Endotoxin-induced endothelial injury and subendothelial accumulation of fibronectin in rat aorta

Endotoxin (lipopolysaccharide, LPS) induces endothelial injury in arterial vessels. Fibronectin is known to be involved in cell attachment and wound repair. The present study was designed to elucidate the effect of LPS on the production and distribution of fibronectin in relation to injury and repair in rat aortic endothelium. Male Sprague-Dawley rats were sacrificed for ultrastructural and immunocytochemical evaluations at 1, 3, 6, 24, and 48 hr after a single intravenous injection of 1.5 or 3 mg/kg body weight E. coli LPS. Apparent morphological signs of endothelial injury, including cell detachment, denudation, cell death, and edema were observed 1-48 hr after injection. Parietal thrombosis and leukocyte diapedesis were also observed in the aorta. A profound increase in subendothelial fibronectin was found following LPS treatment. However, no distinct change in intracellular fibronectin was observed in the same endothelium until 24 hr after injection. Using horseradish peroxidase (HRP) and anti-fibronectin-HRP antibody as tracers, LPS was also found to increase permeability and extravasation of plasma proteins (fibronectin) of the aortic endothelium. The increase of subendothelial fibronectin possibly resulted from increased influx and sequestration of plasma fibronectin. This increase may provide a firm substratum for reendothelialization after vascular injury.     

Repair in arterial tissue. Endothelial regrowth, subendothelial tissue changes and permeability in the healing rabbit thoracic aorta.

The role of the endothelium and subendothelial connective tissue in the permeability of a healing intima was studied by vital staining with Evans blue and transmission electron microscopy after severe mechanical lesion of the rabbit aorta. Reendothelialization, decreasing permeability, and organization of neointimal connective tissue were concomitant events in the healing processes. The determinant factor in decreasing permeability was reendothelialization with the formation of endothelial flaps and junctions. Changes in the subendothelial connective tissue seemed also to be factors that influenced the permeability.     

Contact guidance of rat fibroblasts on various implant materials.

Providing a substrate surface with micrometer-sized parallel grooves influences the behavior of cells growing on such substrates in vitro. Cells elongate in the direction of the groove and migrate guided by the grooves. It has been suggested that cellular alignment on microgrooves is predominantly dependent on groove dimensions and that surface chemical variation of the substrate material has little effect. Therefore we seeded primary rat dermal fibroblasts (RDF) on smooth and microgrooved (groove width 1-10 microm, depth 0.5 microm) polystyrene (PS), poly-L-lactic acid (PLA), silicone (SIL), and titanium (Ti) substrates. The production process was found to be more accurate for PS and PLA than for SIL and Ti substrates. A proliferation study, scanning electron microscopy, confocal laser scanning microscopy, and transmission electron microscopy revealed differences between RDF behavior on the materials. Our conclusions are (1) the accuracy of microtexture production by casting depends greatly on the material used; (2) even if no sharp discontinuities are present, microtextures still are potent tools for inducing contact guidance; and (3) besides surface texture, surface chemistry has a definitive influence on cell morphology.     

Components of subendothelial aorta basement membrane. Immunohistochemical localization and role in cell attachment

Cryosections of fetal and adult bovine aorta were stained with purified, cross-absorbed antibodies against various connective tissue components. The antibodies to the basement membrane components, laminin, heparan sulfate proteoglycan, and type IV collagen, gave a sharp reaction in the subendothelial layer. Antibodies against type III procollagen showed a broad endothelial staining, and staining was also seen in the media layer. A similar staining reaction was seen with antibodies against fibronectin. Bovine fetal aortic endothelial (BAE) cells were isolated and cultured in vitro. The cells became stained by the indirect immunofluorescence method with antibodies against laminin and heparan sulfate proteoglycan and also with antibodies against types III and IV collagen and type I procollagen, as in previously reported experiments. The attachment properties of endothelial cells to the same extracellular matrix components were also studied. BAE cells became attached most readily to surfaces coated with fibronectin or type III or type IV collagen. Laminin and collagen types I and V served as less effective substrates. Attachment to heparan sulfate proteoglycan was slowest of the tested components. The results of the study demonstrate that the BAE cells are associated with basement membranes in vivo. The BAE cells in culture produced interstitial connective tissue components in addition to basement membrane components and showed no clear specific preference in their attachment to any of these.     

Effect of eicosanoids on cholesterol accumulation and subendothelial cell proliferation in the human aorta

Research Institute of Experimental Cardiology, All-Union Cardiologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. Erevan Medical Institute. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 105, No. 1, pp. 35–37, January, 1988.     

Growth of microvessels in serum-free matrix culture of rat aorta. A quantitative assay of angiogenesis in vitro.

We report here that rings of rat aorta embedded in gels of fibrin or collagen and cultured in MCDB 131, an optimized growth medium for microvascular endothelial cells, generate branching microvessels in the absence of serum or other soluble protein supplements. The angiogenic response is self-limited and can be quantitated by counting the newly formed microvessels daily in the living cultures. The microvascular growth curves are characteristic for each gel. Growth of microvessels in collagen gel peaks at the end of the 1st week and is followed by a rapid regression in the 2nd week. Fibrin gels, as compared with collagen, stimulate angiogenesis by 170%, support growth during the 2nd week, and protect the newly formed microvessels from early regression. Angiogenesis is inhibited by adding hydrocortisone to the culture medium. Conversely, a 230% stimulation of angiogenesis is obtained when aortic rings are cultured in collagen gels floating in serum-free medium conditioned by sarcoma 180 cells. Our results demonstrate that: (a) angiogenesis can be obtained reproducibly in serum-free culture; (b) serum-free culture is a sensitive method for testing the inhibitory or stimulatory effects of soluble or matrix factors on angiogenesis; (c) the aortic ring model can be used as a quantitative assay for the study of angiogenesis under chemically defined culture conditions.     

Myoendothelial contacts in the thoracic aorta of rat fetuses.

There is increasing documentation of the presence of endothelial-smooth muscle communication in both small and large arteries. We have found that morphological evidence of these contacts appears early in development. Intimate contacts between endothelial and muscle cells in the thoracic aorta of normal rat fetuses have been studied with transmission electron microscopy. These myoendothelial contacts were seen in the form of cytoplasmic projections passing through fenestrae in the elastic lamellae. Most of these cell processes seem to arise from the muscle cells and they usually have a club-shaped configuration. The specific nature of the cell-cell contact was predominantly via simple appositions with an intercellular space of 6-15 nm. Myoendothelial contacts are potential sites for electrical and metabolic communication between elements of the intima and media, ensuring that the vessel wall works as a unit. The demonstrated necessity of intact endothelium for smooth muscle cell response to load may depend on this type of contact.     

Contact guidance induced organization of extracellular matrix

The scarring response following injury to the central nervous system disrupts the anatomical organization of nervous tissue posing a barrier to the regeneration of axons. In the present study, using materials with nanometer level surface features we examined whether matrix organization could be controlled by engineering meningeal cell asymmetry. Following 5 days in culture, the organization of meningeal cells along with their cytoskeletal elements and extracellular matrix proteins was evaluated. Meningeal cell morphology was markedly affected by nanometer level substrate topography. Cell alignment increased with increasing surface roughness. In addition, linear arrays of extracellular matrix were expressed that appeared related to cellular orientation. When cultured on substrates with topographical features of less than 10 nm neither cells nor their extracellular matrix showed organizational asymmetry. However, as oriented surface roughness increased, cellular and matrix asymmetrical organization became more pronounced reaching a threshold at 345 nm. These results suggest that biomaterial surface topography or other methods of altering the orientation of cells may be used to engineer orientation into the secreted extracellular matrix and as such may be a potential strategy for developing organized cell-derived matrix as a bridging material for nerve repair or other regenerative applications.     

Contact guidance and collective migration in the advancing epithelial monolayer

At the edge of a confluent cell layer, cell-free empty space is a cue that can drive directed collective cellular migration. Similarly, contact guidance is also a robust mechanical cue that can drive cell migration. However, it is unclear which of the two effects is stronger, and how each mechanism affects collective migration. To address this question, here we explore the trajectories of cells migrating collectively on a substrate containing micropatterned grooves (10–20 μm in periodicity, 2 μm in height) compared with unpatterned control substrates. Compared with unpatterned controls, the micropatterned substrates attenuated path variance by close to 70% and augmented migration coordination by more than 30%. Together, these results show that contact guidance can play an appreciable role in collective cellular migration. Also, our result can provide insights into tissue repair and regeneration with the remodeling of the connective tissue matrix.     

Hepatocellular prolapse of hepatic portal tracts and subendothelial space of central veins in idiopathic portal hypertension

We report a case of idiopathic portal hypertension (IPH) with unusual liver pathology. The liver showed changes similar to these previously reported in IPH and, in addition, we observed the unusual features of prolapse of hepatocytes into portal tracts and also into the subendothelial space of hepatic veins. Hepatocyte prolapse into hepatic veins has previously been reported only in patients with a history of androgenic steroid therapy and immunosuppressive therapy. We speculate that, in our case, prolapse of hepatocytes could be related to the abnormal intrahepatic blood flow or to intrahepatic vasculopathy.     

Concanavalin A, but not glycated albumin, increases subendothelial deposition of von Willebrand factor in vitro.

Diabetes is associated with augmentation of prothrombogenic von Willebrand factor (vWF) content in plasma. Earlier, the author and colleagues have shown that high glucose and insulin do not appreciably influence deposition of vWF into the subendothelial extracellular matrix (SECM) produced by cultured human umbilical vein endothelial cells (HUVECs). In the present work, the author used this model to test the effects of nonenzymatically glycated albumin (Glyc-HSA) and two lectins, concanavalin A (ConA) and wheat germ agglutinin (WGA), on vWF deposition into the SECM. First-passage HUVECs were seeded into gelatin-coated 96-well plates and cultured for 6 to 7 days. HSA or Glyc-HSA (at concentrations 25, 50, and 100 microg/mL), and WGA or ConA (4, 8, and 16 microg/mL) were added 3 h after seeding. Cell viability was tested by the MTT method. To determine vWF contents in the SECM, HUVECs were detached by treatment with NH4OH and the residual material was used as a solid phase in an enzyme-linked immunosorbent assay (ELISA)-like assay with primary (anti-vWF) and secondary (peroxidase-conjugated) antibodies. Addition of Glyc-HSA did not essentially influence VWF contents in the SECM (A490 was 0.226 versus 0.268 at 0 and 100 microg/mL, respectively; p > .05, n = 16). Cultivation in the presence of WGA led to the deterioration of cell viability, which was accompanied by a significant decrease of vWF in the SECM (0.248 versus 0.128 at 0 and 16 microg/mL, respectively; p < .001, n = 16). ConA did not influence viability of HUVECs, but this lectin at all concentrations consistently increased the deposition of vWF (up to 164% relative to control, p <.001; n = 16). These data indicate that endothelial carbohydrate determinants and corresponding ligands (namely, mannose-specific lectins) may be involved in the regulation of production and deposition of vWF.     

Cellular pathology of the rat aorta

Summary The aortic intima of adult rats, studied by electron microscopy, showed several changes indicative of spontaneous cellular pathology. These changes occurred almost exclusively at the level of fenestrae in the internal elastic membrane. The initial event was the formation of club-shaped cell processes arising from the smooth muscle cells and protruding into the fenestrae; this phenomenon gave rise to four types of images: (a) shortpseudopodia reaching through the fenestrae; (b) long pseudopodia that pushed their way into the body of the overlying endothelial cells, giving rise tomyo-endothelial herniae (reminiscent of the cell-to-cell herniae previously described in small, normal muscular arteries); (c) membrane-bound cellular parts apparently lying free beneath the endothelium, for which the current termghost bodies in convenient; and (d) intraendothelial structures lined by two membranes, clearly arising through the mechanism of herniation, and best referred to aspseudo-vacuoles. Some of the myo-endothelial herniae become very large and stretch the endothelium to such an extent that it could easily burst, especially during tissue processing. This mechanism should account for many of the endothelial bulges and craters often seen by scanning electron microscopy. The formation of such craters (arising from the collapse of myo-endothelial herniae as well as of endothelial blebs) offers a plausible explanation for the stomata and stigmata that have been described in silver nitrate preparations of the endothelium for over a century.This work was supported in part by Grant HL-16952 from the National Institutes of Health.     

Contribution of nitric oxide to the endothelium-dependent hyperpolarization in rat aorta.

1. The effect of endogenous and exogenous nitric oxide on the membrane potential (Em) of smooth muscle cells of the thoracic aorta of rats was investigated. 2. In tissues with intact endothelium, application of ACh or carbachol generated a change of the membrane potential consisting of an initial hyperpolarization by 10-12 mV, followed by a partial recovery toward a level which was at 10 min still 6-8 mV more negative than in control conditions. 3. Application of NG-nitro-L-arginine methylester (L-NAME), an inhibitor of endogenous NO production, had no significant effect on the resting membrane potential. The initial peak endothelium-dependent hyperpolarization elicited by ACh or carbachol was not significantly diminished. However, the recovery was more accentuated. Similarly, NG-monomethyl-L-arginine (L-NMMA) significantly diminished the second component of the endothelium-dependent hyperpolarization without affecting the magnitude of the first transient peak Em change. 4. Nitroglycerin produced a small sustained hyperpolarization of 1-2 mV, and the NO donor SIN-1, the active metabolite of molsidomine, similarly increased Em by about 1 mV. Infusion of high doses of acidified NaNO2 solution caused a hyperpolarization smaller than that evoked by ACh or carbachol. 5. 8-Bromo-cyclic GMP caused little change of membrane potential. In the presence of 8-Br-cGMP, ACh evoked a membrane electrical response similar to that observed in the absence of the nucleotide. 6. It is concluded that, in the rat aorta, the initial peak endothelium-dependent hyperpolarization observed under the influence of ACh or carbachol is not directly related to the synthesis of NO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Paracellular permeability of extracellular space markers across rat jejunum in vitro. Indication of a transepithelial fluid circuit.

1. The characteristics of [51Cr]EDTA, [3H]methoxyinulin ([3H]MI), [14C]polyethylene glycol-4000 ([14C]PEG), and [3H]mannitol as markers of the extracellular space (e.c.s.) of isolated mucosa from the rat small intestine have been examined. 2. Unidirectional transmural fluxes across the rat jejunum of [3H]MI, [14C]PEG and [3H]mannitol have been measured in the absence of glucose or in the presence of 28 mM glucose. 3. Assuming that [14C]PEG does not enter the cells, [51Cr]EDTA and [3H]mannitol seem to have access to approximately 50 and 90% of the intracellular water respectively. 4. The commercially availabel [3H]MI had access to a space which exceeded th [14C]PEG space by 10%. Upon purification by gel filtration the high molecular weight fraction of the [3H]MI provided estimates of the e.c.s. identical with the estimates obtained with [14C]PEG. 5. For all the markers used the e.c.s. estimates remained constant between the 40th and 80th min of incubation. 6. In the absence of glucose the transepithelial net fluxes of each of the different markers were zero. In the presence of 28 mM glucose the serosato-mucosa fluxes of all markers were dramatically increased. The ratio between the serosa-to-mucosa and the mucosa-to-serosa fluxes increased in the order [3H]mannitol greater than [3H]MI greater than [14C]PEG. 7. The effect of glucose on the flux ratio of the marker substances suggests that glucose-induced net water transport to the serosa side of the gut wall represents the difference between a transcellular net water transport to the serosal side and a significant paracellular net water transport through the lateral intercellular spaces to the mucosal solution.     

Contact guidance enhances the quality of a tissue engineered corneal stroma

Corneal stroma is a very complex structure, composed of 200 lamellae of oriented collagen fibers. This highly complex nature of cornea is known to be important for its transparency and mechanical integrity. Thus, an artificial cornea design has to take into account this complex structure. In this study, behavior of human corneal keratocytes on collagen films patterned with parallel channels was investigated. Keratocytes proliferated well on films and reached confluency after 7 days in the incubation medium. Nearly all of the cells responded to the patterns and were aligned in contrast to the cells on unpatterned surfaces. Collagen type I and keratan sulfate secreted by keratocytes on patterned films appeared to be aligned in the direction of the patterns. The films showed an intermediate degradation over the course of a month. On the whole, transparency of the films increased with degradation and decreased by the presence of the cells. The decrease was, however, low and transparency level was maintained on the patterned films while on the unpatterned films a sharp decrease in transparency was followed by an improvement. This was due to the more organized distribution of cells and the oriented secretion of extracellular matrix molecules on patterned collagen films. Thus, these results suggest that application of contact guidance in cornea tissue engineering may facilitate the remodeling process, hence decrease the rehabilitation period.     

Effect of temporary hypoxia on the permeability of the rat aorta.

Hypoxia was produced experimentally in the infrarenal portion of rat abdominal aorta by ligating it at both ends for one hour. Permeability changes of the injured segment were followed up by means of a colloidal iron tracer after different periods of recirculation. The tracer could be demonstrated in intercellular junctions and subendothelial space already after one hour of recirculation. The permeability disorder was at its peak two days after the injury and it ceased by the tenth day. Insudation of the media by plasma increased proportionally with the endothelial permeability change and it was accompanied by marked ultrastructural alterations.     

Acid esterase in the aorta of the hyperlipidemic rat: a histochemical study.

In rats maintained for about three weeks on a diet inducing hyperlipidemia E-600 resistant acid esterase activities were markedly reduced in the aorta in comparison with untreated animals. In rats which were maintained on an ordinary diet containing the same amount of thiouracil as given to the hyperlipidemic animals, only a slight reduction of acid esterase activities was noted. Another group of animals was fed an ordinary diet for three weeks after three weeks on a hyperlipidemic diet. In the aorta of these animals the acid esterase activities were almost normal. There was little fat deposition in the aortas of animals given the various diets. No such effects on esterase activities were observed in the liver and lung of animals of the various groups. Inhibition of acid esterase activity was also observed in rats kept on a hyperlipidemic diet for 65 days. In these animals patchy deposition of partly anisotropic lipid was observed in the intima and media.