Expermental contribution on the genesis of arteriosclerosis caused by hypertension]

Autoradiographic tests carried out on rats with renal hypertension using 3H-proline resulted in an acclerated collagen synthesis by media cells of aorta and coronary arteries. Electronmicroscopically an increased content of collagen fibers and an enrichment of ruthenium-red-positive substances in the extracellular space were found. The 35S-sulfate-incorporation in aorta and coronary arteries of animals with hypertension is also increased. These changes in the extracellular space of the vascular wall have an atherosclerosis promoting effect, probably caused by a distrubance of the permeability.     

Quantitative study of aortic wall permeability

Fluorescent microscopy with trypan blue was used to assay aortal wall permeability. The dye permeation to the vascular wall was assayed by photometry with graphic data recording. The technique described enabled exploring aortal wall permeability in rabbits with experimental hypertension associated with induced atherosclerosis.     

Vascular smooth muscle in hypertension

The cause of the elevated arterial pressure in most forms of hypertension is an increase in total peripheral resistance. This brief review is directed toward an assessment of recent investigations contributing information about the factors responsible for this increased vascular resistance. Structural abnormalities in the vasculature that characterize the hypertensive process are 1) changes in the vascular media, 2) rarefication of the resistance vessels, and 3) lesions of the intimal vascular surface. These abnormalities are mainly the result of an adaptive process and are secondary to the increase in wall stress and/or to pathological damage to cellular components in the vessel wall. Functional alterations in the vascular smooth muscle are described as changes in agonist-smooth muscle interaction or plasma membrane permeability. These types of changes appear to play a primary, initiating role in the elevation of vascular resistance of hypertension. These alterations are not the result of an increase in wall stress and they often precede the development of high blood pressure. The functional changes are initiated by abnormal function of neurogenic, humoral, and/or myogenic changes that alter vascular smooth muscle activity.     

Geometrical,functional, and histomorphometric adaptation of rat carotid artery in induced hypertension

Acute and long-term (up to 56 days) evolution of geometry, structural properties, vascular smooth muscle (VSM) tone and histomorphometric properties of the rat common carotid arteries under induced hypertension were investigated. Hypertension was induced in 8-week old male Wistar rats by total ligation of the aorta between the two kidneys. Rats were sacrificed 2, 4, 8 and 56 days postsurgery. The arterial wall layers thicken non-uniformly during the adaptation process, the inner layers thicken more in the acute phase of hypertension, whereas the outer layers of the wall are thicker than the inner layers at the end of the adaptation phase. Collagen content in the wall media exhibits a non-linear evolution, with a rapid increase in the acute hypertension phase followed by a slower increase at long-term. The elastin content increase is slight and steady, whereas VSM shows a steady but considerable increase which outdoes the collagen increase in long-term phase. VSM tone increases rapidly in the acute phase of remodelling (0-8 days) and this increase in tone contributes to a considerable increase in arterial compliance in the operating pressure range. At long-term (56 days) VSM tone returns to near control level, but compliance is even further increased, which suggests that at long-term the compliance increase is attributed primarily to structural remodelling.     

Pharmacological modification of pulmonary vascular injury: possible role of cAMP

Experiments were designed to test the hypothesis that drugs which increase adenosine 3',5'-cyclic monophosphate (cAMP) in the lung would prevent the pulmonary hypertension and the increase in vascular permeability caused by the infusion of the oxidant lipid peroxide, tert-butyl hydroperoxide (t-bu-OOH), in isolated rabbit lungs perfused with Krebs-Henseleit buffer. Pretreatment with indomethacin or verapamil was also studied, since these drugs block the increase in pulmonary arterial pressure caused by t-bu-OOH. Indomethacin or verapamil prevented the pulmonary hypertension but did not prevent the increase in permeability caused by t-bu-OOH. Consequently, indomethacin or verapamil treatment partially reduced the gain in lung weight caused by t-bu-OOH. In contrast, pretreatment with isoproterenol, prostaglandin E1, or a cAMP analogue not only prevented the pulmonary hypertension but also inhibited the increase in vascular permeability caused by t-bu-OOH. Consequently, these drugs completely blocked the gain in lung weight caused by t-bu-OOH. Posttreatment with aminophylline or the cAMP analogue also significantly reduced the gain in lung weight caused by t-bu-OOH. These results indicate that pharmacological therapy can reduce the pulmonary hypertension and the increase in vascular permeability caused by the infusion of a lipid hydroperoxide. Since isoproterenol, aminophylline, prostaglandin E1, and a cAMP analogue all had similar effects, the results suggest that the likely common mechanism for their protective effect is an increase in cAMP.     

Blood pressure regulates the activity and function of the Na-K-2Cl cotransporter in vascular smooth muscle

The Na-K-2Cl cotransporter (NKCC1) is one of several transporters that have been linked to hypertension, and its inhibition reduces vascular smooth muscle tone and blood pressure. NKCC1 in the rat aorta is stimulated by vasoconstrictors and inhibited by nitrovasodilators, and this is linked to the contractile state of the smooth muscle. To determine whether blood pressure also regulates NKCC1, we examined the acute effect of hypertension on NKCC1 in rats after aortic coarctation. In the hypertensive aorta (28-mmHg rise in mean blood pressure), an increase in NKCC1 activity (measured as bumetanide-sensitive (86)Rb efflux) was apparent by 16 h and reached a plateau of 62% greater than control at 48 h. In contrast, there was a slight decrease in NKCC1 activity in the hypotensive aorta (21% decrease in mean blood pressure). Measurement of NKCC1 mRNA by real-time PCR revealed a fivefold increase in the hypertensive aorta compared with the hypotensive aorta or sham aorta. The inhibition by bumetanide of isometric force response to phenylephrine was significantly greater in the hypertensive aorta than in the control aorta or hypotensive aorta. We conclude that NKCC1 in rat aortic smooth muscle is regulated by blood pressure, most likely through changes in transporter abundance. This upregulation of NKCC1 is associated with a greater contribution to force generation in the hypertensive aorta. This is the first demonstration that NKCC1 in vascular smooth muscle is regulated by blood pressure and indicates that this transporter is important in the acute response of vascular smooth muscle to hypertension.     

Proteomic analysis permits the identification of new biomarkers of arterial wall remodeling in hypertension

Hypertension represents one of the main risk factors for vascular diseases. Genetic susceptibility may influence the rate of its development and the associated vascular remodeling. To explore markers of hypertension-related morbidity, we have used surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry to study changes in proteins released by the aorta of two rat strains with different susceptibilities to hypertension. Fischer and Brown Norway (BN) rats were divided into a control group and a group receiving low-dose N(Omega)-nitro-L-arginine methyl ester (L-NAME), a hypertensive drug, interfering with endothelial function. In spite of a significant elevation of blood pressure in both strains in response to L-NAME, BN rats exhibited a lower vascular remodeling in response to hypertension. Proteomic analysis of secreted aortic proteins by SELDI-TOF MS allowed detection of four mass-to-charge ratio (m/z) peaks whose corresponding proteins were identified as ubiquitin, smooth muscle (SM) 22alpha, thymosin beta4, and C-terminal fragment of filamin A, differentially secreted in Fischer rats in response to L-NAME. We have confirmed a strain-dependent difference in susceptibility to L-NAME-induced hypertension between BN and Fischer rats. The greater susceptibility of Fischer rats is associated with aortic wall hypertrophic remodeling, reflected by increased aortic secretion of four identified biomarkers. Similar variations in one of them, SM22alpha, also were observed in plasma, suggesting that this marker could be used to assess vascular damage induced by hypertension.     

Tissue differences in vascular permeability induced by leukotriene B4 and prostaglandin E2 in the rat

The activity of synthetic LTB4 and PGE2, in increasing vascular permeability was tested simultaneously in seventeen different organs in the rat. Rats were injected in the aortic arch through a cannula in the carotid artery with 125I-albumin, 51Cr-erythrocytes, and 57Co-EDTA. The rats were then injected through the carotid artery cannula with LTB4, PGE2 or a combination of LTB4 and PGE2. Eight minutes later the rats were killed and the activity of 125I, 51Cr, and 57Co measured in different organs. Changes in vascular permeability were inferred from changes in the ratios of the isotope activities. LTB4 (15 micrograms/kg) induced enhanced permeability in caecum, small bowel, skin, fat pad, stomach, pancreas, and aorta, but not in the heart, brain, colon, testes, diaphragm, forelimb, cremaster muscle, lung, kidney or eye. A lower dose of LTB4, 3 micrograms/kg, enhanced vascular permeability in caecum, small bowel, skin, stomach, and aorta. PGE2 (1 microgram/kg) enhanced vascular permeability only in the caecum. A combination of LTB4 (3 micrograms/kg) and PGE2 (1 microgram/kg) was more potent than either alone. Rats depleted of neutrophils with anti-neutrophil serum were less sensitive to LTB4 than intact rats. These findings suggest that the vasculatures of different tissues in the rat vary markedly in their susceptibility to LTB4 induced increases in permeability.     

Hypoxia-induced increase of endostatin in murine aorta and lung

In the lung, hypoxia induces pulmonary hypertension caused by vasoconstriction and vascular remodeling. Additionally, hypoxia is an inducer of angiogenesis, which is assumed to counteract pulmonary hypertension. We asked whether the anti-angiogenic factor endostatin—a cleavage product of collagen XVIII—participates in the vascular alterations induced by hypoxia. By employing Western blotting of tissue extracts of murine brain, liver and heart an endostatin fragment of 22 kDa was detectable, whereas in lung and aorta additional bands of 24 and 26 kDa were found. The amount of these larger fragments was increased in tissues obtained from mice housed for 4 days or 3 weeks at hypobaric hypoxia. By immunohistochemistry endostatin was detected in association with elastic fibers and in close neighborhood to smooth muscle cells of intrapulmonary vessels and the aorta. In the lung, the activity of matrix metalloproteinases (MMP) known to generate endostatin by cleavage of collagen XVIII was increased (MMP-2) and decreased (proMMP-9), respectively, by hypoxia. Elevated amounts of endostatin within the aortic wall of mice exposed to hypobaric hypoxia may stabilize the vascular wall by inhibition of microvascular sprouting. The surprising finding of increased endostatin in the lung presumably contributes to the development of pulmonary hypertension by reduction of angiogenesis.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Arterial receptors for adrenal steroids and transport of electrolytes in vascular smooth muscle

The role of arterial receptors to mineralocorticoids (MC) and glucocorticoids (GC) in the induction by MC and GC of changes in transmembrane transport of sodium (Na+) and water was investigated. Implantation of Silastic rubber strips impregnated with 11-desoxycorticosterone acetate (DOCA) in rabbits was followed by a marked increase in vascular smooth muscle cell membrane permeability to Na+ and hypertension. Both of these effects were preventable with progesterone, an anti-MC at the steroid-receptor level, implanted in relative excess simultaneously with DOCA, in approximately 50% of the implanted animals. The other 50% were hydroxylating in vivo progesterone to 11-desoxycorticosterone (DOC) efficiently enough not to yield the necessary ratio of progesterone to DOC for the sufficient MC receptor blockage. In vascular smooth muscle cell culture, grown in the presence of steroids, GC but not MC increased intracellular water space. This increase was preventable by a potent synthetic anti-GC,RU 38486, a steroid with high affinity for GC receptors, added to culture medium along with GC. These results provide evidence that both the in vivo effect of MC on Na+ permeability and the induction of hypertension, and the in vitro effect of GC on water transport in cultured vascular smooth muscle cells are elicited through the receptor-mediated molecular mechanism(s) for action of these steroids in the arterial wall.     

Type III collagen can be present on banded collagen fibrils regardless of fibril diameter

Monoclonal antibodies that recognize an epitope within the triple helix of type III collagen have been used to examine the distribution of that collagen type in human skin, cornea, amnion, aorta, and tendon. Ultrastructural examination of those tissues indicates antibody binding to collagen fibrils in skin, amnion, aorta, and tendon regardless of the diameter of the fibril. The antibody distribution is unchanged with donor age, site of biopsy, or region of tissue examined. In contrast, antibody applied to adult human cornea localizes to isolated fibrils, which appear randomly throughout the matrix. These studies indicate that type III collagen remains associated with collagen fibrils after removal of the amino and carboxyl propeptides, and suggests that fibrils of skin, tendon, and amnion (and presumably many other tissues that contain both types I and III collagens) are copolymers of at least types I and III collagens.     

Mast cell-independent mechanisms of immediate hypersensitivity: a role for platelets

Mast cells have been implicated as the central effectors in allergic responses, yet a fatal anaphylactic response can be induced in mast cell-deficient mice. In this study, we examined the immediate hypersensitivity response in wild-type (WT) and mast cell-deficient mice (W/W(v)) in two different tissues (skin and skeletal muscle). Vascular permeability and leukocyte recruitment were studied after immediate challenge or 4 h postchallenge in OVA-sensitized mice. In skin, immediate challenge induced a significant increase in vascular permeability (75%) within 30 min and was accompanied by increased leukocyte adhesion 4 h postchallenge. In the absence of mast cells, no changes in vascular permeability or leukocyte recruitment were observed in skin. In WT skeletal muscle, immediate challenge induced a rapid increase (80%) in vascular permeability within 5 min and significant leukocyte recruitment after 4 h. Surprisingly, in W/W(v), a gradual increase in vascular permeability was observed, reaching a maximum (50%) within 30 min. Despite the absence of mast cells, subsequent leukocyte emigration was similar to that observed in WT mice. Pretreatment with anti-platelet serum in W/W(v) returned Ag-induced vascular permeability and leukocyte recruitment to baseline. Platelets were shown to interact with endothelium in skeletal muscle, but not dermal microvasculature. These data illustrate that mast cells play a prominent role in vascular permeability and leukocyte recruitment in skin in response to Ag, however, in skeletal muscle; these changes can occur in the absence of mast cells, and are mediated, in part, by the presence of platelets.     

A quantitative histochemical study of the microvasculature of irradiated skin

Short-term X-ray damage to the microvasculature of the skin of newborn rats has been quantitated using Horseradish Peroxidase as a tracer. Image analysis of thick sections on which peroxidase was demonstrated histochemically revealed a radioinduced increase in vascular volume coupled with a decrease in vascular length and an altered frequency distribution of blood vessel calibers which resulted in early telangiectasia. The results afforded by direct counting of peroxidase positive macrophagic cells and microphotometric evaluation of peroxidase present in the connective tissue indicate a progressive increase in capillary permeability as a function of dose and time post-irradiation. The accuracy with which the affected region of blood vessels coincided with the area exposed to the beam favours the hypothesis of direct damage to the vessel wall as a major cause of radioinduced lesion.     

Basic studies of cryochemotherapy in a murine tumor system

The combined effect of cryosurgery and anticancer drugs (cryochemotherapy) was studied in an experimental B16 melanoma/BDF1 tumor system. Vascular volume and vascular permeability after cryosurgery of normal skin and the tumor were measured by using 51Cr-labeled red blood cells and 125I-labeled serum albumin. The vascular volume and vascular permeability of both the normal vessels and the tumor vessels greatly increased immediately after cryosurgery, and their vascular volume decreased to less than the normal level within a few hours. However, the tumor vessels showed less dilatation and increase in permeability than the vessels of normal tissue. There was a difference in functional characteristics in response to cryoinjury between the normal vessels and the tumor vessels. The anticancer drugs, peplomycin and adriamycin, were administered intraperitoneally in combination with cryosurgery. When peplomycin was administered 5 min, 1 hr, and 3 hr after cryosurgery, the drug concentration in the frozen tumor was higher than that in the untreated tumor. But when administered 1 hr before cryosurgery, peplomycin was not trapped in the tumor. Trapping of adriamycin was not observed after the same treatment. In cryochemotherapy, it is necessary to administer the appropriate drug at the appropriate time. However, the trapping of the anticancer drug results in a high concentration and lasts for a long time, so that cryochemotherapy is expected to be a new mode of cancer therapy, particularly as a multidisciplinary treatment for cancer.     

Red wine polyphenols affect the collagen composition in the aorta after oxidative damage induced by chronic administration of CCl(4)

Increased amount of collagen type I and decreased amount of type III is described in various pathological processes in the vascular wall. Polyphenols were shown to have protective effect on endothelium, decrease blood pressure and prevent oxidative damage induced by various stimuli. Tetrachlormethane (CCl(4)) is a toxic substance with known negative systemic effects induced by free radicals. Chronic administration of CCl(4) for 12 weeks led to an increase of collagen type I and a decrease of type III in the wall of aorta. Parallel administration of red wine polyphenols significantly reduced the increase of collagen type I, at the same time the content of type III rose to the level above controls. After 4 weeks of spontaneous recovery no changes were observed. If polyphenols were administered during the recovery period, there was a decrease of type I and an increase of type III collagen content in the aorta. It can be concluded that polyphenols have a tendency to lower the amount of type I and to increase the proportion of type III collagen in the wall of the aorta. These changes are significant in prevention or in regression of changes induced by chronic oxidative stress. This effect of polyphenols is most likely the result of their influence on MMP-1 and TIMP activities through which they positively influence the collagen types I and III content ratio in the vascular wall in favor of the type III collagen.     

Sympathetic innervation of blood vessels in rats with aortic constriction hypertension

The adrenergic innervation of blood vessel wall was studied in various vascular beds of adult rats with experimental hypertension induced by the constriction of the aorta between the origins of both renal arteries. A moderate expansion of body fluids was demonstrated in this hypertensive model. The decrease of the density of adrenergic plexus in the vessel wall as well as the diminished catecholamine fluorescence were found only in renal vessels. These changes were pronounced in the left renal artery and vein even if the left kidney was not subjected to elevated blood pressure. Thus the alteration of vascular adrenergic innervation in hypertensive rats is not a consequence of high blood pressure but it seems to be a part of neurohumoral pathogenetic mechanisms.     

Effect of chromocarb diethylamine on the permeability of the blood-brain barrier

Intravenously injected collagenase, detectable in brain microvessels by immunological methods, partially degrades the constituents of the vascular wall and so increases the permeability of the blood-brain barrier (BBB). Intravenous administration of collagenase is a model for diseases in which the concentration of endogenous proteases is increased. Peroral treatment of rats with chromocarb diethylamine (CD) significantly reduced the degradation of the vascular wall by intravenous collagenase, as demonstrated by a lesser permeability increase of the BBB, a shorter recovery time, lower hydroxyproline levels in the cerebrospinal fluid and a lesser decrease of the collagen content of the brain capillary basal lamina.     

Primary versus secondary structural changes of the blood vessels in hypertension

Various researchers have hypothesized that the thickening of the vascular wall plays an important role in the maintenance of hypertension. Such an alteration can increase the vascular resistance by exerting two effects. A thickened vascular wall could occlude the lumen of the blood vessel and (or) cause the artery to hyperreact to contractile stimuli. Until recently, it has been a general conclusion that such alterations were a secondary adaptation produced by the elevation of blood pressure. Consistent with this view, certain classes of larger arteries do exhibit a thickened vascular wall late during hypertension development and such changes can be prevented from occurring by antihypertensive treatment. However, recent studies involving the mesenteric and renal arteries of Wistar-Kyoto spontaneously hypertensive rats have shown that wall thickening of the vasculature occurs prior to hypertension development and is present even under conditions where the blood pressure has been normalized throughout the animal's life. These latter observations suggest that some structural alterations in the blood vessels observed in hypertension are pressure independent and could be of etiological importance in the initiation of hypertension.     

Transient diffusion of albumin in aortic walls: effects of binding to medial elastin layers

The goal of this study was to measure diffusive transport of albumin through artery walls experimentally and to analyze the results theoretically, taking into account the binding of albumin to elastic lamellae. Segments of rabbit aorta were placed in solutions of fluorescently labeled albumin for periods of 30, 60, 90, and 120 min, and the distributions of fluorescence intensity through the arterial media were observed. On average, intensity increased almost linearly with time. Bands of high intensity were observed corresponding to elastin layers within the media. The temporal and spatial variations of intensity were compared with predictions of theoretical models, including effects of albumin binding and hindered diffusion resulting from the complex wall structure. Based on these analyses, it was concluded that the spatial distribution of free albumin within the media equilibrated relatively rapidly, and that the observed linear increase in intensity reflected gradual accumulation of albumin bound to medial elastin layers. The results imply that previous theoretical analyses, in which binding was neglected, substantially underestimated albumin diffusivity in the aortic wall. With respect to stent-associated delivery of inhibitors of vascular cell proliferation, the results suggest that albumin might serve as an "affinity vehicle" for drug delivery to the aorta, by attaching the drug to an abundant component of the artery wall.     

Inhibitory effect of a novel bradykinin B1 receptor antagonist,R-954, on enhanced vascular permeability in type 1 diabetic mice

The morbidity and mortality associated with type 1 diabetes are essentially related to the micro- and macrovascular complications that develop over time and lead to several diabetic complications, including hypertension, atherosclerosis, and retinopathy, as well as coronary and renal failure. Normally absent in physiological conditions, the bradykinin B1 receptor (BKB1-R) was recently found to be overexpressed in pathological conditions, including type 1 diabetes. In the present study, we evaluated the effect of the new BKB1-R antagonist, R-954 (Ac-Orn-[Oic2, alpha-MePhe5, D-betaNal7, Ile8]desArg9-bradykinin, on the increase in vascular permeability in streptozotocin (STZ)-diabetic mice. The capillary permeability to albumin was measured by quantifying the extravasation of albumin-bound Evans blue dye in selected target tissues (liver, pancreas, duodenum, ileum, spleen, heart, kidney, stomach, skin, muscle, and thyroid gland). Acute single administration of R-954 (300 microg/kg, i.v.) to type 1 diabetic mice 4 weeks after STZ significantly inhibited the enhanced vascular permeability in most tissues. These data provide further experimental evidence for the implication of BKB1-R in the enhanced vascular permeability associated with type 1 diabetes.