Mechanoresponse of stem cells for vascular repair

Stem cells have shown great potential in vascular repair. Numerous evidence indicates that mechanical forces such as shear stress and cyclic strain can regulate the adhesion, proliferation, migration, and differentiation of stem cells via serious signaling pathways. The enrichment and differentiation of stem cells play an important role in the angiogenesis and maintenance of vascular homeostasis. In normal tissues, blood flow directly affects the microenvironment of vascular endothelial cells (ECs); in pathological status, the abnormal interactions between blood flow and vessels contribute to the injury of vessels. Next, the altered mechanical forces are transduced into cells by mechanosensors to trigger the reformation of vessels. This process occurs when signaling pathways related to EC differentiation are initiated. Hence, a deep understanding of the responses of stem cells to mechanical stresses and the underlying mechanisms involved in this process is essential for clinical translation. In this the review, we provide an overview of the role of stem cells in vascular repair, outline the performance of stem cells under the mechanical stress stimulation, and describe the related signaling pathways.     

Vascular endothelial growth factor and substrate mechanics regulate in vitro tubulogenesis of endothelial progenitor cells

Endothelial progenitor cells (EPCs) in the circulatory system have been suggested to maintain vascular homeostasis and contribute to adult vascular regeneration and repair. These processes require that EPCs break down the extracellular matrix (ECM), migrate, differentiate and undergo tube morphogenesis. Evidently, the ECM plays a critical role by providing biochemical and biophysical cues that regulate cellular behaviour. Using a chemically and mechanically tunable hydrogel to study tube morphogenesis in vitro, we show that vascular endothelial growth factor (VEGF) and substrate mechanics co‐regulate tubulogenesis of EPCs. High levels of VEGF are required to initiate tube morphogenesis and activate matrix metalloproteinases (MMPs), which enable EPC migration. Under these conditions, the elasticity of the substrate affects the progression of tube morphogenesis. With decreases in substrate stiffness, we observe decreased MMP expression while increased cellular elongation, with intracellular vacuole extension and coalescence to open lumen compartments. RNAi studies demonstrate that membrane type 1‐MMP (MT1‐MMP) is required to enable the movement of EPCs on the matrix and that EPCs sense matrix stiffness through signalling cascades leading to the activation of the RhoGTPase Cdc42. Collectively, these results suggest that coupled responses for VEGF stimulation and modulation of substrate stiffness are required to regulate tube morphogenesis of EPCs.     

A genome-wide screen in yeast identifies specific oxidative stress genes required for the maintenance of sub-cellular redox homeostasis

Maintenance of an optimal redox environment is critical for appropriate functioning of cellular processes and cell survival. Despite the importance of maintaining redox homeostasis, it is not clear how the optimal redox potential is sensed and set, and the processes that impact redox on a cellular/organellar level are poorly understood. The genetic bases of cellular redox homeostasis were investigated using a green fluorescent protein (GFP) based redox probe, roGFP2 and a pH sensitive GFP-based probe, pHluorin. The use of roGFP2, in conjunction with pHluorin, enabled determination of pH-adjusted sub-cellular redox potential in a non-invasive and real-time manner. A genome-wide screen using both the non-essential and essential gene collections was carried out in Saccharomyces cerevisiae using cytosolic-roGFP2 to identify factors essential for maintenance of cytosolic redox state under steady-state conditions. 102 genes of diverse function were identified that are required for maintenance of cytosolic redox state. Mutations in these genes led to shifts in the half-cell glutathione redox potential by 75-10 mV. Interestingly, some specific oxidative stress-response processes were identified as over-represented in the data set. Further investigation of the role of oxidative stress-responsive systems in sub-cellular redox homeostasis was conducted using roGFP2 constructs targeted to the mitochondrial matrix and peroxisome and E(GSH) was measured in cells in exponential and stationary phase. Analyses allowed for the identification of key redox systems on a sub-cellular level and the identification of novel genes involved in the regulation of cellular redox homeostasis.     

Mechanical homeostasis in tissue equivalents: a review

There is substantial evidence that growth and remodeling of load bearing soft biological tissues is to a large extent controlled by mechanical factors. Mechanical homeostasis, which describes the natural tendency of such tissues to establish, maintain, or restore a preferred mechanical state, is thought to be one mechanism by which such control is achieved across multiple scales. Yet, many questions remain regarding what promotes or prevents homeostasis. Tissue equivalents, such as collagen gels seeded with living cells, have become an important tool to address these open questions under well-defined, though limited, conditions. This article briefly reviews the current state of research in this area. It summarizes, categorizes, and compares experimental observations from the literature that focus on the development of tension in tissue equivalents. It focuses primarily on uniaxial and biaxial experimental studies, which are well-suited for quantifying interactions between mechanics and biology. The article concludes with a brief discussion of key questions for future research in this field.

     

Integrative Modeling of Small Artery Structure and Function Uncovers Critical Parameters for Diameter Regulation

Organ perfusion is regulated by vasoactivity and structural adaptation of small arteries and arterioles. These resistance vessels are sensitive to pressure, flow and a range of vasoactive stimuli. Several strongly interacting control loops exist. As an example, the myogenic response to a change of pressure influences the endothelial shear stress, thereby altering the contribution of shear-dependent dilation to the vascular tone. In addition, acute responses change the stimulus for structural adaptation and vice versa. Such control loops are able to maintain resistance vessels in a functional and stable state, characterized by regulated wall stress, shear stress, matched active and passive biomechanics and presence of vascular reserve. In this modeling study, four adaptation processes are identified that together with biomechanical properties effectuate such integrated regulation: control of tone, smooth muscle cell length adaptation, eutrophic matrix rearrangement and trophic responses. Their combined action maintains arteries in their optimal state, ready to cope with new challenges, allowing continuous long-term vasoregulation. The exclusion of any of these processes results in a poorly regulated state and in some cases instability of vascular structure.     

Differentiation of stem/progenitor cells into vascular cells in response to fluid mechanical forces

Vascular functions are regulated not only by chemical mediators, such as hormones, cytokines, and neurotransmitters, but by mechanical hemodynamic forces generated by blood flow and blood pressure. The mechanical force-mediated regulation is based on the ability of vascular cells, including endothelial cells and smooth muscle cells, to recognize fluid mechanical forces, i.e., the shear stress produced by flowing blood and the cyclic strain generated by blood pressure, and to transmit the signals into the cell interior, where they trigger cell responses that involve changes in cell morphology, cell function, and gene expression. Recent studies have revealed that immature cells, such as endothelial progenitor cells (EPCs) and embryonic stem (ES) cells, as well as adult vascular cells, respond to fluid mechanical forces. Shear stress and cyclic strain promote the proliferation and differentiation of EPCs and ES cells into vascular cells and enhance their ability to form new vessels. Even more recently, attempts have been made to apply fluid mechanical forces to EPCs and ES cells cultured on polymer tubes and develop tissue-engineered blood vessel grafts that have a structure and function similar to that of blood vessels in vivo. This review summarizes the current state of knowledge concerning the mechanobiological responses of stem/progenitor cells and its potential applications to tissue engineering.     

Tumor Cells Genetically Labeled with GFP in the Nucleus and RFP in the Cytoplasm for Imaging Cellular Dynamics

Dual-color fluorescent cells with one color fluorescent protein in the nucleus and another color fluorescent protein in the cytoplasm were genetically engineered. The dual-color cancer cells enable real-time nuclear-cytoplasmic dynamics to be visualized in living cells in vivo as well as in vitro. To obtain the dual-color cells, red fluorescent protein (RFP) was expressed in the cytoplasm of a series of human and rodent cancer cells, and green fluorescent protein (GFP) linked to histone H2B was expressed in the nucleus. Nuclear GFP expression enabled visualization of nuclear dynamics, whereas simultaneous cytoplasmic RFP expression enabled visualization of nuclear-cytoplasmic ratios as well as simultaneous cell and nuclear shape changes. Using the Olympus OV100 Whole-Mouse Imaging System, total sub-cellular dynamics can be visualized in the living dual-color cells in real time in the live mouse after cell injection. Highly elongated cancer cells and nuclei in narrow capillaries were visualized where both the nuclei and cytoplasm deform. Both cytoplasm and nuclei were visualized to undergo extreme deformation during extravasation with cytoplasmic processing exiting vessels first and nuclei following along these processes. The dual-color cells described here thus enable the sub-cellular dynamics of cancer cell trafficking to be imaged in the living animal.     

Epigenetic Control of Immunity

Immunity relies on the heterogeneity of immune cells and their ability to respond to pathogen challenges. In the adaptive immune system, lymphocytes display a highly diverse antigen receptor repertoire that matches the vast diversity of pathogens. In the innate immune system, the cell''s heterogeneity and phenotypic plasticity enable flexible responses to changes in tissue homeostasis caused by infection or damage. The immune responses are calibrated by the graded activity of immune cells that can vary from yeast-like proliferation to lifetime dormancy. This article describes key epigenetic processes that contribute to the function of immune cells during health and disease.     

Spontaneous contractions of dispersed vascular muscle in cell culture.

Dispersed vascular muscle cells from chick omphalomesenteric vessels maintained in primary cell culture contracted spontaneously. Six methods which produced contracting isolated muscle cells are described and compared. The combination of dispersion method and culture conditions to produce contracting muscle cells was more critical for vascular than for heart muscle. These findings of continuing pacemaker function demonstrate that functional integrity of isolated vascular muscle cells is possible to maintain. Further indication of the full functional state of the isolated vascular muscle cells was demonstrated by the sensitivity to norepinephrine at a physiological concentration (0.1 muM). Spontaneous contraction frequencies were similar to the range found in situ, and spontanious or norepinephrine-induced contractions had time courses corresponding to intact vessel contractions. This is the first report that isolated vascular muscle cells in primary cell culture retain functional characteristics found in situ and are suitable for pharmacological characterization of individual muscle cells.     

Coxsackie- and adenovirus receptor (CAR) is expressed in lymphatic vessels in human skin and affects lymphatic endothelial cell function in vitro

Lymphatic vessels play an important role in tissue fluid homeostasis, intestinal fat absorption and immunosurveillance. Furthermore, they are involved in pathologic conditions, such as tumor cell metastasis and chronic inflammation. In comparison to blood vessels, the molecular phenotype of lymphatic vessels is less well characterized. Performing comparative gene expression analysis we have recently found that coxsackie- and adenovirus receptor (CAR) is significantly more highly expressed in cultured human, skin-derived lymphatic endothelial cells (LECs), as compared to blood vascular endothelial cells. Here, we have confirmed these results at the protein level, using Western blot and FACS analysis. Immunofluorescence performed on human skin confirmed that CAR is expressed at detectable levels in lymphatic vessels, but not in blood vessels. To address the functional significance of CAR expression, we modulated CAR expression levels in cultured LECs in vitro by siRNA- and vector-based transfection approaches. Functional assays performed with the transfected cells revealed that CAR is involved in distinct cellular processes in LECs, such as cell adhesion, migration, tube formation and the control of vascular permeability. In contrast, no effect of CAR on LEC proliferation was observed. Overall, our data suggest that CAR stabilizes LEC-LEC interactions in the skin and may contribute to lymphatic vessel integrity.     

Regulating the immune system via IL-15 transpresentation

Transpresentation has emerged as an important mechanism mediating IL-15 responses in a subset of lymphocytes during the steady state. In transpresentation, cell surface IL-15, bound to IL-15Rα is delivered to opposing lymphocytes during a cell-cell interaction. The events most dependent on IL-15 include the development and homeostasis of memory CD8 T cells, Natural Killer cells, invariant Natural Killer T cells, and intraepithelial lymphocytes. As lymphocyte development and homeostasis involve multiple steps and mechanisms, IL-15 transpresentation can have diverse roles throughout. Moreover, distinct stages of lymphocyte differentiation require IL-15 transpresented by different cells, which include both hematopoietic and non-hematopoietic cell types. Herein, we will describe the points where IL-15 transpresentation impacts these processes, the specific cells thought to drive IL-15 responses, as well as their role in the course of development and homeostasis.     

Mechanotransduction and endothelial cell homeostasis: the wisdom of the cell

Vascular endothelial cells (ECs) play significant roles in regulating circulatory functions. Mechanical stimuli, including the stretch and shear stress resulting from circulatory pressure and flow, modulate EC functions by activating mechanosensors, signaling pathways, and gene and protein expressions. Mechanical forces with a clear direction (e.g., the pulsatile shear stress and the uniaxial circumferential stretch existing in the straight part of the arterial tree) cause only transient molecular signaling of pro-inflammatory and proliferative pathways, which become downregulated when such directed mechanical forces are sustained. In contrast, mechanical forces without a definitive direction (e.g., disturbed flow and relatively undirected stretch seen at branch points and other regions of complex geometry) cause sustained molecular signaling of pro-inflammatory and proliferative pathways. The EC responses to directed mechanical stimuli involve the remodeling of EC structure to minimize alterations in intracellular stress/strain and elicit adaptive changes in EC signaling in the face of sustained stimuli; these cellular events constitute a feedback control mechanism to maintain vascular homeostasis and are atheroprotective. Such a feedback mechanism does not operate effectively in regions of complex geometry, where the mechanical stimuli do not have clear directions, thus placing these areas at risk for atherogenesis. The mechanotransduction-induced EC adaptive processes in the straight part of the aorta represent a case of the "Wisdom of the Cell," as a part of the more general concept of the "Wisdom of the Body" promulgated by Cannon, to maintain cellular homeostasis in the face of external perturbations.     

Cell mechanics and mechanotransduction: pathways, probes, and physiology

Cells face not only a complex biochemical environment but also a diverse biomechanical environment. How cells respond to variations in mechanical forces is critical in homeostasis and many diseases. The mechanisms by which mechanical forces lead to eventual biochemical and molecular responses remain undefined, and unraveling this mystery will undoubtedly provide new insight into strengthening bone, growing cartilage, improving cardiac contractility, and constructing tissues for artificial organs. In this article we review the physical bases underlying the mechanotransduction process, techniques used to apply controlled mechanical stresses on living cells and tissues to probe mechanotransduction, and some of the important lessons that we are learning from mechanical stimulation of cells with precisely controlled forces. cytoskeleton; micromanipulation; cell signaling     

The interface between biochemical signaling and cell mechanics shapes T lymphocyte migration and activation

tT cells migrate to lymphoid organs to become activated through specific contacts with antigen-presenting cells bearing foreign antigens. During migration and activation, T lymphocytes are exposed not only to diverse biochemical inputs, but also to different mechanical conditions. Passage from the blood or lymph to solid tissues involves lymphocyte rolling, firm arrest and diapedesis through endothelial monolayers. Throughout this process, cells are subjected to diverse fluid flow regimes. After extravasation, T lymphocytes crawl through viscoelastic media of different biochemical and mechanical properties and geometries. In lymph nodes, T cell contact with antigen-presenting cells is guided by rigidity cues and ligand-receptor interactions. T lymphocyte adaptation to diverse mechanical regimes involves multiple signaling and morphological modifications, many of which enable the conversion of mechanical forces into biochemical signals and vice-versa. These components enable T lymphocyte survival, homing and activation. Here, we review the mechanisms that enable T lymphocytes to survive and thrive under the different mechanical conditions they encounter during their life cycle. These processes require the integration of diverse signaling networks that convert extracellular mechano-chemical cues into force, movement and activation.     

Effects of soluble factors and extracellular matrix components on vascular cell behavior in vitro and in vivo: models of de-endothelialization and repair.

Vessel walls are comprised of several different cell populations residing in and on complex extracellular matrices. Each of the vascular cell types has diverse and sometimes unique functions and morphologies, and each has roles in repair processes following injury. Large vessel endothelial cells are known to respond to denudation injury by sheet migration and proliferation. This is in contrast to the migration through soft tissues with tube formation and subsequent lumen formation exhibited by microvascular endothelial cells in response to injury. Vascular smooth muscle cells of larger vessels respond to injury by migration from the arterial media into the intima, proliferation, and matrix biosynthesis, ultimately causing intimal thickening. Both these cell types exhibit "dysfunctional" phenotypes during their responses to injury. Microvascular cell responses to injury, while extremely variable, are less well documented. Specifically, responses to injury by microvascular endothelial vascular cells appear to be modulated, in part, by the composition and organization of the surrounding matrix as well as by the various soluble factors and cytokines found at sites of injury, suggesting that the extracellular matrix and soluble factors modulate each other's effects on local vascular cell populations following injury.     

自噬在急性心肌梗死发病中作用的研究进展

自噬是生物细胞内普遍存在且高度保守的一种生理过程,其通过溶酶体融合降解细胞内的大分子组分、受损的细胞器以及侵入胞内的病原菌,以达到维持细胞稳态的目的。自噬在多种疾病的发生发展中也发挥十分重要的作用,尤其是心血管疾病。自噬对其病程的发展可以发挥两种截然不同的作用。适当的自噬作用可以降低炎症反应和氧化应激促进细胞的存活,以及通过减少泡沫细胞的形成而对维持心血管的正常功能起一个保护作用;但过度的自噬作用会对细胞造成不可逆的损伤,诱导细胞发生不依赖于caspase的自噬性细胞死亡,增加局部的炎症反应,从而促进动脉粥样硬化病变的发展。本文就自噬在急性心肌梗死发生发展中作用的研究进展进行了综述,探讨自噬成为预防及治疗心血管疾病新靶标的可能性。     

结合代谢组学和转录组学分析恶臭假单胞菌Y-9主动稳定胞内外pH的机制

【目的】揭示恶臭假单胞菌(Pseudomonas putida) Y-9在氨氧化过程中主动调节胞外和胞内pH稳态机制。【方法】在初始pH为7.19和9.40的硝化培养基中培养Y-9生长48 h，利用代谢组学对比分析Y-9氨氧化过程中的显著差异代谢产物并预测解离常数(pKa)；结合转录组学对比分析Y-9氨氧化过程中的显著差异调控基因。【结果】Y-9在初始pH为7.19的相对酸性条件下，产生麦芽糖醇提高胞外pH；通过上调脱氨酶、脱亚胺酶和阳离子转运相关基因在相对酸性环境中的表达来维持细胞内pH稳定性。在初始pH为9.40的碱性条件下，产5-氨基戊酸3和草氨酸等有机酸及酸性物质降低胞外pH；通过调控NADH脱氢酶、细胞色素、ATP合酶和氨基酸转运相关基因的表达来维持细胞内酸度，应对碱性环境。【结论】本研究结果首先发现了Y-9具有稳定胞外pH的能力，探讨了其胞内pH稳态机制，拓展了对微生物与环境相互作用的认知，为进一步认识微生物脱氮过程中系统pH稳定机理提供了理论依据。     

Tensional Homeostasis in Single Fibroblasts

Adherent cells generate forces through acto-myosin contraction to move, change shape, and sense the mechanical properties of their environment. They are thought to maintain defined levels of tension with their surroundings despite mechanical perturbations that could change tension, a concept known as tensional homeostasis. Misregulation of tensional homeostasis has been proposed to drive disorganization of tissues and promote progression of diseases such as cancer. However, whether tensional homeostasis operates at the single cell level is unclear. Here, we directly test the ability of single fibroblast cells to regulate tension when subjected to mechanical displacements in the absence of changes to spread area or substrate elasticity. We use a feedback-controlled atomic force microscope to measure and modulate forces and displacements of individual contracting cells as they spread on a fibronectin-patterned atomic-force microscope cantilever and coverslip. We find that the cells reach a steady-state contraction force and height that is insensitive to stiffness changes as they fill the micropatterned areas. Rather than maintaining a constant tension, the fibroblasts altered their contraction force in response to mechanical displacement in a strain-rate-dependent manner, leading to a new and stable steady-state force and height. This response is influenced by overexpression of the actin crosslinker α-actinin, and rheology measurements reveal that changes in cell elasticity are also strain- rate-dependent. Our finding of tensional buffering, rather than homeostasis, allows cells to transition between different tensional states depending on how they are displaced, permitting distinct responses to slow deformations during tissue growth and rapid deformations associated with injury.     

Tensional Homeostasis in Single Fibroblasts

Adherent cells generate forces through acto-myosin contraction to move, change shape, and sense the mechanical properties of their environment. They are thought to maintain defined levels of tension with their surroundings despite mechanical perturbations that could change tension, a concept known as tensional homeostasis. Misregulation of tensional homeostasis has been proposed to drive disorganization of tissues and promote progression of diseases such as cancer. However, whether tensional homeostasis operates at the single cell level is unclear. Here, we directly test the ability of single fibroblast cells to regulate tension when subjected to mechanical displacements in the absence of changes to spread area or substrate elasticity. We use a feedback-controlled atomic force microscope to measure and modulate forces and displacements of individual contracting cells as they spread on a fibronectin-patterned atomic-force microscope cantilever and coverslip. We find that the cells reach a steady-state contraction force and height that is insensitive to stiffness changes as they fill the micropatterned areas. Rather than maintaining a constant tension, the fibroblasts altered their contraction force in response to mechanical displacement in a strain-rate-dependent manner, leading to a new and stable steady-state force and height. This response is influenced by overexpression of the actin crosslinker α-actinin, and rheology measurements reveal that changes in cell elasticity are also strain- rate-dependent. Our finding of tensional buffering, rather than homeostasis, allows cells to transition between different tensional states depending on how they are displaced, permitting distinct responses to slow deformations during tissue growth and rapid deformations associated with injury.