Zaheer Ali

Regulatory and functional connection of microphthalmia-associated transcription factor and anti-metastatic pigment epithelium derived factor in melanoma.

Pigment epithelium-derived factor (PEDF), a member of the serine protease inhibitor superfamily, has potent anti-metastatic effects in cutaneous melanoma through its direct actions on endothelial and melanoma cells. Here we show that PEDF expression positively correlates with microphthalmia-associated transcription factor (MITF) in melanoma cell lines and human samples. High PEDF and MITF expression is characteristic of low aggressive melanomas classified according to molecular and pathological criteria, whereas both factors are decreased in senescent melanocytes and naevi. Importantly, MITF silencing down-regulates PEDF expression in melanoma cell lines and primary melanocytes, suggesting that the correlation in the expression reflects a causal relationship. In agreement, analysis of Chromatin immunoprecipitation coupled to high throughput sequencing (ChIP-seq) data sets revealed three MITF binding regions within the first intron of SERPINF1, and reporter assays demonstrated that the binding of MITF to these regions is sufficient to drive transcription. Finally, we demonstrate that exogenous PEDF expression efficiently halts in vitro migration and invasion, as well as in vivo dissemination of melanoma cells induced by MITF silencing. In summary, these results identify PEDF as a novel transcriptional target of MITF and support a relevant functional role for the MITF-PEDF axis in the biology of melanoma.

Factors regulating capillary remodeling in a reversible model of inflammatory corneal angiogenesis

Newly formed microcapillary networks arising in adult organisms by angiogenic and inflammatory stimuli contribute to pathologies such as corneal and retinal blindness, tumor growth, and metastasis. Therapeutic inhibition of pathologic angiogenesis has focused on targeting the VEGF pathway, while comparatively little attention has been given to remodeling of the new microcapillaries into a stabilized, functional, and persistent vascular network. Here, we used a novel reversible model of inflammatory angiogenesis in the rat cornea to investigate endogenous factors rapidly invoked to remodel, normalize and regress microcapillaries as part of the natural response to regain corneal avascularity. Rapid reversal of an inflammatory angiogenic stimulus suppressed granulocytic activity, enhanced recruitment of remodelling macrophages, induced capillary intussusception, and enriched pathways and processes involving immune cells, chemokines, morphogenesis, axonal guidance, and cell motility, adhesion, and cytoskeletal functions. Whole transcriptome gene expression analysis revealed suppression of numerous inflammatory and angiogenic factors and enhancement of endogenous inhibitors. Many of the identified genes function independently of VEGF and represent potentially new targets for molecular control of the critical process of microvascular remodeling and regression in the cornea.

A microarray whole-genome gene expression dataset in a rat model of inflammatory corneal angiogenesis

In angiogenesis with concurrent inflammation, many pathways are activated, some linked to VEGF and others largely VEGF-independent. Pathways involving inflammatory mediators, chemokines, and micro-RNAs may play important roles in maintaining a pro-angiogenic environment or mediating angiogenic regression. Here, we describe a gene expression dataset to facilitate exploration of pro-angiogenic, pro-inflammatory, and remodelling/normalization-associated genes during both an active capillary sprouting phase, and in the restoration of an avascular phenotype. The dataset was generated by microarray analysis of the whole transcriptome in a rat model of suture-induced inflammatory corneal neovascularisation. Regions of active capillary sprout growth or regression in the cornea were harvested and total RNA extracted from four biological replicates per group. High quality RNA was obtained for gene expression analysis using microarrays. Fold change of selected genes was validated by qPCR, and protein expression was evaluated by immunohistochemistry. We provide a gene expression dataset that may be re-used to investigate corneal neovascularisation, and may also have implications in other contexts of inflammation-mediated angiogenesis.

Selective IKK2 inhibitor IMD0354 disrupts NF-κB signaling to suppress corneal inflammation and angiogenesis

Corneal neovascularization is a sight-threatening condition caused by angiogenesis in the normally avascular cornea. Neovascularization of the cornea is often associated with an inflammatory response, thus targeting VEGF-A alone yields only a limited efficacy. The NF-κB signaling pathway plays important roles in inflammation and angiogenesis. Here, we study consequences of the inhibition of NF-κB activation through selective blockade of the IKK complex IκB kinase β (IKK2) using the compound IMD0354, focusing on the effects of inflammation and pathological angiogenesis in the cornea. In vitro, IMD0354 treatment diminished HUVEC migration and tube formation without an increase in cell death and arrested rat aortic ring sprouting. In HUVEC, the IMD0354 treatment caused a dose-dependent reduction in VEGF-A expression, suppressed TNFα-stimulated expression of chemokines CCL2 and CXCL5, and diminished actin filament fibers and cell filopodia formation. In developing zebrafish embryos, IMD0354 treatment reduced expression of Vegf-a and disrupted retinal angiogenesis. In inflammation-induced angiogenesis in the rat cornea, systemic selective IKK2 inhibition decreased inflammatory cell invasion, suppressed CCL2, CXCL5, Cxcr2, and TNF-α expression and exhibited anti-angiogenic effects such as reduced limbal vessel dilation, reduced VEGF-A expression and reduced angiogenic sprouting, without noticeable toxic effect. In summary, targeting NF-κB by selective IKK2 inhibition dampened the inflammatory and angiogenic responses in vivo by modulating the endothelial cell expression profile and motility, thus indicating an important role of NF-κB signaling in the development of pathologic corneal neovascularization.

Adjustable delivery of pro-angiogenic FGF-2 by alginate:collagen microspheres

ABSTRACT Therapeutic induction of blood vessel growth (angiogenesis) in ischemic tissues holds great potential for treatment of myocardial infarction and stroke. Achieving sustained angiogenesis and vascular maturation has, however, been highly challenging. Here, we demonstrate that alginate:collagen hydrogels containing therapeutic, pro-angiogenic FGF-2, and formulated as microspheres, is a promising and clinically relevant vehicle for therapeutic angiogenesis. By titrating the amount of readily dissolvable and degradable collagen with more slowly degradable alginate in the hydrogel mixture, the degradation rates of the biomaterial controlling the release kinetics of embedded pro-angiogenic FGF-2 can be adjusted. Furthermore, we elaborate a microsphere synthesis protocol allowing accurate control over sphere size, also a critical determinant of degradation/release rate. As expected, alginate:collagen microspheres were completely biocompatible and did not cause any adverse reactions when injected in mice. Importantly, the amount of pro-angiogenic FGF-2 released from such microspheres led to robust induction of angiogenesis in zebrafish embryos similar to that achieved by injecting FGF-2-releasing cells. These findings highlight the use of microspheres constructed from alginate:collagen hydrogels as a promising and clinically relevant delivery system for pro-angiogenic therapy. Summary: The development of alginate:collagen composite hydrogel microspheres of adjustable size and degradation speed is described as a new platform for delivery of pro-angiogenic FGF-2 or pro-angiogenic cells.

Hypoxia-Induced Retinal Angiogenesis in Adult Zebrafish

Hypoxia – which refers to insufficient amounts of oxygen in tissues – is an important pathophysiological driver of angiogenesis through a complicated web of signaling pathways, which are still incompletely understood. Zebrafish are as other fish species highly hypoxia-tolerant compared to mammals. Furthermore their skin and gills are highly permeable to orally active drugs added to the water, including the large number of small synthetic activators or inhibitors of various signaling pathways available today. This enables the specific study of hypoxia-induced signaling pathways leading to angiogenesis in living adult animals, a feature that is of high relevance in medical research and unique for this model system. In this chapter we provide detailed protocols for how to set up hypoxia systems, expose adult zebrafish to hypoxia or hyperoxia (elevated tissue oxygen levels), extract images of hypoxia-induced angiogenesis from the adult retina and methods for quantifying changes in angiogenesis from such images.

Angiogenesis in the Regenerating Adult Zebrafish Tail Fin

Angiogenesis is critical for regeneration and wound healing. Poor angiogenic responses to tissue injury lead to scarring or loss of tissue viability, which can be detrimental, sometimes even fatal in for example myocardial infarction or stroke. It is therefore of the outmost importance to find ways in which to speed the natural regenerative angiogenic response or improve it in patients exhibiting poor regeneration such as people suffering from diabetes. Zebrafish exhibit potent regenerative responses and are even able to regenerate tissues such as heart, brain and muscle tissue, which mammals cannot. Therefore, the zebrafish has recently become a popular model to study mechanisms behind regenerative angiogenesis and to find drugs that aid in or interfere with this process. Here we provide detailed protocols on how to study regenerative angiogenesis in the adult zebrafish tail fin. We give a detailed walk-through of the tail fin amputation itself as well as how to interfere with regeneration at the molecular level by injection of morpholinos or mRNA/expression vectors followed by electroporation. Finally we give suggestions on how to record the results and quantify the regeneration-induced angiogenesis. These protocols are quick and easy to use, and can be done with minimal training using equipment which is available in most zebrafish laboratories.

Methods for Studying Developmental Angiogenesis in Zebrafish

Angiogenesis is a very complicated biological process in which the inter-communication between various cell types in a time-dependent manner is crucial for correct formation and perfusion of new vessels to expand the developing vasculature. Zebrafish have turned out to be incredibly well suited for the study of developmental angiogenesis, as the embryo develop outside of the mother, is transparent and develop very fast enabling highly dynamic visualizations (such as video time-lapse recordings) of the entire developing vasculature. Such a unique advantage of this model has enabled researchers to delineate the spatiotemporal involvement of signaling factors important for the formation and maintenance of tip cells, cues important for vascular patterning and new modes of blood vessel and lumen formation that could not have been delineated in rodent or other models. Here we will thoroughly describe how to use embryonic zebrafish models in studies of developmental angiogenesis. We will describe how to interfere genetically with the process by injection of morpholinos or mRNA into the embryo which would lead to positive or negative regulation of particular genes under investigations. We will also address how to establish gradients of angiogenic factors in non-vascularized areas of the embryo by injection of recombinant growth factors or cells in the perivitelline space, which will then lead to ectopic angiogenesis toward the stimuli – a process that can give information related to the angiogenic properties of compounds. Finally we will go through how to draw maximal benefit from such investigations by confocal imaging. This chapter will end with a trouble-shooting section in which common problems and solutions will be discussed, helping you – the reader – to as smoothly as possible get started on your research on developmental angiogenesis in the zebrafish embryo.