In this work I present conclusions of clinical-laboratory research focused on the patients with diabetic macular edema (DME). We performed biochemical and immunochemical analyses of vitreous samples that were collected during the pars plana vitrectomy. Moreover, at patients with non-proliferative diabetic retinopathy (NPDR) we assessed morphological characteristics of DME using optical coherence tomography (OCT). According to our findings, the vitreous and serum concentrations of uric acid and glucose were significantly higher in patients with diabetic retinopathy and DME compared to controls. Also total ratio (serum/ vitreous concentration) of uric acid and glucose was in diabetics significantly higher than in controls. The most important determinant of increasing concentration of both uric acid and glucose in the vitreous was the grade of diabetic retinopathy. Moreover, we demonstrated significant correlation between vitreous concentration of uric acid and concentration of the vascular endothelial growth factor (VEGF) in patients with DME and NPDR. We found further, that the volume of the macula (cube volume - CV) computed with the software of Cirrus HD-OCT correlates in diabetics significantly with the vitreous VEGF concentration, but not with uric acid. This OCT parameter could be used to...

Introduction: Relation of diabetes mellitus (DM) to the diabetic keratopathy and various stages of corneal nerve fiber damage has been well accepted. A possible association between changes in the cornea of diabetic patients and diabetic retinopathy (DR), DM duration, and age at the time of DM diagnosis were evaluated. Neuropathies are among the most common long-term complications of diabetes mellitus. Good glycemic control is essential in prevention of this complication. DM patients with similar mean glucose levels or glycated hemoglobin (HbA1c) levels often exhibit differences in evaluation of diabetic complications. One reason for these differences may be the differences in glucose variability. DM patients with similar mean glucose levels or HbA1c levels often exhibit differences in glucose variability Hypothesis: Diabetes mellitus damages the subbasal nerve fibers of the corneal and affects the density of epithelial, endothelial and stromal cells. Corneal changes in patients with DM are dependent on the degree of diabetic retinopathy (DR), age at diagnosis, duration of DM, and compensation parameters. Purpose: To compare changes in cell density in individual layers of cornea and status of subbasal nerve fibers in patients with type 1 DM (DM 1) and in healthy subjects. To evaluate the dependence...

Le système kallicréine-kinines est un système peptidergique complexe impliqué dans les processus inflammatoires, le contrôle du tonus et de la perméabilité vasculaire. Les effets biologiques des kinines sont accomplis par l’intermédiaire de deux types de récepteurs couplés aux protéines G, soit le récepteur B1 (B1R) et le récepteur B2 (B2R). Alors que le B2R est un récepteur constitutif, le B1R est faiblement exprimé en situation physiologique; il est induit par le stress oxydatif, les cytokines pro-inflammatoires (interleukine-1β (IL-1β) et le facteur de nécrose tumorale-α (TNF-α)) ou par des endotoxines bactériennes à la fois au niveau systémique et local, notamment dans la rétine. Des études récentes de notre laboratoire ont montré l’implication du B1R dans la pathogenèse et la progression de la rétinopathie diabétique et de la dégénérescence maculaire liée à l’âge (DMLA). Les objectifs des travaux présentés dans cette thèse consistent à déterminer : 1) le mécanisme par lequel le B1R est impliqué dans la rétinopathie diabétique chez le rat; 2) l’implication de la iNOS en aval dans la cascade inflammatoire activée par le B1R; 3) l’expression et la localisation cellulaire du B1R dans les rétines humaines atteintes de DMLA exsudative et atrophique. Nos résultats ont permis de démontrer une implication du B1R dans la rétinopathie diabétique via l’activation de l’enzyme de synthèse du monoxyde d’azote inductible (iNOS) dans un modèle de diabète de type 1 induit par la streptozotocine (STZ) chez le rat. En plus de sa localisation généralisée dans toute la rétine, le B1R est exprimé dans la couche de l’épithélium pigmentaire qui forme la barrière hémato-rétinienne externe. Les taux d’expression (protéique et ARNm) du B1R, de la iNOS, de la carboxypeptidase M (impliquée dans la biosynthèse des agonistes B1R), de l'IL-1β, du TNF-α, du facteur de croissance de l'endothélium vasculaire A (VEGF-A) et de son récepteur, le VEGF-R2, ainsi que des protéines nitrosylées augmentent à deux semaines dans la rétine diabétique. Ces augmentations ainsi que l’hyperperméabilité vasculaire rétinienne induite par le diabète et par l’injection intravitréenne d’un agoniste du B1R (R-838) sont bloquées par un inhibiteur de la iNOS (1400W) appliqué topiquement à la surface de l’œil pendant 1 semaine (premier article). Les résultats du deuxième article montrent une augmentation significative de l'immunoréactivité du B1R dans les rétines humaines prélevées de patients atteints de DMLA exsudative. Toutefois, les changements d’immunoexpression du B1R ne sont pas significatifs dans les rétines des patients atteints de DMLA atrophique. La réactivité des cellules gliales est plus marquée dans la forme exsudative que dans la forme atrophique de DMLA. Une colocalisation du B1R est observée avec des marqueurs des cellules de Müller, des astrocytes, de la microglie, de la iNOS et de la fibrose, suggérant une implication du B1R dans le processus inflammatoire et la formation de fibrose dans la DMLA exsudative. En revanche, l’expression du B2R demeure stable dans les rétines de DMLA exsudative et atrophique par rapport aux rétines témoins; ce résultat ne supporte pas la possibilité que ce récepteur puisse être impliqué dans la DMLA chez l’humain. / The kallikrein-kinins system is a peptidergic system involved in inflammatory processes, the control of the vascular tone and permeability. These effects are mediated by two G proteincoupled receptors, the Bradykinin type 1 (B1R) and type 2 (B2R) receptors. While the B2R is a constitutive receptor, B1R is almost undetectable in physiological condition; it is, however, induced by oxidative stress, pro-inflammatory cytokines (interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α)) or by bacterial endotoxins at both systemic and local levels, notably in the retina. Recent studies from our laboratory supported an implication of B1R in the pathogenesis and progression of diabetic retinopathy and age-related macular degeneration (AMD). This thesis aims at unraveling: 1) the mechanism by which B1R is involved in diabetic retinopathy in rats; 2) the involvement of iNOS in the inflammatory cascade downstream to the B1R; and, 3) the expression and cellular localization of B1R in human retinae with exudative and atrophic AMD. Our results have shown the implication of B1R in diabetic retinopathy via the activation of the inducible nitric oxide synthase (iNOS) in a type 1 model of diabetes induced by streptozotocin (STZ) in rats. In addition to its generalized localization throughout the retina, B1R is expressed in the retinal pigment epithelium which forms the outer blood-retinal barrier. The protein and transcript expression of inflammatory markers; iNOS, carboxypeptidase M, IL-1β, TNF-α, vascular endothelium growth factor A (VEGF-A) and its receptor, VEGF-R2, including B1R as well as nitrosylated proteins are increased in the retina of diabetic rats at 2 weeks post-STZ. These upregulations, as well as the retinal vascular hyperpermeability induced by diabetes and by the intravitreal injection of an B1R agonist (R-838) are blocked by a topical one-week treatment by eye-drop with the selective iNOS inhibitor (1400W) (first manuscript). The results of the second manuscript show significant increases in the immunoreactivity of B1R in exudative AMD retinae. Despite a slight increase, B1R immunostaining does not reach statistical significance in the retina of donors with atrophic AMD. The reactivity of glial cells is more impressive in the exudative than in the atrophic form of AMD. B1R is co-expressed with markers of Müller cells, astrocytes, microglia, iNOS and fibrosis, suggesting an involvement of B1R in the inflammatory events and the formation of fibrosis in exudative AMD. On the other hand, the expression of B2R remains stable in the retinae of exudative and atrophic AMD, supporting a secondary role of this receptor in AMD in humans.

In this work I present conclusions of clinical-laboratory research focused on the patients with diabetic macular edema (DME). We performed biochemical and immunochemical analyses of vitreous samples that were collected during the pars plana vitrectomy. Moreover, at patients with non-proliferative diabetic retinopathy (NPDR) we assessed morphological characteristics of DME using optical coherence tomography (OCT). According to our findings, the vitreous and serum concentrations of uric acid and glucose were significantly higher in patients with diabetic retinopathy and DME compared to controls. Also total ratio (serum/ vitreous concentration) of uric acid and glucose was in diabetics significantly higher than in controls. The most important determinant of increasing concentration of both uric acid and glucose in the vitreous was the grade of diabetic retinopathy. Moreover, we demonstrated significant correlation between vitreous concentration of uric acid and concentration of the vascular endothelial growth factor (VEGF) in patients with DME and NPDR. We found further, that the volume of the macula (cube volume - CV) computed with the software of Cirrus HD-OCT correlates in diabetics significantly with the vitreous VEGF concentration, but not with uric acid. This OCT parameter could be used to...

Indiana University-Purdue University Indianapolis (IUPUI) / Prevention of pathological angiogenesis is a key strategy for treatment of common blinding ocular diseases such as retinopathy of prematurity, proliferative diabetic retinopathy, and wet age-related macular degeneration. The current treatment strategies are associated with partial vision loss and are ineffective in a significant patient population. Hence novel drugs as well as new ways to target ocular angiogenesis are needed for treating these diseases. I pursued a natural antiangiogenic compound, cremastranone, to develop novel drug leads and to find new targets. The objective of my doctoral thesis project was to elucidate cremastranone’s molecular mechanism of action and optimize its structureactivity relationship (SAR). In order to achieve this goal, with the help of chemistry collaborators cremastranone was synthesized for the first time. I showed that cremastranone has 50-fold more potency against endothelial cells as compared to nonendothelial cells, and also tested a novel active isomer, SH-11052. By SAR studies I identified a potent molecule, SH-11037, that has 10-fold more selectivity against retinal endothelial cells as compared to macrovascular endothelial cells. I then elucidated cremastranone’s molecular mechanism using a chemical proteomic approach. I identified ferrochelatase (FECH) as a specific interacting protein partner of cremastranone using photoaffinity chromatography. Hence, I hypothesized that cremastranone exerts its antiangiogenic activities through modulation of the functions of FECH. Cremastranone inhibited the enzymatic activity FECH in endothelial cells. Therefore, I investigated the role of FECH in ocular angiogenesis. Partial loss of FECH, using a siRNA-based knock down approach, decreased retinal angiogenesis both in vitro and in vivo in mouse models. Knock down of FECH decreased the expression levels of key proangiogenic proteins HIF-1α, eNOS, and VEGFR2. This work suggests that ferrochelatase plays an important, previously undocumented role in angiogenesis and that targeting of this enzyme by cremastranone might be exploited to inhibit pathological angiogenesis in ocular diseases.

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The aim of this study was to determine the prevalence and causes as well as the risk factors of visual impairment (VI) and blindness among Black South Africans with diabetes mellitus (DM) aged 40 years and older in Mopani District, Limpopo province, South Africa. This was a cross-sectional study in which Black South Africans with DM aged ≥40 years old were examined for VI and blindness. In addition, anthropometric as well as risk factors for VI and blindness were studied. A total of 225 participants were selected from seven Public Health Facilities in Mopani District. Data was collected using standard optometric instruments, anthropometric instruments and structured interviews. Data analysis was done using the Statistical Analysis System (SAS) and Microsoft Excel software packages. The ages of the participants ranged from 40 to 90 years with a mean of 61.5±10.49 years. There were more females (71.5%) than males (28.4%). The prevalence of uncorrected VI and blindness in the right eyes of the participants was 70.7% and 3.6%, respectively. In the left eyes, it was 72% and 3.1%, respectively. However, following optical correction, the prevalence in right eyes was 41.3% and 3.6%, respectively. In the left eyes, it was 42.2% and 3.1%, respectively. Risk factors that were individually associated with VI and blindness include age, educational qualification, monthly income, knowledge of DM types, oral DM treatment (pills), losing weight, compliance to losing weight, family history of DM, physical activity, and date of last eye examination .When logistic regression was used, knowledge of DM types, pills, and compliance to losing weight, family history of DM, monthly income and physical activity remained associated with VI and blindness. The high prevalence of VI in this diabetes population was not primarily due to DM itself, but due to refractive error and cataract, conditions which have effective and easy treatments. A total of 84% of the participants were visually impaired due to either refractive error or cataract or both and only 3.8% due to diabetes retinopathy. It is therefore recommended that appropriate and affordable refraction and cataract surgical services be made available and accessible to this population / Health Studies / D. Litt. et Phil. (Health Studies)

Diabetic vasculopathy (DV) is the most important consequence of chronic hyperglycemia in patients with diabetes mellitus (DM). This thesis explores the interaction of blood, bone and kidney in the pathogenesis of DV by i) reviewing the current understanding of pathogenesis of macrovascular and microvascular diseases in DM to identify gaps in literature and generate hypotheses relating to various facets of DV ii) undertaking a series of prospective studies to examine these hypotheses iii) analysing the findings and integrating any new information obtained from the clinical studies into the current knowledge base and iv) generating hypotheses upon which future work might be based. The literature search was carried out with the aim of understanding current concepts of pathogenesis of DV and its potential modulators. The original reviews resulting from this process are presented in chapters 2 to 4. A series of pilot studies reported in chapters 7 to 11, were then carried out to interrogate hypotheses originating from this process. The first study was carried out in healthy individuals to define the biological variation of potential modulators of DV, namely erythropoietin (EPO), parathyroid hormone, 25 hydroxyvitamin D and 1, 25-dihydroxyvitamin D to facilitate the design and interpretation of subsequent studies. It revealed a wide biological variation of these modulators in the healthy population thus,emphasizing the need to have a control group in the subsequent study population. To examine whether tubulointerstitial dysfunction occurs before the onset of microalbuminuria, a measurement of the above mentioned parameters was carried out along with markers of tubulointerstitial injury in patients with type 1 and type 2 DM without microalbuminuria and in non-diabetic controls. It was found that tubulointerstitial dysfunction with low levels of EPO and 1, 25-dihydroxyvitamin D and higher excretion of tubular injury markers, occurs before the onset of microalbuminuria. Subsequently, diabetic and nondiabetic chronic kidney disease (CKD) patients with EPO deficiency anaemia were examined to study the effects of EPO therapy on the excretion of tubular injury markers. However, in these patient groups, we were unable to demonstrate an effect of EPO therapy on the markers of tubular injury in spite of a beneficial haematological response. To examine whether vascular calcification (VC) and bone mineral density (BMD) were linked in patients with diabetes mellitus and to explore their relationship to modulators of DV, an assessment of VC and BMD was undertaken in patients with type 2 DM with different degrees of proteinuria and normoalbuminuria. VC was assessed by CT scan and BMD by a DEXA scan. Modulators of DV were measured including serum Osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-b-ligand (RANKL). The findings were i) a high prevalence of VC and osteopenia in normoalbuminuric type 2 DM patients with normal serum creatinine ii) a weak inverse relationship between VC and osteopenia iii) proteinuric patients had worse VC but not osteopenia iv) weak relationships between OPG levels and both VC and osteopenia, masked by age in multivariate analysis. The final study examined the relationship between modulators of DV, including OPG and RANKL, and the degree of CKD. It was found that abnormalities of OPG and RANKL occur before the onset of microalbuminuria and progress with deterioration of renal function. Compared to nondiabetics, DM patients have higher OPG levels in the predialysis phase and lower levels in haemodialysis phase, a phenomenon that might indicate endothelial exhaustion in dialysis patients with DM. The derangements associated with DV seem to occur earlier than previously thought. Further work is required to untangle these complexities and to define the contribution of factors such as the adverse blood milieu, the vasculature, abnormal bone and mineral metabolism, and early tubulointerstitial damage. The findings from the studies reported here may help in the formulation of new hypotheses, which might contribute to future work in this area.

Introdução: Inúmeros estudos estabeleceram correlações entre o índice tornozelo-braço (ITB), um marcador de aterosclerose subclínica, e o prognóstico cardiovascular em diferentes populações. No entanto, poucos estudos avaliaram a correlação entre os valores do ITB e lesões cardiovasculares e renais, exclusivamente, em pacientes com hipertensão arterial e diabetes. Objetivo: Estudar a prevalência de alterações morfofuncionais cardíacas, carotídeas, retinianas e renais de acordo com a presença ou não de valores de ITB alterados (ITB <= 0,9 ou ITB > 1,4) em pacientes hipertensos com diabetes tipo 2. Métodos: Foram incluídos no estudo 99 pacientes hipertensos diabéticos com idade entre 50 e 80 anos. A aferição do ITB foi realizada em todos os pacientes por método validado e estes foram classificados em Grupo 1 (ITB normal, n = 49) ou Grupo 2 (ITB alterado, n =50). Todos os pacientes foram submetidos, em até 06 meses, à realização de ecodopplercardiograma, ultrassonografia de carótidas, retinografia colorida, aferição da taxa de filtração glomerular (TFG) e da albuminúria de 24h. Os pacientes foram analisados para a ocorrência ou não de um desfecho-composto ecocardiográfico que incluiu alterações morfológicas e funcionais cardíacas relevantes para a prática clínica. Os pacientes dos grupos 1 e 2 foram também comparados quanto à prevalência de placas carotídeas com ou sem repercussão hemodinâmica, TFG < 60 ml/mim/m2, albuminúria de 24h > 30mg e presença ou não de retinopatia. Por fim, foram comparadas as frequências médias das seguintes lesões de órgãos-alvo de ambos os grupos, considerando-se valor unitário para a presença de cada uma delas: hipertrofia do ventrículo esquerdo, retinopatia hipertensiva, TFG < 60 ml/min/m2 e estenose da artéria carótida interna > 50% do seu diâmetro. Resultados: A média de idade dos pacientes foi 65,4 ± 7 anos, sendo 61,6% deles do sexo feminino. A presença de níveis elevados de pressão arterial sistólica (153,4 ± 18 versus 170 ± 26 mmHg), de albuminúria de 24h > 30mg (55,3% versus 82,6%) e de TFG < 60 ml/min/m2 (12,8% versus 33,3%) foi significativamente maior (p < 0.05) entre os pacientes do Grupo 2. O desfecho-composto ecocardiográfico foi mais prevalente no grupo 2 (84,0% versus 59,2%; p = 0,006) e a frequência média de lesões de órgãos-alvo também foi maior nos pacientes do grupo 2 (0,36 ± 0,31 versus 0,19 ± 0,19; p = 0,001). Análise por regressão logística binária revelou que o ITB foi uma das variáveis preditoras independentes para o desfecho-composto ecocardiográfico (OR = 3,43; IC 95% = 1,07 - 11,0; p = 0,04). A partir da análise por regressão linear obteve-se um modelo final no qual o ITB foi uma das três variáveis preditoras independentes para a estimativa da frequência média de lesões de órgãos-alvo com coeficiente beta = 13,22 (1,81 - 24,63), ao lado da idade e do infarto prévio. Conclusão: Nossos dados mostram que valores de ITB alterados estão associados à maior prevalência de lesões em órgãos-alvo, principalmente alterações ecocardiográficas, em pacientes com hipertensão arterial e diabetes / Introduction: A lot of studies have established strong correlations between the ankle-brachial index (ABI), a marker of subclinical atherosclerosis and cardiovascular prognosis in different populations. However, few studies have assessed the correlation between the values of the ABI and cardiovascular and renal lesions in patients with hypertension and diabetes. Objective: To study the prevalence of cardiac, carotid, renal and retinal morphological and functional changes according to the presence or not of altered ABI values (ABI <= 0.9 or ABI > 1.4) in hypertensive patients with type 2 diabetes. Methods: It was included 99 diabetic hypertensive patients aged between 50 and 80 years. The measurement of the ABI was performed in all patients by validated method and they were classified in Group 1 (normal ABI, n = 49) or group 2 (altered ABI, n = 50). All patients were submitted, up to 6 months, to Doppler echocardiography, carotid ultrasound, color retinography, assessment of glomerular filtration rate (GFR) and 24h albuminuria. Patients were analyzed for the occurrence or not of a composite echocardiographic outcome which included morphological and functional cardiac alterations relevant to clinical practice. Patients in groups 1 and 2 were compared regarding the prevalence of carotid plaques with or without hemodynamic repercussion, TFG < 60 ml/min/m2, 24h albuminuria > 30 mg and the presence or not of retinopathy. Finally, we compared the prevalence of mean frequency of the following end-organ lesions of both groups, considering unit value for each one: left ventricular hypertrophy, hypertensive retinopathy, TFG < 60 ml/min/m2 and internal carotid artery stenosis > 50%. Results: The mean age of the patients was 65.4 ± 7 years, with 61.6% of them female. The presences of elevated levels of systolic blood pressure (153.4 ± 18 versus 170.0 ± 26 mmHg), of 24h albuminuria > 30 mg (55.3% versus 82.6%) and TFG < 60 ml/min/m2 (12.8% vs. 33.3%) were significantly greater (p < 0.05) among the patients of Group 2. The composite echocardiographic outcome was more prevalent in Group 2 (84.0% versus 59.2%, p = 0.006) and the average frequency of subclinical injury of target organs was also greater in patients of Group 2 (0.36 ± 0.31 versus 0.19 ± 0.19; p = 0.001). Binary logistic regression analysis revealed that the ABI was one of the independent predictors of composite echocardiographic outcome (OR = 3.43; IC 95% = 1.07 - 11.0; p = 0.04). From the linear regression analysis it was obtained a final model in which the ABI was one of three independent predictors for the estimation of the average frequency of end-organ damage with ? coefficient = 13.22 (1.81-24.63), besides age and previous myocardial infarction. Conclusion: Our data demonstrates that changed ABI values are associated with higher prevalence of subclinical end-organ lesions, principally changes in echocardiographic parameters, in patients with hypertension and diabetes