Protein nanomechanics in biological context

How proteins respond to pulling forces, or protein nanomechanics, is a key contributor to the form and function of biological systems. Indeed, the conventional view that proteins are able to diffuse in solution does not apply to the many polypeptides that are anchored to rigid supramolecular structures. These tethered proteins typically have important mechanical roles that enable cells to generate, sense, and transduce mechanical forces. To fully comprehend the interplay between mechanical forces and biology, we must understand how protein nanomechanics emerge in living matter. This endeavor is definitely challenging and only recently has it started to appear tractable. Here, I introduce the main in vitro single-molecule biophysics methods that have been instrumental to investigate protein nanomechanics over the last 2 decades. Then, I present the contemporary view on how mechanical force shapes the free energy of tethered proteins, as well as the effect of biological factors such as post-translational modifications and mutations. To illustrate the contribution of protein nanomechanics to biological function, I review current knowledge on the mechanobiology of selected muscle and cell adhesion proteins including titin, talin, and bacterial pilins. Finally, I discuss emerging methods to modulate protein nanomechanics in living matter, for instance by inducing specific mechanical loss-of-function (mLOF). By interrogating biological systems in a causative manner, these new tools can contribute to further place protein nanomechanics in a biological context.     

Pannexin channel and connexin hemichannel expression in vascular function and inflammation

Control of blood flow distribution and tissue homeostasis depend on the tight regulation of and coordination between the microvascular network and circulating blood cells. Channels formed by connexins or pannexins that connect the intra- and extracellular compartments allow the release of paracrine signals, such as ATP and prostaglandins, and thus play a central role in achieving fine regulation and coordination of vascular function. This review focuses on vascular connexin hemichannels and pannexin channels. We review their expression pattern within the arterial and venous system with a special emphasis on how post-translational modifications by phosphorylation and S-nitrosylation of these channels modulate their function and contribute to vascular homeostasis. Furthermore, we highlight the contribution of these channels in smooth muscle cells and endothelial cells in the regulation of vasomotor tone as well as how these channels in endothelial cells regulate inflammatory responses such as during ischemic and hypoxic conditions. In addition, this review will touch on recent evidence implicating a role for these proteins in regulating red blood cell and platelet function.     

Real-time differential labeling of blood, interstitium, and lymphatic and single-field analysis of vasculature dynamics in vivo

Lymph nodes are highly organized structures specialized for efficient regulation of adaptive immunity. The blood and lymphatic systems within a lymph node play essential roles by providing functionally distinct environments for lymphocyte entry and egress, respectively. Direct imaging and measurement of vascular microenvironments by intravital multiphoton microscopy provide anatomical and mechanistic insights into the essential events of lymphocyte trafficking. Lymphocytes, blood endothelial cells, and lymphatic endothelial cells express sphingosine 1-phosphate receptor 1, a key G protein-coupled receptor regulating cellular egress and a modulator of endothelial permeability. Here we report the development of a differential vascular labeling (DVL) technique in which a single intravenous injection of a fluorescent dextran, in combination with fluorescent semiconductor quantum dot particles, differentially labels multiple blood and lymphatic compartments in a manner dependent on the size of the fluorescent particle used. Thus DVL allows measurement of endothelial integrity in multiple vascular compartments and the affects or pharmacological manipulation in vascular integrity. In addition, this technique allows for real-time observation of lymphocyte trafficking across physiological barriers differentiated by DVL. Last, single-field fluid movement dynamics can be derived, allowing for the simultaneous determination of fluid flow rates in diverse blood and lymphatic compartments.     

Vascular biology: the role of sphingosine 1‐phosphate in both the resting state and inflammation

The vascular and immune systems of mammals are closely intertwined: the individual components of the immune system must move between various body compartments to perform their function effectively. Sphingosine 1-phosphate (S1P), a bioactive lipid mediator, exerts effects on the two organ systems and influences the interaction between them. In the resting state, the vascular S1P gradient contributes to control of lymphocyte recirculation through the blood, lymphoid tissue and lymphatic vasculature. The high level of S1P in blood helps maintain endothelial barrier integrity. During the inflammatory process, both the level of S1P in different immune compartments and S1P receptor expression on lymphocytes and endothelial cells are modified, resulting in functionally important changes in endothelial cell and lymphocyte behaviour. These include transient arrest of lymphocytes in secondary lymphoid tissue, crucial for generation of adaptive immunity, and subsequent promotion of lymphocyte recruitment to sites of inflammation. This review begins with an outline of the basic biochemistry of S1P. S1P receptor signalling is then discussed, followed by an exploration of the roles of S1P in the vascular and immune systems, with particular focus on the interface between them. The latter part concerns crosstalk between S1P and other signalling pathways, and concludes with a look at therapies targeting the S1P-S1P receptor axis.     

EG-VEGF and the concept of tissue-specific angiogenic growth factors

The endothelium of the vascular beds is extremely diverse and exquisitely distinct with respect to the specific tissue compartment served by the vessels. The molecular identity and function of the instructive signals that tailor the tissue-specific endothelial phenotype have been largely undefined. Presumably, a complex, integrated network of signals derived from the tissue parenchyma and/or stromal compartments is responsible. Recently, we identified a novel angiogenic mitogen, endocrine-gland-derived vascular endothelial growth factor, EG-VEGF, with a selective activity and very distinct expression pattern. Human EG-VEGF is expressed by steroid producing cells in the adrenal gland, placenta, testis and ovary, and is a mitogen for endothelial cells derived from these microvascular beds. EG-VEGF may represent the first of a novel class of tissue-specific angiogenic factors that function to regulate and fine-tune endothelial cell growth, structural and functional properties. The identification of other selective angiogenic molecules will allow insight into exciting, basic developmental issues and increase our armamentarium of factors for therapeutic angiogenic and anti-angiogenic strategies.     

皂苷类成分对血管内皮细胞功能的调节作用研究进展

血管内皮细胞在维持血管生理稳态中发挥了重要的作用,其功能障碍是动脉粥样硬化、冠心病、脑卒中、肿瘤等多种重大疾病发生发展的病理基础,调节血管内皮细胞功能是防治上述疾病的主要途径之一。大量研究表明,皂苷类成分可通过改善血管内皮功能达到治疗疾病的目的。综述了近年来报道的皂苷类成分调节血管内皮功能的研究进展,旨在为皂苷类成分作用机制的阐明和相关重大疾病的防治提供一定参考。     

Flow-activated ion channels in vascular endothelium

The ability of vascular endothelial, cells (ECs) to respond to fluid mechanical forces associated with blood flow is essential for flow-mediated vasoregulation and arterial wall remodeling. Abnormalities in endothelial responses to flow also play a role in the development of atherosclerosis. Although our understanding of the endothelial signaling pathways stimulated by flow has greatly increased over the past two decades, the mechanisms by which ECs sense flow remain largely unknown. Activation of flow-sensitive ion channels is among the fastest known endothelial responses to flow; therefore, these ion channels have been proposed as candidate flow sensors. This review focuses on: 1) describing the various types of flow-sensitive ion channels that have been reported in ECs, 2) discussing the implications of activation of these ion channels for endothelial function, and 3) proposing candidate mechanisms for activation of flow-sensitive ion channels.     

Synergistic targeting with bone marrow-derived cells and PDGF improves diabetic vascular function

Diabetes mellitus is associated with an increased risk of vascular disease, with significant alterations in systemic endothelial progenitor cells (EPCs) and peripheral vascular function. To identify the contribution of the different vascular compartments in the diabetic impairment of vascularization, we employed streptozotocin- and control-treated 3-mo-old C57Bl/6 mice in an isogeneic pinnal cardiac allograft model, revealing a significant delay in vascularization of wild-type cardiac tissue transplanted into diabetic mice. To investigate the basis of this impairment, the function of diabetic bone marrow cells was tested by transplantation of bone marrow cells isolated from diabetic and control mice into intact, unirradiated 18-mo-old C57Bl/6 mice, which have impaired function of both EPCs and peripheral endothelial cells. Importantly, cells derived from control, but not diabetic, bone marrow integrated into transplanted cardiac allografts. To assess the contribution of diabetic changes in the local vasculature, diabetic mice were treated with pinnal injections of platelet-derived growth factor (PDGF)-AB, which promotes cardiac angiogenesis in wild-type mice. However, whereas PDGF-AB enhanced allograft function in control mice, the activity of the cardiac transplants in the PDGF-AB-treated diabetic mice was significantly decreased. To decipher the potential interactions between systemic bone marrow-derived cells and local vascular pathways, diabetic mice were transplanted with wild-type bone marrow cells with or without PDGF-AB pinnal pretreatment, resulting in improved allograft function and donor cell recruitment only in the combination treatment arm. Overall, these studies show that the diabetic impairment in cardiac angiogenesis can be reversed by targeting the synergism between local trophic pathways and systemic cell function.     

Intrinsic tyrosine kinase activity is required for vascular endothelial growth factor receptor 2 ubiquitination, sorting and degradation in endothelial cells

The human endothelial vascular endothelial growth factor receptor 2 (VEGFR2/kinase domain region, KDR/fetal liver kinase-1, Flk-1) tyrosine kinase receptor is essential for VEGF-mediated physiological responses including endothelial cell proliferation, migration and survival. How VEGFR2 kinase activation and trafficking are co-coordinated in response to VEGF-A is not known. Here, we elucidate a mechanism for endothelial VEGFR2 response to VEGF-A dependent on constitutive endocytosis co-ordinated with ligand-activated ubiquitination and proteolysis. The selective VEGFR kinase inhibitor, SU5416, blocked the endosomal sorting required for VEGFR2 trafficking and degradation. Inhibition of VEGFR2 tyrosine kinase activity did not block plasma membrane internalization but led to endosomal accumulation. Lysosomal protease activity was required for ligand-stimulated VEGFR2 degradation. Activated VEGFR2 codistributed with the endosomal hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)/signal-transducing adaptor molecule (STAM) complex in a ligand and time-dependent manner, implying a role for this factor in sorting of ubiquitinated VEGFR2. Increased tyrosine phosphorylation of the Hrs subunit in response to VEGF-A links VEGFR2 activation and Hrs/STAM function. In contrast, VEGFR2 in quiescent cells was present on both the endothelial plasma membrane and early endosomes, suggesting constitutive recycling between these two compartments. This pathway was clathrin-linked and dependent on the AP2 adaptor complex as the A23 tyrphostin inhibited VEGFR2 trafficking. We propose a mechanism whereby the transition of endothelial VEGFR2 from a constitutive recycling itinerary to a degradative pathway explains ligand-activated receptor degradation in endothelial cells. This study outlines a mechanism to control the VEGF-A-mediated response within the vascular system.     

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.     

Dynamics of pulmonary endothelial barrier function in acute inflammation: mechanisms and therapeutic perspectives

The lungs provide a large inner surface to guarantee respiration. In lung alveoli, a delicate membrane formed by endo- and epithelial cells with their fused basal lamina ensures rapid and effective gas exchange between alveolar and vascular compartments while concurrently forming a robust barrier against inhaled particles and microbes. However, upon infectious or sterile inflammatory stimulation, tightly regulated endothelial barrier leakiness is required for leukocyte transmigration. Further, endothelial barrier disruption may result in uncontrolled extravasation of protein-rich fluids. This brief review summarizes some important mechanisms of pulmonary endothelial barrier regulation and disruption, focusing on the role of specific cell populations, coagulation and complement cascades and mediators including angiopoietins, specific sphingolipids, adrenomedullin and reactive oxygen and nitrogen species for the regulation of pulmonary endothelial barrier function. Further, current therapeutic perspectives against development of lung injury are discussed.     

Differential responsiveness of vascular endothelial cells to different types of fluid mechanical shear stress

Early atherosclerotic lesions localize preferentially, in arterial regions exposed to low flow, oscillatory flow, or both; however, the cellular basis of this observation remains to be determined. Atherogenesis involves dysfunction of the vascular endothelium, the cellular monolayer lining the inner surfaces of blood vessels. How low flow, oscillatory flow, or both may lead to endothelial dysfunction remains unknown. Over the past two decades, fluid mechanical shear (or frictional) stress has been shown to intricately regulate the structure and function of vascular endothelial cells (ECs). Furthermore, recent data indicate that beyond being merely responsive to shear stress, ECs are able to distinguish among and respond differently to different types of shear stress. This review focuses on EC differential responses to different types of steady and unsteady shear stress and discusses the implications of these responses for the localization of early atherosclerotic lesions. The mechanisms by which endothelial differential responsiveness to different types of flow may occur are also discussed.     

Tissue engineering of vascular grafts.

The challenge of tissue engineering blood vessels with the mechanical properties of native vessels, and with the anti-thrombotic properties required is immense. Recent advances, however, indicate that the goal of providing a tissue-engineered vascular graft that will remain patent in vivo for substantial periods of time, is achievable. For instance, collagen gels have been used to fabricate a tissue in vitro that is representative of a native vessel: an acellular collagen tubular structure, when implanted as a vascular graft, was able to function, and to become populated with host cells. A completely cellular approach culturing cells into tissue sheets and wrapping these around a mandel was able to form a layered tubular structure with impressive strength. Culture of cells onto a biodegradable scaffold within a dynamic bioreactor, generated a tissue-engineered vascular graft with substantial stiffness and, when lined with endothelial cells, was able to remain patent for up to 4 weeks in vivo. In our experiments, use of a non-degradable polyurethane scaffold and culture with smooth muscle cells generated a construct with mechanical properties similar to native vessels. This composite tissue engineered vascular graft with an endothelial layer formed using fluid shear stress to align the endothelial cells, was able to remain patent with an neointima for up to 4 weeks. These results show that tissue engineering of vascular grafts has true potential for application in the clinical situation.     

Vascular development: from precursor cells to branched arterial and venous networks

The adult vascular system is composed of an arterial, a venous and a lymphatic compartment. These different compartments respectively provide oxygen and nutrients to peripheral organs, remove carbon dioxide and waste products and maintain an immune barrier to defend the host against foreign organisms. Malfunctions of the vascular system represent a major cause of mortality and disease in developed countries. Understanding of the molecular mechanisms regulating vascular system development and maintenance is thus crucial for the design of therapies to cure vascular diseases. The molecules implicated in the control of physiological and pathological angiogenesis in the adult already function during embryonic development. Indeed, the survival of the embryo also critically depends on the establishment of a functional circulatory loop. Here we review our current knowledge about the emergence of endothelial precursor cells in the embryo, of their assembly into the primary vascular plexus and of the remodeling of this plexus into arteries and veins. We also focus on the molecular mechanisms controlling the development of arteries, veins and lymphatic vessels.     

Incorporation of the elderberry anthocyanins by endothelial cells increases protection against oxidative stress

The objective of this study was to investigate the ability of endothelial cells (EC) to incorporate anthocyanins and to examine their potential benefits against various oxidative stressors. Endothelial dysfunction has been proposed to play an important role in the initiation and development of vascular disease, with studies having shown that administration of antioxidants improves endothelial function. Elderberry extract contains 4 anthocyanins, which where incorporated into the plasma membrane and cytosol of EC following 4 h incubation at 1 mg.ml(-1). However, incorporation within the cytosol was considerably less than that in the membrane. Uptake within both regions appeared to be structure dependent, with monoglycoside concentrations higher than that of the diglucosides in both compartments. The enrichment of EC with elderberry anthocyanins conferred significant protective effects in EC against the following oxidative stressors: hydrogen peroxide (H(2)O(2)); 2, 2'-azobis(2-amidinopropane) dihydrochloride (AAPH); and FeSO(4)/ascorbic acid (AA). These results show for the first time that vascular EC can incorporate anthocyanins into the membrane and cytosol, conferring significant protective effects against oxidative insult. These findings may have important implications on preserving EC function and preventing the initiation of EC changes associated with vascular diseases.     

Cyclic strain-mediated matrix metalloproteinase regulation within the vascular endothelium: a force to be reckoned with

The vascular endothelium is a dynamic cellular interface between the vessel wall and the bloodstream, where it regulates the physiological effects of humoral and biomechanical stimuli on vessel tone and remodeling. With respect to the latter hemodynamic stimulus, the endothelium is chronically exposed to mechanical forces in the form of cyclic circumferential strain, resulting from the pulsatile nature of blood flow, and shear stress. Both forces can profoundly modulate endothelial cell (EC) metabolism and function and, under normal physiological conditions, impart an atheroprotective effect that disfavors pathological remodeling of the vessel wall. Moreover, disruption of normal hemodynamic loading can be either causative of or contributory to vascular diseases such as atherosclerosis. EC-matrix interactions are a critical determinant of how the vascular endothelium responds to these forces and unquestionably utilizes matrix metalloproteinases (MMPs), enzymes capable of degrading basement membrane and interstitial matrix molecules, to facilitate force-mediated changes in vascular cell fate. In view of the growing importance of blood flow patterns and mechanotransduction to vascular health and pathophysiology, and considering the potential value of MMPs as therapeutic targets, a timely review of our collective understanding of MMP mechanoregulation and its impact on the vascular endothelium is warranted. More specifically, this review primarily summarizes our current knowledge of how cyclic strain regulates MMP expression and activation within the vascular endothelium and subsequently endeavors to address the direct and indirect consequences of this on vascular EC fate. Possible relevance of these phenomena to vascular endothelial dysfunction and pathological remodeling are also addressed.     

Neutrophil-dependent mediation of microvascular permeability

Macromolecular extravasation induced by the chemoattractants N-formyl-methionylleucylphenylalanine (FMLP), complement fragment C5a, and leukotriene B4 (LTB4) has been reported to be neutrophil dependent. A review of experimental evidence argues against mechanical disruption of the endothelial barrier as a likely mechanism for enhanced microvascular permeability. Other proposed mechanisms attribute macromolecular efflux to extracellular liberation of granule constituents or granule-independent neutrophil release products (e.g., oxygen radicals) that can undermine vascular integrity by direct or indirect actions on endothelial cells or other components of vascular walls (glycocalyx, basement membrane). The cytotoxic potential of neutrophil-release products on endothelial cells prompts consideration of a transcellular pathway for macromolecular transport. Further studies are needed to clarify the precise nature of endothelial injury and its resolution to better understand the physiology behind the transient effects of these mediators on vascular permeability.     

Caveolae and non-caveolae lipid raft microdomains of human umbilical vein endothelial cells contain utrophin-associated protein complexes

Several studies have shown the importance of dystrophin-associated protein complex in the development of muscular dystrophies and dilated cardiomyopathy associated to vascular dysfunction. In vascular endothelium, dystrophin is substituted for utrophin (autosomal homolog of dystrophin); however, its role in this tissue is unknown. Therefore, it is important to obtain a more extensive knowledge of utrophin and its associated proteins in endothelial cells. In a previous study, we demonstrated the presence of utrophin-associated protein complex (UAPC) in human umbilical vein endothelial cells HUVEC, which interacts with caveolin-1 (Cav-1) and endothelial nitric oxide synthase (eNOS). Also, some of our observations suggested the presence of this complex in distinct membrane domains. Therefore, the aim of this study was to analyze the presence of the UAPC in caveolae and non-caveolae lipid rafts domains of HUVEC at baseline and with a mechanical stimulus. It was demonstrated, by subcellular fractionation and co-immunoprecipitation assays, the association of UAPC with Cav-1 and eNOS in caveolae domains, as well as its interaction with eNOS in non-caveolae lipid raft domains. Additionally, it was also observed that mechanical stress on endothelial cells induced activation and release of eNOS from both caveolae and non-caveolae lipid raft associated to UAPC. Together these results suggest that UAPC located in caveolae and non-caveolae lipid raft domains of HUVECs may have a mechanosensory function that could participate in the control of eNOS activity.     

How can microbial interactions with the blood–brain barrier modulate astroglial and neuronal function?

The vascular endothelium of the blood-brain barrier (BBB) is regarded as a part of the neurovascular unit (NVU). This emerging NVU concept emphasizes the need for homeostatic signalling among the neuronal, glial and vascular endothelial cellular compartments in maintaining normal brain function. Conversely, dysfunction in any component of the NVU affects another, thus contributing to disease. Brain endothelial activation and dysfunction is observed in various neurological diseases, such as (ischemic) stroke, seizure, brain inflammation and infectious diseases and likely contributes to or exacerbates neurological conditions. The role and impact of brain endothelial factors on astroglial and neuronal activation is unclear. Similarly, it is not clear which stages of BBB endothelial activation can be considered beneficial versus detrimental. Although the BBB plays an important role in context of encephalopathies caused by neurotropic microbes that must first penetrate into the brain, a crucial role of the BBB in contributing to neurological dysfunction may be seen in cerebral malaria (CM), where the Plasmodium parasite remains sequestered in the brain vasculature, does not enter the brain parenchyma, and yet causes coma and seizures. In this minireview some of the scenarios and factors that may play a role in BBB as a relay station to modulate astroneuronal functioning are discussed.