Immunolocalization of collagen types I and III in the arterial wall of the rat

Summary Collagen types I and III were located by immunofluorescence procedures in the aorta and coronary arteries of the rat. Type I collagen was most prevalent in the adventitia of the aorta with only small amounts present in the intima and media. Type III collagen appeared to be a significant component in the media of the aorta and also in the adventitia of both blood vessels. The intima and media of the coronary arteries did not stain strongly for either type I or III collagen. Neither staining procedure was altered with preincubation of the sections with hyaluronidase or chondroitinase ABC. These studies indicate that type III collagen is a major component of the adventitia which has previously not been recognized by immunohistochemical techniques, possibly due to masking of collagen staining with glycosaminoglycans.     

Sodium kinetics in aorta of spontaneously hypertensive rats.

Transport rate constants (kij) for Na exchanges in isolated aorta of normotensive and spontaneously hypertensive rats (SHR) were determined with the use of 33Na as a tracer and the aid of digital computer simulation. A three-compartment model consisting of 1) extracellular, 2) intracellular, and 3) "endointracellular" spaces (compartments) was found to describe adequately the kinetics of 22Na. Results show that in SHR: I) K01, which is related to the overall Na outflow from tissue, was increased by 41%; ii) k12, describing Na movements from intra- to extracellular compartment, was increased by 67%; iii) k21, representative of Na movements from extra-to intracellular compartment, was decreased by 39%. These results indicate a faster turn-over of Na and a relative accumulation or translocation of Na into the extracellular space in aorta of SHR. The findings are interpreted in the light of recent reports on the role of Na in contractile response or reactivity of arteries. A humoral mechanism operative at the arterial wall level for the development of hypertension is at the arterial wall level for the development of hypertension is suggested. The main significance of the methodology employed in this work is that the values found for the kij are not subject to fluctuations intrinsic to auxiliary indicators of extracellular space.     

Preliminary observations, at the ultrastructural level, on the reactivity of cerebral arteries from New Zealand rabbits to combined atherogenic stimuli (hypercholesterol diet and hypertension)

Both in monkeys (Rhesus and Cynomolgus) and in New Zealand rabbits fed an atherogenic diet, a marked delay in the appearance of atherosclerotic lesions of the cerebral arteries in comparison with other arterial districts has been observed. This appearance has been described in monkeys as relatively earlier if hypertension is added to the atherogenic diet. Preliminary observations on a little group of rabbits on a 3 months hypercholesterolic diet, subjected to Goldblatt aortic coarctation, have shown an increase of blood pressure and a severe gross atherosclerotic involvement of aorta, resembling the one obtainable after 6 months of atherogenic diet. Histologically, the aorta predominantly shows lesions of the fatty streaks type with necrotic areas in the deep; the carotid lesions show some lipid in smooth muscle cells disseminated in a sub-endothelial "edematous" space (rich in protein). The cerebral arteries do not show any lesion. At TEM, the aortic lesions look sometimes as advanced plaques with an initial fibrosis at the surface; the carotid lesions are characterized by a granular deposit in the sub-endothelial space in which some smooth muscle cells (with lipid in the cytoplasm) are present; in the cerebral arteries only the presence of collagen fibers among the smooth muscle cells of the media, never observed in the animals fed the atherogenic diet alone, has sometimes been noted.     

Alterations in canine coronary arteries produced by chronic cigarette smoking

Segments of left circumflex coronary artery were obtained from beagles that had been exposed to cigarette smoke for 2 years and from nonsmoking controls. A ring sample from each was used to determine passive (0-Ca2+ and 2 mM EGTA) and active (75 mM K+) force-length relations. Other contiguous samples were used to determine collagen and elastin content, water content and distribution, and total electrolyte content. No significant differences were found in passive mechanical properties or the collagen and elastin content of arteries from the two groups of animals. Maximum values of active force development were found to be increased in arteries from smoking dogs as were values on the ascending part of the active force-length curve. Total water content and the distribution space of an extracellular marker (60Co-EDTA) were not significantly different among arteries from the two groups. This along with the similarity of total electrolyte contents suggests that values of cell volume were not different between the two groups and that the above differences in active force development represent alterations in smooth muscle cells at the subcellular level. These results suggest that the contribution of chronic smoking as one of a group of cardiac risk factors may be due to an effect of augmenting coronary artery smooth muscle contractility.     

Contractile capacity of isolated flaps from the aorta of rats with stable arterial hypertension caused by the prolonged administration of cerebrosides

A study was made of the contractility of smooth muscle cells of abdominal aorta strips of noninbred white rats with stable arterial hypertension induced by protracted intraperitoneal injection of cerebrosides isolated from cattle brain. It was demonstrated that as compared with normotensive animals, smooth muscle cells of the animals' arteries are characterized by the increased influx of extracellular calcium via slow potential-dependent calcium channels and hypersensitivity to noradrenaline and serotonin.     

Collagen characteristics and organization during the progression of cholesterol-induced atherosclerosis in Japanese quail

This study reports the concentration of collagen and its hydroxypyridinoline crosslinks, collagen fibril organization in the dorsal aortas, and systolic blood pressure during the progression of atherosclerosis in Japanese quail selected for cholesterol-induced atherosclerosis. The quail were placed on either a control or 0.5% cholesterol-added diet at approximately 16 weeks of age. The concentration of total collagen did not change in the control arteries during the course of the study, whereas at 5 and 10 weeks of cholesterol feeding, collagen levels decreased in the cholesterol-fed birds. Hydroxypyridinoline concentration increased during the duration of the study in the cholesterol-fed birds and by 15 and 20 weeks of cholesterol feeding, levels were significantly increased over those observed in the control arteries. Transmission electron microscopy showed changes in the organization of collagen fibrils. Increased systolic blood pressure was noted beginning at 10 weeks of cholesterol feeding, which is suggestive of other systemic changes induced by hypercholesterolemia. These results demonstrated remodeling of the collagen component of the dorsal aorta extracellular matrix during the progression of atherosclerosis and are suggestive of other systemic cardiovascular system changes.     

Collagen and the myocardium: fibrillar structure,biosynthesis and degradation in relation to hypertrophy and its regression

The extracellular matrix of the myocardium contains an elaborate structural matrix composed mainly of fibrillar types I and III collagen. This matrix is responsible for the support and alignment of myocytes and capillaries. Because of its alignment, location, configuration and tensile strength, relative to cardiac myocytes, the collagen matrix represents a major determinant of myocardial stiffness. Cardiac fibroblasts, not myocytes, contain the mRNA for these fibrillar collagens. In the hypertrophic remodeling of the myocardium that accompanies arterial hypertension, a progressive structural and biochemical remodeling of the matrix follows enhanced collagen gene expression. The resultant significant accumulation of collagen in the interstitium and around intramyocardial coronary arteries, or interstitial and perivascular fibrosis, represents a pathologic remodeling of the myocardium that compromises this normally efficient pump. This report reviews the structural nature, biosynthesis and degradation of collagen in the normal and hypertrophied myocardium. It suggests that interstitial heart disease, or the disproportionate growth of the extracellular matrix relative to myocyte hypertrophy, is an entity that merits greater understanding, particularly the factors regulating types I and III collagen gene expression and their degradation.     

Prevention of aortic fibrosis by N-acetyl-seryl-aspartyl-lysyl-proline in angiotensin II-induced hypertension

Fibrosis is an important component of large conduit artery disease in hypertension. The endogenous tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) has anti-inflammatory and antifibrotic effects in the heart and kidney. However, it is not known whether Ac-SDKP has an anti-inflammatory and antifibrotic effect on conduit arteries such as the aorta. We hypothesize that in ANG II-induced hypertension Ac-SDKP prevents aortic fibrosis and that this effect is associated with decreased protein kinase C (PKC) activation, leading to reduced oxidative stress and inflammation and a decrease in the profibrotic cytokine transforming growth factor-beta1 (TGF-beta1) and phosphorylation of its second messenger Smad2. To test this hypothesis we used rats with ANG II-induced hypertension and treated them with either vehicle or Ac-SDKP. In this hypertensive model we found an increased collagen deposition and collagen type I and III mRNA expression in the aorta. These changes were associated with increased PKC activation, oxidative stress, intercellular adhesion molecule (ICAM)-1 mRNA expression, and macrophage infiltration. TGF-beta1 expression and Smad2 phosphorylation also increased. Ac-SDKP prevented these effects without decreasing blood pressure or aortic hypertrophy. Ac-SDKP also enhanced expression of inhibitory Smad7. These data indicate that in ANG II-induced hypertension Ac-SDKP has an aortic antifibrotic effect. This effect may be due in part to inhibition of PKC activation, which in turn could reduce oxidative stress, ICAM-1 expression, and macrophage infiltration. Part of the effect of Ac-SDKP could also be due to reduced expression of the profibrotic cytokine TGF-beta1 and inhibition of Smad2 phosphorylation.     

Can the heart initiate some forms of hypertension?

An active role for the heart in the initiation of hypertension can be postulated in two different sets of conditions. 1) Activation of pressor reflexes from the ventricles, coronary arteries, or aorta has been shown to produce substantial rises of arterial pressure; experience with postcoronary bypass hypertension suggests that these reflexes could be responsible for some types of paroxysmal hypertension. 2) Increased cardiac action caused by either neural or humoral factors can initiate a rise in cardiac output and blood pressure; sustained hypertension could be produced experimentally in conscious dogs by electrical stimulation of the stellate ganglion or by continuous infusion of dobutamine in the left coronary artery. Evidence suggesting that this could occur in humans was derived from a study of the relationship of cardiac output to cardiopulmonary volume in essential hypertension.     

Mass ratios of magnesium to calcium and phosphorus in the arteries of Japanese and Thai

To elucidate quantitative changes of Ca, P, and Mg in the arteries with aging, the authors investigated changes of the mass ratios of Mg to Ca and P in the arteries of Japanese and Thai by inductively coupled plasma-atomic emission spectrometry. The arteries of Japanese that were used were the thoracic and abdominal aortas, coronary, common iliac, internal iliac, external iliac, and femoral arteries, in which very high accumulations of Ca and P occurred in old age. The arteries of Thai that were used were the abdominal aorta, ramifying site of the abdominal aorta, coronary, common iliac, internal iliac, and external iliac arteries. It was found that there were extremely significant correlations both between Ca and Mg contents and between P and Mg contents in all of the arteries of the Japanese and the Thai. With regard to the mass ratio, the mass ratios of Mg to Ca ranged from 1.5% to 2.1% in the six arteries of the Japanese, except for the thoracic aorta at 3.1%, at an advanced stage of atherosclerosis, being similar to each other. In the arteries of the Thai, the mass ratios of Mg to Ca ranged from 1.9% to 3.0%, except for the coronary artery at 0.5%, at an advanced stage of atherosclerosis. The mass ratios of Mg to P ranged from 2.5% to 2.7% in the six arteries of the Japanese, except for the coronary artery at 1.8%, at an advanced stage of atherosclerosis. With regard to the arteries of the Thai, the mass ratios of Mg to P ranged from 1.9% to 3.3%, except for the coronary artery at 0.7%, at an advanced stage of atherosclerosis. These results revealed that both the mass ratios of Mg to Ca and Mg to P were almost similar among the arteries of Japanese and Thai, except for the coronary arteries. Therefore, these results suggested that the inorganic deposits in the coronary arteries of Japanese and Thai were similar to those in the intimal tunica of the thoracic aorta, whereas in the other arteries, they were similar to those in the middle tunica of the thoracic aorta.     

Effect of hypertension on fibronectin expression in the rat aorta

Interactions between extracellular fibronectin and vascular cells are thought to influence the phenotype of those cells. To determine if changes in fibronectin expression accompany the phenotypic changes of vascular tissue characteristic of experimental hypertension, steady state mRNA levels for fibronectin were determined in aortae of normotensive and hypertensive rats. A 3-6-fold increase in fibronectin mRNA was observed in aortic tissue of hypertensive rats following 3 weeks of treatment with deoxycorticosterone and salt, whereas if rats were treated only with deoxycorticosterone or salt alone, no changes occurred. The changes were reversed by normalization of blood pressure. The increases observed were localized to aorta and not to the periaortic tissue. Angiotensin II infusion using osmotic minipumps also caused an increase in fibronectin expression. Age-dependent increases in aortic fibronectin mRNA occurred in several rat strains, and the combined effects of hypertension and aging were greater than either variable alone. A clear distinction between the expression of fibronectin mRNA and that for collagen or tropoelastin were found in hypertensive and aging models. Aortic fibronectin was also increased in the hypertensive rats as determined by Western blot analysis. The findings indicate that elevation in blood pressure increases fibronectin expression in rat aorta and suggest that such changes may influence the aortic cellular responses to hypertension.     

Response of aorta connective tissue matrix to injury caused by vassopressin-induced hypertension or hypercholesterolemia.

The aim of the study was to investigate the effect of two various atherogenic stimuli (vasopressin-induced hypertension or hypercholesterolemia) on the collagen and glycosaminoglycan (GAG) content in the internal or external part of both thoracic and abdominal aorta, which are differently susceptible to atherosclerosis. Experimental rabbits were divided into four groups: controls, animals injected with physiological saline or vasopressin at the dose of 1 IU/kg from the 1 st to the 25 th day of experiment, respectively. The animals from group 4 were maintained on food, containing 0.25% cholesterol. Only in the vasopressin-treated group, the systolic blood pressure was elevated from 110 mmHg at the beginning, to 166 mmHg at the end of the study. After 14 weeks the aorta was dissected into internal and external parts. GAG fractions were separated and estimated as uronic acids. Collagen was evaluated as the hydroxyproline content in the tissue. Augmented total GAG and heparan sulphate (HS) level, plus no changes in the collagen content were seen in the internal part of the thoracic aorta in rabbits with hypercholesterolemia or hypertension. In the hypertensive animals, the changes were extended to the external part of the aorta and, additionally, comprised the elevation of the chondroitin-4 sulphate (C-4S) content. The two atherogenic stimuli increased the collagen level with no elevation of the GAG content in the abdominal aorta. A convergent effect of the injury, caused by hypertension or hypercholesterolemia on the collagen, total GAG and HS content was shown in the respective parts of the rabbit aortas. The common GAG, increased in the thoracic aorta, stand for the HS, in both hypertensive and hypercholesterolemic rabbits. As the sensitivity to atherosclerosis development in different segments of the aorta varies, they express various responses of the connective tissue matrix to injuries, caused by hypertension or hypercholesterolemia.     

Micrometer scale ex vivo multiphoton imaging of unstained arterial wall structure.

BACKGROUND: We characterize the application of multiphoton microscopy to the observation of the extracellular matrix of fresh unstained vessels. METHOD: Combined two-photon-excited fluorescence (2PEF) and second harmonic generation (SHG) imaging of large arteries reveals the architecture of elastin and collagen fibers in the vessel wall with remarkable specificity. RESULTS: We present elastin/collagen imaging in unstained rat vessels at both micrometer and whole vessel scales, and we characterize the optical properties of rat carotid artery and aorta walls. We apply this method to evidence deleterious effects of residual doses of a pesticide on the vessel wall. CONCLUSION: This study illustrates the potential of 2PEF/SHG microscopy for pharmacological studies in unlabeled arteries.     

Site specific inelasticity of arterial tissue

Understanding the mechanical behaviour of arterial tissue is vital to the development and analysis of medical devices targeting diseased vessels. During angioplasty and stenting, stress softening and permanent deformation of the vessel wall occur during implantation of the device, however little data exists on the inelastic behaviour of cardiovascular tissue and how this varies through the arterial tree. The aim of this study was to characterise the magnitude of stress softening and inelastic deformations due to loading throughout the arterial tree and to investigate the anisotropic inelastic behaviour of the tissue. Cyclic compression tests were used to investigate the differences in inelastic behaviour for carotid, aorta, femoral and coronary arteries harvested from 3-4 month old female pigs, while the anisotropic behaviour of aortic and carotid tissue was determined using cyclic tensile tests in the longitudinal and circumferential directions. The differences in inelastic behaviour were correlated to the ratio of collagen to elastin content of the arteries. It was found that larger inelastic deformations occurred in muscular arteries (coronary), which had a higher collagen to elastin ratio than elastic arteries (aorta), where the smallest inelastic deformations were observed. Lower magnitude inelastic deformations were observed in the circumferential tensile direction than in the longitudinal tensile direction or due to radial compression. This may be as a result of non-collagenous components in the artery becoming more easily damaged than the collagen fibres during loading. Stress softening was also found to be dependent on artery type. In the future, computational models should consider such site dependant, anisotropic inelastic behaviour in order to better predict the outcomes of interventional procedures such as angioplasty and stenting.     

Role of calcium-activated potassium channels in the regulation of basal and agonist-elevated tones in isolated conduit arteries. Short communication

Functional role of calcium-activated potassium (KCa) channels on the basal and agonist-elevated arterial tones was investigated in isolated rabbit aorta, porcine and canine coronary arteries as well as in human internal mammary artery. The vascular tones enhanced by contractile agents were increased further by preincubation of these conduit blood vessels with selective (charybdotoxin or iberiotoxin) or nonselective (tetraethylammonium) inhibitors of KCa channels. The basal tone (without an agonist) was increased only in the canine coronary artery. The results indicate a feed-back regulatory role of KCa channels counteracting the vasospasm of conduit arteries.     

The role of prostaglandins in hypertension. I. The release of prostaglandins by aorta strips of renal, DOCA-salt, and spontaneously hypertensive rats

The release of prostaglandin-like (PG-like) material by aorta strips of normotensive and hypertensive rats has been studied in vitro. When incubated in an oxygenated Krebs solution kept at 37 degrees C, aorta strips removed from 8- and 12-week-old spontaneously hypertensive (SH) rats generate 1.2-2.5 times more PG-like material than aorta strips from age-matched normotensive Wistar (NW) rats. The overproduction of PG-like material by aorta strips of SH rats did not precede the development of hypertension in SH rats. Aorta strips derived from renal and DOCA-salt hypertensive rats produced 1.5-3 times more PG-like material than aorta strips from NW rats. The production of PG-like material by aorta strips of renal and DOCA-salt hypertensive rats was largely reduced when hypertension was interrupted in these animals, thus suggesting that the alteration taking place in the arteries of hypertensive rats (namely increased production of PGs) during the development of hypertension was reversible. The production of PG-like material by aorta strips of hypertensive rats was inhibited by indomethacin. Analysis of the PG-like material by bioassays and thin-layer chromatography suggests the presence of PGE2 and PGE1. The possible involvement of these PGs in the pathogenesis of hypertension in rats is discussed.     

Accumulation of elements in the arteries and cardiac valves of Thai with aging

To elucidate whether the extent of element accumulation in the arteries and cardiac valves with aging was different between different races, the authors investigated the accumulation of elements in the arteries and cardiac valves of the Thai with aging and the relationships among elements in the cardiac valves. After ordinary dissection at Chiang Mai University was finished, 16 arteries and 4 cardiac valves were resected and element contents were determined by inductively coupled plasma-atomic emission spectrometry. In the 16 arteries, the average content of calcium was the highest in the site of the abdominal aorta ramifying into the common iliac arteries, and it decreased in the order internal iliac, coronary, abdominal aorta, common iliac, external iliac, superior mesenteric, inferior mesenteric, thoracic aorta, brachial, radial, common carotid, subclavian, ulnar, axillary, renal, and internal thoracic arteries. The average contents of phosphorus and magnesium in respective arteries were parallel with the average contents of calcium, except for the coronary artery. In comparison with the arteries of the Japanese, the trend of calcium accumulation in the arteries of the Thai was almost similar to that in the arteries of the Japanese, except for the coronary artery and thoracic aorta. The calcium accumulation in the coronary artery was much higher in the Thai than in the Japanese, whereas that in the thoracic aorta was lower in the Thai than in the Japanese. Regarding elements in the cardiac valves, the calcium content increased remarkably in the seventies in the aortic valve and in the nineties in the pulmonary valve, but it hardly increased in both the mitral and tricuspid valves with aging. The average content of calcium was the highest in the aortic valve, and it decreased in the order pulmonary, tricuspid, and mitral valves. Regarding the relationship among elements in the aortic valves, it was found that there were extremely significant direct correlations among the contents of calcium, phosphorus, and magnesium, whereas there were significant direct correlations between zinc and either calcium or phosphorus contents. Although significant correlations were found between sulfur and the other element contents in the aortic valves of the Japanese, no significant correlations were found between them in the aortic valves of the Thai. In the mitral valves, extremely or very significant direct correlations were found among the contents of calcium, phosphorus, magnesium, and sulfur, with some exceptions that there were no significant correlations between phosphorus and either magnesium or sulfur contents. In addition, no significant correlation was found in the calcium content between the aortic valve and coronary artery in the same individuals.     

Accumulation of calcium and phosphorus accompanied by increase of magnesium and decrease of sulfur in human arteries

To elucidate the accumulation of elements in the arteries with aging, the authors investigated age-related changes of elements in human arteries, such as the thoracic aorta, femoral, basilar, coronary, radial, and common iliac arteries by inductively coupled plasma-atomic emission spectrometry. The subjects consisted of 17 men and 9 women, ranging in age from 55 to 92 yr in the cases of the five arteries, except for the common iliac arteries, in which the subjects consisted of 16 men and 8 women, ranging in age from 65 to 93 yr. It was found that there were significantly direct correlations between calcium and phosphorus contents and between calcium and magnesium contents in all of the six arteries: thoracic aorta, femoral, basilar, coronary, radial, and common iliac arteries. Significantly direct correlations were also found between phosphorus and magnesium contents in the five arteries, except for the basilar artery. In contrast, significantly inverse correlations were found between calcium and sulfur contents and between phosphorus and sulfur contents in the four arteries, except for the coronary and radial arteries. These revealed that the accumulation of calcium and phosphorus in the arteries was accompanied by an increase of magnesium in the arteries and by a decrease of sulfur in the arteries.     

High accumulation of elements in the human femoral artery

The relative contents (RCs) of elements in the femoral arteries as well as the thoracic aorta, coronary, basilar, and radial arteries from 26 subjects within the age range between 55 and 92 yr old, were analyzed by inductively coupled plasma atomic emission spectrometry. The RCs of calcium and phosphorus in the femoral arteries started to increase before the age of 60 yr. The RCs of magnesium increased after the age of 70 yr. However, the RCs of sulfur did not change significantly within the age range between 55 and 92 yr. With regard to localization of the mineral accumulations in the femoral arterial wall, it was found that the accumulations of calcium and phosphorus occurred only in the tunica media, only in the tunica intima, or in both the tunica media and the tunica intima. The manner of accumulation of calcium and phosphorus in the femoral arterial wall was different from that in the aortic wall. The average RCs of calcium in the 26 specimens were the highest in the femoral artery, followed in descending order by the thoracic aorta, coronary, basilar, and radial arteries. The average RCs of phosphorus were highest in the thoracic aorta, followed by the coronary, femoral, basilar, and radial arteries. It is noted that the accumulation of mineral elements never occurred uniformly in all the arteries.     

Age-related alterations in epicardial arteries of spontaneously hypertensive rats

Proximal portions of the left coronary arteries were examined microscopically in aging female normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). The age-related intimal alterations in SHR were largely limited to endothelial cells, which demonstrated a proliferation of organelles (most notably Weibel-Palade bodies). In the media, degenerative alterations appeared to be most marked near the medio-adventitial junction. Compared to WKY and increasing with age, the media of the SHR epicardial artery demonstrated an accumulation of extracellular elements that included basement membrane-like material, collagen fibers and debris. Complex carbohydrates, as determined with the silver methenamine reaction, were noted to accumulate in a lamellar fashion. Smooth muscle cells demonstrated an age-related tendency (which was exaggerated in SHR) toward development of invaginations and irregular profiles. These observations indicate that age-related structural alterations in epicardial arteries of SHR are progressive, and they support the concept that structural alterations in SHR coronary arteries may represent accelerated aging phenomena.