Vassiliki Poulaki

A central role for inflammation in the pathogenesis of diabetic retinopathy

Diabetic retinopathy is a leading cause of adult vision loss and blindness. Much of the retinal damage that characterizes the disease results from retinal vascular leakage and nonperfusion. Diabetic retinal vascular leakage, capillary nonperfusion, and endothelial cell damage are temporary and spatially associated with retinal leukocyte stasis in early experimental diabetes. Retinal leukostasis increases within days of developing diabetes and correlates with the increased expression of retinal intercellular adhesion molecule‐1 (ICAM‐1) and CD18. Mice deficient in the genes encoding for the leukocyte adhesion molecules CD18 and ICAM‐1 were studied in two models of diabetic retinopathy with respect to the long‐term development of retinal vascular lesions. CD18−/− and ICAM‐1−/− mice demonstrate significantly fewer adherent leukocytes in the retinal vasculature at 11 and 15 months after induction of diabetes with STZ. This condition is associated with fewer damaged endothelial cells and lesser vascular leakage. Galactosemia of up to 24 months causes pericyte and endothelial cell loss and formation of acellular capillaries. These changes are significantly reduced in CD18‐ and ICAM‐1‐deficient mice. Basement membrane thickening of the retinal vessels is increased in long‐term galactosemic animals independent of the genetic strain. Here we show that chronic, low‐grade subclinical inflammation is responsible for many of the signature vascular lesions of diabetic retinopathy. These data highlight the central and causal role of adherent leukocytes in the pathogenesis of diabetic retinopathy. They also underscore the potential utility of anti‐inflammatory treatment in diabetic retinopathy.

Molecular sequelae of proteasome inhibition in human multiple myeloma cells

The proteasome inhibitor PS-341 inhibits IκB degradation, prevents NF-κB activation, and induces apoptosis in several types of cancer cells, including chemoresistant multiple myeloma (MM) cells. PS-341 has marked clinical activity even in the setting of relapsed refractory MM. However, PS-341-induced apoptotic cascade(s) are not yet fully defined. By using gene expression profiling, we characterized the molecular sequelae of PS-341 treatment in MM cells and further focused on molecular pathways responsible for the anticancer actions of this promising agent. The transcriptional profile of PS-341-treated cells involved down-regulation of growth/survival signaling pathways, and up-regulation of molecules implicated in proapoptotic cascades (which are both consistent with the proapoptotic effect of proteasome inhibition), as well as up-regulation of heat-shock proteins and ubiquitin/proteasome pathway members (which can correspond to stress responses against proteasome inhibition). Further studies on these pathways showed that PS-341 decreases the levels of several antiapoptotic proteins and triggers a dual apoptotic pathway of mitochondrial cytochrome c release and caspase-9 activation, as well as activation of Jun kinase and a Fas/caspase-8-dependent apoptotic pathway [which is inhibited by a dominant negative (decoy) Fas construct]. Stimulation with IGF-1, as well as overexpression of Bcl-2 or constitutively active Akt in MM cells also modestly attenuates PS-341-induced cell death, whereas inhibitors of the BH3 domain of Bcl-2 family members or the heat-shock protein 90 enhance tumor cell sensitivity to proteasome inhibition. These data provide both insight into the molecular mechanisms of antitumor activity of PS-341 and the rationale for future clinical trials of PS-341, in combination with conventional and novel therapies, to improve patient outcome in MM.

Inhibition of the insulin-like growth factor receptor-1 tyrosine kinase activity as a therapeutic strategy for multiple myeloma, other hematologic malignancies, and solid tumors.

Insulin-like growth factors and their receptor (IGF-1R) have been implicated in cancer pathophysiology. We demonstrate that IGF-1R is universally expressed in various hematologic (multiple myeloma, lymphoma, leukemia) and solid tumor (breast, prostate, lung, colon, thyroid, renal, adrenal cancer, retinoblastoma, and sarcoma) cells. Specific IGF-1R inhibition with neutralizing antibody, antagonistic peptide, or the selective kinase inhibitor NVP-ADW742 has in vitro activity against diverse tumor cell types (particularly multiple myeloma), even those resistant to conventional therapies, and triggers pleiotropic antiproliferative/proapoptotic molecular sequelae, delineated by global transcriptional and proteomic profiling. NVP-ADW742 monotherapy or its combination with cytotoxic chemotherapy had significant antitumor activity in an orthotopic xenograft MM model, providing in vivo proof of principle for therapeutic use of selective IGF-1R inhibitors in cancer.

Activation of NF-kappaB and upregulation of intracellular anti-apoptotic proteins via the IGF-1/Akt signaling in human multiple myeloma cells: therapeutic implications.

Interleukin-6 (IL-6) and insulin-like growth factor-1 (IGF-1) promote the proliferation of multiple myeloma (MM) cells and protect them against dexamethasone (Dex)-induced apoptosis. We have previously shown that Apo2 ligand/TNF-Related apoptosis inducing ligand (Apo2L/TRAIL) induces apoptosis of MM cells, including cells either sensitive or resistant to Dex and cytotoxic drugs, and overcomes the growth and survival effect of IL-6; conversely, NF-κB transcriptional activity attenuates their Apo2L/TRAIL-sensitivity. In the current study, we demonstrate that IGF-1 stimulates sustained activation of NF-κB and Akt; induces phosphorylation of the FKHRL-1 Forkhead transcription factor; upregulates a series of intracellular anti-apoptotic proteins including FLIP, survivin, cIAP-2, A1/Bfl-1, and XIAP; and decreases Apo2L/TRAIL-sensitivity of MM cells. In contrast, IL-6 does not cause sustained NF-κB activation, induces less pronounced Akt activation and FKHRL-1 phosphorylation than IGF-1, and increases the expression of only survivin. Forced overexpression of constitutively active Akt in MM-1S cells reduced their sensitivity to Apo2L/TRAIL and to doxorubicin (Doxo). In contrast, the Akt inhibitor IL-6-Hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate induced cell death of both Dex- and Doxo-sensitive and -resistant cells; opposed the protective effect of constitutive Akt activity against Apo2L/TRAIL; and abrogated the NF-κB activation, increase of anti-apoptotic proteins and protection against Apo2L/TRAIL induced by IGF-1. These findings therefore define an important role of the Akt pathway in modulating tumor cell responsiveness to Apo2L/TRAIL, delineate molecular mechanisms for the survival effects of IGF-1, and characterize differential pathophysiologic sequelae of IGF-1 vs IL-6 on MM cells. Importantly, they provide the basis for future clinical trials in MM combining conventional or novel agents with strategies designed to neutralize IGF-1.

Nonsteroidal anti-inflammatory drugs prevent early diabetic retinopathy via TNF-alpha suppression.

Leukocyte adhesion to the diabetic retinal vasculature results in blood‐retinal barrier breakdown, capillary nonperfusion, and endothelial cell injury and death. Intercellular adhesion molecule‐1 (ICAM‐1) and the leukocyte integrin CD18 are required for these processes. Diabetes was induced in Long Evans rats, resulting in a two‐ to threefold increase in retinal leukocyte adhesion. Following one week of diabetes, neutrophil CD11a, CD11b, and CD18 expression was increased significantly, as were retinal ICAM‐1 levels. Animals were treated with aspirin, a cyclooxygenase 2 (COX‐2) inhibitor (meloxicam), or a soluble tumor necrosis factor α (TNF‐α) receptor/Fc construct (TNFR‐Fc, etanercept). High‐dose aspirin, etanercept, and high‐dose meloxicam each reduced leukocyte adhesion and suppressed blood‐retinal barrier breakdown. High‐dose aspirin also reduced the expression of CD11a, CD11b, and CD18, whereas meloxicam and etanercept did not. High‐dose aspirin, etanercept, and high‐dose meloxicam each reduced retinal ICAM‐1 expression. Aspirin and meloxicam both lowered retinal TNF‐α levels. Notably, aspirin, meloxicam, and etanercept did not change retinal vascular endothelial growth factor levels. High‐dose aspirin, etanercept and high‐dose meloxicam, each suppressed the retinal expression of eNOS and the DNA‐binding capacity of retinal nuclear factor‐κΒ. High‐dose aspirin also suppressed Erk kinase activity, which is involved in CD18 up‐regulation. Taken together, these data identify COX‐2 and TNF‐α as operative in the early signature pathologies of diabetic retinopathy, a newly recognized inflammatory disease.

Retinal Vascular Endothelial Growth Factor Induces Intercellular Adhesion Molecule-1 and Endothelial Nitric Oxide Synthase Expression and Initiates Early Diabetic Retinal Leukocyte Adhesion in Vivo

Leukocyte adhesion to the diabetic retinal vasculature results in early blood-retinal barrier breakdown, capillary nonperfusion, and endothelial cell injury and death. Previous work has shown that intercellular adhesion molecule-1 (ICAM-1) and CD18 are required for these processes. However the relevant in vivo stimuli for ICAM-1 and CD18 expression in diabetes remain unknown. The current study investigated the causal role of endogenous vascular endothelial growth factor (VEGF) and nitric oxide in initiating these events. Diabetes was induced in Long-Evans rats with streptozotocin, resulting in a two- to threefold increase in retinal leukocyte adhesion. Confirmed diabetic animals were treated with a highly specific VEGF-neutralizing Flt-Fc construct (VEGF TrapA(40)). Retinal ICAM-1 mRNA levels in VEGF TrapA(40)-treated diabetic animals were reduced by 83.5% compared to diabetic controls (n = 5, P < 0.0001). VEGF TrapA(40) also potently suppressed diabetic leukocyte adhesion in retinal arterioles (47%, n = 11, P < 0.0001), venules (36%, n = 11, P < 0.0005), and capillaries (36%, n = 11, P < 0.001). The expression of endothelial nitric oxide synthase (eNOS), a downstream mediator of VEGF activity, was increased in diabetic retina, and was potently suppressed with VEGF TrapA(40) treatment (n = 8, P < 0.005). Further, VEGF TrapA(40) reduced the diabetes-related nitric oxide increases in the retinae of diabetic animals. The inhibition of eNOS with N-omega-nitro-L-arginine methyl ester also potently reduced retinal leukocyte adhesion. Although neutrophil CD11a, CD11b, and CD18 levels were increased in 1-week diabetic animals, VEGF TrapA(40) did not alter the expression of these integrin adhesion molecules. Taken together, these data demonstrate that VEGF induces retinal ICAM-1 and eNOS expression and initiates early diabetic retinal leukocyte adhesion in vivo. The inhibition of VEGF bioactivity may prove useful in the treatment of the early diabetic retinopathy.

Acute intensive insulin therapy exacerbates diabetic blood-retinal barrier breakdown via hypoxia-inducible factor-1α and VEGF

Acute intensive insulin therapy is an independent risk factor for diabetic retinopathy. Here we demonstrate that acute intensive insulin therapy markedly increases VEGF mRNA and protein levels in the retinae of diabetic rats. Retinal nuclear extracts from insulin-treated rats contain higher hypoxia-inducible factor-1alpha (HIF-1alpha) levels and demonstrate increased HIF-1alpha-dependent binding to hypoxia-responsive elements in the VEGF promoter. Blood-retinal barrier breakdown is markedly increased with acute intensive insulin therapy but can be reversed by treating animals with a fusion protein containing a soluble form of the VEGF receptor Flt; a control fusion protein has no such protective effect. The insulin-induced retinal HIF-1alpha and VEGF increases and the related blood-retinal barrier breakdown are suppressed by inhibitors of p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositol (PI) 3-kinase, but not inhibitors of p42/p44 MAPK or protein kinase C. Taken together, these findings indicate that acute intensive insulin therapy produces a transient worsening of diabetic blood-retinal barrier breakdown via an HIF-1alpha-mediated increase in retinal VEGF expression. Insulin-induced VEGF expression requires p38 MAPK and PI 3-kinase, whereas hyperglycemia-induced VEGF expression is HIF-1alpha-independent and requires PKC and p42/p44 MAPK. To our knowledge, these data are the first to identify a specific mechanism for the transient worsening of diabetic retinopathy, specifically blood-retinal barrier breakdown, that follows the institution of intensive insulin therapy.

Suppression of diabetic retinopathy with angiopoietin-1.

Diabetic retinopathy remains a leading cause of irreversible blindness. A critical early pathology in the disease is the adhesion of leukocytes to the retinal vasculature, a process that occurs, in part, via intercellular adhesion molecule-1. Once leukocyte adhesion occurs, endothelial cell injury ensues, as does blood-retinal barrier breakdown. Here we show that angiopoietin-1 can prevent and reverse these diabetic retinal vascular changes in both new and established diabetes. Angiopoietin-1, when given intravitreally to newly diabetic rats, normalized retinal vascular endothelial growth factor (VEGF) and intercellular adhesion molecule-1 mRNA and protein levels, leading to reductions in leukocyte adhesion, endothelial cell injury, and blood-retinal barrier breakdown. When an adenovirus coding for angiopoietin-1 was given systemically to mice with established diabetes, it similarly inhibited leukocyte adhesion and endothelial cell injury and blood-retinal barrier breakdown. These changes coincided with reductions in retinal eNOS, nitric oxide, Akt (protein kinase B), and MAP kinase activity, known mediators of VEGF bioactivity and leukocyte adhesion. When endogenous VEGF bioactivity was inhibited with a soluble Flt-1/Fc chimera, retinal Akt kinase activity was significantly reduced in vivo. Taken together, these data document new vascular and anti-inflammatory bioactivities for angiopoietin-1 and identify it as the first naturally occurring protein that directly protects the retinal vasculature in diabetes.

Suppression of Fas-FasL-induced endothelial cell apoptosis prevents diabetic blood–retinal barrier breakdown in a model of streptozotocin-induced diabetes

Diabetic macular edema, resulting from increased microvascular permeability, is the most prevalent cause of vision loss in diabetes. The mechanisms underlying this complication remain poorly understood. In the current study, diabetic vascular permeability (blood‐retinal barrier breakdown) is demonstrated to result from a leukocyte‐mediated Fas‐FasL‐dependent apoptosis of the retinal vasculature. Following the onset of streptozotocin‐induced diabetes, FasL expression was increased in rat neutrophils (P<0.005) and was accompanied by a simultaneous increase in Fas expression in the retinal vasculature. Static adhesion assays demonstrated that neutrophils from diabetic, but not control, rats induced endothelial cell apoptosis in vitro (P<0.005). The latter was inhibited via an antibody‐based FasL blockade (P<0.005). In vivo, the inhibition of FasL potently reduced retinal vascular endothelial cell injury, apoptosis, and blood‐retinal barrier breakdown (P<0.0001) but did not diminish leukocyte adhesion to the diabetic retinal vasculature. Taken together, these data are the first to identify leukocyte‐mediated FasFasL‐dependent retinal endothelial cell apoptosis as a major cause of blood‐retinal barrier breakdown in early diabetes. These data imply that the targeting of the Fas‐FasL pathway may prove beneficial in the treatment of diabetic retinopathy.

Insulin-Like Growth Factor-I Plays a Pathogenetic Role in Diabetic Retinopathy

Diabetic retinopathy is a leading cause of blindness in the Western world. Aberrant intercellular adhesion molecule-1 expression and leukocyte adhesion have been implicated in its pathogenesis, raising the possibility of an underlying chronic inflammatory mechanism. In the current study, the role of insulin-like growth factor (IGF)-I in these processes was investigated. We found that systemic inhibition of IGF-I signaling with a receptor-neutralizing antibody, or with inhibitors of PI-3 kinase (PI-3K), c-Jun kinase (JNK), or Akt, suppressed retinal Akt, JNK, HIF-1alpha, nuclear factor (NF)-kappaB, and AP-1 activity, vascular endothelial growth factor (VEGF) expression, as well as intercellular adhesion molecule-1 levels, leukostasis, and blood-retinal barrier breakdown, in a relevant animal model. Intravitreous administration of IGF-I increased retinal Akt, JNK, HIF-1alpha, NF-kappaB, and AP-1 activity, and VEGF levels. IGF-I stimulated VEGF promoter activity in vitro, mainly via HIF-1alpha, and secondarily via NF-kappaB and AP-1. In conclusion, IGF-I participates in the pathophysiology of diabetic retinopathy by inducing retinal VEGF expression via PI-3K/Akt, HIF-1alpha, NF-kappaB, and secondarily, JNK/AP-1 activation. Taken together, these in vitro and in vivo signaling studies thus identify potential targets for pharmacological intervention to preserve vision in patients with diabetes.