrogliosis and glial scar formation associated with morphological changes and proliferation. This review paper discusses the recent advances in spatial and temporal dynamics of morphology and proliferation of reactive astrocytes after ischemic stroke based on results from experimental animal studies. As reactive astrocytes exhibit stem cell-like properties, knowledge of dynamics of reactive astrocytes and glial scar formation will provide important insights for astrocyte-based cell therapy in stroke.

Cerebral ischemia leads to reactive astrogliosis and glial scar formation. Glial scarring can impede functional restoration during the recovery phase of stroke. Salidroside has been shown to have neuroprotective effects after ischemic stroke, but its impact on long-term neurological recovery, especially whether it regulates reactive astrogliosis and glial scar formation, is unclear. In this study, male adult C57/BL6 mice were subjected to transient cerebral ischemia injury followed by intravenous salidroside treatment. Primary astrocytes were treated with lipopolysaccharide (LPS) or conditioned medium from cultured primary neurons subjected to oxygen-glucose deprivation (CM-OGD). Salidroside significantly improved long-term functional outcomes following ischemic stroke in the rotarod and corner tests. It also reduced brain glial scar volume and decreased expression of the glial scar marker, glial fibrillary acidic protein (GFAP) and inhibited astrocyte proliferation. In primary astrocyte cultures, salidroside protected astrocytes from CM-OGD injury-induced reactive astroglial proliferation, increasing the percentage of cells in G0/G1 phase and reducing the S populations. The inhibitory effect of salidroside on the cell cycle was related to downregulation of cyclin D1 and cyclin-dependent kinase 4 (CDK4) mRNA expression and increased p27Kip1 mRNA expression. Similar results were found in the LPS-stimulated injury model in astroglial cultures. Western blot analysis demonstrated that salidroside attenuated the CM-OGD-induced upregulation of phosphorylated Akt and glycogen synthase kinase 3β (GSK-3β). Taken together, these results suggested that salidroside can inhibit reactive astrocyte proliferation, ameliorate glial scar formation and improve long-term recovery, probably through its effects on the Akt/GSK-3β pathway.