Anna Cancarini

Diabetic Retinopathy: Vascular and Inflammatory Disease

Diabetic retinopathy (DR) is the leading cause of visual impairment in the working-age population of the Western world. The pathogenesis of DR is complex and several vascular, inflammatory, and neuronal mechanisms are involved. Inflammation mediates structural and molecular alterations associated with DR. However, the molecular mechanisms underlying the inflammatory pathways associated with DR are not completely characterized. Previous studies indicate that tissue hypoxia and dysregulation of immune responses associated with diabetes mellitus can induce increased expression of numerous vitreous mediators responsible for DR development. Thus, analysis of vitreous humor obtained from diabetic patients has made it possible to identify some of the mediators (cytokines, chemokines, and other factors) responsible for DR pathogenesis. Further studies are needed to better understand the relationship between inflammation and DR. Herein the main vitreous-related factors triggering the occurrence of retinal complication in diabetes are highlighted.

Predicting the risk of diabetic retinopathy in type 2 diabetic patients.

AIMS Diabetic retinopathy (DR) is often asymptomatic even in its more advanced stages. Timely and repeated screening for DR avoids a late diagnosis of DR, but the high number of diabetic patients precludes a frequent screening; thus, the need for a method to identify patients at higher risk for DR becomes crucial. METHODS A prospective analysis of 5034 type 2 diabetic patients followed from 1996 to 2007 and not affected by retinopathy at the time of the recruitment was performed. Patients were randomly divided (ratio 2:1) into two groups: the train data set and the test set (3327 and 1707 patients, respectively). Factors associated with the occurrence of DR were assessed by the Coxs proportional hazard model. RESULTS Duration of diabetes, glycosylated hemoglobin, systolic blood Pressure, male gender, albuminuria and diabetes therapy other than diet were all significantly associated with the occurrence of DR. CONCLUSIONS The nomogram could help in ranking the type 2 diabetic patients at higher risk to develop DR and thus with a need for more frequent ophthalmologic checks, without enhancing neither the time nor the costs.

Relationship of Wall-to-Lumen Ratio of Retinal Arterioles With Clinic and 24-Hour Blood Pressure

Wall-to-lumen ratio of retinal arterioles might serve as an in vivo parameter of vascular damage. We analyzed the impact of brachial clinic blood pressure (BP), of central BP, and of 24-hour BP on wall-to-lumen ratio (WLR) of retinal arterioles. In 295 subjects (147 men; age range, 22–72 years; mean age, 54±7 years), WLR of retinal arterioles was assessed in vivo using scanning laser Doppler flowmetry. In addition, clinic and 24-hour BP values were measured. Central hemodynamics was assessed by pulse wave analysis. In treated patients with essential hypertension (n=100), a higher WLR (0.29±0.18 versus 0.23±0.13; P=0.009) was observed in comparison with normotensive individuals (n=119); no significant differences were observed between treated and untreated hypertensive patients (0.29±0.18 versus 0.28±0.18; P=0.7). WLR of retinal arterioles was significantly related to clinic systolic (r=0.18; P=0.002) and pulse pressure (r=0.20; P=0.001), to 24-hour systolic (r=0.25; P=0.0001) and pulse pressure (r=0.17; P=0.005), and to central systolic (r=0.16; P=0.006) and pulse pressure (r=0.18; P=0.002). Multiple regression analysis revealed that only mean systolic 24-hour BP was independently associated with an increased WLR of retinal arterioles. In this large group of hypertensive patients and normotensive individuals, 24-hour systolic BP seems to be the strongest determinant of increased WLR of retinal arterioles.

Effect of antihypertensive treatment on microvascular structure, central blood pressure and oxidative stress in patients with mild essential hypertension.

Background: It has been previously demonstrated that dihydropyridine calcium channel blockers may possess antioxidant properties and might improve vascular structure. Combination treatment with an angiotensin-converting enzyme inhibitor may have additional advantages, compared with a thiazide diuretic, in this regard. The aim of the present study was, therefore, to investigate the effects of a short-term treatment with lercanidipine, and to compare two combination treatments: lercanidipine + enalapril vs. lercanidipine + hydrochlorothiazide on structural alterations in retinal arterioles, on skin capillary density and on large artery distensibility. Patients and methods: Twenty essential hypertensive patients were included in the study and treated for 4 weeks with lercanidipine 20 mg per day orally. Then they were treated for 6 months with lercanidipine + enalapril (n = 10) or lercanidipine + hydrochlorothiazide (n = 10) combinations. Investigations were performed in basal condition, after appropriate washout of previous treatments, after 4 weeks of lercanidipine monotherapy treatment, and at the end of the combination treatment. Non-invasive measurements of wall-to-lumen ratio (W/L) and other morphological parameters of retinal arterioles using scanning laser Doppler flowmetry were performed (Heidelberg Retina Flowmeter, Heidelberg Engineering). Capillary density was evaluated by capillaroscopy, whereas pulse wave velocity and central blood pressure were assessed by the Sphygmo-Cor device (AtCor Medical West Ryde, Australia). Results: A significant improvement of W/L and of other indices of retinal artery structure was observed after treatment with lercanidipine alone, with a further improvement after treatment with lercanidipine + enalapril, whereas after treatment with lercanidipine + hydrochlorothiazide the improvement was no longer observed. A similar behaviour was observed for central SBP and DBP. Capillary density was increased only after treatment with lercanidipine + enalapril. Conclusion: Lercanidipine both in monotherapy and in combination with enalapril, was able to improve microvascular structure and to decrease central blood pressure, being thus a useful approach for both reducing blood pressure and improving vascular alterations in hypertension.

Serum and intraocular concentrations of erythropoietin and vascular endothelial growth factor in patients with type 2 diabetes and proliferative retinopathy

AIM This study compared systemic and intraocular concentrations of erythropoietin (EPO) and vascular endothelial growth factor (VEGF) in patients with type 2 diabetes (T2D) and proliferative diabetic retinopathy (PDR) with levels in patients without diabetes, and looked for possible correlations between the concentrations found and other variables analyzed. METHODS Concentrations of EPO and VEGF were measured in the aqueous and vitreous humours and serum of patients undergoing vitrectomy for PDR (33 patients) or for macular holes or puckers (20 control patients). EPO was assayed by radioimmunoassay, with a lower limit of detection (LOD) of 1.0 mIU/mL. VEGF was assayed using enzyme-linked immunosorbent assay (ELISA), with a lower LOD of 10.0 pg/mL. RESULTS EPO concentrations in serum did not differ significantly between the two groups, whereas EPO in vitreous and aqueous were higher in diabetic than in non-diabetic patients. VEGF in serum was lower in diabetic patients than in non-diabetics; conversely, VEGF concentrations in vitreous were significantly higher in diabetic patients. A direct correlation was found between vitreous and aqueous EPO concentrations, and between vitreous EPO and blood glucose concentrations. A significant, negative correlation between vitreous EPO concentration and age was also recorded. CONCLUSION High EPO concentrations in the vitreous of patients with PDR and its correlation with blood glucose suggest that EPO could play a role in the pathogenesis of PDR. All possible factors affecting serum and ocular concentrations of EPO and VEGF should be determined to identify compounds able to prevent and control this serious microvascular complication of diabetes.

Antiangiogenic Effectiveness of the Urokinase Receptor-Derived Peptide UPARANT in a Model of Oxygen-Induced Retinopathy

PURPOSE Pharmacologic control of neovascularization is a promising approach for the treatment of retinal angiogenesis. UPARANT, an inhibitor of the urokinase-type plasminogen activator receptor (uPAR), inhibits VEGF-driven angiogenesis in vitro and in vivo. This study investigates for the first time the effectiveness of UPARANT in counteracting pathologic neovascularization in the retina. METHODS Murine retinal fragments and a mouse model of oxygen-induced retinopathy (OIR) were used. In mice with OIR, UPARANT-treated retinas were analyzed for avascular area and neovascular tuft formation. Levels of transcription and proangiogenic factors were determined. UPARANT effects on the blood-retinal barrier (BRB), visual function, retinal cytoarchitecture, and inflammatory markers were also assessed. Human umbilical vein endothelial cells (HUVECs) and chick embryo chorioallantoic membrane (CAM) in which angiogenesis was induced by the vitreous fluid from patients with proliferative diabetic retinopathy (PDR) were also used. RESULTS UPARANT reduced VEGF-induced angiogenesis in retinal fragments. In mice with OIR, UPARANT decreased neovascular response, VEGF, and VEGF receptor-2 activity. Transcription factors regulating VEGF expression were also reduced. UPARANT restored BRB integrity, recovered visual loss, and reduced levels of inflammatory markers. Restored electroretinogram does not involve any rescue in the retinal cytoarchitecture. Finally, UPARANT blocked PDR vitreous fluid-induced angiogenesis in HUVEC and CAM assays. CONCLUSIONS The finding that UPARANT is effective against neovascularization may help to establish uPAR as a target in the treatment of proliferative retinopathies. The potential application of UPARANT in retinal diseases is further supported by UPARANT capacity to counteract the angiogenic activity of PDR vitreous fluid.

Pharmacokinetic and Pharmacodynamic Properties of Anti-VEGF Drugs After Intravitreal Injection

Subretinal neovascularization and pathologic ocular angiogenesis are common causes of progressive, irreversible impairment of central vision, and dramatically affect quality of life. Anti-vascular endothelial growth factor (anti-VEGF) therapy has improved the quality of life for many patients with age-related macular degeneration, diabetic retinopathy, and other ocular diseases involving neovascularization and edema. In these pathologies, the inhibition of intraocular VEGF is the only therapy that can preserve vision. Four anti-VEGF drugs are currently used to treat ocular neovascularization; pegaptanib, ranibizumab, and aflibercept have been approved for this condition, while bevacizumab can be used off-label. Anti-VEGF therapy is administered regularly for many months or years because its suspension or discontinuation may cause recurrence of neovascularization. On the other hand, VEGF is necessary for the survival of retinal and choroidal endothelial cells. Experimental studies in animal models have shown that local inhibition of VEGF causes thinning and atrophy of the choriocapillaris and degeneration of photoreceptors, primarily cones. These studies combined with clinical experience indicated that prolonged VEGF inhibition could impair retinal function. Moreover, anti-VEGF compounds can cross the blood-retina barrier, enter the systemic circulation, and inhibit serum VEGF. Since circulating VEGF protects blood vessel integrity, prolonged anti-VEGF treatment could induce thromboembolic adverse events from vascular causes such as heart attack and stroke, and even death. The ocular dosing regimen and systemic toxicity of anti-VEGF compounds are therefore central concerns. A better understanding of this topic requires knowledge of the metabolism, tissue distribution, and clearance of anti-VEGF compounds. This manuscript reviews the properties of anti-VEGF compounds following intravitreal administration.

Therapeutic Potential of Anti-Angiogenic Multi-Target N,O-Sulfated E. Coli K5 Polysaccharide in Diabetic Retinopathy

Vascular endothelial growth factor (VEGF) blockers have been developed for the treatment of proliferative diabetic retinopathy (PDR), the leading cause of visual impairments in the working-age population in the Western world. However, limitations to anti-VEGF therapies may exist because of the local production of other proangiogenic factors that may cause resistance to anti-VEGF interventions. Thus, novel therapeutic approaches targeting additional pathways are required. Here, we identified a sulfated derivative of the Escherichia coli polysaccharide K5 [K5-N,OS(H)] as a multitarget molecule highly effective in inhibiting VEGF-driven angiogenic responses in different in vitro, ex vivo, and in vivo assays, including a murine model of oxygen-induced retinopathy. Furthermore, K5-N,OS(H) binds a variety of heparin-binding angiogenic factors upregulated in PDR vitreous humor besides VEGF, thus inhibiting their biological activity. Finally, K5-N,OS(H) hampers the angiogenic activity exerted in vitro and in vivo by human vitreous fluid samples collected from patients with PDR. Together, the data provide compelling experimental evidence that K5-N,OS(H) represents an antiangiogenic multitarget molecule with potential implications for the therapy of pathologic neovessel formation in the retina of patients with PDR.

Inflammation and N-formyl peptide receptors mediate the angiogenic activity of human vitreous humour in proliferative diabetic retinopathy

Aims/hypothesisAngiogenesis and inflammation characterise proliferative diabetic retinopathy (PDR), a major complication of diabetes mellitus. However, the impact of inflammation on the pathogenesis of PDR neovascularisation has not been elucidated. Here, we assessed the capacity of PDR vitreous fluid to induce pro-angiogenic/proinflammatory responses in endothelium and the contribution of the inflammation-related pattern recognition N-formyl peptide receptors (FPRs) in mediating these responses.MethodsPooled and individual pars plana vitrectomy-derived PDR vitreous fluid (‘PDR vitreous’) samples were assessed in endothelial cell proliferation, motility, sprouting and morphogenesis assays, and for the capacity to induce proinflammatory transcription factor activation, reactive oxygen species production, intercellular junction disruption and leucocyte-adhesion molecule upregulation in these cells. In vivo, the pro-angiogenic/proinflammatory activity of PDR vitreous was tested in murine Matrigel plug and chick embryo chorioallantoic membrane (CAM) assays. Finally, the FPR inhibitors Boc-Phe-Leu-Phe-Leu-Phe (Boc-FLFLF) and Ac-l-Arg-Aib-l-Arg-l-Cα(Me)Phe-NH2 tetrapeptide (UPARANT) were evaluated for their capacity to affect the biological responses elicited by PDR vitreous.ResultsPDR vitreous activates a pro-angiogenic/proinflammatory phenotype in endothelial cells. Accordingly, PDR vitreous triggers a potent angiogenic/inflammatory response in vivo. Notably, the different capacity of individual PDR vitreous samples to induce neovessel formation in the CAM correlates with their ability to recruit infiltrating CD45+ cells. Finally, the FPR inhibitor Boc-FLFLF and the novel FPR antagonist UPARANT inhibit neovessel formation and inflammatory responses triggered by PDR vitreous in the CAM assay.Conclusions/interpretationThis study provides evidence that inflammation mediates the angiogenic activity of PDR vitreous and paves the way for the development of FPR-targeting anti-inflammatory/anti-angiogenic approaches for PDR therapy.

Defining reference values of trace elements in the tear film: Diagnostic methods and possible applications

The study has been performed on tears of apparently healthy subjects who live and work in urban and rural areas, respectively. After the collection the following elements were investigated: chromium (Cr); arsenic (As); copper (Cu); zinc (Zn); selenium (Se); rubidium (Rb); barium (Ba); lead (Pb) and cobalt (Co). Significantly higher values of As were found in subjects living and working in rural areas as compared to those found in urban area residents (0.290 vs. 0.025; p<0.001). Conversely, Ba and Pb were significantly lower in rural area residents (1.10 vs. 2.50, p=0.027 and 1.70 vs. 1.10, p=0.057, respectively). Our data show that trace elements analysis in tears is possible; further studies could define if it could be a reliable biomarker in persons exposed to high concentration of trace elements due to working or environmental reasons.