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1.  VEGF Levels in Plasma in Relation to Platelet Activation, Glycemic Control, and Microvascular Complications in Type 1 Diabetes 
Diabetes Care  2013;36(6):1629-1634.
Increased levels of vascular endothelial growth factor (VEGF) in human plasma samples have suggested that circulating VEGF is a cause of endothelial dysfunction in diabetes mellitus. However, artificial release of VEGF from platelets as a source of VEGF in plasma samples, as also occurs in serum samples, has not been ruled out in these studies.
We determined VEGF levels in plasma collected in both citrate and PECT, a medium that inactivates platelets, in a cross-sectional cohort of 21 healthy subjects and 64 patients with type 1 diabetes. In addition, we evaluated whether VEGF levels in both types of plasma correlated with the presence of diabetes, glycemic control, markers of in vivo or ex vivo platelet activation, and degree of diabetic retinopathy and nephropathy.
VEGF levels were invariably low in PECT plasma of both nondiabetic and diabetic subjects and were unrelated to any other diabetes-related variable studied. In contrast, VEGF levels in citrate plasma were 150% higher in diabetic patients than in control subjects and correlated with diabetes-related variables. Multiple linear regression analysis showed that levels of platelet factor 4, a marker for ex vivo platelet activation, and HbA1c were the independent predictors of VEGF levels in citrate plasma. Platelet activation, in vivo and ex vivo, was similar in diabetic persons and control subjects.
Like serum, citrate plasma is not suitable for reliable measurements of circulating VEGF. The low levels of VEGF in vivo, as represented by measurements in PECT plasma in our study, do not support a role of circulating VEGF in endothelial dysfunction in type 1 diabetes. Higher levels of VEGF in citrate plasma samples of diabetic persons do not represent the in vivo situation, but mainly originate from higher artificial ex vivo release from platelets correlating with the degree of glycemic control.
PMCID: PMC3661842  PMID: 23321217
2.  Endothelial Tip Cells in Ocular Angiogenesis 
Endothelial tip cells are leading cells at the tips of vascular sprouts coordinating multiple processes during angiogenesis. In the developing retina, tip cells play a tightly controlled, timely role in angiogenesis. In contrast, excessive numbers of tip cells are a characteristic of the chaotic pathological blood vessels in proliferative retinopathies. Tip cells control adjacent endothelial cells in a hierarchical manner to form the stalk of the sprouting vessel, using, among others, the VEGF-DLL-Notch signaling pathway, and recruit pericytes. Tip cells are guided toward avascular areas by signals from the local extracellular matrix that are released by cells from the neuroretina such as astrocytes. Recently, tip cells were identified in endothelial cell cultures, enabling identification of novel molecular markers and mechanisms involved in tip cell biology. These mechanisms are relevant for understanding proliferative retinopathies. Agents that primarily target tip cells can block pathological angiogenesis in the retina efficiently and safely without adverse effects. A striking example is platelet-derived growth factor, which was recently shown to be an efficacious additional target in the treatment of retinal neovascularization. Here we discuss these and other tip cell-based strategies with respect to their potential to treat patients with ocular diseases dominated by neovascularization.
PMCID: PMC3636692  PMID: 23092791
angiogenesis; endothelial tip cell; proliferative retinopathy; anti-angiogenesis therapy; retinal neovascularization; vascular sprouts; endothelial stalk cell; molecular mediators of angiogenesis; pericytes
3.  A shift in the balance of vascular endothelial growth factor and connective tissue growth factor by bevacizumab causes the angiofibrotic switch in proliferative diabetic retinopathy 
In proliferative diabetic retinopathy (PDR), vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) may cause blindness by neovascularisation followed by fibrosis of the retina. It has previously been shown that a shift in the balance between levels of CTGF and VEGF in the eye is associated with this angiofibrotic switch. This study investigated whether anti-VEGF agents induce accelerated fibrosis in patients with PDR, as predicted by this model.
CTGF and VEGF levels were measured by ELISA in 52 vitreous samples of PDR patients, of which 24 patients had received intravitreal bevacizumab 1 week to 3 months before vitrectomy, and were correlated with the degree of vitreoretinal fibrosis as determined clinically and intra-operatively.
CTGF correlated positively, and VEGF correlated negatively with the degree of fibrosis. The CTGF/VEGF ratio was the strongest predictor of fibrosis. Clinically, increased fibrosis was observed after intravitreal bevacizumab.
These results confirm that the CTGF/VEGF ratio is a strong predictor of vitreoretinal fibrosis in PDR, and show that intravitreal anti-VEGF treatment causes increased fibrosis in PDR patients. These findings provide strong support for the model that the balance of CTGF and VEGF determines the angiofibrotic switch, and identify CTGF as a possible therapeutic target in the clinical management of PDR.
PMCID: PMC3308470  PMID: 22289291
Angiogenesis; choroid; CTGF; diabetic retinopathy; drugs; fibrosis; imaging; macula; retina; VEGF; vitreous
4.  Decreased optical coherence tomography‐measured pericentral retinal thickness in patients with diabetes mellitus type 1 with minimal diabetic retinopathy 
The British Journal of Ophthalmology  2007;91(9):1135-1138.
A comparison of retinal thickness (RT) measurements with optical coherence tomography (OCT) in patients with type 1 diabetes mellitus (DM) and no or minimal diabetic retinopathy (DR) versus healthy controls.
Fifty‐three patients with type 1 DM with no or minimal DR underwent full ophthalmic examination, fundus photography and OCT. Mean RT measured by OCT was calculated for the central fovea, the fovea, the pericentral and the peripheral area of the macula, and compared to healthy controls.
Mean RT in the pericentral area was lower in patients with minimal DR (267 µm ± 20 µm; n = 23) compared to healthy controls (281 µm ±13 µm; p = 0.005; n = 28). Mean pericentral RT in patients without DR (276 µm ±14 µm; n = 30) was less than pericentral RT in healthy controls, but higher than in patients with minimal DR, without being statistically significant. None of the other regions showed a significant change.
In this study a significantly decreased pericentral RT was measured in patients with minimal DR compared to healthy controls. This could be explained by a loss of intraretinal neural tissue in the earliest stage of DR.
PMCID: PMC1954913  PMID: 17383994
5.  Vitreous TIMP-1 levels associate with neovascularization and TGF-β2 levels but not with fibrosis in the clinical course of proliferative diabetic retinopathy 
In proliferative diabetic retinopathy (PDR), vascular endothelial growth factor (VEGF) and CCN2 (connective tissue growth factor; CTGF) cause blindness by neovascularization and subsequent fibrosis. This angio-fibrotic switch is associated with a shift in the balance between vitreous levels of CCN2 and VEGF in the eye. Here, we investigated the possible involvement of other important mediators of fibrosis, tissue inhibitor of metalloproteinases (TIMP)-1 and transforming growth factor (TGF)-β2, and of the matrix metalloproteinases (MMP)-2 and MMP-9, in the natural course of PDR. TIMP-1, activated TGF-β2, CCN2 and VEGF levels were measured by ELISA in 78 vitreous samples of patients with PDR (n = 28), diabetic patients without PDR (n = 24), and patients with the diabetes-unrelated retinal conditions macular hole (n = 10) or macular pucker (n = 16), and were related to MMP-2 and MMP-9 activity on zymograms and to clinical data, including degree of intra-ocular neovascularization and fibrosis. TIMP-1, CCN2 and VEGF levels, but not activated TGF-β2 levels, were significantly increased in the vitreous of diabetic patients, with the highest levels in PDR patients. CCN2 and the CCN2/VEGF ratio were the strongest predictors of degree of fibrosis. In diabetic patients with or without PDR, activated TGF-β2 levels correlated with TIMP-1 levels, whereas in PDR patients, TIMP-1 levels, MMP-2 and proMMP-9 were associated with degree of neovascularization, like VEGF levels, but not with fibrosis. We confirm here our previous findings that retinal fibrosis in PDR patients is significantly correlated with vitreous CCN2 levels and the CCN2/VEGF ratio. In contrast, TIMP-1, MMP-2 and MMP-9 appear to have a role in the angiogenic phase rather than in the fibrotic phase of PDR.
PMCID: PMC3590360  PMID: 23054594
Diabetic retinopathy; CCN2; VEGF; TGF-β; TIMP-1; MMP-2; MMP-9; Neovascularization; Fibrosis
6.  Early Neurodegeneration in the Retina of Type 2 Diabetic Patients 
The purpose of this study was to determine whether diabetes type 2 causes thinning of retinal layers as a sign of neurodegeneration and to investigate the possible relationship between this thinning and duration of diabetes mellitus, diabetic retinopathy (DR) status, age, sex, and glycemic control (HbA1c).
Mean layer thickness was calculated for retinal layers following automated segmentation of spectral domain optical coherence tomography images of diabetic patients with no or minimal DR and compared with controls. To determine the relationship between layer thickness and diabetes duration, DR status, age, sex, and HbA1c, a multiple linear regression analysis was used.
In the pericentral area of the macula, the retinal nerve fiber layer (RNFL), ganglion cell layer (GCL), and inner plexiform layer (IPL) were thinner in patients with minimal DR compared to controls (respective difference 1.9 μm, 95% confidence interval [CI] 0.3–3.5 μm; 5.2 μm, 95% CI 1.0–9.3 μm; 4.5 μm, 95% CI 2.2–6.7 μm). In the peripheral area of the macula, the RNFL and IPL were thinner in patients with minimal DR compared to controls (respective difference 3.2 μm, 95% CI 0.1–6.4 μm; 3.3 μm, 95% CI 1.2–5.4 μm). Multiple linear regression analysis showed DR status to be the only significant explanatory variable (R = 0.31, P = 0.03) for this retinal thinning.
This study demonstrated thinner inner retinal layers in the macula of type 2 diabetic patients with minimal DR than in controls. These results support the concept that early DR includes a neurodegenerative component.
This study demonstrated thinning of the inner retinal layers in type 2 diabetic patients compared to controls. The findings conform to previously reported changes in type 1 diabetes and support the concept that diabetic retinopathy includes a neurodegenerative component.
PMCID: PMC3366721  PMID: 22427582
7.  Association of visual function and ganglion cell layer thickness in patients with diabetes mellitus type 1 and no or minimal diabetic retinopathy 
Vision research  2010;51(2):224-228.
Diabetic retinopathy (DR) classically presents with micro-aneurysms, small haemorrhages and/or lipoprotein exudates. Several studies have indicated that neural loss occurs in DR even before vascular damage can be observed. This study evaluated the possible relationship between structure (spectral domain- optical coherence tomography) and function (Rarebit visual field test) in patients with type 1 diabetes mellitus and no or minimal diabetic retinopathy (DR). Results demonstrated loss of macular visual function and corresponding thinning of the ganglion cell layer (GCL) in the pericentral area of the macula of diabetic patients (Rs = 0.65, p < 0.001). In multivariable logistic regression analysis, GCL thickness remained an independent predictor of decreased visual function (OR 1.5, 95% CI 1.1 – 2.1). Early DR seems to include a neurodegenerative component.
PMCID: PMC3017636  PMID: 20801146
Diabetes Mellitus; Neurodegeneration; Ganglion cells; Visual function test; Retina
8.  CD34 marks angiogenic tip cells in human vascular endothelial cell cultures 
Angiogenesis  2012;15(1):151-163.
The functional shift of quiescent endothelial cells into tip cells that migrate and stalk cells that proliferate is a key event during sprouting angiogenesis. We previously showed that the sialomucin CD34 is expressed in a small subset of cultured endothelial cells and that these cells extend filopodia: a hallmark of tip cells in vivo. In the present study, we characterized endothelial cells expressing CD34 in endothelial monolayers in vitro. We found that CD34-positive human umbilical vein endothelial cells show low proliferation activity and increased mRNA expression of all known tip cell markers, as compared to CD34-negative cells. Genome-wide mRNA profiling analysis of CD34-positive endothelial cells demonstrated enrichment for biological functions related to angiogenesis and migration, whereas CD34-negative cells were enriched for functions related to proliferation. In addition, we found an increase or decrease of CD34-positive cells in vitro upon exposure to stimuli that enhance or limit the number of tip cells in vivo, respectively. Our findings suggest cells with virtually all known properties of tip cells are present in vascular endothelial cell cultures and that they can be isolated based on expression of CD34. This novel strategy may open alternative avenues for future studies of molecular processes and functions in tip cells in angiogenesis.
Electronic supplementary material
The online version of this article (doi:10.1007/s10456-011-9251-z) contains supplementary material, which is available to authorized users.
PMCID: PMC3274677  PMID: 22249946
Angiogenesis; Endothelial tip cell; Vascular endothelial cell cultures; CD34
9.  Microcirculation and atherothrombotic parameters in prolactinoma patients: a pilot study 
Pituitary  2011;15(4):472-481.
Atherothrombosis is a multifactorial process, governed by an interaction between the vessel wall, hemodynamic factors and systemic atherothrombotic risk factors. Recent in vitro, human ex vivo and animal studies have implicated the hormone prolactin as an atherothrombotic mediator. To address this issue, we evaluated the anatomy and function of various microvascular beds as well as plasma atherothrombosis markers in patients with elevated prolactin levels. In this pilot study, involving 10 prolactinoma patients and 10 control subjects, sidestream dark field (SDF) imaging revealed a marked perturbation of the sublingual microcirculation in prolactinoma patients compared to control subjects, as attested to by significant changes in microvascular flow index (2.74 ± 0.12 vs. 2.91 ± 0.05, respectively; P = 0.0006), in heterogeneity index (0.28 [IQR 0.18–0.31] vs. 0.09 [IQR 0.08–0.17], respectively; P = 0.002) and lower proportion of perfused vessels (90 ± 4.0% vs. 95 ± 3.0%, respectively; P = 0.016). In the retina, fluorescein angiography (FAG) confirmed these data, since prolactinoma patients more often have dilatated perifoveal capillaries. In plasma, prolactinoma patients displayed several pro-atherogenic disturbances, including a higher endogenous thrombin potential and prothrombin levels as well as decreased HDL-cholesterol levels. Prolactinoma patients are characterized by microvascular dysfunction as well as plasma markers indicating a pro-atherothrombotic state. Further studies are required to assess if prolactin is causally involved in atherothrombotic disease.
PMCID: PMC3493673  PMID: 21993601
Prolactinoma; Microcirculation; Coagulation; Inflammation; Atherothrombosis
10.  Vitamin K supplementation increases vitamin K tissue levels but fails to counteract ectopic calcification in a mouse model for pseudoxanthoma elasticum 
Pseudoxanthoma elasticum (PXE) is an autosomal recessive disorder in which calcification of connective tissue leads to pathology in skin, eye and blood vessels. PXE is caused by mutations in ABCC6. High expression of this transporter in the basolateral hepatocyte membrane suggests that it secretes an as-yet elusive factor into the circulation which prevents ectopic calcification. Utilizing our Abcc6−/− mouse model for PXE, we tested the hypothesis that this factor is vitamin K (precursor) (Borst et al. 2008, Cell Cycle). For 3 months, Abcc6−/− and wild-type mice were put on diets containing either the minimum dose of vitamin K required for normal blood coagulation or a dose that was 100 times higher. Vitamin K was supplied as menaquinone-7 (MK-7). Ectopic calcification was monitored in vivo by monthly micro-CT scans of the snout, as the PXE mouse model develops a characteristic connective tissue mineralization at the base of the whiskers. In addition, calcification of kidney arteries was measured by histology. Results show that supplemental MK-7 had no effect on ectopic calcification in Abcc6−/− mice. MK-7 supplementation increased vitamin K levels (in skin, heart and brain) in wild-type and in Abcc6−/− mice. Vitamin K tissue levels did not depend on Abcc6 genotype. In conclusion, dietary MK-7 supplementation increased vitamin K tissue levels in the PXE mouse model but failed to counteract ectopic calcification. Hence, we obtained no support for the hypothesis that Abcc6 transports vitamin K and that PXE can be cured by increasing tissue levels of vitamin K.
PMCID: PMC3195265  PMID: 21725681
Pseudoxanthoma elasticum; ABC transporter; Vitamin K; Ectopic calcification; Vascular calcification; Connective tissue; Cardiovascular; Vitamins; Calcium metabolism; Mouse models
11.  Vascular Endothelial Growth Factor in the Circulation in Cancer Patients May Not Be a Relevant Biomarker 
PLoS ONE  2011;6(5):e19873.
Levels of circulating vascular endothelial growth factor (VEGF) have widely been used as biomarker for angiogenic activity in cancer. For this purpose, non-standardized measurements in plasma and serum were used, without correction for artificial VEGF release by platelets activated ex vivo. We hypothesize that “true” circulating (c)VEGF levels in most cancer patients are low and unrelated to cancer load or tumour angiogenesis.
We determined VEGF levels in PECT, a medium that contains platelet activation inhibitors, in citrate plasma, and in isolated platelets in 16 healthy subjects, 18 patients with metastatic non-renal cancer (non-RCC) and 12 patients with renal cell carcinoma (RCC). In non-RCC patients, circulating plasma VEGF levels were low and similar to VEGF levels in controls if platelet activation was minimized during the harvest procedure by PECT medium. In citrate plasma, VEGF levels were elevated in non-RCC patients, but this could be explained by a combination of increased platelet activation during blood harvesting, and by a two-fold increase in VEGF content of individual platelets (controls: 3.4 IU/106, non-RCC: 6.2 IU/106 platelets, p = 0.001). In contrast, cVEGF levels in RCC patients were elevated (PECT plasma: 64 pg/ml vs. 21 pg/ml, RCC vs. non-RCC, p<0.0001), and not related to platelet VEGF concentration.
Our findings suggest that “true” freely cVEGF levels are not elevated in the majority of cancer patients. Previously reported elevated plasma VEGF levels in cancer appear to be due to artificial release from activated platelets, which in cancer have an increased VEGF content, during the blood harvest procedure. Only in patients with RCC, which is characterized by excessive VEGF production due to a specific genetic defect, were cVEGF levels elevated. This observation may be related to limited and selective success of anti-VEGF agents, such as bevacizumab and sorafenib, as monotherapy in RCC compared to other forms of cancer.
PMCID: PMC3102663  PMID: 21637343
12.  Decreased Retinal Ganglion Cell Layer Thickness in Patients with Type 1 Diabetes 
This study demonstrates ganglion cell layer thinning in the pericentral area and corresponding loss of retinal nerve fiber layer thickness in the peripheral macula in patients with type 1 diabetes with no or minimal diabetic retinopathy compared with control subjects. These results support the concept that diabetes has an early neurodegenerative effect on the retina, which occurs even though the vascular component of diabetic retinopathy is still minimal.
To determine which retinal layers are most affected by diabetes and contribute to thinning of the inner retina and to investigate the relationship between retinal layer thickness (LT) and diabetes duration, diabetic retinopathy (DR) status, age, glycosylated hemoglobin (HbA1c), and the sex of the individual, in patients with type 1 diabetes who have no or minimal DR.
Mean LT was calculated for the individual retinal layers after automated segmentation of spectral domain-optical coherence tomography scans of patients with diabetes and compared with that in control subjects. Multiple linear regression analysis was used to determine the relationship between LT and HbA1c, age, sex, diabetes duration, and DR status.
In patients with minimal DR, the mean ganglion cell layer (GCL) in the pericentral area was 5.1 μm thinner (95% confidence interval [CI], 1.1–9.1 μm), and in the peripheral macula, the mean retinal nerve fiber layer (RNFL) was 3.7 μm thinner (95% CI, 1.3–6.1 μm) than in the control subjects. There was a significant linear correlation (R = 0.53, P < 0.01) between GCL thickness and diabetes duration in the pooled group of patients. Multiple linear regression analysis (R = 0.62, P < 0.01) showed that DR status was the most important explanatory variable.
This study demonstrates GCL thinning in the pericentral area and corresponding loss of RNFL thickness in the peripheral macula in patients with type 1 diabetes and no or minimal DR compared with control subjects. These results support the concept that diabetes has an early neurodegenerative effect on the retina, which occurs even though the vascular component of DR is minimal.
PMCID: PMC2904016  PMID: 20130282
13.  Selective Loss of Inner Retinal Layer Thickness in Type 1 Diabetic Patients with Minimal Diabetic Retinopathy 
To determine whether type 1 diabetes preferentially affects the inner retinal layers by comparing the thickness of six retinal layers in type 1 diabetic patients who have no or minimal diabetic retinopathy (DR) with those of age- and sex-matched healthy controls.
Fifty-seven patients with type 1 diabetes with no (n = 32) or minimal (n = 25) DR underwent full ophthalmic examination, stereoscopic fundus photography, and optical coherence tomography (OCT). After automated segmentation of intraretinal layers of the OCT images, mean thickness was calculated for six layers of the retina in the fovea, the pericentral area, and the peripheral area of the central macula and were compared with those of an age- and sex-matched control group.
In patients with minimal DR, the mean ganglion cell/ inner plexiform layer was 2.7 μm thinner (95% confidence interval [CI], 2.1– 4.3 μm) and the mean inner nuclear layer was 1.1 μm thinner (95% CI, 0.1–2.1 μm) in the pericentral area of the central macula compared to those of age-matched controls. In the peripheral area, the mean ganglion cell/inner plexiform layer remained significantly thinner. No other layers showed a significant difference.
Thinning of the total retina in type 1 diabetic patients with minimal retinopathy compared with healthy controls is attributed to a selective thinning of inner retinal layers and supports the concept that early DR includes a neurode-generative component.
PMCID: PMC2937215  PMID: 19151397
14.  The Angio-Fibrotic Switch of VEGF and CTGF in Proliferative Diabetic Retinopathy 
PLoS ONE  2008;3(7):e2675.
In proliferative diabetic retinopathy (PDR), vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) cause blindness by neovascularization and subsequent fibrosis, but their relative contribution to both processes is unknown. We hypothesize that the balance between levels of pro-angiogenic VEGF and pro-fibrotic CTGF regulates angiogenesis, the angio-fibrotic switch, and the resulting fibrosis and scarring.
Methods/Principal Findings
VEGF and CTGF were measured by ELISA in 68 vitreous samples of patients with proliferative DR (PDR, N = 32), macular hole (N = 13) or macular pucker (N = 23) and were related to clinical data, including degree of intra-ocular neovascularization and fibrosis. In addition, clinical cases of PDR (n = 4) were studied before and after pan-retinal photocoagulation and intra-vitreal injections with bevacizumab, an antibody against VEGF. Neovascularization and fibrosis in various degrees occurred almost exclusively in PDR patients. In PDR patients, vitreous CTGF levels were significantly associated with degree of fibrosis and with VEGF levels, but not with neovascularization, whereas VEGF levels were associated only with neovascularization. The ratio of CTGF and VEGF was the strongest predictor of degree of fibrosis. As predicted by these findings, patients with PDR demonstrated a temporary increase in intra-ocular fibrosis after anti-VEGF treatment or laser treatment.
CTGF is primarily a pro-fibrotic factor in the eye, and a shift in the balance between CTGF and VEGF is associated with the switch from angiogenesis to fibrosis in proliferative retinopathy.
PMCID: PMC2443281  PMID: 18628999
15.  Value of routine funduscopy in patients with hypertension: systematic review 
BMJ : British Medical Journal  2005;331(7508):73.
Objective To evaluate the additional value of funduscopy in the routine management of patients with hypertension.
Design Systematic review.
Participants Adults aged 19 or more with hypertensive retinopathy.
Data sources Medline, Embase, and the Cochrane Library from 1990.
Review methods Studies were included that assessed hypertensive retinopathy with blinding for blood pressure and cardiovascular risk factors. Studies on observer agreement had to be assessed by two or more observers and expressed as a κ statistic. Studies on the association between hypertensive retinopathy and hypertensive organ damage were carried out in patients with hypertension. The association between hypertensive retinopathy and cardiovascular risk was carried out in unselected normotensive and hypertensive people without diabetes mellitus.
Results The assessment of microvascular changes in the retina is limited by large variation between observers. The positive and negative predictive values for the association between hypertensive retinopathy and blood pressure were low (47% to 72% and 32% to 67%, respectively). Associations between retinal microvascular changes and cardiovascular risk were inconsistent, except for retinopathy and stroke. The increased risk of stroke, however, was also present in normotensive people with retinopathy. These studies did not adjust for other indicators of hypertensive organ damage.
Conclusion Evidence is lacking that routine funduscopy is of additional value in the management of hypertensive patients.
PMCID: PMC558610  PMID: 16002881

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