De-endothelialized Areas Research Articles

Role of ferroptosis in the process of diabetes-induced endothelial dysfunction.

BACKGROUNDEndothelial dysfunction, a hallmark of diabetes, is a critical and initiating contributor to the pathogenesis of diabetic cardiovascular complications. However, the underlying mechanisms are still not fully understood. Ferroptosis is a newly defined regulated cell death driven by cellular metabolism and iron-dependent lipid peroxidation. Although the involvement of ferroptosis in disease pathogenesis has been shown in cancers and degenerative diseases, the participation of ferroptosis in the pathogenesis of diabetic endothelial dysfunction remains unclear.AIMTo examine the role of ferroptosis in diabetes-induced endothelial dysfunction and the underlying mechanisms.METHODSHuman umbilical vein endothelial cells (HUVECs) were treated with high glucose (HG), interleukin-1β (IL-1β), and ferroptosis inhibitor, and then the cell viability, reactive oxygen species (ROS), and ferroptosis-related marker protein were tested. To further determine whether the p53-xCT (the substrate-specific subunit of system Xc-)-glutathione (GSH) axis is involved in HG and IL-1β induced ferroptosis, HUVECs were transiently transfected with p53 small interfering ribonucleic acid or NC small interfering ribonucleic acid and then treated with HG and IL-1β. Cell viability, ROS, and ferroptosis-related marker protein were then assessed. In addition, we detected the xCT and p53 expression in the aorta of db/db mice.RESULTSIt was found that HG and IL-1β induced ferroptosis in HUVECs, as evidenced by the protective effect of the ferroptosis inhibitors, Deferoxamine and ferrostatin-1, resulting in increased lipid ROS and decreased cell viability. Mechanistically, activation of the p53-xCT-GSH axis induced by HG and IL-1β enhanced ferroptosis in HUVECs. In addition, a decrease in xCT and the presence of de-endothelialized areas were observed in the aortic endothelium of db/db mice.CONCLUSIONFerroptosis is involved in endothelial dysfunction and p53-xCT-GSH axis activation plays a crucial role in endothelial cell ferroptosis and endothelial dysfunction.

Protective Effect of Policosanol on Endothelium and Intimal Thickness Induced by Forceps in Rabbits

Policosanol is a cholesterol-lowering drug isolated from sugar cane wax with concomitant antiplatelet effects that prevents lipofundin-induced atherosclerotic lesions in rabbits and rats, including foam cell formation, and also reduces foam cell formation in carrageenan-induced granulomas in rats, while it inhibits proliferation of smooth muscle cells induced in rabbit cuffed artery. This study was undertaken to determine whether policosanol prevents endothelium damage and increase in arterial wall thickness in rabbits with arterial walls damaged with a forceps. Artery forceps were placed over the central artery of the right ear of all rabbits, and each artery was injured eight times. Animals were randomly distributed into four groups: a positive control group treated with Tween 20/H2O vehicle, two groups treated with policosanol (5 and 25 mg/kg, respectively), and a group treated with aspirin (8 mg/kg). Treatments were given for 30 days. Damaged arteries were examined by light and electron (transmission and scanning) microscopy. To evaluate intimal thickening, areas of intima were measured, and a significant reduction in policosanol-treated animals was observed. The endothelial surface, studied with scanning electron microscopy, revealed several types of damage. In control group, the endothelial surface was severely damaged. De-endothelialized areas were reduced in policosanol-treated animals. Platelet adhesion to subendothelium was seen in all animals of the control group, whereas policosanol-treated groups exhibited significantly reduced platelet adhesion. Policosanol also reduced, dose-dependently, the platelet sequestration induced in the damaged vessel wall, partially preventing the reduction in platelet count. It is concluded that policosanol prevents endothelium injury and reduces significantly intimal thickness of rabbit arteries damaged with forceps.

Effect of gene delivery of NOS isoforms on intimal hyperplasia and endothelial regeneration after balloon injury

Endothelial cell loss is a critical event in the pathological repair of the injured blood vessel. Impaired endothelial function results in reduced production of key vascular mediators such as nitric oxide (NO) within the vessel wall leading to enhanced smooth muscle cell proliferation and migration and ultimately intimal hyperplasia. The aim of the present study was to directly compare the effects of adenoviral-mediated gene delivery of two nitric oxide synthase (NOS) isoforms, eNOS and iNOS on endothelial regeneration and intimal hyperplasia following endothelial injury in the rabbit carotid artery. The right carotid arteries of male New Zealand white rabbits were denuded by passing a 3French Fogarty balloon catheter along the artery three times. In all, 1 x 10(9) PFU of adenoviral(Ad)eNOS, AdiNOS or Adbeta-galactosidase (Adbeta-Gal) was then delivered intraluminally and allowed to dwell for 20 min. Transgene expression was sought after 3 days by immunohistochemistry and at 7 days by quantitative reverse transcriptase PCR. The effect on intimal hyperplasia was sought using histological staining after 14 days. Evans blue staining was used to determine the effect on endothelial regeneration. eNOS and iNOS expression was detected in transduced arteries. Neointima/media ratios were significantly reduced in eNOS (0.07+/-0.044) and iNOS (0.087+/-0.086) transduced arteries compared with Adbeta-Gal (0.332+/-0.14) transduced arteries (n=7). In addition, AdeNOS treatment (4.21+/-3.12% de-endothelialized area) enhanced endothelial regeneration compared to Adbeta-Gal treatment (10.05+/-4.98), while treatment with AdiNOS (25.17+/-11.92) inhibited endothelial regeneration in the injured rabbit carotid artery (n=7-8). These results highlight the potential of NOS gene therapy, in particular, eNOS gene therapy as a potential therapeutic strategy for the prevention of restenosis after vascular injury.

Role of endothelial cells in restenosis after coronary angioplasty.

Percutaneous transluminal coronary angioplasty (PTCA) is today a procedure of choice in many patients with atherosclerotic coronary artery disease. Despite high rates of initial success, restenosis, occurring in 30 to 40 percent of patients within the first six months, remains the major problem limiting the long-term efficacy of the procedure. Animal models have enhanced our knowledge in the understanding of the mechanisms involved in the restenotic process after experimental angioplasty. In fact, the two known determinants of restenosis are the proliferative and migrative response of underlying smooth muscle cells with production of extracellular matrix and the recently highlighted vascular remodeling. Endothelium, which regenerates from the leading edge of the de-endothelialized area within the weeks following arterial injury, is of particular interest in the modulation of the healing process after the procedure. Endothelial dysfunction, as an imbalance between relaxing and contracting factors, between anti- and pro-coagulant mediators or growth-inhibiting and growth-promoting factors, occurs at sites of regenerating endothelium. Experimental studies, using drugs that enhance endothelium-derived relaxing factors release or drugs that diminish endothelium-derived contracting factors production, have often been shown to be effective in the restenosis prevention. Thus, impairment in endothelial cell function may be considered as one of the major regulatory element in the restenotic process. This review discusses the interactions between endothelial and smooth muscle cells and has for aim to point out the major role of endothelial cells in the development of neointimal thickening and arterial remodeling.

Severe damage of cultured vascular endothelial cell monolayer after simultaneous exposure to cadmium and lead.

This study evaluates the toxicity of lead in the presence or absence of cadmium on the maintenance of a monolayer of cultured bovine aortic endothelial cells. It was histologically shown that lead at 50 microM or less induced a slight formation of de-endothelialized areas on the monolayer after a 24-h incubation. Although lead accumulated in endothelial cells in a dose-dependent manner, the cytotoxicity (evaluated by the [3H]adenine release assay) was not detected. However, cadmium (1.0 or 2.0 microM)-induced de-endothelialized areas on the monolayer was considerably larger in the presence of lead at 10 microM; the cytotoxicity of cadmium was significantly enhanced by lead. It was concluded that vascular endothelial cell monolayer is severely destroyed by a greater cytotoxicity of combination of cadmium with lead.

Zinc-induced tolerance to cadmium cytotoxicity without metallothionein induction in cultured bovine aortic endothelial cells

We investigated the cytotoxicity of cadmium in cultured vascular endothelial cells from bovine aorta pretreated with zinc. The cytotoxicity was evaluated by the [ 3H]jadenine release assay. It was shown that the increase in the [ 3H]adenine release after challenge with cadmium was significantly prevented by pretreatment with zinc. It was histologically observed that cadmium-induced occurrence of the de-endothelialized areas was reduced in zincpretreated cell layer, Intracellular accumulation of cadmium was significantly less but that of zinc was more in zincpretreated cells. The content of cadmium in both the particulate and the cytosol fraction was decreased by zinc pretreatment; however, metallothionein content after challenge with cadmium was not changed. It was therefore concluded that zinc is capable of inducing a tolerance to cadmium cytotoxicity in cultured vascular endothelial cells; this tolerance is postulated to be due to a decrease in the intracellular cadmium accumulation rather than a metallothionein induction.

Injury to the arterial wall of rabbits produces proteoglycan variants with enhanced low-density lipoprotein-binding property

The effect of arterial injury on proteoglycans (PG) and their ability to bind low-density lipoprotein (LDL) were studied in rabbits 12 weeks after balloon injury. Following biosynthetic labeling in an organ culture system, PG were isolated under dissociative conditions from deendothelialized areas (DEA), reendothelialized areas (REA), and uninjured areas (control) of the aortic tissue. DEA and REA tissues yielded 42–52% more PG and incorporated 39–67% more 35S-label into proteoglycans than control tissues. Ion-exchange chromatography of PG from DEA and REA tissues yielded PG-I, PG-II, and PG-III, while from control tissue only PG-I and PG-II. PG-II formed major portion (74–84%) of the isolated PG in all three tissue types. PGI preparations comprised entirely of heparan sulfate (HS)-PG of similar hydrodynamic size ( K av = 0.45−0.47). PG-II from DEA and REA tissues consisted of PGII-A ( K av = 0.02−0.04) and PGII-B ( K av = 0.32), while PG-II from control tissue contained only PGII-B with relatively smaller hydrodynamic size ( K av = 0.40). PGII-A preparations contained predominantly chondroitin sulfate (CS)-PG with no dermatan sulfate (DS); whereas PGII-B consisted mainly of CS/DS-PG, with relatively high proportion of DS in DEA and REA tissues vs. control tissue (50–54% vs. 43%). Further, the glycosaminoglycan chains of CS/DS-PG from DEA and REA tissues were 1.7-fold longer than those from control tissue. PG-III contained about 80% CS/DS-PG and 20% HS-PG; CS/DS-PG was similar to those found in PGII-B from DEA and REA tissues. HS-PG from PG-II and PG-III, unlike those from PG-I, was enriched with N-sulfated residues. PGI from all the three tissue types bound poorly to LDL. On the other hand, PGII-A, PGII-B, and PG-III from DEA and REA tissues showed enhanced ability to bind LDL, in that order. For example, the LDL-binding ability of PGII-B from DEA and REA was 2.9- to 3.1-fold above that from control tissue. Thus, arterial injury with or without regenerated endothelium produces proteoglycan variants with altered characteristics and enhanced LDL-binding ability.

Zinc protection against cadmium-induced destruction of the monolayer of cultured vascular endothelial cells

The effect of zinc on the destruction of vascular endothelial cell monolayer caused by cadmium was investigated using bovine aortic endothelial cells in a culture system. It was histologically revealed that cadmium-induced de-endothelialized area formation was markedly prevented by zinc. Zinc prevented cadmium-induced detachment of [ 3H]thymidine-labeled endothelial cells from the monolayer. Although cadmium increased the activity of lactate dehydrogenase in the medium, a marker of nonspecific cell damage, zinc markedly suppressed it. On the other hand, zinc significantly prevented cadmium-induced decrease in the incorporation of [ 3H]thymidine into the acid-insoluble fraction of the growing cells. From these results, it was suggested that impairment of the endothelial cell monolayer caused by cadmium is protected by zinc; this protection was postulated to be due to both a reduction of the cell detachment caused by cadmium-induced nonspecific cell damage and a decrease in cadmium inhibition of the cell proliferation which would help the repair of de-endothelialized areas.

Protective effect of copper against cadmium cytotoxicity on cultured vascular endothelial cells

We investigated the effect of copper on cadmium-induced cytotoxicity on vascular endothelial cells from bovine aorta in a culture system. Cytotoxicity was evaluated by the [ 3H]adenine release assay and the histological observation. After a 24-h incubation, cadmium exhibited a significant cytotoxicity on confluent cultures of endothelial cells in a dose-dependent manner, while copper only slightly did after a 24-h incubation. It was found that copper (5 μM) significantly decreased cadmium (1 and 2 μM) cytotoxicity; histologically, formation of de-endothelialized areas in the cell layer caused by cadmium was reduced by copper. The accumulation of cadmium in the cell layer was significantly decreased by copper; however, that of copper was unaffected by cadmium. It was therefore suggested that copper significantly protects cadmium-induced cytotoxicity on cultured endothelial cells primarily through decreasing the cellular cadmium accumulation.

Effects of antithrombotic drug, Y-20811 on mural thrombus formation and intimal thickening following intimal injury

Inhibitory effect of Y-20811 on platelet thrombus formation induced by mechanical intimal injury and subsequent intimal fibrous thickening was studied. In the short term experiment, polyethylene tubing was inserted into the rabbit aorta and was drawn out one hour after with or without Y-20811 administration, then the rabbits were sacrificed. In the experiment for the quantitative analysis of platelet adhesion, 51Cr-labeled platelets were used. Radioactivities of 2cm length of the injured segment of the thoracic aorta and the proximal 2cm of the normal segment were measured. Radioactivity of the injured segment was significantly lower in rabbits treated with Y-20811 than the control ones. The mean thickness(area of thrombi/length of injured intima) and the maximal thickness of the mural thrombi in the Y-20811-treated rabbits were significantly lower than those in control rabbits. De-endothelialized area showed raised platelet thrombi in the control group and diffuse thin-layered platelet sheets in Y-20811-treated rabbits. In the long term experiment, polyethylene tubing was indwelled for 24 hours. Rabbits were sacrificed 10 days after drawing out the tubing. Through the experiment Y-20811 was injected intravenously every 24 hours. There was no significant difference between the control group and the Y-20811-treated one in both mean thickness and maximal thickness of the intimal fibromuscular thickening. The experiments indicate that Y-20811 has an inhibitory effect on platelet thrombus formation following intimal injury, but subsequent myointimal thickening is not inhibited by the drug.

Actin stress fiber content of regenerated endothelial cells correlates with intramural retention of intermediate plus low density lipoproteins in rat aorta after balloon injury

The rat aortic model of endothelial injury (balloon catheter induced) has been used to establish whether changes in protein intramural penetration in specific areas of the injured aorta were accompanied by phenotypic modifications of the regenerated endothelial cells covering these particular regions. Iodinated lipoproteins ( IDL LDL fraction) and albumin were used as tracers to localize protein permeability and retention in the aorta. Lipoproteins, but not albumin, were retained in the thickened areas covered with regenerated endothelium (i.e., 60 days after balloon induced injury). Neither lipoproteins nor albumin were retained in the other aortic areas studied, including the intimal thickening of de-endothelialized areas (15 days after injury). The relative volume of cytoplasmic stress fibers was significantly increased in regenerated endothelium covering thickened areas as compared with the other regions of the injured or normal aorta. The accumulation of lipids usually observed in atherosclerotic lesions, compatible with the trapping of lipoproteins by the matrix component of the intimal thickening, may be related to modulated features of endothelial cells regenerated over thickened areas of the aorta.

The tachyphylactic effect of arachidonic acid on in-vivo ADP induced arterial platelet thrombosis

The continuous superfusion of arachidonic acid over a branch of the mesenteric artery in an animal in-vivo model results in an initial increase followed by a decrease to control values of ADP induced platelet thrombogenesis. This phenomenon is observed in normal rats as well as in rats submitted to the intravenous administration of ASA three hours prior to the investigation. In the latter group the cyclooxygenase of the platelets is permanently affected while the cyclooxygenase of the endothelial cells is regenerated at the time the investigation is started. The tachyphylactic-like phenomenon observed with arachidonic acid is probably related to the dynamics of the enzymatic reactions of the arachidonate cascade involving the platelets and the endothelial cells surrounding the local de-endothelialized area.

Altered glycosaminoglycan metabolism in injured arterial wall

Glycosaminoglycans (GAG) are believed to be important in the pathogenesis of atherosclerosis. We have previously demonstrated that areas of injured aorta that have been reendothelialized accumulate increased amounts of lipid and GAG when compared to areas remaining de-endothelialized. We have now examined the net incorporation of labeled precursors into the individual GAG present in both re-endothelialized and de-endothelialized areas of rabbit aorta. Aortic tissue was examined at 2–3 and 10–14 weeks after a denuding injury by incubating tissue minces with [3H]glucosamine and sodium [35S]sulfate for 24 hr. Following incubation, the aortic GAG were isolated and assayed for uronic acid concentration and radioactivity. Results indicate that the total GAG concentration was significantly greater (P < 0.001) in the re-endothelialized (9.46 ± 0.29 μg/mg lipid-free dry residues (LFDR), mean ± SE) as compared to de-endothelialized (7.89 ± 0.43 μg/mg LFDR) areas. The concentration in uninjured aorta was 9.01 ± 0.69. The difference between the injured tissues was attributable to increased concentrations of sulfated GAG. Hyaluronic acid and chondroitin sulfate were the most metabolically active of the GAG in either uninjured or injured aorta, together accounting for over 75% of the 3H label. The 3H specific radioactivities of the four GAG in the short-term, re-endothelialized subgroup were all increased nearly twice that found in uninjured and de-endothelialized tissues. With the exception of heparan sulfate, no significant differences were noted in the 3H specific radioactivities between the re-endothelialized and de-endothelialized areas in the long-term subgroup. These results indicate that, relative to adjacent areas of de-endothelialization, GAG preferentially accumulated in re-endothelialized areas even as early as 2–3 weeks following a denuding injury. Overall, metabolic data suggest that increased synthesis is responsible for this effect, although the net contribution of degradative processes cannot be overlooked since GAG turnover was not specifically examined. Thus, it is possible that regenerated endothelium may modify the GAG metabolism of the arterial wall following arterial injury.

Selective accumulation of low density lipoproteins in damaged arterial wall.

To determine whether damaged arterial wall selectively accumulates lipoproteins, normocholesterolemic rabbits were injected with human radiolabeled low density lipoproteins, high density lipoproteins, and/or albumin 24 hr to 12 weeks after balloon-catheter de-endothelialization of the abdominal aorta. When 125I-labeled low density lipoproteins and 99mTc-labeled albumin were injected simultaneously, the amount of 125I-low density lipoprotein present 24 hr later in abdominal aortas increased steadily, for several weeks, above the amount present at 24 hr in control animals. The increase correlated closely with the degree of re-endothelialization and correlated closely with the degree of re-endothelialization and reached an average maximum for the whole abdominal aorta of three times control when re-endothelialization was between 75 and 85% complete. By contrast, the amounts of 99mTc-albumin or 125I-labeled high density lipoprotein in balloon-damaged abdominal aortas, and the amounts of 125I-low density lipoprotein, 125I-high density lipoprotein, or 99mTc-albumin in undamaged thoracic aortas of injured animals showed no such increase. As early as 2 weeks after de-endothelialization, en face radioautographs made following injection of 125I-labeled low density lipoproteins revealed localized areas of greatest radioactivity around the leading edges of regenerating endothelial islands, broad areas of intermediate radioactivity corresponding to the de-endothelialized areas, and very like radioactivity in the re-endothelialized areas. This pattern occurred rarely with 125I-labeled high density lipoproteins and not at all with 125I-labeled albumin. The results suggest that low density lipoproteins are selectively accumulated by the healing rabbit aorta and that the accumulation is greatest in regions where the endothelium is actively regenerating.

Recovery of prostacyclin production by de-endothelialized rabbit aorta. Critical role of neointimal smooth muscle cells.

Prostacyclin (PGI2) synthetic capacity was assayed at the surface of aortas at various intervals after removal of endothelium with a balloon catheter. Results were correlated with morphologic changes in the vessel wall seen by light microscopy, scanning and transmission electron microscopy. To assay PGI2 synthetic capacity, we applied an incubation chamber to the luminal surface of the aortas; after arachidonic acid stimulation we assayed the PGI2 synthesized with a bioassay and radioimmunoassay. PGI2 synthesis in de-endothelialized aortas was determined immediately after balloon-catheter injury and at intervals of 1 h and 2, 4, 15, 35, and 70 d. PGI2 synthesis was low at 1 h and increased over time with levels at 35 and 70 d reaching that of normal artery. Scanning and transmission electron microscopy of de-endothelialized areas showed persistent absence of endothelium with formation of a neointima composed of smooth muscle cells. De-endothelialized aorta was covered with adherent platelets shortly after injury, however several days later only a few platelets adhered to the denuded surface. Results indicated that (a) endothelium is responsible for nearly all PGI2 production at the luminal surface of the normal aorta, (b) de-endothelialized muscular neointima synthesized increasing quantities of PGI2 with time after injury, and (c) increase of PGI2 production at the luminal surface of de-endothelialized aorta correlates with formation of a neointima and with the acquired thromboresistance of the aorta.