Peripheral artery disease (PAD) and the consecutive build-up of an atherosclerotic plaque restricting blood flow to the lower limbs lead to critical limb ischaemia, one of the most common circulation problems in the world. Although a small number of interventions (such as surgery or revascularization treatments) are available, patients with this condition are often too ill for these procedures, giving a poor prognosis for the disease. Several strategies to promote neovascularization using different stem cell populations with angiogenic potential have been proposed as plausible therapies. Perivascular cells (PCs), key structural components of the wall of small and large blood vessels have numerous advantages over other cell types since they are highly abundant, easy to obtain from the stromal vascular fraction (SVF) of human adipose tissue (an ethically approved source) and have mesenchymal and angiogenic properties. The work described in this thesis addressed the hypothesis that PCs isolated from human white adipose tissue would promote the recovery of blood flow in an ischaemic hindlimb by increasing blood vessel number and blood perfusion to the foot. To investigate whether PCs from human white adipose tissue could rapidly increase neovascularization and, therefore, be used as a possible therapeutic treatment for PAD and critical limb ischaemia, the initial aim was to validate, characterise and demonstrate the properties of the murine equivalent of these cells, in order to establish a direct link between the injected cells and the ones natively found in the mouse. This was then followed by the use of murine models of angiogenesis to determine whether transplanted human PCs stimulate angiogenesis in vivo. Initial studies using immunohistochemistry, fluorescence-activated cell sorting (FACS) and in vitro mesodermal differentiation demonstrated that perivascular cells (namely pericytes and adventitial cells) are present in multiple mouse organs, can be sorted to purity, and have mesenchymal stem cell (MSC) properties. These cells had similar characteristics to their human counterparts, thus validating the mouse as a suitable model for determining whether transplanted human PCs could stimulate angiogenesis. Using in vitro and two in vivo (sponge implantation and hindlimb ischaemia) models, it was shown that human PCs have angiogenic properties being capable of tube formation and interaction with endothelial cells, as well as promoting angiogenesis within sponges. Contrary to expectations, PCs did not increase blood perfusion to the mouse ischaemic hindlimb, despite increasing microcirculation within the skeletal muscle and myofibre regeneration. This work showed that PCs obtained from human adipose tissue have important therapeutic implications in promoting angiogenesis and skeletal muscle regeneration but failed to increase arteriogenesis which is the key mechanism allowing the restoration of blood perfusion.

Background - Healthy perivascular adipose tissue (PVAT) exerts an anticontractile effect in response to various vasoconstrictor agonists and this is lost in obesity. A recent study reported that bariatric surgery reverses the damaging effects of obesity on PVAT function. However, PVAT function has not been previously characterised following weight loss induced by caloric restriction, which is often the first line treatment for obesity. This study investigated the mechanisms by which PVAT modulates vascular tone in health and how these are altered in animal models of obesity and diet-induced weight loss. Methods - Male Sprague Dawley rats were fed a 45% fat diet ad libitum for 16 weeks to induce obesity, they were then randomly allocated into two groups; obese rats maintained on the 45% fat diet and weight loss rats that were subjected to 50% caloric restriction for a further four weeks. A weight maintenance group was also established where a cohort of weight loss rats were provided with 70 kcal/day for a further four weeks at the end of the caloric restriction period. A control group was also provided with a 10% fat diet during the 20 week period. The effect of PVAT on the contractility of isolated mesenteric arteries in response to norepinephrine in the presence of pharmacological tools was investigated by wire myography Changes in the PVAT environment were assessed also using western blotting, immunohistochemistry and assays of secretion. Results - PVAT from healthy control rats elicited an anticontractile effect in response to norepinephrine through release of relaxing factors, one of which was nitric oxide. The anticontractile effect was abolished in diet-induced obesity through a mechanism involving local inflammation and reduced nitric oxide bioavailability within PVAT. In addition, reduced KCNQ expression and enhanced COX activation contributed to the loss of anticontractility. Four-week caloric restriction did not restore PVAT anticontractile capacity. However, sustained weight loss led to restoration of PVAT anticontractile function associated with restoration of adipocyte size, reduced inflammation and increased nitric oxide synthase expression. Sustained weight loss and the restoration of PVAT function was associated with reversal of obesity-induced hypertension and normalisation of adipokine levels, including leptin and insulin. Conclusions - Sustained weight loss reverses obesity-induced PVAT damage through a mechanism involving reduced inflammation and reduced nitric oxide bioavailability. Reduced KCNQ activity and enhanced COX activation also contribute to the obesity-associated loss of PVAT anticontractile effect. These data reveal the beneficial effects of weight loss induced by dietary restriction on PVAT function and identify several potential targets for the treatment of PVAT dysfunction associated with obesity.

Pericytes represent a population of potential mesenchymal stem cells (MSC) that reside within a perivascular niche until they are required in normal homeostasis and the response to injury. Their mesenchymal capacities for multipotent differentiation, immune modulation and release of trophic factors hold great promise for regenerative therapies. Pathological expression of these potentials has been described in disease states, while acute or chronic inflammation following injury can lead to the production of signalling molecules that ultimately drive these progenitors to a fibrotic fate. The aim of this work was to explore how fate decisions of pericytes are regulated by their niche (in the setting of osteogenesis), and in the response to acute and chronic injury (in the setting of fibrosis). It was hypothesized that interactions between pericytes and endothelial cells (EC) within their perivascular niche are responsible for regulating mesenchymal differentiation. The osteogenic, adipogenic and chondrogenic potential of pericytes following isolation from multiple human organs was confirmed. The interactions between pericytes and EC in 2D and 3D coculture and the production of basement membrane proteins in these settings were confirmed. The osteogenic differentiation of pericytes was accelerated by EC but no influence of EC on the adipogenic and chondrogenic differentiation of pericytes was detected. Furthermore, data indicated that the influence on pericyte osteogenic potential by EC may occur through wnt signaling. The activation of TGFβ (transforming growth factor beta) through αv integrins has been suggested as central mediator of fibrosis in multiple organs. We hypothesized that selective αv integrin deletions in PDGFRβ (platelet derived growth factor receptor beta) expressing pericytes identifies a targetable pathway regulating fibrosis in skeletal muscle. We report that PDGFRβ-Cre inactivates genes in murine skeletal muscle pericytes with high efficiency. Deletion of the αv integrin subunit in pericytes protected mice from chemical injury induced skeletal muscle fibrosis. Pharmacological blockade of αv integrins by a novel small molecule (CWHM 12) attenuated muscle fibrosis, even when administered after fibrosis was established.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第17816号 / 医博第3814号 / 新制||医||999(附属図書館) / 30631 / 京都大学大学院医学研究科医学専攻 / (主査)教授 小柳 義夫, 教授 一山 智, 教授 武藤 学 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

O consumo crônico de etanol é um importante fator de risco no desenvolvimento de doenças cardiovasculares induzindo elevação da pressão arterial, inflamação, disfunção vascular e aumento do estresse oxidativo em vários tecidos. Além disso, o consumo crônico de etanol induz aumento dos níveis de Fator de necrose tumoral-? (TNF-?). O tecido adiposo perivascular (Perivascular adipose tissue - PVAT) é reconhecido como uma importante fonte de adipocinas e citocinas pró-inflamatórias e esse tecido está envolvido na fisiopatologia de diferentes doenças cardiovasculares. A hipótese deste trabalho é a de que o consumo crônico de etanol estimule a produção de TNF-? no tecido vascular, que por sua vez, induzirá um aumento na produção de espécies reativas de oxigênio (ERO), redução da biodisponibilidade do óxido nítrico (NO), inflamação vascular, prejuízo na função do PVAT, alteração da reatividade vascular e aumento da pressão arterial. Portanto, o objetivo deste estudo foi investigar a participação do TNF-? nas disfunções vasculares induzidas pelo consumo de etanol e avaliar o papel do PVAT em tais danos. O trabalho mostra que o consumo crônico de etanol por 12 semanas induziu aumento da pressão arterial sistólica (PAS) em camundongos machos C57BL/6 (Wild Type -WT) e este aumento foi menor nos animais nocautes para os receptores R1 do TNF-? (TNFR1-/-). O tratamento com etanol não alterou o relaxamento vascular induzido por acetilcolina e nitroprussiato de sódio (NPS). O consumo de etanol induziu aumento da geração de ânion superóxido (O2-), aumento das espécies reativas ao ácido tiobarbitúrico (TBARS) e redução dos níveis de peróxido de hidrogênio (H2O2) em aortas com e sem PVAT (PVAT+ e PVAT-, respectivamente) de animais WT, mas não em camundongos TNFR1-/-. Houve aumento da atividade da catalase (CAT) e da superóxido dismutase (SOD) em aorta PVAT- e PVAT+ e redução dos níveis plasmáticos da glutationa reduzida (GSH) em animais WT, mas não em TNFR1-/-, após o consumo crônico de etanol. Não houve alteração na atividade da glutationa peroxidase (GPx) na aorta dos grupos estudados após o consumo de etanol. Observou-se redução dos níveis de nitrato/nitrito (NOx) em aortas de animais WT após o consumo de etanol, porém não em camundongos TNFR1-/-. O consumo de etanol gerou aumento tecidual das citocinas TNF-? e IL-6 e aumento da atividade da mieloperoxidase (MPO), evidenciando uma exacerbada inflamação vascular e indicando migração de neutrófilos para o tecido aórtico. Tais alterações não foram observadas em animais TNFR1-/-. Os resultados mostram pela primeira vez a participação do TNF-? no aumento da pressão arterial, no aumento do estresse oxidativo e nos prejuízos vasculares induzidos pelo consumo crônico de etanol. O tecido adiposo perivascular não apresentou qualquer efeito benéfico nas alterações supracitadas. / Chronic ethanol consumption is an important risk factor in the development of cardiovascular diseases, inducing increased blood pressure, inflammation, vascular dysfunction and enhanced oxidative stress in several tissues. Furthermore, chronic ethanol consumption induces the production of Tumor necrosis factor-? (TNF-?). Perivascular adipose tissue (PVAT) is known as an important source of adipokines and proinflammatory cytokines. This tissue is involved in the pathophysiology of different cardiovascular diseases. The hypothesis of this work is that the chronic ethanol consumption stimulates the production of TNF-?, which in turn, will induce an increase in reactive oxygen species (ROS) generation, nitric oxide (NO) reduction, vascular inflammation, impairment of PVAT function, alterations of vascular reactivity and increased blood pressure. Therefore, the aim of this study was to investigate the role of TNF-? in chronic ethanol consumption-induced vascular dysfunctions and to evaluate the role of PVAT in such damages. This study demonstrated that chronic ethanol consumption for 12 weeks induced an increase in systolic blood pressure (SBP) in C57BL/6 mice (wild type-WT) and this increase was blunted in TNF-? receptor 1 knockout mice (TNFR1-/-). There was no change in vascular relaxation induced by acetylcholine and sodium nitroprusside (NPS). Ethanol consumption increased the superoxide anion (O2-) generation, thiobarbituric acid reactive species (TBARS) and reduction of hydrogen peroxide (H2O2) levels in aorta without and with PVAT (PVAT- and PVAT+, respectively) from WT animals, but not from TNFR1-/- mice. There was an increase in catalase (CAT) and superoxide dismutase (SOD) activities in aorta PVAT- and PVAT+, decrease on plasma reduced-glutathione (GSH) levels from ethanol-treated WT but not in TNFR1-/-. Ethanol consumption did not change glutathione peroxidase (GPx) activity in any group. Nitrate/nitrite (NOx) aortic levels were decreased in WT animals, but not in TNFR1-/- after chronic ethanol consumption. Ethanol consumption increased TNF-?, IL-6 cytokines and myeloperoxidase activity (MPO) which suggest a strong vascular inflammation and migration of neutrophils into the aortic tissue. Such changes were not observed in TNFR1-/- mice. The results show for the first time the participation of TNF-? in the increase of blood pressure, increase of oxidative stress and vascular dysfunction induced by the chronic ethanol consumption. The perivascular adipose tissue had no beneficial effect on these changes.

A obesidade é caracterizada por inflamação no tecido adiposo. A angiotensina II via receptor AT1 induz estresse oxidativo e inflamação vascular. O tecido adiposo perivascular (PVAT) circunda os vasos sanguíneos, possui ação parácrina na parede vascular e é classificado como marrom e branco, sendo que este último é mais propenso à inflamação. No presente estudo, avaliamos os compenentes do sistema renina-angiotensina no PVAT marrom (da aorta torácica - AT) e branco (do leito mesentérico LM) de camundongos controles (CT) e obesos (OB). O RNAm para angiotensinogênio tanto no PVAT marrom quanto no PVAT branco foi reduzido em camundongos OB. A atividade enzimática da ECA 1 foi reduzida no PVAT-marrom do grupo OB em comparação ao grupo CT e foi semelhante no PVAT branco dos dois grupos. No PVAT marrom, o conteúdo proteico do receptor AT1 (AT1R) foi semelhante nos grupos CT e OB e o receptor AT2 (AT2R) não foi detectado no PVAT dos dois grupos. No PVAT branco, o conteúdo proteico dos receptores AT1 e AT2 foi aumentado no grupo OB. A expressão de RNAm do receptor CCR2 de MCP-1 (proteína quimiotáxica de monócitos) no PVAT marrom foi semelhante nos dois grupos, mas foi aumentada no PVAT branco do grupo OB. O conteúdo proteico da MCP1 foi aumentado no PVAT branco do grupo OB em relação ao grupo CT. O tratamento de camundongos OB com antagonista de receptor AT1 de angiotensina II (losartana; 10 mg/kg) por 30 dias, não reverteu os parâmetros que caracterizam a obesidade e não diminuiu a expressão de MCP-1 no PVAT branco. Em relação aos componentes do sistema renina-angiotensina, o PVAT marrom e o PVAT branco respondem de maneira diferente à obesidade induzida por dieta hiperlipídica. / Obesity is characterized by inflammation in adipose tissue. Angiotensin II/AT1 receptor pathway induces oxidative stress and vascular inflammation. Perivascular adipose tissue surrounds blood vessels and has paracrine actions in the vascular wall. It is classified as brown and white being the last one more willing to inflammation. In the present study, we evaluated the components of renin angiotensin-system in brown (thoracic aortic - TA) and white (mesenteric bed MB) PVAT in lean (CT) as well as in obese mice (OB). RNAm content of angiotensinogen in brown and white PVAT was reduced in obese mice. ACE1 activity was lower in brown PVAT of OB group in comparison to CT group and it was similar to white PVAT in both groups. In brown PVAT, protein content of AT1 receptor (AT1R) was similar to CT and OB groups and AT2 receptor (AT2R) was not detected in both groups. In white PVAT, protein contents of AT1R and AT2R were increased in obese group. RNAm expression of CCR2 receptor from MCP1 (monocytes chemoctatic protein 1) in brown PVAT was similar to both groups; however, it was increased in white PVAT in OB group. Protein content of MCP1 was increased in white PVAT in OB group. Treatment in obese mice with angiotensin II receptor blocker (ARB) (losartan; 10 mg/kg) for 30 days did not reverse the parameters which characterize obesity and did not diminish MCP-1 expression in white PVAT. In relation to the components of renin angiotensinsystem, brown and white PVAT responded differently to high-fat diet obesity.

O consumo crônico de etanol é um importante fator de risco no desenvolvimento de doenças cardiovasculares induzindo elevação da pressão arterial, inflamação, disfunção vascular e aumento do estresse oxidativo em vários tecidos. Além disso, o consumo crônico de etanol induz aumento dos níveis de Fator de necrose tumoral-? (TNF-?). O tecido adiposo perivascular (Perivascular adipose tissue - PVAT) é reconhecido como uma importante fonte de adipocinas e citocinas pró-inflamatórias e esse tecido está envolvido na fisiopatologia de diferentes doenças cardiovasculares. A hipótese deste trabalho é a de que o consumo crônico de etanol estimule a produção de TNF-? no tecido vascular, que por sua vez, induzirá um aumento na produção de espécies reativas de oxigênio (ERO), redução da biodisponibilidade do óxido nítrico (NO), inflamação vascular, prejuízo na função do PVAT, alteração da reatividade vascular e aumento da pressão arterial. Portanto, o objetivo deste estudo foi investigar a participação do TNF-? nas disfunções vasculares induzidas pelo consumo de etanol e avaliar o papel do PVAT em tais danos. O trabalho mostra que o consumo crônico de etanol por 12 semanas induziu aumento da pressão arterial sistólica (PAS) em camundongos machos C57BL/6 (Wild Type -WT) e este aumento foi menor nos animais nocautes para os receptores R1 do TNF-? (TNFR1-/-). O tratamento com etanol não alterou o relaxamento vascular induzido por acetilcolina e nitroprussiato de sódio (NPS). O consumo de etanol induziu aumento da geração de ânion superóxido (O2-), aumento das espécies reativas ao ácido tiobarbitúrico (TBARS) e redução dos níveis de peróxido de hidrogênio (H2O2) em aortas com e sem PVAT (PVAT+ e PVAT-, respectivamente) de animais WT, mas não em camundongos TNFR1-/-. Houve aumento da atividade da catalase (CAT) e da superóxido dismutase (SOD) em aorta PVAT- e PVAT+ e redução dos níveis plasmáticos da glutationa reduzida (GSH) em animais WT, mas não em TNFR1-/-, após o consumo crônico de etanol. Não houve alteração na atividade da glutationa peroxidase (GPx) na aorta dos grupos estudados após o consumo de etanol. Observou-se redução dos níveis de nitrato/nitrito (NOx) em aortas de animais WT após o consumo de etanol, porém não em camundongos TNFR1-/-. O consumo de etanol gerou aumento tecidual das citocinas TNF-? e IL-6 e aumento da atividade da mieloperoxidase (MPO), evidenciando uma exacerbada inflamação vascular e indicando migração de neutrófilos para o tecido aórtico. Tais alterações não foram observadas em animais TNFR1-/-. Os resultados mostram pela primeira vez a participação do TNF-? no aumento da pressão arterial, no aumento do estresse oxidativo e nos prejuízos vasculares induzidos pelo consumo crônico de etanol. O tecido adiposo perivascular não apresentou qualquer efeito benéfico nas alterações supracitadas. / Chronic ethanol consumption is an important risk factor in the development of cardiovascular diseases, inducing increased blood pressure, inflammation, vascular dysfunction and enhanced oxidative stress in several tissues. Furthermore, chronic ethanol consumption induces the production of Tumor necrosis factor-? (TNF-?). Perivascular adipose tissue (PVAT) is known as an important source of adipokines and proinflammatory cytokines. This tissue is involved in the pathophysiology of different cardiovascular diseases. The hypothesis of this work is that the chronic ethanol consumption stimulates the production of TNF-?, which in turn, will induce an increase in reactive oxygen species (ROS) generation, nitric oxide (NO) reduction, vascular inflammation, impairment of PVAT function, alterations of vascular reactivity and increased blood pressure. Therefore, the aim of this study was to investigate the role of TNF-? in chronic ethanol consumption-induced vascular dysfunctions and to evaluate the role of PVAT in such damages. This study demonstrated that chronic ethanol consumption for 12 weeks induced an increase in systolic blood pressure (SBP) in C57BL/6 mice (wild type-WT) and this increase was blunted in TNF-? receptor 1 knockout mice (TNFR1-/-). There was no change in vascular relaxation induced by acetylcholine and sodium nitroprusside (NPS). Ethanol consumption increased the superoxide anion (O2-) generation, thiobarbituric acid reactive species (TBARS) and reduction of hydrogen peroxide (H2O2) levels in aorta without and with PVAT (PVAT- and PVAT+, respectively) from WT animals, but not from TNFR1-/- mice. There was an increase in catalase (CAT) and superoxide dismutase (SOD) activities in aorta PVAT- and PVAT+, decrease on plasma reduced-glutathione (GSH) levels from ethanol-treated WT but not in TNFR1-/-. Ethanol consumption did not change glutathione peroxidase (GPx) activity in any group. Nitrate/nitrite (NOx) aortic levels were decreased in WT animals, but not in TNFR1-/- after chronic ethanol consumption. Ethanol consumption increased TNF-?, IL-6 cytokines and myeloperoxidase activity (MPO) which suggest a strong vascular inflammation and migration of neutrophils into the aortic tissue. Such changes were not observed in TNFR1-/- mice. The results show for the first time the participation of TNF-? in the increase of blood pressure, increase of oxidative stress and vascular dysfunction induced by the chronic ethanol consumption. The perivascular adipose tissue had no beneficial effect on these changes.

A sepse é uma disfunção orgânica causada por uma resposta do hospedeiro à infecção desregulada, com risco de morte. Quando o tratamento da sepse não é efetivo, o quadro pode progredir para hipotensão severa. O tecido adiposo perivascular (perivascular adipose tissue - PVAT) é reconhecido como um elemento regulador na biologia vascular, com implicações na fisiopatologia de doenças cardiovasculares. No entanto, em relação à sepse, pouco se sabe acerca dos efeitos desta sobre a ação modulatória que o PVAT exerce no tônus vascular. Dessa maneira a hipótese do presente trabalho foi a de que a sepse poderia aumentar o efeito anti-contrátil do PVAT. Portanto, o objetivo do trabalho foi avaliar o efeito da sepse experimental na ação modulatória que o PVAT exerce sobre tônus vascular e os possíveis mecanismos envolvidos nessa resposta. Para isso foram utilizados ratos Wistar Hannover com idade entre 60 e 70 dias (270 a 300g). Os ratos foram distribuídos aleatoriamente em 2 grupos: 1) Grupo Sham: foi realizada apenas uma laparotomia sem os procedimentos de ligadura e punção do ceco; 2) Grupo CLP (Cecal Ligation and Puncture) : a sepse letal foi induzida utilizando o modelo CLP no qual foi realizada uma laparotomia para exposição do ceco, onde foi feito uma ligadura e 20 punções intermediárias entre a ligadura e a ponta do ceco com agulha de calibre 18 gauge (G). Os animais foram anestesiados com quetamina/xilasina (80/10 mg/kg, i.p.) e mortos 6 h após a indução da sepse. A aorta torácica foi coletada para realização das análises bioquímicas e funcionais. A sepse letal reduziu a taxa de sobrevida, a pressão arterial média (PAM), não alterou os níveis de leucócitos e neutrófilos, mas aumentou a contagem de bactérias no sangue e no lavado peritoneal, aumentou os níveis plasmáticos de nitrato, uréia e CK-MB. A sepse diminuiu a contração induzida pela fenilefrina e serotonina nas aortas PVAT(-)/Endo(+) ou Endo (-) do grupo CLP, quando comparada ao Sham. Porém nas artérias PVAT(+)/Endo(+) ou Endo (-), o CLP induziu redução mais pronunciada da contração induzida tanto pela fenilefrina, quanto pela serotonina. O aumento do efeito anti-contrátil do PVAT na condição séptica não foi encontrado nas artérias após a incubação com L-NAME, 7-nitroindazol, 1400W, A779, carboxy-PTIO, ODQ, apamina, RO11384552 e indometacina. Tiron, catalase, 4-aminopiridina, glibenclamida e caribdotoxina não alteraram a contração induzida pela fenilefrina no grupo CLP. A sepse aumentou a concentração de H2O2 na aorta, mas não afetou a concentração no PVAT. Aumento dos níveis de ânion superóxido (O2-) e prostaglandina (PG)I2 foram detectados tanto na aorta, quanto no PVAT do grupo CLP. A sepse não alterou os níveis de PGE2 ou angiotensina (1-7) na aorta ou PVAT. Portanto, a sepse letal induzida por CLP aumenta a ação anti-contrátil do PVAT por um mecanismo que envolve a produção de NO pelas enzimas iNOS e nNOS, a participação de canais para KCa de baixa condutância e ativação da enzima guanilato ciclase solúvel. Além disso, sugere-se o envolvimento da PGI2 e angiotensina (1-7) na hiporresponsividade vascular mediada pelo PVAT durante a sepse / Sepsis is an organic dysfunction caused by an unregulated host response to lifethreatening infection. When treatment of sepsis is ineffective, the condition may progress to severe hypotension that is drug-irresponsive. Perivascular adipose tissue (PVAT) is recognized as a regulatory element in vascular biology that is implicated in the pathophysiology of cardiovascular diseases. However, little is known about the effects of sepsis in the modulatory action of PVAT. Thus, the hypothesis of the present study was that sepsis could increase the anti-contractile effect of PVAT. Therefore, the objective of the study was to evaluate the effect of experimental (lethal) sepsis in the modulatory action that PVAT exerts on vascular tone and the possible mechanisms underlying this response. With this purpose, male Wistar Hannover rats (250-300g) were divided in 2 groups: 1) Sham: the cecum was exteriorized without ligation and puncture; 2) CLP: lethal sepsis was induced using the cecal ligation and puncture (CLP) model. The thoracic aorta was isolated 6 h after sepsis for functional and biochemical assays. Lethal sepsis reduced survival rate, mean arterial pressure (MAP), did not alter leukocytes and neutrophils migration, but increased bacterial count in the blood and peritoneal cavity, increased plasma levels of nitrate, urea and CK-MB. We found that in aortas PVAT(-)/Endo(+) or Endo(-), sepsis decreased the contraction induced by phenylephrine or serotonin, when compared to sham. In PVAT(+)/Endo(+) or Endo (-) arteries sepsis induced a more pronounced reduction of phenylephrine-induced contraction. Sepsis-induced increase of anti-contractile action of PVAT was not found after incubation of arteries with L-NAME, 7-nitroindazole, 1400W, A779, carboxyPTIO , ODQ, apamin, indomethacin and RO1138452. Tiron, catalase, charybdotoxin, 4- aminopyridine and glibenclamide did not alter phenylephrine-induced contraction in the CLP group. Sepsis increased H2O2 concentration in the aorta, but did not affect H2O2 concentration in PVAT. Increased levels of superoxide anion (O2-) and prostaglandin (PG) I2 were detected in both aorta and PVAT. Sepsis did not alter the levels of PGE2 or angiotensin (1-7) in the aorta or PVAT. Conclusion: Lethal sepsis increases the anticontractile action of PVAT by a mechanism that involves the production of nitric oxide (NO) by iNOS and nNOS, the participation of calcium-dependent K+ channel of low conductance and activation of the enzyme soluble guanylate cyclase. Angiotensin (1-7) and PGI2 also contribute to the increased anti-contractile effect displayed by PVAT during sepsis. Financial Support: CAPES

Neural crest stem cells (NCSCs) are embryonic multipotent cells that give rise to a wide range of cell types that include those forming the peripheral neural cells and the mesodermal cells of the face including the facial bones. In neonatal and adult skin, skin-derived precursor cells (SKPs) are multipotent dermal precursors that share similarities with NCSCs and can differentiate into peripheral neural and mesodermal cells, such as adipocytes. Based on the similarities between SKPs and NCSCs, I asked, in this thesis, whether rodent or human SKPs can differentiate into skeletal mesodermal cell types by determining their ability to differentiate into osteoblasts and chondrocytes. In culture, rodent and human SKPs differentiated into alkaline phosphatase-, osteopontin- and type-I collagen-positive osteoblasts that produced mineral deposits and into type-II collagen expressing chondrocytes. Clonal analysis showed that SKPs are multipotent for the osteogenic and chondrogenic lineages. To ask whether SKPs can generate these cells in vivo, genetically-tagged naïve rat SKPs were transplanted into a tibia bone fracture model. Six weeks post-transplantation, SKP-derived osteoblasts and osteocytes were present in the newly formed bone, showing their osteogenic differentiation in vivo. At three weeks post-transplantation, some of the injected cells differentiated into hypertrophic chondrocytes in the callus and others into perivascular cells in areas just outside the callus. To test whether it is the local environment that dictates the phenotype of transplanted SKPs, GFP-tagged undifferentiated rat SKPs were injected into the hypodermis of the skin, an adipogenic environment. Four weeks post-transplantation, SKPs differentiated into adipocytes, but not in inappropriate cell types. These results further the known differentiation potential of SKPs, show that local environment of a bone fracture or the hypodermis of the skin is sufficient to induce the differentiation of undifferentiated SKPs into appropriate cell types and suggest the use of SKPs as source of mesodermal precursor cells for cell therapy.

Neural crest stem cells (NCSCs) are embryonic multipotent cells that give rise to a wide range of cell types that include those forming the peripheral neural cells and the mesodermal cells of the face including the facial bones. In neonatal and adult skin, skin-derived precursor cells (SKPs) are multipotent dermal precursors that share similarities with NCSCs and can differentiate into peripheral neural and mesodermal cells, such as adipocytes. Based on the similarities between SKPs and NCSCs, I asked, in this thesis, whether rodent or human SKPs can differentiate into skeletal mesodermal cell types by determining their ability to differentiate into osteoblasts and chondrocytes. In culture, rodent and human SKPs differentiated into alkaline phosphatase-, osteopontin- and type-I collagen-positive osteoblasts that produced mineral deposits and into type-II collagen expressing chondrocytes. Clonal analysis showed that SKPs are multipotent for the osteogenic and chondrogenic lineages. To ask whether SKPs can generate these cells in vivo, genetically-tagged naïve rat SKPs were transplanted into a tibia bone fracture model. Six weeks post-transplantation, SKP-derived osteoblasts and osteocytes were present in the newly formed bone, showing their osteogenic differentiation in vivo. At three weeks post-transplantation, some of the injected cells differentiated into hypertrophic chondrocytes in the callus and others into perivascular cells in areas just outside the callus. To test whether it is the local environment that dictates the phenotype of transplanted SKPs, GFP-tagged undifferentiated rat SKPs were injected into the hypodermis of the skin, an adipogenic environment. Four weeks post-transplantation, SKPs differentiated into adipocytes, but not in inappropriate cell types. These results further the known differentiation potential of SKPs, show that local environment of a bone fracture or the hypodermis of the skin is sufficient to induce the differentiation of undifferentiated SKPs into appropriate cell types and suggest the use of SKPs as source of mesodermal precursor cells for cell therapy.