[show abstract][hide abstract] ABSTRACT: RATIONALE: Tie2 is predominantly expressed by endothelial cells and is involved in vascular integrity control during sepsis. Changes in Tie2 expression during sepsis development may contribute to microvascular dysfunction. Understanding the kinetics and molecular basis of these changes may assist in the development of therapeutic intervention to counteract microvascular dysfunction. OBJECTIVE: To investigate the molecular mechanisms underlying the changes in Tie2 expression upon lipopolysaccharide (LPS) challenge. METHODS AND RESULTS: Studies were performed in LPS and pro-inflammatory cytokine challenged mice as well as in mice subjected to hemorrhagic shock, primary endothelial cells were used for in vitro experiments in static and flow conditions. Eight hours after LPS challenge, Tie2 mRNA loss was observed in all major organs, while loss of Tie2 protein was predominantly observed in lungs and kidneys, in the capillaries. A similar loss could be induced by secondary cytokines TNF-α and IL-1β. Ang2 protein administration did not affect Tie2 protein expression nor was Tie2 protein rescued in LPS-challenged Ang2-deficient mice, excluding a major role for Ang2 in Tie2 down regulation. In vitro, endothelial loss of Tie2 was observed upon lowering of shear stress, not upon LPS and TNF-α stimulation, suggesting that inflammation related haemodynamic changes play a major role in loss of Tie2 in vivo, as also hemorrhagic shock induced Tie2 mRNA loss. In vitro, this loss was partially counteracted by pre-incubation with a pharmacologically NF-кB inhibitor (BAY11-7082), an effect further substantiated in vivo by pre-treatment of mice with the NF-кB inhibitor prior to the inflammatory challenge. CONCLUSIONS: Microvascular bed specific loss of Tie2 mRNA and protein in vivo upon LPS, TNFα, IL-1β challenge, as well as in response to hemorrhagic shock, is likely an indirect effect caused by a change in endothelial shear stress. This loss of Tie2 mRNA, but not Tie2 protein, induced by TNFα exposure was shown to be controlled by NF-кB signaling. Drugs aiming at restoring vascular integrity in sepsis could focus on preventing the Tie2 loss.
European Journal of Intensive Care Medicine 04/2013; · 5.17 Impact Factor
[show abstract][hide abstract] ABSTRACT: Background
In sepsis and various other inflammatory conditions, elevated circulating levels of angiopoietin-2 (Ang2) are detected, but the precise functional role of Ang2 in these conditions is not well understood. Here, we investigated the contribution of Ang2 to the inflammatory response and renal function impairment in a mouse model of endotoxaemia.Methods
Ang2-deficient mice and wild-type littermates were challenged with lipopolysaccharide [LPS; 1500 EU/g, intraperitoneal (i.p.)]. In additional experiments, wild-type C57Bl/6 mice were depleted of circulating neutrophils by antibody treatment (NIMPR14) prior to LPS challenge to study the role of neutrophils in regulating LPS-induced cytokine release. After 8 or 24 h of LPS challenge, the mice were sacrificed and organs were harvested. Quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay were performed for endothelial adhesion molecules (P-selectin, E-selectin, VCAM-1 and ICAM-1) and plasma cytokines (TNF-α, IL-6, KC, MIP-2), respectively. To assess renal function, blood urea nitrogen levels in plasma and albumin-to-creatinine ratio in urine were measured.ResultsUpon LPS challenge, expression levels of various endothelial adhesion molecules in Ang2-deficient mice were reduced in an organ-specific manner. In contrast, in these mice, plasma levels of TNF-α and IL-6 were significantly increased compared with their wild-type littermates, possibly due to decreased neutrophil glomerular influx. Importantly, the absence of Ang2 did not protect the mice from acute kidney injury (AKI) upon LPS challenge.Conclusions
The absence of Ang2 release upon LPS challenge induces pleotropic effects with regard to endothelial activation and systemic inflammation, but does not protect mice from LPS-induced AKI.
[show abstract][hide abstract] ABSTRACT: In established tumors, angiogenic endothelial cells (ECs) coexist next to "quiescent" EC in matured vessels. We hypothesized that angio-gene expression of B16.F10 melanoma would differ depending on the growth stage. Unraveling the spatiotemporal nature thereof is essential for drug regimen design aimed to affect multiple neovascularization stages. We determined the angiogenic phenotype-represented by 52 angio-genes-and vascular morphology of small, intermediate, and large s.c. growing mouse B16.F10 tumors and demonstrated that expression of these genes did not differ between the different growth stages. Yet vascular morphology changed dramatically from small vessels without lumen in small to larger vessels with increased lumen size in intermediate/large tumors. Separate analysis of these vascular morphologies revealed a significant difference in αSMA expression in relation to vessel morphology, while no relation with VEGF, HIF-1α, nor Dll4 expression levels was observed. We conclude that the tumor vasculature remains actively engaged in angiogenesis during B16.F10 melanoma outgrowth and that the major change in tumor vascular morphology does not follow molecular concepts generated in other angiogenesis models.
[show abstract][hide abstract] ABSTRACT: Pericyte loss, vasoregression and neuroglial activation are characteristic changes in incipient diabetic retinopathy. In this study, the effect of the antioxidant and antiglycating dipeptide carnosine was studied on the development of experimental diabetic retinopathy.
STZ-induced diabetic Wistar rats were orally treated with carnosine (1g/kg body weight/day). Retinal vascular damage was assessed by quantitative morphometry. Retinal protein extracts were analyzed for markers of oxidative stress, AGE-formation, activation of the hexosamine pathway and changes in the expression of Ang-2, VEGF and heat shock proteins Hsp27 and HO-1. Glial cell activation was analyzed using Western blot analysis and immunofluorescence of GFAP expression and retinal neuronal damage was histologically examined.
Oral carnosine treatment prevented retinal vascular damage after 6 months of experimental hyperglycemia. The protection was not caused by ROS- or AGE-inhibition, but associated with a significant induction of Hsp27 in activated glial cells and normalization of increased Ang-2 levels in diabetic retinas. A significant reduction of photoreceptors in retinas of carnosine treated animals was noted.
Oral carnosine treatment protects retinal capillary cells in experimental diabetic retinopathy, independent of its biochemical function. The vasoprotective effect of carnosine might be mediated by the induction of protective Hsp27 in activated glial cells and normalization of hyperglycemia-induced Ang-2.
Cellular Physiology and Biochemistry 01/2011; 28(1):125-36. · 3.42 Impact Factor
[show abstract][hide abstract] ABSTRACT: Diabetic retinopathy is the result of increased oxidative and nitrosative stress induced by chronic hyperglycaemia, and affects the vasculature and the neuroglia. Erythropoietin is a neuroprotective and an endothelial survival factor. We assessed the effect of suberythropoietic epoetin delta doses on variables of oxidative stress in target tissues of diabetic complications and on pericyte loss in the diabetic retina.
We administered epoetin delta to streptozotocin-induced diabetic Wistar rats at doses of 384 IU/kg body weight once weekly or 128 IU/kg body weight three times a week. The treatment lasted for 3 months. Oxidative stress and formation of AGEs were assessed by immunoblotting, expression of Ang-2 (also known as Angpt2) by RT-PCR, activation of protein kinase B (AKT) and heat shock protein (HSP)-27 levels by immunofluorescence, and incipient retinal vascular changes by quantitative morphometry of retinal digest preparations.
Diabetes increased variables of oxidative stress and nitrosative stress (N(epsilon)-carboxymethyl-lysine, nitrotyrosine and methylglyoxal-type AGEs) in retina, kidney and heart of diabetic rats. Epoetin delta reduced oxidative and nitrosative stress in all tissues, and AGEs in the retina. It also reduced increased retinal Ang-2 expression and pericyte loss, and ameliorated p-AKT and HSP-27 levels.
Epoetin delta has antioxidative properties in organs affected by diabetes and may prevent incipient microvascular damage in the diabetic retina.
[show abstract][hide abstract] ABSTRACT: Many disabling human retinal disorders involve the central retina, particularly the macula. However, the commonly used rodent models in research, mouse and rat, do not possess a macula. The purpose of this study was to identify small laboratory rodents with a significant central region as potential new models for macular research.
Gerbillus perpallidus, Meriones unguiculatus and Phodopus campbelli, laboratory rodents less commonly used in retinal research, were subjected to confocal scanning laser ophthalmoscopy (cSLO), fluorescein and indocyanine green angiography, and spectral-domain optical coherence tomography (SD-OCT) using standard equipment (Heidelberg Engineering HRA1 and Spectralis™) adapted to small rodent eyes. The existence of a visual streak-like pattern was assessed on the basis of vascular topography, retinal thickness, and the topography of retinal ganglion cells and cone photoreceptors. All three species examined showed evidence of a significant horizontal streak-like specialization. cSLO angiography and retinal wholemounts revealed that superficial retinal blood vessels typically ramify and narrow into a sparse capillary net at the border of the respective area located dorsal to the optic nerve. Similar to the macular region, there was an absence of larger blood vessels in the streak region. Furthermore, the thickness of the photoreceptor layer and the population density of neurons in the ganglion cell layer were markedly increased in the visual streak region.
The retinal specializations of Gerbillus perpallidus, Meriones unguiculatus and Phodopus campbelli resemble features of the primate macula. Hence, the rodents reported here may serve to study aspects of macular development and diseases like age-related macular degeneration and diabetic macular edema, and the preclinical assessment of therapeutic strategies.
PLoS ONE 01/2010; 5(10):e13403. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Proliferative retinopathies remain the most common causes of blindness. Retinal neovascularisation is induced by hypoxic upregulation of angiogenic growth factors, such as vascular endothelial growth factor (VEGF). Thalidomide has been shown to be anti-angiogenic via reduction of VEGF levels. We investigated the effect of intravitreal application of thalidomide on neovascularisation and retinal toxicity in a mouse model of proliferative retinopathy.
C57BL/6J mice were exposed to 75% oxygen from postnatal day (p) 7 to p12. Immediately after transfer to room air at p12, mice received an intravitreal injection of 150 microg/microl thalidomide or control solution. Preretinal neovascularisation was quantified at p17. VEGF levels were assessed in whole retinal lysates at p13 and p17. Retinal toxicity was assessed by measuring retinal layer thickness and by analysing caspase-3 activity and apoptotic cell counts in retinal layers to examine retinal apoptosis.
Intravitreal application of thalidomide significantly reduced preretinal neovascularisation by 62% compared with control treated contralateral eyes (p=0.01). Interestingly, this effect was established without a change in retinal VEGF levels. Intravitreal thalidomide was not toxic, as retinal layer thickness, retinal caspase-3 activity and apoptotic cell counts were unaltered.
These data indicate that intravitreal application of thalidomide can be an effective and safe way to treat retinal neovascularisation.
The British journal of ophthalmology 09/2009; 94(4):504-8. · 2.92 Impact Factor
[show abstract][hide abstract] ABSTRACT: Angiopoietin-2 (Ang-2) antagonises the maturing effect of angiopoietin-1 (Ang-1) on blood vessels, and cooperates with VEGF to induce neovascularisation. In knockout mice, Ang-2 displayed a specific role in postnatal angiogenic remodelling. Here, we demonstrate that mice deficient in Ang-2 fail to form a proper spatial retinal vascular network. The retinal vasculature was characterised by reduced large vessel numbers and defects forming the superficial periphery mostly on the arteriolar site, and the secondary and tertiary deep capillary network. Hypoxia in the retinal periphery induced a four-fold VEGF upregulation and active endothelial proliferation for up to 60 days. Concomitantly, retinal digest preparations showed increased arteriolar (+33%) and capillary diameters (+90%), and fluorescein angiograms revealed leakiness of neovascular front. At one year of age, persistent preretinal vessels were non-leaky in accordance with a relative increase in the ratio of Ang-1 to VEGF. Taken together, the data suggest that Ang-2 has an important function in the spatial configuration of the three-dimensional retinal vasculature. Secondarily, prolonged VEGF activity results in a model of persistent proliferative retinopathy.
Thrombosis and Haemostasis 08/2009; 102(1):120-30. · 6.09 Impact Factor
[show abstract][hide abstract] ABSTRACT: Intravitreal drug administration leads to high intraocular concentrations with potentially toxic effects on ocular tissues. This study was an assessment of the toxicity of triamcinolone and bevacizumab in living retinal explants using two-photon (2P) microscopy.
Wild-type mice received intravitreal injections of triamcinolone, bevacizumab, or vehicle. Ten and 45 days after injection, wholemounted retinal explants were incubated with the fluorescent dye sulforhodamine 101 (SR101) to analyze morphology and tissue damage with 2P microscopy ex vivo. Retinas that received the same treatment were stained for apoptosis (TUNEL) and glial activation (GFAP). An intravitreal injection of NMDA (N-methyl-d-aspartate) was used as a positive control to ensure the fidelity of detection of retinal damage with ex vivo 2P microscopy.
Overall retinal morphology was undisturbed after all procedures and time points. NMDA injection resulted in a strong increase in the number of SR101-labeled cells and increased apoptosis and glial activation when compared with sham-injected eyes. This result was in contrast to exposure to bevacizumab, which caused no appreciable damage. After triamcinolone treatment, marked damage in the inner retina was observed. However, damaged cells were restricted to sharply demarcated areas, and only mild changes in TUNEL-positive cells and GFAP activation was observed when compared to sham-injected eyes.
2P microscopy in combination with SR101 staining allows fast morphologic assessment of living retinal explants and can be used to evaluate adverse effects on retinal viability of test substances. Bevacizumab treatment did not cause any detectable retinal damage, whereas triamcinolone was associated with substantial, although spatially restricted, damage.
[show abstract][hide abstract] ABSTRACT: Our previous data suggested that angiopoietin-2 (Ang-2) is linked to pericyte loss, thereby playing an important role in diabetic retinopathy. In this study, we investigated the effect of retinal overexpression of human Ang-2 (mOpsinhAng2 mouse) on vascular morphology in non-diabetic and streptozotozin-induced diabetic animals. Pericyte (PC) coverage and acellular capillary (AC) formation were quantitated in retinal digest preparations after 3 and 6 months of diabetes duration. The degree of retinopathy in non-diabetic mOpsinhAng2 mice at 3 months (-21% PC, +49% AC) was comparable to age-matched diabetic wild type mice. Diabetic mOpsinhAng2 mice exhibited significantly worse vascular pathology than wild type counterparts at 6 months. Quantitative PCR revealed that human Ang-2 mRNA was highly overexpressed in retinas of transgenic mice. Our data demonstrate that overexpression of Ang-2 in the retina enhances vascular pathology, indicating that Ang-2 plays an essential role in diabetic vasoregression via destabilization of pericytes.
[show abstract][hide abstract] ABSTRACT: The mechanism underlying pericyte loss during incipient diabetic retinopathy remains controversial. Hyperglycemia induces angiopoietin-2 (Ang-2) transcription, which modulates capillary pericyte coverage. In this study, we assessed loss of pericyte subgroups and the contribution of Ang-2 to pericyte migration.
Numbers of total pericytes and their subgroups were quantified in retinal digest preparations of spontaneous diabetic XLacZ mice. Pericytes were divided into subgroups according to their localization, their position relative to adjacent endothelial cells, and the expression of LacZ. The contribution of Ang-2 to pericyte migration was assessed in Ang-2 overexpressing (mOpsinhAng2) and deficient (Ang2LacZ) mice.
Pericyte numbers were reduced by 16% (P < 0.01) in XLacZ mice after 6 months of diabetes. Reduction of pericytes was restricted to pericytes on straight capillaries (relative reduction 27%, P < 0.05) and was predominantly observed in LacZ-positive pericytes (-20%, P < 0.01). Hyperglycemia increased the numbers of migrating pericytes (69%; P < 0.05), of which the relative increase due to diabetes was exclusively in LacZ-negative pericytes, indicating reduced adherence to the capillaries (176%; P < 0.01). Overexpression of Ang-2 in nondiabetic retinas mimicked diabetic pericyte migration of wild-type animals (78%; P < 0.01). Ang-2 deficient mice completely lacked hyperglycemia-induced increase in pericyte migration compared with wild-type littermates.
Diabetic pericyte loss is the result of pericyte migration, and this process is modulated by the Ang-Tie system.
[show abstract][hide abstract] ABSTRACT: The advanced glycation end product (AGE)-receptor for AGE (RAGE) pathway is involved in the pathogenesis of diabetic microvascular damage. The special distribution of RAGE and its engagement has an impact on the development of diabetic retinopathy. In the present study, we used immunofluorescence and confocal laser microscopy to study RAGE expression with special emphasis on Müller glia in Sprague Dawley rats. RAGE expression was low in nondiabetic retinae and was found in ganglion cells and Müller cell end feet. In diabetic retinae, upregulated RAGE was predominantly expressed in retinal glia. Since Müller cells are important in the regulation of important features of early retinal vascular damage, such as vascular permeability, homeostasis, and response to stress, RAGE appears to be a central modulator in diabetic retinopathy.
Annals of the New York Academy of Sciences 05/2008; 1126:42-5. · 4.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: Retinae of aged humans show signs of vascular regression. Vascular regression involves a mismatch between Angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF) expression. We used heterozygous Ang-2 deficient (Ang2LacZ) mice to evaluate murine retinal vascular changes and gene expression of growth factors. Vascular changes were assessed by quantitative retinal morphometry and gene expression levels of growth factors were measured by quantitative PCR. The numbers of endothelial cells and pericytes did not change in the Ang2LacZ retinae with age, whereas they decreased throughout the age spectrum studied in the wild type retinae. Moreover, vascular regression significantly decelerated in the heterozygous Ang2LacZ retinae (200% to 1 month), while the formation of acellular capillaries was significantly increased at 13 months in the wild type retinae (340% to 1 month). Gene expression analysis revealed that VEGF, Ang-1, PDGF-B and Ang2 mRNA levels were decreased in the wild type retinae at 9 month of age. However, the decrease of Ang-2 was smaller compared with other genes. While VEGF levels dropped in wild type mice up to 60% compared to 1 month, VEGF increased in heterozygous Ang-2 deficient retinae at an age of 9 months (141% to 1 month). Similarly, Ang-1 levels decreased in wild type mice (45% to 1 month), but remained stable in Ang2LacZ mice. These data suggest that Ang-2 gene dose reduction decelerates vasoregression in the retina with age. This effect links to higher levels of survival factors such as VEGF and Ang-1, suggesting that the ratio of these factors is critical for capillary cell survival.
Cellular Physiology and Biochemistry 02/2008; 21(1-3):129-36. · 3.42 Impact Factor
[show abstract][hide abstract] ABSTRACT: Vascular complications of chronic hyperglycemia including diabetic retinopathy are an increasing therapeutic and socioeconomic challenge. The epidemiology of diabetic eye disease has been well described, and there is as yet no clear indication for a reduction of incidence of blindness. Due to the complex multifactorial nature of the damage to diabetic vessels, it had been difficult to identify key targets for treatment and prevention. Novel techniques to study molecules and mechanisms involved in retinal vessel development and vascular cell interactions improved the understanding of retinal cell biology and pathobiology. A unifying concept has been proposed which links hyperglycemia-induced mitochondrial overproduction of reactive oxygen species with long-known biochemical alterations such as the formation of advanced glycation end products or the activation of the protein kinase C pathway. Specific inhibitors were identified that inhibited multiple biochemical abnormalities downstream of oxidative stress induced by high glucose.