Degenerative disc disease is a common ailment with enormous medical, psychosocial and economic ramifications. This study was designed to investigate the utility of a thiol-modified hyaluronan(TMHA) and elastin-like polypeptide(EP) composite material as a potential tissue engineering scaffold to reconstitute the nucleus pulposus in early degenerative disc disease. TMHA and EP were combined in various concentrations and cross-linked using poly(ethylene glycol)diacrylate. Resulting materials were evaluated biomechanically and biologically. Confined compression testing revealed that the addition of EP to TMHA-based gels resulted in a stiffer construct, but remained an order of magnitude less stiff than native nucleus. The in vitro cell culture experiments with human intervertebral disc cells demonstrated 70% cell viability at three weeks with apparent maintenance of phenotype. The addition of EP did not have a significant biologic effect. An in vivo pilot study demonstrated biocompatibility of the TMHA-based hydrogels; additional power is required to adequately assess treatment effect.

A cysteine-containing elastin-like polypeptide (ELP2cys) was successfully synthesized and purified, and was shown to behave in a similar fashion to other well-characterized ELPs. Incorporating the ELP2cys as a crosslinking agent into a solution of sulphated hyaluronan (CMHA-S) not only decreased the gelation time of the solution but also increased the crosslinking density of the resultant hydrogel, in turn increasing both the resiliency and stiffness of the construct. Preliminary in vitro work involved culture of human disc cells, followed by their encapsulation within the hydrogel. Unfortunately the results were inconclusive, although it appeared as though the addition of ELP2cys to the matrix did not negatively affect the viability of the cells, as compared to hydrogels with CMHA-S only. This study showed that ELP2cys is a valuable addition to the family of recombinant elastin-like polypeptides, and shows promise as a crosslinking agent in the formation of hyaluronan hydrogels.

An increase in coronary disease prevalence and mortality highlights the growing need for therapies to treat atherosclerotic vessels. While current bypass procedures utilize autologous vessels for small caliber grafts, there is a big push towards the use of engineered tissues to bypass diseased portions of arteries. Cardiovascular tissue engineering is the emerging discipline that aims to create a functional substitute. Ideally, a tissue engineered blood vessel would possess the relevant cells and matrix proteins that interact in a physiologic manner and will respond to the environmental cues of the host. A particular obstacle to achieving appropriate vessel structure is the inclusion of elastin in a tissue engineered media equivalent. Rat arterial smooth muscle cells that were retrovirally mediated to overexpress versican V3 have been shown to have an enhanced expression of tropoelastin in vitro as well as in injury models. The unique tropoelastin expression by these adult cells was studied in the context of tissue engineered media equivalents. Changes to the extracellular matrix architecture and composition, stimulation with medium additives, and cyclic distension, were shown to increase tropoelastin synthesis in V3 versican overexpressing cells. This study not only expanded the characterization of V3 versican overexpressing smooth muscle cells, it also explored the novel use of these cells as a tropoelastin source in a tissue engineered media equivalent.

Incidence of abdominal aortic aneurysm (AAA) is increasing in the aging society of the western world. Development of AAA is mostly asymptomatic and is characterized by a bulge in the abdominal aorta. However, AAA may suddenly rupture, which results in an internal bleeding associated with a high mortality rate. Patients with AAA undergo regular screening until treatment indication. To date, statistical criteria are used to decide whether the risk of rupture exceeds the risk of intervention. Models of AAA development help to understand the disease progression and to yield patient-specific criterion for AAA rupture. Up to date, sophisticated models of AAA development exist. These models assume the abdominal aorta as a thin-walled structure, which saves the computational effort. This thesis aims at investigating the importance of employing a thick-walled model of the aorta. The effects on AAA development that cannot be captured with a thin-walled model are of interest. In Paper A, the thick-walled model of growth and remodeling of one layer of a AAA slice has been extended to a two-layered model. The parameter study has been performed to investigate the influence of mechanical properties and growth and remodeling (G&amp;R) parameters of two individual layers on the gross mechanical response and G&amp;R of the artery. It was concluded that the adventitia acts to protect the arterial wall against rupture even in pathological state. In Paper B, the model was extended to an organ level model of AAA development. Furthermore, the model was incorporated into a so-called Fluid-Solid-Growth (FSG) framework, where the AAA development is loosely coupled to the blood flow conditions such as wall shear stress. One patient-specific geometry of the abdominal aorta is used to illustrate the model capabilities. A transmurally non-uniform distribution of the strains of individual arterial constituents was observed. In addition, an increased aneurysm tortuosity was observed in comparison to a thin-walled approach. These findings signify the importance of a thick-walled approach to model the aneurysm development. Finally, the proposed methodology provides a realistic basis to further explore the growth and remodeling of AAA on a patient-specific basis. / <p>QC 20140311</p>

[Truncated abstract] Urinary desmosines have been proposed as a biomarker for inflammatory lung damage. Desmosine, a breakdown product of elastin, is an effective marker of the degradation of elastin and has been studied in many disease scenarios where there is acute and chronic lung inflammation. Lung matrix degradation has been proven in vitro and in vivo with many experiments showing that the excess proteases degrades lung matrix. The secretion of proteases by neutrophils is an innate response of the body to the invasion by micro organisms and when secreted in excess, the protective anti-protease mechanism is swamped. Chronic inflammation and persistent infection eventually leads to bronchiectasis and respiratory failure. Urinary desmosine has been shown to be elevated in respiratory conditions with acute and chronic inflammation . . . Urinary desmosine levels in a large cohort of healthy children have been established using this method and predictive Z-score formulae have been developed to use in children with lung disease. Exploration of these scores in children with CF have shown that the levels of urinary desmosine appear to be sensitive to the clinical setting, where high urinary desmosine levels were present during exacerbation and significantly reduced when treated for infection with antibiotic therapy and physiotherapy. The study of young children under the age of seven was undertaken to determine if the urinary desmosine levels could indicate when lung damage was occurring and to determine what mechanisms might be involved. Since there appeared to be no apparent relationship between elevated desmosines and proteases in the lung in young children with CF, further studies are required to define the mechanisms behind increased elastin metabolism in those children.

Η HARP (Heparin Affin Regulatory Peptide) είναι ένας αυξητικός παράγοντας με Μ.Β. 18 kDa που ανήκει στην οικογένεια των αυξητικών παραγόντων που έχουν συγγένεια με την ηπαρίνη. Eμπλέκεται στην ανάπτυξη των νευριτών, την επούλωση πληγών και φαίνεται να παίζει σημαντικό επαγωγικό ρόλο στις διαδικασίες της ογκογένεσης, καθώς επάγει την αγγειογένεση και εμφανίζεται σε υψηλές συγκεντρώσεις τόσο σε καρκινικούς ιστούς, όσο και σε κυτταρικές σειρές καρκινικών κυττάρων. Στο πλαίσιο μελέτης της σχέσης δομής/δράσης του αυξητικού αυτού παράγοντα, χρησιμοποιούνται τόσο συνθετικά πεπτίδια, όσο και ανασυνδυασμένες τροποποιημένες μορφές του αυξητικού αυτού παράγοντα. Σε φυσιολογικές συνθήκες, η εκκρινόμμενη HARP πέπτεται από ένζυμα του κυτταρικού μικροπεριβάλλοντος και προκύπτουν πεπτίδια που παρουσιάζουν βιολογικές δράσεις παρόμοιες ή και αντίθετες από αυτές της HARP. Φαίνεται λοιπόν πως η δράση του αυξητικού αυτού παράγοντα ρυθμίζεται τόσο στο επίπεδο βιοσύνθεσης και έκκρισης, όσο και από τη δράση ενζύμων του εξωκυττάριου χώρου. Στην παρούσα εργασία μελετήθηκε η δράση ενός συνθετικού πεπτιδίου το οποίο αντιστοιχεί στα αμινοξέα 65-97 που εντοπίζονται στην ΤSR περιοχή προς το καρβοξυτελικό άκρο της HARP. Με δεδομένο ότι τι πεπτίδιο αυτό εμφανίζει αντιαγγειογενετική δράση, πραγματοποιήθηκαν χρονοεξαρτώμενα και δοσοεξαρτώμενα πειράματα, με σκοπό τη μελέτη της δράσης του στον πολλαπλασιασμό, τη μετανάστευση και την επούλωση πληγών. Στο πλαίσιο αυτών των μελετών, ελέγξαμε τη δράση του στην έκφραση των μεταλλοπρωτεϊνασών ΜΜP-2 και ΜΜP-9, των αναστολέων τους ΤΙMP-1 και ΤΙMP-2 καθώς και του κολλαγόνου και της ελαστίνης σε πρωτογενείς καλλιέργειες ενδοθηλιακών κυττάρων από ομφάλιο λώρο (HUVEC cells). Τα αποτελέσματα έδειξαν πως το συνθετικό αυτό πεπτίδιο καταστέλλει τον πολλαπλασιασμό, την μετανάστευση αλλά και την επούλωση πλήγών των κυττάρων HUVEC με δοδοεξαρτώμενο και στατιστικώς σημαντικό τρόπο. Επιπλέον από τα πειράματά μας δεν παρατηρήθηκε μεταβολή στα πρωτεϊνικά επίπεδα έκφρασης των μεταλλοπρωτεϊνασών ΜΜP-2 και ΜΜP-9 καθώς και των αναστολέων τους ΤΙMP-1 και ΤΙMP-2. Ωστόσο, παρατηρήθηκε στατιστικώς σημαντική μεταβολή στα επίπεδα γονιδιακής έκφρασης των αναστολέων ΤΙMP-1 και ΤΙMP-2 όπως επίσης και της ελαστίνης και του κολλαγόνου IV. / Heparin affin regulatory peptide (HARP) is an 18-kDa secreted growth factor that has a high affinity for heparin and a potent role on tumor growth and angiogenesis. HARP was originally described as a neurite outgrowth promoting molecule, which appears to increases during recovery from injury and is thought to be involved in angiogenesis expression, playing a major role in the cell growth and differentiation that are associated with regeneration in several tissues. HARP is expressed in several human tumors and tumor cell lines and is also indicated in high serum levels of patients with different types of cancer. HARP contains two random coiled clusters of basic residues (N- and C-terminal) and two b-sheet domain. Each b-sheet domain contains a thrombospondin repeat I (TSR-I) motif, which have been suggested to be responsible for the interaction of HARP with heparin. Our project is based on C-TSR-I domain, corresponding to amino acids 65–97 of HARP peptide, respectively, required for the neurite outgrowth activity of HARP. In this study, we investigate the impact of C-TSR on basic biological functions of endothelial cells (HUVEC) such as proliferation, migration, the expression of MMP-2 and MMP-9 and their inhibitors (TIMP-1, TIMP-2) that contribute to the ECM remodeling. Time course and dose-response experiments revealed that CTSR reduces proliferation, migration and wound healing, without affecting the protein levels of MMP-2 and MMP-9 and their inhibitors (TIMP-1, TIMP-2). Moreover, CTSR inhibits the expression of TIMP-1 and TIMP-2 contributing to the ECM remodeling. Concluding, HARP could act as pro- or anti-angiogenic factor, depending on the system used and the cell microenvironment.

Les fibres élastiques sont constituées d'élastine et de microfibrilles riches en fibrilline-1, dont les mutations génétiques respectives conduisent à des pathologies cardiovasculaires graves impliquant l'apparition de sténoses (syndrome de Williams-Beuren, WBS) ou d'anévrismes (syndrome de Marfan, MS) aortiques. Nous avons étudié et comparé les effets de l'élastine (kappa-élastine, kE et tropoélastine recombinante, rTE) et des microfibrilles (MF et le fragment PF14 de la fibrilline-1) sur la signalisation dans les cellules vasculaires. Sur les cellules endothéliales de la veine ombilicale humaine (HUVEC), la kE, la rTE et les MF activent le complexe récepteur de l'élastine (ECR) et les intégrines, la production de messagers intracellulaires et, avec une efficacité variable, des canaux calciques de la membrane cellulaire et du réticulum endoplasmique, mobilisant à la fois le calcium intra- et extra-cellulaire. Les microfilaments d'actine ne sont impliqués que dans le cas de la signalisation liée à l'élastine. Toutes les protéines étudiées augmentent aussi la prolifération et l'adhésion des HUVEC, ainsi que la production ou la dégradation –à travers l'activation de métalloprotéases matricielles (MMP)- de plusieurs composés de la matrice extracellulaire. La capacité de migration des HUVEC est augmentée par la kE, rTE et PF14 alors qu'elle est diminuée par les MF. Aussi, chez le rat, ces protéines induisent une synthèse de l'oxyde nitrique (NO) par les cellules endothéliales, résultant en une vasodilatation aortique. PF14 présente de plus un pouvoir vasocontractant en absence de l'endothélium indiquant qu'il stimule aussi particulièrement les cellules musculaires lisses vasculaires (CMLVs). Pour étudier ces interactions sur modèle cellulaire, un protocole de culture de CMLVs d'aorte de souris adultes, âgées, ou déficientes pour les gènes de l'élastine ou de la fibrilline-1, a été mis au point. Les protéines des fibres élastiques produisent dans ces cellules une montée du niveau de calcium intracellulaire dont les caractéristiques varient suivant la protéine, l'âge ou le génotype des animaux. Ces travaux confirment le rôle majeur de l'élastine et de la fibrilline-1 dans la régulation des fonctions des cellules vasculaires. Malgré quelques différences, les fragments d'élastine et de fibrilline-1 stimulent souvent de manière similaire les cellules vasculaires. Ceci suggère que les symptômes contradictoires observés dans les MS et WBS mettent aussi en cause des facteurs additionnels, probablement liés à d'autres signalisations, par exemple à la voie du TGF-β ou aux différences de mécanisme et de cinétique du dépôt de l'élastine et de la fibrilline-1 lors de l'assemblage des fibres élastiques. / Elastic fibres are composed of elastin and fibrillin-1 rich microfibrils, whose genetic mutations result in severe cardiovascular pathologies characterized by aortic stenosis (Williams syndrome, WS) or aneurysm (Marfan Syndrom, MS), respectively. We studied and compared the effect of elastin (kappa-elastin, kE and recombinant tropoelastin, rTE) and microfibrils (MF and PF14, fibrillin-1 fragment) on vascular cell signaling. In human umbilical vein endothelial cells (HUVEC), kE, rTE and MF activated the elastin complex receptors (ECR) and integrins, the production of intracellular messengers and, with varying efficiencies, membrane and endoplasmic reticulum calcium channels, mobilizing both intra- and extra-cellular calcium. Actin microfilaments were involved only in the case of elastin signaling. All these proteins enhanced the proliferation and adhesion of HUVECs, as well as synthesis or degradation –through matrix metalloproteinanse (MMP) activation- of several extracellular matrix components. HUVEC migration was enhanced by kE, rTE and PF14 and diminished by MF. Also, in rats, these proteins induced nitric oxide (NO) synthesis by endothelial cells, resulting in aortic vasodilatation, although PF14 had an additional constrictor effect on endothelium-free aorta, indicating that PF14 also stimulates vascular smooth muscle cells (VSMCs). To study these interactions in a cellular model, we designed a new protocol to culture VSMCs from aorta of adult, aged, and elastin or fibrillin-1 knock-out mice. Elastic fibre proteins induced an increase in VSMC intracellular calcium level, which characteristics varied with the protein, age and genotype of mice. These findings confirm the major role of elastin and fibrillin-1 in the regulation of vascular cell functions. Despite some differences, elastin and fibrillin-1 often stimulated vascular cells similarly. This suggests that the onset of the contradictory features observed in MS and WS also involve additional factors, probably linked to other signaling pathways, for instance the TGF-β pathway or the differences in the mechanism and kinetic of elastin and fibrillin-1 depositions during elastic fiber assembly.

O objetivo deste trabalho foi avaliar o efeito do exercício físico aeróbio sobre alterações morfológicas ocorridas na parede da aorta abdominal de animais hipertensos (SHR) e normotensos (WKY), sedentários (S) e treinados (T). Ratos SHR e WKY foram utilizados, que consistia de quatro grupos divididos em WKYS, WKYT, SHRS, SHRT. O treinamento durou 13 semanas, sendo 5 horas por semana, 1 hora por dia. Os resultados deste estudo mostraram que o exercício físico foi eficaz em reduzir a pressão arterial, a freqüência cardíaca e a razão parede/luz, bem como em aumentar a quantidade de fibras elásticas e a luz do vaso no grupo SHRT, em comparação ao grupo SHRS. Em relação à expressão protéica de colágeno I e III, os SHR apresentaram um significativo aumento em relação ao grupo WKY e o grupo SHRT apresentou significativa redução em relação ao SHRS. A <font face=\"symbol\">a-actina mostrou maior expressão nos grupos WKYS e SHRS, quando comparado com seus respectivos grupos treinados e a elastina mostrou-se significante aumento na expressão nos grupos treinados em relação aos grupos sedentários. / The aim of this study was to evaluate the effect of aerobic exercises on morphological changes on abdominal aorta wall, in hypertensive (SHR) and normotensive (WKY) animals, sedentary (S) and trained (T). SHR and WKY were used on the experimental protocol that consisted in four groups divided in WKYS, WKYT, SHRS and SHRT. Trained groups were submitted to a training protocol that lasted 13 weeks, 5 hours a week, 1 hour a day. Results showed that physical exercises were effective not only in reducing blood pressure, cardiac frequency and wall-to-lumen ratio, but also in increasing the number of elastic fibers and the internal diameter in SHRT, in comparison to SHRS. According to collagen I and III protein expression, in both, SHR presented a bigger expression than WKY group. Moreover, SHRT group showed a significant reduction of protein expression in comparison to SHRS. <font face=\"symbol\">a-actin showed to be more expressed in WKYS and SHRS, in relation to the WKYT and SHRT groups and elastin showed a significant increased in WKYT and SHRT in relation to the sedentary groups.

Para avaliar as fibras elásticas na junção vésico-uretral de ratos, em diferentes faixas etárias, realizou-se estudos histológicos, microscopia elêtronica e histomorfometria. O estudo histológico do sistema de fibras elásticas mostrou a presença dos três tipos de fibras elásticas, em todas as faixas etárias, a microscopia eletrônica de transmissão, mostrou as diferenças ultra-estruturais entre as fibras elásticas. A avaliação histomerfométrica revelou diminuição na densidade linear das fibras elásticas e oxitalânicas e aumento na das elaunínicas. Na junção vésico-uretral de animais velhos, há uma queda nas propriedades de elasticidade, recuo elástico e ancoragem, que são compensadas pelas fibras elaunínicas. O processo de envelhecimento das fibras elásticas na junção vésico-uretral não contribui sozinha e diretamente no estado de incontinência urinária, mas compensa e dá suporte a muscular, devido ao aumento das fibras elaunínicas, o que torna o sistema elástico na junção vésico-uretral indiretamente responsável pela manutenção da continência urinária. / To evaluate the elastic fibers in the junctio vesico-urethral of the rat, in different age groups, was accomplished studies histologics, electron microcopy and histomorphometrics. Study histologic showed the presence of the three types elastic fibers in all the studied age groups, electron microcopy showed the ultra-structural differences amoung elastic fibers. Histomorphometric studies revealed a decrease the linear density of the mature elastic fibers and oxytalans and an increase elaunin. In the junction vesico-urethral of the old animals there is a fall the properties elastic, elastic recoil and achorage, but they are compensated by elaunins. Processs aging of the elastic system in this place doesn?t contribute alone or directly in urinary incontinence, but it is indirectly responsible for the maintenance urinary continence.

Nas doenças que provocam perdas ósseas, destaca-se a utilização de enxerto ósseo autólogo como tratamento padrão-ouro. Entretanto, devido a comorbidades e limitação quantitativa, apresentam-se como alternativa o uso de biomateriais de elastina e hidroxiapatita, bem como a associação com a proteína morfogenética óssea. O objetivo desta pesquisa foi avaliar de forma qualitativa e quantitativa a contribuição da membrana de elastina utilizada isoladamente ou em combinação com a hidroxiapatita e a proteína morfogenética óssea no reparo de defeitos ósseos provocados experimentalmente no fêmur de ratos. Foram utilizados 77 ratos (Rattus norvegicus, Wistar), machos, com peso aproximado de 330 gramas e 4 meses de idade. Os animais foram submetidos ao procedimento cirúrgico para criação do defeito ósseo na superfície anterior da região supracondiliana do fêmur direito e separados em 7 grupos com 11 animais cada. Os grupos foram divididos da seguinte forma: Grupo controle (G1-C), ratos com defeito crítico induzido no osso femoral, sem preenchimento com implante. Grupo 2 (G2-E24/50), ratos com defeito crítico induzido no osso femoral preenchido com membrana de elastina hidrolisada durante 24h a 50°C; Grupo 3 (G3-E24/50/HA), defeito preenchido com membrana de elastina hidrolisada durante 24h a 50°C e hidroxiapatita. Grupo 4 (G4 E24/50/BMP), defeito preenchido com membrana de elastina hidrolisada durante 24h a 50°C e BMP. Grupo 5 (G5-E96/37), defeito preenchido com membrana de elastina hidrolisada durante 96 h a 37°C. Grupo 6 (G6-E96/37/HA), defeito preenchido com membrana de elastina hidrolisada durante 96 h a 37°C e hidroxiapatita. Grupo 7 (G7-E96/37/BMP), defeito preenchido com membrana de elastina hidrolisada durante 96 h a 37°C e BMP. Após a morte indolor induzida após 6 semanas, as peças anatômicas femorais foram retiradas para análise macroscópica, radiográfica, histológica, morfométrica e biomecânica. As médias e desvios-padrão do volume percentual relativo de osso neoformado no defeito femoral foram respectivamente 36,39±3,86 (G1); 66,40±3,69 (G2); 55,12±2,23 (G3); 58,46±1,79 (G4); 51,36±3,15 (G5); 71,28±4,26 (G6); 45,54±3,98 (G7). Os achados demonstraram biocompatibilidade, biodegradabilidade e osteorregeneração maior que o grupo controle nos biomateriais estudados. Os grupos com maior volume ósseo neoformado foram G2-E24/50 e G6-E96/37/HA. / In diseases that cause bone loss, the use of autologous bone graft is still a goldstandard treatment. However, due to comorbidities and quantitative limitation, the use of biomaterials of elastin, hydroxyapatite as well as the association with the bone morphogenetic protein are presented as alternatives. The objective of this research is to evaluate in a qualitative and quantitative way the contribution of the elastin membrane used alone or in combination with the hydroxyapatite and the bone morphogenetic protein in bone defects experimentally created in femur of rats. A total of 77 male rats (Rattus norvegicus, Wistar) weighing approximately 330 grams and 4 months of age were used. The animals were submitted to the surgical procedure to create the bone defect on the anterior surface of the supracondylar region of the right femur and were separated into 7 groups with 11 animals each. The groups were divided as follows: Control group (G1-C), rats with critical defect induced in the femoral bone without implant filling. Group 2 (G2-E24/50), defect filled with elastin membrane prepared for 24 h at 50°C. Group 3 (G3-E24/50/HA), defect filled with elastin membrane prepared for 24 h at 50°C and hydroxyapatite. Group 4 (G4-E24/50/BMP), defect filled with elastin membrane prepared for 24 h at 50°C and BMP. Group 5 (G5- E96/37), defect filled with elastin membrane prepared for 96 h at 37°C. Group 6 (G6- E96/37/HA), defect filled with elastin membrane prepared for 96 h at 37°C and hydroxyapatite. Group 7 (G7-E96/37/BMP) defect filled with elastin membrane prepared for 96 h at 37°C and BMP. After painless death induced after 6 weeks, the femoral anatomical pieces were removed for macroscopic, radiographic, histological, morphometric and biomechanical analysis. The mean and standard deviations of the relative percentage volume of newly formed bone in the femoral defect were respectively 36,39±3,86 (G1); 66,40±3,69 (G2); 55,12±2,23 (G3); 58,46±1,79 (G4); 51,36±3,15 (G5); 71,28±4,26 (G6); 45,54±3,98 (G7). The findings demonstrated biocompatibility, biodegradability and osseous regeneration greater than the control group in the studied biomaterials. The groups with the highest newly formed bone volume were G2-E24/50 and G6-E96/37/HA.