INTRODUÇÃO: O pênfigo foliáceo (PF) caracteriza-se pela síntese de autoanticorpos contra a desmogleína 1 (Dsg1) com acantólise na epiderme superior. As lesões na face e tronco podem evoluir para eritrodermia (PFE), cuja patogênese é pouco conhecida. Estudos prévios sugerem a participação do fator de crescimento endotelial vascular (VEGF) e da endoglina (Eng). OBJETIVOS: Avaliar dados demográficos, expressão tecidual e níveis séricos do VEGF e Eng, bem como o perfil dos imunocomplexos no PFE. PACIENTES, MATERIAIS E MÉTODOS: Foram selecionados pacientes de PFE (n=31) e indivíduos controles com pênfigo vulgar (PV; n=10), psoríase (PSO; n=10) e saudáveis (CS; n=14) com amostras de soro e pele armazenadas. Os níveis séricos do VEGF, receptor solúvel do VEGF (sVEGFR-1), anti-Dsg1 e -Dsg3 foram avaliados por meio da técnica de ELISA, e a presença do VEGF, Eng, e imunocomplexos in situ foi determinada por imuno-histoquímica (IH) em plataforma automatizada (n=19). Após digitalização das lâminas, a reatividade aos anticorpos supracitados foi classificada manualmente em: 0 (ausente), 1 (discreta-moderada) e 2 (intensa), e analisada por software. RESULTADOS: PFE ocorreu no início da doença em 25/31 pacientes (80,6%), com média de idade de 42,7 anos e predomínio feminino (23/31; 74,2%). Os pacientes foram hospitalizados em média por 41,2 dias; infecção bacteriana foi a principal complicação (30/31; 96,8%), com bacteremia em 10/31 (32,3%) causada por Staphylococcus aureus em 7/10 (70%) dos pacientes. Infecção pelo vírus do herpes simples (HSV) em 11/31 (35,5%) doentes determinou internação prolongada. Valores de imunofluorescência indireta, e anti-Dsg1 e VEGF séricos (ELISA) foram superiores no PFE vs. PF não-eritrodérmico (PFNE) (p < 0,05). Níveis do sVEGFR-1 foram semelhantes no PFE e PFNE, e correlacionaram-se fracamente com a anti-Dsg1 no PFNE (p=0,034). A avaliação manual da IH com anti-VEGF no PFE foi estatisticamente diferente do PFNE (p=0,042) e CS (p=0,004), e similar ao PV (p=0,667) e PSO (p=0,667). Já no PFNE, a expressão do VEGF foi estatisticamente diferente do PV (p=0,049) e PSO (p=0,049) e semelhante ao CS (p=0,247). A contagem automatizada dos vasos marcados com anti-Eng no PFE foi similar ao PFNE (p=0,700) e PSO (p=0,133), e diferente do PV (p=0,0009) e CS (p=0,0009). A avaliação de 6 espécimes de PFE mostrou depósitos de imunocomplexos: intercelulares intraepidérmicos com IgG e C3 (n=6), IgA (n=5) e IgM (n=1); nas células inflamatórias com IgG e C3 (n=6), IgM e IgA (n=1); nos vasos com IgG, C3 e IgA (n=6), e IgM (n=5); e nos anexos com IgG e C3 (n=6), IgA (n=3) e IgM (n=1). CONCLUSÕES: O PFE predomina no início da doença e em mulheres, com maior risco de infecção. O aumento do VEGF sérico e tecidual sugere uma resposta reparadora ao dano tecidual causado pelos níveis elevados de autoanticorpos no PFE. A menor expressão de Eng no PFE indica uma desregulação da angiogênese na eritrodermia. De forma pioneira, a IH automatizada demonstrou a presença de imunocomplexos intraepidérmicos e nas estruturas dérmicas / BACKGROUND: Pemphigus foliaceus (PF) is characterized by the production of autoantibodies against desmoglein 1 (Dsg1), triggering superficial acantholysis. Lesions on the face and trunk may evolve to erythroderma (PFE). The pathogenesis of PFE is not fully understood. Previous studies suggest the role of vascular endothelial growth factor (VEGF) and endoglin (Eng). OBJECTIVES: To evaluate demographic data, VEGF and Eng expression, and immune complexes deposition in patients with PFE. METHODS: This study included patients with PFE (n=31) and controls with pemphigus vulgaris (PV; n=10), psoriasis (PSO; n=10), and health individuals (CS; n=14) that had serum and skin samples stored. Serum levels of VEGF, soluble VEGF receptor (sVEGFR-1), anti-Dsg1 and Dsg3 were measured by ELISA, and the in situ expression of VEGF, Eng, and immune complexes was evaluated utilizing an automated immunohistochemistry (IH) platform (n=19). After digitalizing the slides, the reactivity was manually classified as 0 (negative), 1 (mild-to-moderate) and 2 (intense), and also analyzed by software. RESULTS: PFE occurred at the onset of the disease in 25/31 (80.6%) patients, with a mean age of 42.7 years and a female predominance (23/31; 74.2%). Patients were hospitalized with an average length of stay of 41.2 days. Bacterial infection was the main complication (30/31; 96.8%), with bacteremia in 10/31 (32.3%) due to Staphylococcus aureus in 7/10 (70%) patients. Herpes simplex virus infection in 11/31 (35.5%) PFE patients caused prolonged hospitalization. Indirect immunofluorescence titers and serum anti-Dsg1 and VEGF (ELISA) were increased in PFE vs. non-erythrodermic PF (PFNE) (p < 0.05). Serum levels of sVEGFR-1 were similar in PFE and PFNE, and weakly correlated with anti-Dsg1 in PFNE (p=0.0342). Manual analysis of anti-VEGF positivity in PFE was statistically different from PFNE (p=0.042) and CS (p=0.004), and similar to PSO (p=0.667) and PV (p=0.667). VEGF expression in PFNE was statistically different from PSO (p=0.049) and PV (p=0.049) and similar to CS (p=0.247). The automated positive vessel count with anti-Eng was similar between PFE and PFNE (p=0.700) and PSO (p=0.133), but different from PV (p=0.0009). Immune complex deposits were evaluated in 6 specimens obtained during PFE and exhibited: intraepidermal intercellular deposits with IgG and C3 (n=6), IgA (n=5) and IgM (n=1); reactivity to inflammatory cells with IgG e C3 (n=6), IgM and IgA (n=1); vascular deposits with IgG, C3 and IgA (n=6), and IgM (n=5); and adnexal positivity with IgG and C3 (n=6), IgA (n=3) and IgM (n=1). CONCLUSIONS: Erythroderma predominates at the onset of PF, especially in women, with higher infectious risk. Increased expression of serum and in situ VEGF suggests that healing processes are triggered in response to the tissue damage caused by high levels of circulating autoantibodies in PFE. The reduced expression of Eng in PFE demonstrates a dysregulated angiogenesis during erythroderma. To the best of our knowledge, this is the first study that showed intraepidermal and dermal deposits of multiple immune complexes utilizing automated IH analysis

Bone fractures and pathologic conditions like chronic wounds significantly reduce the quality of life for the patients, which is especially dramatic in an elderly population with considerable multi-morbidity and lead to substantial socio-economic costs. To improve the wound healing capacity of these patients, new strategies for the design of novel multi-functional biomaterials are required: they should be able to decrease extensive pathologic tissue degradation and specifically control angiogenesis in damaged vascularized tissues like bone and skin. Glycosaminoglycans (GAGs) like hyaluronan (HA) and chondroitin sulfate (CS) as important extracellular matrix (ECM) components are involved in several biological processes such as matrix remodeling and growth factor signaling, either by directly influencing the cellular response or by interacting with mediator proteins. This could be useful in functionalizing biomaterials, but native sulfated GAGs (sGAGs) show a high batch-to-batch variability and are limited in their availability. Chemically modified HA and CS derivatives with much more defined characteristics regarding their carbohydrate backbone, sulfate group distribution and sulfation degree are favorable to study the structure-function relationship of GAGs in their interaction with mediator proteins and/or cells and this might be used to precisely modulate activity profiles to stimulate wound healing. By combining collagen type I as the main structural protein of the bone and skin ECM with these GAG derivatives, 2.5-dimensional (2.5D) and 3D artificial ECM (aECM) coatings and hydrogels were developed. These biomaterials as well as the respective GAG derivatives alone were compared to native GAGs and used to analyze how the sulfation degree, pattern and carbohydrate backbone of GAGs influence: i) the activity of tissue inhibitor of metalloproteinase-3 (TIMP-3) and vascular endothelial growth factor-A (VEGF-A) as main regulators of ECM remodeling and angiogenesis, ii) the composition and characteristics of the developed 2.5D and 3D aECMs, iii) the enzymatic degradation of collagen-based aECMs and HA/collagen-based hydrogels, iv) the proliferation and functional morphology of endothelial cells. Surface plasmon resonance (SPR) and enzyme linked immunosorbent assay (ELISA) binding studies revealed that sulfated HA (sHA) derivatives interact with TIMP-3 and VEGF-A in a sulfation-dependent manner. sHA showed an enhanced interplay with these proteins compared to native GAGs like heparin (HEP) or CS, suggesting a further impact of the carbohydrate backbone and sulfation pattern. sGAGs alone were weak modulators of the matrix metalloproteinase-1 and -2 (MMP-1 and -2) activity and did not interfere with the inhibitory potential of TIMP-3 against these proteinases during enzyme kinetic analyses. However, the formation of TIMP 3/GAG complexes reduced the binding of TIMP-3 to cluster II and IV of its endocytic receptor low-density lipoprotein receptor-related protein-1 (LRP-1, mediates the up-take and degradation of TIMP-3 from the extracellular environment) in a sulfation- and GAG type-dependent manner. It is of note that the determined complex stabilities of TIMP-3 with cluster II and IV were almost identical indicating for the first time that both clusters contribute to the TIMP-3 binding. Competitive SPR experiments demonstrated that GAG polysaccharides interfere stronger with the TIMP 3/LRP-1 interplay than GAG oligosaccharides. The importance of the position of sulfation is highlighted by the finding that a sHA tetrasaccharide exclusively sulfated at the C6 position of the N-acetylglucosamine residues significantly blocked the receptor binding, while CS and HEP hexasaccharides had no detectable effects. Thus, sHA derivatives as part of biomaterials could be used to sequester and accumulate TIMP 3 in aECMs in a defined manner where sHA-bound TIMP-3 could decrease the matrix breakdown by potentially restoring the MMP/TIMP balance. GAG binding might extend the beneficial presence of TIMP-3 into wounds characterized by excessive pathologic tissue degradation (e.g. chronic wounds, osteoarthritis). Mediator protein interaction studies with sHA coated surfaces showed the simultaneous binding of TIMP-3 and VEGF-A, even though the sHA/VEGF-A interplay was preferred. Moreover, kinetic analysis revealed almost comparable affinities of both proteins for VEGF receptor-2 (VEGFR-2), explaining their competition that mainly regulates the activation of endothelial cells. Additional SPR measurements demonstrated that the binding of sGAGs to TIMP-3 or VEGF-A decreases the binding of the respective mediator protein to VEGFR-2. Likewise, a sulfation-dependent reduction of the binding signal was observed after pre-incubation of a mixture of TIMP-3 and VEGF-A with sGAG poly- and oligosaccharides. The biological consequences of GAGs interfering with VEGF-A/VEGFR-2 and TIMP-3/VEGFR 2 were assessed in vitro using porcine aortic endothelial cells stably transfected with VEGFR 2 (PAE/KDR cells). The presence of sHA both decreased VEGF-A activity and the activity of TIMP-3 to inhibit the VEGF-A-induced VEGFR-2 phosphorylation. The same decreased activities could be observed for the migration of endothelial cells. However, if sHA, TIMP-3 and VEGF-A were present simultaneously, sHA partially restored the TIMP-3-mediated blocking of VEGF-A activity. These findings provide novel insights into the regulatory potential of sHA during endothelial cell activation as an important aspect of angiogenesis, which could be translated into the design of biomaterials to treat abnormal angiogenesis. These sHA-containing materials might control the angiogenic response by modulating the activity of TIMP 3 and VEGF-A. The in vitro fibrillogenesis of collagen type I in the presence of sHA derivatives led to 2.5D collagen-based aECM coatings with stable collagen contents and GAG contents that resemble the organic part of the bone ECM. A burst release of GAGs was observed during the first hour of incubation in buffer with the GAG content remaining almost constant afterwards, implying that the number of GAG-binding sites of collagen restricts the amounts of associated GAGs. Moreover, two differently sulfated HA derivatives could for the first time be incorporated into one multi-GAG aECM as verified via agarose gel electrophoresis and fluorescence measurements. This illustrates the multiple options to modify the aECM composition and thereby potentially their functionality. Atomic force microscopy showed that the presence of sHA derivatives during fibrillogenesis significantly reduced the resulting fibril diameter in a concentration- and sulfation-dependent manner, indicating an interference of the GAGs with the self-assembly of collagen monomers. In line with enzyme kinetic results, none of the GAGs as part of aECMs altered the enzymatic collagen degradation via a bacterial collagenase. Thus aECMs were proven to be biodegradable independent from their composition, which is favorable concerning a potential biomedical usage of the aECMs e.g. as implant coatings. HA/collagen-based hydrogels containing fibrillar collagen embedded into a network of crosslinked HA and sGAGs were developed as 3D aECMs. Scanning electron microscopy demonstrated a porous structure of the gels after lyophilization, which could favor the cultivation of cells. The presence of collagen markedly enhanced the stability of the gels against the enzymatic degradation via hyaluronidase, something beneficial to clinical use as this is often limited by the generally fast breakdown of HA. Binding and release experiments with lysozyme, as positively charged model protein for e.g. pro-inflammatory cytokines, and VEGF A revealed that the sulfation of GAGs increased the protein binding capacity for pure GAG coatings and retarded the protein release from hydrogels compared to hydrogels without sGAGs. Moreover, the additional acrylation of sHA was shown to strongly reduce the interaction with both proteins when the primary hydroxyl groups were targets of acrylation. This stresses the influence of the substitution pattern on the protein binding properties of the GAG derivatives. However, hydrogel characteristics like the elastic modulus remained unaffected. The different interaction profiles of lysozyme and VEGF-A with GAGs demonstrated a protein-specific preference of different monosaccharide compositions, suggesting that the mediator protein binding could be simultaneously adjusted for several proteins by combining different GAG derivatives. This might allow the scavenging of pro-inflammatory cytokines and at the same time a binding and release of wound healing stimulating growth factors. Since there is a growing demand for biomaterials to regenerate injured vascularized tissues like bone and skin, endothelial cells were used to examine the direct effects of solute GAGs and hydrogels containing these GAGs in vitro. In both cases, sHA strongly enhanced the proliferation of PAE/KDR cells. A VEGFR-2-mediated effect of GAGs on endothelial cells as underlying mechanism is unlikely since GAGs alone did not bind to VEGFR-2 and had no influence on VEGFR-2 phosphorylation. Other factors like GAG-induced alterations of cell-matrix interactions and cell signaling could be responsible. In accordance with SPR results, a decreased endothelial cell proliferation stimulating activity of VEGF-A was observed in the presence of solute GAGs or after binding to hydrogels compared to the respective treatment without VEGF-A. However, tube formation could be observed in the presence of solute VEGF A and GAGs and within hydrogels with sGAGs that released sufficient VEGF-A amounts over time. Overall the presence of GAGs and VEGF-A strongly promoted the endothelial cell proliferation compared to the treatment with GAGs or VEGF-A alone. Thus, HA/collagen-based hydrogels functionalized with sHA derivatives offer a promising option for the design of “intelligent” biomaterials that direct and regulate the cellular behavior instead of simply acting as inert filling material. They could be used for the controlled delivery and/or scavenging of multiple mediator proteins, thus enhancing the local availability or reducing the activity of these GAG-interacting mediator proteins, or by directly influencing the cellular response. This might be applied to a range of pathological conditions by tuning the biomaterial compositions to patient-specific needs. However, extensive in vivo validation is required to show whether these in vitro findings could be used to control the biological activity of for instance TIMP-3 and VEGF-A, especially under the pathological conditions of extended matrix degradation and dysregulated angiogenesis.

This project combines oncolytic Vaccinia virus (VV) with irinotecan (CPT-11) for the treatment of cancer. VV can infect, replicate in and destroy cancer cells, yet leave healthy cells relatively unaffected. CPT-11 is a chemotherapeutic of which ~5% is converted to the more active chemotherapeutic SN-38 by endogenous carboxylesterase (CE) enzymes. SN-38 is a topoisomerase I inhibitor that induces DNA double strand breaks, leading to growth arrest and apoptosis. Consequently, VV has been engineered to express a more effective isoform of the CE enzyme. The virus’ tumour tropism should restrict enhanced conversion of CPT-11 to the tumour. Neither CPT-11 nor SN-38 interfered with VV replication or spread. Engineered recombinants expressed CE enzyme which, when combined with CPT-11, produced DNA double strand breaks and cancer cell death. In vitro, the combination of CE-virus and CPT-11 killed more K-562 cancer cells than its non-CE counterpart and CPT-11.

The endoplasmic reticulum is the primary organelle in the cell which has the responsibility of properly folding proteins belonging to the secretory pathway. Secretory proteins are essential for a variety of functions within the body like metabolism, growth and survival. Hence, proper folding of the proteins in the ER is absolutely essential to maintain cellular and body function. The environment of the ER is substantially different from that of the cytoplasm and is primed essentially to provide the optimum conditions to fold newly synthesized polypeptides following translation by the ribosomes in the cytoplasm and on the surface of the ER. In order for secretory proteins to fold properly, ER homeostasis must be maintained. ER homeostasis is defined by the dynamic balance between the ER protein load and the ER capacity to process this load. The optimum environment of the ER, or ER homeostasis, can be perturbed by pathological processes such as hypoxia, glucose deprivation, viral infections, environmental toxins, inflammatory cytokines, and mutant protein expression, as well as by physiological processes such as aging. Disruption of ER homeostasis causes accumulation of unfolded and misfolded proteins in the ER. This condition is referred to as ER stress. Cells cope with ER stress by activating the unfolded protein response (UPR). The UPR is initiated by three ER transmembrane proteins: Inositol requiring 1 (IRE1), PKR-like ER kinase, and activating transcription factor 6 (ATF6). These three master regulators sense and interpret protein folding conditions in the ER and translate this information across the ER membrane to activate downstream effectors, spliced XBP1, phosphorylated eIF2α and ATF4, and cleaved active ATF6 respectively. These effectors have two distinct outputs, homeostatic and apoptotic. Homeostatic outputs are adaptive responses that function to attenuate ER stress and restore ER homeostasis. These responses include the attenuation of protein translation to reduce ER workload and prevent further accumulation of unfolded proteins, upregulation of molecular chaperones and protein processing enzymes to enhance the ER folding activity, and the increase in ER-associated degradation (ERAD) components to promote clearance of unfolded proteins. When ER stress reaches a point where the cells cannot tolerate the load of unfolded proteins any more, apoptosis sets in. One of the major secretory proteins in mammals, vascular endothelial growth factor VEGF, is essential for either normal or pathological angiogenesis (blood vessel development). VEGFA is the primary member of this family which is expressed in all endothelial cells and is responsible for sprouting and invasion of blood vessels into the interstitium and thus helps in supplying nutrients and oxygen to growing cells. Recent studies have indicated that cells suffering from insufficient blood supply experience ER stress. The ER needs energy and oxygen for the folding process, thus nutrient deprivation (low ATP production) and hypoxia caused by insufficient blood supply leads to inefficient protein folding and ER stress in cells, especially in cancer cells that grow and spread rapidly. This condition also occurs in the development of the mammalian placenta. The placenta is an essential tissue characterized by a lot of blood vessels. It is responsible for the exchange of nutrients and growth factors between maternal and fetal blood vessels and hence is essential for survival of the embryo. Nutrient deprivation and hypoxia stimulate the production of VEGFA and other angiogenic factors, leading to protection against ischaemic injury in both cancer cells as well as the developing placenta. In this dissertation, we report that the three master regulators of the UPR, IRE1α, PERK and ATF6α, mediate transcriptional regulation of VEGFA under ER stress in cancer cells. Inactivation of any of the three master regulators leads to attenuation of VEGFA expression under ER stress. We show that IRE1α is able to regulate VEGFA through its downstream transcription factor XBP1 which activates the VEGFA promoter. IRE1α mediated VEGFA regulation is also essential for normal development of labyrinthine trophoblast cells in the placenta. ATF6α also regulates VEGFA via its promoter. PERK is able to activate VEGFA by preferential activation of its downstream effector, ATF4, which binds intron 1 of the VEGFA gene. Thus our work reveals a twopronged differential regulatory action of the UPR sensors on VEGFA gene expression. This work suggests that a fully active UPR is essential for VEGFA upregulation under ER stress. All three regulators are required in cancer cells for normal VEGFA expression. This tight regulation of VEGFA by the UPR presents a wonderful opportunity for therapeutic intervention into angiogenic growth of tumors.

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...

Both platelet-rich plasma (PRP) and vascular endothelial growth factor (VEGF) can promote regeneration. The aim of this study was to compare the effects of these two elements on bone formation and vascularization in combination with bone marrow stromal cells (BMSC) in a critical-size bone defect in rabbits. The critical-size defects of the radius were filled with: (1) a calcium-deficient hydroxyapatite (CDHA) scaffold + phVEGF165-transfected BMSC (VEGF group), (2) CDHA and PRP, or (3) CDHA, autogenous BMSC, and PRP. As controls served: (4) the CDHA scaffold alone and (5) the CDHA scaffold and autogenous BMSC. The volume of new bone was measured by means of micro-CT scans, and vascularization was assessed in histology after 16 weeks. Bone formation was higher in the PRP + CDHA, BMSC + CDHA, and PRP + BMSC + CDHA groups than in the VEGF group (p < 0.05). VEGF transfection significantly promoted vascularization of the scaffolds in contrast to BMSC and PRP (p < 0.05), but was similar to the result of the CDHA + PRP + BMSC group. The results show that VEGF-transfected BMSC as well as the combination of PRP and BMSC improve vascularization, but bone healing was better with the combination of BMSC and PRP than with VEGF-transfected BMSC. Expression of VEGF in BMSC as a single growth factor does not seem to be as effective for bone formation as expanded BMSC alone or PRP which contains a mixture of growth factors. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Bone fractures and pathologic conditions like chronic wounds significantly reduce the quality of life for the patients, which is especially dramatic in an elderly population with considerable multi-morbidity and lead to substantial socio-economic costs. To improve the wound healing capacity of these patients, new strategies for the design of novel multi-functional biomaterials are required: they should be able to decrease extensive pathologic tissue degradation and specifically control angiogenesis in damaged vascularized tissues like bone and skin. Glycosaminoglycans (GAGs) like hyaluronan (HA) and chondroitin sulfate (CS) as important extracellular matrix (ECM) components are involved in several biological processes such as matrix remodeling and growth factor signaling, either by directly influencing the cellular response or by interacting with mediator proteins. This could be useful in functionalizing biomaterials, but native sulfated GAGs (sGAGs) show a high batch-to-batch variability and are limited in their availability. Chemically modified HA and CS derivatives with much more defined characteristics regarding their carbohydrate backbone, sulfate group distribution and sulfation degree are favorable to study the structure-function relationship of GAGs in their interaction with mediator proteins and/or cells and this might be used to precisely modulate activity profiles to stimulate wound healing. By combining collagen type I as the main structural protein of the bone and skin ECM with these GAG derivatives, 2.5-dimensional (2.5D) and 3D artificial ECM (aECM) coatings and hydrogels were developed. These biomaterials as well as the respective GAG derivatives alone were compared to native GAGs and used to analyze how the sulfation degree, pattern and carbohydrate backbone of GAGs influence: i) the activity of tissue inhibitor of metalloproteinase-3 (TIMP-3) and vascular endothelial growth factor-A (VEGF-A) as main regulators of ECM remodeling and angiogenesis, ii) the composition and characteristics of the developed 2.5D and 3D aECMs, iii) the enzymatic degradation of collagen-based aECMs and HA/collagen-based hydrogels, iv) the proliferation and functional morphology of endothelial cells. Surface plasmon resonance (SPR) and enzyme linked immunosorbent assay (ELISA) binding studies revealed that sulfated HA (sHA) derivatives interact with TIMP-3 and VEGF-A in a sulfation-dependent manner. sHA showed an enhanced interplay with these proteins compared to native GAGs like heparin (HEP) or CS, suggesting a further impact of the carbohydrate backbone and sulfation pattern. sGAGs alone were weak modulators of the matrix metalloproteinase-1 and -2 (MMP-1 and -2) activity and did not interfere with the inhibitory potential of TIMP-3 against these proteinases during enzyme kinetic analyses. However, the formation of TIMP 3/GAG complexes reduced the binding of TIMP-3 to cluster II and IV of its endocytic receptor low-density lipoprotein receptor-related protein-1 (LRP-1, mediates the up-take and degradation of TIMP-3 from the extracellular environment) in a sulfation- and GAG type-dependent manner. It is of note that the determined complex stabilities of TIMP-3 with cluster II and IV were almost identical indicating for the first time that both clusters contribute to the TIMP-3 binding. Competitive SPR experiments demonstrated that GAG polysaccharides interfere stronger with the TIMP 3/LRP-1 interplay than GAG oligosaccharides. The importance of the position of sulfation is highlighted by the finding that a sHA tetrasaccharide exclusively sulfated at the C6 position of the N-acetylglucosamine residues significantly blocked the receptor binding, while CS and HEP hexasaccharides had no detectable effects. Thus, sHA derivatives as part of biomaterials could be used to sequester and accumulate TIMP 3 in aECMs in a defined manner where sHA-bound TIMP-3 could decrease the matrix breakdown by potentially restoring the MMP/TIMP balance. GAG binding might extend the beneficial presence of TIMP-3 into wounds characterized by excessive pathologic tissue degradation (e.g. chronic wounds, osteoarthritis). Mediator protein interaction studies with sHA coated surfaces showed the simultaneous binding of TIMP-3 and VEGF-A, even though the sHA/VEGF-A interplay was preferred. Moreover, kinetic analysis revealed almost comparable affinities of both proteins for VEGF receptor-2 (VEGFR-2), explaining their competition that mainly regulates the activation of endothelial cells. Additional SPR measurements demonstrated that the binding of sGAGs to TIMP-3 or VEGF-A decreases the binding of the respective mediator protein to VEGFR-2. Likewise, a sulfation-dependent reduction of the binding signal was observed after pre-incubation of a mixture of TIMP-3 and VEGF-A with sGAG poly- and oligosaccharides. The biological consequences of GAGs interfering with VEGF-A/VEGFR-2 and TIMP-3/VEGFR 2 were assessed in vitro using porcine aortic endothelial cells stably transfected with VEGFR 2 (PAE/KDR cells). The presence of sHA both decreased VEGF-A activity and the activity of TIMP-3 to inhibit the VEGF-A-induced VEGFR-2 phosphorylation. The same decreased activities could be observed for the migration of endothelial cells. However, if sHA, TIMP-3 and VEGF-A were present simultaneously, sHA partially restored the TIMP-3-mediated blocking of VEGF-A activity. These findings provide novel insights into the regulatory potential of sHA during endothelial cell activation as an important aspect of angiogenesis, which could be translated into the design of biomaterials to treat abnormal angiogenesis. These sHA-containing materials might control the angiogenic response by modulating the activity of TIMP 3 and VEGF-A. The in vitro fibrillogenesis of collagen type I in the presence of sHA derivatives led to 2.5D collagen-based aECM coatings with stable collagen contents and GAG contents that resemble the organic part of the bone ECM. A burst release of GAGs was observed during the first hour of incubation in buffer with the GAG content remaining almost constant afterwards, implying that the number of GAG-binding sites of collagen restricts the amounts of associated GAGs. Moreover, two differently sulfated HA derivatives could for the first time be incorporated into one multi-GAG aECM as verified via agarose gel electrophoresis and fluorescence measurements. This illustrates the multiple options to modify the aECM composition and thereby potentially their functionality. Atomic force microscopy showed that the presence of sHA derivatives during fibrillogenesis significantly reduced the resulting fibril diameter in a concentration- and sulfation-dependent manner, indicating an interference of the GAGs with the self-assembly of collagen monomers. In line with enzyme kinetic results, none of the GAGs as part of aECMs altered the enzymatic collagen degradation via a bacterial collagenase. Thus aECMs were proven to be biodegradable independent from their composition, which is favorable concerning a potential biomedical usage of the aECMs e.g. as implant coatings. HA/collagen-based hydrogels containing fibrillar collagen embedded into a network of crosslinked HA and sGAGs were developed as 3D aECMs. Scanning electron microscopy demonstrated a porous structure of the gels after lyophilization, which could favor the cultivation of cells. The presence of collagen markedly enhanced the stability of the gels against the enzymatic degradation via hyaluronidase, something beneficial to clinical use as this is often limited by the generally fast breakdown of HA. Binding and release experiments with lysozyme, as positively charged model protein for e.g. pro-inflammatory cytokines, and VEGF A revealed that the sulfation of GAGs increased the protein binding capacity for pure GAG coatings and retarded the protein release from hydrogels compared to hydrogels without sGAGs. Moreover, the additional acrylation of sHA was shown to strongly reduce the interaction with both proteins when the primary hydroxyl groups were targets of acrylation. This stresses the influence of the substitution pattern on the protein binding properties of the GAG derivatives. However, hydrogel characteristics like the elastic modulus remained unaffected. The different interaction profiles of lysozyme and VEGF-A with GAGs demonstrated a protein-specific preference of different monosaccharide compositions, suggesting that the mediator protein binding could be simultaneously adjusted for several proteins by combining different GAG derivatives. This might allow the scavenging of pro-inflammatory cytokines and at the same time a binding and release of wound healing stimulating growth factors. Since there is a growing demand for biomaterials to regenerate injured vascularized tissues like bone and skin, endothelial cells were used to examine the direct effects of solute GAGs and hydrogels containing these GAGs in vitro. In both cases, sHA strongly enhanced the proliferation of PAE/KDR cells. A VEGFR-2-mediated effect of GAGs on endothelial cells as underlying mechanism is unlikely since GAGs alone did not bind to VEGFR-2 and had no influence on VEGFR-2 phosphorylation. Other factors like GAG-induced alterations of cell-matrix interactions and cell signaling could be responsible. In accordance with SPR results, a decreased endothelial cell proliferation stimulating activity of VEGF-A was observed in the presence of solute GAGs or after binding to hydrogels compared to the respective treatment without VEGF-A. However, tube formation could be observed in the presence of solute VEGF A and GAGs and within hydrogels with sGAGs that released sufficient VEGF-A amounts over time. Overall the presence of GAGs and VEGF-A strongly promoted the endothelial cell proliferation compared to the treatment with GAGs or VEGF-A alone. Thus, HA/collagen-based hydrogels functionalized with sHA derivatives offer a promising option for the design of “intelligent” biomaterials that direct and regulate the cellular behavior instead of simply acting as inert filling material. They could be used for the controlled delivery and/or scavenging of multiple mediator proteins, thus enhancing the local availability or reducing the activity of these GAG-interacting mediator proteins, or by directly influencing the cellular response. This might be applied to a range of pathological conditions by tuning the biomaterial compositions to patient-specific needs. However, extensive in vivo validation is required to show whether these in vitro findings could be used to control the biological activity of for instance TIMP-3 and VEGF-A, especially under the pathological conditions of extended matrix degradation and dysregulated angiogenesis.

Im Rahmen dieser Arbeit wurde die Rolle des Rezeptors VEGFR-2, Flk-1, im neuronalen Kontext untersucht. In einem ersten Schritt wurde in embryonalen Stammzellen der Maus das fluoreszierende Protein eGFP unter der Kontrolle regulatorischer Sequenzen des flk-1-Promotors, -Enhancers exprimiert. Nach der Differenzierung zu Sphäroiden wurden Endothelzellen nachgewiesen, die sowohl eGFP als auch das zelltypspezifische Oberflächenantigen CD31 ausprägen. Ebenso wurden nach der neuronalen Differenzierung in Gegenwart von Stromazellen eGFP-exprimierende Zellen identifiziert. Diese standen mit Zellen, die das für neuronale Vorläuferzellen charakteristische Protein Nestin ausprägten, in einem räumlichen Zusammenhang. Die Vorgehensweise, die Inaktivierung des flk-1-Gens mit der Differenzierung embryonaler Stammzellen in vitro zu kombinieren, sollte hier die Interpretation des Phänotyps des flk-1-defizienten Mausmodells ermöglichen. Der Rezeptor war während der neuronalen Differenzierung der Stammzellen auf Stromazellen in vitro für die Regulation der Anzahl der Vorläuferzellen essentiell. Ferner spielte der Rezeptor im Rahmen eines weiteren Differenzierungsmodells, das auf der Zugabe relevanter Wachstumsfaktoren beruht, eine instruktive Rolle im Hinblick auf die Identität der Neuronen. Kriterium war hier die differentielle Expression Homeobox-enthaltender Transkriptionsfaktoren. In einem zweiten Schritt wurden mit Hilfe dieses Modells differentiell-exprimierte Gene von Stammzellen des Wildtyps sowie Zellen mit einer Inaktivierung des flk-1-Gens nach der neuronalen Differenzierung durch subtraktive Hybridisierung in Verbindung mit der PCR identifiziert. Tatsächlich wurde das Protein PEA-15 nicht nur differentiell exprimiert sondern auch als Bestandteil des VEGFR-2-vermittelten Signalwegs identifiziert. Die biologischen Funktionen des Proteins PEA-15 wurden durch VEGF-vermittelte Phosphorylierung reguliert. Die Stimulation durch VEGF führte zunächst zu einer Aktivierung des Proteinkinase B-, Akt-Signalwegs. Für die Stimulation des Akt-Signalwegs war die Phosphorylierung der intrazellulären Tyrosinreste Y1052 und Y1057 des Rezeptors essentiell. Damit einhergehend wurde PEA-15 gegenüber der proteasomalen Degradation stabilisiert. Es wurde gezeigt, daß das Protein PEA-15 die Teilungsaktivität von Zellen beeinflusst. Die VEGF- vermittelte Stimulation führte zur Phosphorylierung der Mitogen-aktivierten Proteinkinasen ERK1 und ERK2. Die weitere Phosphorylierung der Substrate dieser Kinasen im Zellkern wurde durch Interaktion mit PEA-15 unterdrückt. Die Regulation des c-fos-Promotors war zugleich Indikator der Inhibition der Phosphorylierung betreffender Substrate sowie der proliferativen Aktivität. Auf diese Weise ist die Phosphorylierung von PEA-15 nach Stimulation durch VEGF für die Selektivität des Flk-1-vermittelten Signalwegs von unmittelbarer Bedeutung. Die Regulation der biologischen Funktion von PEA-15 erklärt die differentielle Ausprägung im Rahmen der neuronalen Differenzierung embryonaler Stammzellen in vitro. So war die Anzahl GFAP- beziehungsweise PEA-15-exprimierender Zellen nach Differenzierung muriner Stammzellen mit einer Inaktivierung des flk-1-Gens deutlich geringer. Die differentielle Expression identifizierter Gene wurde im Mausmodell nach konditionaler Inaktivierung des flk-1-Gens überprüft. Tatsächlich wurde Vimentin in verschiedenen Arealen des Gehirns differentiell ausgeprägt. Ein Zusammenhang zwischen der differentiellen Expression des Proteins PEA-15, der Anzahl GFAP-exprimierender Zellen und der Ausprägung des Rezeptors Flk-1 ergab sich aus der Identifikation einer Zellpopulation in der subgranulären Zone des Gyrus Dentatus. Dort wurde in flk-1-defizienten, adulten Mäusen eine geringere Anzahl GFAP-exprimierender Zellen nachgewiesen. Schließlich wurden sowohl im Cerebellum als auch im Cortex histologische Unterschiede deutlich, die sich im adulten Organismus aus der Inaktivierung des Rezeptors Flk-1 ergeben. Die vorliegende Arbeit zeigt, daß der Rezeptor VEGFR-2, Flk-1, im neuronalen Kontext eine Rolle spielt, die sich nicht ausschließlich auf die Vermittlung eines Schutzmechanismus gegenüber der neuronalen Apoptose beschränkt, sondern auch auf eine Beteiligung an der Neurogenese hinweist. Die Vorgehensweise, mit Hilfe der subtraktiven Hybridisierung Bestandteile Rezeptor-vermittelter Signalwege vor dem Hintergrund der Differenzierung embryonaler Stammzellen zu identifizieren, verdeutlicht die Eignung der Methode auch bei komplexen Zellpopulationen.

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...

Vascular Endothelial Growth Factor Receptor-3 (VEGFR3/Flt4) and its ligand Vegfc are necessary for development of both blood and lymphatic vasculature in vertebrates. In zebrafish, Vegfc/Flt4 signaling is essential for formation of arteries, veins, and lymphatic vessels. Interestingly, Flt4 appears to utilize distinct signaling pathways during the development of each of these vessels. To identify components of this pathway, we performed a transgenic haploid genetic screen in zebrafish that express EGFP under the control of a blood vessel specific promoter. As a result, we indentified a mutant allele of vascular endothelial growth factor c (vegfc), vegfcum18. vegfcum18 mutants display defects in vein and lymphatic vessel development but normal segmental artery (SeA) formation. Characterization of this allele led to the finding that the primary defect in vegfcum18 mutants was a general failure in vein and lymphatic vessel sprouting. Further genetic and biochemical analysis of this mutant revealed profound paracrine defects, which likely result in the observed loss of lymphatic and venous structures. Furthermore, double mutant analysis demonstrated that defects during SeA formation in vegfcum18 mutants were masked by inputs from the Vegfa signaling pathway. Endothelial cell autonomous expression of vegfcum18 induced angiogenic effects on blood vessels while endothelial cells lacking vegfc displayed defects in tip cell occupancy, suggesting a cell autonomous-autocrine role for Vegfc during developmental angiogenesis. Finally, we present genetic evidence that links processing of Vegfc by Furin during the formation of lymphatics in zebrafish. Together the data presented here suggest two discrete modes of signaling during blood and lymphatic vessel development, thus implying that regulation of Vegfc secretion and processing may play a pivotal role in the formation of these distinct vessel types in zebrafish.