ABSTRACT
Cytosolic free calcium concentration ([Ca2+]i) and muscle tension were simultaneously measured in aortic tissue isolated from spontaneously hypertensive rats (SHR), normotensive Wistar-Kyoto (WKY) rats, and SHR chronically treated with a novel angiotensin converting enzyme inhibitor, CS-622. In the presence of 2.5 mM Ca2+ in the bathing solution, aortic [Ca2+]i measured with fura-2 was higher in SHR than in WKY rats, and it was almost the same in CS-622-treated SHR and untreated WKY rats. Increase of external Ca2+ concentration from zero to 2.5 mM elicited a contraction in SHR aortas but not in aortas from both CS-622-treated SHR and untreated WKY rats. When the aortas were contracted by 60 mM K+, however, [Ca2+]i as well as developed tension was similar in the three groups. CGP-28392 (10(-6) M), a Ca2+ channel activator, induced a rhythmic activity superimposed on a gradual increase of [Ca2+]i and tension in SHR aortas but not in the aortas of CS-622-treated SHR or untreated WKY rats. Nicardipine (10(-7) M) decreased the resting [Ca2+]i and the resting tone in SHR aortas, but not in WKY rat aortas. These results suggest that SHR aortas have a higher myogenic tone due to increased [Ca2+]i than WKY rat aortas and that the increased [Ca2+]i is attributed to alterations of dihydropyridine-sensitive Ca2+ channels in SHR aortas. Further, the decrease of the vascular tone induced by long-term administration of the angiotensin converting enzyme inhibitor may be due to a reduction of increased [Ca2+]i in SHR.
Subject(s)
Angiotensin-Converting Enzyme Inhibitors/pharmacology , Aorta/analysis , Calcium/analysis , Hypertension/metabolism , Thiazepines/pharmacology , Animals , Aorta/drug effects , Cytosol/analysis , Male , Muscle Contraction/drug effects , Nicardipine/pharmacology , Pyridines/pharmacology , Rats , Rats, Inbred SHR , Rats, Inbred WKYABSTRACT
We have studied the interaction of 125I-antithrombin (125I-AT) with microvascular endothelial cells (RFPEC) to localize the cellular site of anticoagulantly active heparan sulfate proteoglycans (HSPG). The radiolabeled protease inhibitor bound specifically to the above HSPG with a Kd of approximately 50 nM. Confluent monolayer RFPEC cultures exhibited a linear increase in the amount of AT bound per cell for up to 16 d, whereas suspension RFPEC cultures possessed a constant number of protease inhibitor binding sites per cell for up to 5 d. These results suggest that monolayer RFPEC cultures secrete anticoagulantly active HSPG, which then accumulate in the extracellular matrix. This hypothesis was confirmed by quantitative light and EM level autoradiography which demonstrated that the AT binding sites are predominantly located in the extracellular matrix with only small quantities of protease inhibitor complexed to the cell surface. We have also pinpointed the in vivo position of anticoagulantly active HSPG within the blood vessel wall. Rat aortas were perfused, in situ, with 125I-AT, and bound labeled protease inhibitor was localized by light and EM autoradiography. The anticoagulantly active HSPG were concentrated immediately beneath the aortic and vasa vasorum endothelium with only a very small extent of labeling noted on the luminal surface of the endothelial cells. Based upon the above data, we propose a model whereby luminal and abluminal anticoagulantly active HSPG regulate coagulation mechanism activity.
Subject(s)
Antithrombins/metabolism , Blood Coagulation/physiology , Chondroitin Sulfate Proteoglycans/analysis , Endothelium, Vascular/analysis , Glycosaminoglycans/analysis , Heparitin Sulfate/analysis , Proteoglycans/analysis , Animals , Aorta/analysis , Autoradiography , Cell Membrane/analysis , Extracellular Matrix/analysis , Heparan Sulfate Proteoglycans , In Vitro Techniques , Iodine Radioisotopes , Male , Microscopy, Electron , Perfusion , Protein Binding , Rats , Rats, Inbred StrainsABSTRACT
Foi realizada uma experiência durante 80 dias, usando 32 coelhas em fase de crescimento. Os animais foram distribuídos em quatro grupos (A, B, C e D) de oito animais cada. Os animais dos grupos A e C permaneceram soltos, sendo que os do grupo A foram alimentados com raçäo basal e os do grupo C com raçäo aterogênica (raçäo basal + 0,5% de colesterol). Os animais dos grupos B e D permaneceram praticamente imobilizados em gaiolas estreitas e foram alimentados, respectivamente, com raçäo basal e raçäo aterogênica. Os resultados obtidos permitem inferir que a inatividade física tem efeito nocivo no processo ateromatoso. De fato, foram observados aumentos (relacionados à quantidade de colesterol ingerido), maiores nos animais engaiolados em relaçäo aos soltos, de lipídios totais no plasma sangüineo e no fígado (significativo); e de colesterol total no fígado, na aorta e no músculo (significativo). Além disso, o grau de ateromatose aórtica foi significativamente mais severo nos animais engaiolados
Subject(s)
Rabbits , Atherosclerosis/etiology , Diet, Atherogenic , Restraint, Physical/adverse effects , Aorta/analysis , Cholesterol/analysis , Cholesterol/blood , Liver/analysis , Lipids/analysis , Lipids/blood , Muscles/analysisABSTRACT
Cell necrosis and reactive cellular processes in and near the atherosclerotic core region might result from short-range interactions with toxic lipids. To model these interactions in cell culture, focal crystalline deposits of cholestane-3 beta,5 alpha,6 beta-triol, 25-OH cholesterol, and cholesterol were overlaid by a collagen gel, on which canine aortic smooth muscle cells were seeded. Oxysterols, but not cholesterol, caused focally decreased plating efficiency and cell death, leading to the formation of a persistent circular gap in the cell culture. Cholestanetriol was largely removed from the culture dishes over 3 to 4 weeks, whereas cholesterol and 25-OH cholesterol were largely retained. Smooth muscle cells were motile even in proximity to oxysterol crystals, with occasional suicidal migration toward the crystals. Chemoattraction, however, could not be demonstrated. Despite toxicity, cholestanetriol did not appear to alter the fraction of cells exhibiting 3H-thymidine uptake, even in areas close to the crystals. Thus, oxysterols may be toxic to some cells, without causing major impairment of the migration and proliferation of nearby cells. This would allow the simultaneous occurrence of cell death and proliferation evident in atherosclerosis.
Subject(s)
Cholestanols/toxicity , Hydroxycholesterols/toxicity , Hypolipidemic Agents/toxicity , Muscle, Smooth, Vascular/cytology , Animals , Aorta/analysis , Aorta/cytology , Aorta/drug effects , Arteriosclerosis/pathology , Cell Movement/drug effects , Cell Survival/drug effects , Cells, Cultured , Chemotaxis/drug effects , Cholestanols/analysis , DNA/biosynthesis , DNA/drug effects , Disease Models, Animal , Dogs , Hydroxycholesterols/analysis , Hypolipidemic Agents/analysis , Microscopy, Phase-Contrast , Muscle, Smooth, Vascular/analysis , Muscle, Smooth, Vascular/drug effectsABSTRACT
Lipoprotein metabolism and cholesterol accumulation in atherosclerotic lesions was studied using enzymatically isolated primary cell cultures from aortas of rabbits made atherosclerotic by cholesterol feeding. The cultures consisted of macrophages and smooth muscle cells, thus resembling, in composition, fatty streak lesions. The mean (+/- SD) cholesteryl ester content of the dispersed cells was 1059 +/- 445 micrograms/mg cell protein, but it declined steeply during 1 week in primary culture. The uptake of low-density lipoprotein (LDL), beta-migrating very low-density lipoprotein (beta-VLDL), and acetylated LDL (acetyl-LDL), labeled with 125I or with the fluorescent probe 1,1'-dioctadecyl-3,3,3',3'- tetramethylindocarbocyanine (DiI), was studied in 2-day-old primary cultures. DiI-acetyl-LDL was avidly taken up by the macrophages and, to a lesser extent, by some smooth muscle cells. The uptake of DiI-beta-VLDL by the macrophages was weaker and less homogeneous than that of DiI-acetyl-LDL. The degradation rates of 125I-labeled beta-VLDL, LDL and acetyl-LDL were 135 +/- 54, 195 +/- 20, and 697 +/- 14 ng/mg cell protein/8 hours, respectively. Incubation with unlabeled acetyl-LDL enhanced the incorporation of [3H]oleate into cholesteryl esters and increased the cellular cholesteryl ester content. These results suggest that arterial macrophages and, to some extent, smooth muscle cells from cholesterol-fed rabbits actively metabolize acetyl-LDL and are thus capable of accumulating cholesteryl esters by uptake of modified forms of LDL.
Subject(s)
Arteriosclerosis/pathology , Cholesterol/metabolism , Lipoproteins, LDL/metabolism , Macrophages/pathology , Muscle, Smooth, Vascular/pathology , Animals , Aorta/analysis , Aorta/metabolism , Aorta/pathology , Arteriosclerosis/metabolism , Cells, Cultured , Cholesterol/analysis , Esterification , Fluorescent Antibody Technique , Foam Cells/metabolism , Foam Cells/pathology , Lipoproteins, LDL/pharmacokinetics , Macrophages/metabolism , Male , Muscle, Smooth, Vascular/metabolism , RabbitsABSTRACT
In bovine aortic smooth muscle, about 50% of total GTP-binding activity was present in the cytosol fraction. A major GTP-binding protein (G protein) with a Mr value of about 21,000 (21K G) in this fraction was purified to near homogeneity and characterized. 21K G bound maximally about 0.8 mol of [35S]guanosine 5'-(3-O-thio)triphosphate/mol of protein with a Kd value of about 20 nM. 21K G showed GTPase activity with a turnover number of about 0.007 min-1. 21K G was ADP-ribosylated by botulinum ADP-ribosyltransferase and about 0.4 mol of ADP-ribose was maximally incorporated into 1 mol of 21K G. 21K G and the bovine brain rhoA gene product (rhoA p21) were eluted at the same retention time on C4 reversed-phase high performance liquid chromatography and migrated at the same positions on two-dimensional gel electrophoresis. These results indicate that the major G protein in bovine aortic smooth muscle cytosol is rhoA p21.
Subject(s)
GTP-Binding Proteins/analysis , Membrane Proteins/analysis , Muscle, Smooth, Vascular/analysis , Adenosine Diphosphate Ribose/metabolism , Animals , Aorta/analysis , Cattle , Chromatography , Chromatography, High Pressure Liquid , Cytosol/analysis , Electrophoresis, Gel, Two-Dimensional , rhoA GTP-Binding ProteinABSTRACT
An age-related decrease in elasticity of arteries has been found in clinical and experimental studies done during the past two decades. We have investigated molecular and endocrine aspects of that decrease by examining the effects of age and insulin-like growth factor-I (IGF-I) on rat aorta elastogenesis. For comparison, pulmonary elastogenesis was examined in the same experimental animals. Different aged groups of male Fischer 344 rats (barrier protected) were implanted with minipumps for a two-week infusion of either 0.1 N acetic acid (vehicle solution) or IGF-I (1.2 mg/kg/day). The DNA content (micrograms DNA/g tissue) decreased with age in aorta but remained fairly constant in lung. Administration of IGF-I increased the aortic DNA content in all but the oldest rats. Conversely, the DNA content of pulmonary tissue was significantly increased in only the youngest animals. The steady-state levels of tropoelastin mRNA decreased dramatically in both aorta and lung with increased age. The decrease was greater in lung than aorta. Administration of IGF-I elevated aortic tropoelastin mRNA steady-state levels, whereas lung tropoelastin mRNA levels were unaffected by IGF-I administration. Aortic tissue synthesized decreased amounts of insoluble elastin with increased age. These results establish a direct relationship between aortic tropoelastin mRNA levels and the synthesis of insoluble elastin in aging. Administration of IGF-I increased aortic elastin synthesis throughout the life span of the rat, although the proportionate increase diminished with age.
Subject(s)
Aging/drug effects , Aorta/drug effects , Elastin/biosynthesis , Gene Expression/drug effects , Insulin-Like Growth Factor I/pharmacology , Somatomedins/pharmacology , Animals , Aorta/analysis , Aorta/metabolism , DNA/analysis , Elastin/genetics , Infusion Pumps , Insulin-Like Growth Factor I/administration & dosage , Insulin-Like Growth Factor I/analysis , Lung/analysis , Lung/drug effects , Lung/metabolism , Male , RNA/analysis , Radioimmunoassay , Rats , Rats, Inbred F344ABSTRACT
A cytochemical study was performed on the lamprey ventral aorta with special reference to disulfide and sulfhydryl groups of microfibrils, using the high-iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) method combined with several other types of treatment. The HID-TCH-SP staining observed was classified into three categories: 1) weak staining in the periphery of collagen fibrils, 2) moderate staining in the boundaries of collagen fibrils, microfibrils and smooth muscle cells, and 3) intense staining of microfibrils. The first and second categories of staining were considered to represent chondroitin and/or heparan sulfate because of sensitivity of the staining to chondroitinase ABC (ChABC) and its specific localization. By contrast, the third category of staining was considered to represent disulfide and sulfhydryl groups of microfibrillar glycoprotein, because it was disclosed only after Oxone oxidation or thiosulfation and was not removed by ChABC digestion. Although this staining reactivity was not apparently altered by SH blockade prior to oxidation or thiosulfation, it was markedly diminished or completely inhibited by S-S reduction followed by SH blockade. These results indicate that lamprey aortic microfibrils contain more S-S groups than SH groups.
Subject(s)
Actin Cytoskeleton/ultrastructure , Aorta/ultrastructure , Cytoskeleton/ultrastructure , Disulfides/analysis , Fishes/anatomy & histology , Lampreys/anatomy & histology , Sulfhydryl Compounds/analysis , Actin Cytoskeleton/analysis , Animals , Aorta/analysis , Histocytochemistry , Hydrazines , Indicators and Reagents , Lampreys/metabolism , Silver , Staining and Labeling , Sulfuric Acids , ThiosulfatesABSTRACT
Type VIII collagen was isolated from bovine Descemet's membranes by pepsin treatment and salt fractionation, as described by Kapoor et al. [(1986) Biochemistry 25, 3930-3937]. Contaminating type IV collagen was removed by ion-exchange chromatography. Purified type VIII collagen consisted of two different polypeptide chains and, compared to the fiber forming collagens, showed a higher thermal stability. Corresponding fractions isolated from pepsinized human Ewing's sarcoma and fetal calf aorta reacted immunologically with a protein of similar molecular mass. After extraction of Descemet's membranes with guanidine hydrochloride, a peptide of about 60 kDa was obtained. This seems to be the tissue form of type VIII collagen.
Subject(s)
Collagen/isolation & purification , Amino Acids/analysis , Animals , Aorta/analysis , Aorta/embryology , Basement Membrane/analysis , Cattle , Chromatography, Ion Exchange , Collagen/ultrastructure , Cornea/analysis , Humans , Immunoblotting , Microscopy, Electron , Models, Molecular , Sarcoma, Ewing/analysis , TrypsinABSTRACT
The development and the validation of a general strategy for the simple and accurate analysis of desmosines (isodesmosine and desmosine) in tissues coupled with the determination of collagen (as hydroxyproline) is described. The method is based on simplified sample (i.e., lung) pretreatment which involves, in a PTFE screw-capped Pyrex tube, homogenization, collagen extraction with hot 5% trichloroacetic acid and hydrolysis of the elastin-containing residue with 6 M hydrochloric acid, followed by cellulose minicolumn purification of desmosines from the hydrolysates, dansyl chloride pre-column derivatization of the purified desmosines and reversed-phase high-performance liquid chromatographic (HPLC) analysis of the dansyl derivatives using a Spherisorb ODS-2 column, an on-column enrichment sample device and a linear gradient of organic modifier (acetonitrile) in phosphate buffer. The simple sample pretreatment, the optimized chromatographic conditions and the short HPLC analysis time (less than 15 min) allow the accurate and rapid determination of desmosine and isodesmosine, thus permitting the determination of elastin in several kinds of tissues with a minimum of sample manipulation.
Subject(s)
Amino Acids/analysis , Chromatography, High Pressure Liquid/methods , Desmosine/analysis , Animals , Aorta/analysis , Elastin/analysis , Isodesmosine/analysis , Liver/analysis , Lung/analysis , Lung/embryology , Rabbits , RatsABSTRACT
To investigate the influence of diabetes mellitus on vascular relaxation response, acetylcholine (ACh)-induced relaxation and production of cyclic GMP and cyclic AMP in aortic rings with endothelium were compared between alloxan-induced diabetic and control rabbits. ACh-induced relaxation was significantly attenuated in the aortic rings of diabetic rabbits. Concentration-response curve for ACh-induced relaxation in the aortic rings of control rabbits was shifted to the right by the pretreatment with hemoglobin, and this concentration-response curve was almost identical to that in the aorta from diabetic rabbits. Sodium nitroprusside (SNP)-induced relaxation in the aortic rings without endothelium from diabetic rabbits was similar to that in the aortic rings without endothelium from control rabbits. Basal levels of cyclic GMP and ACh-induced production of cyclic GMP were markedly lower in diabetic rabbits than those in control rabbits. On the other hand, there were no differences in basal and ACh-induced production of cyclic AMP between diabetic and control aorta. These results suggest that impairment of endothelium but not guanylate cyclase activity may be occurred in the aorta of diabetic rabbits. This impairment leads to the decrease in production of cyclic GMP through the attenuation of endothelium-derived relaxing factor (EDRF) release, and this may be responsible for the decreased endothelium-dependent relaxation of ACh.
Subject(s)
Acetylcholine/pharmacology , Aorta/metabolism , Cyclic AMP/biosynthesis , Cyclic GMP/biosynthesis , Diabetes Mellitus, Experimental/metabolism , Vasodilation/drug effects , Alloxan , Animals , Aorta/analysis , Aorta/drug effects , Cyclic AMP/analysis , Cyclic GMP/analysis , Diabetes Mellitus, Experimental/chemically induced , Endothelium, Vascular , Male , RabbitsABSTRACT
We utilized platelet factor 4 (PF4) conjugated to fluorescein to stain the proteoglycans of permeabilized fixed bovine aorta endothelial cells in monolayer culture. Treatment of the monolayers with chondroitin ABC lyase and/or a preparation from Flavobacterium heparinum was used to remove chondroitin sulfate and/or heparan sulfate before staining, with resultant separate identification and partial localization of these glycosaminoglycans. When PF4-fluorescein was utilized with untreated control monolayers, fairly uniform reticular, perinuclear, and cell surface fluorescence was seen. After treatment with chondroitin ABC lyase, fluorescence was retained only on the cell surface. In contrast, treatment with the F. heparinum preparation resulted in the loss of all cell surface fluorescence. Use of both glycosaminoglycan lyases together resulted in loss of essentially all the fluorescence. The cell surface heparan sulfate observed by fluorescence after removal of cell surface chondroitin sulfate appeared to be unevenly distributed, with a heavier accumulation at one pole of each cell. This technique offers a specific method for identification and partial localization of cell surface heparan sulfate.
Subject(s)
Aorta/cytology , Endothelium, Vascular/cytology , Glycosaminoglycans/analysis , Animals , Aorta/analysis , Aorta/metabolism , Cattle , Cells, Cultured , Endothelium, Vascular/analysis , Endothelium, Vascular/metabolism , Fluoresceins/metabolism , Glycosaminoglycans/metabolism , Histocytochemistry/methods , Microscopy, Fluorescence , Platelet Factor 4/metabolismABSTRACT
A method for the co-purification of calponin and caldesmon from bovine aorta smooth muscle was established involving heat treatment, ammonium sulfate fractionation (0-30 and 30-50%), CM52 column chromatography, and Ultrogel AcA44 gel filtration. The yields of calponin and caldesmon from 200 g of smooth muscle were 17 and 32 mg, respectively. This simplified, fast, and efficient method for the purification of calponin and caldesmon constitutes a useful tool for studying thin filament-linked regulation of smooth muscle contraction.
Subject(s)
Calcium-Binding Proteins/isolation & purification , Calmodulin-Binding Proteins/isolation & purification , Muscle, Smooth, Vascular/analysis , Animals , Aorta/analysis , Cattle , Electrophoresis, Polyacrylamide Gel , Microfilament Proteins , CalponinsABSTRACT
The effects of 9 months of orally administered captopril (25-50 mg/kg body wt/day) on aortic atherosclerosis was examined in normotensive Watanabe heritable hyperlipidemic rabbits. Captopril caused a significant decrease in aortic atherosclerosis. Total aortic surface involvement by lesions was reduced from 48 +/- 3.6% in control Watanabe rabbits to 30 +/- 3.9% with captopril treatment (p less than 0.01). Most of the decrease could be accounted for by a marked reduction in atherosclerosis of descending thoracic aortas from 49 +/- 5.2% to 15 +/- 3.9% in control and captopril-related groups, respectively (p less than 0.001). Significant decrease in cholesterol content of descending thoracic aorta was also observed in captopril-treated rabbits. Microscopic examination of the arterial lesions in captopril-treated animals suggested a relative decrease in cellularity and increase in extracellular matrix as compared with untreated animals. These studies indicate that captopril has a potent antiatherosclerotic action in the Watanabe heritable hyperlipidemic rabbit.
Subject(s)
Arteriosclerosis/prevention & control , Captopril/therapeutic use , Animals , Aorta/analysis , Aorta/pathology , Cholesterol/analysis , Female , Male , RabbitsABSTRACT
We have studied rat vascular smooth muscle (VSM) cells in culture for the presence of key elements of the glandular kallikrein-kinin system. Direct radioimmunoassay (RIA) using antiserum against rat urinary kallikrein detected a glandular kallikrein-like enzyme (GKLE) in VSM cells and in media. VSM homogenates and culture media had kininogenase activity, generating kinins from dog kininogen. About half of the GKLE was enzymatically inactive which could be activated with trypsin. Kininogenase activity was inhibited completely by aprotinin but only 20% by soybean trypsin inhibitor (SBTI). Trypsin liberated kinins from homogenates and media, demonstrating that VSM cells contain kininogen. Homogenates and media rapidly degrade bradykinin. GKLE, kininogen, and bradykininase activity were all present in VSM cells grown in defined media that contain no serum, thus eliminating any contamination or artefacts from fetal calf serum in standard culture media. Blood vessels of the rat have been reported to contain GKLE. Our observations indicate that GKLE is synthesized by VSM cells, not deposited from plasma. Furthermore, VSM cells synthesize kininogen and bradykininase(s), the other key elements of the glandular kallikrein-kinin system. Thus it is possible that the system functions as an autocoid mechanism that regulates local vascular tone.
Subject(s)
Carboxypeptidases/analysis , Kallikreins/analysis , Kininogens/analysis , Lysine Carboxypeptidase/analysis , Muscle, Smooth, Vascular/analysis , Animals , Aorta/analysis , Cells, Cultured , Kallikreins/biosynthesis , Kininogens/biosynthesis , Lysine Carboxypeptidase/biosynthesis , Male , Muscle, Smooth, Vascular/metabolism , Rats , Rats, Inbred StrainsABSTRACT
Very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL) were isolated from the blood of healthy subjects and CHD patients. LDL from the blood of healthy individuals did not raise the intracellular lipid values within 24 h of cultivation. During intracellular lipid values within 24 h of cultivation. During the same incubation period. LDL obtained from the blood of CHD patients caused a 2- to 5-fold rise in cholesterol esters as well as a 1.5- to 3-fold rise in free cholesterol and triglycerides, while the intracellular phospholipid levels remained unchanged. In one of the three cases, the ability to raise the intracellular level of cholesterol esters was demonstrated by VLDL (500 micrograms/ml) derived from CHD patients blood. HDL did not affect the lipid levels in smooth muscle cells cultured from unaffected intima. The obtained data suggests that circulating LDL and, possibly, VLDL in the blood of CHD patients are capable of inducing the accumulation of fat in vascular wall cells.
Subject(s)
Aorta/analysis , Coronary Disease/blood , Lipids/analysis , Lipoproteins, LDL/blood , Adult , Cells, Cultured , Cholesterol/analysis , Cholesterol Esters/analysis , Humans , Lipoproteins, LDL/pharmacology , Lipoproteins, VLDL/blood , Lipoproteins, VLDL/pharmacology , Male , Middle Aged , Triglycerides/analysisABSTRACT
Recent evidence suggests that thrombin interacts with various cell types, stimulating cellular proliferation and protein and prostanoid production. To further delineate its role in vascular healing, we have studied the effects of thrombin on proliferation and matrix production by the cells of the vessel wall. The addition of thrombin (1 unit/ml) to cultures of bovine aortic smooth muscle cells resulted in an increase in cell proliferation (p less than 0.01) and number (p less than 0.03), whereas in cultures of bovine aortic endothelial cells thrombin produced a decrease in cell proliferation (p less than 0.01) and number (p less than 0.02). Thrombin also altered matrix composition in cultures of these cells. In both bovine aortic endothelial cells and bovine aortic smooth muscle cell cultures grown in the presence of thrombin, total protein content was significantly increased when compared to controls (p less than 0.03). In bovine aortic endothelial cell cultures the addition of thrombin resulted in a decrease in collagen content (p less than 0.01) and an increase in sulfated glycosaminoglycan content (p less than 0.02). In contrast, in bovine aortic smooth muscle cell cultures thrombin resulted in an increase in collagen content (p less than 0.03), whereas glycosaminoglycan content was unaffected. These findings suggest that thrombin may significantly influence vascular healing and function by altering cell number and matrix composition.
Subject(s)
Endothelium, Vascular/drug effects , Muscle, Smooth, Vascular/drug effects , Thrombin/pharmacology , Animals , Aorta/analysis , Aorta/cytology , Aorta/drug effects , Cattle , Cell Count/drug effects , Cell Division/drug effects , Cells, Cultured/analysis , Cells, Cultured/drug effects , Collagen/analysis , Endothelium, Vascular/analysis , Endothelium, Vascular/cytology , Glycosaminoglycans/analysis , Mitogens/pharmacology , Muscle, Smooth, Vascular/analysis , Muscle, Smooth, Vascular/cytology , Time FactorsABSTRACT
To clarify effecting sites of endothelin (ET) in a circulation system, we have identified specific receptors for porcine ET(ET-1) and investigated the distribution in the porcine cardiovascular tissues. Scatchard analysis of 125I-porcine ET binding indicated the presence of a single class of high-affinity binding sites. The binding was highly specific for ET-1, because (1) none of the other various peptides or Ca2+-channel antagonists affected the binding, (2) the scission of disulfide bonds, the digestion of the C-terminal 6-amino acid residues, or nitrophenylsulfenylization of the C-terminal Trp21 of ET-1 markedly reduced the binding ability and, (3) ET-1 showed the highest affinity for the vascular receptor among three ET isopeptides. Cardiac atria possessed the highest density (2.7 pmol/mg protein) of ET receptors of all the tissues examined, including thoracic aorta, cardiac atria and cardiac ventriculi, basilar, renal, coronary and pulmonary branch arteries, coronary, renal and jugular veins, and small vessels of pia mater encephali. Small vessels, renal and coronary arteries also showed relatively high density (0.8-1.4 pmol/mg protein). Various veins examined also showed considerable density (0.45-0.74 pmol/mg protein). The apparent Kd of cardiac ET receptors (0.76 nM) was significantly greater than that of the receptors of the other tissue (0.06-0.14 nM). The extensive distribution and the local enrichment of ET receptor in a cardiovascular system strongly suggests that ET is one of the essential endogenous substances to control the tone of the vasculature.
Subject(s)
Aorta/metabolism , Myocardium/metabolism , Peptides/pharmacokinetics , Receptors, Cell Surface/metabolism , Aged , Animals , Aorta/analysis , Aorta, Thoracic , Binding, Competitive , Endothelins , Female , Humans , Male , Middle Aged , Myocardium/analysis , Receptors, Cell Surface/analysis , Receptors, Endothelin , Sodium Iodide/metabolism , Substrate Specificity , SwineABSTRACT
Increased pulmonary artery pressure is known to result in enhanced collagen deposition in the pulmonary artery. Here we investigate how changes in collagen metabolism may bring about this increased deposition in the pulmonary artery of animals with pulmonary hypertension induced by bleomycin. Rabbits were injected intratracheally with bleomycin sulfate or with saline. After 14 days the animals were injected with L-[U-14C]proline plus a "flooding" dose of unlabeled proline. Uptake into arterial collagens and release of labeled hydroxyproline were then measured after 2.5 h. The relative amounts of types I and III collagens were assessed from the levels of cyanogen-bromide-derived peptides alpha 1(I)CB8 and alpha 1(III)CB5, respectively, after sodium dodecyl sulfate polyacrylamide gel electrophoresis. Collagen synthesis rates of about 3%/day were found in the control pulmonary artery and aorta, and about one-half of the newly synthesized collagen was degraded rapidly. Fourteen days after bleomycin, there was a fivefold increase in collagen synthesis rate (p less than 0.01) and a marked decrease in the percentage of newly synthesized collagen degraded rapidly. There was no change in collagen metabolism in the aorta of these animals. Pulmonary artery collagen from control rabbits consisted of 26.5 +/- 1.0% type III collagen. There was no change in composition in bleomycin-treated animals. This study demonstrates quite rapid turnover rates for collagen in normal blood vessels. Our results also indicate that remodeling of arterial connective tissue matrix during pulmonary hypertension involves marked but commensurate increases in type I and III collagens brought about by changes in both synthesis and degradative processes.
Subject(s)
Aorta/metabolism , Collagen/metabolism , Hypertension, Pulmonary/metabolism , Pulmonary Artery/metabolism , Animals , Aorta/analysis , Aorta/pathology , Bleomycin/toxicity , Collagen/analysis , Electrophoresis, Polyacrylamide Gel , Hydroxyproline/analysis , Hydroxyproline/metabolism , Hypertension, Pulmonary/chemically induced , Hypertension, Pulmonary/pathology , Male , Proline/administration & dosage , Pulmonary Artery/analysis , Pulmonary Artery/pathology , Rabbits , Spectrophotometry , Time FactorsABSTRACT
Ultraviolet excited laser induced fluorescence (LIF) was studied in normal and atherosclerotic human arterial wall in vitro. Using excitation wavelengths from 306 to 310 nm, two distinct emission bands were observed in the LIF of both normal and pathologic aorta: a short wavelength band, peaking at 340 nm emission, which was attributed to tryptophan; and a long wavelength band, peaking at 380 nm emission, which was assigned to a combination of collagen and elastin. The intensity of the short wavelength band was quite sensitive to the choice of excitation wavelength, while the long wavelength band was not, so that the relative contributions of the bands could be controlled by the precise choice of excitation wavelength. A valley in the spectra at 418 nm was attributed to fluorescence reabsorption by oxy-hemoglobin. By using 308 nm excitation to observe emission simultaneously from both the short and long wavelength bands, normal and atherosclerotic aorta were spectrally distinct. Two LIF emission intensity ratios were defined to characterize both the relative tryptophan fluorescence content as well as the ratio of elastin to collagen fluorescence in each spectrum. The differences in these two emission ratios among the various histologic tissue types correlated qualitatively with the histologic and biochemical compositions of these tissues. By combining these parameters in a binary classification scheme, normal and atherosclerotic aorta were correctly distinguished in 56 of 60 total cases. Furthermore, atherosclerotic plaques, atheromatous plaques, and exposed calcifications could be classified individually with sensitivities/predictive values of 90%/90%, 100%/75%, and 82%/82%, respectively.