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The endothelium: roles in thrombosis and hemostasis.

The renewed interest in endothelial function is based partly on success with tissue culture of endothelial cells. Endothelium functions primarily in the control of blood vessel wall permeability and in the provision of a blood-compatible lining surface. Recent findings indicate that endothelial cells are active metabolically in ways that may help prevent thrombosis. Endothelium actively degrades several different vasoactive compounds that circulate in blood and that can serve as platelet-aggregating agents. Endothelium also contains an inhibitor of platelet function and an activator of plasminogen, both of which can be released from the cell in response to appropriate stimuli. While intact endothelium functions primarily in prevention of thrombosis, damaged endothelium can contribute greatly to thrombus formation. Release of prostaglandins, adenine nucleotides, and other intracellular components from damaged endothelium can enhance platelet aggregation. Damaged endothelium may not function effectively in removal of vasoactive agents and may not release effective quantities of the inhibitor of platelet function or the activator of plasminogen. Altered endothelium exhibits tissue-factor activity, which can activate the extrinsic blood coagulation-system cascade. Finally, altered endothelial cells may contract and expose basement membrane to blood, thus enhancing thrombosis.

Blood Coagulation

Repair in arterial tissue. The role of endothelium in the permeability of a healing intimal surface. Vital staining with Evans blue and silver-staining of the aortic intima after a single dilatation trauma.

The role of the endothelium as a factor determining the permeability of a healing aortic intima after a severe mechanical lesion in the rabbit was studied by vital staining with Evans blue, by surface light microscopy after silver-staining, fluorescence microscopy and by other methods. Endothelium to develop from pre-existing endothelium in the mouths of intercostal arteries and at the borders of the lesion, remained unstained by vital staining, whereas denuded surfaces as well as pseudo-endothelium, probably derived from smooth muscle cells in the neointima, were intensely coloured. There was a general tendency towards a decreasing permeability of the aortic wall throughout an experimental period of 6 months. The determinant factor in the decreasing permeability was the presence of genuine endothelium, but changes in the endothelium, probably in the form of differentiation of intercellular junctions and changes in the connective tissue in the deeper layers, seemed to be additional factors which influenced the permeability.

Animals

Effect of the ophthalmic preservative thimerosal on rabbit and human corneal endothelium.

Widespread use of the mercurial-containing preservative thimerosal as an antibacterial agent in ophthalmic drugs and solutions warranted an investigation into its possible cytotoxic effects on the functional and ultrastructural integrity of the corneal endothelium. No changes in corneal thickness were observed during 5 hours' perfusion of the endothelium of rabbit and human corneas with 0.0001 and 0.0005 percent thimerosal in glutathione bicarbonate Ringer's solution (GBR). Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) of the endothelium of the 0.0001 percent group revealed normal ultrastructure. SEM and TEM of the endothelium of corneas perfused with 0.0005 percent thimerosal for 5 hours revealed condensed mitochondria, cytoplasmic vacuoles, and cytoplasmic flaps at the apical end of the cellular junctions. Perfusion of higher concentrations (0.001 and 0.005 perecnt) of thimerosal in GBR resulted in increases in corneal thickness after 2 hours and irreversible ultrastructural damage to the endothelial cells by 5 hours. Corneas perfused with 0.01 and 0.1 percent thimerosal in GBR showed a rapid and immediate increase in corneal thickness and endothelial cell death and necrosis within 1 hour. It is postulated that the mercury in thimerosal becomes bound to the cell membrane protein sulfhydryl groups, causing an increase in cellular permeability; These results suggest that the prolonged exposure of the corneal endothelium to thimerosal in the accepted antimicrobial dosage of 0.005 to 0.001 percent may result in functional and structural damage to the endothelium.

Animals

Role of endothelium and hypercholesterolemia in intimal thickening and lipid accumulation.

In experiments reported here we tested the hypothesis that persistent absence of endothelium favors intimal thickening, lipid accumulation, and atherosclerosis. Rabbit aortas were de-endothelialized with a balloon catheter at Day 0. Initially, all rabbits were fed a diet low in lipid. Some rabbits (Group I) were continued on a diet low in lipid for 8 to 20 weeks after de-endothelialization. Beginning 4 to 9 weeks after de-endothelialization, other rabbits were fed semisynthetic lipid-rich diets (Group II) or cholesterol-supplemented diets (Groups III) for 4 to 20 weeks. Lipid accumulation in all groups was significantly greater in the re-endothelialized intima than in adjacent intima lacking an endothelial lining. In aortas of Groups I, II, and III the degree of intimal thickening was significantly greater in re-endothelialized areas than in adjacent areas lacking endothelium. Intimal thickness was enhanced in re-endothelialized areas of hypercholesterolemic rabbits of Group III compared with normocholesterolemic rabbits of Group I but not in areas lacking endothelium. Thus, results of these experiments do not support the hypothesis that the absence of endothelium particularly favors intimal thickening and intimal lipid accumulation. Results indicate that intima covered by regenerated endothelium is significantly thicker and more likely to accumulate lipid.

Animals

Gene expression profiles of endothelium, microglia and oligodendrocytes in hippocampus of post-stroke depression rat at single cell resolution.

Post-stroke depression (PSD) is a common but severe mental complication after stroke. However, the cellular and molecular understanding of PSD is still yet to be illustrated. In current study, we prepared PSD rat model (MD) via unilateral middle cerebral artery occlusion (MCAO) and chronic stress stimulation (DEPR), and isolated hippocampal tissues for single cell sequencing of 10x Genomics Chromium. First, we determined the presence of the increased cell population of endothelium and microglia and the compromised oligodendrocytes in MD compared to NC, MCAO and DEPR. The enriched functions of highly variable genes (HVGs) of endothelium and microglia suggested a reinforced blood-brain barrier in MD. Next, cell clusters of endothelium, microglia and oligodendrocytes were individually analyzed, and the subtypes with distinct functions were identified. The presence of expression profiles, intercellular communications and signaling pathways of these three cell populations of PSD displayed a similar but more aggressive appearance with DEPR compared to MCAO and NC. Taken together, this study characterized the specific gene profile of endothelium, microglia and oligodendrocytes of hippocampal PSD by single cell sequencing, emphasizing the crosstalk among them to provide theoretical basis for the in-depth mechanism research and drug therapy of PSD.

Animals

Effect of regenerated endothelium on lipid accumulation in the arterial wall.

We tested the hypothesis that loss of endothelium results in increased transport of lipoprotein into the arterial wall, favors accumulation of lipid, and thus predisposes to atherosclerosis. In rabbits initially fed a diet low in lipid, the aortas were de-endothlialized with an intraarterial balloon catheter; 28 days later, the animals were divided into two groups. Group I animals were continued on a diet low in lipid and sacrificed at 8, 11, 13, and 15 weeks after de-endothelialization. Group II animals were fed the same diet supplemented with 0.5% cholesterol and sacrificed at comparable intervals. Aortas of group I animals revealed proliferative fibromuscular intimal thickening in both de-endothelialized and re-endothelialized areas, with little or no fatty change in the intima. In contrast, aortas of group II animals revealed slight to marked fatty change in the intima, characterized by accumulation of oil red O-positive material with anisotropic lipid inclusions. The greatest quantity of lipid was present in intimal thickening beneath regenerated endothelium, and not in adjacent intimal thickening lacking an endothelial lining. These results do not support the hypothesis that the absence of endothelium favors accumulation of lipid and predisposes to atherosclerosis. The experiments indicate that lipid accumulates preferentially in areas of intimal thickening covered by regenerated endothelium.

Animals

Seeding arterial prostheses with vascular endothelium. The nature of the lining.

Arterial prostheses seeded with autogenous vascular endothelium demonstrate a well-organized, cellular, inner lining. To determine the nature of the lining cells, six animals underwent replacement of the infrarenal aorta with Dacron prostheses. During the preparation of three such grafts, endothelium was scraped from the saphenous vein with a steel wool pledget, suspended in chilled Sack's solution, and mixed with blood used to preclot the graft. This suspension was omitted from the three control grafts. After six weeks, the grafts were removed, rinsed and examined. Fluorescent Factor VIII related antigen (F VIII-RA) strongly stained the lining cells. Silver nitrate Haütchen and electron microscopy preparations revealed a lining pattern characteristic of vascular endothelium. Endothelial cell-specific Weibel-Palade bodies were identified in the lining cell cytoplasm. Masson's trichrome staining revealed a relatively collagen-poor connective tissue within the seeded fabric. Transmission electron microscopy disclosed vascular smooth muscle cells between the seeded graft fabric and the lining cells. Vasa vasorum, arising from the outer capsule, penetrated the fabric to supply the inner capsules of the seeded grafts. It is concluded that the cells lining seeded canine arterial prostheses are true vascular endothelium supported by vascular smooth muscle cells, that the lining contains minimal connective tissue, and that vasa vasorum develop. Unseeded control grafts lacked these features.

Animals

Effects of epinephrine, benzalkonium chloride, and intraocular miotics on corneal endothelium.

Drugs formulated for use both inside and outside of the eye were tested for their potential toxic effects on the corneal endothelium. Commercially available epinephrine 1:1000 was toxic to the corneal endothelium, but solutions diluted fivefold caused no endothelial damage. The toxic agent was the sodium bisulfite 0.1% preservative. Benzalkonium chloride is highly toxic to corneal endothelium in its commonly used concentration of 0.01% and had to be diluted one thousand times to prevent endothelial damage. Ophthalmic medications for extraocular use should never be used intracamerally. Miotics commonly used in surgery during intraocular lens implantation are generally nontoxic to the cornea, though caution is advised in the use of carbachol in patients with preexisting endothelial disease and in patients having procedures in which substantial mechanical trauma to the endothelium may occur.

Animals

[Histology and function of the cryopreserved corneal endothelium].

This work consists of a histological study of cryopreserved corneal endothelium before and after perfusion, and of measurements of the corneal thickness during perfusion. 1) Histoligical study: a) After 7 hours of perfusion, the endothelium of fresh corneas is unchanged. b) The preserving solutions used for freezing do not modify the histological structure of the endothelium. c) All the freezing and thawing processes described in this paper substantially alter the histological structure of the endothelium. Latent alterations are revealed during perfusion. 2) Pachymetric study: a) In the experimental conditions described, the fresh corneas keep their normal thickness for 7 hours of perfusion. b) The preserving solutions do not modify this property. c) After freezing and thawing the thickness of the perfused cornea increases rapidly. The thickness of the perfused cornea appears more slowly under the following conditions: 33 to 66 p. 100 of serum in the preserving solution, volume less than 1 ml; eutectic point between - 4 and - 6 degrees C; the duration of the plateau less than 4 min. speed of freezing after the plateau about 5 degrees C/min.

Animals

[Ciliated cells of corneal endothelium. Functional and morphological aspects compared to cilia of other organs].

The presence of ciliated cells has been demonstrated by scanning and transmission electron microscopy in corneal endothelium. This study has been carried out on human cornea in normal and pathological cases and on rabbit's normal cornea. Another study has been lead on regeneration of endothelium after freeze destruction. The cilia cells described have a normal structure. The cilia has nine peripheral groups of tubules without any tubule in the center. The cilia seem to be more numerous in case of corneal disease and during the regeneration of corneal endothelium. The authors compare the presence of ciliated cells in different tissues. This suggest that the potency to form single cilia is present in almost every kind of tissue but there is no proof of their pathological importance. In the corneal endothelium, as in the kidney, cilia seem to have no function of fluid transportation but the possibility for cilia to be a receptor is discussed.

Animals

[Vascular endothelium (author's transl)].

Studies during recent years have shown the importance of the vascular endothelium in several physiological and pathological circumstances. The culture of endothelial cells has permitted the direct study of endothelial functions. The endothelium is a selective barrier between blood and tissues: the molecules cross it, according to their size, either through the intercellular junctions or through the cells by pinocytotic vesicles. The permeability is modulated by vasomotor agents and modified during endothelial regeneration, especially for the lipids. The endothelium plays a prominent part in the maintenance of the blood flow through its nonthrombogenic properties. It metabolizes circulating thrombogenic substances (arachidonic acid, adenosine diphosphate) and produces potent antiaggregating agents (prostacyclin and adenosine). It may also release a plasminogen activator promoting thrombolysis. The endothelial cells contribute to the formation of the basement membrane by synthesizing collagen and fibronectin, which are involved in platelet adhesion and aggregation to exposed subendothelium. On the other hand, the endothelium has a modulating influence on the local blood flow by producing vasoconstrictors (angiotensin II and III) and vasodilating agents (adenosine and prostacyclin). It is not necessary to elucidate the coordination of these functions and their relationship to the endothelial disorders in vascular diseases.

Actins

Fate of endothelium in a corneal graft.

An 81-year-old white man with bullous keratopathy received a corneal graft from a 13-month-old donor. The postoperative course has been uneventful. The endothelium of the donor cornea was studied on specular microscopy both before grafting and up to 9 months after surgery. There has been no change in the morphologic characteristics of the corneal endothelium nor has there been any decrease in cell density. The donor endothelium seems to persist for at least 9 months unchanged. This case provides a good illustration since it is not too often that a cornea from a 13-month-old donor is used for grafting in an 81-year-old individual. This experience also gives an indication of the value of specular microscopy as a very useful method to monitor the changes in the endothelium of a corneal graft.

Aged

Structural aspects of the permeability of the microvascular endothelium.

This article reviews recent findings and current views concerning the structural aspects of microvascular permeability. The vascular endothelium is considered as a simple squamous epithelium which has acquired a remarkably high permeability to water and water soluble solutes (including macromolecules) through a characteristic process of differentiation of its cells. In terms of cellular structures, this differentiation involves an unusually large population of plasmalemmal vesicles. The evidence so far obtained indicates that these vesicles function as (1) mass-carriers of fluid and solutes across the endothelium and as (2) generators of transendothelial channels by concomitant fusion (followed by fission) with both domains (luminal and tissular) of the plasmalemma. The endothelial fenestrae of visceral capillaries are initially transendothelial channels subsequently collapsed to minimal length. The intercellular junctions of the endothelium are not detectably permeable to tracers of diam. greater than or equal to 18--20 A in capillaries, but are focally open to probes of 50--60 A diam. in postcapillary (pericytic) venules. A correlation is attempted between transendothelial channels (and fenestrae) and the pore systems postulated by the pore theory of capillary permeability. The channels appear to function as either small or large pores depending on the porosity of their associated diaphragms and on the size of local strictures along their pathway. Two main components are recognized in the analysis of capillary permeability: 1) a basic component comparable to that of other simple epithelia and involving transport across the plasmalemma and probably along the intercellular junctions (for molecules of diam. greater than or equal to 10 A); and a differentiated component which involves plasmalemmal vesicles and their derivatives (transendothelial channels and fenestrae). The postulated pores of the capillary endothelium are part of this differentiated component. The special situation found in postcapillary venules (focally open junctions) seems to be related to the role played by these vessels in inflammatory reactions.

Animals

The effect of estrogen on the vascular endothelium and its possible relation to thrombosis.

The effect of estrogen on the permeability of the endothelium of the aorta has been studied in the rat. The endothelium was subjected to different forms of chemical injury, and its permeability to silver ions was estimated in rats in a control group and in rats after estrogen treatment. In rats subjected to estrogen treatment, the degree of silver penetration through the endothelium was significantly higher than in untreated rats. This was interpreted as an increased vulnerability of the endothelium after estrogen treatment. Thrombosis in females using oral contraceptives often has an aberrant localization. These findings lead to a theory that an endothelial factor may be involved in the pathogenesis of this form of thrombosis.

Animals

Regeneration of endothelium in rat aorta after local freezing. A scanning electron microscopic study.

Immediately after freezing the abdominal aorta of rats by applying a cold probe, the endothelium is desquamated or necrotic and platelets adhere to desquamated areas (at the earliest observable time) but not to necrotic endothelium or endothelium adjacent to the injury. Preferential adhesion of platelets to fenestrations of the internal elastic laimina appears to lead to their active migration into the media, where a large number may be present up to 8 days after injury. Platelets seems to be removed from the surface by the action of the advancing edge of the regenerating endothelium, moving at a maximum mean rate of 590 mu daily. An increase in area of individual cells appears to relate better than an increase in their number to the advance of the edge of the regenerating sheet.

Animals

Transplantation of cultured bovine corneal endothelial cells to species with nonregenerative endothelium. The cat as an experimental model.

The in vivo transplantation of cultured bovine corneal endothelial cells has been attempted in the cat. Cat corneas denuded of their endothelium were coated with bovine corneal endothelial cells previously maintained in tissue culture. When grafted back into cat recipients, the corneal buttons remained clear with no edema. Alizarin red staining of the endothelial side of the corneal transplant demonstrated that the coated bovine corneal endothelial cells reorganized themselves into a highly organized cell monolayer within eight days in vivo. In contrast, corneas denuded of their endothelium became opaque and edematous within seven days and remained so thereafter. These results demonstrate that cultured corneal endothelial cells remain functional in vitro and can replace a damaged or nonfunctional endothelium i

Animals

Scanning electron-microscope studies of the endothelium of aortic allografts in the rabbit: morphological observations.

Segments of aorta were transplanted between male rabbits using a cuff technique for anastomosis. The endothelial surfaces of recipient and donor aortas were observed by scanning electron microscopy following silver staining and pressure fixation at 1/2, 5, 24, 48 hr and 10 and 20 days after transplantation. Five hours after transplantation the endothelium was intact and only occasional cells were adherent to the surface. After 24 hr the endothelium was still intact, but showed breaks and small holes in the intercellular junction. There were many round cells adherent to the surface and lying in the sub-endothelium especially in association with the holes. Light microscopy showed that the intima was packed with cells, but they had not penetrated the internal elastic lamella into the media. Twenty days after transplantation the outlines of endothelial cells were no longer discernible and the surface was carpeted by leukocytes, platelets and thrombotic material.

Animals

Biology in vitro of corneal epithelium and endothelium.

Four main areas were explored: 1) the proper medium for culturing corneal tissue; 2) the effect of serum on tissue growth in vitro; 3) the interrelationships in vitro between corneal epithelium and endothelium, and 4) the biology of cultures of whole corneas (organ cultures). Modified Eagle's minimal essential medium (MEM) proved to be an excellent culture fluid. Corneal tissue could be grown in MEM without serum or clot, thus providing a defined culture medium. The biology in vitro of outgrowths of multilayered corneal epithelium and monolayered corneal endothelium are discussed. Contact inhibition between epithelium and endothelium is demonstrated in whole corneal (organ) cultures.

Animals