Regulation of inflammation and angiogenesis in the cornea

Inflammation and angiogenesis, the growth of new blood vessels from pre-existing ones, are involved in tumor growth, ocular diseases and wound healing. In ocular angiogenesis, new pathological vessels grow into a specific eye tissue, leak fluid, and disrupt vision. The development of safe and effective therapies for ocular angiogenesis is of great importance for preventing blindness, given that current treatments have limited efficacy or are associated with undesirable side effects. The search for alternative treatment targets requires a deeper understanding of inflammation and how it can lead to angiogenesis in the eye in pathologic situations. This thesis provides new insights into the regulation of inflammation and angiogenesis, particularly at the gene expression and phenotypic levels, in different situations characterized by angiogenesis of the cornea, often called corneal neovascularization. For instance, specific genes and pathways are either endogenously activated or suppressed during active inflammation, wound healing, and during resolution of inflammation and angiogenesis, serving as potential targets to modulate the inflammatory and angiogenic response. In addition, as part of the healing response to restore corneal transparency, inflammation and angiogenesis subside with time in the cornea. In this context, LXR/RXR signaling was found to be activated in a time-dependent manner, to potentially regulate resolution of inflammation and angiogenesis. During regression of new angiogenic capillaries, ghost vessels and empty basement membrane sleeves are formed, which can persist in the cornea for a long time. Here, ghost vessels were found to facilitate subsequent revascularization of the cornea, while empty basement membrane sleeves did not revascularize. The revascularization response observed here was characterised by vasodilation, increased inflammatory cell infiltration and by sprouting at the front of the reperfused vessels. Importantly, reactive oxygen species and nitrous oxide signaling among other pro-inflammatory pathways were activated, and at the same time anti-inflammatory LXR/RXR signaling was inhibited. The interplay between activation and inhibition of these pathways highlights potential mechanisms that regulate corneal revascularization. When treating corneal neovascularization clinically, corticosteroids are in widespread use due to their effectiveness. To minimize the many undesirable side effects associated with corticosteroid use, however, identifying new and more selective agents is of great importance. Here, it was observed that corticosteroids not only suppressed pro-inflammatory chemokines and cytokines, but also activated the classical complement pathway. Classical complement may represent a candidate for further selective therapeutic manipulation to investigate its effect on treatment of corneal neovascularization. In summary, this thesis identifies genes, pathways, and phenotypic responses involved in sprouting and remodeling of corneal capillaries, highlights novel pathways and factors that may regulate inflammation and angiogenesis in the cornea, and provides insights into regulation of capillary regression and reactivation. Further investigation of these regulatory mechanisms may offer alternative and effective treatment targets for the treatment of corneal inflammation and angiogenesis

Transcriptome-wide analysis of differentially expressed chemokine receptors, SNPs, and SSRs in the age-related macular degeneration

Abstract Background Age-related macular degeneration (AMD) is the most common, progressive, and polygenic cause of irreversible visual impairment in the world. The molecular pathogenesis of the primary events of AMD is poorly understood. We have investigated a transcriptome-wide analysis of differential gene expression, single-nucleotide polymorphisms (SNPs), indels, and simple sequence repeats (SSRs) in datasets of the human peripheral retina and RPE-choroid-sclera control and AMD. Methods and results Adaptors and unbiased components were removed and checked to ensure the quality of the data sets. Molecular function, biological process, cellular component, and pathway analyses were performed on differentially expressed genes. Analysis of the gene expression datasets identified 5011 upregulated genes, 11,800 downregulated genes, 42,016 SNPs, 1141 indels, and 6668 SRRs between healthy controls and AMD donor material. Enrichment categories for gene ontology included chemokine activity, cytokine activity, cytokine receptor binding, immune system process, and signal transduction respectively. A functional pathways analysis identified that chemokine receptors bind chemokines, complement cascade genes, and create cytokine signaling in immune system pathway genes (p value < 0.001). Finally, allele-specific expression was found to be significant for Chemokine (C-C motif) ligand (CCL) 2, 3, 4, 13, 19, 21; C-C chemokine receptor (CCR) 1, 5; chemokine (C-X-C motif) ligand (CXCL) 9, 10, 16; C-X-C chemokine receptor type (CXCR) 6; as well as atypical chemokine receptor (ACKR) 3,4 and pro-platelet basic protein (PPBP). Conclusions Our results improve our overall understanding of the chemokine receptors’ signaling pathway in AMD conditions, which may lead to potential new diagnostic and therapeutic targets

Photoreceptor degeneration accompanies vascular changes in a Zebrafish model of diabetic retinopathy

Purpose: Diabetic retinopathy (DR) is a leading cause of vision impairment and blindness worldwide in the working-age population, and the incidence is rising. Until now it has been difficult to define initiating events and disease progression at the molecular level, as available diabetic rodent models do not present the full spectrum of neural and vascular pathologies. Zebrafish harboring a homozygous mutation in the pancreatic transcription factor pdx1 were previously shown to display a diabetic phenotype from larval stages through adulthood. In this study, pdx1 mutants were examined for retinal vascular and neuronal pathology to demonstrate suitability of these fish for modeling DR. Methods: Vessel morphology was examined in pdx1 mutant and control fish expressing the fli1a:EGFP transgene. We further characterized vascular and retinal phenotypes in mutants and controls using immunohistochemistry, histology, and electron microscopy. Retinal function was assessed using electroretinography. Results: Pdx1 mutants exhibit clear vascular phenotypes at 2 months of age, and disease progression, including arterial vasculopenia, capillary tortuosity, and hypersprouting, could be detected at stages extending over more than 1 year. Neural-retinal pathologies are consistent with photoreceptor dysfunction and loss, but do not progress to blindness. Conclusions: This study highlights pdx1 mutant zebrafish as a valuable complement to rodent and other mammalian models of DR, in particular for research into the mechanistic interplay of diabetes with vascular and neuroretinal disease. They are furthermore suited for molecular studies to identify new targets for treatment of early as well as late DR

Genome-wide expression differences in anti-Vegf and dexamethasone treatment of inflammatory angiogenesis in the rat cornea

Angiogenesis as a pathological process in the eye can lead to blindness. In the cornea, suppression of angiogenesis by anti-VEGF treatment is only partially effective while steroids, although effective in treating inflammation and angiogenesis, have broad activity leading to undesirable side effects. In this study, genome-wide expression was investigated in a suture-induced corneal neovascularization model in rats, to investigate factors differentially targeted by dexamethasone and anti-Vegf. Topical treatment with either rat-specific anti-Vegf, dexamethasone, or normal goat IgG (sham) was given to sutured corneas for 48 hours, after which in vivo imaging, tissue processing for RNA microarray, and immunofluorescence were performed. Dexamethasone suppressed limbal vasodilation (P amp;lt; 0.01) and genes in PI3K-Akt, focal adhesion, and chemokine signaling pathways more effectively than anti-Vegf. The most differentially expressed genes were confirmed by immunofluorescence, qRTPCR and Western blot. Strong suppression of Reg3g and the inflammatory chemokines Ccl2 and Cxcl5 and activation of classical complement pathway factors C1r, C1s, C2, and C3 occurred with dexamethasone treatment, effects absent with anti-Vegf treatment. The genome-wide results obtained in this study provide numerous potential targets for specific blockade of inflammation and angiogenesis in the cornea not addressed by anti-Vegf treatment, as possible alternatives to broad-acting immunosuppressive therapy.Funding Agencies|Swedish Research Council [2012-2472]; Swedish Foundation Stiftelsen Synframjandets Forskningsfond/Ogonfonden</p

Genome-wide expression datasets of anti-VEGF and dexamethasone treatment of angiogenesis in the rat cornea

Therapeutics against pathologic new blood vessel growth, particularly those targeting vascular endothelial growth factor (VEGF) are of enormous clinical interest. In the eye, where anti-VEGF agents are in widespread clinical use for treating retinal and corneal blindness, only partial or transient efficacy and resistance to anti-VEGF agents are among the major drawbacks. Conversely, corticosteroids have long been used in ophthalmology for their potency in suppressing inflammation and angiogenesis, but their broad biological activity can give rise to side effects such as glaucoma and cataract. To aid in the search for more targeted and effective anti-angiogenic therapies in the eye, we present here a dataset comparing gene expression changes in dexamethasone versus anti-Vegfa treatment of inflammation leading to angiogenesis in the rat cornea. Global gene expression analysis with GeneChip Rat 230 2.0 microarrays was conducted and the metadata submitted to Expression Omnibus repository. Here, we present a high-quality validated dataset enabling genome-wide comparison of genes differentially targeted by dexamethasone and anti-Vegf treatments, to identify potential alternative therapeutic targets for evaluation.Funding Agencies|Swedish Research Council [2012- 2472]; Swedish Ophthalmological Society Stiftelsen Synframjandets Forskningsfond/Ogonfonden</p

Time-dependent LXR/RXR pathway modulation characterizes capillary remodeling in inflammatory corneal neovascularization

Inflammation in the normally immune-privileged cornea can initiate a pathologic angiogenic response causing vision-threatening corneal neovascularization. Inflammatory pathways, however, are numerous, complex and are activated in a time-dependent manner. Effective resolution of inflammation and associated angiogenesis in the cornea requires knowledge of these pathways and their time dependence, which has, to date, remained largely unexplored. Here, using a model of endogenous resolution of inflammation-induced corneal angiogenesis, we investigate the time dependence of inflammatory genes in effecting capillary regression and the return of corneal transparency. Endogenous capillary regression was characterized by a progressive thinning and remodeling of angiogenic capillaries and inflammatory cell retreat in vivo in the rat cornea. By whole-genome longitudinal microarray analysis, early suppression of VEGF ligand-receptor signaling and inflammatory pathways preceded an unexpected later-phase preferential activation of LXR/RXR, PPAR alpha/RXR alpha and STAT3 canonical pathways, with a concurrent attenuation of LPS/IL-1 inhibition of RXR function and Wnt/beta-catenin signaling pathways. Potent downstream inflammatory cytokines such as Cxcl5, IL-1 beta, IL-6 and Ccl2 were concomitantly downregulated during the remodeling phase. Upstream regulators of the inflammatory pathways included Socs3, Sparc and ApoE. A complex and coordinated time-dependent interplay between pro- and anti-inflammatory signaling pathways highlights a potential anti-inflammatory role of LXR/RXR, PPAR alpha/RXR alpha and STAT3 signaling pathways in resolving inflammatory corneal angiogenesis.Funding Agencies|Swedish Research Council [2012-2472]</p

Revascularization after angiogenesis inhibition favors new sprouting over abandoned vessel reuse

Inhibiting pathologic angiogenesis can halt disease progression, but such inhibition may offer only a temporary benefit, followed by tissue revascularization after treatment stoppage. This revascularization, however, occurs by largely unknown phenotypic changes in pathologic vessels. To investigate the dynamics of vessel reconfiguration during revascularization, we developed a model of reversible murine corneal angiogenesis permitting longitudinal examination of the same vasculature. Following 30 days of angiogenesis inhibition, two types of vascular structure were evident: partially regressed persistent vessels that were degenerate and barely functional, and fully regressed, non-functional empty basement membrane sleeves (ebms). While persistent vessels maintained a limited flow and retained collagen IV+ basement membrane, CD31+ endothelial cells (EC), and alpha-SMA+ pericytes, ebms were acellular and expressed only collagen IV. Upon terminating angiogenesis inhibition, transmission electron microscopy and live imaging revealed that revascularization ensued by a rapid reversal of EC degeneracy in persistent vessels, facilitating their phenotypic normalization, vasodilation, increased flow, and subsequent new angiogenic sprouting. Conversely, ebms were irreversibly sealed from the circulation by excess collagen IV deposition that inhibited EC migration and prevented their reuse. Fully and partially regressed vessels therefore have opposing roles during revascularization, where fully regressed vessels inhibit new sprouting while partially regressed persistent vessels rapidly reactivate and serve as the source of continued pathologic angiogenesis.Funding Agencies|Swedish Research CouncilSwedish Research Council [2012-2472]</p

Discovery of novel L-type voltage-gated calcium channel blockers and application for the prevention of inflammation and angiogenesis

Background The ways in which microglia activate and promote neovascularization (NV) are not fully understood. Recent in vivo evidence supports the theory that calcium is required for the transition of microglia from a surveillance state to an active one. The objectives of this study were to discover novel L-type voltage-gated channel (L-VGCC) blockers and investigate their application for the prevention of inflammation and angiogenesis. Methods Pharmacophore-based computational modeling methods were used to screen for novel calcium channel blockers (CCBs) from the ZINC compound library. The effects of CCBs on calcium blockade, microglial pro-inflammatory activation, and cell toxicity were validated in BV-2 microglial cell and freshly isolated smooth muscle cell (SMC) cultures. Laser-induced choroidal neovascularization (NV) and the suture-induced inflammatory corneal NV models of angiogenesis were used for in vivo validation of the novel CCBs. CX3CR1(gfp/+) mice were used to examine the infiltration of GFP-labeled microglial cells. Results We identified three compounds from the ZINC database (Zinc20267861, Zinc18204217, and Zinc33254827) as new blockers of L-type voltage-gated calcium channels (L-VGCC) using a structure-based pharmacophore approach. The effects of the three CCBs on Ca2+ influx into cells were verified in BV-2 microglial cells using Fura-2 fluorescent dye and in freshly isolated SMCs using the voltage-patch clamp. All three CCBs reduced microglial cell migration, activation stimulated by lipopolysaccharide (LPS), and reduced the expression of the inflammatory markers NF-kappa B (phospho-I kappa B alpha) and cyclooxygenase-2 (COX-2) as well as reactive oxygen species. Of the three compounds, we further examined the in vivo activity of Zinc20267861. Topical treatment with Zinc20267861 in a rat model of suture-induced inflammatory cornea neovascularization demonstrated efficacy of the compound in reducing monocyte infiltration and overall corneal NV response. Subconjunctival administration of the compound in the choroidal NV mouse model effectively prevented CNV and microglial infiltration. Conclusions Our findings suggest that the novel CCBs identified here are effective anti-inflammatory agents that can be further evaluated for treating NV disorders and can be potentially applied in the treatment of ocular inflammatory and pathological angiogenetic disorders.Funding Agencies|NIH R01 grantUnited States Department of Health &amp; Human ServicesNational Institutes of Health (NIH) - USA [EY027824]; University of Missouri start-up fund (Hu Huang research group); Ogonfonden award (Neil Lagali research group, Linkoping University, Linkoping, Sweden)</p

Interconnectedness of Ecosystem Services Potential with Land Use/Land Cover Change Dynamics in Western Uganda

Understanding the evolution of land use/land cover change (LULCC) and how it shapes current and future ecosystem services (ES) supply potential remains critical in sustainable natural resource management. Community perception of historic LULCC was reconciled with previous study via remote sensing/geographical information systems using recall data in the Budongo–Bugoma landscape in Uganda. Then, a CA-Markovian prediction model of a LULC situation in 2040 under business as usual (BAU) and forest restoration scenarios was constructed. Additionally, we assessed the perceived proximate and underlying drivers of LULCC, and how LULCC shapes ecosystem services potential using household surveys. The perceived LULCC trend for the past three decades (1990–2020) corresponded with previous studies showing grassland, bushland, tropical high forest, and wetland cover declined greatly, while subsistence farmland, commercial farmland, and built-up areas had a great increment. The predicted LULC under (i) the business as usual scenario showed a continued decline of natural LULC while anthropogenic LULC increased greatly, tending to cover half of the landscape area; (ii) forest restoration under different levels showed an improvement of forest cover and other native LULC classes with a decline in mostly subsistence farmland. The proximate drivers were in three principal components (soil infertility, subsistence farming, drought; infrastructural development, commercial farming, overstocking of livestock, pest and disease challenges; tree planting), while underlying drivers were in two principal components (technology adoption, corruption of environment stewards, policy implementation gaps; cultural gaps). Food and cash crops were perceived to be the most important ecosystem services in the landscape. Generally, the landscape ES supply potential was dwindling and predicted to continue with a similar trend under BAU, despite the increment in ES contribution of subsistence and commercial farmland. Forest restoration would slightly improve the landscape ES potential but would cause a decline in subsistence farmland, which would result in either a threat to food/livelihood security or a livelihood shift. We recommend combined interventions that seek to achieve a progressive frontier that achieves development needs and priorities based on national need such as food security through local level production with recognition for sustainable availability of ecosystem services