Insulin-initiated PI3K–Akt–eNOS–NO survival signalling and cardiovascular protection. Insulin binds to cell membrane insulin receptor, leading to the activation of mainly two signalling pathways: Ras–MAPK, which results in cell proliferation; and PI3K–Akt–eNOS, which results in metabolic modulation and cardiovascular protection. Among the insulin-activated signalling cascades, PI3K–Akt–eNOS–NO represents a special link between insulin and the cardiovascular system with regard to health and pathology. Activation of this signalling cascade, together with other Akt-activated molecules (such as GSK-3β, mTOR and p70S6 kinase), elicits pro-survival and cardiovascular protective effects, including vasodilatation, anti-apoptosis, anti-inflammation, and anti-oxidative/nitrative stress.
Abbreviations: Akt, protein kinase B; eNOS, endothelial nitric oxide synthase; ET-1, endothelin-1; GLUT4, glucose transporter 4; GSK, glycogen synthase kinase; IRS, insulin receptor substrate; MAPK, mitogen-activated protein kinase; mTOR, the mammalian target of rapamycin; PI3K, phosphatidylinositol 3′-kinase; PMN, polymorphonuclear neutrophil; and ROS, reactive oxygen species.
Nitrite in pulmonary arterial hypertension: therapeutic avenues in the setting of dysregulated arginine/nitric oxide synthase signalling
The classic arginine–nitric oxide synthase–nitric oxide pathway. This figure illustrates the ‘classic’ nitric oxide pathway and both cyclic guanosine monophosphate-dependent and -independent signalling. Furthermore, the figure highlights the multiple levels of this pathway that can be taken advantage of for therapeutic benefit. One strategy is to increase nitric oxide synthase substrate availability via l-arginine supplementation or arginase inhibitors. Alternative strategies are to increase nitric oxide synthase enzymes via gene or protein therapy as well as direct deliver of nitric oxide gas via inhalation or pharmacological donors. Additionally, therapeutics take advantage of cyclic guanosine monophosphate-dependent signalling including phosphodiesterase inhibitors, such as sildenafil, and the direct guanylate cyclase activators such as riociguat.
(A) The concept of lethal reperfusion injury. During ischaemia, irreversible cell injury leading to cell death occurs within the ischaemic risk zone in a time-dependent manner. In the absence of reperfusion, ischaemic injury would progressively kill more and more cells, eventually accounting for near total cell death (broken line). Reperfusion halts the process of ischaemic cell death but in its early stages imposes injury that results in further cell death, beyond that due to the ischaemic period: this is lethal reperfusion injury. The net result, however, is that the reperfused tissue sustains less cell death than would occur in ischaemic tissue without reperfusion. Hence, targeting cell death due to reperfusion has the potential to maximize cell salvage. Postconditioning applied at the onset of reperfusion limits the extent of reperfusion injury and maximizes reperfusion salvage. Adapted from Garcia-Dorado and Piper209.
(B) Attenuation of lethal reperfusion injury as a function of the duration of the preceding index ischaemia. Infarct size reduction by protective interventions performed after reperfusion (e.g. ischaemic postconditioning) may vary according to the duration of the preceding ischaemia. Experimental studies show that with prolonged periods of index ischaemia, the potential to minimize tissue salvage (infarct size reduction) may be limited probably because the extent of lethal ischaemic injury is so severe. With briefer lethal periods of ischaemia, interventions at reperfusion are able to confer marked reduction in infarct size. When the index ischaemic event is so brief that no infarction occurs (sub-lethal ischaemia), the application of ischaemic postconditioning at the onset of reperfusion may prove injurious and cause a small degree of infarction.
Traditional versus revised view of trans-endothelial fluid movement and the driving forces.
Fluid movement across microvascular endothelium (flow Jv) underlies plasma/interstitial fluid volume regulation, lymph generation, and clinical oedema. According to the traditional Starling principle (A), endothelium is a symmetrical, semipermeable membrane perforated by protein-reflecting, water-conducting small pores. Proteins in plasma exert colloid osmotic pressure Πp, which partially offsets the filtration force, namely capillary blood pressure Pc minus interstitial fluid pressure Pi. Conversely, the osmotic pressure of escaped plasma proteins in interstitial fluid (Πi should boost filtration. However, this was not the case in recent experiments. Moreover, the model predicts lymph formation rates that far exceed reality.
The glycocalyx-cleft model (B) is asymmetrical because the glycocalyx has emerged as the semipermeable component containing small pores. The wider, underlying intercellular cleft conducts plasma ultrafiltrate outwards. Grey shading denotes the low, sub-glyocalyx concentration of plasma protein, with a colloid osmotic pressure Πg. The disequilibrium between Πg and Πi is maintained by the outward filtration stream. The effective, down-side osmotic pressure is Πg not Πi. This explains why changing Πi experimentally had little effect on filtration and helps explain the low lymph flow paradox. The osmotic reflection coefficient σ determines the fraction of Πp exerted across the membrane; σ falls during inflammation, due to large pore formation.
Mechanisms underlying barrier regulation by small GTPases. Barrier stabilization is considered to be primarily mediated by Rac1 and Cdc42. Mechanisms hereby involve stabilization of junctional complexes as well as strengthening of cortical actin via inhibition of cofilin and recruitment of cortactin. Also, Rap1 and possibly RhoA utilize these pathways. However, RhoA, via its effector Rho kinase, is thought to predominantly destabilize endothelial barrier properties by promoting MLC-dependent contraction of stress fibres and reducing VE-cadherin-mediated adhesion.
Mechanisms of endothelial barrier failure following ischaemia–reperfusion (I/R).
The endothelial barrier failure elicited by I/R is accompanied by oxidative stress and the recruitment of leucocytes and platelets. The blood cells that are recruited into the post-ischaemic microvasculature appear to contribute to both the endothelial barrier dysfunction and enhanced production of reactive oxygen species (ROS) via mechanisms that require adhesive interactions between blood cells and vascular endothelium. The available evidence suggests that products of blood cell activation, including ROS, cytokines, and chemokines, play a major role in mediating the adhesion-dependent increase in vascular permeability caused by I/R. Whether these agents act directly on endothelial cells or do so indirectly by activating perivascular cells such as mast cells and macrophages remain unclear. Despite these uncertainties about the nature of the involvement of different cell populations and molecular mediators in the I/R-induced endothelial barrier dysfunction, there is mounting evidence that the well-established risk factors for cardiovascular disease amplify the inflammatory and oxidative responses elicited by I/R, with a corresponding exacerbation of the barrier failure.
Abbreviations: O2-, superoxide; H2O2, hydrogen peroxide; IL-12, interleukin-12; IFN-γ, interferon--γ PSGL-1, P-selectin glycoprotein ligand-1; IL-1, interleukin-1; TNF-α, tumor necrosis factor-α ONOO-, peroxynitrite anion; MIP-1α, monocyte inhibitory peptide-1; IL-6, interleukin-6
Vasodilator-stimulated phosphoprotein: crucial for activation of Rac1 in endothelial barrier maintenance
VASP stabilizes endothelial barrier functions by regulation of small GTPase Rac1.
It is well established in the meantime that vasodilator-stimulated phosphoprotein (VASP) is required to maintain endothelial barrier properties. Under conditions of acute inflammation and hypoxia, VASP was shown to be down-regulated, leading to increased endothelial permeability. Recent data provide evidence for a completely new mechanism by which VASP stabilizes the endothelial barrier, i.e. by facilitating activation of Rac1 downstream of PKA and PKG. According to this model VASP-dependent endothelial barrier stabilization was shown to act via cAMP- and cGMP-mediated signalling pathways. VASP is associated with actin filaments via zyxin and vinculin while paxillin is linked to endothelial adherens junctions via β-catenin. We found that VASP was required for cAMP-mediated Rac1 activation and barrier enhancement. Serine-157 phosphorylation of VASP via PKA induced translocation of VASP to cell junctions and binding of VASP to ZO-1. In line with this, PKG-mediated serine-239 phosphorylation was also shown to be required for Rac1 activation. Thus, we propose that cAMP- and cGMP-mediated signalling facilitates Rac1 activation close to cell junctions in VASP-dependent manner, which in turn appears to be crucial for endothelial barrier stabilization.
Abbreviations: FAK= focal adhesion kinase; PKA= cAMP-dependent protein kinase A; PKG= cGMP-dependent protein kinase G; EPAC= exchange protein directly activated by cAMP; ZO-1/2/3= zonula occludens protein1/2/3; α, β = α- and β-catenin; ECM= extracellular matrix.
High glucose, NO, and adenosine: a vicious circle in chronic hyperglycaemia.
HUVEC isolated from gestational diabetic pregnancies show a reduced adenosine transport activity via hENT1. This effect of gestational diabetes leads to extracellular accumulation and a higher bioavailability of this nucleoside to activate the A2a adenosine receptor subtype. The intracellular signalling cascade triggered by A2a purinoreceptor activation by adenosine results in an increased l-arginine transport activity via hCATs and increased NO synthesis by eNOS. The intracellular second messengers involved in the effect of adenosine include activation of protein kinase C (PKC) and 42/44 kDa mitogen-activated protein kinases (P42/44mapk), which then activate (+) l-arginine transport. The up-regulation in the endothelial l-arginine/NO pathway by adenosine is associated with an increase in NO. NO activates hCHOP and C/EBPα transcription factor complex formation, which migrates to the nucleus of the endothelial cells and binds, as a complex, to a consensus sequence located on the promoter region of the SLC29A1 gene (for hENT1).
This phenomenon results in reduced transcriptional activity of the SLC29A1 promoter, leading to reduced levels of the hENT1 mRNA and protein. As a consequence, a decreased hENT1 transport-like activity could result in reducing the removal of the endogenous nucleoside adenosine from the extracellular medium in HUVEC. The reduced adenosine transport via hENT1 detected in HUVEC from gestational diabetes could also result from the inhibition (−) by PKC or P42/44mapk. Notably, hyperglycaemia (glucose) may be proposed as a regulator of the illustrated vicious circle since it might increase (+) both eNOS and NO levels. hCHOP, a key transcriptional regulator of the SLC29A1 gene, has been demonstrated to be increased (+) by high glucose and diabetes.
Editor's choice: Targeting cancer vasculature via endoglin/CD105: a novel antibody-based diagnostic and therapeutic strategy in solid tumours
Endoglin (CD105), a vascular target within the TGF-β receptor complex
CD105 is a homodimeric transmembrane protein that belongs to the TGF-β receptor complex and plays a key role in angiogenesis. Its expression is highly restricted to endothelial cells where its levels affect the response to TGF-β by modulating key cellular processes including proliferation, differentiation, and migration. Through its interactions with the TGF-β receptors type I and II, CD105 regulates their phosphorylation status and signalling ability. In endothelial cells, TGF-β activates two type I receptor pathways with opposite effects: ALK-5, which induces the phosphorylation of the intracellular mediators Smad 2/3, and ALK-1, which promotes the phosphorylation of Smad 1/5. CD105 binds TGF-β by associating with its type II signalling receptors: this tri-molecular complex activates endothelial cells via ALK-1 or inhibits their proliferation and migration via ALK-5.
Downstream signals include phosphorylation of Smads, which form heteromeric complexes with the common mediator Smad 4 and accumulate in the nucleus, acting as transcription factors regulating the expression of target genes. Based on its biological features and on its overexpression on highly proliferating tumor-associated endothelial cells, CD105 represents a powerful vascular target for cancer therapy. Along this line, anti-CD105 monoclonal antibodies are under investigation within phase I/II trials in metastatic cancer patients with promising evidence of clinical activity.
Abbreviations: TGF-β, transforming growth factor-β; ALK1, activin receptor-like kinases-1; ALK5, activin receptor-like kinases-5; ECs, endothelial cells; TβR-II, TGF-β receptor type II.
Schematic diagram of strategies for site-targeted imaging of stem/progenitor cells using contrast-enhanced ultrasound (CEU)
With the rapid progression of research into stem or progenitor cell therapy, there is a growing need to develop imaging modalities to track progenitor cells in vivo after their delivery. The ability to track delivered cells using contrast-enhanced ultrasound (CEU) has only been recently investigated. This schematic diagram shows the potential strategies for CEU-targeted imaging of stem/progenitor cells, such as endothelial progenitor cells (EPCs). One strategy involves alteration/transfection of EPCs to express a specific marker protein on the cell surface. Microbubbles (MB) targeted to engrafted EPCs could be constructed by the attachment of the ligand/antibody targeted against the specific cell surface marker protein on the microbubble surface (left panel, inset). When this EPC-targeted MB is administered intravenously, the MBs circulate to target sites where they can bind to EPCs that are engrafted within the vasculature and subsequently be imaged by CEU imaging techniques. The other described strategy involves manipulating EPCs to fully engulf MB prior to cell delivery. Once delivered and engrafted, ultrasonic imaging could then detect MBs present and retained within engrafted EPCs (right panel, inset). Future work will likely focus on 1) the refinement of these two strategies; 2) pre-clinical testing in relevant animal models of disease, and 3) the development of new techniques for progenitor cell-targeted CEU imaging.
Abbreviations: MB – microbubble; EPC – endothelial progenitor cell; PEG – polyethylene glycol; CEU – contrast-enhanced ultrasound
Nitrite homeostasis is determined by nitric oxide (NO) generation from NO synthases and dietary consumption of nitrate. Nitrate enters the stomach and then circulates in the blood and is converted into nitrite via salivary bacteria containing nitrate reductase. Nitrite derived from the diet and NOS activity rapidly accumulates in the plasma and is transported into tissues such as the heart. Nitrite is then stored in the myocardium and is metabolized into NO during hypoxia or ischaemia.
Agonist-induced impairment of glycocalyx exclusion properties: contribution to coronary effects of adenosine
Cartoons illustrating the concept of agonist-stimulated glycocalyx modulation as means of increasing functionally perfused blood volume in the capillaries. Top and middle panels: longitudinal (top) and cross (middle) sections of two capillaries depicting proposed relations between glycocalyx exclusion of flowing RBCs/plasma (yellow) and capillary tube haematocrit/vascular volume available for perfusion, in control conditions (left), and during agonist stimulation (right). Under control conditions, a solid glycocalyx (grey), which excludes circulating blood and macromolecules (a: see article references 10,23,24,33,60; b: article references 47,48) lines the endothelium (black), causing capillary tube haematocrit to be low (represented by the low number of RBCs in the left section; c: article references 16,17,19,23,26,33). During stimulation with vasoactive substances, the glycocalyx becomes accessible for large dextrans with a very limited effect on RBC accessibility (d: article references 33,60), and capillary tube haematocrit increases (e: article references 19,33). These data suggest that these agonists can ‘recruit’ capillary volume for perfusion by increasing accessibility of the glycocalyx for flowing plasma, but μ-PIV measurements are needed for definite proof. Vasodilators such as adenosine, bradykinin, and SNP (f: article references 33,60) have been indicated to induce recruitment of blood-excluding glycocalyx volume in capillaries. We propose that the induced increase
The insets at the bottom give a simplified illustration of the proposed constitution of the glycocalyx under control conditions and during agonist stimulation. The scaffold of the glycocalyx is formed by a mesh of anionic polysaccharide structures (proteoglycans, glycosaminoglycans, and glycoproteins) produced by the EC. In addition, association of the glycans with plasma proteins from the circulating blood permits the glycocalyx to hinder accessibility of flowing plasma and large dextrans. Agonists may increase shedding of glycosaminoglycan components (g: article references 30,82) and of plasma proteins, possibly via nitric oxide (NO) and reactive oxygen species (ROS), resulting in an increased permeation of large dextrans and presumably allowing axial plasma flow through a larger cross-section of the vessel.
Endothelial cell-borne platelet bridges selectively recruit monocytes in human and mouse models of vascular inflammation
The mechanisms by which secretory SMCs promote the recruitment of monocytes from flowing blood.
The routes by which inflammatory leucocytes are recruited to the artery wall and are enriched within the atherosclerotic environment are poorly described. Here, observations using co-culture and animal models of the diseased artery wall, allied with data from previous studies, show that interactions between cells of the diseased artery wall can generate signals that coordinate the preferential recruitment of monocytes from flowing blood. This process depends upon crosstalk between secretory smooth muscle cells (SMCs) and endothelial cells (ECs), which leads to the plasmin-dependent generation of active transforming growth factor-β1 (TGF-β1). This agent induces von Willebrand factor (vWF) expression on ECs so that platelets are recruited and activated and act as an adhesive bridge between the EC surface and the flowing blood. Monocytes are preferentially recruited from blood by tethering to platelet P-selectin and preferentially activated by CCL2 (MCP-1), which is released from ECs by the action of the platelet chemokine CXCL4 (PF4; platelet factor 4), stimulating the endothelial cell receptor CXCR3b.
This process greatly enriches for monocytes at the EC surface and supports monocyte transmigration. Thus, a series of cellular and molecular interactions initiated by cells resident within the diseased artery are shown to result in the specific recruitment of the predominant population of inflammatory leucocytes recruited during atherogenesis.