Adipositas und Typ-2-Diabetes sind häufige Erkrankungen des Stoffwechsels. Zur Basistherapie der Adipositas und des Typ-2-Diabetes gehören eine gesunde Ernährungs- weise und die Erhöhung der körperlichen Aktivität unter anderem mit dem Ziel der Gewichtsreduktion. Vermehrte Bewegung führt neben der Verbesserung der körperlichen Leistungsfähigkeit zur Fettmassenreduktion, Verbesserungen der Hyperglykämie, Lipo- proteinstoffwechsels und des Adipokinprofils. Bone morphogenetic proteins (BMPs) werden im Fettgewebe produziert und spielen eine wichtige Rolle in der Adipogenese und Transdifferenzierung von Adipozyten. Während ein Zusammenhang zwischen der BMP-7-Serumkonzentration und Adipositas vor kurzem belegt wurde, ist bisher nicht bekannt, ob weitere BMPs wie BMP-2 und -4 mit Adipositas und Typ-2-Diabetes assoziiert sind. Ziel dieser Arbeit war es deshalb zu untersuchen, ob die BMP-2 und -4 Serumkonzentrationen im Zusammenhang mit Körpergewicht, Fett- verteilung und Parametern des Glukosestoffwechsels bei Patienten mit Adipositas und Typ-2-Diabetes (n=213) stehen. Im Rahmen von drei Interventionsstudien wurde der Einfluss einer hypokalorischen Ernährungsweise über sechs Monate (n=19), eines 45,3 ± 7,4 kg Gewichtsverlustes ein Jahr nach bariatrischer Chirurgie (n=32) sowie eines zwölf- wöchigen Trainingsprogramms (n=60) auf die BMP-2- und -4-Serumkonzentrationen untersucht. Zusätzlich wurde die BMP-2-und -4-mRNA-Expression in humanen omentalen und subkutanen Fettgewebsproben von 161 Patienten charakterisiert. Die BMP-2- und -4-Serumkonzentrationen und die BMP-2- und -4-mRNA-Expression im viszeralen Fettgewebe korrelieren signifikant mit dem BMI und dem Körperfettgehalt. Zirkulierende BMP-4-Spiegel sind geschlechtsabhängig und bei Patienten mit T2D signifikant niedriger als bei gesunden Kontrollpatienten. Sowohl eine moderate Gewichts- reduktion durch kalorienreduzierte Ernährung als auch ein Gewichtsverlust von 45,3 ± 7,4 kg nach bariatrischer Chirurgie führen zu einer signifikanten Reduktion der zirkulierenden BMP-2- und -4-Spiegel. Das zwölfwöchige Trainingsprogramm führte lediglich zu einer signifikanten Reduktion der BMP-2-Serumkonzentration und zu signifikanten Ver- besserung der Leistungsfähigkeit, von Parametern des Glukosestoffwechsels und der Serumkonzentrationen von Adiponektin und Interleukin-6. Zusammengefasst zeigen die Daten, dass erhöhte Serumkonzentrationen von BMP-2 und 4 mit Adipositas assoziiert sind und durch Gewichtsreduktion und Erhöhung der körperlichen Aktivität verringert werden können. Die BMP-2- und -4-mRNA-Expression im viszeralen Fettgewebe kann zu erhöhten Serumkonzentrationen dieser Adipokine bei viszeraler Fettverteilung beitragen.

The cardiovascular system is an essential component of mammalian biology. It is a complex network of various tissues and structures with unique functions. The function of the cardiovascular system is to supply nutrients including oxygen to the various cells, tissues and organs within the body, and remove waste products from them. Given the importance of this role, it is not surprising that there are countless regulatory mechanisms at the molecular, cellular and tissue levels that are required to support this functional system. Perturbations in parts of this system are likely to lead to abnormalities, and thus give rise to cardiovascular-related diseases. Despite the currently expanding list of genes reported to be involved in a variety of cardiovascular-related diseases, including calcific aortic valve disease (CAVD), the functions and associated pathways of these factors in both normal and pathological physiology have yet to be fully understood, such as at the transcriptomic level. In this thesis, a genome-wide transcriptomic atlas of the healthy mammalian cardiovascular system was generated using the sheep as a large animal model. This atlas was generated using RNA-seq, with the aim of further understanding normal gene expression patterns in the context of the known physiology of healthy mammalian tissues. Through this work, I identified novel gene networks and detailed functional clustering of co-expressed genes with region-specific expression and specialised cardiovascular roles. One interesting cluster was highly expressed in the cardiac valves, and shared genes found in physiological bone development, such as bone morphogenetic protein 4 (BMP4), collagen type I alpha 2 (COL1A2), Sry homeobox 8 (SOX8) and bone gamma-carboxyglutamate protein (BGLAP), some of which have been implicated in vascular calcification. Further to this work, I studied the expression profiles of these key cardiovascular genes during development in the sheep from foetal to adult stages. In addition, I investigated the gene expression patterns of various key vascular calcification genes. These studies showed differential expression of genes in the different cardiovascular tissues, demonstrating transcriptional differences between these different tissues known to have different functions. CAVD involves progressive valve leaflet thickening and severe calcification, resulting in impaired leaflet motion. The in vitro calcification of primary rat, human, porcine and bovine aortic valve interstitial cells (VICs) is commonly employed to examine the mechanisms of CAVD. However, to date, no published studies have utilised cell lines to investigate this process Thus, in this project, I generated and evaluated the calcification potential of an immortalised cell line derived from sheep aortic VICs (SAVICs). This novel large animal in vitro model of CAVD was demonstrated to calcify under high calcium and phosphate conditions. Changes in the expression of key calcification genes during VIC calcification was also observed, including increased mRNA expression of bone markers Runt-related transcription factor 2 (RUNX2) and sodium-dependent phosphate transporter 1 (PiT1), and a concomitant decrease in matrix Gla protein (MGP) mRNA expression. In addition, the role of extracellular nucleotides and their receptors (P2 receptors), which have been previously shown to be important in bone and vascular calcification, were investigated using SAVICs in vitro. This study has shown that extracellular nucleotides, particularly adenosine 5’-triphosphate (ATP) and uridine 5’-triphosphate (UTP) and other agonists of P2 receptors, reduced VIC calcification in vitro. Moreover, the cutting-edge gene-editing technology, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9), was successfully applied to generate large animal models of cardiovascular-related diseases. In this project, I applied the CRISPR/Cas9 technology to edit ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) and fibrillin 1 (FBN1) to generate two models of vascular calcification and Marfan Syndrome (MFS), respectively. In the ENPP1-edited animals, soft tissue calcification has been observed in the biallelic mutant and homozygous pigs. In this project, I have developed a range of novel in vitro and in vivo tools to advance the study of cardiovascular disease. These studies demonstrate that large animal models are highly valuable in the field of cardiovascular biology. The in vivo and in vitro experimental models described should facilitate detailed analysis of cardiovascular molecular biology and ultimately lead to therapies which will minimise the morbidity and mortality currently arising from cardiovascular pathology.

Thesis (Ph.D. in Neuroscience) -- University of Colorado Denver, 2008. / Includes bibliographical references (leaves 112-120). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Adipositas und Typ-2-Diabetes sind häufige Erkrankungen des Stoffwechsels. Zur Basistherapie der Adipositas und des Typ-2-Diabetes gehören eine gesunde Ernährungs- weise und die Erhöhung der körperlichen Aktivität unter anderem mit dem Ziel der Gewichtsreduktion. Vermehrte Bewegung führt neben der Verbesserung der körperlichen Leistungsfähigkeit zur Fettmassenreduktion, Verbesserungen der Hyperglykämie, Lipo- proteinstoffwechsels und des Adipokinprofils. Bone morphogenetic proteins (BMPs) werden im Fettgewebe produziert und spielen eine wichtige Rolle in der Adipogenese und Transdifferenzierung von Adipozyten. Während ein Zusammenhang zwischen der BMP-7-Serumkonzentration und Adipositas vor kurzem belegt wurde, ist bisher nicht bekannt, ob weitere BMPs wie BMP-2 und -4 mit Adipositas und Typ-2-Diabetes assoziiert sind. Ziel dieser Arbeit war es deshalb zu untersuchen, ob die BMP-2 und -4 Serumkonzentrationen im Zusammenhang mit Körpergewicht, Fett- verteilung und Parametern des Glukosestoffwechsels bei Patienten mit Adipositas und Typ-2-Diabetes (n=213) stehen. Im Rahmen von drei Interventionsstudien wurde der Einfluss einer hypokalorischen Ernährungsweise über sechs Monate (n=19), eines 45,3 ± 7,4 kg Gewichtsverlustes ein Jahr nach bariatrischer Chirurgie (n=32) sowie eines zwölf- wöchigen Trainingsprogramms (n=60) auf die BMP-2- und -4-Serumkonzentrationen untersucht. Zusätzlich wurde die BMP-2-und -4-mRNA-Expression in humanen omentalen und subkutanen Fettgewebsproben von 161 Patienten charakterisiert. Die BMP-2- und -4-Serumkonzentrationen und die BMP-2- und -4-mRNA-Expression im viszeralen Fettgewebe korrelieren signifikant mit dem BMI und dem Körperfettgehalt. Zirkulierende BMP-4-Spiegel sind geschlechtsabhängig und bei Patienten mit T2D signifikant niedriger als bei gesunden Kontrollpatienten. Sowohl eine moderate Gewichts- reduktion durch kalorienreduzierte Ernährung als auch ein Gewichtsverlust von 45,3 ± 7,4 kg nach bariatrischer Chirurgie führen zu einer signifikanten Reduktion der zirkulierenden BMP-2- und -4-Spiegel. Das zwölfwöchige Trainingsprogramm führte lediglich zu einer signifikanten Reduktion der BMP-2-Serumkonzentration und zu signifikanten Ver- besserung der Leistungsfähigkeit, von Parametern des Glukosestoffwechsels und der Serumkonzentrationen von Adiponektin und Interleukin-6. Zusammengefasst zeigen die Daten, dass erhöhte Serumkonzentrationen von BMP-2 und 4 mit Adipositas assoziiert sind und durch Gewichtsreduktion und Erhöhung der körperlichen Aktivität verringert werden können. Die BMP-2- und -4-mRNA-Expression im viszeralen Fettgewebe kann zu erhöhten Serumkonzentrationen dieser Adipokine bei viszeraler Fettverteilung beitragen.

A síndrome dos ovários policísticos (SOP) é um distúrbio endócrino complexo e heterogêneo, caracterizado por hiperandrogenismo e anovulação crônica. Dentre as manifestações clínicas do hiperandrogenismo, o hirsutismo é o mais frequente e pode estar presente em cerca de 70% das pacientes com SOP. Sabe-se que o crescimento e diferenciação do pelo são regulados por fatores locais, endócrinos, parácrinos e genéticos. No entanto, a fisiopatologia do hirsutismo ainda é pouco conhecida. O gene BMP4 (que codifica a Proteína Morfogenética Óssea-4) relaciona-se ao controle do crescimento e diferenciação do pelo, porém não há estudos sobre seu papel no hirsutismo em mulheres com síndrome dos ovários policísticos. Foram estudadas 245 mulheres, 142 SOP e 103 controles em que analisaram-se os polimorfismos rs4898820 e 538 T/C, para verificar frequências genotípicas e alélicas. Nas pacientes com SOP foi investigada a existência de associação entre esses polimorfismos e hirsutismo e parâmetros laboratoriais e clínicos. Não houve diferença significante na frequência dos polimorfismos entre os grupos. Não houve associação dos polimorfismos com hirsutismo. Quando analisados os exames laboratoriais das mulheres com SOP, houve associação do genótipo mutado do polimorfismo 538 T/C (CC) com níveis mais altos de SHBG, menores de glicemia e maior sensibilidade à insulina. Houve também associação entre os alelos mutados (CC ou TC), com menores níveis de testosterona livre. Encontrou-se diferença significante para o FSH nas portadoras do genótipo mutado para o polimorfismo rs4898820 (TT). Não houve associação dos polimorfismos com hirsutismo / Polycystic ovary syndrome (PCOS) is a complex and heterogeneous endocrine disorder characterized by chronic anovulation and hiperandrogesnism. Among the clinical manifestations of hyperandrogenism, hirsutism is the most frequent and may be present in approximately 70% of patients with PCOS. It is known that the hair growth and differentiation are coordinated by local, endocrine, paracrine and genetic factors. However, the pathophysiology of hirsutism is poorly understood. BMP-4 (Bone Morphogenetic Protein-4) is a gene involved in the hair growth and differentiation, but there are no studies about its action in hirsutism in women with polycystic ovary syndrome. A total of 245 women, 142 with PCOS diagnostic and 103 control women were studied to investigate the the allelic frequency of the single nucleotide polymorfisms rs4898820 and 538 T/C in PCOS in comparison with the control group. In PCOS group, we sought to investigate a possible association between the genetic variations and the hirsutism. There were no differences for the polymorphisms between groups. There was no association between the genotypes and the presence of hirsutism in PCOS women. When the polymorphisms were analyzed in the PCOS group, those who had homozygous genotype for 538 T/C (CC) had lower levels of SHBG, lower levels of glucose and better insulin sensitivity. Mutated allele (CC or TC), were associated with lower levels of free testosterone. Those who had the mutated genotype for the polymorphism rs4898820 (TT) had higher levels of FSH

A síndrome dos ovários policísticos (SOP) é um distúrbio endócrino complexo e heterogêneo, caracterizado por hiperandrogenismo e anovulação crônica. Dentre as manifestações clínicas do hiperandrogenismo, o hirsutismo é o mais frequente e pode estar presente em cerca de 70% das pacientes com SOP. Sabe-se que o crescimento e diferenciação do pelo são regulados por fatores locais, endócrinos, parácrinos e genéticos. No entanto, a fisiopatologia do hirsutismo ainda é pouco conhecida. O gene BMP4 (que codifica a Proteína Morfogenética Óssea-4) relaciona-se ao controle do crescimento e diferenciação do pelo, porém não há estudos sobre seu papel no hirsutismo em mulheres com síndrome dos ovários policísticos. Foram estudadas 245 mulheres, 142 SOP e 103 controles em que analisaram-se os polimorfismos rs4898820 e 538 T/C, para verificar frequências genotípicas e alélicas. Nas pacientes com SOP foi investigada a existência de associação entre esses polimorfismos e hirsutismo e parâmetros laboratoriais e clínicos. Não houve diferença significante na frequência dos polimorfismos entre os grupos. Não houve associação dos polimorfismos com hirsutismo. Quando analisados os exames laboratoriais das mulheres com SOP, houve associação do genótipo mutado do polimorfismo 538 T/C (CC) com níveis mais altos de SHBG, menores de glicemia e maior sensibilidade à insulina. Houve também associação entre os alelos mutados (CC ou TC), com menores níveis de testosterona livre. Encontrou-se diferença significante para o FSH nas portadoras do genótipo mutado para o polimorfismo rs4898820 (TT). Não houve associação dos polimorfismos com hirsutismo / Polycystic ovary syndrome (PCOS) is a complex and heterogeneous endocrine disorder characterized by chronic anovulation and hiperandrogesnism. Among the clinical manifestations of hyperandrogenism, hirsutism is the most frequent and may be present in approximately 70% of patients with PCOS. It is known that the hair growth and differentiation are coordinated by local, endocrine, paracrine and genetic factors. However, the pathophysiology of hirsutism is poorly understood. BMP-4 (Bone Morphogenetic Protein-4) is a gene involved in the hair growth and differentiation, but there are no studies about its action in hirsutism in women with polycystic ovary syndrome. A total of 245 women, 142 with PCOS diagnostic and 103 control women were studied to investigate the the allelic frequency of the single nucleotide polymorfisms rs4898820 and 538 T/C in PCOS in comparison with the control group. In PCOS group, we sought to investigate a possible association between the genetic variations and the hirsutism. There were no differences for the polymorphisms between groups. There was no association between the genotypes and the presence of hirsutism in PCOS women. When the polymorphisms were analyzed in the PCOS group, those who had homozygous genotype for 538 T/C (CC) had lower levels of SHBG, lower levels of glucose and better insulin sensitivity. Mutated allele (CC or TC), were associated with lower levels of free testosterone. Those who had the mutated genotype for the polymorphism rs4898820 (TT) had higher levels of FSH

Yung, Lai Hang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 93-115). / Abstract also in Chinese. / Declaration --- p.i / Acknowledgements --- p.ii / Abbreviations --- p.iii / Abstract in English --- p.v / Abstract in Chinese --- p.ix / Contents --- p.xi / Chapter Chapter I - --- Introduction / Chapter 1.1) --- Endothelial cells function --- p.1 / Chapter 1.2) --- Oxidative stress in the vascular wall --- p.2 / Chapter 1.2.1) --- Sources of ROS --- p.3 / Chapter 1.2.2) --- Actions of ROS --- p.3 / Chapter 1.2.2.1) --- Impaired endothelium-dependent vasodilatation --- p.3 / Chapter 1.2.2.2) --- VSMC migration --- p.4 / Chapter 1.2.2.3) --- Programmed cell death (cell apoptosis) --- p.4 / Chapter 1.3) --- Endothelial cell apoptosis --- p.7 / Chapter 1.3.1) --- Apoptosis and cardiovascular diseases --- p.7 / Chapter 1.3.2) --- Mechanisms of endothelial cells apoptosis --- p.7 / Chapter 1.3.2.1) --- What are caspases? --- p.8 / Chapter 1.3.2.2) --- Death receptor-mediated apoptosis --- p.9 / Chapter 1.3.2.3) --- Mitochondria-dependent pathway --- p.9 / Chapter 1.3.3) --- Regulations of endothelial cells apoptosis --- p.10 / Chapter 1.3.3.1) --- Oxidative stress --- p.10 / Chapter 1.3.3.2) --- Shear Stress --- p.11 / Chapter 1.3.3.3) --- Growth factors --- p.12 / Chapter 1.3.3.4) --- NO --- p.12 / Chapter 1.3.3.5) --- Inflammatory mediators --- p.13 / Chapter 1.4) --- Mitogen activated kinases signaling in apoptosis --- p.15 / Chapter 1.5) --- Bone morphogenic proteins (BMPs) --- p.17 / Chapter 1.5.1) --- BMPs functions and cardiovascular system --- p.17 / Chapter 1.5.2) --- BMPs signaling pathways --- p.18 / Chapter 1.5.2.1) --- Smad-dependent pathway --- p.18 / Chapter 1.5.2.2) --- MAPKs and SAPKs pathways --- p.19 / Chapter 1.5.2.3) --- Antagonists of BMPs signaling --- p.20 / Chapter 1.5.3) --- BMP4 and cardiovascular diseases --- p.20 / Chapter 1.6) --- "Justification, long-term significance and objectives of the present project" --- p.23 / Chapter Chapter II - --- Methods and Materials / Chapter 2.1) --- Animal handling --- p.24 / Chapter 2.2) --- Endothelial cell isolation and culture --- p.24 / Chapter 2.2.1) --- Primary culture of rat endothelial cells --- p.24 / Chapter 2.2.2) --- Culture of human umbilical cord vein endothelial cells… --- p.25 / Chapter 2.3) --- Apoptosis assessment --- p.25 / Chapter 2.3.1) --- Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay --- p.25 / Chapter 2.3.2) --- Cell death detection ELISA kit --- p.26 / Chapter 2.3.3) --- Flow cytometry --- p.27 / Chapter 2.4) --- Western blot analysis --- p.28 / Chapter 2.4.1) --- Sample preparation --- p.28 / Chapter 2.4.2) --- SDS-PAGE and transfer --- p.28 / Chapter 2.5) --- DHE fluorescence --- p.29 / Chapter 2.6) --- "Drugs, chemicals and other reagents" --- p.30 / Chapter 2.6.1) --- Drugs and chemicals used in the present experiments --- p.30 / Chapter 2.6.2) --- Reagents for Western blot analysis --- p.30 / Chapter 2.6.3) --- Primary antibodies --- p.33 / Chapter 2.7) --- Small interfering RNA experiment --- p.34 / Chapter 2.8) --- Statistical analysis --- p.34 / Chapter Chapter III - --- BMP4 induces endothelial cell apoptosis in ROS related p38 MAPK and JNK mediated caspase-3 dependent pathway / Chapter 3.1) --- Introduction --- p.35 / Chapter 3.2) --- Methods and materials --- p.39 / Chapter 3.2.1) --- Isolation and culture of endothelial cells --- p.39 / Chapter 3.2.2) --- Drugs treatment --- p.39 / Chapter 3.2.3) --- Assay for cell apoptosis --- p.40 / Chapter 3.2.3.1) --- Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay --- p.40 / Chapter 3.2.3.2) --- Cell death detection ELISA kit --- p.41 / Chapter 3.2.3.3) --- Flow cytometric analysis --- p.41 / Chapter 3.2.4) --- Western blot analysis --- p.41 / Chapter 3.2.5) --- Dihydroethidium (DHE) staining --- p.42 / Chapter 3.2.6) --- Statistical analysis --- p.42 / Chapter 3.3) --- Results --- p.43 / Chapter 3.3.1) --- Dose- and time-dependent effect of BMP4 --- p.43 / Chapter 3.3.2) --- Role of caspases in apoptosis of RAECs and HUVECs --- p.43 / Chapter 3.3.3) --- Roles of BMP4 and ROS in endothelial cell apoptosis --- p.44 / Chapter 3.3.3.1) --- Noggin antagonism of BMP4-induced effect --- p.44 / Chapter 3.3.3.2) --- NAD(P)H oxidase-mediated ROS production --- p.44 / Chapter 3.3.3.3) --- Inhibition of endothelial cell apoptosis by ROS scavengers --- p.45 / Chapter 3.3.4) --- Roles of MAPKs/SAPKs in BMP4-induced endothelial cell apoptosis --- p.45 / Chapter 3.3.5) --- Relationship between ROS and MAPKs/SAPKs --- p.46 / Chapter 3.3.6) --- Relationship between p38 MAPK and JNK --- p.46 / Chapter 3.4) --- Discussion --- p.82 / Chapter 3.4.1) --- Caspase-dependent pathways --- p.82 / Chapter 3.4.2) --- Oxidative stress --- p.85 / Chapter 3.4.3) --- Role of MAPKs activation in BMP4-induced endothelial cell apoptosis --- p.87 / Chapter 3.4.4) --- ROS mediates BMP4-induced activation of MAPKs --- p.88 / Chapter 3.4.5) --- Role of p38 MAPK in the activation of JNK 1 --- p.89 / Chapter 3.5) --- Concluding remarks --- p.91 / References --- p.93 / Publications and Awards --- p.116

Este estudo avaliou a influência da fototerapia a laser (FTL) na proliferação e diferenciação de células-tronco da polpa dentária humana (DPSCs; do inglês, Dental Pulp Stem Cells ) encapsuladas em carreador injetável e termoresponsivo (PL; Pluronic® F-127, Sigma-Aldrich, MO, EUA) com incorporação de proteína morfogenética óssea 4 recombinante humana (rhBMP4) (sistema PL/rhBMP4). O biomaterial foi caracterizado de acordo com seus perfis de embebição e dissolução, liberação de rhBMP4 e sua estrutura morfológica. DPSCs foram isoladas, caracterizadas e encapsuladas em PL para confirmar sua viabilidade e seu potencial de diferenciação (adipo e osteogênico) em comparação com células-tronco mesenquimais de medula óssea (BMMSCs; do inglês, Bone Marrow Mesenchymal Stem Cells). Quando encapsuladas no sistema PL/rhBMP4, DPSCs foram irradiadas com duas densidades de energia diferentes utilizando laser de diodo de fosfeto de índio-gálio-alumínio (InGaAlP), modos contínuo, pontual e em contato [660 nm, 0,028 cm2, 20 mW, 0,71 W/cm2, 3 J/cm2 (4 s) ou 5 J/cm2 (7 s)]. Os ensaios de PKH26 (do inglês, Red Fluorescent Cell Linker), CFU-F (do inglês, Coloning Forming Units - Fibroblastic), e MTT (do inglês, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide)) foram utilizados para avaliar adesão/proliferação, diferenças na capacidade formadora de colônias e viabilidade das DPSCs (neste último caso sob estresse nutricional), respectivamente. Finalmente, a diferenciação odonto/osteogênica foi analisada por qRT-PCR e confirmada por ensaio de vermelho de alizarina. O biomaterial embebeu e dissolveu rapidamente; densa rede tubular e reticular com poros interconectados foi observada. DPSCs e BMMSCs apresentaram alta viabilidade celular quando encapsuladas em PL. Ambas as linhagens celulares tiveram êxito em se diferenciar em tecidos adiposo e ósseo. De acordo com o PKH26, DPSCs puderam aderir e proliferar no sistema PL/rhBMP4. DPSCs irradiadas encapsuladas tanto em PL como em PL/rhBMP4 formaram mais CFU-F que os controles não irradiados. Sob estresse nutricional, DPSCs semeadas no PL e irradiadas com 5 J/cm2 exibiram maior taxa de viabilidade celular em relação aos grupos não irradiados e irradiados com 3 J/cm2. Na presença de rhBMP4, os grupos irradiados tanto com 3 J/cm2 quanto com 5 J/cm2 apresentaram deposição mineral precoce quando comparados aos grupos não irradiados. Ainda, após 21 dias de diferenciação odonto/osteogênica, DPSCs irradiadas produziram maior quantidade de nódulos mineralizados. A irradiação com 5 J/cm2 levou ao aumento significativo da expressão de genes envolvidos na diferenciação odonto/osteogênica, como colágeno tipo I (COL1A1), osteocalcina (OCN), proteína da matriz dentinária 1 (DMP1), sialofosfoproteina dentinária (DSPP) e proteína heat shock 27 kDa (HSPB1). A associação entre rhBMP4 e FTL promove proliferação e diferenciação odonto/osteogênica de DPSCs acelerando e aumentando notavelmente a formação de tecido mineralizado, em especial quando a densidade de energia de 5 J/cm2 é aplicada. / This study evaluated the influence of laser phototherapy (LPT) on dental pulp stem cells (DPSCs) proliferation and differentiation upon encapsulation in an injectable and thermo-responsive cell carrier (PL; Pluronic® F-127, Sigma-Aldrich, MO, USA) loaded with human recombinant bone morphogenetic protein 4 (rhBMP4)(PL/rhBMP4 system). The biomaterial was characterized according to its swelling and dissolution profiles, release of rhBMP4 and morphological structure. DPSCs were isolated, characterized and encapsulated in PL to confirm their viability and multilineage differentiation potential (adipo and osteogenic) in comparison to bone marrow mesenchymal stem cells (BMMSCs). When encapsulated in the PL/rhBMP4 system, DPSCs were irradiated with two different energy densities using a continuous-wave indium-gallium-aluminum-phosphide (InGaAlP) diode laser [660 nm, 0.028 cm2, 20 mW, 0.71 W/cm2, 3 J/cm2 (4 s) or 5 J/cm2 (7 s)] in punctual and contact modes. The PKH26 (Red Fluorescent Cell Linker), the CFU-F (Coloning Forming Units - Fibroblastic), and the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] assays were used to assess differences in cell adhesion/proliferation, colony forming units formation ability, and cell viability of DPSCs (in this case under nutritional stress), respectively. Then, alizarin red and qRT-PCR analyzes were used to evaluate odonto/osteogenic differentiation. The biomaterial swelled and dissolved rapidly; dense tubular and reticular network morphology with well-interconnected pores was observed. DPSCs and BMMSCs presented high cell viability when encapsulated in PL. Both cell lineages successfully differentiated into bone or adipose tissues. According to PKH26, DPSCs were able to adhere and proliferate in the PL/rhBMP4 system. Irradiated DPSCs encapsulated in either PL or PL/rhBMP4 system formed more CFU-F than non-irradiated controls. Under nutritional stress, DPSCs encapsulated in the hydrogels with no rhBMP4 and irradiated at 5 J/cm2 exhibited higher cell viability than the other groups. In the presence of rhBMP4, the groups irradiated both at 3 and 5 J/cm2 energy densities displayed earlier mineral deposition than the non-irradiated groups. Moreover, after 21 days of odonto/osteogenic differentiation, irradiated DPSCs produced greater nodule formation than the control groups. At the energy density of 5 J/cm2, there were significant upregulation of genes involved in odonto/osteoblast differentiation, such as type I collagen (COL1A1), osteocalcin (OCN), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP) and heat shock protein 27 kDa (HSPB1). The association between rhBMP4 and LPT promotes cell proliferation and odonto/osteogenic differentiation of DPSCs accelerating and increasing the formation of mineralized tissue, in particular when the energy density of 5 J/cm2 is applied.

Introduction: Idiopathic Pulmonary arteriole hypertension (IPAH) is a rare but severely debilitating disease that strikes women to men at a ratio of 3:1. Endothelial cell (EC) dysfunction is a hallmark of the disease. This includes rapid growth of the ECs until the occlusion of the vasculature as well as decreased blood levels of vasodilators. Markedly increased levels of endothelin-1, a potent vasoconstrictor and smooth muscle mitogen, have been noted in IPAH patients. / Recently mutations in the bone morphogenic protein receptor type II (BMPRII) have been linked to the disease. Interestingly mutations in activin-like kinase-1 (ALK-1) and endoglin have been linked to hereditary haemorrhagic telangiectasia (HHT), a disease that results in PAH clinically indistinguishable from IPAH. All of these proteins are either receptors or co-receptors to members of the TGFbeta superfamily. The connection of these mutations to the disease still remains largely a mystery to researchers and the effects of either bone morphogenic proteins 2, 4, 7 or TGFbeta levels on endothelin-1(ET-1) production in human microvascular endothelial cells cultured from normal lungs (HMVEC-LBI) are unknown. / Methods: HMVEC-LBI cells were cultured in the presence of various concentrations of BMP 2,4,7 and TGFbeta, in complete media or serum starved conditions. After allotted time points the media was collected and assayed by ELISA, meanwhile the cells were lysed and protein content assayed for normalization purposes. Small Mothers against Decapentaplegic (SMAD) 1/5 phosphorylation was also measured. / Results and Conclusions: Despite evidence that all BMPs used were biologically active, namely through SMAD phosphorylation studies, only BMP7 at very high dosages increased ET-1 production levels. TGFbeta had a more pronounced effect at earlier time points with lower concentrations. The results provide insights on the effects of an important group of proteins, the BMPs and TGFbeta, on lung microvascular ECs and which are likely the key cellular player In IPAH development. These findings may have clinical relevance in terms of control of the disease and understanding the normal response of these cells BMPs and TGFbeta.

Este estudo avaliou a influência da fototerapia a laser (FTL) na proliferação e diferenciação de células-tronco da polpa dentária humana (DPSCs; do inglês, Dental Pulp Stem Cells ) encapsuladas em carreador injetável e termoresponsivo (PL; Pluronic® F-127, Sigma-Aldrich, MO, EUA) com incorporação de proteína morfogenética óssea 4 recombinante humana (rhBMP4) (sistema PL/rhBMP4). O biomaterial foi caracterizado de acordo com seus perfis de embebição e dissolução, liberação de rhBMP4 e sua estrutura morfológica. DPSCs foram isoladas, caracterizadas e encapsuladas em PL para confirmar sua viabilidade e seu potencial de diferenciação (adipo e osteogênico) em comparação com células-tronco mesenquimais de medula óssea (BMMSCs; do inglês, Bone Marrow Mesenchymal Stem Cells). Quando encapsuladas no sistema PL/rhBMP4, DPSCs foram irradiadas com duas densidades de energia diferentes utilizando laser de diodo de fosfeto de índio-gálio-alumínio (InGaAlP), modos contínuo, pontual e em contato [660 nm, 0,028 cm2, 20 mW, 0,71 W/cm2, 3 J/cm2 (4 s) ou 5 J/cm2 (7 s)]. Os ensaios de PKH26 (do inglês, Red Fluorescent Cell Linker), CFU-F (do inglês, Coloning Forming Units - Fibroblastic), e MTT (do inglês, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide)) foram utilizados para avaliar adesão/proliferação, diferenças na capacidade formadora de colônias e viabilidade das DPSCs (neste último caso sob estresse nutricional), respectivamente. Finalmente, a diferenciação odonto/osteogênica foi analisada por qRT-PCR e confirmada por ensaio de vermelho de alizarina. O biomaterial embebeu e dissolveu rapidamente; densa rede tubular e reticular com poros interconectados foi observada. DPSCs e BMMSCs apresentaram alta viabilidade celular quando encapsuladas em PL. Ambas as linhagens celulares tiveram êxito em se diferenciar em tecidos adiposo e ósseo. De acordo com o PKH26, DPSCs puderam aderir e proliferar no sistema PL/rhBMP4. DPSCs irradiadas encapsuladas tanto em PL como em PL/rhBMP4 formaram mais CFU-F que os controles não irradiados. Sob estresse nutricional, DPSCs semeadas no PL e irradiadas com 5 J/cm2 exibiram maior taxa de viabilidade celular em relação aos grupos não irradiados e irradiados com 3 J/cm2. Na presença de rhBMP4, os grupos irradiados tanto com 3 J/cm2 quanto com 5 J/cm2 apresentaram deposição mineral precoce quando comparados aos grupos não irradiados. Ainda, após 21 dias de diferenciação odonto/osteogênica, DPSCs irradiadas produziram maior quantidade de nódulos mineralizados. A irradiação com 5 J/cm2 levou ao aumento significativo da expressão de genes envolvidos na diferenciação odonto/osteogênica, como colágeno tipo I (COL1A1), osteocalcina (OCN), proteína da matriz dentinária 1 (DMP1), sialofosfoproteina dentinária (DSPP) e proteína heat shock 27 kDa (HSPB1). A associação entre rhBMP4 e FTL promove proliferação e diferenciação odonto/osteogênica de DPSCs acelerando e aumentando notavelmente a formação de tecido mineralizado, em especial quando a densidade de energia de 5 J/cm2 é aplicada. / This study evaluated the influence of laser phototherapy (LPT) on dental pulp stem cells (DPSCs) proliferation and differentiation upon encapsulation in an injectable and thermo-responsive cell carrier (PL; Pluronic® F-127, Sigma-Aldrich, MO, USA) loaded with human recombinant bone morphogenetic protein 4 (rhBMP4)(PL/rhBMP4 system). The biomaterial was characterized according to its swelling and dissolution profiles, release of rhBMP4 and morphological structure. DPSCs were isolated, characterized and encapsulated in PL to confirm their viability and multilineage differentiation potential (adipo and osteogenic) in comparison to bone marrow mesenchymal stem cells (BMMSCs). When encapsulated in the PL/rhBMP4 system, DPSCs were irradiated with two different energy densities using a continuous-wave indium-gallium-aluminum-phosphide (InGaAlP) diode laser [660 nm, 0.028 cm2, 20 mW, 0.71 W/cm2, 3 J/cm2 (4 s) or 5 J/cm2 (7 s)] in punctual and contact modes. The PKH26 (Red Fluorescent Cell Linker), the CFU-F (Coloning Forming Units - Fibroblastic), and the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] assays were used to assess differences in cell adhesion/proliferation, colony forming units formation ability, and cell viability of DPSCs (in this case under nutritional stress), respectively. Then, alizarin red and qRT-PCR analyzes were used to evaluate odonto/osteogenic differentiation. The biomaterial swelled and dissolved rapidly; dense tubular and reticular network morphology with well-interconnected pores was observed. DPSCs and BMMSCs presented high cell viability when encapsulated in PL. Both cell lineages successfully differentiated into bone or adipose tissues. According to PKH26, DPSCs were able to adhere and proliferate in the PL/rhBMP4 system. Irradiated DPSCs encapsulated in either PL or PL/rhBMP4 system formed more CFU-F than non-irradiated controls. Under nutritional stress, DPSCs encapsulated in the hydrogels with no rhBMP4 and irradiated at 5 J/cm2 exhibited higher cell viability than the other groups. In the presence of rhBMP4, the groups irradiated both at 3 and 5 J/cm2 energy densities displayed earlier mineral deposition than the non-irradiated groups. Moreover, after 21 days of odonto/osteogenic differentiation, irradiated DPSCs produced greater nodule formation than the control groups. At the energy density of 5 J/cm2, there were significant upregulation of genes involved in odonto/osteoblast differentiation, such as type I collagen (COL1A1), osteocalcin (OCN), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP) and heat shock protein 27 kDa (HSPB1). The association between rhBMP4 and LPT promotes cell proliferation and odonto/osteogenic differentiation of DPSCs accelerating and increasing the formation of mineralized tissue, in particular when the energy density of 5 J/cm2 is applied.