Dr Malgorzata Rózanowska
The general aim of my research is to better understand the sources of oxidative damage, and mechanisms responsible for protection from oxidative damage in the eye, especially in the retina. The outer retina is constantly exposed to high oxygen tensions and large concentrations of polyunsaturated lipids extremely susceptible to oxidation. Moreover, it contains several photosensitisers, that is molecules that upon absorption of visible light generate damaging reactive species, such as singlet oxygen and free radicals. The protective mechanisms against photo-oxidative damage are extremely efficient in the retina. For most of us, the retina performs its function properly throughout the whole lifetime. Only during the exposure to intensive light, such us during gazing directly at the sun, watching solar eclipse without proper filters, sometimes during eye surgery, these mechanisms fail, and the retina becomes damaged.
An increased chronic exposure to light has been considered as one of the risk factors for development of age-related macular degeneration (AMD) – the leading cause of blindness of people above 60. About 30% of people above the age of 60 develop at least first signs of the disease but its aetiology is not clear, and the therapeutic options to delay vision loss are limited.
In my research, I investigate mostly the roles of the melanin, carotenoids, retinoids, and lipofuscin in the dysfunction of the retina and development of AMD.
Endogenous retinal photosensitizers and photoprotectants
Retinaldehyde (vitamin A aldehyde)
One of photosensitizers present in the retina is all-trans-retinaldehyde, a reactive aldehyde, which accumulates in the photoreceptor outer segments due to photobleaching of visual pigment, rhodopsin. Upon exposure to blue light, all-trans-retinaldehyde generates reactive oxygen species, such as singlet oxygen, and undergoes photodegradation. The photodegradation products retain photoreactivity of all-trans-retinaldehyde and exhibit greater toxicity and phototoxicity than all-trans-retinaldehyde. In the intimate proximity to the photoreceptor outer segments, there is a monolayer of retinal pigment epithelial (RPE) cells. Due to interactions between the photoreceptors and RPE, all-trans-retinaldehyde may impose a risk of photo-oxidative damage not only to photoreceptors but also to the RPE cells. In my research, I test the hypothesis that all-trans-retinaldehyde is the major factor responsible for the acute light-induced damage to the retina, as well as for the accumulation of lipofuscin, which then propagates the chronic damage.
Hypothetical pathways by which all-trans-retinaldehyde (atRal) accumulated in photoreceptor outer segments (POS) as a result of photobleaching of visual pigments leads to photodamage of photoreceptors and retinal pigment epithelium (RPE). AtRal can mediate the generation of superoxide radical anion (O2●─) in the dark, and O2●─, singlet oxygen (1O2), and peroxides (ROOH) when irradiated with UV-A or blue light. Unless effective antioxidants and repair enzymes counteract it, these reactive oxygen species produced by atRal induce oxidative damage to lipids and proteins, which may affect their structures and functions, including inactivation of enzymes involved in atRal removal (such as ABCR). The tips of the outer segments are phagocytosed daily by the RPE, and are meant to undergo lysosomal degradation. However, oxidatively damaged lipids and proteins may no longer be susceptible to the degradation by lysosomal enzymes, and/or may inactivate the enzymes. As a result of incomplete lysosomal degradation of the outer segments, the residual bodies, called lipofuscin (LF) accumulate in the RPE. LF photoactivated by blue or green light can also generate reactive oxygen species, and induces further oxidation of intragranular components, some of which may leak out of the granule and cause damage to the cellular components of the RPE, leading to RPE dysfunction or even death. Some of the oxidation products affect gene expression in the RPE, resulting in a release of pro-inflammatory and pro-angiogenic cytokines. The exocytosed lipofuscin may contribute to the formation of age-related deposits, such as drusen, accumulating between the RPE and Bruch's membrane, which separates the RPE from the choroidal blood supply. Some components of those deposits exhibit photosensitizing properties and include oxidation products with pro-angiogenic and pro-inflammatory properties. Moreover, oxidation leads to formation of crosslinks in the Bruch's membrane contributing to loss of its permeability. From http://www.photobiology.info/Rozanowska.html.
Linda teaches Michael how to use the rotary evaporator for preparation of lipid vesicles (liposomes).
Lipofuscin, also called an age pigment, accumulates in different tissues with ageing. In the retina, lipofuscin is believed to be formed from an incomplete digestion of photoreceptor outer segments, which are constantly shed and phagocytosed by the RPE. Retinal lipofuscin is a mixture of lipids, highly modified proteins, photosensitizers and fluorophores. The fluorophores make the lipofuscin easily visible in human eyes due to its characteristic golden-yellow fluorescence. The photosensitizers make the lipofuscin at least in part responsible for the age-related increase in the susceptibility of RPE cells to photooxidation. Upon excitation with visible light, an unknown photosensitizer of lipofuscin undergoes an intersystem crossing to form a triplet state. In the presence of oxygen, the energy of the triplet state is transferred to oxygen resulting in generation of an excited state of molecular oxygen, singlet oxygen. In addition to generation of singlet oxygen, lipofuscin generates other reactive oxygen species: superoxide, lipid hydroperoxides and hydrogen peroxide. Photoexcitation lipofuscin leads to oxidation of lipids and proteins, both intra- and extragranular. The observed photoreactions of lipofuscin are dependent on the irradiation wavelength, being more pronounced at shorter wavelengths. However, it is still unknown what the identity of the photosensitizer(s) within the lipofuscin granule is.
Linda measures light from the solar simulator before exposure of cultured RPE cells.
Melanin is a dark pigment of the eye, hair and skin. In the skin, melanin is synthesised in response to exposure to UV-A light. In the eye melanin synthesis starts early during foetal development and is completed within a few years after birth. Melanin constitutes the major pigment in young RPE cells is, where it is present within melanosomes. Although melanin is photoreactive, its pro-oxidant potential is significantly smaller compared to all-trans-retinal or lipofuscin. Only in the presence of ascorbate, aerobic photoexcitation of melanin induces substantial generation of superoxide and hydrogen peroxide. Due to the optical screening, sequestration of metal ions, free radical scavenging and quenching of excited states of photosensitisers, melanin exhibits antioxidant properties. Melanin can also 'repair' oxidised carotenoids by an efficient electron transfer. However, melanosomes undergo age-related changes – their photoreactivity increases with age; the total melanin concentration and number of melanosomes decreases; and there is age-related increase in a number of complex granules – melanolysosomes and melanolipofuscin.
Antioxidant protection against oxidative damage
In addition to melaninand retinoids, there is a number of endogeneously synthesized and dietary antioxidants, which may protect the retina from reactive oxygen species. Carotenoids, such as those constituting macular pigment – lutein and zeaxanthin, effectively quench singlet oxygen in a safest way possible: they accept the excess energy from oxygen molecule and dissipate it thermally. Antioxidants react with radicals but as a result they become radicals themselves, accumulation of which may have pro-oxidant effects. Antioxidants may increase their protective effects if present as a right combination, such as a combination of a singlet oxygen quencher (carotenoid) and a free radical scavenger (vitamin C or vitamin E), which offer better protection against photo-oxidative damage than increasing concentrations of individual antioxidants.
In my studies I use direct electron spin resonance (ESR) spectroscopy, ESR oximetry and spin trapping, nanosecond laser flash photolysis, time-resolved detection of characteristic phosphorescence of singlet oxygen at 1270 nm, pulse radiolysis, and fluorescence and absorption spectroscopy. To study (photo)toxicity and antioxidant protection against (photo)oxidative damage, I use cells in culture and established tests of cell function and viability.
10/2013-9/2016 Light therapy for mitochondrial optic neuropathy; co-supervisor (PI: Prof. Marcela Votruba) in PhD studentship sponsored by Fight for Sight, UK.
10/2013-09/2014 Idebenone for mitochondrial optic neuropathy; co-investigator (PI: Prof. Marcela Votruba); project sponsored by the National Eye Research Centre, UK.
07/2013-08/2014 “Evaluation of fluorescence characteristics of pigment granules as a marker of oxidative stress in the retina” ref. no. CUSBO001909; 15 days of access to LASERLAB-EUROPE facility Centre for Ultrafast Science and Biomedical Optics, Politecnico di Milano, Dipartimento di Fisica, Milano, Italia.
1/2011-12/2013 Bioenergetics and reactive oxygen species in a mouse model of dominant optic atrophy. Co-applicant and co-supervisor (PI: Prof. Andrew Quantock; primary supervisor: Prof. Marcela Votruba) in BBSRC-funded studentship, UK.
the National Eye Research Centre,
the Council for the Central Laboratory of the Research Councils (CCLRC)
the Royal Society
the Polish-US Fulbright Commission,
the Wellcome Trust,
the State Committee for Scientific Research (KBN), Poland
the Foundation for Polish Science