Objective - The majority of apoptotic cells in atherosclerotic lesions are macrophages. However, the pathogenic role of macrophage apoptosis in the development of atherosclerosis remains unclear. Elevated expression of Bax, one of the pivotal proapoptotic proteins of the Bcl-2 family, has been found in human atherosclerotic plaques. Activation of Bax also occurs in free cholesterol-loaded and oxysterol-treated mouse macrophages. In this study, we examined the effect of Bax deficiency in bone marrow-derived leukocytes on the development of atherosclerosis in low-density lipoprotein receptor-null (LDLR-/-) mice. Methods and Results - Fourteen 8-week-old male LDLR-/- mice were lethally irradiated and reconstituted with either wild-type (WT) C57BL6 or Bax-null (Bax-/-) bone marrow. Three weeks later, the mice were challenged with a Western diet for 10 weeks. No differences were found in the plasma cholesterol level between the WT and Bax-/- group. However, quantitation of cross sections from proximal aorta revealed a 49.2% increase (P=0.0259) in the mean lesion area of the Bax-/- group compared with the WT group. A 53% decrease in apoptotic macrophages in the Bax-/- group was found by TUNEL staining (P<0.05). Conclusions - The reduction of apoptotic activity in macrophages stimulates atherosclerosis in LDLR-/- mice, which is consistent with the hypothesis that macrophage apoptosis suppresses the development of atherosclerosis.

No description available.

Background-Previously we found that smooth muscle cell (SMC)-specific knockout of miR-17 similar to 92 attenuates hypoxia-induced pulmonary hypertension. However, the mechanism underlying miR-17 similar to 92-mediated pulmonary artery SMC (PASMC) proliferation remains unclear. We sought to investigate whether miR-17 similar to 92 regulates hypoxia-inducible factor (HIF) activity and PASMC proliferation via prolyl hydroxylases (PHDs). Methods and Results-We show that hypoxic sm-17 similar to 92(-/-) mice have decreased hematocrit, red blood cell counts, and hemoglobin contents. The sm-17 similar to 92 (-/-) mouse lungs express decreased mRNA levels of HIF targets and increased levels of PHD2. miR-17 similar to 92 inhibitors suppress hypoxia-induced levels of HIF1 alpha, VEGF, Glut1, HK2, and PDK1 but not HIF2 alpha in vitro in PASMC. Overexpression of miR-17 in PASMC represses PHD2 expression, whereas miR-17/20a inhibitors induce PHD2 expression. The 3'-UTR of PHD2 contains a functional miR-17/20a seed sequence. Silencing of PHD2 induces HIF1a and PCNA protein levels, whereas overexpression of PHD2 decreases HIF1 alpha and cell proliferation. SMC-specific knockout of PHD2 enhances hypoxia-induced vascular remodeling and exacerbates established pulmonary hypertension in mice. PHD2 activator R59949 reverses vessel remodeling in existing hypertensive mice. PHDs are dysregulated in PASMC isolated from pulmonary arterial hypertension patients. Conclusions-Our results suggest that PHD2 is a direct target of miR-17/20a and that miR-17 similar to 92 contributes to PASMC proliferation and polycythemia by suppression of PHD2 and induction of HIF1 alpha.

BACKGROUND: Cell migration is a vital process for growth and repair. In vitro migration assays, utilized to study cell migration, often rely on physical scraping of a cell monolayer to induce cell migration. The physical act of scrape injury results in numerous factors stimulating cell migration - some injury-related, some solely due to gap creation and loss of contact inhibition. Eliminating the effects of cell injury would be useful to examine the relative contribution of injury versus other mechanisms to cell migration. Cell exclusion assays can tease out the effects of injury and have become a new avenue for migration studies. Here, we developed two simple non-injury techniques for cell exclusion: 1) a Pyrex® cylinder - for outward migration of cells and 2) a polydimethylsiloxane (PDMS) insert - for inward migration of cells. Utilizing these assays smooth muscle cells (SMCs) and human umbilical vein endothelial cells (HUVECs) migratory behavior was studied on both polystyrene and gelatin-coated surfaces. RESULTS: Differences in migratory behavior could be detected for both smooth muscle cells (SMCs) and endothelial cells (ECs) when utilizing injury versus non-injury assays. SMCs migrated faster than HUVECs when stimulated by injury in the scrape wound assay, with rates of 1.26 % per hour and 1.59 % per hour on polystyrene and gelatin surfaces, respectively. The fastest overall migration took place with HUVECs on a gelatin-coated surface, with the in-growth assay, at a rate of 2.05 % per hour. The slowest migration occurred with the same conditions but on a polystyrene surface at a rate of 0.33 % per hour. CONCLUSION: For SMCs, injury is a dominating factor in migration when compared to the two cell exclusion assays, regardless of the surface tested: polystyrene or gelatin. In contrast, the migrating surface, namely gelatin, was a dominating factor for HUVEC migration, providing an increase in cell migration over the polystyrene surface. Overall, the cell exclusion assays - the in-growth and out-growth assays, provide a means to determine pure migratory behavior of cells in comparison to migration confounded by cell wounding and injury.

Surgical intervention can result in endothelial denudation, driving growth factor-stimulated vascular smooth muscle cell (VSMC) migration towards the intima, leading to luminal narrowing and restenosis. Clinically approved PGI₂ analogues, including beraprost, activate the cyclic adenosine monophosphate (cAMP) signaling pathway to inhibit VSMC migration in vitro. This pathway is a potential therapeutic target, however the downstream proteins involved in the inhibitory effects of cAMP on migration remain unknown. The aims of this study were to determine the signalling pathways involved in inhibiting VSMC migration through cAMP downstream mediators, protein kinase A (PKA) and the more recently characterised exchange protein activated by cAMP (Epac), and delineate the mechanisms involved. In human saphenous vein VSMCs, Epac activation using an Epac analogue inhibited VSMC migration. Therapeutic concentrations of beraprost (1 nM) also resulted in an inhibition of VSMC migration. The use of fluorescence resonance energy transfer (FRET) confirmed 1 nM beraprost activated Epac, but not PKA. Epac is a guanine nucleotide exchange factor (GEF) for Rap1 thus Rap1 siRNA was used to inhibit the Epac pathway. This blocked the inhibitory effects of beraprost on VSMC migration. Epac1 was localised to the leading edge of migrating VSMCs. Another G-protein, RhoA, was investigated since it is essential for cell migration and is involved in several processes including actin regulation. Epac signaling inhibited PDGF-induced RhoA activation and disassembled F-actin at the leading edge, where Epac1 was previously located. This indicates that beraprost activated the Epac pathway, which inhibited RhoA to decrease VSMC migration. The clinical relevance of this study has discovered the mechanisms of Epac's inhibitory action on VSMC migration and this pathway could be targeted therapeutically to reduce restenosis. In the future the potential use of beraprost on a drug eluting stent might be beneficial to prevent restenosis formation following surgical intervention.

Intimal hyperplasia (IH) is characteristic of a cell population increase within the innermost layer of the arterial wall. It is hypothesized that extracellular matrix vascular remodeling secondary vascular injury is dependent upon the Th17 subset of the CD4+ lymphocytes. Male C57BL/6J and FVB/NJ murine strains underwent complete left common carotid artery ligation for periods of 14 and 28 days. A therapeutic simvastatin model was carried out in the FVB/NJ strain and involved a daily subcutaneous injection regimen of 40 mg/kg/mouse beginning 72 hours prior to and daily following a 14 day carotid ligation period. Histological and RT-PCR analysis was carried out with harvested carotid artery samples. The FVB/NJ 14 day and 28 day histological stains of the left common carotid artery following ligation injury developed evident structured and disassembled intimal hyperplasia, respectively. A gene array demonstrated dramatic expression of immune and cytokine transcription markers particularly in the FVB/NJ strain at both ligation time points. IL-17 and IL-6 transcriptional gene expression was upregulated greater than 20-fold in the FVB/NJ 28 day injury model. IL-17 transcription was significantly expressed by a change of 50.06 ± 0.19 (p = 0.004) in this strain at 28 days versus the control. Lastly, the simvastatin treatment model was found to exacerbate the immune response to ligation injury. These results revealed that the immune system elicits a role in the vascular remodeling that potentiates intimal hyperplasia.

As fosfolipases A2 secretadas (sFLA2) de veneno de serpente apresentam homologia estrutural e funcional com as sFLA2s do GIIA de mamíferos, cujos níveis estão elevados em doenças inflamatórias, como a aterosclerose. Nesta doença, as células de músculo liso vascular (CMLVs) acumulam corpúsculos lipídicos (CLs) e se diferenciam em células espumosas. Porém, o papel das sFLA2s neste fenômeno não é conhecido. Neste estudo foram avaliados os efeitos das FLA2 MT-III, cataliticamente ativa, e da BthTx-II, sem atividade catalítica, em CMLVs, com ênfase na formação de CLs e a participação de fatores da homeostasia lipídica. Os resultados obtidos demonstraram que a MT-III e a BthTx-II induziram a formação de CMLVs espumosas. Para tanto, estas enzimas recrutaram diferentes fatores envolvidos na síntese e acúmulo de lipídios. Nesta condição, os CLs constituem um local de síntese de prostaglandinas. Ainda, a MT-III induziu a diferenciação de CMLVs para fenótipo e função de macrófagos. A atividade catalítica não é relevante para a formação de CLs induzida por FLA2s. / Bothrops snake venom secreted phospholipases A2 (sPLA2s) share structural and functional features with mammalian GIIA sPLA2s, which are highly expressed during inflammatory diseases, such as atherosclerosis. In this disease, vascular smooth muscle cells (VSMCs) are loaded with lipid droplets (LDs) differentiating into foam cells. However, the role of these enzymes in this process is still unknown. In this study the effects of snake venom PLA2s MT-III with catalytic activity and BthTx-II, devoid of catalytic activity in VSMCs, with focus on LDs formation and mechanisms involved were investigated. Results here obtained show that both MT-III and BthTx-II induce formation of foam VSMCs and recruit distinct factors of synthesis and storage of lipids in these cells. In this condition, LDs constitute sites for synthesis of prostaglandins. Moreover, MT-III showed the ability to modulate VSMCs functions, leading them to a phenotipic switch to macrophage-like cells. In addition, the catalytic activity is not relevant to sPLA2-induced LDs formation.

Every year, 400,000 coronary artery bypasses (CABG) are performed in the United States. However, one third of all patients who need a CABG cannot undergo the procedure because of the lack of suitable autologous blood vessels. Both synthetic and tissue engineered vascular grafts have been used clinically for vascular grafts or other surgical applications, but no small- diameter engineered vessels have yet been successfully used for CABG. The success of vascular tissue engineering is strongly dependent on being able to control tissue contractility and extracellular matrix (ECM) production to achieve balance between tissue strength and physiological function. Smooth muscle cells (SMCs), the main contributor of contractility in blood vessels, retain phenotypic plasticity, meaning they possess the ability to switch between a contractile and synthetic phenotype. In 2D culture, a number of biochemical and mechanical cues have been shown to promote the switch to a contractile phenotype in SMCs. However, achieving a stable contractile phenotype in 3D tissue has proven difficult. The work in this dissertation describes an investigation of how passive and dynamic environmental cues influence the smooth muscle phenotype. We studied the effects of substrate modulus in conjunction with changes in cell culture media composition on SMC phenotype in 2D and 3D cultures. Culturing SMCs in a low-serum culture medium resulted in an increase in SMC contractility in 2D cell culture but not in 3D cell-derived tissue. We found that, in SMCs cultured on soft substrates, the ability to modulate SMC phenotype in response to changes in media was diminished. Passively crosslinking the ECM of our cell-derived tissues with genipin resulted in modest increases in elastic modulus, though not enough to observe changes in SMC phenotype. Additionally, we investigated how dynamic cyclic mechanical stretch, in conjunction with cell culture medium, modified SMC contractility in cell and tissue cultures. SMCs increased contractile protein expression when exposed to dynamic stretch in 2D culture, even on soft substrates, which have previously been shown to inhibit phenotypic modulation. In 3D tissue rings, after mechanical stimulation, SMCs became more aligned, the tissue became tougher, and SMCs exhibited a measurable increase in contractile protein expression. In summary, we found that increasing substrate modulus, culturing in low serum cell culture medium, and imparting cyclic mechanical stretch can promote SMC differentiation and cellular alignment, and improve tissue mechanical properties. This information can be used to more accurately recapitulate vascular tissue for use in modeling or in the creation of tissue engineered blood vessels.

Orientador: Willian Fernando Zambuzzi / Resumo: As células endoteliais (ECs) e células musculares lisas (AoSMCs) são os principais componentes celulares do endotélio. As interações entre estes tipos celulares desempenham funções na homeostase e na estrutura vascular. Como uma interface entre o sangue e a parede do vaso, as ECs ocupam um local único diretamente exposto ao shear stress (SS), a força mecânica de atrito lateral produzido pelo fluxo de sangue na membrana apical da célula endotelial, que pode influenciar o comportamento de ambas ECs e AoSMCs. Geralmente, AoSMCs não sofrem diretamente às forcas de cisalhamento, no entanto, estas são diretamente expostas ao fluxo sanguíneo quando ocorre alguma injúria vascular, como por exemplo em algumas lesões ateroscleróticas ou por técnicas invasivas, como a angioplastia. As forças hemodinâmicas influenciam as propriedades funcionais do endotélio, porém estas não são profundamente compreendidas quanto aos mecanismos bioquímicos de respostas de células endoteliais e de musculatura lisa. Assim, a proposta desta dissertação foi estabelecer um modelo de cultivo in vitro que mimetize as forças tensionais de cisalhamento (shear stress), buscando compreender mecanismos celulares, bioquímicos e epigenéticos. Cultura de células primárias endoteliais e de musculatura lisa humanas foram obtidas da empresa LONZA e mantidas conforme recomendações do fabricante. Estas células foram mantidas rotineiramente em condições convencionais em incubadora de CO2. Para mimetizar o fluxo sanguíneo, esta... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Na+/Ca2+ exchanger (NCX) is a membrane protein which can mediate either Ca2+ entry (reverse mode) or exit (forward mode) in cells. As one of the major Ca2+ transport systems, NCX is postulated to play a critical role in the vascular smooth muscle cell. The aims of the present study are to firstly demonstrate the functional existence of NCX in vascular smooth muscle (including aorta and arteriole); to clarify the modulation of NCX; to explore the selectivity of NCX inhibitor KB-R7943; and lastly to investigate the role of NCX in the myogenic response. KB-R7943 has been widely used as a NCX inhibitor. The study investigated its pharmacological actions in rat aorta on a variety of Ca2+ dependent systems. Rat aortic rings were used. The constriction to low extracellular [Na+] is a functional response mediated by NCX operating in reverse mode. The data demonstrate that 10 µM KB-R7943 inhibited L-type Ca2+ channel, the capacitative Ca2+ entry and  adrenergic receptor pathway. Nevertheless, KB-R7943 can be used as a selective inhibitor of NCX at the lower concentration of 1 µM in rat aortic rings. The study investigated whether the endothelium could modulate NCX in rat aortic rings. Lowering extracellular [Na+] to 1.18 mM induced constriction in endothelium denuded rat aortic rings, but only a small constriction in endothelium intact rat aortic rings. In endothelium intact rat aortic rings, the guanylate cyclise inhibitor ODQ (1 µM) and the nitric oxide synthase inhibitor L-NAME (50 µM) greatly amplified the vasoconstriction to lowering extracellular [Na+], but had no effect when the endothelium was removed. The adenylate cyclise inhibitor SQ 22536 (100 µM) and the cyclooxygenase inhibitor indomethacin (10 M) showed no significant effect on the low-Na+ induced vasoconstriction in either endothelium denuded or intact aortic rings. The results suggest that endothelium modulated the NCX operation via the nitric oxide/guanylate cyclase, not the adenylate cyclase system; further prostanoids including prostacyclin was not involved. The interaction between nitric oxide and NCX was furt her explored using the nitric oxide donor sodium nitroprusside. Endothelium denuded rat aortic rings were preconstricted to the same extent with either low Na+ (1.18 mM), or the thromboxane A2 agonist U46619 (0.1 µM) or high K+ (80 mM). The vasorelaxation of SNP (30 nM) in low Na+ constriction was significantly larger compared to other agents. This indicates that NO has a special antagonism of low Na+ constriction and a hypothesis is proposed involving Na+/K+ ATPase. The investigation of NCX is mainly conducted in large vessels; much less evidence is available for small resistance vessels. The study investigated the role of NCX on myogenic response in pressurized cremaster muscle arterioles. Reducing extracellular [Na+] resulted in graded vasoconstriction which was inhibited by NCX inhibitor SEA0400 (1 µM). Myogenic vasoconstriction and the concomitant rise in internal [Ca2+] were induced by a transmural pressure increase from 70 to 120 mmHg which was prevented by NCX inhibitor: SEA0400 (1 µM). In conclusion, the present study suggests that NCX contributes to the myogenic response in cremaster arteriole.