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BACKGROUND: There are sex differences in risk for stroke and small vessel ischemic disease in the brain. Microvesicles (MV) derived from activated cells vary by cell of origin and the stimulus initiating their release. MV released from cells activated by inflammatory and thrombotic factors have the potential to disrupt endothelial cells of the brain microvasculature. Therefore, experiments were designed to identify sex differences in the phenotype of MV released from cultured human brain microvascular endothelial cells (HBMEC) in response to inflammatory and thrombotic stimuli. METHODS: Cultured HBMEC derived from 20- to 30-year-old male and female donors were treated for 20 h with medium supplemented with tumor necrosis factor alpha (TNFα; 20 ng/ml), thrombin (THR; 2 U/ml), or vehicle (i.e., control). MV were isolated from the conditioned media by high-speed centrifugation and quantified by digital flow cytometry by labeling with fluorophore-conjugated primary antibodies against PECAM-1, integrin αvß3, ICAM-1, E-selectin, or MCAM. In addition, temporal uptake of labeled MV into control HBMEC was examined by confocal microscopy. RESULTS: Under control conditions, male HBMEC released fewer MV expressing each antigen, except for PECAM-1, than female cells (P < 0.05). Neither TNFα nor THR reduced cell viability. However, TNFα induced apoptosis in female and male cells, whereas THR increased apoptosis marginally only in male cells. TNFα increased expression of all antigens tested on MV in male cells, but only increased expression of integrin αvß3, ICAM-1, and E-selectin on MV from female cells. THR increased expression of PECAM-1, ICAM-1, and MCAM-1 on MV from male but not female cells. MV were internalized and localized to lysosomes within 90 min after their application to HBMEC. CONCLUSIONS: There are sex differences in expression of cell adhesion molecules on MV released from HBMEC under control conditions and upon activation by TNFα or THR. MV taken up by unstimulated HBMEC may impact the integrity of the brain microvasculature and account, in part, for sex differences in vascular pathologies in the brain.
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Moléculas de Adhesión Celular/metabolismo , Micropartículas Derivadas de Células/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Caracteres Sexuales , Trombina/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Adulto , Encéfalo/citología , Supervivencia Celular/efectos de los fármacos , Micropartículas Derivadas de Células/metabolismo , Células Cultivadas , Células Endoteliales/metabolismo , Femenino , Humanos , Inflamación , Masculino , Microvasos , Trombosis , Adulto JovenRESUMEN
BACKGROUND: Nano-sized complexes of calcium phosphate mineral and proteins (calcifying nanoparticles [CNPs]) serve as mineral chaperones. Thus, CNPs may be both a result and cause of soft tissue calcification processes. This study determined if CNPs could augment calcification of arterial vascular smooth muscle cells in vitro. METHODS: CNPs 210 nm in diameter were propagated in vitro from human serum. Porcine aortic smooth muscle cells were cultured for up to 28 days in medium in the absence (control) or presence of 2 mM phosphate ([P] positive calcification control) or after a single 3-day exposure to CNPs. Transmission electron-microscopy was used to characterize CNPs and to examine their cellular uptake. Calcium deposits were visualized by light microscopy and von Kossa staining and were quantified by colorimetry. Cell viability was quantified by confocal microscopy of live-/dead-stained cells and apoptosis was examined concurrently by fluorescent labeling of exposed phosphatidylserine. RESULTS: CNPs, as well as smaller calcium crystals, were observed by transmission electron-microscopy on day 3 in CNP-treated but not P-treated cells. By day 28, calcium deposits were visible in similar amounts within multicellular nodules of both CNP- and P-treated cells. Apoptosis increased with cell density under all treatments. CNP treatment augmented the density of apoptotic bodies and cellular debris in association with mineralized multicellular nodules. CONCLUSION: Exogenous CNPs are taken up by aortic smooth muscle cells in vitro and potentiate accumulation of smooth-muscle-derived apoptotic bodies at sites of mineralization. Thus, CNPs may accelerate vascular calcification.
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Nanopartículas Calcificantes/farmacología , Calcio/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Animales , Aterosclerosis/metabolismo , Células Cultivadas , Humanos , PorcinosRESUMEN
Abstract Menopausal hormone therapies (MHT) may increase thrombotic risk but modulate endothelial function and reduce development of vascular lesions. This study compared effects of MHT on prostanoid-modulated adenosine triphosphate (ATP) secretion from platelets in relationship with endothelial reactive hyperemia (RH) index and carotid intima medial thickness (CIMT). Participants were healthy, recently menopausal women of the Kronos Early Estrogen Prevention Study (KEEPS) randomized to one of three treatments: oral conjugated equine estrogen (oCEE, 0.45 mg/day), transdermal 17ß-estradiol (tE2, 50 µg/day) each with intermittent oral progesterone or placebo pills and patch (PL). Prostacyclin and thromboxane A2 were assessed by quantification of their stable metabolites (6-keto-prostaglandin F1α, 6-k-PGF1α; thromboxane B2, TXB2), using ELISA. Dense granule ATP secretion from activated platelets was determined by bioluminescence; RH and CIMT were determined by fingertip tonometry and ultrasound, respectively. After 48 months of treatment, platelet content of 6-k-PGF1α and TXB2 was significantly lower in oCEE compared to the PL. Inhibition of ATP secretion by exogenous activation of cAMP associated with platelet 6-k-PGF1α (r = -0.41, P = 0.04) and TXB2 (r = 0.71, P = 0.0005) only in the oCEE group. Serum and platelet content of 6-k-PGF1α and TXB2 associated positively in the PL and tE2 groups. Serum 6-k-PGF1α positively associated with RH in the oCEE group (r = 0.73, P = 0.02), while serum TXB2 positively associated with CIMT in the tE2 group (r = 0.64, P = 0.01). Thus, oCEE and tE2 differentially affect prostanoid-mediated platelet secretory pathways but alone would not account for an increased thrombotic risk for oral MHT. Furthermore, platelet-derived prostanoids may contribute to RH and vascular remodeling in healthy menopausal women.
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BACKGROUND: Pro-coagulant membrane microvesicles (MV) derived from platelets and leukocytes are shed into the circulation following receptor-mediated activation, cell-cell interaction, and apoptosis. Platelets are sentinel markers of toll-like receptor 4 (TLR4) activation. Experiments were designed to evaluate the time course and mechanism of direct interactions between platelets and leukocytes following acute activation of TLR4 by bacterial lipopolysaccharide (LPS). METHODOLOGY/PRINCIPAL FINDINGS: Blood from age-matched male and female wild type (WT) and TLR4 gene deleted (dTLR4) mice was incubated with ultra-pure E. coli LPS (500 ng/ml) for up to one hour. At designated periods, leukocyte antigen positive platelets, platelet antigen positive leukocytes and cell-derived MV were quantified by flow cytometry. Numbers of platelet- or leukocyte-derived MV did not increase within one hour following in vitro exposure of blood to LPS. However, with LPS stimulation numbers of platelets staining positive for both platelet- and leukocyte-specific antigens increased in blood derived from WT but not dTLR4 mice. This effect was blocked by inhibition of TLR4 signaling mediated by My88 and TRIF. Seven days after a single intravenous injection of LPS (500 ng/mouse or 20 ng/gm body wt) to WT mice, none of the platelets stained for leukocyte antigen. However, granulocytes, monocytes and apoptotic bodies stained positive for platelet antigens. CONCLUSIONS/SIGNIFICANCE: Within one hour of exposure to LPS, leukocytes exchange surface antigens with platelets through TLR4 activation. In vivo, leukocyte expression of platelet antigen is retained after a single exposure to LPS following turn over of the platelet pool. Acute expression of leukocyte antigen on platelets within one hour of exposure to LPS and the sustained expression of platelet antigen on leukocytes following a single acute exposure to LPS in vivo explains, in part, associations of platelets and leukocytes in response to bacterial infection and changes in thrombotic propensity of the blood.
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Plaquetas/metabolismo , Vesículas Citoplasmáticas/efectos de los fármacos , Vesículas Citoplasmáticas/metabolismo , Leucocitos/metabolismo , Lipopolisacáridos/farmacología , Receptor Toll-Like 4/metabolismo , Animales , Plaquetas/efectos de los fármacos , Células Cultivadas , Femenino , Antígenos de Histocompatibilidad/metabolismo , Leucocitos/efectos de los fármacos , Masculino , RatonesRESUMEN
Calcifying biologic nanoparticles (NPs) have been implicated as nucleation points for a number of -pathologic events that include vascular calcification and the formation of kidney stones. In order to study these potential relationships, reproducible isolation of well-characterized biologic NPs is a necessity. Our group has isolated and propagated calcifying NPs from several human tissues and renal stones. Specific proteins that could nucleate a calcium phosphate shell under physiologic conditions have been identified as part of their structure, including elongation factor Tu (EF-Tu) and fetuin-A. Visualization, using advanced transmission electron microscopy (TEM), immunofluorescence microscopy, and nuclear and antibody staining in conjunction with flow cytometry, can further elucidate NPs composition and their role in pathology. In order to allow uniform investigation by others, the isolation, culture, and handling procedures for biologic NPs from human calcified vascular tissue and kidney stones are reported in detail.
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Fraccionamiento Químico/métodos , Nanopartículas/análisis , Nanopartículas/química , Western Blotting , Calcinosis/metabolismo , Oxalato de Calcio/metabolismo , Fosfatos de Calcio/metabolismo , Cloroformo/química , ADN/química , ADN/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Citometría de Flujo , Humanos , Inmunohistoquímica , Cálculos Renales/metabolismo , Espectrometría de Masas , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Fenol/químicaRESUMEN
Implanted silicone medical prostheses induce a dynamic sequence of histologic events in adjacent tissue resulting in the formation of a fibrotic peri-prosthetic capsule. In some cases, capsular calcification occurs, requiring surgical intervention. In this study we investigated capsules from silicone gel-filled breast prostheses to test the hypothesis that this calcification might be regulated by the small vitamin K-dependent protein, matrix Gla protein (MGP), a potent inhibitor of arterial calcification, or by Fetuin-A, a hepatocyte-derived glycoprotein also implicated as a regulator of pathologic calcification. Immunolocalization studies of explanted capsular tissue, using conformation-specific antibodies, identified the mineralization-protective γ-carboxylated MGP isomer (cMGP) within cells of uncalcified capsules, whereas the non-functional undercarboxylated isomer (uMGP) was typically absent. Both were upregulated in calcific capsules and co-localized with mineral plaque and adjacent fibers. Synovial-like metaplasia was present in one uncalcified capsule in which MGP species were differentially localized within the pseudosynovium. Fetuin-A was localized to cells within uncalcified capsules and to mineral deposits within calcific capsules. The osteoinductive cytokine bone morphogenic protein-2 localized to collagen fibers in uncalcified capsules. These findings demonstrate that MGP, in its vitamin K-activated conformer, may represent a pharmacological target to sustain the health of the peri-prosthetic tissue which encapsulates silicone breast implants as well as other implanted silicone medical devices.
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Implantes de Mama , Calcinosis , Proteínas de Unión al Calcio/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Geles de Silicona , Proteína Morfogenética Ósea 2/metabolismo , Proteína Morfogenética Ósea 4/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Isomerismo , Microscopía Electrónica de Rastreo , Persona de Mediana Edad , alfa-2-Glicoproteína-HS/metabolismo , Proteína Gla de la MatrizRESUMEN
Alkaline phosphatase (ALP) is an enzyme critical for physiological and pathological biomineralization. Experiments were designed to determine whether ALP participates in the formation of calcifying nanometer sized particles (NPs) in vitro. Filtered homogenates of human calcified carotid artery, aorta and kidney stones were inoculated into cell culture medium containing 10% fetal bovine serum in the absence or presence of inhibitors of ALP or pyrophosphate. A calcific NP biofilm developed within 1 week after inoculation and their development was reduced by pyrophosphate and inhibitors of ALP. ALP protein and enzymatic activity were detected in washed NPs, whether calcified or decalcified. Therefore, ALP activity is required for the formation of calcifying NPs in vitro, as has previously been implicated during pathological calcification in vivo.
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Fosfatasa Alcalina/metabolismo , Nanopartículas , Western Blotting , Medios de Cultivo , Humanos , Microscopía Electrónica de TransmisiónRESUMEN
Calcifying biological nanoparticles (NPs) develop under cell culture conditions from homogenates of diverse tissue samples displaying extraosseous mineralization, including kidney stones and calcified aneurysms. Probes to definitively identify NPs in biological systems are lacking. Therefore, the aim of this study was to begin to establish a proteomic biosignature of NPs in order to facilitate more definitive investigation of their contribution to disease. Biological NPs derived from human kidney stones and calcified aneurysms were completely decalcified by overnight treatment with ethylenediaminetetraacetic acid or brief incubation in HCl, as evidenced by lack of a calcium shell and of Alizarin Red S staining, by transmission electron microscopy and confocal microscopy, respectively. Decalcified NPs contained numerous proteins, including some from bovine serum and others of prokaryotic origin. Most prominent of the latter group was EF-Tu, which appeared to be identical to EF-Tu from Staphylococcus epidermidis. A monoclonal antibody against human EF-Tu recognized a protein in Western blots of total NP lysate, as well as in intact NPs by immunofluorescence and immunogold EM. Approximately 8% of NPs were quantitatively recognized by the antibody using flow cytometry. Therefore, we have defined methods to reproducibly decalcify biological NPs, and identified key components of their proteome. These elements, including EF-Tu, can be used as biomarkers to further define the processes that mediate propagation of biological NPs and their contribution to disease.
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Arterias/patología , Calcinosis/patología , Cálculos Renales/química , Nanopartículas/análisis , Proteoma/análisis , Secuencia de Aminoácidos , Animales , Biomarcadores/metabolismo , Bovinos , Humanos , Datos de Secuencia Molecular , Factor Tu de Elongación Peptídica/genética , Factor Tu de Elongación Peptídica/metabolismoRESUMEN
Platelets derived from aged (reproductively senescent) female mice with genetic deletion of estrogen receptor beta (betaER) are more thrombogenic than those from age-matched wild-type (WT) mice. Intracellular processes contributing to this increased thrombogenicity are not known. Experiments were designed to identify subcellular localization of estrogen receptors and evaluate both glycolytic and mitochondrial energetic processes which might affect platelet activation. Platelets and blood from aged (22-24 months) WT and estrogen receptor beta knockout (betaERKO) female mice were used in this study. Body, spleen weight, and serum concentrations of follicle-stimulating hormone and 17beta-estradiol were comparable between WT and betaERKO mice. Number of spontaneous deaths was greater in the betaERKO colony (50% compared to 30% in WT) over the course of 24 months. In resting (nonactivated) platelets, estrogen receptors did not appear to colocalize with mitochondria by immunostaining. Lactate production and mitochondrial membrane potential of intact platelets were similar in both groups of mice. However, activities of NADH dehydrogenase, cytochrome bc ( 1 ) complex, and cytochrome c oxidase of the electron transport chain were reduced in mitochondria isolated from platelets from betaERKO compared to WT mice. There were a significantly higher number of phosphatidylserine-expressing platelet-derived microvesicles in the plasma and a greater thrombin-generating capacity in betaERKO compared to WT mice. These results suggest that deficiencies in betaER affect energy metabolism of platelets resulting in greater production of circulating thrombogenic microvesicles and could potentially explain increased predisposition to thromboembolism in some elderly females.
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Plaquetas/metabolismo , Receptor beta de Estrógeno/metabolismo , Mitocondrias/metabolismo , Animales , Peso Corporal , Complejo III de Transporte de Electrones/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Metabolismo Energético/fisiología , Ensayo de Inmunoadsorción Enzimática , Estradiol/sangre , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Hormona Folículo Estimulante/sangre , Lactatos/sangre , Ratones , Ratones Endogámicos , NADH Deshidrogenasa/metabolismo , Tamaño de los Órganos , Oxígeno/metabolismo , Agregación PlaquetariaRESUMEN
AIM: Microbial biofilm matrix contains polysaccharides and proteins and can require extracellular nucleic acids for initial formation. Experiments were designed to identify infectious pathogens in human aneurysms and to characterize biofilm formed by calcified human arterial-derived nanoparticles. MATERIALS & METHOD: A total of 26 different microbial pathogens were isolated from 48 inflammatory aneurysms. Consistent amounts (0.49 McFarland units) of nanoparticles derived from similar tissue were seeded into 24-well plates and cultured for 21 days in the absence (control) or presence of RNase, tetracycline or gentamicin. RESULTS: Control biofilm developed within 14 days, as detected by concanavalin A and BacLight Green staining. The formation of biofilm in wells treated with RNase was not different from the control; however, gentamicin partially inhibited and tetracycline completely inhibited biofilm formation. Therefore, nanoparticle biofilm retains some characteristics of conventional bacterial biofilm and requires protein-calcium interactions, although extracellular RNA is not required. CONCLUSION: This model system may also allow study of nanosized vesicles derived from donor tissue, including any microbes present, and could provide a useful tool for in vitro investigation of nanoparticle biofilm formation.
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Aneurisma/microbiología , Biopelículas/crecimiento & desarrollo , Nanopartículas/microbiología , Humanos , Técnicas In VitroRESUMEN
AIM: Nanosized particles (NPs) enriched in hydroxyapatite and protein isolated from calcified human tissue accelerate occlusion of endothelium-denuded arteries when injected intravenously into rabbits. Since platelet aggregation and secretory processes participate in normal hemostasis, thrombosis and vascular remodeling, experiments were designed to determine if these biologic NPs alter specific platelet functions in vitro. METHODS: Platelet-rich plasma was prepared from citrate anticoagulated human blood. Platelet aggregation and ATP secretion were monitored in response to thrombin receptor agonists peptide (10 microM) or convulxin (50 microg/ml) prior to and following 15 min incubation with either control solution, human-derived NPs, bovine-derived NPs or crystals of hydroxyapatite at concentrations of 50 and 150 nephelometric turbidity units. RESULTS: Incubation of platelets for 15 min with either human- or bovine-derived NPs reduced aggregation induced by thrombin receptor activator peptide and convulxin in a concentration-dependent manner. Hydroxyapatite caused a greater inhibition than either of the biologically derived NPs. Human-derived NPs increased ATP secretion by unstimulated platelets during the 15 min incubation period. CONCLUSION: Effects of bovine-derived and hydroxyapatite NPs on basal release of ATP were both time and concentration dependent. These results suggest that biologic NPs modulate both platelet aggregation and secretion. Biologically derived NPs could modify platelet responses within the vasculature, thereby reducing blood coagulability and the vascular response to injury.
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Materiales Biocompatibles/farmacología , Durapatita/farmacología , Nanomedicina/métodos , Nanopartículas/química , Agregación Plaquetaria/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Adulto , Anciano , Animales , Materiales Biocompatibles/química , Materiales Biocompatibles/uso terapéutico , Bovinos , Durapatita/química , Durapatita/uso terapéutico , Femenino , Humanos , Masculino , Microscopía Confocal , Microscopía Fluorescente , Microscopía de Túnel de Rastreo , Persona de Mediana Edad , Nanopartículas/uso terapéutico , Nanopartículas/ultraestructura , Plasma Rico en Plaquetas/efectos de los fármacos , Plasma Rico en Plaquetas/metabolismo , ConejosRESUMEN
Experiments were designed to test the hypothesis that the systemic delivery of planktonic forms of nanoparticles (NPs) derived from calcified, diseased human tissue or bovine blood are transmissible particles that exacerbate arterial response to injury. New Zealand White rabbits in which the endothelium was mechanically removed from one carotid artery were injected intravenously with either saline (control), lipopolysaccharide (LPS; surrogate for subclinical infection), hydroxyapatite crystals (HA; surrogate for NP shell), HA crystals exposed to culture media, or planktonic forms of bovine- or human-derived NPs. Carotid arteries were monitored by ultrasonography for 5 wk and then removed for histological examination. Uninjured arteries from all animals in each group remained patent with a normal anatomy. Injured arteries from 6 of 11 animals injected with human-derived NPs occluded and/or calcified; none of the injured arteries from animals in the other groups occluded (n = 28; P < or = 0.05). Injured arteries of rabbits injected with LPS or HA crystals developed eccentric hyperplasia. Discontinuous internal elastic laminae and thinning media characterized arteries from animals injected with bovine-derived NPs or cultured HA crystals. In conclusion, the systemic administration of planktonic forms of human-derived NPs exacerbated arterial response to injury distinct from that of bovine-derived NPs and other inflammatory agents.
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Aneurisma de la Aorta/patología , Calcinosis/patología , Traumatismos de las Arterias Carótidas/patología , Durapatita/toxicidad , Nanopartículas/toxicidad , Albúmina Sérica Bovina/toxicidad , Animales , Traumatismos de las Arterias Carótidas/diagnóstico por imagen , Traumatismos de las Arterias Carótidas/etiología , Bovinos , Modelos Animales de Enfermedad , Durapatita/administración & dosificación , Tejido Elástico/patología , Humanos , Hiperplasia , Inyecciones Intravenosas , Lipopolisacáridos/administración & dosificación , Masculino , Nanopartículas/administración & dosificación , Conejos , Albúmina Sérica Bovina/administración & dosificación , Factores de Tiempo , Técnicas de Cultivo de Tejidos , Túnica Media/patología , UltrasonografíaRESUMEN
Vascular complications are the leading cause of morbidity and mortality in autosomal dominant polycystic kidney disease. Although evidence suggests an abnormal vascular reactivity, contractile function in Pkd mutant vessels has not been studied previously. Contractile response to phenylephrine (PE; 10(-10) to 10(-4)M), an alpha1-adrenergic receptor agonist, was examined. De-endothelialized Pkd2(+/-) aortic rings generated a higher maximum force (F(max)) than that in wild-type (wt; 5.78 +/- 0.73 versus 2.69 +/- 0.43 mN; P < 0.001) and a significant left shift in PE dosage-response curve. On simultaneous recordings, Pkd2(+/-) aortic helical strips also responded to PE with a greater F(max) but a lesser [Ca(2+)](i) rise, resulting in a greatly enhanced Deltaforce/DeltaCa(2+) ratio than that in wt. At F(max), a higher elevation in the phosphorylated regulatory myosin light chain was observed in Pkd2(+/-) strips. Ca(2+)-dependent calmodulin/myosin light-chain kinase-mediated contraction was examined by direct Ca(2+) (pCa8-5) stimulation to beta-escin permeabilized aortic strips; the pCa-force curve in Pkd2(+/-) strips was not shifted, thereby indicating that PE induced dosage-response alteration that resulted from Ca(2+)-independent mechanisms. Quantitative analyses of contractile proteins demonstrated elevated expressions in smooth muscle alpha-actin and myosin heavy chain in Pkd2(+/-) arteries, changes that likely contribute to the higher F(max). Similar to those in aortas, de-endothelialized Pkd2(+/-) resistance (fourth-order mesenteric) arteries responded to PE with a stronger contraction but a lesser [Ca(2+)](i) rise than in wt. Taken together, the arterial vasculature in Pkd2(+/-) mice exhibits an exaggerated contractile response and increased sensitivity to PE. An enhanced Ca(2+)-independent force generation and elevated contractile protein expression likely contribute to these abnormalities.
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Músculo Liso Vascular/fisiopatología , Fenilefrina/farmacología , Canales Catiónicos TRPP , Vasoconstricción/fisiología , Animales , Aorta/efectos de los fármacos , Aorta/fisiología , Calcio/fisiología , Escina/farmacología , Ratones , Ratones Noqueados , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Músculo Liso Vascular/enzimología , Vasoconstricción/efectos de los fármacosRESUMEN
BACKGROUND AND PURPOSE: The purpose of this study was to determine whether implanting exogenous fibroblasts on platinum coils could enhance intra-aneurysmal fibrosis. Hypotheses included: (1) fibroblast-coated (FBC) platinum coils can improve angiographic results after embolization; and (2) FBC platinum coils can accelerate histological healing of embolized aneurysms. METHODS: Experimental aneurysms in rabbits were embolized with control platinum coils (n=18) or FBC coils (n=18). Subjects were euthanized at 14 days, 1 month, 3 months and 6 months after implantation. Digital subtraction angiography was used to evaluate stability after embolization. Histological samples were examined with a grading system (range, 0 to 12) based on neck and dome healing. RESULTS: Histology total scores and fibrosis ratio at 14 days were significantly greater in the FBC coil group compared with controls (6.6+/-1.9 versus 2.5+/-1.1, 1.2+/-0.6% versus 0.2+/-0.3%, respectively; P=0.0090). Cavities embolized with FBC coils showed cellular proliferation and thrombus organization, with an endothelialized membrane bridging the neck. There were no differences between groups in the later timepoints. The FBC coil group showed radiographic stability in 11 (61%) cases, coil compaction in 2 (11%) cases, and progressive occlusion in 5 (28%) cases. No progressive occlusion was seen in controls; 3 (17%) of 18 control cases exhibited coil compaction (P=0.0546). CONCLUSIONS: FBC coils can accelerate early histological healing compared with control coils in the rabbit aneurysm model.
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Embolización Terapéutica/instrumentación , Embolización Terapéutica/métodos , Fibroblastos/trasplante , Aneurisma Intracraneal/terapia , Prótesis e Implantes/tendencias , Actinas/metabolismo , Animales , Biomarcadores , Células Cultivadas , Angiografía Cerebral , Modelos Animales de Enfermedad , Femenino , Fibroblastos/citología , Fibroblastos/fisiología , Aneurisma Intracraneal/diagnóstico por imagen , Aneurisma Intracraneal/patología , Masculino , Mioblastos del Músculo Liso , Platino (Metal)/uso terapéutico , Prótesis e Implantes/normas , Conejos , Resultado del Tratamiento , Vimentina/metabolismo , Cicatrización de Heridas/fisiologíaRESUMEN
Cardiovascular complications are the leading cause of morbidity and mortality in autosomal dominant polycystic kidney disease. Pkd2+/- vascular smooth muscle cells (VSMCs) have an abnormal phenotype and defective intracellular Ca2+ ([Ca2+]i) regulation. We examined cAMP content in vascular smooth muscles from Pkd2+/- mice because cAMP is elevated in cystic renal epithelial cells. We found cAMP concentration was significantly increased in Pkd2+/- vessels compared with wild-type vessels. Furthermore, reducing the wild-type VSMC [Ca2+]i by Verapamil or BAPTA-AM significantly increased cellular cAMP concentration (mainly by phosphodiesterase [PDE] inhibition), the rate of VSMC proliferation (determined by direct cell counting, 3H-incorporation, FACS analysis of cells entering S phase, and quantitative Western PCNA and ERK1/2 analyses), and the rate of apoptosis (by Hoechst staining, FACS analysis of the Annexin-V positive cells, and quantitative Western Bax, cytochrome c, and activated caspase 9 and 3 analyses). The low [Ca2+]i induced VSMC proliferation was independent of cAMP/B-Raf signaling, while that of apoptosis was promoted by cAMP. In summary, Pkd2+/- VSMCs have elevated cAMP levels. This elevation can also be induced by reducing [Ca2+]i in wild-type VSMCs. The [Ca2+]i reduction and cAMP accumulation can cause an increase in both cellular proliferation and apoptosis, resembling Pkd mutant phenotype.
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Apoptosis , Calcio/metabolismo , Proliferación Celular , Proteínas de la Membrana/genética , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , 3',5'-AMP Cíclico Fosfodiesterasas/genética , Animales , Células Cultivadas , AMP Cíclico/análisis , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Proteínas de la Membrana/fisiología , Ratones , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas B-raf/análisis , Proteínas Proto-Oncogénicas c-akt , Canales Catiónicos TRPPRESUMEN
Autosomal-dominant polycystic kidney disease is a multiorgan disease and its vascular manifestations are common and life-threatening. Despite this, little is known about their pathogenesis. Somatic mutations to the normal PKD allele in cystic epithelia and cyst development associated with the unstable Pkd2(WS25) allele suggest a two-hit model of cystogenesis. However, it is unclear if this model can account for the cardiovascular pathology or if haploinsufficiency alone is disease-associated. In the present study, we found a decreased polycystin-2 (PC2, protein encoded by Pkd2 gene) expression in Pkd2( +/-) vessels, roughly half the wild-type level, and an enhanced level of intracranial vascular abnormalities in Pkd2 (+/-) mice when induced to develop hypertension. Consistent with these observations, freshly dissociated Pkd2 (+/-) vascular smooth muscle cells have significantly altered intracellular Ca(2+) homeostasis. The resting [Ca(2+)](i) is 17.1% lower in Pkd2 (+/-) compared with wild-type cells (P=0.0003) and the total sarcoplasmic reticulum Ca(2+) store (emptied by caffeine plus thapsigargin) is decreased (P<0.0001). The store operated Ca(2+) (SOC) channel activity is also decreased in Pkd2 (+/-) cells (P=0.008). These results indicate that inactivation of just one Pkd2 allele is sufficient to significantly alter intracellular Ca(2+) homeostasis, and that PC2 is necessary to maintain normal SOC activity and the SR Ca(2+) store in VSMCs. Based on these findings, and the fact that [Ca(2+)](i) signaling is essential to the regulation of contraction, production and secretion of extracellular matrix, cellular proliferation and apoptosis, we propose that the abnormal intracellular Ca(2+) regulation associated with Pkd2 haploinsufficiency is directly related to the vascular phenotype.
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Alelos , Señalización del Calcio , Proteínas de la Membrana/deficiencia , Músculo Liso Vascular/metabolismo , Riñón Poliquístico Autosómico Dominante/genética , Animales , Western Blotting , Calcio/metabolismo , Cartilla de ADN , Modelos Animales de Enfermedad , Técnica del Anticuerpo Fluorescente , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Mutantes , Reacción en Cadena de la Polimerasa , Canales Catiónicos TRPPAsunto(s)
Anestésicos por Inhalación/farmacología , Corazón/fisiología , Intercambiador de Sodio-Calcio/fisiología , Envejecimiento , Animales , Animales Recién Nacidos , Calcio/metabolismo , Halotano/farmacología , Corazón/efectos de los fármacos , Corazón/crecimiento & desarrollo , Ventrículos Cardíacos , Isoproterenol/farmacología , Células Musculares/efectos de los fármacos , Células Musculares/fisiología , Ratas , Ratas Sprague-Dawley , Sodio/metabolismo , Intercambiador de Sodio-Calcio/efectos de los fármacosRESUMEN
BACKGROUND: In neonatal heart, plasma membrane Na+-Ca2+ exchange (NCX) and Ca2+ influx channels play greater roles in intracellular Ca2+ concentration [Ca2+]i regulation compared with the sarcoplasmic reticulum (SR). In neonatal (aged 0-3 days) and adult (aged 84 days) rat cardiac myocytes, we determined the mechanisms underlying greater sensitivity of the neonatal myocardium to inhibition by volatile anesthetics. METHODS: The effects of 1 and 2 minimum alveolar concentration halothane and sevoflurane on Ca2+ influx during electrical stimulation in the presence or blockade of NCX and the Ca2+ channel agonist BayK8644 were examined. [Ca2+]i responses to caffeine were used to examine anesthetic effects on SR Ca2+ release (via ryanodine receptor channels) and reuptake (via SR Ca2+ adenosine triphosphatase). Ca2+ influx via NCX was examined during rapid activation in the presence of the reversible SR Ca2+ adenosine triphosphatase inhibitor cyclopiazonic acid and ryanodine to inhibit the SR. Efflux mode NCX was examined during activation by extracellular Na+ in the absence of SR reuptake. RESULTS: Intracellular Ca2+ concentration transients during electrical stimulation were inhibited to a greater extent in neonates by halothane (80%) and sevoflurane (50%). Potentiation of [Ca2+]i responses by BayK8644 (160 and 120% control in neonates and adults, respectively) was also blunted by anesthetics to a greater extent in neonates. [Ca2+]i responses to caffeine in neonates ( approximately 30% adult responses) were inhibited to a lesser extent compared with adults (35 vs. 60% by halothane). Both anesthetics inhibited Ca2+ reuptake at 2 minimum alveolar concentration, again to a greater extent in adults. Reduction in NCX-mediated influx was more pronounced in neonates (90%) compared with adults (65%) but was comparable between anesthetics. Both anesthetics also reduced NCX-mediated efflux to a greater extent in neonates. Potentiation of NCX-mediated Ca2+ efflux by extracellular Na+ and NCX-mediated Ca2+ influx by intracellular Na+ were both prevented by halothane, especially in neonates. CONCLUSIONS: These data indicate that greater myocardial depression in neonates induced by volatile anesthetics may be mediated by inhibition of NCX and Ca2+ influx channels rather than inhibition of SR Ca2+ release.
Asunto(s)
Anestésicos por Inhalación/farmacología , Corazón/efectos de los fármacos , Miocardio/metabolismo , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Factores de Edad , Animales , Animales Recién Nacidos , Cafeína/farmacología , Calcio/metabolismo , Estimulación Eléctrica , Ratas , Ratas Sprague-Dawley , Retículo Sarcoplasmático/metabolismo , Sodio/metabolismo , Intercambiador de Sodio-Calcio/fisiologíaRESUMEN
We hypothesized that 1) hypothyroidism (Hyp) decreases myosin heavy chain (MHC) content per half-sarcomere in diaphragm muscle (Dia(m)) fibers, 2) Hyp decreases the maximum specific force (F(max)) of Dia(m) fibers because of the reduction in MHC content per half-sarcomere, and 3) Hyp affects MHC content per half-sarcomere and F(max) to a greater extent in fibers expressing MHC type 2X (MHC(2X)) and/or MHC type 2B (MHC(2B)). Studies were performed on single Triton X-permeabilized fibers activated at pCa 4.0. MHC content per half-sarcomere was determined by densitometric analysis of SDS-polyacrylamide gels and comparison with a standard curve of known MHC concentrations. After 3 wk of Hyp, MHC content per half-sarcomere was reduced in fibers expressing MHC(2X) and/or MHC(2B). On the basis of electron-microscopic analysis, this reduction in MHC content was also reflected by a decrease in myofibrillar volume density and thick filament density. Hyp decreased F(max) across all MHC isoforms; however, the greatest decrease occurred in fibers expressing fast MHC isoforms (approximately 40 vs. approximately 20% for fibers expressing slow MHC isoforms). When normalized for MHC content per half-sarcomere, force generated by Hyp fibers expressing MHC(2A) was reduced compared with control fibers, whereas force per half-sarcomere MHC content was higher for fibers expressing MHC(2X) and/or MHC(2B) in the Hyp Dia(m) than for controls. These results indicate that the effect of Hyp is more pronounced on fibers expressing MHC(2X) and/or MHC(2B) and that the reduction of F(max) with Hyp may be at least partially attributed to a decrease in MHC content per half-sarcomere but not to changes in force per cross bridge.