RESUMO
Subacute ruminal acidosis is induced by high concentrations of short-chain fatty acids (SCFA, mainly acetate, propionate, and butyrate) that release protons to decrease the pH of the ruminal digesta. This low pH, in turn, is thought to damage epithelial barrier function. The present study applied a model of simulated ruminal acidosis ex vivo to investigate if SCFA directly contribute to epithelial barrier failure beyond their role as proton donors. Epithelial tissues from the rumen of slaughtered sheep were mounted in Ussing chambers and incubated under 3 different conditions. Two groups were incubated in the absence of SCFA at mucosal pH 6.1 (control) and pH 5.1, respectively, for 7 h. A third group was first incubated in a mucosal solution containing 100 mM SCFA at pH 5.1 for 2 h and, thereafter, in a mucosal solution without SCFA at pH 6.1 for the remaining 5 h. Transepithelial conductance (Gt), short-circuit current (Isc), and fluorescein fluxes were determined. After 7 h of incubation, the expression levels of claudin-1, claudin-4, claudin-7, and occludin were measured by quantitative reverse-transcription PCR and Western blot. Furthermore, the local distribution of these tight junction (TJ) proteins was examined by confocal laser scanning microscopy. A 7-h incubation at pH 5.1 in the absence of SCFA did not influence either Gt or fluorescein flux rates of ruminal tissues ex vivo compared with the control. In contrast, incubation at pH 5.1 with SCFA for only 2 h induced increases in Gt and fluorescein flux rates that continued even after tissues were returned back to pH 6.1. Expression analysis showed that pH 5.1 without SCFA for 7 h induced no changes in mRNA expression of claudin-1, claudin-4, claudin-7, and occludin and a selective decrease in protein expression of only claudin-4 compared with the control. However, a 2-h incubation at pH 5.1 in the presence of SCFA decreased the mRNA-expression of claudin-7, as well as the protein expression of claudin-4, claudin-7, and occludin. The decreased expression of these TJ proteins in the group incubated with SCFA was also evident in immunohistochemistry. Immunohistochemistry additionally evidenced a considerable retraction of all tested TJ proteins out of the TJ in that group. We conclude that a low mucosal pH of 5.1 is tolerated well by ruminal epithelia for several hours. However, a low pH in combination with SCFA induces damage to the TJ and disturbs barrier function, which is not immediately reversible upon the removal of the acidotic insult.
Assuntos
Acidose/veterinária , Ácidos Graxos Voláteis/fisiologia , Rúmen/metabolismo , Doenças dos Ovinos/fisiopatologia , Gastropatias/veterinária , Acidose/fisiopatologia , Animais , Epitélio/fisiopatologia , Concentração de Íons de Hidrogênio , Rúmen/química , Rúmen/fisiopatologia , Ovinos , Gastropatias/fisiopatologiaRESUMO
Dairy cows can have different degrees of hypocalcaemia around calving. Lowering dietary Ca availability before calving can prevent it. Rice bran, treated for lower rumen degradability of phytic acid can reduce dietary availability of Ca. During 3 periods of 3 weeks, 113 multiparous cows calved in a single close-up group, which was fed first a control diet, then 140 g/kg DM of rumen-protected rice bran, and at last the control diet again. Cows joined the group 3 weeks before expected calving date and left it at calving. Blood samples were taken weekly before parturition and 0, 6 and 12 h after calving, as well as 3 and 28 d in lactation. Serum was analysed for Ca, Mg, and P. Rice bran introduction produced a transient serum Ca decrease. Rice bran feeding reduced serum P and its withdrawal reduced serum Mg. Serum Ca at calving, nadir of serum Ca and serum Ca the first 3 d after calving was higher in cows calving during rice bran feeding. Serum P decreased less and recovered faster after calving when cows had been fed rice bran. Rumen-protected rice bran reduced dietary availability of Ca and induced adaptation of Ca metabolism resulting in improved Ca and P homoeostasis at calving.
Assuntos
Ração Animal , Doenças dos Bovinos/prevenção & controle , Hipocalcemia/veterinária , Oryza , Rúmen/metabolismo , Animais , Cálcio/sangue , Cálcio da Dieta/administração & dosagem , Cálcio da Dieta/farmacocinética , Bovinos , Indústria de Laticínios , Feminino , Hipocalcemia/prevenção & controle , Lactação , Paridade , Parto , Fósforo/sangue , Ácido Fítico/metabolismo , GravidezRESUMO
Ruminal fermentation products such as short-chain fatty acids (SCFA) and CO2 acutely stimulate urea transport across the ruminal epithelium in vivo, whereas ammonia has inhibitory effects. Uptake and signaling pathways remain obscure. The ruminal expression of SLC14a1 (UT-B) was studied using polymerase chain reaction (PCR). The functional short-term effects of ammonia on cytosolic pH (pHi) and ruminal urea transport across native epithelia were investigated using pH-sensitive microelectrodes and via flux measurements in Ussing chambers. Two variants (UT-B1 and UT-B2) could be fully sequenced from ovine ruminal cDNA. Functionally, transport was passive and modulated by luminal pH in the presence of SCFA and CO2, rising in response to luminal acidification to a peak value at pH 5.8 and dropping with further acidification, resulting in a bell-shaped curve. Presence of ammonia reduced the amplitude, but not the shape of the relationship between urea flux and pH, so that urea flux remained maximal at pH 5.8. Effects of ammonia were concentration dependent, with saturation at 5 mmol/l. Clamping the transepithelial potential altered the inhibitory potential of ammonia on urea flux. Ammonia depolarized the apical membrane and acidified pHi, suggesting that, at physiological pH (< 7), uptake of NH4 (+) into the cytosol may be a key signaling event regulating ruminal urea transport. We conclude that transport of urea across the ruminal epithelium involves proteins subject to rapid modulation by manipulations that alter pHi and the cytosolic concentration of NH4 (+). Implications for epithelial and ruminal homeostasis are discussed.
Assuntos
Amônia/farmacologia , Prótons , Rúmen/metabolismo , Ovinos/metabolismo , Ureia/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Dióxido de Carbono/metabolismo , Relação Dose-Resposta a Droga , Ácidos Graxos Voláteis/metabolismo , Feminino , Concentração de Íons de Hidrogênio , Proteínas de Membrana Transportadoras/metabolismo , Rúmen/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transportadores de UreiaRESUMO
Periparturient hypocalcaemia (milk fever) is a disorder of Ca metabolism in dairy cattle primarily affecting multiparous cows. The major reasons for the rapid decrease of blood Ca concentration after calving are the prompt increase of Ca secretion into the colostrum and the delayed activation of Ca regulation mechanisms including calcitriol, a metabolite of vitamin D. In man, vitamin D receptor (VDR) gene polymorphisms are reported to be associated with disturbances of Ca metabolism, whereas data confirming the same in dairy cows are still missing. Moreover, polymorphisms that only affect non-coding regions are sometimes difficult to ascribe to a specific disorder as pathways and unequivocal links remain elusive. Therefore, the idea of the present study was to investigate in a small group of dairy cows with documented clinical records whether polymorphisms in the coding regions of the VDR gene existed and whether these potentially found variations were correlated with the incidence of periparturient hypocalcaemia. For this purpose, blood DNA was isolated from 26 dairy cows in their 4th to 6th lactation, out of which 17 had experienced hypocalcaemia at least once, whereas 9 cows had never undergone periparturient hypocalcaemia in their lifetime. The 10 VDR exons and small parts of adjacent introns were sequenced and compared with the Bos taurus VDR sequence published on NCBI based on the DNA of one Hereford cow. In total, 8 sequence alterations were detected in the fragments, which were primarily heterozygous. However, only 4 of them were really located on exons thereby potentially causing changes of the encoded amino acid of the VDR protein, but were not correlated with the incidence of periparturient hypocalcaemia. Certainly, this lack of statistical correlation could be due to the small number of animals included; anyhow, it was not encouraging enough to initiate a larger study with hundreds of cows and document blood Ca levels post partum for at least four lactations.
Assuntos
Alelos , Doenças dos Bovinos/genética , Predisposição Genética para Doença/genética , Hipocalcemia/veterinária , Complicações na Gravidez/veterinária , Receptores de Calcitriol/genética , Animais , Bovinos , DNA/sangue , Feminino , Variação Genética/genética , Hipocalcemia/genética , Parto , Polimorfismo Genético/genética , Gravidez , Complicações na Gravidez/genéticaRESUMO
[This corrects the article DOI: 10.1016/j.vas.2017.04.001.].
RESUMO
A protein of approximately 70-kDa was identified as a candidate Na+/Mg2+ exchanger in rumen epithelial cells (REC). Melastatin-related Transient Receptor Potential 7 (TRPM7) and Magnesium Transporter 1 (MagT1) transcripts and, from them, encoded proteins were also detected. The regulation of these Mg transport pathways by extracellular [Mg] changes was the main focus of this study. Therefore, a 24-h pre-incubation of ovine REC in control (1.2 mM), low (0.12 mM)-Mg, and high (5 mM)-Mg medium was performed. Na+/Mg2+ exchangers, TRPM7 and MagT1 abundance and activity were investigated by Western blot analysis, flow cytometry, immunocytochemistry and fluorescence spectroscopic measurements of [Mg2+]i changes. Inhibitors were employed to differentiate Na+/Mg2+ exchanger-mediated (imipramine) and channel-mediated (cobalt(III)hexaammine, nitrendipine) Mg transport. Basal [Mg2+]i (0.40 +/- 0.02 mM) was not influenced by pre-incubation in low- or high-Mg medium. However, compared with control REC (4.1 +/- 0.7 microM/min), such cells showed reduced (2.8 +/- 0.6 microM/min) or elevated (6.4 +/- 0.9 microM/min) Mg extrusion rates that correlated with a decreased (25%) and increased (38%) expression of the putative Na+/Mg2+ exchanger protein, respectively. Low- and high-Mg pre-incubated REC were both characterized by an increased (30-40%) influx capacity. In low-Mg REC, the latter resulted mainly from a strong activation of the TRPM7-related transport component. The data thus clearly demonstrate the intrinsic regulation of REC transmembrane Mg transport.
Assuntos
Células Epiteliais/metabolismo , Magnésio/metabolismo , Rúmen/citologia , Animais , Células Cultivadas , Citometria de Fluxo , Imuno-Histoquímica , Magnésio/química , Proteínas Serina-Treonina Quinases , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rúmen/metabolismo , Transdução de Sinais , Canais de Cátion TRPM/genéticaRESUMO
OBJECTIVE: Despite the success of antiproliferative therapies, restenosis remains a common problem after percutaneous transluminal coronary angioplasty (PTCA). Longer-term clinical results of brachytherapy (intracoronary radiation), the lack of long-term clinical results after implantation of drug eluting stents, and the occurrence of late thrombosis after both procedures leave room for skepticism. Neointimal proliferation is not substantially inhibited at late time points after brachytherapy, and late lumen loss with a "catch-up" proliferation can occur. We hypothesized that the transcription factors nuclear factor-{kappa}B (NF-kappaB) and activator protein-1 (AP-1) are involved in these processes. We addressed the role of these mediators in a porcine model of coronary restenosis. METHODS: Thirty-nine pigs underwent PTCA in two major coronary arteries. One of the two balloon-injured arteries was randomly assigned to receive immediate 20 Gy beta-irradiation (Brachy group) using a noncentered source train ((90)Sr/Y Beta-Cath, Novoste). Animals were sacrificed after 1 day, 14 days, or 28 days. Proliferating cells were labeled prior to euthanasia. RESULTS: At late time points, lumen area was significantly smaller and the inflammatory response was more pronounced in the Brachy group than in the PTCA group. These findings coincided with sustained activation of MMP-9 and transcription factors like NF-kappaB and AP-1. Initially, cell proliferation was reduced in the Brachy group; however, at late time points, differences between the two treatment groups were no longer significant. CONCLUSIONS: Brachytherapy initially inhibits cell proliferation; however, cellular and molecular inflammatory processes (e.g. activation of NF-kappaB) are enhanced within the arterial wall. This proinflammatory side effect may be responsible for the observed delayed proliferation and the resulting lumen loss.
Assuntos
Reestenose Coronária/metabolismo , Reestenose Coronária/prevenção & controle , Vasos Coronários/metabolismo , NF-kappa B/metabolismo , Fator de Transcrição AP-1/metabolismo , Angioplastia Coronária com Balão , Animais , Partículas beta , Braquiterapia , Proliferação de Células , Terapia Combinada , Doença das Coronárias/metabolismo , Doença das Coronárias/radioterapia , Doença das Coronárias/terapia , Reestenose Coronária/patologia , Vasos Coronários/química , Vasos Coronários/patologia , Ensaio de Desvio de Mobilidade Eletroforética , Imuno-Histoquímica , Macrófagos/patologia , Metaloproteinase 9 da Matriz/análise , Metaloproteinase 9 da Matriz/metabolismo , Modelos Animais , NF-kappa B/análise , Distribuição Aleatória , Radioisótopos de Selênio , Sus scrofa , Suínos , Linfócitos T/patologia , Fatores de Tempo , Fator de Transcrição AP-1/análiseRESUMO
The aim of this study was to test whether a combination of plant bioactive lipid compounds (also termed 'essential oils') and biotin (PBLC+B) could decrease the mobilization of body reserves and ketosis incidence in postpartum dairy cows. We compared non-supplemented control (CON) cows with cows receiving monensin (MON) as a controlled-release capsule at d -21, and with cows receiving PBLC+B from day (d) -21 before calving until calving (Phase 1) and further until d 37 after calving (Phase 2), followed by PBLC+B discontinuation from d 38 to d 58 (Phase 3). The PBLC+B cows had higher body weight and higher back fat thickness than CON cows and lesser body weight change than MON and CON cows in Phase 3. Body condition score was not different among groups. Milk protein concentration tended to be higher on the first herd test day in PBLC+B vs. CON cows. Milk fat concentration tended to be highest in PBLC+B cows throughout Phases 2 and 3, with significantly higher values in PBLC+B vs. MON cows on the second herd test day. Yields of energy-corrected milk were higher in PBLC+B vs. CON and MON cows in Phase 2 and higher in PBLC+B and MON cows vs. CON cows in Phase 3. The incidence of subclinical ketosis was 83%, 61% and 50% in CON, PBLC+B and MON cows, respectively, with lower mean ß-hydroxybutyrate values in MON than in PBLC+B cows in Phase 1 prepartum. The serum triglyceride concentration was higher in PBLC+B vs. CON cows on d 37. No differences were observed in serum glucose, urea, non-esterified fatty acids, cholesterol and bilirubin concentrations. Aspartate transaminase and γ-glutamyltranspeptidase but not glutamate dehydrogenase activities tended to be highest in MON and lowest in PBLC+B in Phase 2. We conclude that PBLC+B prevent body weight loss after parturition and are associated with similar ketosis incidence and partly higher yields of energy-corrected milk compared to MON supplementation of dairy cows.
Assuntos
Biotina/farmacologia , Indústria de Laticínios , Metabolismo Energético/efeitos dos fármacos , Lipídeos/farmacologia , Leite/metabolismo , Monensin/farmacologia , Plantas/química , Ração Animal/análise , Animais , Análise Química do Sangue , Peso Corporal/efeitos dos fármacos , Bovinos , Interações Medicamentosas , Feminino , Cetose/metabolismo , Lactação/efeitos dos fármacosRESUMO
BACKGROUND: The short-term results for the prevention of coronary restenosis after intravascular brachytherapy (IVBT) and use of drug-eluting stents (DESs) are excellent. The long-term results either lack or present with late complications (e.g., late thrombosis and late catch-up phenomenon leading to late restenosis even years after the initial procedure). Both IVBT and DESs mediate their potent antirestenotic effects via a cytostatic mechanism, but the cardiovascular pathology at late time points after the use of these antiproliferative therapies is incompletely understood. This study investigated the long-term effects of antiproliferative beta-irradiation in a clinically relevant porcine coronary model to address the pathophysiology of late coronary restenosis after antiproliferative vascular interventions. METHODS: We performed percutaneous transluminal coronary angioplasty (PTCA) in two major coronary arteries in 12 domestic crossbred pigs. One of the two balloon-injured segments was randomly assigned to receive immediate beta-irradiation (PTCA+IVBT group) using a noncentered delivery catheter (20 Gy; Novoste Beta-Cath System, Novoste, Norcross, GA, USA). The animals were sacrificed after 14 days (n=6) or 3 months (n=6). RESULTS: The luminal area in the PTCA+IVBT group decreased significantly 3 months after the intervention as compared with that in the PTCA group (PTCA 3.45+/-0.46 mm2 vs. PTCA+IVBT 1.22+/-0.26 mm2; P=.0017). This lumen loss was primarily due to shrinkage of the external elastic lamina area (negative arterial remodeling; PTCA 5.22+/-0.27 mm2 vs. PTCA+IVBT 3.42+/-0.45 mm2; P=.0064), which was accompanied by an increase in the adventitial area (PTCA 3.07+/-0.2 mm2 vs. PTCA+IVBT 5.41+/-0.5 mm2; P=.0049). CONCLUSIONS: The application of antiproliferative radiation in a porcine coronary model caused an early beneficial effect (reduction of intimal-medial lesion and luminal gain) that was followed by a late lumen loss primarily due to negative arterial remodeling. This mechanism may in part help us understand the pathophysiology of late adverse events occurring after IVBT.
Assuntos
Angioplastia Coronária com Balão/efeitos adversos , Braquiterapia/efeitos adversos , Proliferação de Células/efeitos da radiação , Reestenose Coronária/radioterapia , Vasos Coronários/efeitos da radiação , Túnica Íntima/efeitos da radiação , Túnica Média/efeitos da radiação , Animais , Partículas beta , Braquiterapia/métodos , Tecido Conjuntivo/patologia , Tecido Conjuntivo/efeitos da radiação , Reestenose Coronária/etiologia , Reestenose Coronária/patologia , Vasos Coronários/lesões , Vasos Coronários/patologia , Modelos Animais de Doenças , Tecido Elástico/patologia , Tecido Elástico/efeitos da radiação , Projetos de Pesquisa , Sus scrofa , Fatores de Tempo , Túnica Íntima/lesões , Túnica Íntima/patologia , Túnica Média/lesões , Túnica Média/patologiaRESUMO
OBJECTIVE: We determined the effect of prolonged treatment with clopidogrel on C-reactive protein (CRP) concentrations and blood thrombogenicity after percutaneous transluminal coronary angioplasty followed by intracoronary brachytherapy in the porcine model. ANIMAL MODEL: All 48 pigs received antiplatelet therapy, including aspirin (325 mg, daily) and clopidogrel (300 mg, loading dose) 1 day before PCI, followed by a daily dose of clopidogrel (75 mg/day) in addition to aspirin. During PCI, one of two balloon-injured arteries was randomly assigned to receive immediate radiation treatment. Animals were sacrificed after 24 h, 1 month, and 3 months post-PCI. The pigs, which were sacrificed 3 months post-PCI, were divided into two groups. The first group received clopidogrel in addition to aspirin for 3 months, and the second group received clopidogrel in addition to aspirin for only 1 month after PCI and then aspirin alone. METHODS: Blood was taken from all pigs before intervention, immediately after intervention, and before sacrifice. Serum CRP was measured by enzyme-linked immunosorbent assay. To analyze the procoagulant effects of PCI on blood thrombogenicity, a one-stage clotting assay was performed. RESULTS: Clopidogrel treatment for 3 months reduced CRP levels more than did clopidogrel therapy for 1 month only at 3 months post-PCI (27.9+/-3.9 vs. 56.6+/-11.3 microg/ml; P=.019). Baseline CRP levels were found to be 50.4+/-4.8 microg/ml. Plasma clotting was not affected by prolonged clopidogrel therapy (322.8+/-59.3 s vs. 295.2+/-52.5 s; P=ns). CONCLUSIONS: Prolonged treatment with clopidogrel reduced CRP levels post-PCI.
Assuntos
Angioplastia Coronária com Balão/efeitos adversos , Anti-Inflamatórios/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Braquiterapia/efeitos adversos , Proteína C-Reativa/metabolismo , Inflamação/prevenção & controle , Inibidores da Agregação Plaquetária/farmacologia , Ticlopidina/análogos & derivados , Animais , Anti-Inflamatórios/uso terapêutico , Aspirina/farmacologia , Clopidogrel , Inflamação/sangue , Inflamação/etiologia , Modelos Animais , Inibidores da Agregação Plaquetária/uso terapêutico , Sus scrofa , Ticlopidina/farmacologia , Ticlopidina/uso terapêutico , Fatores de Tempo , Resultado do TratamentoRESUMO
The ability of enteropathogenic Escherichia coli (EPEC) to express virulence factor genes and develop attaching and effacing (AE) lesions is inhibited in acidic environmental conditions. This inhibition is due to the activation of transcription factor GadX, which upregulates expression of glutamic acid decarboxylase (Gad). Gad, in turn, produces γ-aminobutyric acid (GABA), which was recently shown to have a beneficial effect on the jejunal epithelium in vitro due to increased mucin-1 levels. In the present study, we sought to test whether forced GadX activation/overexpression abolishes virulence associated features of EPEC and provokes increased GABA production. EPEC strains were isolated from diarrheic pigs and submitted to activation of GadX by acidification as well as gadX overexpression via an inducible expression vector plasmid. GABA concentrations in the growth medium, ability for adhesion to porcine intestinal epithelial cells (IPEC-J2) and virulence gene expression were determined. Growth in acidified media led to increased GABA levels, upregulated gadA/B expression and downregulated mRNA synthesis of the bacterial adhesin intimin. EPEC strains transformed with the gadX gene produced 2.1-3.4-fold higher GABA levels than empty-vector controls and completely lost their ability to adhere to IPEC-J2 cells and to induce actin accumulation. We conclude that intensified gadX activation can abolish the ability of EPEC to adhere to the intestinal epithelium by reducing the expression of major virulence genes.
RESUMO
Negative arterial remodeling still plays an important role in the pathogenesis of coronary restenosis even in the era of interventional stenting (e.g. arterial narrowing occurs proximal and distal of a stented segment). Previous studies suggest that increased angiogenesis and inhibited regression of injury-induced adventitial microvessels prevents negative remodeling. We have examined the effect of local vascular endothelial growth factor (VEGF(165)) gene transfer on adventitial microvessel angiogenesis/regression and arterial remodeling after coronary angioplasty. Twenty pigs underwent angioplasty, each one in two major coronary arteries, followed by plasmid liposome gene transfer with either VEGF(165) or control gene LacZ (50 microg DNA with 50 microg of Lipofectine) into the (peri)adventitial space using a needle injection catheter. Arteries were examined at days 1, 7, 14, and 28. Local delivery of VEGF(165) gene into the outer compartments of balloon-injured porcine coronary arteries reduced lumen area loss due to distinct positive remodeling (arterial enlargement). Prevention of adventitial microvessel regression, enhanced adventitial elastin accumulation, reduced adventitial myofibroblast numbers, and a pronounced adventitial inflammatory response considered as a part of arterial healing seem to be the main VEGF-mediated mechanisms indicating the therapeutic potential of VEGF for restenosis prevention.
Assuntos
Angioplastia Coronária com Balão/efeitos adversos , Reestenose Coronária/prevenção & controle , Vasos Coronários/patologia , Expressão Gênica , Técnicas de Transferência de Genes , RNA Mensageiro/genética , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Reestenose Coronária/genética , Reestenose Coronária/patologia , Modelos Animais de Doenças , Terapia Genética/métodos , Imuno-Histoquímica , Hibridização In Situ , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SuínosRESUMO
OBJECTIVE: Experimental studies have provided evidence that neovascularization is an important feature of plaque growth, and angiogenic gene therapy may, therefore, increase plaque growth. This study examined the effect of local (peri)adventitial vascular endothelial growth factor165 (VEGF) gene transfer on vascular thickening after coronary balloon injury. METHODS: Two coronary arteries of 15 pigs were subjected to balloon injury followed by either (peri)adventitial VEGF165 or beta-galactosidase (LacZ) plasmid/liposome-mediated gene transfer via needle injection catheter. At days 3, 14 and 28, histologic sections of coronary arteries were analyzed. RESULTS: Transferred VEGF165 gene and increased adventitial neovascularization were detected in coronary arteries after balloon injury and VEGF injection. The mean intima+media (I+M) area increased after coronary balloon injury and VEGF (1.13+/-0.17 and 2.54+/-0.52 mm(2)) or LacZ (1.37+/-0.19 and 2.96+/-0.41 mm(2)) gene transfer, with no significant difference between both groups at 3 and 28 days, respectively. No significant difference in I+M neovascularization was observed at day 28 between the treatment groups (microvessel area density 0.24+/-0.08% with VEGF and 0.26+/-0.14% with LacZ, respectively). I+M endothelial cell proliferation index ranged from 7% to 22% (VEGF) and 18% to 24% (LacZ). CONCLUSIONS: Catheter-mediated (peri)adventitial VEGF165 gene transfer induces adventitial neovascularization but not an increase of vascular thickening/I+M growth and vascularization in a porcine model of coronary artery injury.
Assuntos
Angioplastia Coronária com Balão/efeitos adversos , Vasos Coronários/lesões , Terapia Genética/métodos , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Divisão Celular , Vasos Coronários/patologia , Endotélio Vascular/patologia , Injeções , Óperon Lac , Modelos Animais , Neovascularização Patológica , Suínos , Transfecção/métodosRESUMO
The inhibitory neurotransmitter GABA (γ-aminobutyric acid) is synthesized by glutamic acid decarboxylase, which is expressed in the central nervous system and in various other tissues including the intestine. Moreover, GABA can be ingested in vegetarian diets or produced by bacterial commensals in the gastrointestinal tract. As previous studies in lung have suggested a link between locally increased GABA availability and mucin 5AC production, the present study sought to test whether the presence or lack of GABA (and its precursor glutamine) has an effect on intestinal mucin expression. Porcine jejunum epithelial preparations were incubated with two different amounts of GABA or glutamine on the mucosal side for 4 h, and changes in the relative gene expression of seven different mucins, enzymes involved in mucin shedding, GABA B receptor, enzymes involved in glutamine/GABA metabolism, glutathione peroxidase 2, and interleukin 10 were examined by quantitative PCR (TaqMan(®) assays). Protein expression of mucin-1 (MUC1) was analyzed by Western blot. On the RNA level, only MUC1 was significantly up-regulated by both GABA concentrations compared with the control. Glutamine-treated groups showed the same trend. On the protein level, all treatment groups showed a significantly higher MUC1 expression than the control group. We conclude that GABA selectively increases the expression of MUC1, a cell surface mucin that prevents the adhesion of microorganisms, because of its size and negative charge, and therefore propose that the well-described positive effects of glutamine on enterocytes and intestinal integrity are partly attributable to effects of its metabolite GABA.
RESUMO
BACKGROUND: Late thrombotic events are important complications associated with intracoronary brachytherapy (ICBT) using ionizing radiation (IR) or with antiproliferative treatment modalities such as drug-eluting stents (DES). The mechanism mediating these thrombotic events is not well understood. This study assessed the effect of prolonged clopidogrel treatment on tissue factor (TF) expression in coronary arteries and on the circulating TF level after percutaneous transluminal coronary angioplasty /ICBT in a porcine coronary model. METHODS: Pigs were treated with aspirin plus a 300 mg loading dose of clopidogrel one day before percutaneous coronary intervention (PCI), followed by a daily dose of clopidogrel and aspirin. During PCI one of the two balloon-injured arteries was treated by brachytherapy. Animals were sacrificed at different time points. The pigs, which were sacrificed 3 months post-PCI, were divided into two groups (Group I: clopidogrel for 3 months; Group II: clopidogrel for 1 month). Plasma TF was measured by enzyme-linked immunosorbent assay in blood samples taken from all pigs before and immediately after intervention and before sacrifice. Morphometric analysis was performed on digitalized images employing the software LUCIA G for TF staining. Vascular TF expression levels were assessed by quantitative real-time polymerase chain reaction. RESULTS: Prolonged clopidogrel application significantly reduced coronary TF at the protein (Group I vs. II, 8.975 ± 3.947% vs. 26.44 ± 5.375%, P = .007) and mRNA level [Group I vs. II, (0.3501 ± 0.0519) × 10(-3) vs. (0.7073 ± 0.0436) × 10(-3), P<.0005]. Circulating TF protein tended to be lower after 3 months than after 1 month clopidogrel treatment post-PCI (Group I vs. Group II, 488.3 ± 35.37 pg/ml vs. 572.3 ± 39.9 pg/ml, P = .130). CONCLUSIONS: Prolonged clopidogrel treatment reduced coronary TF expression and tended to reduce the blood TF level post-PCI, thus possibly modulating the risk of late thrombosis.
Assuntos
Angioplastia Coronária com Balão , Trombose Coronária/prevenção & controle , Vasos Coronários/efeitos dos fármacos , Inibidores da Agregação Plaquetária/administração & dosagem , Tromboplastina/metabolismo , Ticlopidina/análogos & derivados , Angioplastia Coronária com Balão/efeitos adversos , Animais , Aspirina/administração & dosagem , Braquiterapia/efeitos adversos , Clopidogrel , Trombose Coronária/etiologia , Trombose Coronária/metabolismo , Trombose Coronária/patologia , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Esquema de Medicação , Quimioterapia Combinada , Ensaio de Imunoadsorção Enzimática , Fibrina/metabolismo , Fibrinogênio/metabolismo , Imuno-Histoquímica , Modelos Animais , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sus scrofa , Tromboplastina/genética , Ticlopidina/administração & dosagem , Fatores de Tempo , Regulação para CimaRESUMO
BACKGROUND: The incidence of coronary artery disease (CAD) is still increasing in industrialized countries and it is even higher in diabetic patients. For experimental studies investigating the pathophysiology of CAD, the use of an animal model comparable with the pathological situation in patients is crucial. OBJECTIVE: To develop a model of advanced coronary atherosclerosis with induction of hyperlipidemia and hyperglycemia in domestic pigs. METHODS: Six pigs were fed a standard pig chow (controls), two were fed a 2% cholesterol and 17% coconut fat diet (Chol group), and two pigs received a 4% cholesterol and 17% coconut fat diet combined with streptozotocin (STZ) injections to induce diabetes (High Chol+STZ group). Serum lipid and plasma glucose values were analyzed, and histochemical staining for morphometric analysis and immunohistochemistry were performed. RESULTS: Pigs on the hyperlipidemic diet had elevated mean (+/- SD) serum lipid levels (total cholesterol 5.05+/-1.45 mmol/L [Chol] and 5.03+/-2.41 mmol/L [High Chol+STZ] versus 2.09+/-0.23 mmol/L [controls]). Histopathological evaluation revealed an initial stage of coronary atherosclerosis. None of the STZ-treated pigs showed a sustained elevation of plasma glucose (mean glucose before STZ injection was 5.11+/-0.94 mmol/L and thereafter was 6.03+/-2.39 mmol/L) or a decline in pancreatic beta cells. CONCLUSIONS: The current data suggest that the domestic porcine model is not suitable to create severe CAD using an atherogenic diet in combination with STZ injections for experimental interventional vascular research. This may be due to different STZ sensitivities among species. However, hyperlipidemia induced early pathological lesions in coronary arteries resembling initial stages of atherosclerosis without severe luminal narrowing.