Menno van Lookeren Campagne

VEGF antagonism reduces edema formation and tissue damage after ischemia/reperfusion injury in the mouse brain

VEGF is mitogenic, angiogenic, and a potent mediator of vascular permeability. VEGF causes extravasation of plasma protein in skin bioassays and increases hydraulic conductivity in isolated perfused microvessels. Reduced tissue oxygen tension triggers VEGF expression, and increased protein and mRNA levels for VEGF and its receptors (Flt-1, Flk-1/KDR) occur in the ischemic rat brain. Brain edema, provoked in part by enhanced cerebrovascular permeability, is a major complication in central nervous system pathologies, including head trauma and stroke. The role of VEGF in this pathology has remained elusive because of the lack of a suitable experimental antagonist. We used a novel fusion protein, mFlt(1-3)-IgG, which sequesters murine VEGF, to treat mice exposed to transient cortical ischemia followed by reperfusion. Using high-resolution magnetic resonance imaging, we found a significant reduction in volume of the edematous tissue 1 day after onset of ischemia in mice that received mFlt(1-3)-IgG. 8-12 weeks after treatment, measurements of the resultant infarct size revealed a significant sparing of cortical tissue. Regional cerebral blood flow was unaffected by the administration of mFlt(1-3)-IgG. These results demonstrate that antagonism of VEGF reduces ischemia/reperfusion-related brain edema and injury, implicating VEGF in the pathogenesis of stroke and related disorders.

CRIg: A Macrophage Complement Receptor Required for Phagocytosis of Circulating Pathogens

The complement system serves an important role in clearance of pathogens, immune complexes, and apoptotic cells present in the circulation. Complement fragments deposited on the particle surface serve as targets for complement receptors present on phagocytic cells. Although Kupffer cells, the liver resident macrophages, play a dominant role in clearing particles in circulation, complement receptors involved in this process have yet to be identified. Here we report the identification and characterization of a Complement Receptor of the Immunoglobulin superfamily, CRIg, that binds complement fragments C3b and iC3b. CRIg expression on Kupffer cells is required for efficient binding and phagocytosis of complement C3-opsonized particles. In turn, Kupffer cells from CRIg-deficient mice are unable to efficiently clear C3-opsonized pathogens in the circulation, resulting in increased infection and mortality of the host. CRIg therefore represents a dominant component of the phagocytic system responsible for rapid clearance of C3-opsonized particles from the circulation.

Macrophage complement receptors and pathogen clearance

Phagocytosis, an important mechanism of the host‐defence system and a primary function of macrophages, is facilitated by opsonization, a process by which serum components tag pathogens for recognition by neutrophils and macrophages. Complement component C3 is central to opsonization. Its first cleavage product, C3b, forms the multisubunit enzyme, C3bBb, which proteolytically cleaves additional C3 molecules on the pathogen surface. C3b is further degraded to iC3b, C3c and C3dg, products that serve as ligands for selective complement receptors on leukocytes. This receptor–ligand interaction subsequently modulates immune responses or directly targets the pathogen for clearance by phagocytosis. Although a central role for C3 in phagocytosis of certain pathogens is well accepted, the receptors orchestrating the phagocytic response have not been well characterized. The recent structures of C3 and its breakdown products have increased our insights into the molecular basis of complement activation and recognition by their receptors. Here we review the biology of macrophage receptors for C3 fragments and discuss their role in the host response to pathogens.

Structure of C3b in complex with CRIg gives insights into regulation of complement activation.

The complement system is a key part of the innate immune system, and is required for clearance of pathogens from the bloodstream. After exposure to pathogens, the third component of the complement system, C3, is cleaved to C3b which, after recruitment of factor B, initiates formation of the alternative pathway convertases. CRIg, a complement receptor expressed on macrophages, binds to C3b and iC3b mediating phagocytosis of the particles, but it is unknown how CRIg selectively recognizes proteolytic C3-fragments and whether binding of CRIg to C3b inhibits convertase activation. Here we present the crystal structure of C3b in complex with CRIg and, using CRIg mutants, provide evidence that CRIg acts as an inhibitor of the alternative pathway of complement. The structure shows that activation of C3 induces major structural rearrangements, including a dramatic movement (>80 Å) of the thioester-bond-containing domain through which C3b attaches to pathogen surfaces. We show that CRIg is not only a phagocytic receptor, but also a potent inhibitor of the alternative pathway convertases. The structure provides insights into the complex macromolecular structural rearrangements that occur during complement activation and inhibition. Moreover, our structure–function studies relating the structural basis of complement activation and the means by which CRIg inhibits the convertases provide important clues to the development of therapeutics that target complement.

A Crohn/'s disease variant in Atg16l1 enhances its degradation by caspase 3

Crohn’s disease is a debilitating inflammatory bowel disease (IBD) that can involve the entire digestive tract. A single-nucleotide polymorphism (SNP) encoding a missense variant in the autophagy gene ATG16L1 (rs2241880, Thr300Ala) is strongly associated with the incidence of Crohn’s disease. Numerous studies have demonstrated the effect of ATG16L1 deletion or deficiency; however, the molecular consequences of the Thr300Ala (T300A) variant remains unknown. Here we show that amino acids 296–299 constitute a caspase cleavage motif in ATG16L1 and that the T300A variant (T316A in mice) significantly increases ATG16L1 sensitization to caspase-3-mediated processing. We observed that death-receptor activation or starvation-induced metabolic stress in human and murine macrophages increased degradation of the T300A or T316A variants of ATG16L1, respectively, resulting in diminished autophagy. Knock-in mice harbouring the T316A variant showed defective clearance of the ileal pathogen Yersinia enterocolitica and an elevated inflammatory cytokine response. In turn, deletion of the caspase-3-encoding gene, Casp3, or elimination of the caspase cleavage site by site-directed mutagenesis rescued starvation-induced autophagy and pathogen clearance, respectively. These findings demonstrate that caspase 3 activation in the presence of a common risk allele leads to accelerated degradation of ATG16L1, placing cellular stress, apoptotic stimuli and impaired autophagy in a unified pathway that predisposes to Crohn’s disease.

Tumor-suppressor p53 is expressed in proliferating and newly formed neurons of the embryonic and postnatal rat brain: comparison with expression of the cell cycle regulators p21Waf1/Cip1, p27Kip1, p57Kip2, p16Ink4a, cyclin G1, and the proto-oncogene Bax.

The tumor‐suppressor protein p53 has been implicated in cell cycle arrest and apoptotic cell death in dividing cells (Yonish‐Rouach et al. [1991] Nature 352:342–347). To elucidate possible functions of p53 in the regulation of cell division and cell death during development of the rat central nervous system, we compared the spatial and temporal expressions of p53 mRNA and protein with those of its transcriptional targets Bax, p21Waf1, and cyclin G1 and with the cyclin‐dependent kinase inhibitors p27Kip1, p57Kip2, and p16Ink4a.

Geographic Atrophy: Clinical Features and Potential Therapeutic Approaches

In contrast to wet age-related macular degeneration (AMD), where loss of vision is typically acute and treatment leads to a relatively rapid reduction in retinal fluid and subsequent improvements in visual acuity (VA), disease progression and vision loss in geographic atrophy (GA) owing to AMD are gradual processes. Although GA can result in significant visual function deficits in reading, night vision, and dark adaptation, and produce dense, irreversible scotomas in the visual field, the initial decline in VA may be relatively minor if the fovea is spared. Because best-corrected VA does not correlate well with GA lesions or progression, alternative clinical endpoints are being sought. These include reduction in drusen burden, slowing the enlargement rate of GA lesion area, and slowing or eliminating the progression of intermediate to advanced AMD. Among these considerations, slowing the expansion of the GA lesion area seems to be a clinically suitable primary efficacy endpoint. Because GA lesion growth is characterized by loss of photoreceptors, it is considered a surrogate endpoint for vision loss. Detection of GA can be achieved with a number of different imaging techniques, including color fundus photography, fluorescein angiography, fundus autofluorescence (FAF), near-infrared reflectance, and spectral-domain optical coherence tomography. Previous studies have identified predictive characteristics for progression rates including abnormal patterns of FAF in the perilesional retina. Although there is currently no approved or effective treatment to prevent the onset and progression of GA, potential therapies are being evaluated in clinical studies.

Mechanisms of age-related macular degeneration and therapeutic opportunities.

As the age of the population increases in many nations, age‐related degenerative diseases pose significant socioeconomic challenges. One of the key degenerative diseases that compromise quality of life is age‐related macular degeneration (AMD). AMD is a multi‐faceted condition that affects the central retina, which ultimately leads to blindness in millions of people worldwide. The pathophysiology and risk factors for AMD are complex, and the symptoms manifest in multiple related but distinct forms. The ability to develop effective treatments for AMD will depend on a thorough understanding of the underlying pathophysiology, risk factors, and driver molecular pathways, as well as the ability to develop useful animal models. This review provides an overview of the aforementioned aspects in AMD. Copyright

Secondary Reduction in the Apparent Diffusion Coefficient of Water, Increase in Cerebral Blood Volume, and Delayed Neuronal Death After Middle Cerebral Artery Occlusion and Early Reperfusion in the Rat

It has been reported recently that very delayed damage can occur as a result of focal cerebral ischemia induced by vascular occlusion of short duration. With use of diffusion-, T2-, and contrast-enhanced dynamic magnetic resonance imaging (MRI) techniques, the occlusion time dependence together with the temporal profile for this delayed response in a rat model of transient focal cortical ischemia have been established. The distal branch of the middle cerebral artery was occluded for 20, 30, 45, or 90 minutes. Twenty minutes of vascular occlusion with reperfusion exhibited no significant mean change in either the apparent diffusion coefficient of water (ADC) or the T2 relaxation time at 6, 24, 48, or 72 hours after reperfusion (P = 0.97 and 0.70, respectively). Ninety minutes of ischemia caused dramatic tissue injury at 6 hours, as indicated by an increase in T2 relaxation times to 135% of the contralateral values (P < 0.01). However, at intermediate periods of ischemia (30 to 45 minutes), complete reversal of the ADC was seen at 6 hours after reperfusion but was followed by a secondary decline over time, such that a 25% reduction in tissue ADC was seen at 24 as compared with 6 hours (P < 0.02). This secondary response was accompanied by an increase in cerebral blood volume (CBV), as shown by contrast-enhanced dynamic MRI (120% of contralateral values; P < 0.001), an increase in T2 relaxation time (132%; P < 0.01), together with clear morphological signs of cell death. By day 18, the mean volume of missing cortical tissue measured with high-resolution MRI in animals occluded for 30 and 45 minutes was 50% smaller than that in 90-minute occluded animals (P < 0.005). These data show that ultimate infarct size is reduced after early reperfusion and is occlusion time dependent. The early tissue recovery that is seen with intermediate occlusion times can be followed by cell death, which has a delayed onset and is accompanied by an increase in CBV.

A role of macrophage complement receptor CRIg in immune clearance and inflammation

Complement receptor of the immunoglobulin superfamily (CRIg), also referred to as Z39Ig and V-set and Ig domain-containing 4 (VSIG4), has recently been implicated in the clearance of systemic pathogens and autologous cells. CRIg is exclusively expressed on tissue resident macrophages and binds to multimers of C3b and iC3b that are covalently attached to particle surfaces. Next to functioning as an important clearance receptor, CRIgs extracellular domain inhibits complement activation through the alternative, but not the classical, pathway, providing a novel tool to selectively block this pathway in vivo. Here, we review a role for CRIg in immune clearance, T-cell responses and complement regulation, and discuss the implications for disease manifestation.