Electron microscopic study on the medial defect at the apex of human cerebral arterial bifurcations

Summary It is a well known fact that the berry aneurysms, which are the direct cause of subarachnoid hemorrhage, develop at the apices of bifurcations of larger cerebral arteries. In order to elucidate the morphogenesis of the aneurysms in these sites, electron microscopic observation was made on one of their predilective regions, that is, the bifurcation of the first temporal branches from the middle cerebral arteries. Human autopsy cases from newborn and elderly patients were studied.The apices of the cerebral arterial bifurcations exhibited a medial defect immediately after birth, where collagen fibrils, ground substance and increased basement membrane-like substance were observed, suggesting that the defect might have resulted from necrosis of medial muscle cells. The tunica media adjacent to the defect became tapered into a wedge-like shape with the thin and towards the defect and the medial muscle cells were decreased in number. In young cases, medial muscle cells near the defect displayed focal cytoplasmic necrosis, and granulovesicular cell debris and lamellar increase of basement membrane-like substance were seen around the muscle cells with irregular profiles. With aging the defect gradually enlarged and the adjacent part of the media composed of lamellarly or reticularly increased basement membrane-like substance and granulovesicular cell debris, without muscle cells. In the media away from this area muscle cells were irregular and surrounded by granulovesicular cell debris, similarly increased basement membrane-like substance, and increased collagen fibrils, with enlarged intercellular spaces.The internal elastic lamina at the apices of bifurcations showed fragmentation and lumpy degradation both of which increased with age.Necrosis of medial muscle cells and subsequent enlargement of the medial defect together with degenerative changes in the internal elastic lamina, which are age-induced and presumed to be due to haemodynamic factors, are all considered to be important in the formation of berry aneurysms.     

颈总动脉损伤大鼠成纤维细胞生长因子受体1、p21ras表达与辛伐他汀的抑制效应**

背景：辛伐他汀可通过抑制血管平滑肌细胞增殖发挥抗血管狭窄作用,但其作用机制尚不完全清楚。 目的：观察辛伐他汀对血管损伤后狭窄时成纤维细胞生长因子受体1、p21ras蛋白表达的影响。 方法：建立SD大鼠颈总动脉损伤模型,随机分为假手术组、模型组、低剂量和高剂量辛伐他汀组,后2组分别于术前3 d灌胃辛伐他汀5,10 mg/(kg•d)直至术后14 d。 结果与结论：大鼠颈总动脉损伤后：①苏木精-伊红染色显示,血管中膜血管平滑肌增殖显著,排列紊乱,管腔严重狭窄;应用辛伐他汀后血管内膜相对光滑,中膜血管平滑肌细胞排列趋于规整,管腔狭窄程度有不同程度减轻。②Western blot检测显示,颈总动脉成纤维细胞生长因子受体1、p21ras表达明显升高(P < 0.05),低剂量辛伐他汀干预后两种蛋白表达无明显变化,应用高剂量辛伐他汀干预后两种蛋白表达明显下降(P < 0.05)。表明辛伐他汀可能通过抑制成纤维细胞生长因子受体1、p21ras蛋白的表达抑制血管平滑肌细胞增殖,减轻血管损伤后狭窄。 关键词：辛伐他汀;颈总动脉损伤;狭窄;成纤维细胞生长因子受体1;p21rasS doi:10.3969/j.issn.1673-8225.2012.07.031     

The physiological closure of ductus arteriosus in the rat

Summary The evolution of morphological changes in the wall of the ductus arteriosus during its physiological closure in newborn rats was examined by electron microscopy. The contraction of smooth muscle cells in the tunica media seems to be the primary mechanism which leads to the physiological closure of the ductus arteriosus. For this reason our attention was centred mainly on the morphology of the tunica media.No important changes in the ultrastructure of smooth muscle cells can be observed in the early phases of the closure. Most of them exhibit ultrastructural features of cells with enhanced synthetic activity during all phases of the closure.The permanent contraction of smooth muscle cells results in their morphological changes. The most striking is the herniation of smooth muscle cell cytoplasm into the endothelial and later into adjoining muscle cells. These changes together with signs of degeneration of the smooth muscle cells are already clearly discernible 120 min after birth.The elastic component of the tunica media exhibits surprisingly fast changes. As soon as 60 min after birth, the fragmentation of elastic membranes and their structural changes provided evidence about the degradation of elastic material. The matrix vesicles, probably derived from the lysosomal apparatus of the muscle cells, may play an essential role in this process.     

Model system to study interaction of platelets with damaged arterial wall. II. Inhibition of smooth muscle cell proliferation by dipyridamole and AH-P719

A new in vivo model for the initial events in atherogenesis was employed to investigate drugs which may inhibit intimal muscle cell proliferation following repeated limited endothelial cell injury. An artery forceps was placed over the central artery of the ear of an anesthetized rabbit for 30 min. The forceps were removed, blood flow resumed in the vessel, and platelets contacted the damaged vessel wall. When a vessel was injured two or more times the smooth muscle cells of the media migrated into the intima and proliferated there between 1 and 3 weeks after the last injury despite restoration of an apparently intact endothelium. The intima of control undamaged vessels sometimes contained a few individual smooth muscle cells while vessels injured two, four, or six times showed correspondingly increasing numbers of layers of intimal smooth muscle cells covering increasing amounts of the intima. Arteries from thrombocytopenic rabbits showed, at most, a single layer of smooth muscle cells covering a small area. In rabbits pretreated with dipyridamole (1.5 mg/kg) for 3 days before each injury, proliferation was also limited to a small area. Neither aspirin (8 mg/kg) nor ticlopidine (40 mg/kg, 5X over 3 days), which inhibit platelet aggregation ex vivo, nor the continuous presence of heparin (800 U/kg, bid), reported to inhibit smooth muscle cell growth in vitro and in vivo, prevented smooth muscle cell proliferation in response to two injuries. However, a potent inhibitor of platelet cyclic-adenosine monophosphate (cAMP) phosphodiesterase, AH-P719 (1.5 or 2.1 mg/kg), was able to inhibit intimal smooth muscle cell proliferation in doses that inhibited platelet aggregation ex vivo.     

An electron microscopic study of plasmatic arterionecrosis in the human cerebral arteries

Summary An electron microscopic study of the intracerebral arteries from 9 hypertensive cases was performed in order to elucidate the morphogenesis of the plasmatic arterionecrosis which was considered to be the direct cause of hypertensive intracerebral hemorrhage. In the preceding stage of the arterial lesions, marked necrosis of medial smooth muscle cells and increase of basement membrane-like substance in the intima and media were observed. The lumina of these arteries were slightly dilated. The dilatation and hemodynamic factors were supposed to cause endothelial injury resulting in blood plasma insudation into the intima through the opened spaces between endothelial cells. The insudated blood plasma dispersed and dissolved the basement membrane-like substance, collagen and elastic fibers in the arterial wall, leading to the development of the plasmatic arterionecrosis.This study was supported by a Grant-in-Aid for Scientific Research of the Japanese Ministry of Education (No. 857052).     

Aortic changes induced by hypercholesterolemia and hypercalcemia in rats

The effects of vitamin D and cholesterol-rich diets on rat aortas were examined. Examination by electron microscopy showed a widening of the subendothelial space and the presence in this space of amorphous substances as early as the second week into mild hypercholesterolemia. After further time elapsed, basement membrane-like substances, microfilaments, migration of mononuclear cells, and modified smooth muscle cells were seen in the subendothelial space. In the vitamin D group, the main electron microscopic finding was of medial smooth muscle cell changes, such as focal degeneration and matrix vesicles. However, calcification was not observed in our present study. In rats fed a cholesterol-rich diet plus vitamin D, changes associated with both the cholesterol-rich diet only and vitamin D only were seen.     

Histogenesis of tunica media of the chick aorta.

We investigated the formation of the aortic tunica media in developing chicks from Hamburger-Hamilton stage (st) 12 to 1-day after hatching. At st 12, some cells of the lateral plate and the somite adjacent to the aorta extended processes toward the aortic endothelial tube. This is the first sign of the tunica media formation of the aorta. Then, these cells detached from the lateral plate and the somite attached to the aortic endothelial tube and covered it almost entirely by st 18. At this stage, bundles of microfilaments and spots of dense material were found in the peripheral region of cells of the tunica media (media cells); suggesting the earliest differentiation of smooth muscle cells. Subsequently, the tunica media increased in the number of cell-layers, reaching approximately 20 layers at st 34 (8-day incubation). The outer layers of the tunica media consisted of undifferentiated cells, whereas typical smooth muscle cells were found within the inner 4 to 5 layers. Elastic fibers first appeared at this st 34. With there was an increase in the elastic component, the tunica media assumed the lamella structure consisting of the alternating myocytes and elastic laminae. The aortic media 1-day after hatching essentially exhibited the same mature state as seen in the adult chickens.     

Myoendothelial contacts in arteriolosclerosis.

Human renal biopsies were examined electron microscopically to investigate close contacts between endothelial and smooth muscle cells in small arterioles. These myoendothelial contacts were seen in the form of cytoplasmic projections passing through fenestrae in the basal lamina. Most of these cell processes seem to arise from the endothelial cells. In the control vessels, the separation between the endothelial cells and smooth muscle cells of the tunica media is 0.09-0.27 microns. With arteriolosclerosis there is an increasing separation between the elements of the intima and the media, from 1.0 to 2.42 microns. In spite of this increasing separation, myoendothelial contacts maintain an intercellular space of 10-15 nm, as observed in the control vessels. At 2.42 microns of separation, the amount of extracellular material accumulated is such that the cells can no longer keep in contact. Break up of the myoendothelial contacts may be responsible for impairment of communication between the tunica intima and media in the vessel wall in arteriolosclerosis.     

Serum from diabetic patients enhances synthesis of arterial basement membrane-like material in cultured smooth muscle cells

The effect of serum from both type I and type II diabetic subjects on the metabolism of arterial basement membrane (BM)-like material was studied in cultures of rabbit aortic smooth muscle cells. The basement membrane-like material was isolated from the cell-layer by a combined sonication and centrifugation technique. Serum from type I diabetic persons added to the incubation medium increased statistically significantly the incorporation of L-[4,5]-3H-leucine into the basement membrane-like material as compared to serum from non-diabetic subjects (2P less than 0.05). The same effect was seen with serum from type II diabetic patients as compared to serum from nondiabetic subjects (2P less than 0.05). No effect of serum from type I diabetic persons was seen in degradation experiments. Incubation medium supplemented with normal serum and extra glucose neither changed the production of basement membrane-like material nor the disappearance rate of radioactive leucine from the basement membrane-like material in degradation experiments. The present study indicates that serum from diabetic subjects enhances the production of arterial basement membrane-like material from arterial smooth muscle cells in culture. The obtained data may be relevant for the understanding of the development of macroangiopathy among diabetic patients.     

Fibroblast growth factor receptor-1 expression is associated with neointimal formation in vitro.

Neointimal formation was studied in a porcine aortic organ culture model that exhibits intimal smooth muscle cell accumulation after a brief time in culture. This in vitro model is dependent upon an intact endothelium, as removal of the endothelium at the time of harvesting results in the failure to develop a neointima. We previously showed that conditioned media from intact cultures induce neointimal formation in denuded aortic explants, and we speculated that basic fibroblast growth factor was the endothelial-derived factor in conditioned media promoting neointimal formation. However, the concentration of basic fibroblast growth factor in conditioned media from both intact and denuded explants, measured by an enzyme-linked immunosorbent assay, was not significantly different and, in fact, steadily decreased over the first 7 days of culture. Furthermore, the amount and intensity of immunoreactive basic fibroblast growth factor in tissue sections, also similar in both groups, decreased over the same time course. Nonetheless, exogenous basic fibroblast growth factor (1 ng/ml) induced neointimal formation in intact explants but was unable to do so in denuded explants. Western blot analysis of intimal lysates prepared from both intact and denuded explants showed a time-dependent increase in fibroblast growth factor receptor-1 expression over the first 7 days of culture, with higher levels seen in intimal lysates from intact explants at each time point examined. Immunoreactive fibroblast growth factor receptor-1 was detected in both endothelial cells and intimal smooth muscle cells of intact explant sections. These data indicate that, in the presence of the endothelium, neointimal formation may in part be mediated by upregulation of fibroblast growth factor receptor-1 in the intimal cells of porcine aortic explants.     

Endothelial heparan sulfate proteoglycan. I. Inhibitory effects on smooth muscle cell proliferation

Proliferation of smooth muscle cells is an important component of pulmonary arterial morphogenesis, both during normal development and pathologic remodeling. However, little is known of the factors that regulate smooth muscle proliferation in these vessels. To investigate the hypothesis that factors produced by endothelial cells may regulate smooth muscle cell growth, we studied the effects of culture medium conditioned by fetal bovine pulmonary arterial endothelium on proliferation of smooth muscle cells in culture. This conditioned medium contains an inhibitor of smooth muscle proliferation that is degraded by nitrous acid, heparinase, and heparitinase, but resists degradation by protease, boiling, and chondroitin ABC lyase, indicating that the inhibitor is structurally similar to heparin. Inhibitor release occurs in both growing and confluent endothelial cell cultures and in the presence and absence of serum. A growth-inhibiting proteoglycan purified to homogeneity from endothelial cell-conditioned medium has physicochemical characteristics similar to those of the prototypic basement membrane heparan sulfate proteoglycan of the Englebreth-Holm-Swarm tumor: an overall size of approximately 10(6) D, heparan sulfate chains of 60,000 D, and a buoyant density of 1.33 g/ml. Antibody raised against the tumor basement proteoglycan recognizes this endothelial heparan sulfate proteoglycan, and Western blotting after SDS-PAGE demonstrates that the core proteins of both proteoglycans migrate as a doublet at apparent molecular weights of 450,000 and 360,000 D. Heparan sulfate glycosaminoglycan prepared from purified medium proteoglycan is a potent inhibitor of smooth muscle cell growth, exhibiting activity approximately 1,000 times greater than that of heparin. These results indicate that endothelial cells cultured from fetal bovine pulmonary arteries produce a basement membrane heparan sulfate proteoglycan that is a potent inhibitor of smooth muscle proliferation. This proteoglycan may mediate endothelial regulation of smooth muscle growth during development or pathologic pulmonary arterial remodeling.     

Histological pattern and changes in extracellular matrix in aortic dissections.

Samples from 34 patients were studied both histologically and immunocytochemically by the indirect biotin-avidin peroxidase technique to analyse the distribution of the extracellular matrix components (type IV collagen, fibronectin, types I and III collagens) in dissection of the aorta. Most showed defects in type IV collagen around medial smooth muscle cells. Defects in smooth muscle cell basement membrane were found throughout the media in cystic medial degeneration and in medionecrosis, whereas in atherosclerosis such unlabelled areas were found only above advanced atherosclerotic plaques. In aortitis other defects in the smooth muscle cell basement membrane were found in areas of inflammatory infiltrates. In all of these conditions similar defects in fibronectin expression were also found. No defects in the expression of interstitial collagens type I and III were seen in the dissecting aortas. Moreover, cystic medial degeneration, medionecrosis, and atherosclerosis were characterised by intense staining of these interstitial matrix components. In the pathogenesis of the aortic dissection local changes in the basement membranes of the medial layer may be important.     

Nicotine potentiates vascular endothelial growth factor expression in balloon-injured rabbit aortas

Both nicotine and vascular endothelial growth factor (VEGF) have been proposed to play an important role in the development and progression of atherosclerosis. In vitro and ex vivo studies have demonstrated that nicotine significantly stimulates VEGF expression in several cell types. This study examined the effects and the mechanisms of nicotine on the expression of VEGF in a rabbit model of balloon-injured aortas. Forty-eight male New Zealand white rabbits were randomly divided into sham, control, nicotine, and nicotine plus hexamethonium (nicotine-hex) groups. Balloon catheter denuding injury iliac artery was performed in control, nicotine, and nicotine-hex animals fed with a high-cholesterol diet beginning 2 weeks before operation. Twenty-four hours after surgery, nicotine (0.05 microg/kg) or nicotine (0.05 microg/kg) and hexamethonium (6 mg/kg) was administered daily by intramuscular injection for 3 weeks in nicotine and nicotine-hex groups, respectively. Sham and control rabbits received an identical volume of phosphate-buffered saline injection, but without nicotine or hexamethonium. VEGF protein expression and intimal cell proliferation in balloon-injured aortas were determined by enzyme-link immunosorbent assay, immunohistochemistry, and Western blot analysis. Six rabbits died during the experiment. The remaining 42 rabbits were included in the study. VEGF protein expression in nicotine group was significantly higher than that in control group (P < 0.01). VEGF positive staining was seen in vascular endothelial cells, vascular smooth muscle cells, and infiltrative inflammatory cells. The number of the proliferative cells in intima was also significantly higher in nicotine group than in control group (P < 0.01). Hexamethonium, a nonselective antagonist of nicotinic acetylcholine receptors (nAChRs), significantly inhibited nicotine-induced VEGF protein expression (P < 0.01). The present study shows that intramuscular administration of nicotine markedly potentiates the expression of VEGF protein in balloon-injured rabbit aortas, which appears to be mediated through nAChRs.     

Angiogenesis in the postovulatory primate endometrium: the coiled arteriolar system

The postovulatory period in the primate endometrium of the menstrual cycle is characterized by rapid growth of the coiled arterioles. A great variety of developing microvascular components occurs among a well-differentiated microvasculature of coiled arterioles, capillaries, and venules. Endometrial biopsies were obtained by hysterotomy during progesterone dominance at 5, 6, 7, 10, 12, and 14 days following the peak of the estrogen surge as determined by serum radioimmunoassay. Arteriolar ultrastructural differentiation is remarkably similar on each of these days. Ultrastructural evidence of elastogenesis in the extracellular matrix adjacent to certain endothelial tubes provides the initial sign of coiled arteriolar formation. The cellular primordia of the tunica intima and media are identified by spatial location and glycogen storage in smooth muscle cells. Endothelial projections span the incipient internal elastic membrane to make contact with the surfaces of the innermost vascular smooth muscle cells. Subsequent arteriolar differentiation centers on formation of a muscular media composed of 1 or 2 muscle layers separated by a spiraling lamellar elastic matrix that appears initially between the endothelial tube and the first muscle layer. Vascular smooth muscle cells are highly branched and linked across the elastic matrix by surface contacts. Definitive coiled arterioles consist of interlinked endothelial and smooth muscle cells within a thick, spiraling elastic matrix that provides flexibility for rapid changes in shape. Progressive differentiation of coiled arterioles continues up to the premenstrual stage. This abundant angiogenesis may reflect preparation and maintenance of a suitable uterine environment for the possibility of implantation and pregnancy during each menstrual cycle.     

Electron microscopic study on the proximal portions of the anterior cerebral arteries in rats with long-term hypertension

Proximal portions of the anterior cerebral arteries in rats with long-term hypertension were electron microscopically studied. The abnormal substances such as granules, vesicles, vacuoles and other membranous structures, relatively homogeneous masses, and basement membrane-like substances were observed around the medial smooth muscle cells. Basement membrane-like substances were increased with advancing age and the majority of these substances are thought to be turned from the granulo-vesicles. Abnormal substances deposited in the media are thought to be "hyalino-fibrous materials". The majority of our materials had a clear-cut border between the media and the adventitia. The subendothelial spaces were slightly distended with fibrillar and/or finely granular substances with age, but neither cellular element nor fibrinoid substance was found in them.     

The selective estrogen receptor-beta agonist biochanin A shows vasculoprotective effects without uterotrophic activity

OBJECTIVE: Current hormone therapy in postmenopausal women is associated with uterotrophic activity and cancer-promoting effects. In this experimental study, we compared the effects of the selective estrogen-receptor (ER) beta agonist biochanin A, and the selective ERalpha agonist ethinylestradiol, on the development of intimal hyperplasia after balloon injury and on uterus morphology. DESIGN: Female F344 rats with or without prior ovariectomy were used for aortic denudations. Animals remained untreated or received oral biochanin A (100 mg/kg) or ethinylestradiol (100 microg/kg). After 14 days, aortas and uteri were harvested for histologic and immunohistochemical analyses. Computerized assessments of aortic adhesion molecule expression, and isometric relaxation experiments, and uteri were analyzed. In vitro studies with smooth muscle cells and endothelial cells were performed to further investigate the effects of hormone treatment on cell proliferation, migration and adhesion molecule expression. RESULTS: Among untreated rats, ovariectomized animals tended to show greater neointimal hyperplasia and increased expression of the adhesion molecules 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1). Biochanin A treatment reduced neointima formation, inhibited VCAM-1 up-regulation, and improved the vascular relaxation response. No effect was observed on uterus growth or histology. Ethinylestradiol also reduced aortic neointima formation and inhibited VCAM-1 up-regulation, but failed to improve endothelial function and significantly induced uterus growth. Both agents showed antiproliferative and weak antimigratory effects on smooth muscle cells, and reduced VCAM-1 expression on stimulated endothelial cells in vitro. CONCLUSIONS: The ERbeta agonist biochanin A shows vasculoprotective effects without uterotrophic activity. Because hormone therapy may have cancer-promoting side effects, administration of ERbeta-selective agents might be alternatively used to reduce the risk of cardiovascular disease in postmenopausal women.     

Enhanced production of the chemotactic cytokines interleukin-8 and monocyte chemoattractant protein-1 in human abdominal aortic aneurysms.

Inflammatory leukocytes play a central role in the pathogenesis of human atherosclerotic disease, from early atherogenesis to the late stages of atherosclerosis, such as aneurysm formation. We have shown previously that human abdominal aortic aneurysms are characterized by the presence of numerous chronic inflammatory cells throughout the vessel wall (Am J Pathol 1990, 137: 1199-1213). The signals that attract lymphocytes and monocytes into the aortic wall in aneurysmal disease remain to be precisely defined. We have studied the production of the chemotactic cytokines interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) by aortic tissues obtained from 47 subjects. We compared the antigenic production of these cytokines by explants of: 1) human abdominal aneurysmal tissue, 2) occlusive (atherosclerotic) aortas, and 3) normal aortas. IL-8, which is chemotactic for neutrophils, lymphocytes, and endothelial cells was liberated in greater quantities by abdominal aortic aneurysms than by occlusive or normal aortas. Using immunohistochemistry, macrophages, and to a lesser degree endothelial cells, were found to be positive for the expression of antigenic IL-8. Similarly, MCP-1, a potent chemotactic cytokine for monocytes/macrophages, was released by explants from abdominal aortic aneurysms in greater quantities than by explants from occlusive or normal aortas. Using immunohistochemistry, the predominant MCP-1 antigen-positive cells were macrophages and to a lesser extent smooth muscle cells. Our results indicate that human abdominal aortic aneurysms produce IL-8 and MCP-1, both of which may serve to recruit additional inflammatory cells into the abdominal aortic wall, hence perpetuating the inflammatory reaction that may result in the pathology of vessel wall destruction and aortic aneurysm formation.     

The fine structure and innervation of the cushion veins of the human nasal respiratory mucosa

Cushion veins of the human nasal lining were studied in eight patients of both sexes ranging in age from 11 to 59 years. It was found that the subendothelial cushions were part of the tunica media and consisted of smooth muscle cells, collagen and elastic fibers and occasional fibrocytes. The muscle fibers of the cushion nearest to the endothelium were circular. They extended processes towards the endothelium through gaps in the endothelial basement membrane and formed appositional junctions with the endothelial cells. The rest of the cushion consisted of longitudinal muscle fibers. The sarcoplasm of the muscle cells was characterized by large areas filled with vesicles of various sizes. In addition, these cells possessed cytoplasmic processes which were devoid of a basement membrane and which did not show the regular structure of sarcoplasm. The subendothelial cushion possessed a rich, intrinsic nerve supply of adrenergic and cholinergic axons. It is suggested that the cushion veins regulate the drainage of the cavernous tissue and are under nervous and humoral control. The increase in girth of the subendothelial cushion is effected by contraction of the longitudinal muscle cells and probably by uptake of extracellular fluid by means of the specialized cytoplasmic processes. The single layer of circular muscle cells situated between the endothelial lining and the longitudinal musculature, may provide protection to the endothelium against distension when the cushion expands.