Adventitial cells do not contribute to neointimal mass after balloon angioplasty of the rat common carotid artery.

BACKGROUND: Previous studies suggest that the migration of adventitial cells into the neointima after balloon angioplasty might have an important role in vascular lesion formation. The current experiments were designed to study the migration of adventitial cells in response to mechanical injury of the rat carotid artery. METHODS AND RESULTS: Adventitial cells were stained in situ with PKH26, a fluorescent dye, after balloon angioplasty of the rat common carotid artery. Animals were killed at different time points, and tissue sections were examined under light and fluorescence microscopy. PKH26-labeled cells were detected exclusively in the adventitia. No labeled cells were present in the media or the neointima at any time point examined. A highly cellular neoadventitial layer composed of myofibroblasts exhibited an extensive proliferative response 3 days after injury over the entire adventitial circumference. CONCLUSIONS: Despite the prominent role that adventitial myofibroblasts seem to have in the postangioplasty remodeling process, they do not migrate to the medial or intimal layers in the rat carotid artery angioplasty model.

Transformation of nonvascular acellular tissue matrices into durable vascular conduits.

BACKGROUND: Prosthetic grafts commonly used for vascular reconstruction are limited to synthetics and cross-linked tissue grafts. Within these devices, graft infections are common, compliance mismatch is significant, and handling qualities are poor. Natural biological tissues that are unfixed have been shown to resist infections and be durable and compliant. A natural biological matrix that could be remodeled appropriately after implantation would be a desirable graft for vascular reconstruction. METHODS: SynerGraft tissue engineering strategies have been used to minimize antigenicity and produce stable unfixed vascular grafts from nonvascular bovine tissues. These grafts have replaced the abdominal aortas of 8 dogs that have been followed for up to 10 months. RESULTS: Early evaluation indicates rapid recellularization by recipient smooth muscle actin positive cells, which become arranged circumferentially, into the media. Arterioles were present in the adventitial areas and endothelial cells were seen to cover lumenal surfaces. After 10 months, grafts were patent and not aneurysmal. CONCLUSIONS: These data indicate that SynerGraft processing of animal tissues is capable of producing stable vascular conduits that exhibit long-term functionality in other species.

Recellularization of heart valve grafts by a process of adaptive remodeling.

The objective of this study was to investigate if function and durability of connective tissue grafts stems from in vivo revascularization and recellularization. Viability is important for durable valve performance, demonstrated by pulmonary autografts. A pattern of in vivo recellularization occurs in xenogeneic or allogeneic heart valves decellularized prior to implantation, dictated by the tissue matrix and functional biomechanics. Porcine or sheep heart valves were decellularized with the SynerGraft antigen reduction process (a common treatment process to remove all histologically demonstrable leaflet cells), and implanted as pulmonary (n = 11) or aortic valve (n = 9) replacements in sheep. Sheep allograft pulmonary valves (n = 4) were implanted as pulmonary valve replacements. Recellularization was evaluated histologically after 3, 4, 5, 6, and 11 months, with cell phenotypes identified using specific antibodies. SynerGraft heart valves were progressively recellularized beginning with an initial cellular infiltrate, and subsequent repopulation with mature interstitial cells. This process occurs in the conduit and then in the leaflet, and is associated with revascularization of the graft. Functional, fully developed fibrocytes, actively synthesizing type I procollagen (antibody probe) were present within 3 months. As the process matured cell density and distribution became similar to native valve leaflets with localization of smooth muscle actin positive cells at the ventricularis/spongiosa interface. After 11 months, leaflet explants had no detectable inflammatory cells, were as much as 80% repopulated, and had a distribution of smooth muscle actin positive cells similar to that of the natural leaflet. SynerGraft- treated heart valve implants are repopulated by a process typical of adaptive remodeling following implantation. This antigen reduction treatment is the first successful tissue engineering effort obtaining an implant with mature recipient cells capable of matrix protein synthesis. Normal early valve function and durability is maintained.

Successful small diameter arterial grafting using cryopreserved allograft arteries.

Intimal hyperplasia (IH) limits the long-term success of veins as arterial grafts. IH occurs in veins partly as an adaptive process to arterial pressure conditions. The authors have previously reported early success with cryopreserved (CP) saphenous veins as aortocoronary bypass grafts, and they have hypothesized that CP arterial segments were already structurally adapted for arterial conditions. Six femoral arterial segments were harvested from three adult donor dogs, and cryopreserved. The segments were thawed and implanted into six recipient dogs, in end-to-end fashion, as interpositional grafts in the femoral artery. A similar length of native femoral artery was removed from the implant site and grafted in the contralateral femoral artery of the same animal to serve as native autograft-matched controls. Grafts were harvested bilaterally after 2 (n = 3) and 4 weeks (n = 3), perfusion fixed (80 mmHg, 15 min), and analyzed histologically. All grafts were patent at harvest, and flows distal to the grafted segments were not significantly different between grafts within an animal either at implant or subsequent harvest. Although CP arterial grafts still showed slight but significant dilation compared with native autograft, the dilation was much less than seen previously with either CP or native venous segments. No evidence of inflammation or IH was seen in CP arterial grafts. The absence of early IH or inflammation suggests that CP small diameter arteries may perform better than many currently available allograft tissues and synthetic prosthetics.

Agonist-induced phosphorylation of the vascular type 1 angiotensin II receptor.

Agonist-induced receptor phosphorylation plays a role in transmembrane signal transduction systems. Although the cDNA for the rat vascular type 1 angiotensin II receptor (AT1AR) encodes a G protein-coupled receptor with several potential phosphorylation sites for serine/threonine and tyrosine kinases, little is known about the phosphorylation of this receptor. The aim of this study was to determine the effects of angiotensin II (Ang II) on phosphorylation of the AT1AR in rat aortic vascular smooth muscle cells. Using [32P]orthophosphate-labeled cells, immunoprecipitates with anti-AT1AR antibody revealed a labeled band of molecular weight 52 kD, corresponding to the Ang II receptor. Ang II induced a rapid and significant increase in phosphorylation of the Ang II receptor, with a peak at 20 minutes. Phosphoamino acid analysis showed that the major phosphoamino acid is serine, in both the basal and Ang II-stimulated states. Constitutive and agonist-stimulated tyrosine phosphorylation is also observed to a lesser extent. Immunoblotting of anti-phosphotyrosine immunoprecipitates with anti-AT1AR antibody showed that Ang II caused a delayed tyrosine phosphorylation of the receptor with a peak at 20 minutes in a dose-dependent manner. Forskolin increased total phosphorylation of AT1AR but had no effect on tyrosine phosphorylation. Neither phorbol 12-myristate-13-acetate nor ionomycin altered receptor phosphorylation. These findings suggest that Ang II induces the phosphorylation of its own G protein-coupled receptor through both serine and tyrosine kinases and raise the possibility that phosphorylation of the AT1AR is an important regulator of receptor function.

Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells.

The signaling pathways involved in the long-term metabolic effects of angiotensin II (Ang II) in vascular smooth muscle cells are incompletely understood but include the generation of molecules likely to affect oxidase activity. We examined the ability of Ang II to stimulate superoxide anion formation and investigated the identity of the oxidases responsible for its production. Treatment of vascular smooth muscle cells with Ang II for 4 to 6 hours caused a 2.7 +/- 0.4-fold increase in intracellular superoxide anion formation as detected by lucigenin assay. This superoxide appeared to result from activation of both the NADPH and NADH oxidases. NADPH oxidase activity increased from 3.23 +/- 0.61 to 11.80 +/- 1.72 nmol O2-/min per milligram protein after 4 hours of Ang II, whereas NADH oxidase activity increased from 16.76 +/- 2.13 to 45.00 +/- 4.57 nmol O2-/min per milligram protein. The NADPH oxidase activity was stimulated by exogenous phosphatidic and arachidonic acids and was partially inhibited by the specific inhibitor diphenylene iodinium. NADH oxidase activity was increased by arachidonic and linoleic acids, was insensitive to exogenous phosphatidic acid, and was inhibited by high concentrations of quinacrine. Both of these oxidases appear to reside in the plasma membrane, on the basis of migration of the activity after cellular fractionation and their apparent insensitivity to the mitochondrial poison KCN. These observations suggest that Ang II specifically activates enzyme systems that promote superoxide generation and raise the possibility that these pathways function as second messengers for long-term responses, such as hypertrophy or hyperplasia.

Angiotensin-II-and endothelin-induced protein phosphorylation in cultured vascular smooth muscle cells.

In cultured vascular smooth muscle cells, angiotensin II and endothelin stimulate a variety of intracellular signals, including generation of inositol trisphosphate and diacylglycerol, mobilization of intracellular calcium, and activation of protein kinase C. These latter two events have been shown to mediate the phosphorylation of numerous proteins, but these substrates and the specific pathways mediating their phosphorylation have not been identified in vascular smooth muscle. Angiotensin II (100 nM, 10 min) induced a characteristic pattern of protein phosphorylation, which included the phosphorylation of many proteins, ranging in molecular mass from 20 to 76 kD. Three of these proteins have been identified as vimentin (M(r) 57,000), a specific protein kinase C substrate (M(r) 76,000) and the myosin light chain (M(r) 20,000). The 76-kD protein was one of the most highly phosphorylated proteins after agonist treatment. Endothelin-1 produced an identical pattern of phosphorylation. Five of these substrates were also phosphorylated by phorbol-12-myristate-13-acetate, and 5 were also phosphorylated after treatment with ionomycin. In general, the protein-kinase-C-dependent phosphorylations were sustained, while those mediated by calcium were rapid. Since these experiments were performed in cultured, phenotypically modulated cells stimulated with agents that promote cellular hypertrophy or hyperplasia, this pattern of phosphorylation may be representative of that seen during the growth response.

The effect of short chain fatty acid supplementation on membrane electrolyte transport and blood pressure.

Eleven normotensive subjects with no family history of essential hypertension took part in a double-blind randomized placebo-controlled crossover study to examine the effects of supplementing a normal omnivore diet with miglyol. This resulted in a fall in diastolic blood pressure in both the supine and standing positions, achieving statistical significance for the standing diastolic pressures, following miglyol treatment. Miglyol is rich in caprylic (8:0) and capric acids (10:0), both short chain saturated fatty acids, and supplementation with this produced a significant fall in erythrocyte membrane oleic and linoleic acid (P less than 0.01 compared to placebo for each fatty acid), as well as a fall in the saturated fat palmitic acid (16:0) (P less than 0.01). These changes were not associated with any alterations in total erythrocyte sodium influx, bumetanide sensitive influx or sodium red cell intracellular or potassium content. In addition, body weight and urinary excretion of sodium and potassium did not change. These data indicate that this dietetic manipulation with an oil rich in short chain saturated fatty acids lowers diastolic blood pressure but not as a result of changes in membrane sodium handling. It is possible that the short chain fats displace the longer carbon chain fatty acids which are metabolically important to cellular integrity and it is in this way that blood pressure falls.

Resistance artery phosphoinositide metabolism in genetic hypertension.

Precapillary resistance arteries from spontaneously hypertensive rats (SHR) and Wistar-Kyoto normotensive rats (WKY) were found to contain three inositol lipids and to produce five inositol phosphate peaks. These were assessed by a highly sensitive procedure which involved the separation of radiolabelled inositol-containing components by anion-exchange high-performance liquid chromatography. Basal levels of radiolabelled inositol lipids were found to be significantly increased in SHR at 5 weeks of age, and also increased at 12 weeks, although this was only statistically significant for glycerophosphoinositol. At 5 weeks of age, exposure to a maximal concentration of noradrenaline brought about a significant increase in lipid radioactivity in WKY and in glycerophosphoinositol and glycerophosphoinositol 4-phosphate in SHR. The levels of these lipids remained significantly raised in the SHR at this time. At 12 weeks of age, exposure to noradrenaline produced reductions in radioactivity associated with inositol lipids. These new levels were elevated in SHR and significant for glycerophosphoinositol 4-phosphate. Basal levels of four inositol phosphates were not different between the two rat strains at 5 weeks of age. Inositol 1,3,4-trisphosphate was only present at minute levels and could not be measured. At 12 weeks of age, basal radiolabelling of inositol phosphates was not different between the two strains. The application of noradrenaline caused age-dependent changes in arterial inositol-containing compounds in both strains, which resulted in increased levels of inositol 1,4,5-trisphosphate in young SHR and also in adult rats in which the blood pressure level had become established.(ABSTRACT TRUNCATED AT 250 WORDS)

Linoleic acid supplementation, membrane lipids and leucocyte sodium transport in normotensive humans.

Ten normotensive subjects had their omnivore diet supplemented with increasing doses of linoleic acid in the form of safflower seed oil in order to examine the effects of this polyunsaturated fat upon leucocyte sodium transport. Increasing the dose ingested to the limits of tolerance produced a significant decrease in ouabain resistant sodium efflux (P less than 0.05) but no significant change in total or ouabain-sensitive sodium extrusion. Intraleucocytic sodium content was correlated with erythrocyte membrane oleic acid % (r = 0.368, P less than 0.05); leucocyte ouabain resistant flux was correlated with oleic acid % (r = 0.453, P less than 0.01) and linoleic acid % (r = -0.319, P less than 0.05). No such associations were observed with ouabain sensitive sodium extrusion. No changes in body weight or urinary electrolyte excretion were observed. It is concluded that, at physiological concentrations, membrane linoleic acid content influences transmembrane sodium fluxes but not through modulation of sodium pump activity. Furthermore, the beneficial effect of this dietary manoeuvre, observed previously with small increments of safflower seed oil, was not seen in this experiment so the hypotensive activity of this manipulation must be regarded as limited.

The effect of dietary linoleic acid on blood pressure and erythrocyte sodium transport.

The influence of an increase in the polyunsaturated fat linoleic acid on blood pressure and erythrocyte membrane sodium transport was investigated in normotensive first degree relatives of hypertensive patients and controls by the double blind administration of safflower oil or paraffin oil (placebo) capsules for four weeks separated by a four week washout period. Systolic blood pressure fell in the controls with linoleic acid supplementation but there was no significant change in total sodium efflux rate constant. When the pattern of response was compared the changes in supine systolic blood pressure, plasma renin activity, total and ouabain-sensitive sodium efflux rate constant were significantly different in the controls compared to the relatives. These results show that dietary linoleic acid supplementation may have effects on ionic fluxes across cell membranes and cause a modest fall in blood pressure. In addition, since the response to the change in fat intake was different in the relatives and the controls, this provides further evidence of differences in the physicochemical structure of the plasma membrane in hypertensive subjects and their offspring.

The effects of coarctation hypertension upon vascular inositol phospholipid hydrolysis in Wistar rats.

In order to examine the effects of imposing an acute pressure load upon vascular structure and phosphoinositide hydrolysis, aortic medial thickness, cross-sectional area, cell nuclear counts and inositol phosphate accumulation were measured in Wistar rats with experimental coarctation and sham-operated controls at 3, 9 and 20 days after surgery. Compared to sham-operated animals, rats with coarctation had significantly higher carotid artery pressures throughout the study. Below the ligature, femoral arterial pressure was significantly lower in animals with coarctation. Proximal to the coarctation, there was an increase in aortic medial cross-sectional area and medial thickness but no increase in nuclear counts, suggesting the induction of hypertrophy; statistically significant changes were present at 9 and 20 days. Distal to the ligature only small structural changes were observed indicating some atrophy had occurred. Inositol phosphate accumulation had increased slightly at 3 days in proximal aortae from ligatured rats, and was significantly raised at 9 and 20 days; no such changes were recorded below the coarctation. These data indicate that the increased load upon the proximal aorta initiates increased inositol phosphate production through local mechanisms. Whilst a causal link has not been conclusively established between phosphoinositide turnover and hypertrophy, the association between the two in these studies is consistent with the hypothesis that the induction of increased cell phosphoinositide metabolism contributes, at least in part, to the proximal aortic structural changes observed in this model.

Noradrenaline stimulation of the phosphoinositide system: evidence for a novel hydrophobic inositol-containing compound in resistance arterioles.

1. Five inositol phosphates were extracted from adult rat resistance arterioles and separated by ion-exchange high performance liquid chromatography. 2. By use of this technique, inositol phosphates liberated were identified as inositol 1-phosphate, inositol 1,4-bisphosphate, inositol 1,3,4-trisphosphate, inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate. Stimulation of phosphoinositide hydrolysis with noradrenaline produced increases in inositol phosphate production. 3. Three inositol-containing phospholipids extracted from resistance arterioles were measured as their glycerol esters following deacylation, thereby permitting an analysis of both membrane and cytosolic components of the phosphoinositide signalling system. 4. A substantial agonist-sensitive pool of a previously undescribed inositol but not glycerol-containing lipid extract component was also identified in this tissue. 5. These experiments for the first time allow a precise description of phosphoinositide metabolism in resting and agonist-stimulated resistance arterioles and provide data on a novel compound possibly similar to that recently described in other tissues.

Erythrocyte phosphoinositide metabolism in essential hypertensive patients and their normotensive offspring.

The metabolism of phosphoinositides was investigated in erythrocyte membranes of essential hypertensive patients, normotensive offspring of hypertensive patients and matched controls. Measurement of 32P-labelling of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate revealed no differences in the rate of incorporation of the isotope in essential hypertensive patients compared with controls. In the normotensive offspring of essential hypertensive patients there was a highly significant increase in the rate of 32P incorporation (P less than 0.01), compared with matched controls, indicating a higher rate of metabolic turnover in these subjects. These data demonstrate that phosphoinositide metabolism is enhanced in subjects genetically at risk of hypertension, before the blood pressure has risen, but once the blood pressure is established, it is no different from control values. Abnormal phosphoinositide metabolism may be a further manifestation of a genetically determined defect of membrane physicochemical function underlying essential hypertension.

Abnormalities of erythrocyte membrane fatty acid composition in human essential hypertension.

The fatty acid content of erythrocyte plasma membrane was estimated in cells of 17 untreated hypertensive patients, and compared to that of 16 control subjects matched for age, sex and weight. Mean linoleic acid (18:2) content was significantly lower in hypertensives than controls, whereas arachidonic acid (20:4) and oleic acid (18:1) were significantly higher. These results provide further evidence for an alteration in the physiochemical structure of the plasma membrane in cells of hypertensive patients and may be related to the multiple disturbances in erythrocyte ion fluxes which have been observed.

Abnormal vascular phosphoinositide hydrolysis in the spontaneously hypertensive rat.

The production of [3H]-inositol phosphates was studied in labelled segments of aorta from spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) controls at 5 and 19 weeks, either unstimulated or in the presence of noradrenaline. Basal hydrolysis of inositol phospholipids was significantly enhanced in young SHR (P less than 0.05) compared to controls but this difference was no longer detected at 19 weeks. Noradrenaline increased [3H]-inositol phosphate accumulation in both SHR and WKY, but maximal hydrolysis was significantly greater in WKY (P less than 0.01), although the ED50 was similar in both groups of animals. These data demonstrate that phosphatidylinositide hydrolysis is enhanced in the young hypertensive rat at the time blood pressure is rising, but that this activity has declined by the time hypertension has reached an established phase. In addition, alpha 1-agonist induction of inositol phospholipid hydrolysis differs in the two species of animals, being reduced in genetically mature hypertensive rats.

Influence of dietary linoleic acid on leucocyte sodium transport and blood pressure.

In a randomised double blind study to determine whether an increase in the polyunsaturated fat linoleic acid might influence leucocyte membrane sodium transport 22 normotensive volunteers received an oral supplement of linoleic acid or placebo daily for four weeks. Mean total sodium efflux rose significantly during supplementation with linoleic acid compared with placebo. In addition, all components of lying and standing blood pressure fell, though only the fall in supine systolic pressure was significant. Dietary supplementation with linoleic acid may alter ion fluxes across the cell membrane, presumably through changes in its physicochemical structure. In addition, the change in fat intake may lower blood pressure, though to only a very modest extent.

Should the acetylator phenotype be determined when prescribing hydralazine for hypertension?

The role of acetylator phenotype in determining the response to hydralazine when it was added to diuretic and beta-blocker at doses not exceeding 200 mg daily was examined in 57 hypertensive patients. 81% of rapid acetylators needed 200 mg hydralazine daily compared to 38% of slow acetylators (p less than 0.01). Despite higher doses of hydralazine the blood pressure was controlled in only 27% of rapid acetylators compared to 65% of slow acetylators (p less than 0.02). The relation of acetylator phenotype to blood pressure response was statistically independent of initial blood pressure, age, sex, body weight and serum creatinine (p less than 0.005). Current recommendations on hydralazine dosage are unsatisfactory for the 40% of hypertensive patients who are rapid acetylators. We suggest measurement of the acetylator phenotype in patients who respond incompletely to 200 mg hydralazine daily. About 70% of these patients will be rapid acetylators in whom the dose of hydralazine can be increased safely.