Purushottam Jha

Role of Complement and Complement Membrane Attack Complex in Laser-Induced Choroidal Neovascularization

Choroidal neovascularization (CNV), or choroidal angiogenesis, is the hallmark of age-related macular degeneration and a leading cause of visual loss after age 55. The pathogenesis of new choroidal vessel formation is poorly understood. Although inflammation has been implicated in the development of CNV, the role of complement in CNV has not been explored experimentally. A reliable way to produce CNV in animals is to rupture Bruch’s membrane with laser photocoagulation. A murine model of laser-induced CNV in C57BL/6 mice revealed the deposition of C3 and membrane attack complex (MAC) in the neovascular complex. CNV was inhibited by complement depletion using cobra venom factor and did not develop in C3−/− mice. Anti-murine C6 Abs in C57BL/6 mice inhibited MAC formation and also resulted in the inhibition of CNV. Vascular endothelial growth factor, TGF-β2, and β-fibroblast growth factor were elevated in C57BL/6 mice after laser-induced CNV; complement depletion resulted in a marked reduction in the level of these angiogenic factors. Thus, activation of complement, specifically the formation of MAC, is essential for the development of laser- induced choroidal angiogenesis in mice. It is possible that a similar mechanism may be involved in the pathophysiology of other angiogenesis essential diseases.

Complement Activation via Alternative Pathway Is Critical in the Development of Laser-Induced Choroidal Neovascularization: Role of Factor B and Factor H

The objective of this study was to explore the role of classical, lectin, and alternative pathways of complement activation in laser-induced choroidal neovascularization (CNV). The classical and alternative pathways were blocked in C57BL/6 mice by small interfering RNAs (siRNA) directed against C1q and factor B, respectively. C4−/− mice developed CNV similar to their wild-type controls and inhibition of C1q by siRNA had no effect on the development of CNV. In contrast, CNV was significantly inhibited (p < 0.001) in C5−/− mice and C57BL/6 mice treated with factor B siRNA. Inhibition of the alternative pathway by factor B siRNA resulted in decreased levels of membrane attack complex and angiogenic factors–vascular endothelial growth factor and TGF-β2. Furthermore, factor B was up-regulated in complement sufficient C57BL/6 mice at day 1 postlaser and remained elevated at day 7. Significantly reduced levels of factor H were observed at day 3 in these animals. In conclusion, our results demonstrate that activation of the factor B-dependent alternative pathway, but not the classical or lectin pathways, was essential for the development of CNV in mouse model of laser-induced CNV. Thus, specific blockade of the alternative pathway may represent a therapeutically relevant strategy for the inhibition of CNV.

CD59, a Complement Regulatory Protein, Controls Choroidal Neovascularization in a Mouse Model of Wet-Type Age-Related Macular Degeneration

We have shown that membrane attack complex (MAC) formation via the activation of the alternative pathway plays a central role in the laser-induced choroidal neovascularization (CNV). This study was undertaken to understand the role of a complement regulatory protein, CD59, which controls MAC assembly and function, in this model. CNV was induced by laser photocoagulation in C57BL/6 and Cd59a−/− mice using an argon laser. Animals from each group were sacrificed on day 1, 3, 5, and 7 postlaser. Retinal pigment epithelium-choroid-scleral tissue was examined to determine the incidence and size of CNV complex, and semiquantitative RT-PCR and Western blot analysis for CD59a was studied. Recombinant soluble mouse CD59a-IgG2a fusion (rsCD59a-Fc) protein was injected via i.p. or intravitreal routes 24 h before laser. Our results demonstrated that CD59a (both mRNA and protein) was down-regulated during laser-induced CNV. Cd59a−/− mice developed CNV complex early in the disease process. Increased MAC deposition was also observed in these Cd59a−/− mice. Administration of rsCD59a-Fc inhibited the development of CNV complex in the mouse model by blocking MAC formation and also inhibited expression of angiogenic growth factors. These data provide strong evidence that CD59a plays a crucial role in regulating complement activation and MAC formation essential for the release of growth factors that drive the development of laser-induced CNV in mice. Thus, our results suggest that the inhibition of complement by soluble CD59 may provide a novel therapeutic alternative to current treatment.

The role of complement in ocular pathology.

Functionally active complement system and complement regulatory proteins are present in the normal human and rodent eye. Complement activation and its regulation by ocular complement regulatory proteins contribute to the pathology of various ocular diseases including keratitis, uveitis and age-related macular degeneration. Furthermore, a strong relationship between age-related macular degeneration and polymorphism in the genes of certain complement components/complement regulatory proteins is now well established. Recombinant forms of the naturally occurring complement regulatory proteins have been exploited in the animal models for treatment of these ocular diseases. It is hoped that in the future recombinant complement regulatory proteins will be used as novel therapeutic agents in the clinic for the treatment of keratitis, uveitis, and age-related macular degeneration.

Suppression of Complement Regulatory Proteins (CRPs) Exacerbates Experimental Autoimmune Anterior Uveitis (EAAU)

This study was undertaken to explore the role of complement regulatory proteins (CRPs) in experimental autoimmune anterior uveitis (EAAU). We observed that the levels of CRPs, Crry and CD59, in the eyes of Lewis rats increased during EAAU and remained elevated when the disease resolved. The in vivo role of these CRPs in EAAU was explored using neutralizing mAbs, antisense oligodeoxynucleotides (AS-ODNs), and small interfering RNAs against rat Crry and CD59. Suppression of Crry in vivo at days 9, 14, or 19 by neutralizing mAb or AS-ODNs resulted in the early onset of disease, the exacerbation of intraocular inflammation, and delayed resolution. Suppression of CD59 was only effective when the Abs and ODNs were given before the onset of disease. The most profound effect on the disease was observed when a mixture of Crry and CD59 mAbs or AS-ODNs was administered. A similar effect was observed with a combination of Crry and CD59 small interfering RNA. There was no permanent histologic damage to ocular tissue after the inflammation cleared in these animals. Increased complement activation as determined by increased deposition of C3, C3 activation fragments, and membrane attack complex was observed in the eyes of Lewis rats when the function and/or expression of Crry and CD59 was suppressed. Thus, our results suggest that various ocular tissues up-regulate the expression of Crry and CD59 to avoid self-injury during autoimmune uveitis and that these CRPs play an active role in the resolution of EAAU by down-regulating complement activation in vivo.

Recombinant membrane-targeted form of CD59 inhibits the growth of choroidal neovascular complex in mice.

This study was designed to explore the effect of recombinant, membrane-targeted CD59 (rCD59-APT542) on the growth and size of fully developed neovascular complex using the murine model of laser-induced choroidal neovascularization (CNV). CNV was induced by laser photocoagulation in C57BL/6 mice using an argon laser, and the animals received rCD59-APT542 via intravitreal (ivt) route. Western blot analysis, immunohistochemistry, and total complement hemolytic assay demonstrated that exogenously administered rCD59-APT542 was incorporated as well as retained in RPE and choroid and was functionally active in vivo. Single ivt injection during the growth of the CNV (i.e. at day 3 post-laser) resulted in ∼79% inhibition of the further growth of neovascular complex. The size of the CNV complex was significantly (p < 0.05) reduced by the administration of rCD59-APT542 after the CNV complex has fully developed (i.e. at day 7 post-laser). Treatment with rCD59-APT542 blocked the formation of membrane attack complex (MAC), increased apoptosis and decreased cell proliferation in the neovascular complex. On the basis of results presented here we conclude that recombinant membrane targeted CD59 inhibited the growth of the CNV complex and reduced the size of fully developed CNV in the laser-induced mouse model. We propose that a combination of two mechanisms: increased apoptosis and decreased cell proliferation, both resulting from local inhibition of MAC, may be responsible for inhibition of CNV by rCD59-APT542.

Vaccinia complement control protein: multi-functional protein and a potential wonder drug.

Vaccinia virus complement control protein (VCP) was one of the first viral molecules demonstrated to have a role in blocking complement and hence in the evasion of host defense. Structurally it is very similar to the human C4b-BP and the other members of complement control protein. Functionally it is most similar to the CR1 protein. VCP blocks both major pathways of complement activation. The crystal structure of VCP was determined a little over a year ago and it is the only known structure of an intact and complete complement control protein. In addition to binding complement, VCP also binds to heparin. These two binding abilities can take place simultaneously and contribute to its many function and to its potential use in several inflammatory diseases, e.g. Alzheimer’s disease (AD), CNS injury, xenotransplantation, etc. making it a truly fascinating molecule and potential drug.

Role of ocular complement factor H in a murine model of choroidal neovascularization.

The objective of this study was to explore the relationship between local (ie, ocular) complement factor H (CFH) and choroidal neovascularization (CNV) associated with wet age-related macular degeneration (AMD), a leading cause of irreversible blindness, in laser-treated C57BL/6 mice. Immunohistochemical and RT-PCR analysis of retinal pigmented epithelium (RPE)-choroid sclera revealed that the expression of CFH was down-regulated on day 1 with a dramatic increase on days 5 and 7 postlaser injury. Flat mount and Western blot analysis further revealed that membrane attack complex (MAC) expression was up-regulated on days 1 and 3 postlaser injury; however, MAC was down-regulated on days 5 and 7 postinjury but was still higher than in non-injured mice. Similar patterns for CFH and MAC were observed for RPE cells when serial paraffin sections of the laser spots were analyzed. Subretinal injection of siRNA directed against CFH resulted in a threefold suppression of CFH in the RPE and choroid without affecting either CFH levels in the liver or the functional activity of the alternative pathway in the peripheral blood. Ocular knock-down of CFH resulted in increased MAC deposition, which leads to the early onset as well as exacerbation of laser-induced CNV. In conclusion, our findings provide evidence that CFH present on RPE and choroid regulates local MAC formation that is critical for the development of laser-induced CNV.

Relationship between Complement Membrane Attack Complex, Chemokine (C-C Motif) Ligand 2 (CCL2) and Vascular Endothelial Growth Factor in Mouse Model of Laser-induced Choroidal Neovascularization

The present study investigated the interactions among the complement membrane attack complex (MAC), CCL2, and VEGF that occur in vivo during the development of choroidal neovascularization (CNV). We first investigated the sequential expression of MAC, CCL2, and VEGF during laser-induced CNV in C57BL/6 mice. Increased MAC deposition was detected at 1 h, CCL2 increased at 3 h, and VEGF was up-regulated at day 3 post-laser treatment. These results suggested that during laser-induced CNV, MAC, CCL2 and VEGF are formed and/or expressed in the following order: MAC → CCL2 → VEGF. To determine the cross-talk between MAC, CCL2, and VEGF during laser-induced CNV, neutralizing antibodies were injected both systemically and locally to block the bioactivity of each molecule. Blocking MAC formation inhibited CCL2 and VEGF expression and also limited CNV formation, whereas neutralization of CCL2 bioactivity did not affect MAC deposition; however, it reduced VEGF expression and CNV formation. When bioactivity of VEGF was blocked, CNV formation was significantly inhibited, but MAC deposition was not affected. Together, our results demonstrate that MAC is an upstream mediator and effect of MAC on the development of laser-induced CNV can be attributed to its direct effect on VEGF as well as its effect on VEGF that is mediated by CCL2. Understanding the interplay between immune mediators is critical to gain insight into the pathogenesis of CNV.

Complement mediated apoptosis leads to the loss of retinal ganglion cells in animal model of glaucoma.

This study investigated the role of complement in the protection of retinal ganglion cells (RGCs) in chronic ocular hypertension model of glaucoma. Intraocular pressure (IOP) was elevated in the right eye of Lewis rats by laser photocoagulation (two treatments, 7days apart) of episcleral and limbal veins. Left eye did not receive laser treatment and served as control. Animals were injected with cobra venom factor every fifth day starting day 7 after first laser, to deplete the complement system. Animals were sacrificed at 6-week post-laser. Levels of C3 split products and membrane attack complex (MAC) were elevated in the retina of eyes with increased IOP and complement depletion reduced the loss of Brn3a(+) RGCs accompanied by decreased expression of GFAP and reduced MAC deposition. In complement depleted rats with increased IOP, reduced TUNEL(+) cells in ganglion cell layer, and decreased levels of active caspase-8 and active caspase-9 was observed compared to PBS treated complement sufficient rats with increased IOP. Interestingly, complement depletion also resulted in reduction of calcium influx and levels of BAD in the retinal cells of the eyes with increased IOP. Together, our results provide evidence that complement mediated apoptosis plays a pivotal role in the loss of RGCs in chronic ocular hypertension model of glaucoma.