RESUMO
Hemorrhagic shock is a leading cause of morbidity and mortality worldwide. Significant blood loss may lead to decreased blood pressure and inadequate tissue perfusion with resultant organ failure and death, even after replacement of lost blood volume. One reason for this high acuity is that the fundamental mechanisms of shock are poorly understood. Proteomic and metabolomic approaches have been used to investigate the molecular events occurring in hemorrhagic shock but, to our knowledge, a systematic analysis of the transcriptomic profile is missing. Therefore, a pilot analysis using paired-end RNA sequencing was used to identify changes that occur in the blood transcriptome of rats subjected to hemorrhagic shock after blood reinfusion. Hemorrhagic shock was induced using a Wigger's shock model. The transcriptome of whole blood from shocked animals shows modulation of genes related to inflammation and immune response (Tlr13, Il1b, Ccl6, Lgals3), antioxidant functions (Mt2A, Mt1), tissue injury and repair pathways (Gpnmb, Trim72) and lipid mediators (Alox5ap, Ltb4r, Ptger2) compared with control animals. These findings are congruent with results obtained in hemorrhagic shock analysis by other authors using metabolomics and proteomics. The analysis of blood transcriptome may be a valuable tool to understand the biological changes occurring in hemorrhagic shock and a promising approach for the identification of novel biomarkers and therapeutic targets. Impact statement This study provides the first pilot analysis of the changes occurring in transcriptome expression of whole blood in hemorrhagic shock (HS) rats. We showed that the analysis of blood transcriptome is a useful approach to investigate pathways and functional alterations in this disease condition. This pilot study encourages the possible application of transcriptome analysis in the clinical setting, for the molecular profiling of whole blood in HS patients.
Assuntos
Células Sanguíneas/patologia , Perfilação da Expressão Gênica , Choque Hemorrágico/patologia , Animais , Modelos Animais de Doenças , Masculino , Ratos Wistar , Análise de Sequência de RNARESUMO
One of the key features of cardiovascular complications, such as hypertension or diabetes, is that they often appear at the same time in the same individual together with other forms of co-morbidities. While clinically a recognized phenomenon, no molecular mechanism for such co-morbidities has received universal acceptance. We propose a new hypothesis that provides a molecular basis for co-morbidities in hypertension due to unchecked proteolytic activity and receptor destruction. Testing of the hypothesis in the spontaneously hypertensive rat reveals an unchecked matrix metalloproteinase and serine protease activity in plasma and on several cardiovascular and parenchymal cells. The elevated proteolytic activity causes extracellular cleavage of multiple receptor types, such that cleavage of one receptor type leads to loss of the function carried out by this receptor. Proteolytic cleavage of the extracellular domain of the ß(2) adrenergic receptor in arteries and arterioles causes vasoconstriction and elevation of the central blood pressure while cleavage of the extracellular domain of the insulin receptor leads to insulin resistance and lack of transmembrane glucose transport. A diverse set of cell dysfunctions in the spontaneously hypertensive rat are accompanied by cleavage of the membrane receptors that are involved in these functions. Chronic inhibition of the unchecked protease activity in the spontaneously hypertensive rat serves to restore the extracellular receptor density and alleviates the corresponding cell dysfunctions. The mild unchecked proteolytic activity in the spontaneously hypertensive rat points towards a chronic autodigestion process as a contributor to the end organ injury encountered in this rat strain. The presence of various soluble receptors, which consist of extracellular fragments of membrane receptors, in the plasma of hypertensive and diabetic patients suggest that the autodigestion process may also be present in man.
RESUMO
One mechanism for organ damage in individuals with arterial hypertension may be due to oxygen free radical production. This study was designed to localize free radicals in a microvascular network of mature spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats. Because glucocorticoids play a role in pressure elevation of SHRs, we investigated their role in microvascular free radical formation. Oxygen radical production in mesentery was detected by tetranitroblue tetrazolium reduction to formazan aided by digital light-absorption measurements. Formazan deposits were observed in the endothelial cells and lumens of all microvessels and in lymphatic endothelia but were fewer in tissue parenchyma. The formazan distribution in younger (14-16 wk old) WKY rats and SHRs was heterogeneous with low values in capillaries and small arterioles/venules (<30 microm) but enhanced deposits in larger venules. Adrenalectomy served to reduce the formazan density in SHRs to the level of WKY rats, whereas dexamethasone supplementation of the adrenalectomized rats caused elevation in the larger venules of SHRs. In older (40 wk old) SHRs, formazan levels were elevated in all hierarchies of microvessels. After pressure reduction was employed with chronic hydralazine treatment, the formazan deposits were reduced in all locations of the microcirculation in both WKY rats and SHRs. Elevated formazan deposits were also found in lymphatic endothelium. These results suggest that oxygen free radical production is elevated in both high- and low-pressure regions of SHR microcirculation via a process that is controlled by glucocorticoids. Older SHRs have higher formazan levels than younger SHRs in all microvessels. Chronic hydralazine treatment, which serves to reduce arterial blood pressure, attenuates tetranitroblue tetrazolium reduction in WKY rats and SHRs even in venules of the microcirculation, which has no micropressure elevation. Free radical production may be a more global condition in SHRs and may not be limited to arteries and arterioles.
Assuntos
Endotélio Linfático/irrigação sanguínea , Endotélio Linfático/metabolismo , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Estresse Oxidativo/fisiologia , Córtex Suprarrenal/fisiologia , Adrenalectomia , Animais , Pressão Sanguínea/fisiologia , Radicais Livres/metabolismo , Hidralazina/farmacologia , Hipertensão/tratamento farmacológico , Masculino , Microcirculação/efeitos dos fármacos , Microcirculação/fisiologia , Nitroazul de Tetrazólio , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Circulação Esplâncnica/efeitos dos fármacos , Circulação Esplâncnica/fisiologia , Superóxidos/metabolismo , Vasodilatadores/farmacologiaRESUMO
The mechanism for interstitial fluid uptake into the lymphatics remains speculative and unresolved. A system of intralymphatic valves exists that prevents reflow along the length of the lymphatic channels. However, these valves are not sufficient to provide unidirectional flow at the level of the initial lymphatics. We investigate here the hypothesis that initial lymphatics have a second, separate valve system that permits fluid to enter from the interstitium into the initial lymph channels but prevents escape back out into the tissue. The transport of fluorescent microspheres (0.31 microm) across endothelium of initial lymphatics in rat cremaster muscle was investigated with micropipette manipulation techniques. The results indicate that microspheres can readily pass from the interstitium across the endothelium into the lumen of the initial lymphatics. Once inside the lymphatic lumen, the microspheres cannot be forced out of the lumen even after elevation of the lymphatic pressure by outflow obstruction. Reaspiration of the microspheres inside the lymphatic lumen with a micropipette is blocked by the lymphatic endothelium. This blockade exists whether the aspiration is carried out at the microsphere entry site or anywhere along the initial lymphatics. Nevertheless, puncture of the initial lymphatic endothelium with the micropipette leads to rapid aspiration of intralymphatic microspheres. Investigation of lymphatic endothelial sections fixed during lymph pumping shows open interendothelial junctions not found in resting initial lymphatics. These results suggest that initial lymphatics have a (primary) valve system at the level of the endothelium. In conjunction with the classical (secondary) intralymphatic valves, the primary valves provide the mechanism that facilitates the unidirectional flow during periodic compression and expansion of initial lymphatics.
Assuntos
Sistema Linfático/anatomia & histologia , Músculos Abdominais/anatomia & histologia , Animais , Transporte Biológico , Endotélio/anatomia & histologia , Espaço Extracelular/fisiologia , Corantes Fluorescentes , Microesferas , Pressão , Ratos , Ratos Wistar , SucçãoRESUMO
Spontaneously hypertensive rats (SHRs) have elevated numbers of apoptotic cells. However, the extent and pattern of cell death at the microvascular level is unexplored. We developed a technique to determine early forms of cell death in vivo in the mesentery by use of the life/death indicator ethidium bromide (EB). The mesenteric microvasculature was superfused with 5 microM EB for a period of 3 min, rinsed and immediately viewed by digital fluorescence microscopy. EB-positive cell structures were observed both in the wall of microvessels as well as in the tissue parenchyma. The microvessels had about 2--4 EB-positive cell structures per 100 microm of vessel length. Larger arterioles (>25 microm) in the SHR had an increased EB-positive structure density. After normalization of the blood pressure in the SHR with adrenalectomy, no significant differences remained between Wistar-Kyoto (WKY) rats and SHRs. After dexamethasone treatment, the adrenalectomized SHRs had a higher EB-positive cell density in the smaller class of microvessels than the WKY rats. In addition, EB-positive cell fragments (0.5--2 microm) were observed in the mesentery microvessel wall, and with TUNEL labeling, they were demonstrated to represent DNA fragments. The percentage of microvessels with EB-positive fragments was higher in the SHR arterioles and capillaries. Capillaries and larger venules (>30 microm) in the SHR had higher levels of cell fragments per vessel length. After adrenalectomy, no significant differences remained between WKY rats and SHRs in any of the microvessel categories. When adrenalectomized rats were treated with dexamethasone, a higher number of EB-positive fragments was detected in the wall of SHR capillaries. These results indicate that the mesentery microcirculation in both strains is subject to an early and nonuniform pattern of cell death, as detected by EB, but is enhanced in selected individual microvascular segments of the SHR by a glucocorticoid-driven mechanism.
Assuntos
Apoptose , Hipertensão/patologia , Microcirculação/patologia , Adrenalectomia , Animais , Pressão Sanguínea , Morte Celular , Núcleo Celular/ultraestrutura , Dexametasona/farmacologia , Etídio/química , Corantes Fluorescentes/química , Glucocorticoides/farmacologia , Marcação In Situ das Extremidades Cortadas , Masculino , Microcirculação/ultraestrutura , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Circulação EsplâncnicaRESUMO
OBJECTIVE: In vitro studies suggest that microhemorrhages with escape of red cells into the tissue may be cytotoxic to parenchymal cells due to oxygen free radical formation. We examined in the rat mesentery the impact of microhemorrhages on parenchymal cell death, as detected by propidium iodide staining, using an intravital approach. METHODS AND RESULTS: Postcapillary venules were punctured with a closed-end micropipette, permitting escape of blood cells and plasma into the mesentery interstitium. Over a period of 2 h, no significant increase in parenchymal cell death was encountered in tissues with hemorrhagic sites compared with nonhemorrhagic control sites. Interstitial microinjections of plasma derived from whole blood incubated for several hours with and without a combination of sodium azide (2 mM) and hydrogen peroxide (1 mM) led to significantly increased levels of cell death compared to control experiments. Interventions against the hydroxyl radical with dimethylthiourea (DMTU, 2 mM) or 2,2'-dipyridyl (DPD, 2 mM), a lipid soluble iron chelator, provided no protective effect against the parenchymal cell death. DMTU slightly delayed tile cytotoxic reaction. CONCLUSIONS: These observations suggest that a newly formed microhemorrhage is not necessarily cytotoxic to parenchymal tissue cells. Interstitial microinjections of plasma, derived from whole blood after prolonged exposure to oxygen free radicals or just aging under in vitro conditions, may be cytotoxic to mesenteric parenchymal cells without effective blockade by interventions against the hydroxyl radical.
Assuntos
Morte Celular , Hemorragia/patologia , Microcirculação , 2,2'-Dipiridil/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Sequestradores de Radicais Livres/farmacologia , Radicais Livres , Hemoglobinas/química , Peróxido de Hidrogênio/farmacologia , Radical Hidroxila , Quelantes de Ferro/farmacologia , Masculino , Veias Mesentéricas/lesões , Mesentério/irrigação sanguínea , Mesentério/patologia , Microinjeções , Estresse Oxidativo , Plasma , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio , Azida Sódica/farmacologia , Tioureia/análogos & derivados , Tioureia/farmacologia , Vênulas/lesõesRESUMO
One of the hallmarks of venous insufficiency is an elevated venous pressure. While a number of mechanisms have been proposed for vascular and parenchymal cell damage following venous pressure elevation, such as white cell infiltration, a key question remains as to what degree venous occlusion and flow interruption per se may constitute a risk factor in venous disease. To gain an insight into this mechanism, we examined the effect of venous occlusion followed by reperfusion. A draining venule (circa 50 micrometer) in the rat mesentery was occluded with a micropipette (1 h) followed by reperfusion (1 h). The procedure serves to raise the microvascular pressure to about 31 mm Hg during the occlusion while the flow is completely stopped in the local venous and capillary network. Parenchymal cell death in the mesentery was monitored by propidium iodide (PI) labeling. The number of PI-positive cells significantly increased predominantly during reperfusion. A 1-week treatment with a micronized purified flavonoid fraction (100 mg/kg/day) served to significantly reduce parenchymal cell death as well as leukocyte rolling, adhesion to postcapillary venule, and migration into the tissue both during occlusion and reperfusion. The results indicate, that even in an initially symptomless tissue, flow reduction combined with microvascular pressure elevation during venous occlusion results in tissue damage not only during reperfusion (as in arterial occlusion) but also during occlusion.
Assuntos
Flebite/etiologia , Insuficiência Venosa/complicações , Animais , Adesão Celular , Morte Celular , Doença Crônica , Corantes , Constrição , Fluoresceínas , Leucócitos/patologia , Leucócitos/fisiologia , Masculino , Veias Mesentéricas/fisiopatologia , Propídio , Ratos , Ratos Wistar , Reperfusão , Insuficiência Venosa/patologia , Insuficiência Venosa/fisiopatologia , Pressão Venosa , Vênulas/fisiopatologiaRESUMO
OBJECTIVE: To determine if oxygen free radicals derived from xanthine oxidase are involved in the development of salt-induced hypertension. Enhanced production of oxygen free radicals may play a role in hypertension by affecting vascular smooth muscle contraction and provide a mechanism for lesion formation. METHODS: Dahl salt-sensitive (Dahl-S) and salt-resistant (Dahl-R) rats were fed either a low-salt, high-salt or high-salt + tungsten diet for 4 wk. In vivo production of superoxide (O2-) was detected by the reduction of a tetranitroblue tetrazolium (TNBT) dye in the rat mesentery, while plasma hydrogen peroxide (H2O2) production levels were determined using a modified electrochemical electrode technique. RESULTS: The tungsten diet lowered the blood pressure of Dahl-S rats compared to high-salt-treated Dahl-S rats, but had no effect on blood pressure in Dahl-R rats. Light absorption of formazan deposits revealed that tungsten-treated Dahl-S rats had reduced TNBT staining along the endothelium of arterioles and venules compared to hypertensive, high-salt-treated Dahl-S rats. In addition, tungsten-treated Dahl-S rats had a lower plasma H2O2 concentration compared to hypertensive, high-salt-treated Dahl-S rats. CONCLUSIONS: These findings indicate that xanthine oxidase-derived oxygen free radicals are involved in the pathogenesis of salt-induced hypertension.
Assuntos
Ratos Endogâmicos Dahl/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Pressão Sanguínea , Dieta , Peróxido de Hidrogênio/sangue , Indicadores e Reagentes , Masculino , Músculo Esquelético/enzimologia , Ratos , Superóxidos/metabolismo , Sais de Tetrazólio , Compostos de Tungstênio/metabolismo , Xantina Oxidase/antagonistas & inibidores , Xantina Oxidase/metabolismoRESUMO
OBJECTIVE: The objective of this study was to explore the degree of parenchymal cell injury after mast cell degranulation by application of compound 48/80 (CMP 48/80) in the absence of adherent leukocytes in the rat mesentery. METHODS: Rats were rendered leukopenic by injection of an antibody against leukocytes, and the mesentery was superfused with CMP 48/80 during intravital microscopy. The extent of cell injury was determined using a fluorescent cell-viability indicator, propidium iodide (PI). In an additional group, mast cell degranulation with CMP 48/80 was prevented by using the mast cell stabilizer Ketotifen. RESULTS: After a reduction in the number of circulating leukocytes, mast cell degranulation produced a mild increase in parenchymal cell injury. The injury levels significantly increased when individual regions of the mesentery were compared. Stabilization of the mast cells with Ketotifen reduced the injury to below baseline values. CONCLUSIONS: In the absence of leukocyte adhesion to the endothelium, mast cell degranulation contributes to parenchymal cell injury in the mesentery.
Assuntos
Mastócitos/citologia , Mesentério/citologia , Mesentério/lesões , Animais , Degranulação Celular/efeitos dos fármacos , Endotélio/citologia , Cetotifeno/farmacologia , Leucopenia/induzido quimicamente , Masculino , Propídio , Ratos , Ratos Wistar , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
The mechanisms contributing to organ injury in hypertension have been incompletely defined. The thymus gland of the spontaneously hypertensive rat (SHR) shows significant atrophy at the age of 15 wk compared with its normotensive control, the Wistar-Kyoto rat (WKY). The aim of the present study was to examine the thymus of SHR for evidence of DNA nicking as one of the mechanisms for thymic atrophy. SHR and WKY were subjected to adrenalectomy or sham surgery at 12 wk and studied at 15 wk. Adrenalectomy served to normalize the blood pressure in the SHR. DNA nicking was detected by in situ nick-end labeling (ISEL) of fixed tissue sections. Tissue sections were treated with proteolysis, and terminal deoxyribonucleotidyl transferase was used to incorporate biotinylated deoxynucleotides into DNA nick end in situ. Separately, DNA fragmentation was evaluated by measuring the level of released mono- and oligonucleosomes to the cytoplasm. A higher number of thymic ISEL-positive cells and a higher level of cytoplasmic mono- and oligonucleosomes were observed in SHR than in WKY. After adrenalectomy the enhanced level of ISEL and cytoplasmic mono- and oligonucleosomes in SHR was reduced to the level in WKY. Dexamethasone treatment (0.05 mg. kg-1. day-1) in WKY serves to decrease the thymus weight and significantly elevate the level of mono- and oligonucleosomes. Thus increased DNA fragmentation represents one of the mechanisms associated with thymic atrophy, a feature that reflects immune suppression in SHR.
Assuntos
Fragmentação do DNA/fisiologia , Ratos Endogâmicos SHR/genética , Timo/fisiopatologia , Adrenalectomia , Animais , Atrofia , Pressão Sanguínea/fisiologia , Citoplasma/metabolismo , Dexametasona/farmacologia , Técnicas Genéticas , Glucocorticoides/farmacologia , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Nucleossomos/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Endogâmicos WKY , Valores de Referência , Timo/metabolismo , Timo/patologiaRESUMO
Recent evidence in vivo indicates that spontaneously hypertensive rats (SHR) exhibit an increase in oxyradical production in and around microvascular endothelium. This study is aimed to examine whether xanthine oxidase plays a role in overproduction of oxidants and thereby may contribute to hypertensive states as a consequence of the increasing microvascular tone. The xanthine oxidase activity in SHR was inhibited by dietary supplement of tungsten (0.7 g/kg) that depletes molybdenum as a cofactor for the enzyme activity as well as by administration of (-)BOF4272 [(-)-8-(3-methoxy-4-phenylsulfinylphenyl)pyrazolo(1,5-alpha)-1,3, 5-triazine-4-monohydrate], a synthetic inhibitor of the enzyme. The characteristic elevation of mean arterial pressure in SHR was normalized by the tungsten diet, whereas Wistar Koto (WKY) rats displayed no significant alteration in the pressure. Multifunctional intravital videomicroscopy in mesentery microvessels with hydroethidine, an oxidant-sensitive fluoroprobe, showed that SHR endothelium exhibited overproduction of oxyradicals that coincided with the elevated arteriolar tone as compared with WKY rats. The tungsten diet significantly repressed these changes toward the levels observed in WKY rats. The activity of oxyradical-producing form of xanthine oxidase in the mesenteric tissue of SHR was approximately 3-fold greater than that of WKY rats, and pretreatment with the tungsten diet eliminated detectable levels of the enzyme activity. The inhibitory effects of the tungsten diet on the increasing blood pressure and arteriolar tone in SHR were also reproducible by administration of (-)BOF4272. These results suggest that xanthine oxidase accounts for a putative source of oxyradical generation that is associated with an increasing arteriolar tone in this form of hypertension.
Assuntos
Pressão Sanguínea , Endotélio Vascular/fisiopatologia , Hipertensão/fisiopatologia , Músculo Liso Vascular/fisiopatologia , Xantina Oxidase/metabolismo , Animais , Arteríolas/efeitos dos fármacos , Arteríolas/fisiologia , Arteríolas/fisiopatologia , Pressão Sanguínea/efeitos dos fármacos , Suplementos Nutricionais , Endotélio Vascular/enzimologia , Endotélio Vascular/fisiologia , Inibidores Enzimáticos/farmacologia , Frequência Cardíaca/efeitos dos fármacos , Hipertensão/enzimologia , Hipertensão/genética , Microcirculação , Tono Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Triazinas/farmacologia , Tungstênio/administração & dosagem , Tungstênio/farmacologia , Vênulas/efeitos dos fármacos , Vênulas/fisiologia , Vênulas/fisiopatologia , Xantina Oxidase/antagonistas & inibidoresRESUMO
The events responsible for cell injury after a tissue stimulation are only incompletely understood. The purpose of this study was to examine mechanisms of cell injury in two tissues, rat mesentery and cremaster muscle, after tissue stimulation with N-formylmethionyl-leucyl-phenylalanine (FMLP) and platelet-activating factor (PAF). The response was studied in the same animal in random order using normal and leukopenic rats. The tissues were exteriorized after pentobarbital anesthesia. Five to six vascularized areas were chosen in each tissue, and cell injury and hydroperoxide production were assessed visually by continuous superfusion with 1 microM propidium iodide and 5 microM dichlorofluorescin diacetate (DCFH), respectively. FMLP (1 x 10(-8) M) and then PAF (1 x 10(-8) M) were added to the superfusate, and measurements were made at several time points. The second tissue was then examined using the same protocol. In the cremaster, there was little hydroperoxide production, and the tissue injury was eliminated after leukopenia. Leukopenia had no effect on tissue injury in the mesentery. Although hydroperoxide production was observed, there was no correlation between it and the tissue injury. The level of preactivation showed no correlation with either tissue injury or hydroperoxide production. In light of these results, mast cell degranulation may be an important mechanism of tissue injury in the mesentery.
Assuntos
Inflamação/patologia , Mesentério/patologia , Músculos/patologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Fator de Ativação de Plaquetas/farmacologia , Abdome , Animais , Adesão Celular , Endotélio Vascular/metabolismo , Contagem de Leucócitos , Leucopenia/patologia , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Fatores de TempoRESUMO
Enhanced production of oxygen free radicals may play a role in hypertension by affecting vascular smooth muscle contraction, resistance to blood flow, and organ damage. The aim of this study was to determine whether oxygen free radicals are involved in the development of salt-induced hypertension. Dahl salt-sensitive (Dahl-S) and salt-resistant (Dahl-R) rats were fed either a high salt (6.0% NaCl) or low salt (0.3% NaCl) diet for 4 weeks. The high salt diet caused the development of severe hypertension in Dahl-S animals and had no effect on blood pressure in Dahl-R animals. A tetranitroblue tetrazolium dye was used to detect superoxide radicals in microvessels of the mesentery. Light absorption measurements revealed enhanced staining along the endothelium of arterioles and venules in hypertensive Dahl-S animals, with significantly lower values in normotensive animals. In addition, a Clark electrochemical electrode was used to measure hydrogen peroxide levels in fresh plasma. Hypertensive Dahl-S animals had a higher plasma hydrogen peroxide concentration compared with their normotensive counterparts (2.81+/-0.43 versus 2.10+/-0.41 micromol/L), while no difference was detected between high- and low salt-treated Dahl-R animals (1.70+/-0.35 versus 1.56+/-0.51 micromol/L). The plasma hydrogen peroxide levels of all groups correlated with mean arterial pressure (r=.77). These findings demonstrate an enhanced production of oxygen free radicals in the microvasculature of hypertensive Dahl-S rats.
Assuntos
Pressão Sanguínea , Hipertensão/fisiopatologia , Microcirculação/fisiologia , Estresse Oxidativo , Sódio na Dieta , Circulação Esplâncnica/fisiologia , Animais , Arteríolas/patologia , Arteríolas/fisiologia , Arteríolas/fisiopatologia , Frequência Cardíaca , Peróxido de Hidrogênio/sangue , Hipertensão/genética , Hipertensão/patologia , Masculino , Tono Muscular , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiologia , Músculo Liso Vascular/fisiopatologia , Ratos , Ratos Endogâmicos , Análise de Regressão , Superóxidos/metabolismo , Vênulas/patologia , Vênulas/fisiologia , Vênulas/fisiopatologiaRESUMO
OBJECTIVE: Neutrophilic leukocyte rolling on postcapillary endothelium requires membrane interaction between SLex-containing ligands on neutrophils and P-selectin on endothelial cells. The current sequence of studies was performed to explore the hypothesis that the leukocyte-endothelial rolling interaction not only precedes leukocyte migration but also is accompanied by oxygen free radical production and interstitial cell death in the rat mesentery. METHODS: The ratio of leukocyte rolling velocity on the endothelium of postcapillary venules and centerline red cell velocity was determined after topical application of platelet activating factor (PAF; 10(-8) mol/L). Superoxide formation was determined by an in situ nitroblue tetrazolium reduction to dark blue formazan crystals in the in situ mesentery preparation, and cell death was detected by nuclear staining with propidium iodide. RESULTS: Leukocyte rolling and subsequent adhesion was inhibited with a monoclonal antibody against P-selectin, PB1.3. Superoxide formation, as well as parenchymal cell death, was significantly enhanced in the mesentery after stimulation with platelet activating factor, and both could be significantly attenuated by reduction of the rolling leukocyte-endothelial interaction with PB1.3. CONCLUSIONS: These results provide direct evidence that the interaction of leukocytes and endothelium followed by migration of leukocytes into the interstitium is accompanied by enhanced oxidative stress and parenchymal cell death. Early interruption of the interaction provides significant protection even in the presence of a proinflammatory stimulus.
Assuntos
Inflamação/metabolismo , Selectina-P/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Movimento Celular , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Masculino , Mesentério/irrigação sanguínea , Microcirculação/fisiologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/fisiologia , Estresse Oxidativo , Selectina-P/imunologia , Fator de Ativação de Plaquetas/farmacologia , Propídio/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
This study describes miniaturized technology for the in vivo analysis of the volume and function of the embryonic mouse heart and the application of this technology to study the normal embryonic left ventricle (LV) at two stages of development. With the use of microsurgical techniques, embryos from embryonic day (ED) 10.5 (ED10.5) to ED16 were delivered individually from litters of normal dams, and cardiac visualization was achieved with the use of intravital microscopy by transillumination, with the umbilical circulation intact. At ED10.5-11, the heart could be imaged in color in the intact embryo, whereas at ED12.5 it was necessary to open the chest; at ED13.5-14.5, fluorescent imaging with the use of microinjection of fluorescein-conjugated albumin was necessary to visualize the LV chamber. At ED10.5-11, the LV end-diastolic volumes averaged 0.16 microliter (n = 14), and at ED13.5-14.5, they averaged 0.57 microliter (n = 16). At both ages there was a positive linear relationship between the LV end-diastolic volume and the stroke volume despite substantial variations in individual heart rates, reflecting the relative uniformity of the LV ejection fractions within each age group. The average of the individual ejection fractions was 27.4% at ED10.5-11 and 58.4% at ED13.5-14.5, the latter being within the normal range for the adult rodent heart. These methods will be useful for assessing in vivo cardiac function at ED10.5 and older murine embryos in litters of transgenic or gene-targeted mice when the mutation leads to later embryonic lethality.
Assuntos
Coração Fetal/fisiologia , Função Ventricular Esquerda , Animais , Angiografia Coronária , Diástole , Desenvolvimento Embrionário e Fetal , Feminino , Coração Fetal/anatomia & histologia , Idade Gestacional , Frequência Cardíaca Fetal , Ventrículos do Coração , Camundongos , Camundongos Endogâmicos , Microscopia de Vídeo , Gravidez , Análise de RegressãoRESUMO
Leukocyte migration from a hemopoietic pool across marrow endothelium requires active pseudopod formation and adhesion. Leukocytes rarely show pseudopod formation while in circulation. At question then is the mechanism that serves to minimize leukocyte pseudopod formation in the circulation. We tested the hypothesis that fluid shear stress acts to prevent pseudopod formation. When individual human leukocytes (neutrophils, monocytes) spreading on glass surfaces in vitro were subjected to fluid shear stress ( approximately 1 dyn/cm2), an instantaneous retraction of pseudopods was observed. Removal of the fluid shear stress in turn led to the return of pseudopod projection and cell spreading. When steady shear stress was prolonged over several minutes, leukocyte swelling occurs together with an enhanced random motion of cytoplasmic granules and a reduction of cytoplasmic stiffness. The response to shear stress could be suppressed by K+ channel blockers and chelation of external Ca2+. In rat mesentery microvessels after occlusion, circulating leukocytes project pseudopods in free suspension or when attached to the endothelium, even though immediately after occlusion only few pseudopods were present. When flow was restored, pseudopods on adhering leukocytes were retracted and then the cells began to roll and detach from the endothelium. In conclusion, plasma shear stress in the circulation serves to reduce pseudopod projection and adhesion of circulating leukocytes and vice versa reduction of shear stress leads to pseudopod projection and spreading of leukocytes on the endothelium.
Assuntos
Leucócitos/fisiologia , Leucócitos/ultraestrutura , Estresse Mecânico , Amilorida/farmacologia , Análise de Variância , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Adesão Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Colina/farmacologia , Glibureto/farmacologia , Humanos , Masculino , Microcirculação , Monócitos/fisiologia , Monócitos/ultraestrutura , Neutrófilos/fisiologia , Neutrófilos/ultraestrutura , Ouabaína/farmacologia , Bloqueadores dos Canais de Potássio , Ratos , Ratos Wistar , Bloqueadores dos Canais de Sódio , Circulação Esplâncnica , Tetraetilamônio , Compostos de Tetraetilamônio/farmacologia , Fatores de Tempo , Vênulas/fisiologiaRESUMO
The present study was designed to elucidate whether molecular mechanisms for leukocyte adhesion to microvascular endothelium may differ between spontaneously hypertensive rats and Wistar Kyoto rats. Leukocyte rolling and adhesion were investigated while monitoring venular wall shear rates in the mesenteric microcirculation stimulated with histamine or tert-butyl hydroperoxide in the two strains. In Wistar Kyoto rats, 10 microM histamine as well as 500 microM tertbutyl hydroperoxide promoted a significant reduction of venular leukocyte rolling velocity and subsequent adhesion. These changes in leukocyte behavior were blocked by monoclonal antibodies against P-selectin (PB 1.3) and against sialyl Lewis X-like carbohydrates (2H5). However, spontaneously hypertensive rats exhibited a blunted response of the stimulus-elicited leukocyte rolling, which was associated with impairment of venular P-selectin expression as well as a decrease in the expression of sialyl Lewis X-like carbohydrates on circulating neutrophils. No significant differences were detected between the two strains not only in the surface CD11b/CD18 expression but also in the CD18-mediated adhesivity of neutrophils to intracellular adhesion molecule-1 transfectants in vitro. These results suggest that impairment of selectin-mediated leukocyte adhesion is an event responsible for disorders of inflammatory responses in spontaneously hypertensive rats.
Assuntos
Endotélio Vascular/fisiologia , Hipertensão/sangue , Leucócitos/fisiologia , Selectina-P/fisiologia , Animais , Células CHO , Adesão Celular , Cricetinae , Endotélio Vascular/citologia , Histamina/farmacologia , Masculino , Neutrófilos/fisiologia , Selectina-P/análise , Peróxidos/farmacologia , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , terc-Butil HidroperóxidoRESUMO
We have recently characterized a cardiac model of ventricular chamber defects in retinoid X receptor alpha (RXR alpha) homozygous mutant (-/-) gene-targeted mice. These mice display generalized edema, ventricular chamber hypoplasia, and muscular septal defects, and they die at embryonic day 15. To substantiate our hypothesis that the embryos are dying of cardiac pump failure, we have used digital bright-field and fluorescent video microscopy and in vivo microinjection of fluorescein-labeled albumin to analyze cardiac function. The affected embryos showed depressed ventricular function (average left ventricular area ejection fraction, 14%), ventricular septal defects, and various degrees of atrioventricular block not seen in the RXR alpha wild-type (+/+) and heterozygous (+/-) littermates (average left ventricular area ejection fraction, 50%). The molecular mechanisms involved in these ventricular defects were studied by evaluating expression of cardiac-specific genes known to be developmentally regulated. By in situ hybridization, aberrant, persistent expression of the atrial isoform of myosin light chain 2 was identified in the ventricles. We hypothesize that retinoic acid provides a critical signal mediated through the RXR alpha pathway that is required to allow progression of development of the ventricular region of the heart from its early atrial-like form to the thick-walled adult ventricle. The conduction system disturbances found in the RXR alpha -/- embryos may reflect a requirement of the developing conduction system for the RXR alpha signaling pathway, or it may be secondary to the failure of septal development.
Assuntos
Cardiopatias Congênitas/genética , Receptores do Ácido Retinoico/genética , Fatores de Transcrição/genética , Animais , Modelos Animais de Doenças , Feminino , Átrios do Coração/anormalidades , Átrios do Coração/embriologia , Átrios do Coração/fisiopatologia , Sistema de Condução Cardíaco/fisiopatologia , Cardiopatias Congênitas/embriologia , Cardiopatias Congênitas/fisiopatologia , Ventrículos do Coração/anormalidades , Ventrículos do Coração/embriologia , Ventrículos do Coração/fisiopatologia , Homozigoto , Hibridização In Situ , Masculino , Camundongos , Camundongos Mutantes , Microscopia de Vídeo , Contração Miocárdica , Fenótipo , Gravidez , Sondas RNA , Receptores X de Retinoides , Função VentricularRESUMO
The factors responsible for predisposition to progressive organ injury and vascular complications in arterial hypertension are uncertain. Recent evidence shows that leukocytes participate in cardiovascular conditions for which hypertension is a risk factor. Therefore, there is a need to define the properties of circulating leukocytes in hypertensives. There are about twice as many circulating leukocytes in spontaneous hypertensive rats (SHRs) compared with their normotensive controls, the Wistar-Kyoto rats (WKYs). The SHR neutrophils are viscoelastic and similar to neutrophils in WKYs but exhibit lower deformability in short-term elastic deformation. Mature SHRs have elevated levels of spontaneous pseudopod formation. Mild stimulation with N-formyl-Met-Leu-Phe or platelet-activating factor (10(-8) M) results in a significantly enhanced level of neutrophil pseudopod formation in SHRs but not in WKYs. SHRs exhibit higher levels of spontaneous superoxide formation. Alkaline phosphatase content of individual circulating neutrophils in SHRs is on average lower while plasma levels of alkaline phosphatase in the same samples are elevated in the SHRs. Spontaneous degranulation of SHR neutrophils is also detectable with myeloperoxidase measurements. Such activity of circulating leukocytes poses a significant risk for vascular cytotoxicity in the hypertensive rats.
Assuntos
Envelhecimento/fisiologia , Hipertensão/fisiopatologia , Neutrófilos/fisiologia , Animais , Adesão Celular , Movimento Celular , Tamanho Celular , Hipertensão/complicações , Masculino , Microcirculação/fisiologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Fator de Ativação de Plaquetas/farmacologia , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKYRESUMO
Previous evidence has shown that rats with spontaneous hypertension have on average about twice as many circulating leukocytes in comparison with their normotensive counterparts, the Wistar-Kyoto rats. Since such high levels of leukocytes may increase the risk for vascular complications for hypertensive animals, it is useful to ascertain whether a comparable derangement is present in other forms of hypertension. The present study deals with the properties of the circulating leukocytes in rats exhibiting another form of experimental hypertension; Dahl salt-sensitive (Dahl-S) hypertensive rats were compared with Dahl salt-resistant (Dahl-R) control rats. Measurements were performed to determine the following: circulating hematocrit levels, leukocyte counts, differential counts, number of activated leukocytes (by means of nitro blue tetrazolium [NBT] reduction), leukocyte adhesion in vitro and neutrophil CD-18 expression, alkaline phosphatase activity in individual neutrophils and in the plasma, and myeloperoxidase activity in neutrophils. The experimental cohort consisted of Dahl-S and Dahl-R rats maintained for a 6-week period on a 6% NaCl diet. The results show a highly significant elevation in the number of total leukocytes, neutrophil and monocyte counts, and NBT-positive neutrophils and monocytes in Dahl-S but not Dahl-R rats. There was a significant loss of alkaline phosphatase and myeloperoxidase activity in the neutrophils of the salt-treated Dahl-S rats but not in the neutrophils of the untreated Dahl-S or Dahl-R rats. No significant differences were found in neutrophil adhesion under in vitro test conditions between the two strains maintained on the salt diet.(ABSTRACT TRUNCATED AT 250 WORDS)