Sequence of cellular responses in rabbit aortas following one and two injuries with a balloon catheter.

In order to further elucidate the pathogenesis of intimal proliferation and increased thrombogenesis following repeated arterial injuries we studied the sequence of the cellular changes following two injuries of rabbit aortas with a balloon catheter. Following the first injury, the de-endothelialized surface was covered by a platelet monolayer. Polymorphonuclear leucocytes adhered to the inner surface of this monolayer and did not appear to penetrate the vessel wall. By 4 to 7 days, areas of neointima had formed. Within seconds after the reinjury at 7 days after the de-endothelialization small platelet aggregates formed on injured neointimal smooth muscle cells. Within I min platelet thrombi and fibrin strands formed. At 30 min most of the platelet thrombi had become fibrin-rich. Polymorphonuclear leucocytes had accumulated and many had begun to penetrate into the neointimal tissue. The number and extent of penetration of leucocytes into the inner parts of the arterial wall increased with time. Four days after the injury the neointimal cushions were restored and thickened. Both following the first and second injury the formation of neointimal cushions was accompanied by a change in the polarity of the inner layers of medial smooth muscle cells, some of which appeared to have migrated into the neointima.     

Distribution of cellular responses in rabbit aortae following one and two injuries with a balloon catheter

To investigate the mechanisms involved in the cellular reactions to arterial injuries, we studied the distribution of the deposits on the injured intima and the pattern of neointimal thickening following single and double injuries of rabbit aortae with a balloon catheter. Thirty minutes after the first injury most, but not all, of the inner surface of the aortae was covered by adherent, spread platelets. Seven days following the first injury areas of neointima, mainly proliferating smooth muscle cells, had formed around and opposite the orifices of branch vessels. The rest of the inner aortic surface consisted of acellular subendothelial matrix. Thirty minutes after the second injury, 7 days after the first, single platelets adhered once more to parts of the reinjured subendothelium, mostly between the orifices. Numerous fibrin-rich, platelet thrombi were present mainly on the surface of the injured neointima. Thirty minutes after both the first and second injury polymorphonuclear leucocytes adhered to the inner surface downstream from the orifices of branch vessels and in longitudinally oriented zones opposite the orifices. Four days following the second injury, the neointima was restored with the same distribution as before the second injury, and few thrombi, adherent platelets, or leucocytes remained.     

Distribution of cellular responses in rabbit aortae following one and two injuries with a balloon catheter.

To investigate the mechanisms involved in the cellular reactions to arterial injuries, we studied the distribution of the deposits on the injured intima and the pattern of neointimal thickening following single and double injuries of rabbit aortae with a balloon catheter. Thirty minutes after the first injury most, but not all, of the inner surface of the aortae was covered by adherent, spread platelets. Seven days following the first injury areas of neointima, mainly proliferating smooth muscle cells, had formed around and opposite the orifices of branch vessels. The rest of the inner aortic surface consisted of acellular subendothelial matrix. Thirty minutes after the second injury, 7 days after the first, single platelets adhered once more to parts of the reinjured subendothelium, mostly between the orifices. Numerous fibrin-rich, platelet thrombi were present mainly on the surface of the injured neointima. Thirty minutes after both the first and second injury polymorphonuclear leucocytes adhered to the inner surface downstream from the orifices of branch vessels and in longitudinally oriented zones opposite the orifices. Four days following the second injury, the neointima was restored with the same distribution as before the second injury, and few thrombi, adherent platelets, or leucocytes remained.     

Ultrastructural changes in re-endothelialized and non-endothelialized rabbit aortic neo-intima following re-injury with a balloon catheter

The response by normal rabbit aortas to the removal of the endothelium with a balloon catheter, was compared to the response to the removal of regenerated endothelium from rabbit aortas that had been previously de-endothelialized. De-endothelialization results in the formation of a neo-intima. Thrombus formation following a second balloon catheter injury was compared among injured neo-intima that had been re-endothelialized, non-re-endothelialized neo-intima, and the subendothelium of normal vessels following a single injury. Rabbit aortas were examined by scanning electron microscopy of full circumference segments of the aorta and by transmission electron microscopy. Thirty minutes after a single de-endothelialization injury with a balloon catheter the luminal surface is covered by a monolayer of platelets adhering to the subendothelial connective tissues. Two weeks later there is neo-intimal formation and endothelial regeneration around branch vessel orifices. The remainder of the luminal surface is composed of smooth muscle cells (SMC). A balloon catheter injury to a vessel injured 2 weeks previously results in fibrin formation and platelet-fibrin microthrombi on the aortic intimal surface. The response of the aortic wall to re-injury does not seem to be related to the prior existence of endothelium. Both single and repeated injuries result in a distribution of formed elements which may depend, in part, on haemodynamic factors.     

Ultrastructural changes in re-endothelialized and non-endothelialized rabbit aortic neo-intima following re-injury with a balloon catheter.

The response by normal rabbit aortas to the removal of the endothelium with a balloon catheter, was compared to the response to the removal of regenerated endothelium from rabbit aortas that had been previously de-endothelialized. De-endothelialization results in the formation of a neo-intima. Thrombus formation following a second balloon catheter injury was compared among injured neo-intima that had been re-endothelialized, non-re-endothelialized neo-intima, and the subendothelium of normal vessels following a single injury. Rabbit aortas were examined by scanning electron microscopy of full circumference segments of the aorta and by transmission electron microscopy. Thirty minutes after a single de-endothelialization injury with a balloon catheter the luminal surface is covered by a monolayer of platelets adhering to the subendothelial connective tissues. Two weeks later there is neo-intimal formation and endothelial regeneration around branch vessel orifices. The remainder of the luminal surface is composed of smooth muscle cells (SMC). A balloon catheter injury to a vessel injured 2 weeks previously results in fibrin formation and platelet-fibrin microthrombi on the aortic intimal surface. The response of the aortic wall to re-injury does not seem to be related to the prior existence of endothelium. Both single and repeated injuries result in a distribution of formed elements which may depend, in part, on haemodynamic factors.     

Glycosaminoglycan distribution in rabbit aortic wall following balloon catheter deendothelialization. An ultrastructural study

The glycosaminoglycan (GAG), content of rabbit aortic wall was assayed morphometrically using ruthenium red-stained sections viewed by transmission electron microscopy. Two types of ruthenium red-positive granules were identified in the intercellular matrix. Large granules (20 to 50 nm. in diameter) with interconnecting fibrils were removed by digestion of the tissue with testicular hyaluronidase or chondroitinase ABC. Smaller granules (20 nm). were localized mainly in endothelial basement membrane and were resistant to digestion by these enzymes. Both types of granules were removed from the tissue by the action of nitrous acid. At intervals of 11 to 17 weeks after a single balloon deendothelialization of the aortas of normolipemic rabbits, there were significantly more granules of both types in areas where endothelium had regenerated over the neointima than in areas not covered by endothelium. Lipid also accumulated preferentially in the endothelium-covered neointima. Compared to normal aortas, there is an increase in the large granule content in the reendothelialized areas and a decrease in both large and small granule content in the nonendothelialized areas. It is suggested that GAG may trap low density lipoproteins. The low GAG content of the nonendothelialized neointima may account for the low lipid content; additionally, the lack of endothelial cover may allow diffusion of GAG out of these areas carrying low density lipoprotein into the blood stream in the form of GAG-low density lipoprotein complexes or low density lipoprotein removal may be facilitated by high density lipoprotein.     

Contractions in normal and atherosclerotic rabbit aortas

Length-tension relationships and contractile responses to agonists associated with cyclic nucleotide levels in aortas from atherosclerotic rabbits were studied. The same number of tissues from healthy rabbits was also studied. Diffuse lesions were produced by denudation of the vascular endothelium of rabbit aorta using a balloon catheter and then feeding these rabbits a high cholesterol diet for two months. Stretch-passive tension curves and length-active tension curves which developed in the presence of 60 mM KCl significantly shifted to the left. The altered length-tension relationships indicate a decreased distensibility in these strips and this is attributed to an enhanced accumulation of collagen. Dose-response curve analysis revealed a marked hypersensitivity to serotonin as indicated by greater pD2 values (negative logarithm of half maximum dose) than seen in the controls. In addition, the mean absolute values of the maximum contraction induced by serotonin and KCl were significantly higher than in the controls. Although cyclic AMP levels in the strips with a pathology were lower and cyclic GMP/cyclic AMP ratios were higher than the controls in the basal state, serotonin induced no marked alterations in the cyclic nucleotides levels in the strips from either the diseased or control rabbits. These mechanical and pharmacological alterations of atherosclerotic vessels may relate to clinical events such as coronary spasm.     

Hepatitis C virus-specific cellular and humoral immune responses following immunization with a multi-epitope fusion protein

Hepatitis C virus (HCV) is an important causative agent of acute and chronic hepatitis worldwide. We prepared a fusion protein in the vector of pET-11d that included three conserved broadly neutralizing B-cell epitopes and a series of T-cell epitopes located in the HCV NS3 region. In vivo administration of this fusion construct resulted in specific CD8+ cytotoxic lymphocytes in both PBMCs and splenocytes that could recognize specific antigen sites that could be detected by FACS. An HCVcc system was established and applied to detect HCV-specific neutralizing antibodies. These results suggest that the multi-epitope fusion protein is immunogenic and can elicit both humoral and cellular immune responses. In particular, this fusion protein is able to elicit HCV-specific neutralizing antibodies, which are critical for viral clearance. This construct may be significant for vaccine development and could be a potential candidate to be included in the design of a prophylactic and therapeutic vaccine against HCV.     

Optimization and safety of the intra-aortic balloon pumping balloon catheter

Tokai Medical Products developed an intra-aortic balloon pumping (IABP) balloon catheter with the following unique characteristics: the balloon can be applied to any patient irrespective of their physical size, and is therefore suitable for Japanese patients of small stature; a long soft tip is used, which is designed to avoid damage to blood vessels; the size of the catheter is reduced to 7 Fr, and the catheter can be used as a multifunctional balloon catheter, such as the Yoshioka type, that allows simultaneous percutaneous coronary intervention (PCI). The safety of this catheter has been proven in scientific studies. In this review, we report the development of our IABP balloon catheter and give an outline of its characteristics.     

A comparative study of two isolation procedures for elastin and contaminating substances in fetal and adult rabbit aortas.

A quantitative study of the individual steps in the isolation of elastin from mature and fetal aortas of rabbits was carried out. The proportions of insoluble residue and acetone- and ether-soluble substances were determined. The yields of elastin and other associated components of connective tissue derived from the aortas by two different isolation procedures were compared, i.e. the classical Lansing treatment with hot alkali and the less drastic procedure described by Rasmussen et al. Results were found to be strongly influenced by the method used. The purity of the elastin preparations was evaluated by means of amino acid analyses for relative contents of desmosines and dicarboxylic amino acids. The possible reasons for the differences found are discussed.     

Cardiovascular and sympatho-adrenal responses to static handgrip performed with one and two hands

Summary 12 healthy men aged 21–25 years performed, in the sitting position, a sustained handgrip at 25% of their maximum voluntary contraction, first with each hand separately and then with both hands simultanesouly. Heart are (HR), systolic blood pressure (SBP), stroke volume (determined reographically) and plasma catecholamine concentration were measured during each handgrip test. The HR and SBP increased consistently during each handgrip test while stroke volume decreased by approximately 20% of the initial value. Cardiac output did not change significantly. There were no significant differences in the magnitude and dynamics of the cardiovascular responses between the tests with one and with both hands. Plasma noradrenaline and adrenaline levels showed similar elevations in response to handgrip performed with the right hand and with both hands, while during the exercise performed with the left hand the increase in the plasma catecholamine concentration was less pronounced. It was concluded that: (1) during sustained handgrip, performed in the sitting position by young healthy subjects, the stroke volume markedly decreases and cardiac output does not change significantly in spite of the increased HR; (2) the cardiovascular and sympatho-adrenal responses to static handgrip do not depend on the mass of contracting muscle when the same relative tension is developed.     

Local and remote cellular responses following a surgical lesion in the Cebus apella cerebral cortex

Distribution of bromodeoxyuridine immunoreactive (BrdU-IR) cell nuclei was analyzed at proximal and remote cortical sites in adult Cebus apella monkeys after a programmed surgical lesion placed either in the prefrontal or in the striate cerebral cortex. Increased GFAP-IR and vimentin-IR astrocytes, as well as IsolectinB4 labeled microglial cells, were observed both at lesional and perilesional areas. After injury at either location, the BrdU nuclear incorporation spread to supragranular layers in remote cortical areas functionally related to the injured cortex, probably due to involvement of degenerated cortico-cortical association fibers. Double labeling with Ki-67 suggested that remote BrdU-IR nuclei belong to proliferating cells, but the cell type remains to be determined, since these nuclei did not correspond to NeuN, MAP2, GFAP, Vimentin and isolectinB4 labeled cells. This remote effect should be incorporated to current clinical and experimental appraisal of cortical lesions.     

The in vitro interactions between serum lipoproteins and proteoglycans of the neointima of rabbit aorta after a single balloon catheter injury.

The effect of injury-induced alterations in the aortic neointimal proteoglycans on their binding with homologous serum lipoproteins was examined. Proteoglycans of the aortic intimal-medial tissues of rabbits that had undergone denudation with a balloon catheter 12 weeks earlier were isolated after homogenization of the tissues in 0.33 M sucrose, ultracentrifugation and subsequently by gel-exclusion chromatography. Lipoproteins from the plasma of healthy donors were prepared by sequential, ultracentrifugal floatation after density adjustment with KBr. To study the interactions, aliquots of electrophoretically pure very low-density lipoproteins (VLDL, d less than 1.006 g/ml), low-density lipoproteins (LDL, d = 1.019-1.063 g/ml), or high-density lipoproteins (HDL, d = 1.210 g/ml) were incubated with proteoglycans in the presence of Ca++ and Mg++ at 4 C. The amount of cholesterol found in the resulting pellet was measured as a marker of the binding capacity of the proteoglycans. Among lipoprotein fractions both VLDL and LDL showed strong binding with proteoglycans, whereas no appreciable binding was observed when incubation experiments were done with HDL. There were significant differences in the lipoprotein binding capacity of proteoglycan of control and injured animals, indicating that injury induced changes in proteoglycan composition exert profound influences on their ionic interactions.     

Development of atherosclerotic plaque with endothelial disruption in Watanabe heritable hyperlipidemic rabbit aortas.

To better understand the morphogenesis of atherosclerotic plaque, we evaluated temporal distribution of leukocytes, macrophages, foam cells, vascular smooth muscle cells, and subendothelial lipid in Watanabe heritable hyperlipedimic (WHHL) rabbit aortas. Aortas of WHHL (n=20) and New Zealand White (NZW, controls; n=8) rabbits were perfusion fixed at 1, 3, 6, and 12 months of age. At initial gross evaluation of lipid distribution, we identified aortic areas at high risk for lesion development. In WHHL rabbits, the lipid-positive portion of high-risk areas increased from 3% at 1 month to 50% at 12 months; during the same period, adherent cell count increased from <1 leukocyte and monocyte/mm(2) to 25 leukocytes, 44 monocytes, and 10 foam cells/mm(2). Controls showed no increase over time in lipid-positive areas or cellular adherence to the endothelium. One-month-old WHHL rabbit aortas had scattered lipid-positive cells in the intima (primarily branch points). Immunostaining of these areas did not show rabbit macrophages (RAM antibody) but were actin positive. Occasionally, platelets and monocytes adhered to the endothelial surface. By age 3 months, well-defined fatty streaks/atherosclerotic plaques had RAM-positive cells within foam cell core, along core margins, and in focal clusters in the fibrous cap and subendothelium. By age 12 months, isolated RAM-positive cells were on the endothelial surface, and surface morphology showed endothelial cell disruption foci containing clusters of macrophages and foam cells. Our results indicate that lipid accumulation (extra- and intracellular) is important in the early development of atherosclerotic lesions; a corresponding, slower accumulation of adherent cells on the lesion surface promotes lipid conversion from fatty streak to plaque.     

Early cellular and biochemical alveolar responses following intra-tracheal inoculation with low dose of asbestos and quartz

The study of broncho-alveolar lavage harvested from rats intratracheally dusted with chrysotile (0.5 mg), leached chrysotile (0.5 mg), crocidolite (0.5 mg) and quartz (0.5 and 5 mg) indicated: 1. A cytotoxic lysis of alveolar macrophages in relation to phagocytosis of dust particles in the following decreasing order: quartz, crocidolite, chrysotile. 2. Regarding cell intensity and duration while asbestos, even leached chrysotile, gave merely an early and transient response. This cell recruitment concerned mostly PMN leukocytes and at a less extent alveolar macrophage. 3. Cell recruitment was associated with an increased protein and phospholipids alveolar content. The increase of proteins came probably mostly from an inflammatory serum exudation. However, an increase synthesis of complement C3 and phospholipids is not excluded.