Dead Endothelial Cells Research Articles

Экспериментальное обоснование использования 1% раствора гидроксипропилметилцеллюлозы для защиты эндотелия заднего послойного трансплантата роговицы при его выкраивании низкоэнергетическим фемтосекундным лазером по инвертированной методике

Purpose. To evaluate experimentally endothelial cell loss of animal's cornea during inverted posterior lamellar corneal graft preparation technique by low-energy femtosecond laser with and without ophthalmic viscosurgical device (OVD) – 1% hydroxypropyl methylcellulose (HPMC) solution application. Material and methods. Posterior lamellar corneal grafts were created using a femtosecond laser (FSL). In the control group (n=16), a few drops of corneal storage solution were applied to the endothelium just before applanation, in the experimental group (n=16) – 1% HPMС. The quality of the applanation were monitored using an integrated in laser OCT system. The endothelial viability was determined using live/dead cells assay and the grafts were examined on a confocal laser scanning microscope. The obtained images were analyzed to count live and dead endothelial cells (EC). To assess the state of collagen fibers of the stromal side of the graft, samples from the control and experimental groups were examined using a scanning electron microscope (SEM). Results. According to the OCT, in all samples of both groups, a flat profile of the contact zone between the laser head and the endothelial monolayer was observed during applanation. The difference between the number of live and dead ECs was statistically significant between both groups. There were 2854 [2819; 2879] live cells/mm² in the control group, and 3477 [3426; 3719] live cells/mm² in the experimental group (p<0.001). Thus, the number of dead ECs in the control group was 710 [649; 728] cells/mm² . The number of dead ECs in the experimental group was 402 [366; 427] cells/mm² (p<0.001). When analyzing the images obtained on a SEM, the preservation of the architectonics of the graft collagen fibers with their single defibrations in both groups was observed. Conclusion. Application of 1% HPMC provides protection for endothelial cells from damage during posterior laser dissection using an FSL. In the study group, the number of endothelial cells was 9.92% higher than in the control group. The presence of the OVD layer in the interface between the head of the FSL and the EC did not affect the formation of an effective laser cut and did not reduce the quality of the stromal surface of the graft. The inverted technique FSL cut using a 1% solution of HPMC on the surface of the endothelium may be recommended for use in clinical practice. Key words: endothelium, keratocytes, corneal transplantation, keratoplasty, posterior lamellar keratoplasty, femtosecond laser, ophthalmic viscosurgical device, hydroxypropyl methylcellulose, confocal microscopy, scanning electron microscopy

Экспериментальное обоснование применения вископротекции эндотелия роговичного трансплантата для задней послойной кератопластики, сформированного при помощи фемтосекундного лазера

Purpose. To rationale experimentally the use of a viscoprotection of the corneal endothelium on the graft formed with a femtosecond laser for the descemet stripping endothelial keratoplasty. Material and methods. In our study, we used 12 pig's corneoscleral discs. The preservation time before the experiment averaged 12±4 hours. The corneas were divided into 2 groups. In the operating room, the graft was formed using an LDV Z8 femtosecond laser (Ziemer, Switzerland) from the endothelial side. Before applanation of the donor cornea and femtolaser head, a 1% solution of hydroxypropyl methylcellulose (HPMC) was applied to the endothelium - experimental group. The control group was appraised according to the standard technique, with the application of a few drops of a solution for storing the corneas. Then the applanation was monitored and evaluated by laser optical coherence tomography. Then the graft was separated from the bed and transferred to a conservation medium. Under laboratory conditions, to determine the viability of endothelial cells, the graft was stained with a «vital» dye with the commercial name Life and Dead (Abcam, UK) and placed in a confocal laser scanning microscope. Endothelial cells were counted using the ImageJ software. Results. In the experimental and control groups, the number of living endothelial cells (EC) was 91.06±1.49% and 83.86±2.14%, respectively (p<0.001). The number of dead ECs in the control group was 7.2±0.65% more than in the experimental group and amounted to 16.14±2.14% and 8.94±1.49%, respectively (p<0.001). Conclusion. The study demonstrated that the use of viscoprotection of the corneal graft endothelium for posterior lamellar keratoplasty is quite effective, and significantly reduces the loss of EC at the stage of cutting out the graft with a femtosecond laser. Key words: descemet stripping endothelial keratoplasty, femtosecond laser, vital dyes.

Cdc42 has important roles in postnatal angiogenesis and vasculature formation

Cdc42, a Rho family low molecular weight G protein, has important roles in various cell functions, including cytoskeletal rearrangement, cell adhesion and cell proliferation and differentiation. To investigate the involvement of Cdc42 in the activities of vascular endothelial cells, we generated Cdc42 conditional knockout mice in which Cdc42 was time -specifically deficient in vascular endothelial cells (Cdc42 ​fl/fl; VE-Cad CreERT: Cdc42 cKO). When the Cdc42 gene was deleted after birth, Cdc42 cKO mice were smaller than the control mice, and died between postnatal day 8 (P8) and P10. Necropsy findings confirmed that these mice had various pathological aberrances in the vessels of most organs, such as blood flow congestion and blood cell invasion. Electron microscopic observations also revealed that capillary endothelial cells were detached from the basement membrane as well as phagocytosis of dead endothelial cells induced by macrophages. Moreover, vascular sprouting from aortic rings induced by VEGF-A was diminished in samples from the Cdc42 cKO mice because of an endothelial cell proliferation defect. These results suggest that Cdc42 in vascular endothelial cells has important roles in blood vessel formation after birth.

The Relationship of Endothelial Dysfunction with the Development of Diastolic Heart Failure in Patients with Hypertension

Aim . To identify genetic predictors of diastolic dysfunction and signs of endothelial dysfunction in relation to indicators of left ventricular myocardial deformity in patients with hypertension. Material and methods . The study included 74 patients (38 women and 36 men) with hypertension 1-2 stages observed in the cardiology department of the Road clinical hospital at Chita II station. The mean age of patients was 32±9.4 years. Circulating endothelial cells (CEC) were determined by flow cytometry with BT 5000 device and antibodies to CD146+, CD45. Besides, 7AAD ion dye was used to detect living and dead endothelial cells. The number of CD146+, CD45 cells per 500000 blood cells was determined. Changes in CEC, Living/Dead CEC were studied. The data was processed using CXP Cytometer and Kaluza programs. The standard echocardiography, Doppler echocardiography and tissue Doppler echocardiography were used. Frequency distribution of alleles and genotypes: 1 mutation nitric oxide synthase 3 gene NOS3 -786 T>C; ( С -786 Т ) in relation to indicators of left ventricular myocardial deformity was analyzed in hypertensive patients with or without diastolic dysfunction as well as in the control group. Polymerase chain reaction with detection of amplification product in real time (RCR-rt) or electrophoresis in agarose gel on genomic DNA of peripheral blood leukocytes were used for studying gene mutations. Results . Development of diastolic dysfunction in hypertensive patients enhanced with the carrier of the genotype NOS3 -786 CC ( р =0.01 2, x 2 =1 2.53), endothelial dysfunction was manifested by an increase in the number of circulating endotheliocytes and was associated with a decrease in global longitudinal deformation of the left ventricle. Conclusion . Endothelial dysfunction in patients with hypertension is associated with a violation of global longitudinal deformation; and in patients with diastolic dysfunction these abnormalities are more pronounced than in patients without diastolic dysfunction.

Experimental Rationale for Using an Eximer Laser for Preparation of Ultrathin Graft for Posterior Lamellar Keratoplasty

Purpose. To rationale experimentally the use of an excimer laser for forming an ultrathin transplant for posterior lamellar keratoplasty. Materials and methods. Atomic force microscopy was used for examination of 10 samples. Control group was 5 corneal flaps obtained by mechanical microkeratome (Moria SLK-2, France). Main group — 5 corneal flaps, obtained by microkeratome and underwent photoablation by “Microscan 500” (Optosystems, Troick, RF) excimer laser at 50 um depth. For quantification of endothelial loss 10 donor corneas (5 pairs) with viable endothelium, preserved in Borzenok—Moroz media, were used. Two groups were formed: main — 5 ultrathin transplants obtained by consistent application of microkeratome and excimer laser, control (from the paired eyes of same donors) — ultrathin transplants prepared by two cuts of microkeratome. Detection of live and dead endothelial cells (EC) was provided by Calcein Violet 450 and Propidium Iodide “vital” fluorescent dyes. Nonparametric Mann—Whitney test was used for statistical analysis. Coefficient of reliability (p < 0.05) was considered to be significant. Results. RMS (roughnessmean square) of the transplant, prepared by excimer laser was — 24.17 ± 12.4 um, and RMS of the transplant prepared by microkeratome — 22.3 ± 18.3 um. Statistical analysis did not reveal significant differences in RMS values in the mentioned groups (p > 0.05). EC death in excimer laser group was 10.35 ± 5.84 %. EC death in microkeratome group — 8.06 ± 1.31 %. No statistically reliable difference of EC death was revealed in the 2 groups (p > 0.05). Conclusions. The investigation has showed identical RMS values in the examined groups, representing high optical qualities of the transplant prepared by excimer laser. EC loss was also comparable in 2 groups. The received results indicate considerable potential of excimer laser transplants for posterior lamellar keratoplasty.

MiR‐30a‐5p inhibition promotes interaction of Fas+endothelial cells and FasL+microglia to decrease pathological neovascularization and promote physiological angiogenesis

Ischemia-induced angiogenesis contributes to various neuronal and retinal diseases, and often results in neurodegeneration and visual impairment. Current treatments involve the use of anti-VEGF agents but are not successful in all cases. In this study we determined that miR-30a-5p is another important mediator of retinal angiogenesis. Using a rodent model of ischemic retinopathy, we show that inhibiting miR-30a-5p reduces neovascularization and promotes tissue repair, through modulation of microglial and endothelial cell cross-talk. miR-30a-5p inhibition results in increased expression of the death receptor Fas and CCL2, to decrease endothelial cell survival and promote microglial migration and phagocytic function in focal regions of ischemic injury. Our data suggest that miR-30a-5p inhibition accelerates tissue repair by enhancing FasL-Fas crosstalk between microglia and endothelial cells, to promote endothelial cell apoptosis and removal of dead endothelial cells. Finally, we found that miR-30a levels were increased in the vitreous of patients with proliferative diabetic retinopathy. Our study identifies a role for miR-30a in the pathogenesis of neovascular retinal disease by modulating microglial and endothelial cell function, and suggests it may be a therapeutic target to treat ischemia-mediated conditions.

Endothelial Wound Repair of the Organ-Cultured Porcine Corneas

ABSTRACTPurpose: To assess whether injured porcine endothelium of small and large corneoscleral disc differ in its reparative/regenerative capacity under various conditions of organ culture storage.Material and methods: 166 paired porcine corneas were trephined to obtain tissues with diameter 12.0 mm and 17.5 mm (with area neighboring endothelial periphery). In tested discs, central endothelium was mechanically wounded. Density of live endothelial cells (LECD), percentage of dead cells (%DC), coefficient of variation and cell hexagonality were assessed in central and paracentral endothelium following 5- or 9-day incubation in medium with 2% or 10% fetal bovine serum. The parameters were assessed also in fresh and intact cultured discs. Dead endothelial cells (EC) were visualized by trypan blue, cell borders by Alizarin Red S dye. Endothelial imprints were immunoassayed for the proliferation marker Ki-67 and the nucleolar marker fibrillarin.Results: In fresh corneas, the LECD/mm2 (mean ± standard deviation) were 3998.0 ± 215.4 (central area) and 3888.2 ± 363.1 (paracentral area). Only the length of storage had significant effect on wound repair. Lesion was repaired partially after 5-day and fully after 9-day cultivation. After 9-day storage in medium with 10% serum, the mean LECD detected in small discs were 2409.4 ± 881.8 (central area) and 3949.5 ± 275.5 (paracentral area) and in large discs the mean LECD were 2555.0 ± 347.0 (central area) and 4007.5 ± 261.2 (paracentral area). Ki-67 showed cell proliferation associated with healing of EC of both large and small corneas.Conclusions: The lesions were completely repaired within 9 days of storage. Presence of the area, where stem cells appear to be located, contributes to stimulation of endothelial reparation less than serum concentration and time of culture. Both cell migration and proliferation contribute to the wound repair.

The matricellular protein CCN1 regulates TNF-α induced vascular endothelial cell apoptosis.

Due to the epidemic obesity and associated diabetes, the incidence of atherosclerosis is increasing worldwide. Atherosclerosis is a chronic inflammatory disease characterized by the hardening and narrowing of arteries with plaques that consist of inflammatory cells, dead endothelial cells, lipids, and often hyper proliferated vascular smooth muscle cells. During the development of atherosclerosis, vascular endothelial cell (EC) apoptosis induced by the adipokine tumor necrosis factor alpha (TNF-α), is an early event in the plaque formation. However, TNF-α alone is not sufficient to induce apoptosis of endothelial cells. Recent studies suggested that the matricellular protein CCN family member 1 (CCN1) involves in endothelial cell dysfunction besides its well-known angiogenic function during tissue repair by promoting vascular smooth muscle cells proliferation and migration. Herein, we explored the possibility and mechanism of CCN1 in TNF-α induced endothelial cells apoptosis. Both mRNA and protein levels of CCN1 are found up-regulated in endothelial cells after TNF-α treatment. In addition, overexpression of CCN1 promoted endothelial cell apoptosis in the presence of TNF-α. Furthermore, CCN1 directly up-regulated the expression of TNF-α-target genes, and this up-regulation required the activation of P53 and NF-κB both in vivo and in vitro. Taken together, CNN1 regulates TNF-α induced endothelial cells apoptosis that may underlie poor response to TNF-α therapy and hence may be a better therapeutic target for preventing vascular dysfunction in obesity.

New Insights Into The Mechanisms Of Ferric Chloride-Induced Thrombosis: a Reductionist Microfluidic Approach

The ferric chloride (FeCl3) murine model of thrombosis is used extensively in hematology, in which application of ferric chloride (200 mM to 1M) to the adventitial side of mouse arteries results in occlusive thrombosis. This effect has historically been attributed to the denudation of endothelial cells by way of free iron-induced oxidative stress. Recently, a well-designed SEM analysis revealed that endothelial cells actually remain intact, and that erythrocytes adhere to the endothelium prior to platelets (Barr, et al, Blood, 2013). The same study found that washed red blood cells exposed to FeCl3 adhere to endothelial cells in vitro. These novel findings inspired us to comprehensively investigate the mechanisms of FeCl3-induced thrombosis via an in vitro reductionist approach: interrogating the effect of FeCl3 on individual blood components in the absence and presence of endothelial cells.To this end, a microfluidic channel was designed to approximately recreate the in vivo blood/FeCl3 interface (Fig 1). By using a microfluidic platform, we were able to tightly control ferric chloride influx; determine the shear rate in the “artery” ; visualize the effects of ferric chloride on isolated blood components in a controlled environment; and quantitatively track clot characteristics such as composition, size, and time to occlusion. We found that a wide range of ferric chloride concentrations causes aggregation of whole blood in our system, even in the absence of endothelial cells. In addition, FeCl3 causes “clotting” of plasma proteins and blood cells in a dose-dependent manner (Fig 2A). Once aggregation is initiated, however, FeCl3 concentration does not affect the time required for stable clot formation. Interestingly, at low concentrations, the presence of blood cells are the slowest to form clots while cell/plasma mixtures (platelet-rich plasma and whole blood) are the fastest, perhaps due to plasma proteins forming net-like structures that enhances cell binding and aggregation. (Fig 2B) [Display omitted] [Display omitted] This universal aggregation effect of FeCl3 led us to posit a charge-based hypothesis. Mechanistically, as Fe3+ ions bind negatively charged surfaces, such as cell membranes and plasma proteins, the intrinsic charge equilibrium of blood may shift, resulting in protein and cell aggregation. Indeed, erythrocyte suspensions exposed to Al3+, which is chemically similar to Fe3+, resulted in cell aggregation. In contrast, erythrocyte suspensions exposed to Cr3+, which forms substitutionally inert water complexes in solution and thus would not be expected to bind protein and cell surfaces, did not result in aggregation (Fig 2C). This suggests that inner-sphere complexation of Fe(III) to plasma proteins/endothelial or blood membranes may have a mechanistic role in FeCl3-induced thrombosis and further explains how the sheet-like protein aggregates could act as Fe3+ linkers that effectively enhances cell binding.However, a complete understanding of FeCl3-induced thrombosis requires the inclusion of endothelial cells. To that end, we used our previously developed technique to “endothelialized” microfluidic channels (Tsai, et al, JCI, 2012) (Fig 3A). We found that the effect of FeCl3 on endothelial cells is likewise concentration dependent: they remain viable at low FeCl3 concentrations (<50 mM) but have compromised cell membranes at higher concentrations (Fig 3B). However, the dead endothelial cells remain intact, corroborating the results of Barr et al. As shown in Fig 3C, FeCl3-induced clotting of platelet-rich plasma nevertheless occurs when endothelial cells are alive. This would suggest that at varying apparent FeCl3 concentrations, the primary means of clotting will differ. If endothelial cells are dead, it is posited that a normal clotting cascade would proceed, in addition to the Fe3+ based aggregation of negatively charged blood components. Overall, our microfluidic studies reveal that FeCl3 has multiple dose-dependent effects involving aggregation of different components of blood and endothelial cells. In addition, we discovered a novel mechanism in which charge effects may have a role in FeCl3-induced thrombosis that is independent of biological ligand-receptor interactions. Further studies are needed to determine how these charge effects interact with the biological aspects of FeCl3-induced thrombosis and the implications for data interpretation when using this model system. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Toxicity of voriconazole on corneal endothelial cells in an animal model

AimsTo determine the effect of intracamerally injected voriconazole on corneal endothelial cells in rabbit eyes.MethodsVarious concentrations of voriconazole (0%, 0.03%, 0.1%, 0.25%, 0.5% and 1%) were injected intracamerally in 36...

Organ culture, but not hypothermic storage, facilitates the repair of the corneal endothelium following mechanical damage

To evaluate the reparative capacity of the mechanically injured endothelium of corneas stored under organ culture (OC) or hypothermic conditions. The central endothelium of 12 pairs of human corneas with similar endothelial parameters was damaged to create a 1 mm(2) lesion. One cornea from each pair was stored under OC and one under hypothermic conditions. The endothelial cell density (ECD), coefficient of variation, hexagonality and percentage of dead cells were assessed before and after damage and on days 7, 14, 21 and 28 of storage. The mean ECD of corneas subsequently stored under OC or hypothermic conditions was 2764/mm(2). Immediately after damage, a denuded Descemet's membrane with a few remaining dead cells was observed at the injured area. After 7 days of storage under OC conditions, almost no dead cells were observed at the place of injury. A non-significant worsening of the qualitative parameters (polymegatism and pleomorphism) was found. After 14 days, ECD was 1933/mm(2) and 2478/mm(2) centrally and pericentrally, respectively. Similar values were found after 21 and 28 days of storage. The lesions with remnant dead cells persisted throughout hypothermic preservation. From day 14 the corneas became cloudy and in poor condition, while the pericentral ECD was 2523/mm(2). The reparative capacity of the cornea is maintained under OC but not under hypothermic conditions. For corneas containing dead endothelial cells, OC is therefore the method of choice because it may improve the quality of the stored tissue.

Improvement of the cold storage of blood vessels with a vascular preservation solution. Study in porcine aortic segments

Cold-induced injury to various cell types has been shown to be mediated predominantly by chelatable iron. For endothelial cells, this type of injury has so far only been shown in cultured cells. Hypothesizing that this iron-dependent cold-induced injury might also occur in the endothelium of intact vessels, we here set out to optimize the hypothermic storage of blood vessels. Segments of porcine aorta were stored for 2 to 21 days in histidine-tryptophan-ketoglutarate (HTK) solution or in modified solutions with or without the iron chelators deferoxamine or LK 614 at 4 degrees C. Parts of the segments were assayed immediately after cold storage, the other parts after subsequent rewarming. The percentage of dead (propidium iodide-positive) endothelial cells was assessed by "intravital" fluorescence microscopy, mitochondrial membrane potential was assessed by laser scanning microscopy after staining with tetramethylrhodamine methyl ester (TMRM) and thrombocyte adhesion was studied using 5-(and -6)-carboxy SNARF-1-stained thrombocytes. The endothelium of porcine aortic segments sustained moderate injury during the cold incubation itself, but major injury during rewarming. The addition of the iron chelator deferoxamine (1 mmol/L) significantly inhibited cold-induced endothelial cell injury irrespective of the solution used for cold storage (eg, 14 days of cold storage + 3 hours rewarming: HTK 66 +/- 7%, HTK + 1 mmol/L deferoxamine 40 +/- 10% propidium iodide-positive endothelial cells). An amino acid (glycine, alanine, aspartate)-containing base solution with N-acetylhistidine as buffer was optimized. The optimized base solution with pH 7.0 and potassium and chloride as main ions yielded a further decrease of endothelial cell injury. Combination of deferoxamine (in lower concentration, ie, 0.1 mmol/L) with the new, more membrane-permeable iron chelator LK 614 (20 mumol/L) further improved preservation so that even after 3 weeks of cold storage plus 3 hours rewarming only 10 +/- 1% of endothelial cells were propidium iodide positive. In this optimized solution, both endothelial cell survival and mitochondrial membrane potential were significantly better preserved than in the clinically used solutions HTK, University of Wisconsin (UW) and Perfadex, or in physiological saline. Thrombocyte adhesion was also significantly reduced after cold storage in the optimized solution compared with HTK solution. Cold-induced injury to the endothelium of porcine aortic segments is, as the injury to cultured endothelial cells, mediated by chelatable iron. Thus, iron chelators, but also optimized base solutions, are options to improve the storage of vascular endothelium. The optimized solution should now be tested in in vivo animal experiments.

Interaction of Jar choriocarcinoma cells with endothelial cell monolayers

During human pregnancy the uterine spiral arteries are invaded by placental trophoblasts which replace the endothelial cells that line the non-pregnant spiral arteries and transform these vessels into large-bore conduits enabling adequate perfusion of the placenta with maternal blood. Failure of this process may predispose to preeclampsia and fetal growth restriction [Brosens I, Robertson WB, Dixon HG. The physiological response of the vessels of the placental bed to normal pregnancy. Journal of Pathology and Bacteriology 1967;93:569-79; Khong TY, De Wolf F, Robertson WB, Brosens I. Inadequate maternal vascular response to placentation in pregnancies complicated by pre-eclampsia and by small-for-gestational age infants. British Journal of Obstetrics and Gynaecology 1986;93:1049-59]. There is a paucity of data on the role of maternal endothelial cells in this process. In this study we investigated the cellular interactions between trophoblast-derived Jar cells and endothelial cells (HUVECs and HMEC-1). The effect of coculturing Jar cells with endothelial cell monolayers was determined by confocal microscopy, DNA fragmentation assay and flow cytometry. We demonstrated that Jar cells migrate into focal areas in endothelial cell monolayers, where they induce endothelial cell death and, then phagocytose the dead endothelial cells. Our results suggest that endothelial cells may not simply be passive targets for invading trophoblasts during the remodeling of the spiral arteries.

Endothelial cell integrity after phacoemulsification with 2 different handpieces

Purpose: To investigate corneal endothelial cell loss in fresh phakic human autopsy cadaver eyes after phacoemulsification (PE) with a conventional handpiece or a new oscillatory handpiece. Setting: Department of Ophthalmology, Storm Eye Institute, Medical University of South Carolina, Charleston, South Carolina, USA. Methods: Twenty fresh phakic human autopsy globes were randomized to PE with a conventional handpiece (n = 10) or an oscillatory handpiece (n = 10). The main parameters evaluated were age, nucleus density, amount of ophthalmic viscosurgical device (OVD) used, and mean PE time and ultrasound (US) power. After surgery, the corneas were excised and the endothelia were vitally stained with trypan blue and alizarin red. Light microscopy was used to assess the endothelial cell loss. Results: The mean age, density of the nucleus, OVD used, and US power were similar between the groups. The mean PE time was less with the NeoSoniX handpiece (0.71 minutes ± 0.39 [SD]) than with the conventional handpiece (1.05 ± 0.53 minutes). The mean number of dead endothelial cells was significantly lower in the oscillatory handpiece group (31.3 ± 24.2 cells/mm 2) than in the conventional handpiece group (60.4 ± 46.8 cells/mm 2) ( P<.001, Mann-Whitney rank sum test). Conclusions: Less corneal endothelial cell loss occurred after PE with an oscillatory handpiece than with a conventional handpiece. Further randomized clinical trials are recommended to validate this study.

Relationship between endothelial cell turnover and permeability to horseradish peroxidase

The quantitative relations between cell turnover (cell mitosis and death) and macromolecular leakage were studied at the level of individual endothelial cells (ECs) in the thoracic aortae of 32 adult male Sprague-Dawley rats. The experiments were performed on en face preparations of aortic specimens obtained 1, 3, 5 or 10 min after the intravenous administration of horseradish peroxidase (HRP). Mitotic ECs were identified by hematoxylin nuclear staining; dying or dead ECs containing cytoplasmic immunoglobulin G were detected by indirect immunocytochemistry and endothelial leakages to HRP were visualized by light microscopy. The number and size of HRP spots increased with time and the spots fused to form large brown areas in 10 min. Quantitative data on the contributions of EC mitosis and EC death to the transendothelial leakage of HRP were obtained in the same animals. Although mitotic ECs (0.01%) and dying ECs (0.1%) were infrequent in occurrence, the great majority (over 90%) of these ECs were associated with focal HRP uptake. These mitotic and dying ECs, however, accounted for only 17% of the total leakage sites indicating that significant leakage of the 4–5 nm HRP also occurs in normal ECs not morphologically identified as being in mitosis or death. The percentages of leaky spots attributable to mitosis or cell death were greater for the 6 nm albumin and the 22 nm low density lipoprotein (LDL) which probably cannot traverse the normal junctions and use the leaky junctions during cell turnover as the major pathway.

Hypoxia-reoxygenation impairs endothelium-dependent relaxation in isolated rat aorta.

The effects of hypoxia followed by reoxygenation on endothelium-dependent relaxation in isolated rat aorta were investigated. Acetylcholine (ACh, 3 nM-10 microM) and calcium ionophore A-23187 (3 nM-300 nM)-induced endothelium-dependent vasorelaxation of isolated rate aortic vessel rings was impaired after 15 min of hypoxia followed by 30 min of reoxygenation. Impairment of ACh-induced relaxation was prevented by pretreatment with the combination of superoxide dismutase (200 U/ml) and catalase (1,000 U/ml). Hypoxia-reoxygenation did not affect sodium nitroprusside (0.1 nM-1 microM)-induced endothelium-independent relaxation nor the dissociation constant of ACh to endothelial M3 muscarinic receptors. Propidium iodide staining of the vascular endothelium revealed a significant increase in the number of dead endothelial cells on the aortic vessel rings following hypoxia-reoxygenation, but not on those pretreated with superoxide dismutase and catalase. These results suggest that hypoxia-reoxygenation impairs endothelium-dependent relaxation of rat aorta by a mechanism that involves oxidant-mediated endothelial cell death.

Detection by ELISA of laminin released by endothelial cells from a collagen coated layer of Matrigel

We have developed an in vitro model system quantitating the degradation of extracellular matrix by endothelial cells. Collagen is layered over a reconstituted basement membrane matrix (Matrigel) and the release of laminin contained in the Matrigel into the media by endothelial cells is quantitated by ELISA. The cell-specific release of laminin is consistent and reproducible. Incubating an equivalent number of dead endothelial cells in the same manner results in no release of laminin relative to media controls. The cell-specific laminin release is abolished by removing the exogenous growth factor component of the media and plating the cells in media plus 10% serum. Cells plated onto wells coated with collagen alone do not release laminin into the media, indicating that no de novo synthesis and release of laminin occurs during the time frame of the experiment, and that some factor or combination of factors present in the media supplement is essential for the endothelial cells to release laminin degrading enzymes.

Mechanism of Phototoxicity of Protoporphyrin and Uroporphyrin on Bovine Corneal Endothelium

AbstractPorphyrins are potent photosensitizers. This paper reports the mechanisms of phototoxicity produced by protoporphyrin (PP) and uroporphyrin (UP) in corneal endothelium. Corneas from fresh bovine eyes were exposed to ultraviolet- (UV) visible radiation in the presence of PP or UP. The control corneas exposed were either irradiated in the absence of porphyrins or incubated in the dark in the presence of PP or UP. After incubation in the dark or irradiation of the corneas, damage to the endothelium was assessed by morphologic examination after staining with trypan blue and alizarin red S, increase in corneal thickness measured using ultrasound pachymetry, and by determining the quantity of lactate dehydrogenase (LDH) released. The results presented in this paper demonstrate that UV-visible irradiation of bovine corneas in the presence of PP and UP induces endothelial damage. Larger numbers of dead endothelial cells per unit area and larger areas of damage were observed in the corneas irradiated with ...

Increased aortic endothelial cell death and enhanced transendothelial macromolecular transport in streptozotocin-diabetic rats

Hypertension, cigarette smoking and diabetes mellitus are well-known risk factors for atherosclerosis and coronary heart disease. Repeated endothelial cell injury and increased lipid entry have been suggested as initiating events in atherogenesis. Our previous studies have demonstrated that the frequency of endothelial cell death and associated endothelial permeability were significantly increased in the aorta of spontaneously hypertensive rats and chronic oral nicotine-treated rats. In the present investigation, we examined the hypothesis that diabetes also increases the frequency of arterial endothelial cell death and hence transendothelial macromolecular transport, which may have some implications in increasing lipid entry and thus accelerating atherogenesis. Diabetes was induced in 15 male Sprague-Dawley rats by intraperitoneal injection of 60 mg streptozotocin per kg body weight. The duration of diabetes was 6 weeks. A group of 15 age-matched rats, injected only with the buffer and maintained over the same time period, served as the controls. In en face preparations of the thoracic aorta, IgG-containing dead endothelial cells were identified by an indirect immunoperoxidase method, and endothelial leakage to Evans blue-albumin complexes was quantified by fluorescence microscopy. Diabetic rats, compared to control rats, had significantly higher values for the frequency of endothelial cell death (0.77 +/- 0.10% vs 0.38 +/- 0.04%; p < 0.005 by two-tailed, unpaired Student's t-test) and the number density of Evans blue-albumin leaky foci (4.33 +/- 0.48/mm2 vs 2.99 +/- 0.38/mm2; p < 0.05 by two-tailed, unpaired t-test) in the aorta.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term nicotine exposure increases aortic endothelial cell death and enhances transendothelial macromolecular transport in rats.

Repeated endothelial cell injury has been suggested as an initiating factor in atherogenesis. Dying or dead endothelial cells have been shown to make significant contributions to the local enhancement of transendothelial macromolecular transport. Since cigarette smoking is one of the major risk factors for atherosclerosis, we examined the hypothesis that smoking accelerates atherogenesis by increasing the frequency of endothelial cell death and hence transendothelial macromolecular transport. Sixteen male Sprague-Dawley rats were given nicotine at a weight-adjusted dose of 5 mg/kg body wt per day in their drinking water over a period of 6 weeks. A group of 16 age-matched male Sprague-Dawley rats not exposed to nicotine and maintained over the same time period served as the control group. In en face preparations of thoracic aorta, immunoglobulin G-containing dying or dead endothelial cells were identified by the indirect immunoperoxidase method, and endothelial leakage to Evans blue-albumin (EBA) complexes (5 minutes after intravenous injection) was visualized by fluorescence microscopy. The results showed that in nicotine-treated rats, 51% of dead endothelial cells were associated with EBA leakage, which was responsible for 57% of total EBA leaky foci. Both the frequency of endothelial cell death (0.94 +/- 0.11% versus 0.40 +/- 0.04%, p < 0.0001 by two-tailed, unpaired Student's t test) and the number density of EBA leaky foci (6.45 +/- 1.23/mm2 versus 3.30 +/- 0.49/mm2, p < 0.05 by two-tailed, unpaired t test) were significantly greater in nicotine-treated rats than in control rats.(ABSTRACT TRUNCATED AT 250 WORDS)