A rare complication of leukostasis with AML M4 patient: Microcirculatory dysfunction of upper extremities.

BACKGROUND: In children with acute myeloid leukemia, the incidence of hyperleukocytosis is 5-33%. Patients with AML and hyperleukocytosis have a higher early mortality rate than patients with nonhyperleukocytic AML because of the increased risk of severe pulmonary and neurologic complications. Leukapheresis provides rapid cytoreduction and reduces early mortality rates. CASE PRESENTATION: In this report, we present a case with microcirculatory failure of upper extremities as a rare symptom of hyperleukocytic AML M4 at initial presentation. CONCLUSIONS: Early diagnosis and treatment of patients with AML admitted to emergency services with these symptoms is too important to prevent from loss of extremities. Most of the complications of hyperleukocytosis can be reversible with early treatment.

The peroxisome proliferator-activated receptor-ß/δ antagonist GSK0660 mitigates retinal cell inflammation and leukostasis.

Diabetic retinopathy (DR) is triggered by retinal cell damage stimulated by the diabetic milieu, including increased levels of intraocular free fatty acids. Free fatty acids may serve as an initiator of inflammatory cytokine release from Müller cells, and the resulting cytokines are potent stimulators of retinal endothelial pathology, such as leukostasis, vascular permeability, and basement membrane thickening. Our previous studies have elucidated a role for peroxisome proliferator-activated receptor-ß/δ (PPARß/δ) in promoting several steps in the pathologic cascade in DR, including angiogenesis and expression of inflammatory mediators. Furthermore, PPARß/δ is a known target of lipid signaling, suggesting a potential role for this transcription factor in fatty acid-induced retinal inflammation. Therefore, we hypothesized that PPARß/δ stimulates both the induction of inflammatory mediators by Müller cells as well the paracrine induction of leukostasis in endothelial cells (EC) by Müller cell inflammatory products. To test this, we used the PPARß/δ inhibitor, GSK0660, in primary human Müller cells (HMC), human retinal microvascular endothelial cells (HRMEC) and mouse retina. We found that palmitic acid (PA) activation of PPARß/δ in HMC leads to the production of pro-angiogenic and/or inflammatory cytokines that may constitute DR-relevant upstream paracrine inflammatory signals to EC and other retinal cells. Downstream, EC transduce these signals and increase their synthesis and release of chemokines such as CCL8 and CXCL10 that regulate leukostasis and other cellular events related to vascular inflammation in DR. Our results indicate that PPARß/δ inhibition mitigates these upstream (MC) as well as downstream (EC) inflammatory signaling events elicited by metabolic stimuli and inflammatory cytokines. Therefore, our data suggest that PPARß/δ inhibition is a potential therapeutic strategy against early DR pathology.

The effect of therapeutic leukapheresis on early complications and outcomes in patients with acute leukemia and hyperleukocytosis: a propensity score-matched study.

BACKGROUND: Hyperleukocytosis in acute leukemia is associated with higher early mortality due to the major complications of leukostasis, tumor lysis syndrome (TLS), and disseminated intravascular coagulopathy (DIC). Leukapheresis remains an important modality for the management of patients with acute leukemia and hyperleukocytosis. However, the role of leukapheresis in early mortality is controversial. This study sought to evaluate the prognostic impact of leukapheresis and its beneficial effects on TLS and DIC. STUDY DESIGN AND METHODS: We conducted a propensity score-matched study of 166 patients with acute leukemia and hyperleukocytosis admitted between 2006 and 2016. The incidence of TLS and DIC was determined using well-defined Cairo-Bishop criteria for TLS and International Society of Thrombosis and Haemostasis criteria for DIC. RESULTS: Before matching, 27 of 91 patients (30%) with acute myeloid leukemia (AML) and 32 of 75 patients (43%) with acute lymphoblastic leukemia (ALL) underwent leukapheresis. Propensity score matching was performed to adjust for clinical disparities between the leukapheresis and without-leukapheresis groups and resulted in 22 matched pairs of patients with AML and 16 matched pairs of patients with ALL. After matching, we observed no significant difference in early mortality rates or in the incidence of TLS or DIC between the two groups of patients with AML and ALL. CONCLUSION: Although leukapheresis may rapidly reduce white blood cell counts and leukemic blasts, any positive influence of leukapheresis could not be demonstrated by an effect on survival outcome and the incidence of early complications, such as TLS and DIC. These results suggest that a routinely performed, prophylactic leukapheresis cannot be recommended.

Fenofibrate ameliorates diabetic retinopathy by modulating Nrf2 signaling and NLRP3 inflammasome activation.

Oxidative stress and neuroinflammation contribute significantly to the development and progression of diabetic retinopathy. Fenofibrate has received great attention as it benefits diabetic patients by reducing retinal laser requirement. Nuclear factor erythroid-2-related factor 2 (Nrf2) is a master regulator of anti-oxidative defense. Activation of nucleotide binding domain, leucine-rich repeat-containing receptor (NLR), pyrin domain-containing 3 (NLRP3) inflammasome plays a pivotal role in neuroinflammation. The purpose of this study is to determine whether fenofibrate protects retinas from oxidative damage and neuroinflammation via modulating the Nrf2 pathway and blocking NLRP3 inflammasome activation during diabetes. Diabetes is induced by intraperitoneal injection of streptozotocin in mice. Fenofibrate was given to mice in rodent chow. Upregulation of Nrf2 and NLRP3 inflammasome, enhanced ROS formation, and increased leukostasis and vascular leakage were observed in diabetic mouse retinas. Notably, Nrf2 and Caspase-1 were mainly colocalized with glutamine synthetase, one of the Mȕller cell markers. Fenofibrate further increased the expression of Nrf2 and its target gene NQO-1 and HO-1 and reduced ROS formation in diabetic retinas. In addition, retinal expression of NLRP3, Caspase-1 p20, IL-1ß p17, and ICAM-1 were dramatically increased in vehicle-treated diabetic mice, which were abolished by fenofibrate intervention. Moreover, fenofibrate treatment also attenuated diabetes-induced retinal leukostasis and vascular leakage in mice. Taken together, fenofibrate attenuates oxidative stress and neuroinflammation in diabetic retinas, which is at least partially through modulating Nrf2 expression and NLRP3 inflammasome activation.

Exchange Transfusion and Leukapheresis in Pediatric Patients with AML With High Risk of Early Death by Bleeding and Leukostasis.

BACKGROUND: The risk of early death (ED) by bleeding/leukostasis is high in patients with AML with hyperleukocytosis (>100,000/µl). Within the pediatric AML-BFM (Berlin-Frankfurt-Münster) 98/04 studies, emergency strategies for these children included exchange transfusion (ET) or leukapheresis (LPh). Risk factors for ED and interventions performed were analyzed. PATIENTS: Two hundred thirty-eight of 1,251 (19%) patients with AML presented with hyperleukocytosis; 23 of 1,251 (1.8%) patients died of bleeding/leukostasis. RESULTS: ED due to bleeding/leukostasis was highest at white blood cell (WBC) count >200,000/µl (14.3%). ED rates were even higher (20%) in patients with FAB (French-American-British) M4/M5 and hyperleukocytosis >200,000/µl. Patients with WBC >200,000/µl did slightly better with ET/LPh compared to those without ET/LPh (ED rate 7.5% vs. 21.2%, P = 0.055). Multivariate WBC >200,000/µl was of strongest prognostic significance for ED (P(χ(2) ) <0.0001). CONCLUSION: Our data confirm the high risk of bleeding/leukostasis in patients with hyperleukocytosis. ET/LPh shows a trend toward reduced ED rate due to bleeding/leukostasis and is recommended at WBC >200,000/µl, and in FAB M4/M5 even at lower WBC.

The effect of puerarin against IL-1ß-mediated leukostasis and apoptosis in retinal capillary endothelial cells (TR-iBRB2).

PURPOSE: Blood-retinal barrier (BRB) breakdown, the early hallmark of diabetic retinopathy (DR), is thought to depend on retinal inflammation and cell damage. The proinflammatory factor interleukin-1ß (IL-1ß) was demonstrated to cause inflammation as well as cell apoptosis during the process of BRB breakdown. This study extensively evaluated the protective effect of puerarin, a major active component extracted from the traditional herb Radix puerariae, against IL-1ß-induced cell dysfunction in TR-iBRB2 cells, a retinal capillary endothelial cell line. METHODS: TR-iBRB2 cells were pretreated with IL-1ß (10 ng/ml) for 24 h and then exposed to puerarin (0, 10, 25, and 50 µM) for another 24 h. Leukocyte endothelial adhesion was assessed through a cell-based assay using lymphoblastoid cells. Cell apoptosis was evaluated with flow cytometry, and the expression of adhesion molecules and apoptosis-related molecules was assessed with western blot analysis. RESULTS: Our data showed that puerarin attenuated IL-1ß-mediated leukostasis and cell apoptosis in TR-iBRB2 cells. Furthermore, puerarin strikingly prevented IL-1ß-induced molecular events of the upstream and downstream signaling pathways involved in this cellular process. CONCLUSIONS: These findings may significantly contribute to better understanding of the protective effect of puerarin, in particular for DR, as well as provide novel insights into the potential application of this compound in DR therapy.

Pazopanib, a multitargeted tyrosine kinase inhibitor, reduces diabetic retinal vascular leukostasis and leakage.

OBJECTIVE: To determine the efficacy of pazopanib eye drops in the streptozotocin induced diabetic retinopathy rat model. METHODS: A 0.5% w/v pazopanib suspension was prepared in phosphate buffered saline (PBS, pH 7.4) in the presence of 0.5% w/v sodium carboxymethyl cellulose. Brown Norway rats were divided into three groups (n=4) - (1) healthy, (2) diabetic, and (3) diabetic with treatment. The drug suspension was administered twice daily as eye drops to group 3 for 30 days. Efficacy parameters including the number of adherent leukocytes in the retinal vasculature (leukostasis), blood-retinal FITC-dextran leakage, and vitreous-to-plasma protein ratio were measured. RESULTS: Pazopanib suspension in the form of eye drops significantly reduced leukostasis (32%), FITC-dextran leakage (39%), and the vitreous-to-plasma protein ratio (64%) in diabetic animals compared to untreated diabetic group. CONCLUSION: Pazopanib eye drops can alleviate retinal complications of diabetic retinopathy.

Adiponectin suppresses pathological microvessel formation in retina through modulation of tumor necrosis factor-alpha expression.

The fat-derived hormone adiponectin has been shown to have a protective role in macrovascular disorders. However, nothing is known about the function of adiponectin in retinal microvessel disease. Here, we investigated the causal role of adiponectin in retinal vessel formation and inflammation under conditions of hypoxia. When neonatal mice were subjected to ischemia-induced retinopathy, pathological retinal neovascularization during ischemia was exacerbated in adiponectin-knockout (APN-KO) mice compared with wild-type mice (neovascular area: 17.0+/-1.0% versus 11.7+/-0.6%, respectively). APN-KO mice also exhibited increased leukocyte adhesion (2.3+/-0.4-fold) and tumor necrosis factor (TNF)-alpha expression (2.6+/-0.2-fold) in hypoxic retina. Adenovirus-mediated overexpression of adiponectin attenuated hypoxia-induced pathological retinal neovascularization by 35% in wild-type mice and by 40% in APN-KO mice and leukostasis by 64% in wild-type mice and by 75% in APN-KO mice, which were associated with reduced TNF-alpha production. TNF-alpha blockade diminished the enhanced pathological neovascularization in APN-KO mice by 34%, and the inhibitory effects of adiponectin overexpression on retinal neovascularization and leukocyte adhesion were abolished in mice lacking TNF-alpha. These data provide evidence that adiponectin protects against retinal vessel injury following pathological stimuli through modulation of TNF-alpha inflammatory responses.

Identification of a retinal aldosterone system and the protective effects of mineralocorticoid receptor antagonism on retinal vascular pathology.

Blockade of the renin-angiotensin-aldosterone system (RAAS) is being evaluated as a treatment for diabetic retinopathy; however, whether the mineralocorticoid receptor (MR) and aldosterone influence retinal vascular pathology is unknown. We examined the effect of MR antagonism on pathological angiogenesis in rats with oxygen-induced retinopathy (OIR). To determine the mechanisms by which the MR and aldosterone may influence retinal angiogenesis; inflammation and glucose-6-phosphate dehydrogenase (G6PD) were evaluated in OIR and cultured bovine retinal endothelial cells (BRECs) and bovine retinal pericytes (BRPs). In OIR, MR antagonism (spironolactone) was antiangiogenic. Aldosterone may mediate the pathogenic actions of MR in the retina, with 11beta-hydroxysteroid dehydrogenase type 2 mRNA being detected and with aldosterone stimulating proliferation and tubulogenesis in BRECs and exacerbating angiogenesis in OIR, which was attenuated with spironolactone. The MR and aldosterone modulated retinal inflammation, with leukostasis and monocyte chemoattractant protein-1 mRNA and protein in OIR being reduced by spironolactone and increased by aldosterone. A reduction in G6PD may be an early response to aldosterone. In BRECs, BRPs, and early OIR, aldosterone reduced G6PD mRNA, and in late OIR, aldosterone increased mRNA for the NAD(P)H oxidase subunit Nox4. A functional retinal MR-aldosterone system was evident with MR expression, translocation of nuclear MR, and aldosterone synthase expression, which was modulated by RAAS blockade. We make the first report that MR and aldosterone influence retinal vasculopathy, which may involve inflammatory and G6PD mechanisms. MR antagonism may be relevant when developing treatments for retinopathies that target the RAAS.

Vascular Expression of Permeability-Resistant Occludin Mutant Preserves Visual Function in Diabetes.

Diabetic retinopathy is one of the leading causes of vision loss and blindness. Extensive preclinical and clinical evidence exists for both vascular and neuronal pathology. However, the relationship of these changes in the neurovascular unit and impact on vision remains to be determined. Here, we investigate the role of tight junction protein occludin phosphorylation at S490 in modulating barrier properties and its impact on visual function. Conditional vascular expression of the phosphorylation-resistant Ser490 to Ala (S490A) form of occludin preserved tight junction organization and reduced vascular endothelial growth factor (VEGF)-induced permeability and edema formation after intraocular injection. In the retinas of streptozotocin-induced diabetic mice, endothelial-specific expression of the S490A form of occludin completely prevented diabetes-induced permeability to labeled dextran and inhibited leukostasis. Importantly, vascular-specific expression of the occludin mutant completely blocked the diabetes-induced decrease in visual acuity and contrast sensitivity. Together, these results reveal that occludin acts to regulate barrier properties downstream of VEGF in a phosphorylation-dependent manner and that loss of inner blood-retinal barrier integrity induced by diabetes contributes to vision loss.

Inhibition of Hsp90 attenuates inflammation in endotoxin-induced uveitis.

Heat shock protein (Hsp) 90 inhibitors, such as 17-allylamino-17-demethoxy-geldanamycin (17-AAG), constitute promising novel therapeutic agents. We investigated the anti-inflammatory activity of 17-AAG in endotoxin-induced uveitis (EIU) in rats. After the induction of EIU with a footpad injection of lipopolysaccharide (LPS), female Lewis rats received a single intraperitoneal. (i.p.) injection of 17-AAG or vehicle. Twenty-four hours later, the retinas were extracted and assayed for leukocyte adhesion; blood-retinal barrier breakdown; VEGF, TNF-alpha, IL-1beta, and CD14 protein levels; NF-kappaB and HIF-1alpha activity; hsp90 and 70 levels and expression and phosphorylation of the tight junction proteins ZO-1 and occludin. 17-AAG treatment significantly suppressed the LPS-induced increase in retinal leukocyte adhesion; vascular leakage; NF-kappaB, HIF-1alpha, p38, and PI-3K activity; and VEGF, TNF-alpha, and IL-1beta levels. 17-AAG also suppressed phosphorylation of ZO-1 and occludin by inhibiting their association with p38 and PI-3K. Although 17-AAG treatment did not reduce the LPS-induced increase in total CD14 levels in leukocytes, it significantly decreased membrane CD14 levels. These data suggest that Hsp90 inhibition suppresses several cardinal manifestations of endotoxin-induced uveitis in the rat. 17-AAG has demonstrated a favorable safety profile in clinical trials in cancer patients and represents a promising therapeutic agent for the treatment of inflammatory eye diseases.

Inhibition of diabetic leukostasis and blood-retinal barrier breakdown with a soluble form of a receptor for advanced glycation end products.

PURPOSE: The interaction of advanced glycation end products (AGEs) with their receptors is hypothesized to be involved in the development of diabetic retinopathy. In the present study, the role of an AGE receptor, RAGE, was investigated in the development of diabetic retinopathy in vivo. METHODS: C57/BJ6 and RAGE-transgenic mice that carried human RAGE genetic DNA under the control of the murine flk-1 promoter were made diabetic with streptozocin. Three months after the onset of diabetes, the soluble form of RAGE (sRAGE) or mouse serum albumin was injected intraperitoneally at 100 mug/d for 14 consecutive days. After the final injection, blood-retinal barrier breakdown, retinal leukostasis, expression of VEGF and ICAM-1, and expression of RAGE in the retina were investigated. RESULTS: Blood-retinal barrier breakdown and increased leukostasis were associated with the experimental diabetes in the C57/BJ6 mice. These changes were significantly augmented in RAGE-transgenic mice. The blood-retinal barrier breakdown and leukostasis in the diabetic C57/BJ6 and RAGE-transgenic mice were accompanied by increased expression of VEGF and ICAM-1 in the retina. The systemic administration of sRAGE significantly inhibited blood-retinal barrier breakdown, leukostasis, and expression of ICAM-1 in the retina in both the diabetic C57/BJ6 and RAGE-transgenic mice. The expression of RAGE was slightly increased in the retinal vessels in diabetic or RAGE-transgenic mice. Furthermore, a strong induction of RAGE was observed in the retinal vessels of diabetic RAGE-transgenic mice. CONCLUSIONS: This study further demonstrates the role of the AGEs and RAGE axis in blood-retinal barrier breakdown and the retinal leukostasis, which are characteristic clinical symptoms of diabetic retinopathy. Furthermore, these data demonstrate that blocking AGE bioactivity may be effective for the treatment of diabetic retinopathy.

Leukostasis and pigment epithelium-derived factor in rat models of diabetic retinopathy.

PURPOSE: Spontaneously diabetic Torii (SDT) rats, an animal model of type 2 diabetes, have a low incidence of neovascular formation and an absence of non-perfused areas in their retinas at the proliferative stage that presents tractional retinal detachment with fibrous proliferation. The aim of this study was to determine whether leukostasis is present in the retina, to evaluate the levels of pigment epithelium-derived factor (PEDF) and intracellular adhesion molecule-1 (ICAM-1) levels in the blood of SDT rats, and to examine the effects of PEDF on leukostasis. METHODS: SDT rats, streptozotocin-induced diabetic (STZ) rats, and control Sprague-Dawley (SD) rats were studied. The index of leukostasis in the retina was determined immunohistochemically by counting the number of labeled adherent leukocytes. The levels of PEDF and the soluble intracellular adhesion molecule (sICAM)-1 in the plasma were measured. To investigate the effect of PEDF and vascular endothelial growth factor (VEGF) on leukostasis, the adhesion of monocytes to human umbilical vein endothelial cells (HUVECs) was assayed in vitro. RESULTS: SDT and STZ diabetic rats showed a significant increase of retinal leukostasis compared to that of control SD rats, but SDT rats had noteworthy lower levels of leukostasis than STZ rats in long term experiments. The sICAM-1 levels and PEDF expression were up-regulated in both STZ and SDT rats, but the SDT rats showed significantly higher levels of PEDF than STZ rats. In vitro studies showed that exposure of HUVECs to VEGF increased the number of adhering monocytes, and PEDF inhibited the VEGF-induced leukostasis in a dose-dependent manner. CONCLUSIONS: The inhibition of the VEGF-induced leukostasis by PEDF is most likely responsible for the low incidence of capillary occlusion and retinal neovascularization in SDT rats.

Captopril inhibits capillary degeneration in the early stages of diabetic retinopathy.

PURPOSE: This study was conducted to examine the effect of angiotensin-converting enzyme (ACE) inhibitors on the development of early stages of diabetic retinopathy. METHODS: Rats were made diabetic by injection of streptozotocin and treated with the ACE inhibitor captopril and the AT1 antagonist losartan. The extent of capillary degeneration and leukostasis in the retina were determined. RESULTS: Acellular capillaries and pericyte ghosts in the retina of diabetic animals were increased by approximately two-fold after 8 months of diabetes compared with the nondiabetic control, and captopril completely inhibited this capillary degeneration. Captopril and losartan also inhibited hyperglycemia-induced leukostasis at 6 weeks and 1 week in the retinal vasculature, respectively. In cultured retinal endothelial cells, angiotensin II-induced VCAM-1 expression was inhibited by losartan. CONCLUSIONS: Inhibition of the renin-angiotensin system can block retinal capillary degeneration and inflammation in the early stages of diabetic retinopathy.

Leukostasis: Management to Prevent Crisis in Acute Leukemia
.

BACKGROUND: Hyperleukocytosis, a peripheral white blood cell count greater than 100,000/mm3,is most commonly seen in patients with newly diagnosed or relapsed acute lymphoblastic leukemia and acute myeloid leukemia. Leukostasis is a reduction in blood flow related to hyperviscosity. Hyperleukocytosis, causing leukostasis, is an oncologic emergency and requires an exacting assessment and rapid response with appropriate intervention to prevent morbidity and mortality in the first week after diagnosis. OBJECTIVES: The objectives of this article are to equip oncology nurse to identify patients with hyperleukocytosis and to provide nursing interventions that will ensure safe, quality care. METHODS: A case study is used to demonstrate key concepts that are critical in early assessment, identification, and treatment of patients with leukostasis.
. FINDINGS: Oncology nurses well versed in the pathophysiology, clinical presentation, and management of leukostasis can make a significant contribution to the safe management of patients with cancer.

Crisis management in the treatment of childhood acute lymphoblastic leukemia: putting right what can go wrong (emergency complications of disease and treatment).

The improvement in overall survival in children with acute lymphoblastic leukemia (ALL) over the last 5 decades has been considerable, with around 90% now surviving long term. The risk of relapse has been reduced to such an extent that the risk of treatment-related mortality is now approaching that of mortality caused by relapse. Toxicities may also lead to the suboptimal delivery of chemotherapy (treatment delays, dose reductions, dose omissions), potentially increasing relapse risk, and short- and long-term morbidity, adding to the "burden of therapy" in an increasing number of survivors. Thus, the need to reduce toxicity in pediatric ALL is becoming increasingly important. This work focuses on the risk factors, pathogenesis, clinical features, and emergency management of the life-threatening complications of ALL at presentation and during subsequent chemotherapy, including leucostasis, tumor lysis syndrome, infection, methotrexate encephalopathy, thrombosis, and pancreatitis. Potential strategies to abrogate these toxicities in the future are also discussed.

Effects of peroxisome proliferator-activated receptor gamma and its ligand on blood-retinal barrier in a streptozotocin-induced diabetic model.

PURPOSE: To clarify whether endogenous peroxisome proliferator-activated receptor gamma (PPARgamma) and its ligand, rosiglitazone, affect retinal leukostasis and the associated vascular leakage using an experimental diabetic model. METHODS: Diabetes was induced in heterozygous PPARgamma+/- mice and Brown Norway rats with an intraperitoneal streptozotocin (STZ) injection. Retinal leukostasis and leakage, quantified by concanavalin A (Con A) lectin perfusion labeling combined with a fluorophotometric dextran leakage assay, were investigated at 120 days in diabetic PPARgamma+/- and wild-type mice and at 21 days in diabetic rats receiving rosiglitazone or the vehicle. The retinal protein expression levels of vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF)-alpha, and the intercellular adhesion molecule (ICAM)-1 were investigated by means of the ELISA assay. RESULTS: In the diabetic PPARgamma+/- mice, retinal leukostasis and leakage were greater than in the diabetic wild-type mice. In addition retinal leukostasis and leakage were suppressed by treatment with rosiglitazone in experimental diabetic rats. ELISA analysis revealed that the upregulated ICAM-1 expression in the diabetic rat retina was reduced by rosiglitazone treatment. CONCLUSIONS: An endogenous pathway involving PPARgamma provides protection against retinal leukostasis and retinal leakage in diabetes and treatment with PPARgamma specific ligands inhibits retinal leukostasis and retinal leakage in diabetic rats.

Intravitreal injection of corticosteroid attenuates leukostasis and vascular leakage in experimental diabetic retina.

PURPOSE: Recently, intravitreal injection of corticosteroids has been in wide use as a treatment for diabetic macular edema, and the outcomes have been favorable. However, the exact mechanism remains unclear. The hypothesis for the current study was that intravitreal corticosteroids may improve diabetic retinal edema by amelioration of blood-retinal barrier (BRB) breakdown, by inhibiting leukocyte stasis (leukostasis). METHODS: Diabetes was induced in 6-week-old male Long-Evans rats by intraperitoneal injection of streptozotocin (75 mg/kg). Three weeks after induction of diabetes, intravitreal injection of dexamethasone (40 microg/10 microL) was performed. At 2 days after intravitreal injection, accumulated leukocytes were counted in vivo by acridine orange leukocyte fluorography, and BRB breakdown was evaluated by measurement of retinal vascular permeability. The mRNA expression and protein levels of intercellular adhesion molecule (ICAM)-1 in the retina were also studied. RESULTS: The number of leukocytes accumulated in the retina, once increased in the diabetic group, was decreased by 31.6% (P = 0.0001) after dexamethasone injection. The level of BRB breakdown, also elevated in the diabetic group, was suppressed by 61.1% (P = 0.0046) after dexamethasone injection. The level of ICAM-1 mRNA expression and its protein, upregulated in the diabetic group, were downregulated by dexamethasone treatment by 70.0% (P < 0.0001) and 56.4% (P = 0.0003). CONCLUSIONS: Intravitreal injection of corticosteroids improves diabetic retinal edema through inhibiting leukocyte recruitment in the diabetic retina.

VEGF164 is proinflammatory in the diabetic retina.

PURPOSE: The objectives of this study were to characterize the differential potency of two major VEGF isoforms, VEGF(120) and VEGF(164), for inducing leukocyte stasis (leukostasis) within the retinal vasculature and blood-retinal barrier (BRB) breakdown and to determine whether endogenous VEGF(164) mediates retinal leukostasis and BRB breakdown in early and established diabetes. METHODS: Retinal leukostasis and BRB breakdown were simultaneously quantified by combining concanavalin A lectin (ConA) perfusion labeling with a fluorophotometric dextran leakage assay. CD45 immunohistochemistry was performed to confirm that ConA-stained cells within the vasculature were leukocytes. Retinal leukostasis and BRB breakdown were compared in nondiabetic rats receiving intravitreous injections of VEGF(120) or VEGF(164). Retinal intercellular adhesion molecule (ICAM)-1 and VEGF protein levels were studied by Western blot and ELISA, respectively. An anti-VEGF(164(165)) aptamer (EYE001) was administered by intravitreous injection to 2-week and 3-month diabetic rats, and the effect on retinal leukostasis and BRB breakdown was quantified. RESULTS: Compared with VEGF(120), VEGF(164) more potently increased retinal ICAM-1 levels (2.2-fold), leukostasis (1.9-fold), and BRB breakdown (2.1-fold, P < 0.01 for all), despite negligible differences in vitreoretinal VEGF levels at the time of evaluation (P > 0.05). Retinal leukostasis and leakage increased with the duration of diabetes (P < 0.01) and correlated closely (P < 0.01, r = 0.889). The isoform-specific blockade of endogenous VEGF(164) with EYE001 resulted in a significant suppression of retinal leukostasis and BRB breakdown in both early (72.4% and 82.6%, respectively) and established (48.5% and 55.0%, respectively) diabetes (P < 0.01). CONCLUSIONS: On an equimolar basis, VEGF(164) is at least twice as potent as VEGF(120) at inducing ICAM-1-mediated retinal leukostasis and BRB breakdown in vivo. The inhibition of diabetic retinal leukostasis and BRB breakdown with EYE001 in early and established diabetes indicates that VEGF(164) is an important isoform in the pathogenesis of early diabetic retinopathy.

Pathogenetic potential of leukocytes in diabetic retinopathy.

Recently, increasing interest has been directed toward the role of leukocytes in microvascular disorders including diabetic retinopathy because of their large cell volume, high cytoplasmic rigidity, natural tendency to stick to the vascular endothelium, and capacity to generate toxic superoxide radicals and proteolytic enzymes. Leukocytes in diabetes are reported to be less deformable and more activated, and may be involved in capillary non-perfusion, endothelial cell damage, and vascular leakage in the retinal microcirculation. In fact, histological studies show many capillary occlusions by leukocytes and capillary dropout or degeneration associated with leukocytes in the diabetic retina. Serial acridine orange leukocyte fluorography and fluorescein angiography studies also identify trapped leukocytes directly associated with areas of downstream non-perfusion in the diabetic retinal microcirculation. More recent studies suggest that adhesion molecules may mediate retinal leukocyte stasis (leukostasis) in diabetes and a reduction in the leukostasis by anti-adhesion antibodies can suppress retinal vascular leakage. In addition, some agents inhibiting leukostasis are reported to improve retinal abnormalities induced by diabetes. Thus, leukostasis in the retinal microcirculation can be a new promising target in the treatment of diabetic retinopathy.