Tim M. Curtis

Microvascular lesions of diabetic retinopathy: clues towards understanding pathogenesis?

Retinopathy is a major complication of diabetes mellitus and this condition remains a leading cause of blindness in the working population of developed countries. As diabetic retinopathy progresses a range of neuroglial and microvascular abnormalities develop although it remains unclear how these pathologies relate to each other and their net contribution to retinal damage. From a haemodynamic perspective, evidence suggests that there is an early reduction in retinal perfusion before the onset of diabetic retinopathy followed by a gradual increase in blood flow as the complication progresses. The functional reduction in retinal blood flow observed during early diabetic retinopathy may be additive or synergistic to pro-inflammatory changes, leucostasis and vaso-occlusion and thus be intimately linked to the progressive ischaemic hypoxia and increased blood flow associated with later stages of the disease. In the current review a unifying framework is presented that explains how arteriolar dysfunction and haemodynamic changes may contribute to late stage microvascular pathology and vision loss in human diabetic retinopathy.

Fast retinal vessel detection and measurement using wavelets and edge location refinement.

The relationship between changes in retinal vessel morphology and the onset and progression of diseases such as diabetes, hypertension and retinopathy of prematurity (ROP) has been the subject of several large scale clinical studies. However, the difficulty of quantifying changes in retinal vessels in a sufficiently fast, accurate and repeatable manner has restricted the application of the insights gleaned from these studies to clinical practice. This paper presents a novel algorithm for the efficient detection and measurement of retinal vessels, which is general enough that it can be applied to both low and high resolution fundus photographs and fluorescein angiograms upon the adjustment of only a few intuitive parameters. Firstly, we describe the simple vessel segmentation strategy, formulated in the language of wavelets, that is used for fast vessel detection. When validated using a publicly available database of retinal images, this segmentation achieves a true positive rate of 70.27%, false positive rate of 2.83%, and accuracy score of 0.9371. Vessel edges are then more precisely localised using image profiles computed perpendicularly across a spline fit of each detected vessel centreline, so that both local and global changes in vessel diameter can be readily quantified. Using a second image database, we show that the diameters output by our algorithm display good agreement with the manual measurements made by three independent observers. We conclude that the improved speed and generality offered by our algorithm are achieved without sacrificing accuracy. The algorithm is implemented in MATLAB along with a graphical user interface, and we have made the source code freely available.

Diabetes Downregulates Large-Conductance Ca2+-Activated Potassium β1 Channel Subunit in Retinal Arteriolar Smooth Muscle

Retinal vasoconstriction and reduced retinal blood flow precede the onset of diabetic retinopathy. The pathophysiological mechanisms that underlie increased retinal arteriolar tone during diabetes remain unclear. Normally, local Ca2+ release events (Ca2+-sparks), trigger the activation of large-conductance Ca2+-activated K+(BK)-channels which hyperpolarize and relax vascular smooth muscle cells, thereby causing vasodilatation. In the present study, we examined BK channel function in retinal vascular smooth muscle cells from streptozotocin-induced diabetic rats. The BK channel inhibitor, Penitrem A, constricted nondiabetic retinal arterioles (pressurized to 70mmHg) by 28%. The BK current evoked by caffeine was dramatically reduced in retinal arterioles from diabetic animals even though caffeine-evoked [Ca2+]i release was unaffected. Spontaneous BK currents were smaller in diabetic cells, but the amplitude of Ca2+-sparks was larger. The amplitudes of BK currents elicited by depolarizing voltage steps were similar in control and diabetic arterioles and mRNA expression of the pore-forming BK&agr; subunit was unchanged. The Ca2+-sensitivity of single BK channels from diabetic retinal vascular smooth muscle cells was markedly reduced. The BK&bgr;1 subunit confers Ca2+-sensitivity to BK channel complexes and both transcript and protein levels for BK&bgr;1 were appreciably lower in diabetic retinal arterioles. The mean open times and the sensitivity of BK channels to tamoxifen were decreased in diabetic cells, consistent with a downregulation of BK&bgr;1 subunits. The potency of blockade by Pen A was lower for BK channels from diabetic animals. Thus, changes in the molecular composition of BK channels could account for retinal hypoperfusion in early diabetes, an idea having wider implications for the pathogenesis of diabetic hypertension.

Arteriolar Involvement in the Microvascular Lesions of Diabetic Retinopathy: Implications for Pathogenesis

Diabetic retinopathy (DR) is the most widespread complication of diabetes mellitus and a major cause of blindness in the working population of developed countries. The clinicopathology of the diabetic retina has been extensively studied, although the relative contribution of the various biochemical and molecular sequelae of hyperglycemia remains ill defined. Many neural and microvascular abnormalities occur in the retina of short‐term diabetic animals but it remains uncertain how closely these acute changes relate to chronic human disease. It is important to determine the relationship between alterations observed within the first weeks or months in short‐term animal models, and human disease, where clinically manifest retinopathy occurs only after durations of diabetes measured in years. This review is focused on the retinal microvasculature, although it should be appreciated that pathological changes in this system often occur in parallel with abnormalities in the neural parenchyma that may be derivative or even causal. Nevertheless, it is useful to reevaluate the microvascular lesions that are manifest in the retina during diabetes in humans and long‐term animal models, since in addition to providing useful clues to the pathogenic basis of DR as a disease entity, it is in the deterrence of such changes that the efficacy of any novel treatment regimes will be measured. In particular, an emphasis will be placed on the relatively unappreciated role of arteriolar dysfunction in the clinical manifestations and pathology of this disease.

Advances in our understanding of diabetic retinopathy

Diabetic retinopathy remains the most common complication of diabetes mellitus and is a leading cause of visual loss in industrialized nations. The clinicopathology of the diabetic retina has been extensively studied, although the precise pathogenesis and cellular and molecular defects that lead to retinal vascular, neural and glial cell dysfunction remain somewhat elusive. This lack of understanding has seriously limited the therapeutic options available for the ophthalmologist and there is a need to identify the definitive pathways that initiate retinal cell damage and drive progression to overt retinopathy. The present review begins by outlining the natural history of diabetic retinopathy, the clinical features and risk factors. Reviewing the histopathological data from clinical specimens and animal models, the recent paradigm that neuroretinal dysfunction may play an important role in the early development of the disease is discussed. The review then focuses on the molecular pathogenesis of diabetic retinopathy with perspective provided on new advances that have furthered our understanding of the key mechanisms underlying early changes in the diabetic retina. Studies have also emerged in the past year suggesting that defective repair of injured retinal vessels by endothelial progenitor cells may contribute to the pathogenesis of diabetic retinopathy. We assess these findings and discuss how they could eventually lead to new therapeutic options for diabetic retinopathy.

Nifedipine blocks Ca2+ store refilling through a pathway not involving L-type Ca2+ channels in rabbit arteriolar smooth muscle

1 This study assessed the contribution of L‐type Ca2+ channels and other Ca2+ entry pathways to Ca2+ store refilling in choroidal arteriolar smooth muscle. 2 Voltage‐clamp recordings were made from enzymatically isolated choroidal microvascular smooth muscle cells and from cells within vessel fragments (containing < 10 cells) using the whole‐cell perforated patch‐clamp technique. Cell Ca2+ was estimated by fura‐2 microfluorimetry. 3 After Ca2+ store depletion with caffeine (10 mm), refilling was slower in cells held at ‐20 mV compared to ‐80 mV (refilling half‐time was 38 ± 10 and 20 ± 6 s, respectively). 4 To attempt faster refilling via L‐type Ca2+ channels, depolarising steps from ‐60 to ‐20 mV were applied during a 30 s refilling period following caffeine depletion. Each step activated L‐type Ca2+ currents and [Ca2+]i transients, but failed to accelerate refilling. 5 At ‐80 mV and in 20 mm TEA, prolonged caffeine exposure produced a transient Ca2+‐activated Cl− current (ICl(Ca)) followed by a smaller sustained current. The sustained current was resistant to anthracene‐9‐carboxylic acid (1 mm; an ICl(Ca) blocker) and to BAPTA AM, but was abolished by 1 μm nifedipine. This nifedipine‐sensitive current reversed at +29 ± 2 mV, which shifted to +7 ± 5 mV in Ca2+‐free solution. Cyclopiazonic acid (20 μm; an inhibitor of sarcoplasmic reticulum Ca2+‐ATPase) also activated the nifedipine‐sensitive sustained current. 6 At ‐80 mV, a 5 s caffeine exposure emptied Ca2+ stores and elicited a transient ICl(Ca). After 80 s refilling, another caffeine challenge produced a similar inward current. Nifedipine (1 μm) during refilling reduced the caffeine‐activated ICl(Ca) by 38 ± 5 %. The effect was concentration dependent (1‐3000 nm, EC50 64 nm). In Ca2+‐free solution, store refilling was similarly depressed (by 46 ± 6 %). 7 Endothelin‐1 (10 nm) applied at ‐80 mV increased [Ca2+]i, which subsided to a sustained 198 ± 28 nm above basal. Cell Ca2+ was then lowered by 1 μm nifedipine (to 135 ± 22 nm), which reversed on washout. 8 These results show that L‐type Ca2+ channels fail to contribute to Ca2+ store refilling in choroidal arteriolar smooth muscle. Instead, they refill via a novel non‐selective store‐operated cation conductance that is blocked by nifedipine.

Human odontoblasts express functional thermo-sensitive TRP channels: implications for dentin sensitivity

&NA; Odontoblasts form the outermost cellular layer of the dental pulp where they have been proposed to act as sensory receptor cells. Despite this suggestion, evidence supporting their direct role in mediating thermo‐sensation and nociception is lacking. Transient receptor potential (TRP) ion channels directly mediate nociceptive functions, but their functional expression in human odontoblasts has yet to be elucidated. In the present study, we have examined the molecular and functional expression of thermo‐sensitive TRP channels in cultured odontoblast‐like cells and in native human odontoblasts obtained from healthy wisdom teeth. PCR and western blotting confirmed gene and protein expression of TRPV1, TRPA1 and TRPM8 channels. Immunohistochemistry revealed that these channels were localised to odontoblast‐like cells as determined by double staining with dentin sialoprotein (DSP) antibody. In functional assays, agonists of TRPV1, TRPA1 and TRPM8 channels elicited [Ca2+]i transients that could be blocked by relevant antagonists. Application of hot and cold stimuli to the cells also evoked rises in [Ca2+]i which could be blocked by TRP‐channel antagonists. Using a gene silencing approached we further confirmed a role for TRPA1 in mediating noxious cold responses in odontoblasts. We conclude that human odontoblasts express functional TRP channels that may play a crucial role in mediating thermal sensation in teeth. Cultured and native human odontoblasts express functional TRP channels that may play a crucial role in mediating thermal sensation in teeth.

The role of lipids and protein kinase Cs in the pathogenesis of diabetic retinopathy

Diabetic retinopathy is one of the most common complications of diabetes and is a major cause of new blindness in the working‐age population of developed countries. While the exact pathogenic basis of this condition remains ill defined, it is clear that hyperglycaemia is a critical factor in its aetiology. Protein kinase C (PKC) activation is one of the sequelae of hyperglycaemia and it is thought to play an important role in the development of diabetic complications. This review questions the currently held dogma that PKC stimulation in diabetes is solely mediated through the overproduction of palmitate and oleate enriched diacylglycerols. Blood glucose concentrations are closely tracked by changes in the levels of free fatty acids and these, in addition to oxidative stress, may account for the aberrant activation of PKCs in diabetes. Little is known about why PKCs fail to downregulate in diabetes and efforts should be directed towards acquiring such information. Considerable evidence implicates the PKCβ isoform in the pathogenesis of diabetic retinopathy, but other isoforms may also be of relevance. In addition to PKCs, it is evident that novel diacyglycerol‐activated non‐kinase receptors could also play a role in the development of diabetic complications. Therapeutic agents have been developed to inhibit specific PKC isoforms and PKCβ antagonists are currently undergoing clinical trials to test their toxicity and efficacy in suppressing diabetic complications. The likely impact of these drugs in the treatment of diabetic patients is considered. Copyright

Müller glial dysfunction during diabetic retinopathy in rats is linked to accumulation of advanced glycation end-products and advanced lipoxidation end-products

Aims/hypothesisThe impact of AGEs and advanced lipoxidation end-products (ALEs) on neuronal and Müller glial dysfunction in the diabetic retina is not well understood. We therefore sought to identify dysfunction of the retinal Müller glia during diabetes and to determine whether inhibition of AGEs/ALEs can prevent it.MethodsSprague–Dawley rats were divided into three groups: (1) non-diabetic; (2) untreated streptozotocin-induced diabetic; and (3) diabetic treated with the AGE/ALE inhibitor pyridoxamine for the duration of diabetes. Rats were killed and their retinas were evaluated for neuroglial pathology.ResultsAGEs and ALEs accumulated at higher levels in diabetic retinas than in controls (p < 0.001). AGE/ALE immunoreactivity was significantly diminished by pyridoxamine treatment of diabetic rats. Diabetes was also associated with the up-regulation of the oxidative stress marker haemoxygenase-1 and the induction of glial fibrillary acidic protein production in Müller glia (p < 0.001). Pyridoxamine treatment of diabetic rats had a significant beneficial effect on both variables (p < 0.001). Diabetes also significantly altered the normal localisation of the potassium inwardly rectifying channel Kir4.1 and the water channel aquaporin 4 to the Müller glia end-feet interacting with retinal capillaries. These abnormalities were prevented by pyridoxamine treatment.Conclusions/interpretationWhile it is established that AGE/ALE formation in the retina during diabetes is linked to microvascular dysfunction, this study suggests that these pathogenic adducts also play a role in Müller glial dysfunction.

Advanced glycation end products and diabetic retinopathy

Diabetic retinopathy (DR) has a complex pathogenesis which is impacted by a raft of systemic abnormalities and tissue-specific alterations occurring in response to the diabetes milieu. Many pathogenic processes play key roles in retinal damage in diabetic patients. One such pathway is the formation and accumulation of advanced glycation endproducts (AGEs) and advanced lipoxidation end products (ALEs) which are relevant modifications with roles in the initiation and progression of pathology. In this review, AGE/ALE formation in the diabetic retina is discussed alongside their impact on retinal cell function. In addition, various inhibitors of the AGE-RAGE system and their therapeutic utility for DR will also be evaluated.