Hypertensive structural changes in blood vessels: do endothelial cells hold the key?

In recent decades, the complexity of the endothelium and its major role in maintaining or altering blood vessel architecture are being revealed. In contrast, the vascular smooth muscle cell previously received the most attention. I suggest support of the hypothesis that the endothelium is the key to vascular disease. An altered endothelium in diabetes mellitus likewise is likely to be pivotal in vascular complications that develop. We have demonstrated that adherent monocytes, indicators of altered endothelium, occur in deoxycorticosterone acetate induced hypertension in male Wistar rats. The coronary artery and thoracic aorta were investigated using transmission electron microscopy. Details of hypertensive changes were revealed as well as early atherogenic pathology in the absence of dietary modifications. Scanning electron microscopy of thoracic aorta showed details of the luminal endothelial surface and adherent monocyte-macrophages in hypertensive animals. There were two cell types: numerous typical monocytes with upstream tails, and larger cells that may have been free grazing macrophages or macrophages that had returned to the circulation. Debris and amorphous material were particularly evident in vessels from hypertensive animals. Monocytes squeezed between intact endothelial plasma membranes (as seen in section), and were found as subendothelial foam cells and phagocytosing macrophages. The endothelial adherence of monocytes to the aortas from diabetic animals was significantly (p less than 0.05) elevated over that found in controls (but not different from control-hypertensive or diabetic-hypertensive animals) supporting the concept of altered endothelium in diabetes.     

Expression of myogenic constrictor tone in the aorta of hypertensive rats

We investigated the effect of intraluminal pressure or stretch on the development of tone in the descending thoracic aorta from rats with aortic coarctation-induced hypertension of 7-14 days duration. Increments of pressure >100 mmHg decreased the diameter of thoracic aortas from hypertensive but not from normotensive rats. The pressure-induced constriction was not demonstrable in vessels superfused with calcium-free buffer. Stretched rings of aorta from hypertensive rats exhibited a calcium-dependent constrictor tone accompanied by elevated calcium influx that varied in relation to the degree of stretch. Blockers of L-type calcium channels and inhibitors of protein kinase C reduced both basal tone and calcium influx in aortic rings of hypertensive rats. Hence, the thoracic aorta of hypertensive rats expresses a pressure- and stretch-activated constrictor mechanism that relies on increased calcium influx through L-type calcium channels via a protein kinase C-regulated pathway. The expression of such a constrictor mechanism is suggestive of acquired myogenic behavior.     

Development of a pseudointima in a synthetic prosthesis in experimental hypercholesterolemia]

Under study was the dynamics of formation of pseudointima in a synthetic lavsan prosthesis of the aorta of 32 rabbits, 22 of them were on the atherogenic diet. Against the background of experimental hypercholesterolemia the proliferation of cells and the obliteration of the vessel lumen were found to proceed more rapidly than in normal animals.     

Rapid induction of atherosclerosis in rabbits.

Japanese white rabbits fed a restricted amount (100 g/head/day) of an atherogenic diet (AD) containing 0.2% cholesterol and 6% peanut oil showed mild and persistent hypercholesterolemia (338 +/- 79 mg/dl). They developed atherosclerotic lesions 4 weeks after deendothelialization of aorta carried out at the 4th week of AD-feeding. This rabbit model of atherosclerosis has such advantages as being able to be produced in a short period and having similar biochemical and pathological characteristics with those in human atherosclerosis.     

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.     

Combination of Periaortic Elastase Incubation and Cholesterol-Rich Diet: A Novel Model of Atherosclerosis in Rabbit Abdominal Aorta

Atherosclerosis, the leading cause of most cardiovascular disease, is a progressive multifaceted inflammatory disease characterized by extracellular matrix degradation and extensive remodeling of artery wall. However, its mechanism has not been completely understood, and animal models are useful to study its pathogenetic process. An analysis of literature on the nature of atherosclerosis indicates that focal accumulation of smooth muscle cells (SMCs) into the intima by plasma factors is fundamental to the entire process of plaque growth. In our previous study, vascular SMCs proliferation was obvious in elastase-induced aorta by day 15, which led to intimal hyperplasia and regression of rabbit aneurysm. Model induced by combination of balloon injury and an atherogenic diet in rabbits is the conventional, but most largely used experimental model of atherosclerosis. Since proliferation and accumulation of intimal SMCs are found in elastase-induced aorta, and hypercholesterolemia is usually induced by cholesterol-rich diets in rabbits, a novel atherosclerosis model may be induced by combination periaortic elastase incubation and cholesterol-rich diet.     

The role of hypertension and hypercholesterolemia on aortic sudanophilia and carotid distensibility in rabbits

These experiments were designed to determine if male New Zealand white rabbits made mildly hypertensive (20-30 mm Hg increase) with bilateral renal artery clips developed more or less sudanophilic lesions than controls, and if the animals responded differently if hypercholesterolemia was produced soon (one week) or late (eight weeks) after the animals were operated on. Both groups received the diet of 2% cholesterol and 6% corn oil for six weeks. We also studied the distensibility of the carotid artery to determine if altered elastic behaviour played a role in lesion development. The experiments showed that the acute hypertensive group developed most lesions (by area), but that the lesions in all groups had the same shape and location. The carotid arteries from the chronic hypertensives were least distensible, and most of the changes appeared to be in the elastance of collagen. The blood pressure actually dropped slightly in the chronic shams after the diet was started. These experiments suggest that, at least, in the rabbit, the duration of the hypertension may determine how the arterial wall responds to hypercholesterolemia. They show that mild hypertension, like hypercholesterolemia, alters the rate at which lesions develop, rather than altering their distribution. The changes do not appear to be related to altered distensibility.     

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.     

Persistent impairment of endothelium-dependent relaxation to acetylcholine and progression of atherosclerosis following 6 weeks of cholesterol feeding in the rabbit

The synthesis and (or) release of endothelium-dependent relaxant factor released by acetylcholine is impaired in New Zealand white rabbits fed an atherogenic diet. Experiments were designed to investigate whether the synthesis and (or) release of the endothelium-dependent relaxant factor from rabbit aortas are restored after reversal from an atherogenic diet to a non-atherogenic diet. Atherosclerosis was induced by feeding a diet containing lipids and 2% cholesterol for 6 weeks. Rabbits were sacrificed after 6 weeks on the atherogenic diet and 36 weeks after return to a standard laboratory diet. Synthesis and (or) release of the factor from the thoracic aorta was assayed using a bioassay system. The relaxant responses produced in the assay tissue were impaired both in the acute stage and after 36 weeks on non-atherogenic food. This impaired relaxation is probably due to a persistent functional abnormality in the aortic endothelium resulting in the failure to synthesize and (or) release endothelium-dependent relaxation factor 36 weeks after induction of atherosclerosis.     

Characterization of calcium accumulation in the brain of rats administered orally calcium: The significance of energy-dependent mechanism

Primary cultures of rat hepatocytes maintained on different matrix proteins such as collagen (Co IV) fibronectin (Fn), Laminin (Ln) or different tissue biomatrices were metabolically labelled with ³⁵[S]-SO₄ and the synthesis of sulphated proteoglycans was studied. The incorporation of the label into total glycosaminoglycan (GAG) was significantly higher in cells maintained on Co IV compared to those maintained on Fn or Ln. Similarly the incorporation of label was maximum in those cells maintained on the aortic biomatrix compared to liver or mammary gland biomatrix. About 80–95% of the GAG synthesised and secreted by cells maintained on individual matrix proteins and liver biomatrix was heparan sulphate (HS). But in the case of cells maintained on collagen IV aortic or mammary biomatrix in addition to HS, significant amount of chondroitin sulphate (CS) was also found. Nearly 50% of the total ³⁵[S]-GAG was associated with the cell layer after 24 h in culture in the case of cells maintained on individual matrix protein while those maintained on tissue biomatrix, retained about 70% of the ³⁵[S]-labelled proteoglycans (PG) with the cell layer. Analysis of the cell surface ³⁵[S]-labelled proteoglycans isolated from cells maintained on different biomatrix showed that it is a hybrid proteoglycan consisting of CS and HS. While the PG isolated from cells maintained on liver biomatrix consists of HS and CS in the ratio of 3:2 that from cells maintained on aorta or mammary gland matrix was about 2:3 indicating an alteration in the nature of the cell surface PGs produced by cells maintained on different tissue biomatrix. These results indicate that depending on the nature of the matrix substratum with which the cells are in contact, the nature and quantity of sulphated proteoglycans produced by hepatocytes vary.     

Examination of corneal proteoglycans and glycosaminoglycans by rotary shadowing and electron microscopy

Proteoglycans (PGs) from cornea and their relevant glycosaminoglycan (GAG) chains, dermatan sulphate (DS) and keratin sulphate (KS), were examined by electron microscopy following rotary shadowing, and compared with hyaluronan (HA), chondroitin sulphate (CS), alginate, heparin, heparan sulphate (HS) and methyl cellulose. Corneal DS PG had the tadpole shape previously seen in scleral DS FG, and the images from corneal KS PG could be interpreted similarly, although the GAG (KS) chains were very much fainter than those of DS PG GAG. Isolated GAG (KS, DS, CS, HA, etc.) examined in the same way showed images that decreased very significantly in clarity and contrast, in the sequence HA greater than DS greater than CS greater than KS. The presence of secondary and tertiary structures in the GAGs may be at least partly responsible for these variations. HA appeared to be double stranded, and DS frequently self-aggregated, KS and HS showed tendencies to coil into globular shapes. It is concluded that it is unsafe to assume the absence of GAGs, based on these techniques, and quantitative measurements of length may be subject to error. The results on corneal DS PG confirm and extend the hypothesis that PGs specifically associated with collagen fibrils are tadpole shaped.     

Comprehensive Gene Expression Profiling Reveals Synergistic Functional Networks in Cerebral Vessels after Hypertension or Hypercholesterolemia

Atherosclerotic stenosis of cerebral arteries or intracranial large artery disease (ICLAD) is a major cause of stroke especially in Asians, Hispanics and Africans, but relatively little is known about gene expression changes in vessels at risk. This study compares comprehensive gene expression profiles in the middle cerebral artery (MCA) of New Zealand White rabbits exposed to two stroke risk factors i.e. hypertension and/or hypercholesterolemia, by the 2-Kidney-1-Clip method, or dietary supplementation with cholesterol. Microarray and Ingenuity Pathway Analyses of the MCA of the hypertensive rabbits showed up-regulated genes in networks containing the node molecules: UBC (ubiquitin), P38 MAPK, ERK, NFkB, SERPINB2, MMP1 and APP (amyloid precursor protein); and down-regulated genes related to MAPK, ERK 1/2, Akt, 26 s proteasome, histone H3 and UBC. The MCA of hypercholesterolemic rabbits showed differentially expressed genes that are surprisingly, linked to almost the same node molecules as the hypertensive rabbits, despite a relatively low percentage of ‘common genes’ (21 and 7%) between the two conditions. Up-regulated common genes were related to: UBC, SERPINB2, TNF, HNF4A (hepatocyte nuclear factor 4A) and APP, and down-regulated genes, related to UBC. Increased HNF4A message and protein were verified in the aorta. Together, these findings reveal similar nodal molecules and gene pathways in cerebral vessels affected by hypertension or hypercholesterolemia, which could be a basis for synergistic action of risk factors in the pathogenesis of ICLAD.     

Vascular non-protein thiols: prooxidants or antioxidants in atherogenesis?

Abstract-cell-mediated lipoprotein oxidation may be due to generation of non-protein thiols (NP-SH) from cystine with formation of oxidizing species. However, NP-SH, especially GSH, may also exert antioxidant effects in vitro and in vivo. To further investigate whether vascular NP-SH could be prooxidants or antioxidants in atherosclerosis, we have correlated the aortic content of NP-SH with that of lipoperoxides in 10 rabbits fed on a fat-enriched atherogenic diet for 9 weeks. As compared to 7 control rabbits, aortic NP-SH and lipoperoxides were significantly increased in the fat-fed animals. The levels of NP-SH were strongly and inversely correlated with those of lipid peroxidation in the atherosclerotic aorta (r(s) -0.92, P < 0.0001 for thiobarbituric acid reactive substances, and r(s) -0.80, P < 0.01 for fluorescent damage products of lipid peroxidation). A similar trend was evident also in the control rabbits (r(s) -0.60 for both indices of lipid peroxidation). Thus, the present data suggest that vascular NP-SH exert significant antioxidant-antilipoperoxidative effects in vivo especially in fat diet-related atherogenic conditions.     

Effects of atherogenic diet on young farm pigs.

This study was designed to assess the usefulness of young farm pigs as an experimental model for hypercholesterolemia. In order to test this, we investigated both serologic and electrocardiographic effects of atherogenic diet. Four-week-old pigs were fed an atherogenic diet for 8 weeks. No arrhythmia was observed on ECG in all animals. There were no significant difference between control and atherogenic diet group on the values of ECG parameters. However, plasma lipids values of atherogenic diet group were significantly (p less than 0.05) higher than those of control diet group. Thus, hypercholesterolemia was induced in young farm pigs by feeding atherogenic diet in a relatively short time. This fact suggests that young farm pigs may be an useful model for further studies of the effect of hypercholesterolemia on cardiovascular function and the early pathogenesis of atherosclerosis.     

Demonstration of fibronectin in normal and injured aorta by an indirect immunoperoxidase technique

The presence and localization of fibronectin in normal and mechanically injured aorta in rabbits was studied using an indirect immunoperoxidase technique on tissue specimens fixed in formaldehyde, embedded in paraffin and pretreated with pepsin. The effect on staining quality of treatment with testicular hyaluronidase prior to immunoperoxidase staining was also examined. In the intima from normal aorta fibronectin was present in the subendothelial basal layer, along the internal and external elastic laminae, around and between the smooth muscle cells of the media and along the collagen and elastic fibres in the adventitia. Sixteen days after a single mechanical dilatation of the descending thoracic aorta all animals developed gross atherosclerotic-like changes. Microscopic examination revealed prominent neo-intimal hyperplasia with subendothelial, cushion-like thickenings but no medial or adventitial alterations. Fibronectin, in increased amounts, was found between and around the endothelial cells and in the subendothelial thickenings between the proliferating smooth muscle cells in relation to the fine, thin elastic and argyrophilic fibres. In the media and adventitia the amount and distribution of fibronectin was indistinguishable from uninjured control aortas. Treatment with testicular hyaluronidase before immunoperoxidase staining resulted in a higher staining resolution in normal and injured aorta. The conspicuous observation in the present study is that fibronectin exclusively accumulates in areas of tissue repair. The origins and functions of fibronectin during tissue injury and repair are discussed.     

Increased calcium influx mediates increased cardiac stiffness in hyperthyroid rats

Cardiac remodeling (hypertrophy and fibrosis) and an increased left ventricular diastolic stiffness characterize models of hypertension such as the SHR and DOCA-salt hypertensive rats. By contrast, hyperthyroidism induces hypertrophy and hypertension, yet collagen expression and deposition is unchanged or decreased, whereas diastolic stiffness is increased. We determined the possible role of increased calcium influx in the development of increased diastolic stiffness in hyperthyroidism by administering verapamil (15 mg/[kg x d] orally) to rats given triiodothyronine (T3) (0.5 mg/[kg x d] subcutaneously for 14 d). Administration of T3 significantly increased body temperature (control: 36.7 +/- 0.2 degrees C; T3: 39.6 +/- 0.2 degrees C), left ventricular wet weight (control: 2.09 +/- 0.02 mg/kg; T3 3.07 +/- 0.07 mg/kg), systolic blood pressure (control: 128 +/- 5 mmHg; T3: 156 +/- 4 mmHg), and left ventricular diastolic stiffness (control: 20.6 +/- 2.0; T3: 28.8 +/- 1.4). Collagen content of the left ventricle was unchanged. Contractile response to noradrenaline in thoracic aortic rings was reduced. Relaxation in response to acetylcholine (ACh) was also reduced in T3-treated rats, whereas sodium nitroprusside response was unchanged. Verapamil treatment of hyperthyroid rats completely prevented the increased diastolic stiffness and systolic blood pressure while attenuating the increased body temperature and left ventricular weight; collagen content remained unchanged. ACh response in thoracic aortic rings was restored by verapamil. Thus, in hyperthyroid rats, an increased calcium influx is a potential mediator of the increased diastolic stiffness independent of changes in collagen.     

Hypertension and Nephrosclerosis: A Reappraisal and a New Theory of Renal Ischemia

An observation in human autopsy material showing a statistically close relationship between complicated atherosclerosis of the aorta, at or above the renal artery take-off, and nephrosclerosis of usual type (i.e. the “granular kidney” of essential hypertension) led to a study of platelet aggregates as a cause of renal lesions. The renal cortical surface is peculiarly sensitive to ischemic damage. When an embolic source, which sheds repeatedly, was placed in the thoracic aorta of rabbits, they became hypertensive. The hypertension persisted for six months, at which time the kidneys showed nephrosclerosis characterized by surface cortical lesions consisting of shrunken glomeruli and atrophical tubules, subtended by arterioles whose intimas showed fibrous thickening. It is suggested that the renal component of the hypertension so induced is transitory, serving as a trigger mechanism for sustained hypertension.     

The Influence of Fluoride Ions upon Selected Enzymes of Protein Metabolism in Blood Plasma of Rabbits with Hypercholesterolemia

Three-month studies were performed on 18 adult rabbits of New Zealand breed divided into three groups, with six animals in each: a control group on standard diet, a cholesterol group receiving 500 mg of cholesterol/100 g of feed per rabbit per 24 h (CH group), and a cholesterol + fluorine group (CH + F group) receiving 500 mg of cholesterol/100 g of feed per rabbit per 24 h and 3 mg of F(-)/kg of body weight per 24 h. The conducted studies proved that cholesterol in the applied dosage (500 mg cholesterol per rabbit per 24 h) has an atherogenic action. Fluoride ions administered together with a 500-mg cholesterol atherogenic diet inhibit the atheromatosic changes in the aorta. The concentration of plasma cholesterol was elevated in both study groups when compared to the control group but decreased in the CH + F group when compare to the CH group. The influence of fluoride ions has been examined upon the activity of alanine aminotransferase, aspartate aminotransferase, and glutamate dehydrogenase (GLDH) in the plasma in the liver of rabbits in the course of experimental hypercholesterolemia. Increase in the activity of study enzymes has been observed in the blood plasma, which may be due to damage occurring to hepatocytes of the animals examined (a statistically significant increase in the activity of GLDH in the plasma). In the liver, the inhibition of activity for all examined enzymes has been observed in the group of rabbits with hypercholesterolemia, which testifies the disturbances in protein metabolism in examined animals. The addition of sodium fluoride to the diet rich in cholesterol results in "removing the block" on those activities, which increase. We suppose that the permeability of the hepatocyte membrane was elevated, so the activities of examined enzymes increased in the plasma ("escape" to plasma). On the one hand, fluoride ions result in probable lesion of hepatocytes membranes; on the other hand, they inhibit the atheromatosic changes in the aorta.     

Biological implications of glycosaminoglycan interactions with haemopoietic cytokines

Heparan sulphate (HS) glycosaminoglycans (GAGs) are an integral part of the signalling complex of fibroblast derived growth factor (FGF) family members, HS being regarded as a coreceptor. FGFs are also retained in the tissues by binding to HS structures. Early studies on the contribution of the bone marrow stroma to haemopoiesis suggested that cytokines with a role in haemopoiesis were similarly retained in the stroma through interactions with HS. However, the functional outcomes of these cytokines binding HS were poorly understood. Here the GAG-binding properties of cytokines of the four alpha-helical bundle family and the biological consequences of such binding are reviewed. From this analysis it is apparent that although many of these cytokines do bind GAGs, GAG binding is not a consistent feature, nor is the site of GAG binding conserved among these cytokines. The biological outcome of GAG binding depends, in part, on the location of the GAG-binding site on the cytokine. In some cases GAG binding appears to block signalling, whereas in others signalling is likely to be facilitated by binding. It is postulated that the interactions of these cytokines with their receptor complexes evolved independently of GAG binding, with GAG binding being an additional feature for a subset of this cytokine family. Nevertheless, because GAG binding localizes cytokines to sites within tissues, these interactions are likely to be critically important for the biology of these cytokines.     

Influence of serum cholesterol on atherogenesis and intimal hyperplasia after angioplasty: inhibition by amlodipine

The objectives of the present study were to determine whether serum hypercholesterolemia (HC) promotes the development of spontaneous and angioplasty-induced lesions and whether amlodipine inhibits these lesions and cellular processes underlying their genesis. Rabbits were fed normal, 0.5%, or 2% cholesterol diets for 9 wk, which resulted in the development of increasing HC. After week one, balloon dilation of the abdominal aorta was performed while the thoracic aorta was not disturbed and monitored for the development of spontaneous lesions. Lesion size increased with the degree of HC and was accompanied by increased collagen synthesis and smooth muscle cell (SMC) proliferation at each site. Amlodipine (5 mg/kg p.o.) inhibited lesion size by 50% (P < 0.01) at both sites in cholesterol-fed animals but not at angioplasty sites in animals on a normal diet. Local collagen synthesis was inhibited at both sites by amlodipine in the diet animals. The increase in HC was accompanied by a 1.7-fold increase in basal Ca2+ uptake in SMCs in the thoracic aorta, which was not altered by amlodipine, nifedipine, Ni2+, or La3+, revealing an uninhibitable calcium leak during atherogenesis. In culture, cholesterol enrichment increased SMC proliferation, collagen synthesis, and the secretion of a soluble SMC mitogen, which were inhibited by amlodipine (10(-9) M). Finally, in SMC membranes, amlodipine uniquely restored the cholesterol-expanded membrane bilayer width without any effect on membrane fluidity. This study establishes a causal role between serum HC and the development of spontaneous and angioplasty-induced lesions and the ability of amlodipine to disrupt this action by a novel remodelling action on the SMC membrane.