Effects of Cilostazol on Angiogenesis in Diabetes through Adiponectin/Adiponectin Receptors/Sirtuin1 Signaling Pathway.

Cilostazol is an antiplatelet agent with vasodilating effects that functions by increasing the intracellular concentration of cyclic adenosine monophosphate. We have previously shown that cilostazol has favorable effects on angiogenesis. However, there is no study to evaluate the effects of cilostazol on adiponectin. We investigated the effects of cilostazol on angiogenesis in diabetes in vitro and in vivo through adiponectin/adiponectin receptors (adipoRs) and the sirtuin 1 (SIRT1)/AMP-activated protein kinase (AMPK) signaling pathway. Human umbilical vein endothelial cells (HUVECs) and human aortic smooth muscle cells (HASMCs) were cocultured under high glucose (HG) conditions. Adiponectin concentrations in the supernatants were significantly increased when HASMCs were treated with cilostazol but not significantly changed when only HUVECs were treated with cilostazol. Cilostazol treatment enhanced the expression of SIRT1 and upregulated the phosphorylation of AMPK in HG-treated HUVECs. By sequential knockdown of adipoRs, SIRT1, and AMPK, our data demonstrated that cilostazol prevented apoptosis and stimulated proliferation, chemotactic motility, and capillary-like tube formation in HG-treated HUVECs through the adipoRs/SIRT1/AMPK signaling pathway. The phosphorylation of downstream signaling molecules, including acetyl-CoA carboxylase (ACC) and endothelial nitric oxide synthase (eNOS), was downregulated when HUVECs were treated with a SIRT1 inhibitor. In streptozotocin-induced diabetic mice, cilostazol treatment could improve blood flow recovery 21-28 days after inducing hindlimb ischemia as well as increase the circulating of CD34+CD45dim cells 14-21 days after operation; moreover, these effects were significantly attenuated by the knockdown of adipoR1 but not adipoR2. The expression of SIRT1 and phosphorylation of AMPK/ACC and Akt/eNOS in ischemic muscles were significantly attenuated by the gene knockdown of adipoRs. Cilostazol improves HG-induced endothelial dysfunction in vascular endothelial cells and enhances angiogenesis in diabetic mice by upregulating the expression of adiponectin/adipoRs and its SIRT1/AMPK downstream signaling pathway.

Diverse Effects of Cilostazol on Proprotein Convertase Subtilisin/Kexin Type 9 between Obesity and Non-Obesity.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in cholesterol homeostasis. Cilostazol exerts favorable cellular and metabolic effects; however, the effect of cilostazol on the expression of PCSK9 has not been previously reported. Our study aimed to investigate the potential mechanisms of action of cilostazol on the expression of PCSK9 and lipid homeostasis. We evaluated the effects of cilostazol on the expression of PCSK9 in HepG2 cells and evaluated potential molecular mechanisms by measuring signaling molecules in the liver and serum lipid profiles in high-fat diet-induced obese mice and normal chow-fed mice. Cilostazol treatment significantly induced the messenger RNA and protein expression of PCSK9 in HepG2 cells and enhanced PCSK9 promoter activity. Chromatin immunoprecipitation assays confirmed that cilostazol treatment enhanced PCSK9 transcription by binding to peroxisome proliferator-activated receptor-γ (PPARγ) via the PPARγ DNA response element. PPARγ knockdown attenuated the stimulatory effect of cilostazol on PCSK9. In vitro, cilostazol treatment increased PCSK9 expression in vehicle-treated HepG2 cells but decreased PCSK9 expression in palmitic acid-treated HepG2 cells. In vivo, cilostazol treatment increased the serum levels of PCSK9 in normal mice but significantly reduced PCSK9 levels in obese mice. The expressions of PCSK9-relevant microRNAs also showed similar results. Clinical data showed that cilostazol treatment significantly reduced serum PCSK9 levels in patients with obesity. The obesity-dependent effects of cilostazol on PCSK9 expression observed from bench to bedside demonstrates the therapeutic potential of cilostazol in clinical settings.

A Randomized Controlled Trial Evaluating Outcome Impact of Cilostazol in Patients with Coronary Artery Disease or at a High Risk of Cardiovascular Disease.

Previous studies found that cilostazol has a favorable effect on glucose and lipid homeostasis, endothelial function, atherosclerosis, and vasculo-angiogenesis. However, it is poorly understood whether these effects can translate into better clinical outcomes. This study investigated the outcome effect of cilostazol in patients with coronary artery disease (CAD) or at a high risk of cardiovascular (CV) disease. We conducted a randomized, double-blind, placebo-controlled trial involving 266 patients who received cilostazol, 200 mg/day (n = 134) or placebo (n = 132). Pre-specified clinical endpoints including composite major adverse cardiovascular events (MACE) (CV death, non-fatal myocardial infarct, non-fatal stroke, hospitalization for heart failure, or unplanned coronary revascularization), the composite major coronary event (MCE) and major adverse CV and cerebrovascular event (MACCE), were prospectively assessed. The mean duration of follow-up was 2.9 years. Relative to placebo, cilostazol treatment had a borderline effect on risk reduction of MACE (hazard ratio [HR], 0.67; 95% confidence interval (CI), 0.34-1.33), whereas the beneficial effect in favor of cilostazol was significant in patients with diabetes mellitus or a history of percutaneous coronary intervention (p for interaction, 0.02 and 0.06, respectively). Use of cilostazol, significantly reduced the risk of MCE (HR, 0.38; 95% CI, 0.17-0.86) and MACCE (HR, 0.47; 95% CI, 0.23-0.96). A significantly lower risk of angina pectoris (HR, 0.38; 95% CI, 0.17-0.86) was also observed in the cilostazol group. After multi-variable adjustment, cilostazol treatment independently predicted a lower risk of MCE. In conclusion, these results suggest cilostazol may have beneficial effects in patients with CAD or at a high risk of CV disease.

Deletion of MicroRNA-21 Impairs Neovascularization Following Limb Ischemia: From Bedside to Bench.

With an increasing prevalence, peripheral arterial disease (PAD), cause by atherosclerosis is a new threat to public health beyond coronary artery disease and involves aberrant vascular endothelial cell proliferation and angiogenesis. The degree of vascular remodeling is influenced by the processes described. MicroRNA-21 (miR-21) has been found to play a critical role in cellular functions, including angiogenesis. Nevertheless, the effect of miR-21 on endothelial cells in response to hypoxia is largely unknown. Using wild-type C57BL/6J and miR-21-/- mice, we compared the capability of angiogenesis in response to hindlimb hypoxic/ischemia. In an in vitro study, we further studied whether overexpression of miR-21 mitigates hypoxia-induced apoptosis and impaired angiogenesis. Also, we prospectively collected the sera of patients with limb ischemia and followed the clinical information, including major adverse limb events (MALEs). Using laser Doppler perfusion imaging and CD31 staining, compared with miR-21-/- mice, wild-type mice expressed a significantly higher capability of angiogenesis and less apoptosis following 28 days of hindlimb hypoxic/ischemic surgery. In our in vitro study, after 24 h of hypoxia, proliferation, migration, and tube formation were significantly impaired in cells treated with the miR-21 inhibitor but rescued by the miR-21 mimic. Mechanistically, by suppressing PTEN/PI3K/AKT, miR-21 promoted angiogenesis and suppressed apoptosis in endothelial cells post hypoxia. In patients with limb ischemia, the high expression of circulating miR-21 was associated with less subsequent MALE. Collectively, miR-21 could be a biomarker associated with the endogenous ability of angiogenesis and reflect subsequent MALE in patients. Additionally, abolishing miR-21 impairs angiogenesis and promotes apoptosis post limb ischemia. Further studies are required to elucidate the clinical applications of miR-21.

The role of vascular smooth muscle cell membrane-bound thrombomodulin in neointima formation.

BACKGROUND AND AIMS: Thrombomodulin (TM) is an endothelial cell membrane-bound anticoagulant protein expressed in normal arteries. After vascular injury, medial and neointimal smooth muscle cells (SMCs) exhibit large amounts of TM. The purpose of this study was to investigate the physiological significance of vascular SMC-bound TM. METHODS: The morphology, expression of phenotype markers and cell behaviors of cultured aortic SMCs after knockdown of TM were observed. Transgenic mice with SMC-specific TM deletion were generated, and carotid neointima formation was induced by carotid ligation. RESULTS: Cultured human aortic SMCs displayed a synthetic phenotype with a rhomboid-shaped morphology and expressed TM. TM knockdown induced a spindle-shaped change in morphology with an increased expression of contractile phenotype marker and decreased expression of synthetic phenotype marker. TM knockdown not only attenuated the proliferation of SMCs but also reduced tumor necrosis factor-α-induced nuclear factor-κB activation and interlukin-6 production. In a carotid artery ligation model, transgenic mice with SMC-specific TM deletion (SM22-cretg/TMflox/flox) had significantly less cellular proliferation in arterial walls compared with wild type mice (SM22-cretg/TM+/+). The neointima area and neointima/media area ratio were smaller in SM22-cretg/TMflox/flox mice at 4 weeks after ligation. CONCLUSIONS: Our results indicate that vascular SMC-bound TM plays a role in changes of the SMC phenotype. It also influences SMC cell behavior and injury-induced neointima formation.

Effect of cilostazol on plasma levels of proprotein convertase subtilisin/kexin type 9.

The protein complex proprotein convertase subtilisin/kexin type 9 (PCSK9) serves as an important target for the prevention and treatment of atherosclerosis and lipid homeostasis. This study investigated the effect of cilostazol on plasma PCSK9 concentrations. We performed a post hoc analysis of two prospective, double-blind, randomized controlled trials including 115 patients of whom 61 received cilostazol 200 mg/day and 54 received placebo for 12 weeks. Linear regression analysis was performed to determine the associations between several parameters and changes in PCSK9 levels. Use of cilostazol, but not placebo, significantly increased plasma PCSK9 concentrations, high-density lipoprotein cholesterol levels, and number of circulating endothelial progenitor cells (EPCs), and decreased triglyceride levels with a trend toward an increase in total cholesterol (TC) levels. A reduction in hemoglobin A1C and an increase in plasma vascular endothelial growth factor and adiponectin levels with cilostazol treatment were also found. Changes in the number of circulating EPCs were positively correlated and the TC concentrations were inversely correlated with changes in the PCSK9 levels. After adjusting for changes in levels of TC and numbers of circulating EPCs and history of metabolic syndrome, use of cilostazol remained independently associated with changes in plasma PCSK9 levels. In conclusion, cilostazol treatment was significantly and independently associated with an increase in plasma PCSK9 levels in patients with peripheral artery disease or at a high risk of cardiovascular disease regardless of background statin use and caused an improvement in some metabolic disorders and levels of vasculo-angiogenic biomarkers.

Cilostazol Improves Proangiogenesis Functions in Human Early Endothelial Progenitor Cells through the Stromal Cell-Derived Factor System and Hybrid Therapy Provides a Synergistic Effect In Vivo.

This study investigated the effect of cilostazol on proangiogenesis functions in human early endothelial progenitor cells (EPCs) in vitro and the therapeutic implication of hybrid therapy with cilostazol and human early EPCs in vivo. Cilostazol significantly increased colony-forming units and enhanced differentiation of EPCs toward endothelial lineage. Treatments resulted in antiapoptotic effects and stimulated proliferation and migration and in vitro vascular tube formation through activation of stromal cell-derived factor-1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4)/phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway. Blood flow recovery and capillary density in murine ischemic hindlimbs were significantly improved in cilostazol-treated, human early EPCs-treated, and cotreatment groups. The effects were attenuated with SDF-1α inhibition. Plasma SDF-1α levels were significantly higher in 3 active treatment groups after surgery, with greatest effects observed in hybrid therapy. The angiogenic effects of transplanted EPCs pretreated with cilostazol ex vivo were superior to untreated EPCs using in vivo Matrigel assay. Implanted EPCs were incorporated into the capillary, with pretreatment or cotreatment with cilostazol resulting in enhanced effects. Taken together, cilostazol promotes a large number of proangiogenic functions in human early EPCs through activation of SDF-1/CXCR4/PI3K/Akt signaling, and hybrid therapy provides a synergistic effect in vivo. Cotreatment may be beneficial in ischemic disease.

Cilostazol Enhances Mobilization of Circulating Endothelial Progenitor Cells and Improves Endothelium-Dependent Function in Patients at High Risk of Cardiovascular Disease.

This is the first study to investigate the vasculoangiogenic effects of cilostazol on endothelial progenitor cells (EPCs) and flow-mediated dilatation (FMD) in patients at high risk of cardiovascular disease (CVD). This double-blind, placebo-controlled study included 71 patients (37 received 200 mg/d cilostazol and 34 received placebo for 12 weeks). Use of cilostazol, but not placebo, significantly increased circulating EPC (kinase insert domain receptor(+)CD34(+)) counts (percentage changes: 149.0% [67.9%-497.8%] vs 71.9% [-31.8% to 236.5%], P = .024) and improved triglyceride and high-density lipoprotein cholesterol levels (P = .002 and P = .003, respectively). Plasma levels of vascular endothelial growth factor (VEGF)-A165 and FMD significantly increased (72.5% [32.9%-120.4%] vs -5.8% [-46.0% to 57.6%], P = .001; 232.8% ± 83.1% vs -46.9% ± 21.5%, P = .003, respectively) in cilostazol-treated patients. Changes in the plasma triglyceride levels significantly inversely correlated with the changes in the VEGF-A165 levels and FMD. Cilostazol significantly enhanced the mobilization of EPCs and improved endothelium-dependent function by modifying some metabolic and angiogenic markers in patients at high risk of CVD.

Plasma Levels of Proprotein Convertase Subtilisin/Kexin Type 9 Are Elevated in Patients With Peripheral Artery Disease and Associated With Metabolic Disorders and Dysfunction in Circulating Progenitor Cells.

BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is involved in cholesterol homeostasis, inflammation, and oxidative stress. This study investigated the association of plasma PCSK9 levels with the presence and severity of peripheral artery disease (PAD) and with parameters of endothelial homeostasis. METHODS AND RESULTS: A post hoc analysis of 2 randomized trials (115 patients, 44 with PAD and 71 without atherosclerotic disease) was conducted. Patients with PAD had significantly higher plasma PCSK9 levels than those without (471.6±29.6 versus 302.4±16.1 ng/mL, P<0.001). Parameters for glucose homeostasis, endothelial progenitor cell functions, apoptotic circulating endothelial cell counts, and plasma levels of vascular endothelial growth factor-A165 and oxidized low-density lipoprotein were correlated with PCSK9 concentration. By multivariable linear regression analysis, presence of PAD, plasma glucose or hemoglobin A1c levels, apoptotic circulating endothelial cell counts, and vascular endothelial growth factor-A165 concentration were found to be associated with PCSK9 levels after multivariable adjustment. Patients with extensive involvement of PAD or with severe PAD had significantly higher PCSK9 levels than those without PAD. Computed tomographic angiography showed that the numbers of chronic total occlusion sites and vessels involved were positively associated with PCSK9 levels in patients with PAD (r=0.40, P=0.01, and r=0.36, P=0.02, respectively). CONCLUSION: PCSK9 levels were significantly higher in patients with PAD, especially those with advanced PAD. Further large-scale studies examining the effect of PCSK9-targeting therapies or the modification of PCSK9 levels on cardiovascular outcomes in this clinical setting are warranted. CLINICAL TRIAL REGISTRATION: Cohort 1: URL: ClinicalTrials.gov. Unique identifier: NCT01952756; cohort 2: URL: ClinicalTrials.gov. Unique identifier: NCT02194686.

Impact of routine coronary catheterization in low extremity artery disease undergoing percutaneous transluminal angioplasty: study protocol for a multi-center randomized controlled trial.

BACKGROUND: The prevalence of significant obstructive coronary artery disease with complex lesions is high in patients who have low extremity artery disease (LEAD). However, intermediate- or long-term cardiovascular prognosis of LEAD patients undergoing percutaneous transluminal angioplasty (PTA) remains poor. Accordingly, prophylactic coronary revascularization may modify short- and long-term cardiovascular outcomes of LEAD patients receiving PTA. Because myocardial ischemic symptoms are often masked in LEAD and the accuracy of non-invasive stress tests is usually limited, a high-quality randomized controlled trial aimed at the investigation of the prognostic role of coronary evaluation strategies before PTA is warranted. METHODS/DESIGN: The proposed study is designed as a prospective, multi-center, open-label, superiority, randomized controlled trial. The study is conducted in high-volume centers for PTA and coronary revascularization in Taiwan. To meet the inclusion criteria, the patients must be at least 20 years old, have known LEAD, and have been admitted for elective PTA. We plan to enroll 450 participants who are randomly allocated to a routine group (routine coronary angiography without a previous non-invasive stress test before PTA) and a selective group (selective coronary angiography based on the results of non-invasive stress tests before PTA) with 1:1 ratio. Besides, we expect to enroll about 250 additional participants, who are not willing to be randomly assigned, in the registration group. The choice of revascularization procedure depends on the operator's or cardiovascular team's suggestion and the patient's decision. Clinical follow-up will be performed 30 days after PTA and every 6 months until the end of the 1-year follow-up for the last randomly assigned participant. The primary endpoint is the composite major adverse cardiac event on long-term follow-up. Pre-specified secondary and other endpoints are also evaluated. Those assessing biomarkers and clinical endpoints are all blinded after assignment to interventions. DISCUSSION: The results of the trial will, for the first time, support better decision-making for coronary evaluation before PTA in LEAD. If favorable, routine coronary angiography followed by revascularization will improve cardiovascular outcomes in LEAD patients undergoing PTA. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02169258 (registered on 21 June 2014); registry name: Routine Coronary Catheterization in Low Extremity Artery Disease Undergoing Percutaneous Transluminal Angioplasty (PIROUETTE-PTA).

Cilostazol improves high glucose-induced impaired angiogenesis in human endothelial progenitor cells and vascular endothelial cells as well as enhances vasculoangiogenesis in hyperglycemic mice mediated by the adenosine monophosphate-activated protein kinase pathway.

OBJECTIVE: Cilostazol is an antiplatelet agent with vasodilatory effects that works by increasing intracellular concentrations of cyclic adenosine monophosphate (cAMP). This study investigated the effects of cilostazol in preventing high glucose (HG)-induced impaired angiogenesis and examined the potential mechanisms involving activation of AMP-activated protein kinase (AMPK). METHODS: Assays for colony formation, adhesion, proliferation, migration, and vascular tube formation were used to determine the effect of cilostazol in HG-treated endothelial progenitor cells (EPCs) or human umbilical vein endothelial cells (HUVECs). Animal-based assays were performed in hyperglycemic ICR mice undergoing hind limb ischemia. An immnunoblotting assay was used to identify the expression and activation of signaling molecules in vitro and in vivo. RESULTS: Cilostazol treatment significantly restored endothelial function in EPCs and HUVECs through activation of AMPK/acetyl-coenzyme A carboxylase (ACC)-dependent pathways and cAMP/protein kinase A (PKA)-dependent pathways. Recovery of blood flow in the ischemic hind limb and the population of circulating CD34(+) cells were significantly improved in cilostazol-treated mice, and these effects were abolished by local AMPK knockdown. Cilostazol increased the phosphorylation of AMPK/ACC and Akt/endothelial nitric oxide synthase signaling molecules in parallel with or downstream of the cAMP/PKA-dependent signaling pathway in vitro and in vivo. CONCLUSIONS: Cilostazol prevents HG-induced endothelial dysfunction in EPCs and HUVECs and enhances angiogenesis in hyperglycemic mice by interactions with a broad signaling network, including activation of AMPK/ACC and probably cAMP/PKA pathways.

Effects of rosiglitazone on the cardiovascular profile in postmenopausal women without diabetes mellitus: interplay of thiazolidinediones and hormone therapy.

OBJECTIVE: Thiazolidinediones have antiatherothrombotic effects on persons with diabetes. Hormone therapy among postmenopausal women has both positive and negative cardiovascular effects. However, the effects of rosiglitazone with or without concurrent long-term hormone therapy on the cardiovascular profile of nondiabetic postmenopausal women are unknown. METHODS: Thirty-eight nondiabetic postmenopausal women were enrolled in this double-blind and placebo-controlled study. Eighteen participants received 4 mg rosiglitazone, and 20 participants took placebo daily for 12 weeks. Global endothelial function and plasma biomarkers were measured. RESULTS: Baseline characteristics and parameters were similar between the groups. Rosiglitazone, but not placebo, significantly reduced leukocyte count and plasma levels of matrix metalloproteinase-9 and inhibited the elevation of plasma levels of plasminogen activator inhibitor-1 and tissue plasminogen activator (P < 0.05 for all). Most of the favorable effects provided by rosiglitazone were still present in participants with concurrent hormone therapy. Increased body weight and waist size as well as elevation of the plasma levels of total and low-density lipoprotein cholesterol were noted after rosiglitazone treatment among participants without concurrent hormone therapy. No significant change in the global endothelial function occurred in response to treatment in either group. CONCLUSIONS: Rosiglitazone treatment provided both protective and harmful cardiovascular effects in nondiabetic postmenopausal women. Concurrent hormone therapy resulted in the maintenance of the major beneficial effects while neutralizing the unfavorable effects of rosiglitazone.

A novel vasculo-angiogenic effect of cilostazol mediated by cross-talk between multiple signalling pathways including the ERK/p38 MAPK signalling transduction cascade.

Cilostazol is an anti-platelet agent with vasodilatory activity that acts by increasing intracellular concentrations of cAMP. Recent reports have suggested that cilostazol may promote angiogenesis. In the present study, we have investigated the effect of cilostazol in promoting angiogenesis and vasculogenesis in a hindlimb ischaemia model and have also examined its potential mechanism of action in vitro and in vivo. We found that cilostazol treatment significantly increased colony formation by human early EPCs (endothelial progenitor cells) through a mechanism involving the activation of cAMP/PKA (protein kinase A), PI3K (phosphoinositide 3-kinase)/Akt/eNOS (endothelial NO synthase) and ERK (extracellular-signal-regulated kinase)/p38 MAPK (mitogen-activated protein kinase) signalling pathways. Cilostazol also enhanced proliferation, chemotaxis, NO production and vascular tube formation in HUVECs (human umbilical vein endothelial cells) through activation of multiple signalling pathways downstream of PI3K/Akt/eNOS. Cilostazol up-regulated VEGF (vascular endothelial growth factor)-A165 expression and secretion of VEGF-A in HUVECs through activation of the PI3K/Akt/eNOS pathway. In a mouse hindlimb ischaemia model, recovery of blood flow ratio (ipsilateral/contralateral) 14 days after surgery was significantly improved in cilostazol-treated mice (10 mg/kg of body weight) compared with vehicle-treated controls (0.63±0.07 and 0.43±0.05 respectively, P<0.05). Circulating CD34+ cells were also increased in cilostazol-treated mice (3614±670 compared with 2151±608 cells/ml, P<0.05). Expression of VEGF and phosphorylation of PI3K/Akt/eNOS and ERK/p38 MAPK in ischaemic muscles were significantly enhanced by cilostazol. Our data suggest that cilostazol produces a vasculo-angiogenic effect by up-regulating a broad signalling network that includes the ERK/p38 MAPK, VEGF-A165, PI3K/Akt/eNOS and cAMP/PKA pathways.

Distribution of vimentin-expressing cells in pterygium: an immunocytochemical study of impression cytology specimens.

PURPOSE: To investigate the distribution of vimentin-expressing cells in human pterygium by immunocytochemical analysis of impression cytology specimens. METHODS: Using impression cytology, 3 samples of superficial conjunctival epithelial cells, including pterygium and adjacent conjunctiva were obtained from each of 24 eyes of 24 patients with unilateral primary pterygium, 16 eyes of 16 patients with unilateral recurrent pterygium, and the 40 unaffected fellow eyes. Specimens were processed in avidin-biotin complex immunostaining system with monoclonal anti-vimentin antibody and/or anti-CD1a antibody and counterstained with hematoxylin. Under light microscopy, the extent of vimentin-expressing cell infiltration beyond the pterygium margin was measured with a micrometer and the number of vimentin-expressing cells was counted in each of 6 randomly selected high-power fields in the specimens of pterygium and normal conjunctiva. RESULTS: Two types of cells expressed vimentin, epithelioid cells and dendritic cells, whereas cells that expressed CD1a showed only a dendritic pattern. Vimentin-expressing round epithelioid cells (presumed pterygial cells) were present over the entire surface of the pterygium and also on adjacent normal-appearing conjunctiva, up to an average of 3.01 +/- 0.83 mm beyond the superior margin and 3.14 +/- 0.99 mm beyond the inferior margin of primary pterygium and 3.72 +/- 0.85 mm and 3.58 +/- 0.98 mm beyond the superior and inferior margins of recurrent pterygium. For the superior margin but not the inferior margin, the clinically occult extension of vimentin-expressing epithelioid cells was statistically significantly greater for eyes with recurrent pterygium compared with those with primary pterygium (P = 0.016). The density of dendritic (Langerhans) cells, stained by anti-vimentin and anti-CD1a antibodies, was statistically significantly lower in normal fellow eyes than in eyes with either primary (P < 0.001) or recurrent (P = 0.024) pterygium. CONCLUSIONS: The vimentin-expressing epithelioid cells were present not only over the ocular surface of the pterygium but also in the normal-appearing conjunctiva adjacent to primary and recurrent pterygium. The increased density of Langerhans cells in pterygium might reflect a higher level of antigenic and mitogenic exposure in the conjunctiva. However, the significance of these 2 phenomena in the recurrence and pathogenesis of pterygium remains undetermined and merits further studies.

In vitro benzyl alcohol cytotoxicity: implications for intravitreal use of triamcinolone acetonide.

The aim of the study was to investigate the toxicity of benzyl alcohol (BA), the preservative in commercial triamcinolone acetonide (TA) suspensions, on retinal pigment epithelial (RPE) cells. Cultured RPE cells from a human cell line (ARPE-19) and from rabbits were exposed to the balanced salt solution (control) or BA (0.0225, 0.225, 0.9, 3 or 9mg/mL) for 5, 30, 60, or 120min. Morphological changes of RPE cells were evaluated by the trypan blue in situ staining. The proportions of dead cells were quantitatively measured by the trypan blue exclusion assay, and those of functional cells were assessed by a mitochondrial dehydrogenase assay. The mechanism of cytotoxicity was determined by the acridine orange/ethidium bromide staining and DNA laddering technique. Furthermore, ultrastructural changes were observed by transmission electron microscopy. The results showed that RPE cell damage was dose- and time-dependent. BA 0.225mg/mL, the clinically relevant concentration in TA following intravitreal injection, caused ultrastructural damage and impaired human RPE cell function at 2h; but BA 0.0225mg/mL did not. BA 9.0mg/mL, the concentration in commercial TA suspensions, was toxic within 5min on each assay for both human and rabbit RPE cells. The major mechanism of cell death was necrosis. In conclusion, BA in commercial TA suspensions injected intravitreally (0.225-9mg/mL) can damage RPE cells. Our in vitro study on benzyl alcohol cytotoxicity has significant clinical implications for intravitreal use of TA. We suggest that, before a commercial TA solution is used intravitreally, the vehicle should be removed to prevent damaging the RPE layer, particularly during macular hole surgery. Commercial development of a preservative-free TA suspension for intraocular use is urged.

Cytotoxicity of triamcinolone acetonide on human retinal pigment epithelial cells.

PURPOSE: To investigate the toxic effects of triamcinolone acetonide (TA) suspensions on human retinal pigment epithelial (RPE) cells. METHODS: Cultured human RPE cells were exposed for up to 2 hours to one of seven solutions: control (balanced salt solution, BSS; Alcon Laboratories, Ft. Worth TX), commercial TA suspension (cTA), cTA from which the vehicle (which contains the preservative benzyl alcohol) had been removed (vehicle-removed TA, -vTA), vehicle of the cTA (V), or a 1:10 dilution (in BSS; Alcon) of cTA, -vTA or V. Solution effects were evaluated by phase-contrast microscopy of cells stained in situ with trypan blue and in vitro by trypan blue exclusion assay. RPE cell function was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The mechanism of TA toxicity was studied by acridine orange-ethidium bromide staining and epifluorescence microscopy, and ultrastructural changes were examined by transmission electron microscopy (TEM). RESULTS: The effects of vehicle-removed solutions (-vTA and 1:10 -vTA) were similar to those of the control solution. Exposure for 1 hour or longer to a vehicle-containing solution (cTA and V) resulted in similar and significant degrees of cell damage that were dose and time dependent. The major mechanism of cell death was necrosis, and the early ultrastructural change was swelling of organelles in the cytoplasm. CONCLUSIONS: Preserved commercial TA suspensions damaged human RPE cells, but vehicle-free solutions did not. The authors suggest removing the vehicle as completely as possible from TA solutions before they are administered intravitreally. Furthermore, they recommend that a commercial formulation of preservative-free TA suspension be made available for intraocular use.

Triamcinolone acetonide suspension toxicity to corneal endothelial cells.

PURPOSE: To investigate the cytotoxicity of triamcinolone acetonide (TA) suspensions to corneal endothelial cells (CECs). SETTING: Department of Ophthalmology, Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan. METHODS: New Zealand white rabbit CECs were exposed for 1 minute to balanced salt solution (BSS); commercial TA suspension (cTA); vehicle-removed TA (-vTA); pure vehicle (V); 1/10 dilutions of cTA, -vTA, or V in BSS; or benzyl alcohol (BA) (cTA preservative) 9 mg/mL. Corneal endothelial cell toxicity was assessed by light microscopy (trypan blue staining) and transmission electron microscopy. The effects of 3-, 10-, or 30-minute exposures to 1/10 cTA, 1/10 -vTA, or V were also investigated. RESULTS: One-minute exposures to -vTA or 1/10 -vTA did not damage CECs; however, cTA, V, or 1/10 dilutions of cTA or V caused damage and cells exposed to BA showed severe ultrastructural damage/lysis. A 30-minute exposure to 1/10 -vTA did not cause significant cell damage, whereas 3- to 30-minute exposures to 1/10 cTA or V showed significant time-dependent cytotoxicity. CONCLUSIONS: Commercial TA suspension was cytotoxic to cultured rabbit CECs because of the preservative, BA, in the vehicle. Because 1/10 -vTA appeared to be safe for up to 30 minutes of exposure, use of 1/10 dilutions of vehicle-removed TA is suggested to help surgeons visualize prolapsed vitreous during anterior vitrectomy in complicated cataract surgeries.

Cytotoxicity of lidocaine or bupivacaine on corneal endothelial cells in a rabbit model.

PURPOSE: To investigate the corneal endothelial cytotoxicity of commercial formulations of agents used for intracameral anesthesia in cataract and other ocular surgery. METHODS: Cultured corneal endothelial cells (CECs) of New Zealand White rabbits were exposed for 1 minute to balanced salt solution (control); Xylocaine (lidocaine) 1% E (with epinephrine), 2% E, 2%, or 4%; or Marcaine (bupivacaine) 0.5% or 0.5% spinal heavy. The degree of cytotoxicity was determined by in vitro staining with trypan blue and light microscopic evaluation of cell morphology. The effect of longer exposure (up to 16 minutes) to lidocaine 1% E was also investigated. RESULTS: CECs were not significantly damaged by 1-minute exposure to lidocaine 1% E or 2% E; however, significant cytotoxicity was seen after 1-minute exposure to lidocaine 2% or 4% or bupivacaine 0.5% or 0.5% spinal heavy. Exposure to lidocaine 1% E showed a trend toward time-dependent cytotoxicity that reached significance at 16 minutes. CONCLUSIONS: One-minute exposure to lidocaine 1% E or 2% E appears to be safe for cultured rabbit CECs, although longer exposures could cause time-dependent cytotoxicity, which should be considered in planning cataract or other ocular surgery. Because bupivacaine 0.5% and 0.5% spinal heavy cause cytotoxic effects within the first minute of contact with CECs, they should be used with great caution, if at all, in the anterior chamber of human eyes.

The cleavage plane of corneal epithelial adhesion complex in traumatic recurrent corneal erosion.

PURPOSE: To determine the anatomic cleavage plane of the corneal epithelial adhesion complex in eyes with traumatic recurrent corneal erosion (RCE). METHODS: A loosened sheet of corneal epithelium was obtained from corneal epithelial wounds in eight patients with traumatic RCE, before each patient underwent phototherapeutic keratectomy. Three control groups were employed in the study, including normal corneal epithelial sheets obtained by mechanical separation, normal corneal tissues obtained by partial lamellar keratectomy during pterygial surgery, and corneal tissues from three residual corneoscleral rims of corneas donated for transplantation. Immunofluorescence staining was performed using monoclonal antibodies against integrins beta1 and beta4, laminin 5, and collagen VII to identify abnormalities in specific layers of the adhesion complex. RESULTS: In both experimental and control specimens, the suprabasal and basal cells stained positive for integrin beta1, and basal cells stained positive for integrin beta4. Similarly, a continuous line along the base of each epithelial sheet and each control specimen stained positive for laminin 5, a major basement membrane component. In contrast, in all controls there was a continuous linear staining pattern along the basement membrane of stain positive for collagen VII, a marker for the presence of fibrils that anchor corneal basement membrane to Bowman's layer, but epithelial sheets from eyes with RCE showed either a discontinuous line stained positive for collagen VII (three out of eight specimens) or no positively stained areas (five out of eight specimens). The results indicated the cleavage plane of RCE was located at collagen VII layer, between basement membrane and Bowman's layer. CONCLUSIONS: This study identified a defect in collagen fibrils that anchor the corneal epithelium basement membrane to Bowman's layer as the cause of corneal epithelial loss in cases of traumatic RCE. As hemidesmosomes do not seem to be impaired, a treatment specific to restore anchoring fibril function might prove helpful.