Introdução: Micose fungoide poiquilodérmica (MFp) é uma variante clínica de micose fungoide (MF). É mais indolente e caracterizada pela presença da poiquilodermia. As metaloproteinases (MMP) e seus inibidores específicos TIMP (Tissue Inhibitors of Metaloproteinases) estão envolvidos na oncogênese. Especificamente as MMP2 e MMP9 e seus inibidores, TIMP-2 e TIMP-1, respectivamente, foram relacionados ao prognóstico em tumores. Poucos trabalhos estudaram MMP e nenhum estudou a ação dos TIMP na MF. Objetivos: avaliar a relação entre MMP2 e MMP9 e seus inibidores TIMP2 e TIMP1 e a agressividade da MF e descrever a casuística de micose fungoide poiquilodérmica no ambulatório de linfomas cutâneos da Divisão de Clínica Dermatológica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. Métodos: análise retrospectiva de 54 casos de MFp, sendo 25 de MFp localizada 14 de MFp generalizada e 15 de MFp mista. Para análise das MMP e TIMP, os grupos de MFp foram comparados com 7 amostras de pele normal (PN), 10 casos de MF clássica inicial (MFi), 9 casos de MF tumoral não-transformada (MFT nt) e 10 de MF tumoral transformada (MFT t). Resultados: A proporção de mulheres: homens foi 2,44. MFp apresentou maior tempo entre os primeiros sintomas e o diagnóstico. MFpG apresentou maior prevalência de lesões do tipo pitiríase liquenoide crônica (PLC) (79%). Houve alta prevalência de MF hipocromiante (62%) no grupo MFp mista. A histologia da MFp apresentou características típicas de MF e, adicionalmente, atrofia, telangectasias e derrame pigmentar, específicos da forma poiquilodérmica. Na imuno-histoquímica predominou o fenótipo CD3+, CD4+, CD7-, CD8- em todos os grupos, e MFp apresentou significantemente menor predomínio do fenótipo CD8+ que o grupo MFi. O grupo MFpG apresentou baixa positividade para pesquisa de clonalidade T da pele (12,5%). A MMP2 esteve mais presente na epiderme em MFi e MFp relativamente a MFT. Na derme superficial, os grupos MFi e MFp marcaram mais MMP2 que a pele normal, mas sem diferença estatística entre eles. Não houve diferença estatística em MMP2 na derme profunda entre os grupos. À zimografia, houve maior atividade de MMP2 ativa no grupo MFTt. Não houve expressão de TIMP-2 pela epiderme da pele normal. Os grupos MFi e MFp marcaram TIMP-2 na epiderme de forma semelhante, porém menos que os grupos MFT. Na derme superficial, não houve diferença estatística entre os grupos MFi e MFp. TIMP-2 foi mais expresso na derme profunda dos dois grupos de MFT comparativamente a todos os outros grupos. Na epiderme e na derme superficial, MMP9 foi mais expressa no grupo MFi comparativamente a MFp. Na derme profunda, a expressão de MMP9 foi maior nos grupos MFT, seguido por MFi e, por último, MFp. A atividade de MMP9 foi maior no grupo MFT não transformada comparativamente aos outros grupos. TIMP-1, ne epiderme e na derme superficial e na derme profunda foi mais expresso no grupo MFi, comparativamente aos outros grupos. Discussão: MFpG apresentou mais lesões tipo PLC e a forma mista, lesões hipocrômicas. A histologia da MFp foi semelhante à descrita previamente na literatura, mas a baixa positividade de CD8 difere de relatos prévios. A MMP2 pareceu ser um marcador de atividade para MF, principalmente quando a sua presença por imunohistoquímica foi associada a dados de zimografia. A expressão de MMP9 nas amostras foi compatível com os dados prévios de literatura, tendo sido mais expressa nas formas mais agressivas de MF e, histologicamente, mais localizada nos locais de maior atividade do tumor. TIMP-1 foi expresso de forma análoga à MMP9, conforme descrito previamente na literatura. TIMP-2, por sua vez, seguiu o padrão de distribuição de MMP2. No entanto, não foi expresso pela pele normal e foi mais expresso pelos grupos de MFT, o que não ocorreu com a MMP2 na imuno-histoquímica. Conclusões: A expressão de MMP e TIMP correlacionou-se com o local de maior atividade linfocitária e com a agressividade da MF. A atividade da MMP2 e MMP9 foi maior nos grupos MFT comparativamente aos grupos mais indolentes. Separar os casos de MFp de acordo com suas apresentações localizadas, generalizada e mista foi relevante do ponto de vista clínico, laboratorial e evolutivo / Introduction: poikilodermatous mycosis fungoides (pMF) is a clinical variant of mycosis fungoides (MF). It is more indolent than classic MF and is characterized by the presence of poikiloderma. The matrix metalloproteinases (MMPs) and their specific inhibitors TIMP (Tissue Inhibitors of Metalloproteinases) are involved in oncogenesis. Specifically, MMP2 and MMP9 and their inhibitors, TIMP-2 and TIMP-1, respectively, have been related to prognosis in tumors. There are few studies on MMP and none on the role of TIMPs in MF. Objectives: To evaluate if there is a relationship between the presence and activity of MMP2 and MMP9 and their inhibitors TIMP2 and TIMP1, and the aggressiveness of MF. To describe a casuistic of poikilodermatous mycosis fungoides in an outpatient clinic in the Dermatological Division of Hospital das Clinicas of University of Sao Paulo Medical School. Methods: Retrospective analysis of 54 cases of pMF, this included 25 localized pMF (LpMF), 14 generalized pMF (GpMF) and 15 mixed pMF. For the analysis of MMPs and TIMPs, the pMF groups were compared with 7 normal skin samples (NS), 10 cases of initial classical MF (cMF), 9 cases of non-transformed tumor MF (nt MFT) and 10 transformed tumor MF (t MFT). Results: The proportion of women : men was 2.44. The pMFs groups showed a longer period of time from the first symptoms to the diagnosis than the cMF group. The GpMF group had a higher incidence of pityriasis lichenoides chronica-like lesions (PLC) (79%) than the other groups. There was a high incidence of hypopigmented MF (62%) in the mixed pMF group. Histology showed typical characteristics of MF and, additionally, atrophy, telangiectasia and pigmentary alterations compatible with pMF. At immunohistochemistry the cases were predominantly CD3+, CD4+, CD7-, CD8- phenotype in all groups, and the pMF groups had a significantly lower prevalence of CD8+ phenotype than the cMF group. The GPMF group showed low positivity for clonality of the T-cell receptor at the T skin (12.5%) compared to the other groups. The MMP2 was more present in the epidermis for the cMF and pMF groups compared to MFT. In the superficial dermis, the cMF, LpMF and GpMF groups showed more MMP2 than normal skin, however there was no statistical difference between the three groups. There was no statistical difference in the presence of MMP2 in the deep dermis between the groups. The zymography showed higher MMP2 activity in the MFT group. There was no TIMP-2 expression by the normal epidermis. The epidermis of cMF and pMFs groups marked TIMP-2 in a similar way, but at a lower intensity than the MFT groups. In the superficial dermis, there was no statistical difference between the cMF and pMFs groups. TIMP-2 was more expressed in the deep dermis of the two MFT groups compared to all of the other groups. In the epidermis and superficial dermis, the MMP9 was more expressed in cMF compared to pMF groups. In the deep dermis, MMP9 expression was higher in the MFT groups, followed by cMF and finally pMF. The MMP9 activity was higher in the nt MFT group compared to other groups. TIMP-1, in epidermis, superficial dermis and deep dermis was more expressed in the cMF group compared to other groups. Discussion: The study confirmed that the pMF is an indolent form of MF and the time period between the symptoms and the diagnosis in pMF was longer than in classical MF. There were clinical differences amongst the groups of pMF. The GpMF group had a higher prevalence of PLC-like lesions than the mixed form of pMF, which had more hypochromic lesions. Histology of pMF was similar to descriptions provided in other case studies. However, the low CD8 positivity differs from previous reports. The MMP2 appeared to be a marker of activity for MF in our work, especially when their presence by immunohistochemistry was associated with the enzyme activity. The expression of MMP9 in our samples was consistent with previous data from other case studies, being more expressed in the most aggressive forms of MF and histologically more localized in most active sites of the tumor. TIMP-1 was expressed in an analogous manner to MMP9, as previously described in the literature. TIMP-2, in turn, followed the distribution pattern of MMP2. However, it was not expressed by normal skin and was more expressed by the MFT group, which did not occur with the MMP2 in immunohistochemistry. Conclusions: The expression of MMP and TIMP was correlated with the location of higher lymphocyte activity and with the aggressiveness of MF. The activity of MMP2 and MMP9 was higher in the MFT groups than the more indolent groups. It was important to split the pMF cases according to their presentation (GpMF, LpMF and mix pMF) from a clinical, laboratory and prognostic point of view

Os tumores hipofisários mesmo sendo, em sua maioria, benignos, podem apresentar comportamento invasivo, com extensão para seio cavernoso, seio esfenoidal e clivo. As metaloproteinases de matriz tipo 2 (MMP-2) e 9 (MMP-9) e o inibidor tecidual de metaloproteinases-2 (TIMP-2) têm sido estudados em relação ao comportamento invasivo desses tumores, em especial quanto à invasão do seio cavernoso. Esse estudo teve como objetivo avaliar a expressão proteica das MMP-2, MMP-9 e do TIMP-2 nos tumores hipofisários e sua relação com invasão do seio cavernoso e, de maneira inédita, investigar a expressão dessas proteínas em nível plasmático. Adicionalmente, foram avaliadas a expressão dos RNAs mensageiros (RNAm) das MMP-2 e TIMP-2 com intuito de correlacioná-las com a expressão proteica no tecido tumoral, bem como a expressão do marcador de proliferação Ki67. Foram selecionados 77 casos, todos com amostras de tumor emblocado em parafina para análise imuno-histoquímica (IHQ). Destes, foram coletadas amostras de tumor fresco em 29 pacientes e de sangue periférico pré-operatório em outros 29 casos. A expressão proteica plasmática foi detectada de forma semi-quantitativa utilizando um arranjo de anticorpos em membrana comercial. A expressão dos RNAm das MMP-2 e TIMP-2 foi avaliada por reação em cadeia da polimerase quantitativo (qPCR) em tempo real. Do total de casos, 20 pacientes apresentavam tumores com invasão para o seio cavernoso. A expressão proteica tumoral das MMP-2, MMP-9 e TIMP-2 apresentou-se aumentada no grupo invasivo, contudo esta diferença não foi estatisticamente significante em relação ao grupo não- invasivo. A expressão plasmática das MMP-9 e TIMP-2 também não mostrou diferença entre os dois grupos e não se correlacionou com a expressão tumoral. A expressão plasmática da MMP-2 não foi detectada em nenhum caso. Quanto à expressão do RNAm das MMP-2 e TIMP-2, também não houve diferença significante entre os grupos e nem correlação com a expressão proteica tecidual ou plasmática. Foi observada uma diferença significante na dimensão tumoral [3.6 (2.5-5.2) x 2.0 (1.3-2.7); P < 0.001] e no índice do Ki67 [1.05 (0.27-25) x 0.5 (0.2-1.0); P < 0.001] entre os grupos invasivo e não-invasivo respectivamente. Em conclusão, em nossa coorte, não foi encontrada relação entre a expressão tecidual e plasmática das MMP-2, MMP-9 e TIMP-2 e a invasão para o seio cavernoso nos adenomas hipofisários / Pituitary tumors, although mostly benign, may present invasive behavior, with extension to the cavernous sinus, sphenoid sinus and clivus. Type 2 (MMP-2) and type 9 (MMP-9) matrix metalloproteinases and the metalloproteinase tissue inhibitor type 2 (TIMP-2) have been studied in relation to the invasive behavior of these tumors, especially regarding invasion of the cavernous sinus. The aim of this study was to evaluate the protein expression of MMP-2, MMP-9 and TIMP-2 in pituitary tumors and its relation with invasion of the cavernous sinus and, in an unprecedented way, to investigate the expression of these proteins at the plasma level. Additionally, expression of MMP-2 and TIMP-2 messenger RNAs (mRNAs) was evaluated in order to correlate with protein expression in tumor tissue, as well as Ki67 proliferation marker expression. A total of 77 cases were selected, all of them with paraffin embedded tumor samples for immunohistochemical analysis (IHC). Of these, fresh tumor samples were collected in 29 patients and preoperative peripheral blood in another 29 cases. Protein plasma expression was detected semi-quantitatively using a commercial membrane antibody array. Expression of MMP-2 and TIMP-2 mRNAs was evaluated by quantitative realtime polymerase chain reaction (qPCR). Of the total cases, 20 patients presented tumors invasive to the cavernous sinus. Tumor protein expression of MMP-2, MMP-9 and TIMP-2 was increased in the invasive group, not reaching, however, statistically significant difference as compared with the non-invasive group. Plasma expression of MMP-9 and TIMP-2 also did not differ between the two groups and did not correlate with tumor expression. Plasma expression of MMP-2 was not detected in any case. Concerning MMP-2 and TIMP-2 mRNA expression, there was also no significant difference between groups and no correlation with tissue or plasma protein expression was observed. A significant difference was observed in tumor size [3.6 (2.5-5.2) x 2.0 (1.3-2.7); P < 0.001] and in the Ki67 index [1.05 (0.27-25) x 0.5 (0.2-1.0); P < 0.001] between the invasive and non-invasive groups respectively. In conclusion, in our cohort, no relationship was found between the tissue and plasma expression of MMP-2, MMP-9 and TIMP-2 and the invasion of the cavernous sinus in pituitary adenomas

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.

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.

INTRODUÇÃO: A propensão do CaP em gerar metástases decorre de mecanismos moleculares específicos em um processo composto por múltiplas etapas, sendo que o remodelamento do meio extracelular através de ações de enzimas proteolíticas denominadas metaloproteinases da matriz (MMP) é uma etapa fundamental. As MMP degradam vários componentes da matriz extracelular, sendo que seu controle pode ser exercido por outras proteínas denominadas TIMPs. Em nível gênico, outro controle pode ser exercido por moléculas chamadas microRNAs. OBJETIVO: O objetivo deste estudo é avaliar a regulação da MMP-9 por miRNAs. A partir de dados da literatura identificamos que a MMP-9 pode sofrer influência do miR-21 e 338-3p. MÉTODOS: Para os experimentos in vitro, linhagens celulares de CaP (DU145, PC3 e LNCaP) foram transfectadas com os miRNAs de interesse e a expressão de MMP-9 foi avaliada por reação em cadeia de polimerase quantitativa com transcriptase reversa (qRT-PCR). O sobrenadante da transfecção foi usado para ensaios de invasão com matrigel, e ELISA. Nos experimentos in vivo, células da linhagem PC-3-luc foram implantadas no subcutâneo de camundongos Balb-c nude e tratadas com injeções de anti-miR-21, miR-338-3p ou a combinação de ambos. RESULTADOS: O miR-21 aumentou expressão de MMP-9 em 72% na PC3. Houve maior invasão celular tanto na PC3 como DU145. In vivo, o bloqueio do miR-21 reduziu em 10% a expressão de MMP-9 nos tumores implantados (p=0,04). O miR-338-3p reduziu a expressão de MMP-9 em 53% na PC3 (p=0,001), 31% na LnCaP (p=0,23) e 24% na DU145 (p=0,16). No ensaio de invasão, menor número de células e colônias foram capazes de invadir a membrana de matrigel. In vivo, houve redução de 27% na expressão de MMP-9 nos camundongos tratados com o miR-338-3p (p=0,07). A combinação anti-miR-21+miR-338-3p reduz a expressão de MMP-9 em maior intensidade tanto in vitro como in vivo. CONCLUSÕES: A expressão de MMP-9 pode ser regulada pelo miR-21 e miR-338-3p. O primeiro se comporta como um oncomiR ao passo que o segundo como um supressor tumoral. A combinação de miRNAs é uma estratégia plausível para ampliar o efeito sobre expressão de genes de interesse / INTRODUCTION: The propensity of CaP to generate metastases results from specific molecular mechanisms in a multiphase process and the remodeling of the extracellular medium through the actions of proteolytic enzymes called matrix metalloproteinases (MMP) is a fundamental step. MMPs degrade several components of the extracellular matrix, and their control can be exerted by other proteins called TIMPs. At the gene level, another control can be exerted by molecules called microRNAs. OBJECTIVE: The objective of this study is to evaluate the regulation of MMP-9 by miRNAs. From literature data we have identified that MMP-9 may be influenced by miR-21 and 338-3p. METHODS: For in vitro experiments, CaP cell lines (DU145, PC3 and LNCaP) were transfected with the miRNAs of interest and the expression of MMP-9 was assessed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The transfection supernatant was used for matrigel and ELISA invasion assays. For the in vivo experiments, PC3-luc cells were implanted into the subcutaneous Balb-c nude mice and treated with anti-miR-21, miR-338-3p injections or the combination of both. RESULTS: The miR-21 increased MMP-9 expression by 72% in PC3. There was greater cell invasion in both PC3 and DU145. In vivo, miR-21 blockade reduced MMP-9 expression by 10% in implanted tumors (p = 0.04). MiR-338-3p reduced MMP-9 expression by 53% in PC3 (p = 0.001), 31% in LNCaP (p = 0.23), and 24% in DU145 (p = 0.16). In the invasion assay, fewer cells and colonies were able to invade the matrigel membrane. In vivo, there was a 27% reduction in MMP-9 expression in mice treated with miR-338-3p (p = 0.07). The combination of anti-miR-21 + miR-338-3p reduces MMP-9 expression in greater intensity both in vitro and in vivo. CONCLUSIONS: MMP-9 expression can be regulated by miR-21 and miR-338-3p. The former behaves as an oncomyR while the second as a tumor suppressor. The combination of miRNAs is a plausible strategy to extend the effect on gene expression of interest

Indiana University-Purdue University Indianapolis (IUPUI) / The negative heath consequences of smoking are widely recognized, but there are still about 20% of the people in United States using tobacco products. Cigarette smoke condensate (CSC), the particulate matter of cigarette smoke, is comprised of thousands of chemicals (e.g., nicotine). Secondary only to bacterial plaque, cigarette smoking is a major risk factor for periodontal disease. Human gingival fibroblasts (HGFs) are the main cellular component of periodontal connective tissues. During the development of periodontal disease, collagen degradation occurs. Collagen is the major extracellular matrix component of the gingiva. The major extracellular matrix degrading enzymes produced by the HGFs are the matrix metalloproteinases (MMPs). The MMPs are mainly modulated by the tissue inhibitors of metalloproteinases (TIMPs). In this dissertation, three studies aimed at understanding the effects of tobacco on human gingival fibroblasts and their mechanisms have been conducted: the effects of CSC on HGF-mediated collagen degradation; comparison of the effects of CSC on HGFs with that of nicotine; and the combined effects of CSC and bacteria on HGFs. The cell proliferation of HGFs decreased and cytotoxicity increased in HGFs treated with increasing concentrations of CSC. CSC increased the collagen degrading ability of the HGFs by altering the production and localization of MMPs and TIMPs. Nicotine is one of the major components and the most pharmacologically active agent in tobacco. The percentage of nicotine in the CSC was 2.4%. CSC (100 µg/ml) increased the collagen degrading ability of the HGFs by affecting membrane associated MMP-2, MMP-14, and TIMP-2, but the level of nicotine in the CSC may only play a limited role in this process. Porphyromonas gingivalis (P. gingivalis) is an opportunistic pathogen involved in periodontal disease. The combined effects of CSC and P. gingivalis supernatant increased HGF-mediated collagen degradation by destroying the balance between the MMPs and TIMPs at the protein and mRNA levels. This project demonstrated that tobacco (with or without P. gingivalis) increased HGF mediated collagen degradation, as seen in the periodontal disease, through altering the MMPs and TIMPs.