Inflammation in the Neurovascular Unit Flashcards
What is the NVU?
- Now becoming clear that neurons, glia and microvessels are organized into well-structured NVUs
- The BBB is the cerebral microvascular endothelium, which, together with astrocyte end feet pericytes, neurons and the ECM constitute a NVU that is essential for the function of the CNS
- The NVU is a conceptual model by which close interactions of brain cells with the brain endothelium, and ECM contributes to the maintenance of brain homeostasis and functions.
- The idea is that there is reciprocal transmission of information – from the neurons/astrocytes to the endothelia and back.
So the components are:
• Astrocytes (astrocyte end feet), microglia, neurons
• Endothelial cells and pericytes
• ECM
How do endothelial-endothelial interactions contribute to the NVU?
• Endothelial cells are kept together via tight junctions → strong interaction between different classes of adhesion molecule
Luminal (blood) compartment:
• Homodimeric interactions between occludins or claudins 3,5 and 12
• They interact with intracellular adaptor proteins zona occludens ZO1/ZO2
• These adaptors then interact with the cytoskeleton of the cell
• These form the tight junctions
Middle:
• Homodimeric interaction between Junction adhesion molecules
• They interact with ZO-1, and hence with the cell cytoskeleton
Abluminal (brain) compartment:
• Adherans junctions mediated by E-cadherin
• These interact with alpha and beta catenin adaptors, and then these mediate the interactions with the cytoskeleton
Describe the ECM as a component of the NVU.
• Complex network of extracellular glycoproteins and proteoglycans that surround cells
• Structural role → extracellular glue that holds cells together in the shape of the tissue/organ
• Functional role → important modulator of cell survival, differentiation and activation
• The matrix of the BBB provides a barrier to the transmigration or leakage of erythrocytes during haemorrhage and leukocyte subsets in response to inflammatory stimuli
• There are two types of ECM molecule:
− Glycoproteins (long, glycosylated peptides) → laminin, fibronectin and collagen
− Proteoglycans (short core protein with long chains of glycosaminoglycans) → hepartin sulfate proteoglycan, chondroitin sulfate proteoglycan
Laminin:
• Composed of 3 chains that form a cruciform structure
− An alpha chain
− Two smaller chains made up of a beta and gamma chain
• Within the structure are different binding domains:
− Binding domain for collagen IV (on the beta and gamma chain globular domains)
− Binding domains for cell surface receptors → (on the alpha chain globular domain) these are the most important function of laminin, as they are sensed by the adhesion receptors on the epithelium
− Binding domain for heparin (on the alpha globular domain and beta/gamma coiled coil)
• The globular domains promote polymerization into a network
Fibronectin:
• Dimer of two nearly identical monomers linked by disulphide bonds
• Alternative splicing of the pre-MRNA leads to several isoforms
• Soluble plasma fibronectin is a major protein component of blood plasma (300ug/ml) and is produced in the liver by hepatocytes
• Insoluble cellular fibronectin is a major component of the ECM – secreted by primarily fibrobalsts, and is assembled into an insoluble matrix.
• Has various binding domains within its structure similar to laminin
Collagen:
• The main component of BMs is collagen IV
• Made of a triple helix, defined by specific alpha subunits
• The alpha helices have Gly-X-Y repeating motifs – X usually proline, Y usually hydroxyproline.
• A characteristic feature of collagen ist he presence of 21-26 interruptions in this sequence which can provide molecular flexibility
• The NC domains of each alpha helices form specific interactions via their NC domains, out of 76 possible combinations, only 3 have been found:
− a1a1a2
− a3a4a5
− a5a5a6
• The N terminals then interaction to give interchain cross-iinking of 4 triple helical molecules
Describe integrins as a component of the NVU
• Heterodimers of alpha and beta subunits
• Have a large extracellular domain and a single membrane spanning domain and short cytoplasmic domain
• IN mammals, 18 alpha and 8 beta subunits combine to make 24 integrins
• Integrins bind to RGD domains (Arg-Gly-Asp) domains in ECM components
• Certain dimers are ECM-specific receptors:
− Fibronectin → a5b1, avb3
− Laminin → a1b1, a6b1, a6b4
• Integrins exist in an unactivated and activated conformation → inactive, unliganded integrin (ie, if there is no ECM) will adopt a low affinity, closed state
• If there is ECM present, they integrins ‘grab’ onto it and adopt an active conformation
• Integrins have this physical function, but they also have a signaling function
− When integrins bind to the ECM, they induce an intracellular signaling resppnse
− They recruit intracellular adaptor proteins such as vinculin, talin and paxilin and interact with intracellular b-actin stress fibres at points known as focal adhesions
− Not only this, but intracellular signaling pathways such as PI3K/Akt, ERK and SRC are activated → prosurvival
− So if the cells don’t bind to ECM, you don’t get this activation, and they may undergo apoptosis
Cellular adhesion to the ECM
• Astrocyte adhesion to the ECM results in cellular spreading and formation of stress fibres/focal adhesions
Integrins convery outside in and inside out signals:
• Outside in → if integrins bind to the ECM, this results in an intracellular signaling for cell survival
• Inside out → cellular activation can lead to focal adhesion formation, and this leads to ECM assembly. This may be a mechanism by which the cells themselves can dictate how the ECM forms to best mediate binding.
Integrins mediate the integrity of the NVU:
• They are expressed on the abluminal compartment and bind to the BM
• They are also expressed on the astrocyte end feet, and these bind to the BM
• This makes the whole structure very compact and stable
What is the function of the NVU?
• BBB is a selective barrier formed by the endothelial cells that line the cerebral microvessels
• Acts as a physical barrier because complex tight junctions force molecular traffic to take a transcelluar route
− Small lipophilic molecules such as O2, CO2, barbiturate drugs and ethanol can diffuse freely through the memrbanes
− Specific transporters regulates the traffick of other molecules and exludes potentially harmful compounds
• A combination of intracellular and extracellular enzymes provides a metabolic barrier
− Peptidases and nucleotideases metabolise peptides and ATP
− MAO and cytochrome P450 can inactivate many neuroactive and toxic comounds
• The BBB has a much lower degree of transcytosis transporter activity than the peripheral endothelium
• Hence, the term BBB covers a range of passive and active functions of the brain endothelium
• The BBB has several roles:
− Supplies the brain with essential nutrients
− Mediates efflux of many waste products
− Restricts the ionic and fluid movements to produce a prain ISF that provides an optimal medium for neuronal function
How are the BBB properties induced?
• What causes the endothelium of blood vessels growing into the brain during development to become so specialsed?
• the close cell-cell associations of the NVU lead to the suggestion that they could mediate the induction of the specific features of the barrier phenotype
• Astrocytes show a number of different morphologies depending on location and association with other cells
− Of the 11 distinct phenotypes, 8 involve specific interactions with blood vessels
− There is now strong evidence that astrocytes can upregulate many BBB features, leading to tighter tight junctions, the expression and polarized location of transporters and specialized enzyme systems
− Astrocytes are necessary for the correct association of endothelial cells and pericytes in vitro
− The converse induction, where brain endothelium enhances growth and differentiation of astrocytes has also been shown
What is the response of the NVU to injury?
Thornton et al,:
• The first thing you see is a vascular response, the endothelial cells responding to injury
• Endothelial cells respond to inflammation and injury by expressing cell adhesion molecules such as VCAM and ICAM
• You can see this in experimental models of stroke (MCAO)
− Stroke causes a significant increase in expression of ICAM-1 and VCAM-1
− This response is lost of you do it in an IL-1B KO mouse, so IL-1 is the driver for expression of these molecules in acute brain injury.
• Alongside expression of adhesion molecules, you have destabilization of the NVU → loss of interaction between the blood vessel and the BM
− In addition to degradation, you have some overexpression of matrix molecules, eg, fibronectin and laminin
→ The result of these increases in adhesion molecules and ECM breakdown is neutrophil migration into the brain
→ Neutrophils secrete proteases that can further help them enter the brain
Describe the role of neutrophils in acute brain injury
• Stroke induces CXC chemokine production
McColl et al,:
• Systemic inflammation (eg, infection) increases the risk of stroke and associated with poorer clinical outcome
• Brain damage and neurological deficit 24h after MCA occlusion exacerbated by systemic LPS administration.
• Exacerbaton dependent on IL-1, because co-admin of IL-1RA abolished the effect of LPS on brain damage
• Systemic admin of IL-1 increased ischemic damage to a similar extent as LPS, increased levels of IL-6 and acute phase proteins, and increased neutrophil selective chemokines.
• Neutrophil mobolisation were aggravated by IL-1 before increased ischaemic damage seen.
• Neutropenia abolished effects of systemic IL-1
➢ Suggests that IL-1 induced potentiation of neutrophil mobilization via CXC chemokines is detrimental to outcome after stroke
➢ May explain poorer outcome in stroke patients presenting with infection.
→ Chemokine production is exacerbated by systemic inflammation (show in the study by injection of LPS or IL-1)
→ Inflammatory mediators synergise with the injury to increase chemokines
→ Stroke increases circulating neutrophils and neutrophil recruitment.
How important are these neutrophils to injury?
• Can target the things neutrophils produce, eg, MMPs → this is protective
• Deplete neutrophils from an organism using PMN antibody → reduces the brain injury induced by stroke
• Neutrophils are looking for things to phagocytose – but this isn’t present. So they are just causing collateral damage.
Explain the role of inflammation on specific components of the NVU (Endothelium, Astrocytes, ECM,
The Endothelium:
• Under normoxic conditions, the intact resting endothelium provides the anti-inflammatory characteristics of the cerebral microvasculature and contributes to the physical barrier
• Endothelial cells respond to TFNa and IL-1B by translocation of P-selectin stored in Weibel Palade bodies to the luminal surface, and the subsequent translation of E-selectin mRNA. This facilitates firm adhesion of leukocytes.
• Both TNFa and IL-1B have been shown to downregulate the integrin a6b1
• These events could contribute to the development of edema during focal ischaemia → FI stimulates loss in the solute permeability barrier, allowing leakage of electrolytes and plasma proteins
Astrocytes:
• There is considerable evidence that astrocytes can serve immune function, can potentially present antigen, have phagocytic properties and can generate chemokines and cytokines → TNFa, IL-1B, MMP
• MMP are generated in the ischaemic core of the evolving lesion within 1-2 hours following the onset of focal ischaemia
ECM:
• Endothelial cells and astrocytes adhere firmly to matrix proteins that are ligands for adhesion receptors of the integrin and dystroglycan families
• Cellular matrix receptors are central to the integrity of the microvasculature
• The cerebral microvessel wall displays rapid changes in response to focal ischaemia that precede the influx of inflammatory cells. Significant changes in the matrix integrity of the basal lamina and in matrix receptors occur simultaneously with neuron injury
− The expression of the matrix constituents of the basal lamina, including laminin 1 and 5, collagen IV, fibronectin and perlecan decrease substantially following onset of FI.
− The endothelial cell B1-integrin receptor and integrin a6b4 on astrocyte end feet decrease in the first 60 mins following MCAO
− Separation of the end feet from the matrix of select microvessels occurs in this period, coinciding with the accumulated fluid in the extravascular space
Neurons:
• The impact of inflammation initiated by focal ischemia on neuron and axon viability has been debated.
• The concordance of cytokine generation and leukocyte infiltration in the early hours following ischemia onset has suggested that both events can contribute to neuron injury.
• The cytokines TNF-α and IL-1β can derive from endothelial cells, astrocytes, microglia, and neurons themselves.
• Recent work has indicated that in isolated conditions, dorsal root ganglia (DRG) neurons can stimulate isolated PMN leukocytes to initiate their respiratory burst with free radical release leading to an increase in Ca+2 transients in the neurons.(Shaw et al., 2008)
• One consequence is that PMN leukocyte infiltration could aggravate neuron injury.
• While IL-1β expression has been associated with neuron injury, a recent report suggests that it could stimulate neurite outgrowth from DRG neurons, and could support nerve regeneration.
Microglia:
• Microglia can play a number of roles in inflammatory responses of the CNS. While normally quiescent, these cells have Fc receptors, complement receptors, receptors for a number of cytokines, chemokine receptors, CD40, Fas and Fas ligand.
• Microglia can also generate TNF-α and IL-1β in addition to other cytokines, and are known to regulate T-cell mediated immune processes.(Aloisi, 2001
• Within the first 24 hours following the onset of focal ischemia, peripheral blood cells of the monocyte/macrophage lineage enter the neuropil. At the same time microglia become activated, undergo shape change, and express markers of inflammation
Summarise the response of the NVU after acute injury
After injury there is:
• Production of various inflammatory mediators
• Activation of endothelial cells and astrocytes
• Downregulation of integrins, upregulation of adhesion molecules
• Degredation fo the ECM in the BM and parenchyma
• Loosening of physical interactions between endothelia, astrocyte end-feet and the ECM.
➢ Leaky BBB
➢ Infiltration of circulating molecules
➢ Infiltration of neutrophils into the brain tissue
Does the NVU recover after injury?
• Acute injury:
− Integrins are degraded and expression decreasd
− ECM molecules degraded
− Astrocyte end feet detach
• Recovery:
− ECM are increased in astrocyte end feet and in the parenchyma
− Integrin expression increases
− Partial structural and functional recovery of the NVU
