RESUMEN

Abdominal aortic aneurysm (AAA) is a life-threatening vascular disease for which surgical or endovascular repair are the only currently available therapeutic strategies. The development of AAA involves the breakdown of elastic fibers (elastolysis), infiltration of inflammatory cells, and apoptosis of smooth muscle cells (SMCs). However, the specific regulators governing these responses remain unknown. We previously demonstrated that Cysteine and glycine-rich protein 3 (Crp3) sensitizes SMCs to apoptosis induced by stretching. Building upon this finding, we aimed to investigate the influence of Crp3 on elastolysis and apoptosis during AAA development. Using the elastase-CaCl2 rat model, we observed an increase in Crp3 expression, aortic diameter, and a reduction in wall thickness in wild type rats. In contrast, Crp3-/- rats exhibited a decreased incidence of AAA, with minimal or no changes in aortic diameter and thickness. Histopathological analysis revealed the absence of SMC apoptosis and degradation of elastic fibers in Crp3-/- rats, accompanied by reduced inflammation and diminished proteolytic capacity in Crp3-/- SMCs and bone marrow-derived macrophages. Collectively, our findings provide evidence that Crp3 plays a crucial role in AAA development by modulating elastolysis, inflammation, and SMC apoptosis. These results underscore the potential significance of Crp3 in the context of AAA progression and offer new insights into therapeutic targets for this disease.

RESUMEN

Vascular aging is accompanied by the fragmentation of elastic fibers and collagen deposition, leading to reduced distensibility and increased vascular stiffness. A rigid artery facilitates elastin to degradation by MMPs, exposing vascular cells to greater mechanical stress and triggering signaling mechanisms that only exacerbate aging, creating a self-sustaining inflammatory environment that also promotes vascular calcification. In this review, we highlight the role of crosstalk between smooth muscle cells and the vascular extracellular matrix (ECM) and how aging promotes smooth muscle cell phenotypes that ultimately lead to mechanical impairment of aging arteries. Understanding the underlying mechanisms and the role of associated changes in ECM during aging may contribute to new approaches to prevent or delay arterial aging and the onset of cardiovascular diseases.

Asunto(s)

Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiología , Envejecimiento/fisiología , Animales , Matriz Extracelular/metabolismo , Humanos , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/fisiología

RESUMEN

The interleukin-1 family is associated with innate immunity and inflammation. The latter has been linked to the genesis of cardiovascular diseases. We, therefore, investigated whether interleukin-1 beta (IL-1ß) is activated during arterialization of vein grafts. First, we examined the activation of IL-1ß using the rat arterialized jugular vein serially sampled for up to 90 days. IL-1ß expression increased 18 times on day 1 in the arterialized rat jugular vein and remained five times above nonarterialized vein levels for up to 90 days. Similarly, IL-1ß expression increased early (1-5 days) in human vein graft autopsy samples compared with late phases (1-4 years). Activation was also detected in ex vivo arterialized human saphenous veins. Upon stratification of the results, we uncovered a T allele promoter attenuating effect in IL-1ß activation in response to hemodynamic stress. Altogether, the results show that IL-1ß is activated during arterialization of vein grafts in rats and humans, and this response is modulated by -511C/T IL-1ß gene polymorphism. It is tempting to speculate that the activation of IL-1ß, and consequently local inflammation, modulates early vascular remodeling and that the gene polymorphism may be useful in predicting outcomes or assisting in interventions.

RESUMEN

The global expression profile of the arterialized rat jugular vein was established to identify candidate genes and cellular pathways underlying the remodeling process. The arterialized jugular vein was analyzed on days 3 and 28 post-surgery and compared with the normal jugular vein and carotid artery. A gene array platform detected 9846 genes in all samples. A heatmap analysis uncovered patterns of gene expression showing that the arterialized vein underwent a partial transition from vein to artery from day 3 to 28 post-surgery. The same pattern was verified for 1845 key differentially expressed genes by performing a pairwise comparison of the jugular vein with the other groups. Interestingly, hierarchical clustering of 60 genes with altered expression on day 3 and day 28 displayed an expression pattern similar to that of the carotid artery. Enrichment analysis results and the network relationship among genes modulated during vein arterialization showed that collagen might play a role in the early remodeling process. Indeed, the total collagen content was increased, with the augmented expression of collagen I, collagen IV, and collagen V in arterialized veins. Additionally, there was an increase in the expression of versican and Thy-1 and a decrease in the expression of biglycan and ß1-integrin. Overall, we provide evidence that vein arterialization remodeling is accompanied by consistent patterns of gene expression and that collagen may be an essential element underlying extracellular matrix changes that support the increased vascular wall stress of the new hemodynamic environment.

RESUMEN

Vein graft failure limits the long-term patency of the saphenous vein used as a conduit for coronary artery bypass graft. Early graft adaptation involves some degree of intima hyperplasia to sustain the hemodynamic stress, but the progress to occlusion in some veins remains unclear. We have demonstrated that stretch-induced up-regulation of cysteine and glycine-rich protein 3 (Crp3) in rat jugular vein and human saphenous vein in response to arterialization. Here, we developed a Crp3-KO rat to investigate the role of Crp3 in vascular remodeling. After 28 days jugular vein arterialization, the intima layer was 3-fold thicker in the Crp3-KO that showed comparable smooth muscle cells (SMC) proliferation but an absence of early apoptosis observed in the wild-type rat (WT). We then investigated the role of Crp3 in early integrin-mediated signaling apoptosis in isolated jugular SMC. Interestingly, under basal conditions, ceramide treatment failed to induce apoptosis in both WT and Crp3-KO SMC. Under stretch, Crp3 expression increased in WT SMC and ceramide induced apoptosis. Immunoblotting analysis indicated that ceramide stretch-induced apoptosis in SMC is accompanied by a decrease in the phosphorylation status of both Fak and Akt, leading to an increase in Bax expression and caspase-3 cleavage. In contrast, ceramide failed to decrease Fak and Akt phosphorylation in Crp3-KO SMC and, therefore, there was no downstream induction of Bax expression and effector caspase-3 cleavage. Taken together, we provide evidence that stretch-induced Crp3 modulates vein remodeling in response to arterialization by sensitizing SMC to apoptosis.

RESUMEN

Recent evidence suggests that ß-arrestins, which are involved in G protein-coupled receptors desensitization, may influence mechanotransduction. Here, we observed that nitric oxide (NO) production was abrogated in human saphenous vein endothelial cells (SVECs) transfected with siRNA against ß-arrestin 1 and 2 subjected to shear stress (SS, 15 dynes/cm2, 10 min). The downregulation of ß-arrestins 1/2 in SVECs cells also prevented the SS-induced rise in levels of phosphorylation of Akt and endothelial nitric oxide synthase (eNOS, Serine 1177). Interestingly, immunoprecipitation revealed that ß-arrestin interacts with Akt, eNOS and caveolin-1 and these interactions are not influenced by SS. Our data indicate that ß-arrestins and Akt/eNOS downstream signaling are required for early SS-induced NO production in SVECs, which is consistent with the idea that ß-arrestins and caveolin-1 are part of a pre-assembled complex associated with the cellular mechanotransduction machinery.

Asunto(s)

Células Endoteliales/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , beta-Arrestina 1/metabolismo , Arrestina beta 2/metabolismo , Caveolina 1/metabolismo , Células Cultivadas , Regulación hacia Abajo , Humanos , Mecanotransducción Celular/fisiología , Fosforilación , ARN Interferente Pequeño/genética , Transducción de Señal , Estrés Mecánico , beta-Arrestina 1/antagonistas & inhibidores , beta-Arrestina 1/genética , Arrestina beta 2/antagonistas & inhibidores , Arrestina beta 2/genética

RESUMEN

BACKGROUND: Patients with severe aortic stenosis have increased levels of prothrombotic and proinflammatory microparticles (MP), and MPs actively regulate pathological processes that lead to atherothrombotic cardiovascular events. Shear stress is a validated stimulus of MP production, and abnormal shear stress in aortic stenosis increases MP release in ex-vivo studies. We hypothesized that in patients with severe aortic stenosis, percutaneous replacement of the aortic valve (TAVR) would reduce abnormal shear stress and would decrease levels of circulating MPs. FINDINGS: The experimental protocol utilized flow cytometry (FC) and nanoparticle tracking analysis (NTA) to quantify circulating plasma MP levels in aortic stenosis patients at baseline and 5 days after TAVR. The baseline and 5 day MP counts measured by FC were 6.10⋅10(5) ± 1.21⋅10(5) MP/µL and 5.74⋅10(5) ± 9.54⋅10(4) MP/µL, respectively (p = 0.91). The baseline and 5 day MP counts measured by NTA were 9.29⋅10(13) ± 1.66⋅10(13) MP/µL and 3.95⋅10(14) ± 3.11⋅10(14) MP/µL, respectively (p = 0.91). When MPs were stratified by cell source, there was no difference in pre/post TAVR endothelial, platelet, or leukocyte MP levels. CONCLUSION: Levels of circulating MPs do not change acutely following TAVR therapy for aortic stenosis. Trial registered at clinicaltrials.gov NCT02193035 on July 11, 2014.

Asunto(s)

Válvula Aórtica/cirugía , Plaquetas/química , Endotelio Vascular/química , Prótesis Valvulares Cardíacas , Macrófagos/química , Reemplazo de la Válvula Aórtica Transcatéter , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino

RESUMEN

Identifying molecules that serve as markers for cell aging is a goal that has been pursued by several groups. Senescence-associated ß galactosidase (SA-ßgal) staining is broadly used and very easily detected. ß-gal is a lysosomal enzyme strongly correlated to the progression of cell senescence. Here, we describe a simple, fast, and quantitative protocol to quantify SA-ßgal activity in cell lysate extracts by a chemiluminescent method using galacton as substrate.

Asunto(s)

Senescencia Celular , Mediciones Luminiscentes/métodos , beta-Galactosidasa/metabolismo , Humanos , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/enzimología , Factores de Tiempo

RESUMEN

OBJECTIVES: We tested whether angiotensin converting enzyme (ACE) and phosphorylation of Ser(1270) are involved in shear-stress (SS)-induced downregulation of the enzyme. METHODS AND RESULTS: Western blotting analysis showed that SS (18 h, 15 dyn/cm(2)) decreases ACE expression and phosphorylation as well as p-JNK inhibition in human primary endothelial cells (EC). CHO cells expressing wild-type ACE (wt-ACE) also displayed SS-induced decrease in ACE and p-JNK. Moreover, SS decreased ACE promoter activity in wt-ACE, but had no effect in wild type CHO or CHO expressing ACE without either the extra- or the intracellular domains, and decreased less in CHO expressing a mutated ACE at Ser(1270) compared to wt-ACE (13 vs. 40%, respectively). The JNK inhibitor (SP600125, 18 h), in absence of SS, also decreased ACE promoter activity in wt-ACE. Finally, SS-induced inhibition of ACE expression and phosphorylation in EC was counteracted by simultaneous exposure to an ACE inhibitor. CONCLUSIONS: ACE displays a key role on its own downregulation in response to SS. This response requires both the extra- and the intracellular domains and ACE Ser(1270), consistent with the idea that the extracellular domain behaves as a mechanosensor while the cytoplasmic domain elicits the downstream intracellular signaling by phosphorylation on Ser(1270).

Asunto(s)

Mecanotransducción Celular/fisiología , Peptidil-Dipeptidasa A/metabolismo , Fosforilación/fisiología , Estrés Mecánico , Animales , Western Blotting , Células CHO , Células Cultivadas , Cricetinae , Electroforesis en Gel de Poliacrilamida , Células Endoteliales/enzimología , Humanos , Mecanotransducción Celular/genética , Peptidil-Dipeptidasa A/genética , Fosforilación/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Adipose tissue-derived stem cells (ASCs) are among the more attractive adult stem cell options for potential therapeutic applications. Here, we studied and compared the basic biological characteristics of ASCs isolated from humans (hASCs) and mice (mASCs) and maintained in identical culture conditions, which must be examined prior to considering further potential clinical applications. hASCs and mASCs were compared for immunophenotype, differentiation potential, cell growth characteristics, senescence, nuclear morphology, and DNA content. Although both strains of ASCs displayed a similar immunophenotype, the percentage of CD73(+) cells was markedly lower and CD31(+) was higher in mASC than in hASC cultures. The mean population doubling time was 98.08 ± 6.15 h for hASCs and 52.58 ± 3.74 h for mASCs. The frequency of nuclear aberrations was noticeably lower in hASCs than in mASCs regardless of the passage number. Moreover, as the cells went through several in vitro passages, mASCs showed changes in DNA content and cell cycle kinetics (frequency of hypodiploid, G0/G1, G2/M, and hyperdiploid cells), whereas all of these parameters remained constant in hASCs. Collectively, these results suggest that mASCs display higher proliferative capacity and are more unstable than hASCs in long-term cultures. These results underscore the need to consider specificities among model systems that may influence outcomes when designing potential human applications.

Asunto(s)

Tejido Adiposo/citología , Células Madre Adultas/citología , Proliferación Celular , Inestabilidad Genómica , Adipocitos/citología , Adulto , Células Madre Adultas/fisiología , Animales , Antígenos CD/metabolismo , Técnicas de Cultivo de Célula , Ciclo Celular , Diferenciación Celular , Forma del Núcleo Celular , Senescencia Celular , Citometría de Flujo , Humanos , Ratones , Persona de Mediana Edad , Osteocitos/citología , Fenotipo , Ploidias , Especificidad de la Especie , Factores de Tiempo

RESUMEN

It has been demonstrated that human adipose tissue-derived mesenchymal stem cells (hASCs) enhance vascular density in ischemic tissues, suggesting that they can differentiate into vascular cells or release angiogenic factors that may stimulate neoangiogenesis. Moreover, there is evidence that shear stress (SS) may activate proliferation and differentiation of embryonic and endothelial precursor stem cells into endothelial cells (ECs). In this work, we investigated the effect of laminar SS in promoting differentiation of hASCs into ECs. SS (10 dyn/cm(2) up to 96 h), produced by a cone plate system, failed to induce EC markers (CD31, vWF, Flk-1) on hASC assayed by RT-PCR and flow cytometry. In contrast, there was a cumulative production of nitric oxide (determined by Griess Reaction) and vascular endothelial growth factor (VEGF; by ELISA) up to 96 h of SS stimulation ( in nmol/10(4) cells: static: 0.20 +/- 0.03; SS: 1.78 +/- 0.38, n = 6; VEGF in pg/10(4) cells: static: 191.31 +/- v35.29; SS: 372.80 +/- 46.74, n = 6, P < 0.05). Interestingly, the VEGF production was abrogated by 5 mM N(G)-L-nitro-arginine methyl ester (L-NAME) treatment (VEGF in pg/10(4) cells: SS: 378.80 +/- 46.74, n = 6; SS + L-NAME: 205.84 +/- 91.66, n = 4, P < 0.05). The results indicate that even though SS failed to induce EC surface markers in hASC under the tested conditions, it stimulated NO-dependent VEGF production.

Asunto(s)

Células Endoteliales/metabolismo , Células Madre Mesenquimatosas/metabolismo , Óxido Nítrico/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Tejido Adiposo/citología , Adulto , Diferenciación Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células Endoteliales/citología , Inhibidores Enzimáticos/farmacología , Citometría de Flujo , Expresión Génica , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Persona de Mediana Edad , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estrés Mecánico , Factores de Tiempo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Adulto Joven , Factor de von Willebrand/genética , Factor de von Willebrand/metabolismo

RESUMEN

BACKGROUND: Mesenchymal stem cells (MSCs) have been considered for human regenerative therapy applications, and safe culture and expansion protocols are needed especially in the context of interspecies contamination. Human platelet lysate (PL) has been proposed as animal serum substitute during in vitro MSC expansion. In this work, a simplified and efficient method to obtain autologous PL to replace animal serum in cell culture applications is described. STUDY DESIGN AND METHODS: PL obtained by freezing and centrifugation procedures was tested as medium supplement for human adipose mesenchymal stem cell (hASC) culture. Differential proliferation, immunophenotypic changes, and differentiation under PL or fetal bovine serum (FBS) were assessed. RESULTS: In contrast to 10% FBS supplementation, cell population doubling time was significantly lower when hASCs were cultured with the same concentration of PL (PL 22.9 +/- 1.5 hr vs. FBS 106.7 +/- 6.5 hr, t test, p < 0.05). Furthermore, hASCs maintained with 2.5% PL supplementation also showed satisfactory results. Immunophenotypic analysis revealed no differences between hASCs cultivated with PL or FBS supplementation and both cultures retained the potential to differentiate into adipose cells. These results demonstrate that autologous PL obtained from the same donor can be used as animal serum substitute in hASC culture. CONCLUSIONS: Taken together, evidence is provided that platelets provided by a single donor are sufficient to obtain PL for hASC propagation for clinical-scale applications mitigating the potential untoward side effects associated with the use of animal-derived reagents.

Asunto(s)

Tejido Adiposo/citología , Plaquetas , Técnicas de Cultivo de Célula/métodos , Extractos Celulares/farmacología , Medio de Cultivo Libre de Suero/farmacología , Células Madre Mesenquimatosas/citología , Adipocitos/citología , Animales , Bovinos , Diferenciación Celular , División Celular , Humanos , Inmunofenotipificación , Osteocitos/citología , Suero

RESUMEN

Neo-intima development and atherosclerosis limit long-term vein graft use for revascularization of ischaemic tissues. Using a rat model, which is technically less challenging than smaller rodents, we provide evidence that the temporal morphological, cellular, and key molecular events during vein arterialization resemble the human vein graft adaptation. Right jugular vein was surgically connected to carotid artery and observed up to 90 days. Morphometry demonstrated gradual thickening of the medial layer and important formation of neo-intima with deposition of smooth muscle cells (SMC) in the subendothelial layer from day 7 onwards. Transmission electron microscopy showed that SMCs switch from the contractile to synthetic phenotype on day 3 and new elastic lamellae formation occurs from day 7 onwards. Apoptosis markedly increased on day 1, while alpha-actin immunostaining for SMC almost disappeared by day 3. On day 7, cell proliferation reached the highest level and cellular density gradually increased until day 90. The relative magnitude of cellular changes was higher in the intima vs. the media layer (100 vs. 2 times respectively). Cyclin-dependent kinase inhibitors (CDKIs) p27(Kip1) and p16(INKA) remained unchanged, whereas p21(Cip1) was gradually downregulated, reaching the lowest levels by day 7 until day 90. Taken together, these data indicate for the first time that p21(Cip1) is the main CDKI protein modulated during the arterialization process the rat model of vein arterialization that may be useful to identify and validate new targets and interventions to improve the long-term patency of vein grafts.

Asunto(s)

Apoptosis/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Venas Yugulares/trasplante , Actinas/metabolismo , Animales , Derivación Arteriovenosa Quirúrgica , Arteria Carótida Común/cirugía , Proliferación Celular , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/metabolismo , Etiquetado Corte-Fin in Situ , Venas Yugulares/metabolismo , Venas Yugulares/cirugía , Venas Yugulares/ultraestructura , Microscopía Electrónica , Músculo Liso Vascular/metabolismo , Ratas , Ratas Wistar

RESUMEN

AIMS: Cysteine- and glycine-rich protein 3/muscle LIM-domain protein (CRP3/MLP) mediates protein-protein interaction with actin filaments in the heart and is involved in muscle differentiation and vascular remodelling. Here, we assessed the induction of CRP3/MLP expression during arterialization in human and rat veins. METHODS AND RESULTS: Vascular CRP3/MLP expression was mainly observed in arterial samples from both human and rat. Using quantitative real time RT-PCR, we demonstrated that the CRP3/MLP expression was 10 times higher in smooth muscle cells (SMCs) from human mammary artery (h-MA) vs. saphenous vein (h-SV). In endothelial cells (ECs), CRP3/MLP was scarcely detected in either h-MA or h-SV. Using an ex vivo flow through system that mimics arterial condition, we observed induction of CRP3/MLP expression in arterialized h-SV. Interestingly, the upregulation of CRP3/MLP was primarily dependent on stretch stimulus in SMCs, rather than shear stress in ECs. Finally, using a rat vein in vivo arterialization model, early (1-14 days) CRP3/MLP immunostaining was observed predominantly in the inner layer and later (28-90 days) it appeared more scattered in the vessel layers. CONCLUSION: Here we provide evidence that CRP3/MLP is primarily expressed in arterial SMCs and that stretch is the main stimulus for CRP3/MLP induction in veins exposed to arterial haemodynamic conditions.

Asunto(s)

Proteínas Musculares/metabolismo , Reflejo de Estiramiento/fisiología , Vena Safena/metabolismo , Resistencia al Corte/fisiología , Animales , Fenómenos Biomecánicos , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Humanos , Proteínas con Dominio LIM , Masculino , Modelos Animales , Proteínas Musculares/genética , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Ratas , Ratas Wistar , Estrés Mecánico

RESUMEN

INTRODUCTION: Saphenous vein grafting is still widely used to revascularize ischemic myocardium. The effectiveness of this procedure is limited by neointima formation and accelerated atherosclerosis, which frequently leads to graft occlusion. A better understanding of this process is important to clarify the mechanisms of vein graft disease and to aid in the formulation of strategies for prevention and/or therapeutics. OBJECTIVE: To develop an ex vivo flow system that allows for controlled hemodynamics in order to mimic arterial and venous conditions. METHODS: Human saphenous veins were cultured either under venous (flow: 5 ml/min) or arterial hemodynamic conditions (flow: 50 ml/min, pressure: 80 mmHg) for 1-, 2- and 4-day periods. Cell viability, cell density and apoptosis were compared before and after these intervals using MTT, Hoeschst 33258 stain, and TUNEL assays, respectively. RESULTS: Fresh excised tissue segments were well preserved prior to the study. Hoechst 33258 and MTT stains showed progressive losses in cell density and cell viability in veins cultured under arterial hemodynamic conditions from 1 to 4 days, while no alterations were observed in veins cultured under venous conditions. Although the cell density from 1-day cultured veins under arterial conditions was similar to that of freshly excised veins, the TUNEL assay indicated that most of these cells were undergoing apoptosis. CONCLUSION: The results observed resemble the events taking place during early in vivo arterial-vein grafting and provide evidence that an ex vivo perfusion system may be useful for the identification of new therapeutic targets that ameliorate vein graft remodeling and increase graft patency over time.

Asunto(s)

Hemodinámica , Modelos Cardiovasculares , Técnicas de Cultivo de Órganos/métodos , Perfusión/métodos , Vena Safena/patología , Análisis de Varianza , Apoptosis/fisiología , Recuento de Células , Supervivencia Celular/fisiología , Humanos , Etiquetado Corte-Fin in Situ , Vena Safena/trasplante , Vena Safena/ultraestructura , Coloración y Etiquetado

RESUMEN

INTRODUCTION: Saphenous vein grafting is still widely used to revascularize ischemic myocardium. The effectiveness of this procedure is limited by neointima formation and accelerated atherosclerosis, which frequently leads to graft occlusion. A better understanding of this process is important to clarify the mechanisms of vein graft disease and to aid in the formulation of strategies for prevention and/or therapeutics. OBJECTIVE: To develop an ex vivo flow system that allows for controlled hemodynamics in order to mimic arterial and venous conditions. METHODS: Human saphenous veins were cultured either under venous (flow: 5 ml/min) or arterial hemodynamic conditions (flow: 50 ml/min, pressure: 80 mmHg) for 1-, 2- and 4-day periods. Cell viability, cell density and apoptosis were compared before and after these intervals using MTT, Hoeschst 33258 stain, and TUNEL assays, respectively. RESULTS: Fresh excised tissue segments were well preserved prior to the study. Hoechst 33258 and MTT stains showed progressive losses in cell density and cell viability in veins cultured under arterial hemodynamic conditions from 1 to 4 days, while no alterations were observed in veins cultured under venous conditions. Although the cell density from 1-day cultured veins under arterial conditions was similar to that of freshly excised veins, the TUNEL assay indicated that most of these cells were undergoing apoptosis. CONCLUSION: The results observed resemble the events taking place during early in vivo arterial-vein grafting and provide evidence that an ex vivo perfusion system may be useful for the identification of new therapeutic targets that ameliorate vein graft remodeling and increase graft patency over time.

Asunto(s)

Humanos , Hemodinámica , Modelos Cardiovasculares , Técnicas de Cultivo de Órganos/métodos , Perfusión/métodos , Vena Safena/patología , Análisis de Varianza , Apoptosis/fisiología , Recuento de Células , Supervivencia Celular/fisiología , Etiquetado Corte-Fin in Situ , Coloración y Etiquetado , Vena Safena/trasplante , Vena Safena/ultraestructura

Asunto(s)

Humanos , Enfermedades Cardiovasculares/terapia , Proteómica/tendencias , Cromatografía de Gases y Espectrometría de Masas

RESUMEN

Mechanical forces contribute to maintenance of cardiovascular homeostasis via the control of release and production of vasoactive substances. We demonstrated previously that shear stress decreases rat ACE activity and expression. Using a reporter gene approach and mutagenesis, we show now that the classic shear stress responsive element or SSRE (GAGACC) contained within 1,274 bp of this promoter is not functional in response to shear stress (15 dyn/cm2, 18 h) [for the wild-type ACE promoter (WLuc), static control (C) = 107 +/- 6.5%, shear stress (SS) = 65.9 +/- 9.4%, n = 8; for the promoter with the classic SSRE mutated (WSS-mut), C = 100 +/- 8.2%, SS = 60.2 +/- 5.2%, n = 10, respectively]. Analysis of progressive deletion mutants unraveled a 57-bp fragment, position -251 to -195, from the transcription start site, containing functional SSRE (for WLuc, C = 107 +/- 6.5%, SS = 65.9 +/- 9.4%, n = 8; for 378, C = 100 +/- 6.4%, SS = 60.4 +/- 4.3%, n = 11; for 251, C = 99.7 +/- 2.6%, SS = 63.2 +/- 5.5%, n = 7; for 194, C = 104.6 +/- 8.1%, SS = 92.4 +/- 6.9%, n = 9). This fragment responded to shear stress even in the context of a heterologous promoter. Finally, functional analysis of mutated candidate regulatory elements identified by gel shift, DNase I footprint, and conservation of aligned sequences revealed that only the double mutant (Barbie/GAGA-mut) but not isolated disruption of the Barbie (WBarbie-mut) or the GAGA (WGAGA-mut) prevented the shear-stress-induced response (for Barbie/GAGA-mut, C = 97.9 +/- 5%, SS = 99.4 +/- 7.2%, n = 6; for WBarbie-mut, C = 106.1 +/- 8.6%, SS = 65.9 +/- 9.4%, n = 6; for WGAGA-mut, C = 100.1 +/- 2.9%, SS = 66.7 +/- 1.6, n = 6;). Taken together, these data provide direct evidence for the new role of Barbie and GAGA boxes in mediating the shear-stress-induced downregulation of rat ACE expression and demonstrate that the classic SSRE (GAGACC) is not functional under the experimental conditions tested.

Asunto(s)

Peptidil-Dipeptidasa A/genética , Regiones Promotoras Genéticas , Elementos de Respuesta , Animales , Secuencia de Bases , Sitios de Unión , Proteínas de Unión al ADN/metabolismo , Datos de Secuencia Molecular , Conejos , Ratas , Eliminación de Secuencia , Estrés Mecánico , Activación Transcripcional

RESUMEN

Evidências recentes mostram que forças físicas como o "shear stress" têm participação importante na expressão de genes que participam do controle da homeostasia do sistema cardiovascular. Além disto, observa-se que o aparecimento de disfunções cardiovasculares, como a aterosclerose, é mais freqüente em regiões de bifurcações vasculares, onde há diminuição do "shear stress" associada à transição de regime de fluxo laminar para turbulento. A enzima conversara de angiotensina I (ECA) é uma ectoproteína ancorada à membrana da célula endotelial, e por esta razão é um alvo natural para sofrer influência do " shear stress". Em concordância com esta idéia, recentemente em nosso laboratório foi demonstrado que o "shear stress" diminui a atividade e a expressão da ECA "in vitro" e "in vivo". Esta resposta é acompanhada pela redução da atividade da função do fragmento de 1274 pb do promotor da ECA de rato, que contém seqüência idêntica ao elemento responsivo ao "shear stress" (SSRE) descrito inicialmente no gene do PDGF. No presente estudo apresentamos evidências direta de que o SSRE clássico presente no promotor da ECA de rato não é funcional, pois uma mutação nesta seqüência não modificou a resposta apresentada pelo promotor selvagem quando submetido ao "shear stress". Análise funcional de construções contendo deleções progressivas do promotor mostraram que a seqüência regulatória responsiva ao shear stress" no promotor da ECA encontra-se em um fragmento de 57pb, entre as posições -251 e -l94 do sítio de início da transcrição. O fragmento de 57pb do promotor da ECA apresenta várias sequências consensos candidatas, que podem estar interagindo com fatores de transcrição para desempenhar a resposta desencadeada pelo "shear stress". A participação da proteína AP-2, candidata natural, foi excluída, uma vez que o promotor contendo mutação no sítio putativo de lnteração para AP-2 continuou respondendo às alterações de "shear stress". Em conjunto nossos resultados mostram que o SSRE clássico, GAGACC, não é funcional no promotor da ECA de rato. Além disto, foi identificado uma região de 57pb, localizada no intervalo -251 a -l94 pb do sítio de início da transcrição, que contém pelo menos um SSRE do gene da ECA

Asunto(s)

Endotelio Vascular , Peptidil-Dipeptidasa A , Estrés Fisiológico