Intravitreal aflibercept for macular oedema secondary to central retinal vein occlusion in patients with prior treatment with bevacizumab or ranibizumab.

PurposeTo report the visual and anatomic outcomes in eyes with macular oedema (MO) secondary to central retinal vein occlusion (CRVO) that were switched from either intravitreal bevacizumab or ranibizumab to intravitreal aflibercept.MethodsTwo-center retrospective chart review. Eyes with MO secondary to CRVO that received a minimum of three intravitreal injections of bevacizumab or ranibizumab and were switched to intravitreal aflibercept for persistent or recurrent MO not responding to either bevacizumab and/or ranibizumab.ResultsIn all 42 eyes of 42 patients were included in the study. The median visual acuity before the switch was 20/126, 1 month after the first injection of aflibercept 20/89 (P=0.0191), and at the end of the follow-up 20/100 (P=0.2724). The median CRT before the switch was 536 µm, 1 month after the first injection of aflibercept 293.5 µm (P=0.0038), and at the end of the follow-up 279 µm (P=0.0013 compared to before the switch). The median number of weeks between injections before the switch was 5.6 and after the switch was 7.6 (P<0.0001).ConclusionConverting eyes with refractory MO due to CRVO to aflibercept can result in stabilization of the vision, improved macular anatomy, and extension of the injection interval.

Membrane-bound and soluble Fas ligands have opposite functions in photoreceptor cell death following separation from the retinal pigment epithelium.

Fas ligand (FasL) triggers apoptosis of Fas-positive cells, and previous reports described FasL-induced cell death of Fas-positive photoreceptors following a retinal detachment. However, as FasL exists in membrane-bound (mFasL) and soluble (sFasL) forms, and is expressed on resident microglia and infiltrating monocyte/macrophages, the current study examined the relative contribution of mFasL and sFasL to photoreceptor cell death after induction of experimental retinal detachment in wild-type, knockout (FasL-/-), and mFasL-only knock-in (ΔCS) mice. Retinal detachment in FasL-/- mice resulted in a significant reduction of photoreceptor cell death. In contrast, ΔCS mice displayed significantly more apoptotic photoreceptor cell death. Photoreceptor loss in ΔCS mice was inhibited by a subretinal injection of recombinant sFasL. Thus, Fas/FasL-triggered cell death accounts for a significant amount of photoreceptor cell loss following the retinal detachment. The function of FasL was dependent upon the form of FasL expressed: mFasL triggered photoreceptor cell death, whereas sFasL protected the retina, indicating that enzyme-mediated cleavage of FasL determines, in part, the extent of vision loss following the retinal detachment. Moreover, it also indicates that treatment with sFasL could significantly reduce photoreceptor cell loss in patients with retinal detachment.

Macrophage- and RIP3-dependent inflammasome activation exacerbates retinal detachment-induced photoreceptor cell death.

Detachment of photoreceptors from the retinal pigment epithelium is seen in various retinal disorders, resulting in photoreceptor death and subsequent vision loss. Cell death results in the release of endogenous molecules that activate molecular platforms containing caspase-1, termed inflammasomes. Inflammasome activation in retinal diseases has been reported in some cases to be protective and in others to be detrimental, causing neuronal cell death. Moreover, the cellular source of inflammasomes in retinal disorders is not clear. Here, we demonstrate that patients with photoreceptor injury by retinal detachment (RD) have increased levels of cleaved IL-1ß, an end product of inflammasome activation. In an animal model of RD, photoreceptor cell death led to activation of endogenous inflammasomes, and this activation was diminished by Rip3 deletion. The major source of Il1b expression was found to be infiltrating macrophages in the subretinal space, rather than dying photoreceptors. Inflammasome inhibition attenuated photoreceptor death after RD. Our data implicate the infiltrating macrophages as a source of damaging inflammasomes after photoreceptor detachment in a RIP3-dependent manner and suggest a novel therapeutic target for treatment of retinal diseases.

Necrotic enlargement of cone photoreceptor cells and the release of high-mobility group box-1 in retinitis pigmentosa.

Retinitis pigmentosa (RP) refers to a group of inherited retinal degenerations resulting form rod and cone photoreceptor cell death. The rod cell death due to deleterious genetic mutations has been shown to occur mainly through apoptosis, whereas the mechanisms and features of the secondary cone cell death have not been fully elucidated. Our previous study showed that the cone cell death in rd10 mice, an animal model of RP, involves necrotic features and is partly mediated by the receptor interacting protein kinase. However, the relevancy of necrotic cone cell death in human RP patients remains unknown. In the present study, we showed that dying cone cells in rd10 mice exhibited cellular enlargement, along with necrotic changes such as cellular swelling and mitochondrial rupture. In human eyes, live imaging of cone cells by adaptive optics scanning laser ophthalmoscopy revealed significantly increased percentages of enlarged cone cells in the RP patients compared with the control subjects. The vitreous of the RP patients contained significantly higher levels of high-mobility group box-1, which is released extracellularly associated with necrotic cell death. These findings suggest that necrotic enlargement of cone cells is involved in the process of cone degeneration, and that necrosis may be a novel target to prevent or delay the loss of cone-mediated central vision in RP.

Mammalian STE20-like kinase 2, not kinase 1, mediates photoreceptor cell death during retinal detachment.

Photoreceptor cell death is the definitive cause of vision loss in retinal detachment (RD). Mammalian STE20-like kinase (MST) is a master regulator of both cell death and proliferation and a critical factor in development and tumorigenesis. However, to date the role of MST in neurodegeneration has not been fully explored. Utilizing MST1(-/-) and MST2(-/-) mice we identified MST2, but not MST1, as a regulator of photoreceptor cell death in a mouse model of RD. MST2(-/-) mice demonstrated significantly decreased photoreceptor cell death and outer nuclear layer (ONL) thinning after RD. Additionally, caspase-3 activation was attenuated in MST2(-/-) mice compared to control mice after RD. The transcription of p53 upregulated modulator of apoptosis (PUMA) and Fas was also reduced in MST2(-/-) mice post-RD. Retinas of MST2(-/-) mice displayed suppressed nuclear relocalization of phosphorylated YAP after RD. Consistent with the reduction of photoreceptor cell death, MST2(-/-) mice showed decreased levels of proinflammatory cytokines such as monocyte chemoattractant protein 1 and interleukin 6 as well as attenuated inflammatory CD11b cell infiltration during the early phase of RD. These results identify MST2, not MST1, as a critical regulator of caspase-mediated photoreceptor cell death in the detached retina and indicate its potential as a future neuroprotection target.

Programmed necrosis, not apoptosis, is a key mediator of cell loss and DAMP-mediated inflammation in dsRNA-induced retinal degeneration.

There is no known treatment for the dry form of an age-related macular degeneration (AMD). Cell death and inflammation are important biological processes thought to have central role in AMD. Here we show that receptor-interacting protein (RIP) kinase mediates necrosis and enhances inflammation in a mouse model of retinal degeneration induced by dsRNA, a component of drusen in AMD. In contrast to photoreceptor-induced apoptosis, subretinal injection of the dsRNA analog poly(I : C) caused necrosis of the retinal pigment epithelium (RPE), as well as macrophage infiltration into the outer retinas. In Rip3(-/-) mice, both necrosis and inflammation were prevented, providing substantial protection against poly(I : C)-induced retinal degeneration. Moreover, after poly(I : C) injection, Rip3(-/-) mice displayed decreased levels of pro-inflammatory cytokines (such as TNF-α and IL-6) in the retina, and attenuated intravitreal release of high-mobility group box-1 (HMGB1), a major damage-associated molecular pattern (DAMP). In vitro, poly(I : C)-induced necrosis were inhibited in Rip3-deficient RPE cells, which in turn suppressed HMGB1 release and dampened TNF-α and IL-6 induction evoked by necrotic supernatants. On the other hand, Rip3 deficiency did not modulate directly TNF-α and IL-6 production after poly(I : C) stimulation in RPE cells or macrophages. Therefore, programmed necrosis is crucial in dsRNA-induced retinal degeneration and may promote inflammation by regulating the release of intracellular DAMPs, suggesting novel therapeutic targets for diseases such as AMD.

An albumin leader sequence coupled with a cleavage site modification enhances the yield of recombinant C-terminal Mullerian Inhibiting Substance.

Mullerian Inhibiting Substance (MIS) has been shown to inhibit ovarian cancer cells both in-vitro and in-vivo. Furthermore, recent evidence suggests that MIS may effectively target a putative ovarian cancer progenitor cell population enriched by a panel of CD44+, CD24+, Ep-CAM+, and E-cadherin-cell surface markers. In order to accommodate clinical testing of MIS in ovarian cancer patients, the production of recombinant human MIS must be optimized to increase yield and purity. Here we show that, compared to wild type, the substitution of the MIS leader sequence to that of human serum albumin, combined with a modification of the endogenous cleavage site from RAQR/S to a furin/kex2 RARR/S consensus site results in high expression, increased C-terminus cleavage and a reduction in unwanted cryptic internal cleavage products when produced in CHO cells. Purified MIS containing these alterations retains its capacity to induce regression of the Mullerian duct in fetal rat embryonic urogenital ridge assays.

Ras activation of the Raf kinase: tyrosine kinase recruitment of the MAP kinase cascade.

A continuing focus of our work has been an effort to understand the signal transduction pathways through which insulin achieves its cellular actions. In the mid-1970s, we and others observed that insulin promoted an increase in Ser/Thr phosphorylation of a subset of cellular proteins. This finding was unanticipated, inasmuch as nearly all of the actions of insulin then known appeared to result from protein dephosphorylation. In fact, nearly 15 years elapsed before any physiologic response to insulin attributable to stimulated (Ser/Thr) phosphorylation was established. Nevertheless, based on the hypothesis that insulin-stimulated Ser/Thr phosphorylation reflected the activation of protein (Ser/Thr) kinases downstream of the insulin receptor, we sought to detect and purify these putative, insulin-responsive protein (Ser/Thr) kinases. Our effort was based on the presumption that an understanding of the mechanism for their activation would provide an entry into the biochemical reactions through which the insulin receptor activated its downstream effectors. To a degree that, in retrospect, is surprising, this goal was accomplished, much in the way originally envisioned. It is now well known that receptor tyrosine kinases (RTKs) recruit a large network of protein (Ser/Thr) kinases to execute their cellular programs. The first of these insulin-activated protein kinase networks to be fully elucidated was the Ras-Raf-mitogen-activated protein kinase (MAPK) cascade. This pathway is a central effector of cellular differentiation in development; moreover, its inappropriate and continuous activation provides a potent promitogenic force and is a very common occurrence in human cancers. Conversely, this pathway contributes minimally, if at all, to insulin's program of metabolic regulation. Nevertheless, the importance of the Ras-MAPK pathway in metazoan biology and human malignancies has impelled us to an ongoing analysis of the functions and regulation of Ras and Raf. This chapter will summarize briefly the way in which work from this and other laboratories on insulin signaling led to the discovery of the mammalian MAP kinase cascade and, in turn, to the identification of unique role of the Raf kinases in RTK activation of this protein (Ser/Thr) kinase cascade. We will then review in more detail current understanding of the biochemical mechanism through which the Ras proto-oncogene, in collaboration with the 14-3-3 protein and other protein kinases, initiates activation of the Raf kinase.

Exercise diminishes the activity of acetyl-CoA carboxylase in human muscle.

Studies in rats suggest that increases in fatty acid oxidation in skeletal muscle during exercise are related to the phosphorylation and inhibition of acetyl-CoA carboxylase (ACC), and secondary to this, a decrease in the concentration of malonyl-CoA. Studies in human muscle have not revealed a consistent decrease in the concentration of malonyl-CoA during exercise; however, measurements of ACC activity have not been reported. Thus, whether the same mechanism operates in human muscle in response to physical activity remains uncertain. To investigate this question, ACC was immunoprecipitated from muscle of human volunteers and its activity assayed in the same individual at rest and after one-legged knee-extensor exercise at 60, 85, and 100% of knee extensor VO2max. ACC activity was diminished by 50-75% during exercise with the magnitude of the decrease generally paralleling exercise intensity. Treatment of the immunoprecipitated enzyme with protein phosphatase 2A restored activity to resting values, suggesting the decrease in activity was due to phosphorylation. The measurement of malonyl-CoA in the muscles revealed that its concentration is 1/10 of that in rats, and that it is diminished (12-17%) during the higher-intensity exercises. The respiratory exchange ratio increased with increasing exercise intensity from 0.84 +/- 0.02 at 60% to 0.99 0.04 at 100% VO2max. Calculated rates of whole-body fatty acid oxidation were 121 mg/min at rest and 258 +/- 35, 264 +/- 63, and 174 +/- 76 mg/min at 60, 85, and 100% VO2max, respectively. The results show that ACC activity, and to a lesser extent malonyl-CoA concentration, in human skeletal muscle decrease during exercise. Although these changes may contribute to the increases in fat oxidation from rest to exercise, they do not appear to explain the shift from mixed fuel to predominantly carbohydrate utilization when exercise intensity is increased.

Cytosolic citrate and malonyl-CoA regulation in rat muscle in vivo.

In liver, insulin and glucose acutely increase the concentration of malonyl-CoA by dephosphorylating and activating acetyl-CoA carboxylase (ACC). In contrast, in incubated rat skeletal muscle, they appear to act by increasing the cytosolic concentration of citrate, an allosteric activator of ACC, as reflected by increases in the whole cell concentrations of citrate and malate [Saha, A. K., D. Vavvas, T. G. Kurowski, A. Apazidis, L. A. Witters, E. Shafrir, and N. B. Ruderman. Am. J. Physiol. 272 (Endocrinol. Metab. 35): E641-E648, 1997]. We report here that sustained increases in plasma insulin and glucose may also increase the concentration of malonyl-CoA in rat skeletal muscle in vivo by this mechanism. Thus 70 and 125% increases in malonyl-CoA induced in skeletal muscle by infusions of glucose for 1 and 4 days, respectively, and a twofold increase in its concentration during a 90-min euglycemic-hyperinsulinemic clamp were all associated with significant increases in the sum of whole cell concentrations of citrate and/or malate. Similar correlations were observed in muscle of the hyperinsulinemic fa/fa rat, in denervated muscle, and in muscle of rats infused with insulin for 5 h. In muscle of 48-h-starved rats 3 and 24 h after refeeding, increases in malonyl-CoA were not accompanied by consistent increases in the concentrations of malate or citrate. However, they were associated with a decrease in the whole cell concentration of long-chain fatty acyl-CoA (LCFA-CoA), an allosteric inhibitor of ACC. The results suggest that increases in the concentration of malonyl-CoA, caused in rat muscle in vivo by sustained increases in plasma insulin and glucose or denervation, may be due to increases in the cytosolic concentration of citrate. In contrast, during refeeding after starvation, the increase in malonyl-CoA in muscle is probably due to another mechanism.

Malonyl-CoA, fuel sensing, and insulin resistance.

Malonyl-CoA is an allosteric inhibitor of carnitine palmitoyltransferase (CPT) I, the enzyme that controls the transfer of long-chain fatty acyl (LCFA)-CoAs into the mitochondria where they are oxidized. In rat skeletal muscle, the formation of malonyl-CoA is regulated acutely (in minutes) by changes in the activity of the beta-isoform of acetyl-CoA carboxylase (ACCbeta). This can occur by at least two mechanisms: one involving cytosolic citrate, an allosteric activator of ACCbeta and a precursor of its substrate cytosolic acetyl-CoA, and the other involving changes in ACCbeta phosphorylation. Increases in cytosolic citrate leading to an increase in the concentration of malonyl-CoA occur when muscle is presented with insulin and glucose, or when it is made inactive by denervation, in keeping with a diminished need for fatty acid oxidation in these situations. Conversely, during exercise, when the need of the muscle cell for fatty acid oxidation is increased, decreases in the ATP/AMP and/or creatine phosphate-to-creatine ratios activate an isoform of an AMP-activated protein kinase (AMPK), which phosphorylates ACCbeta and inhibits both its basal activity and activation by citrate. The central role of cytosolic citrate links this malonyl-CoA regulatory mechanism to the glucose-fatty acid cycle concept of Randle et al. (P. J. Randle, P. B. Garland. C. N. Hales, and E. A. Newsholme. Lancet 1: 785-789, 1963) and to a mechanism by which glucose might autoregulate its own use. A similar citrate-mediated malonyl-CoA regulatory mechanism appears to exist in other tissues, including the pancreatic beta-cell, the heart, and probably the central nervous system. It is our hypothesis that by altering the cytosolic concentrations of LCFA-CoA and diacylglycerol, and secondarily the activity of one or more protein kinase C isoforms, changes in malonyl-CoA provide a link between fuel metabolism and signal transduction in these cells. It is also our hypothesis that dysregulation of the malonyl-CoA regulatory mechanism, if it leads to sustained increases in the concentrations of malonyl-CoA and cytosolic LCFA-CoA, could play a key role in the pathogenesis of insulin resistance in muscle. That it may contribute to abnormalities associated with the insulin resistance syndrome in other tissues and the development of obesity has also been suggested. Studies are clearly needed to test these hypotheses and to explore the notion that exercise and some pharmacological agents that increase insulin sensitivity act via effects on malonyl-CoA and/or cytosolic LCFA-CoA.

Malonyl CoA as a metabolic switch and a regulator of insulin sensitivity.

Malonyl CoA is a regulator of carnitine palmitoyl transferase 1 (CPT1), the enzyme that controls the transfer of long chain fatty acyl CoA into mitochondria where it is oxidized. Recent studies indicate that in skeletal muscle the concentration of malonyl CoA is acutely (minutes) regulated by changes in its fuel supply and energy expenditure. In response to changes in fuel supply, regulation appears to be due to alterations in the cytosolic concentration of citrate, which is both an allosteric activator of acetyl CoA carboxylase (ACC), the enzyme that catalyzes malonyl CoA synthesis and a source of its precursor, cytosolic acetyl CoA. During exercise and immediately thereafter regulation by citrate appears to be lost and malonyl CoA levels diminish as the result of a decrease in ACC activity secondary to phosphorylation. Sustained increases in the concentration of malonyl CoA have been observed in muscle of a number of insulin-resistant rodents including the Zucker (fa/fa) and GK rats, KKAgy mice, glucose-infused rats and rats in which muscle has been made insulin resistant by denervation. Available data suggest that malonyl CoA could be linked to insulin resistance in these rodents by virtue of its effects on the cytosolic concentration of long chain fatty acyl CoA (LCFA CoA) and one or more protein kinase C isozymes. Whether similar alterations occur in other tissues and contribute to the pathophysiology of the insulin resistance syndrome remains to be determined.

Identification of Nore1 as a potential Ras effector.

The small GTP-binding protein Ras is pivotal in transmitting growth and differentiation signals downstream of cell surface receptors. Many observations have indicated that Ras transmits signals from cell surface receptors into multiple pathways via direct interaction with different effectors in mammalian cells. We have identified a novel potential Ras effector or target named Nore1. Nore1 has no significant sequence similarity to known mammalian proteins and lacks an identifiable catalytic domain, but contains sequence motifs that predict DAG_PE binding and SH3 domain binding. We show that Nore1 directly interacts with Ras in vitro in a GTP-dependent manner, and the interaction requires an intact Ras effector domain. Nore1 becomes associated with Ras in situ following activation of epidermal growth factor receptor in COS-7 and in KB cells.

Contraction-induced changes in acetyl-CoA carboxylase and 5'-AMP-activated kinase in skeletal muscle.

The concentration of malonyl-CoA, a negative regulator of fatty acid oxidation, diminishes acutely in contracting skeletal muscle. To determine how this occurs, the activity and properties of acetyl-CoA carboxylase beta (ACC-beta), the skeletal muscle isozyme that catalyzes malonyl-CoA formation, were examined in rat gastrocnemius-soleus muscles at rest and during contractions induced by electrical stimulation of the sciatic nerve. To avoid the problem of contamination of the muscle extract by mitochondrial carboxylases, an assay was developed in which ACC-beta was first purified by immunoprecipitation with a monoclonal antibody. ACC-beta was quantitatively recovered in the immunopellet and exhibited a high sensitivity to citrate (12-fold activation) and a Km for acetyl-CoA (120 microM) similar to that reported for ACC-beta purified by other means. After 5 min of contraction, ACC-beta activity was decreased by 90% despite an apparent increase in the cytosolic concentration of citrate, a positive regulator of ACC. SDS-polyacrylamide gel electrophoresis of both homogenates and immunopellets from these muscles showed a decrease in the electrophoretic mobility of ACC, suggesting that phosphorylation could account for the decrease in ACC activity. In keeping with this notion, citrate activation of ACC purified from contracting muscle was markedly depressed. In addition, homogenization of the muscles in a buffer free of phosphatase inhibitors and containing the phosphatase activators glutamate and MgCl2 or treatment of immunoprecipitated ACC-beta with purified protein phosphatase 2A abolished the decreases in both ACC-beta activity and electrophoretic mobility caused by contraction. The rapid decrease in ACC-beta activity after the onset of contractions (50% by 20 s) and its slow restoration to initial values during recovery (60-90 min) were paralleled temporally by reciprocal changes in the activity of the alpha2 but not the alpha1 isoform of 5'-AMP-activated protein kinase (AMPK). In conclusion, the results suggest that the decrease in ACC activity during muscle contraction is caused by an increase in its phosphorylation, most probably due, at least in part, to activation of the alpha2 isoform of AMPK. They also suggest a dual mechanism for ACC regulation in muscle in which inhibition by phosphorylation takes precedence over activation by citrate. These alterations in ACC and AMPK activity, by diminishing the concentration of malonyl-CoA, could be responsible for the increase in fatty acid oxidation observed in skeletal muscle during exercise.

Malonyl-CoA regulation in skeletal muscle: its link to cell citrate and the glucose-fatty acid cycle.

Malonyl-CoA is an inhibitor of carnitine palmitoyltransferase I, the enzyme that controls the oxidation of fatty acids by regulating their transfer into the mitochondria. Despite this, knowledge of how malonyl-CoA levels are regulated in skeletal muscle, the major site of fatty acid oxidation, is limited. Two- to fivefold increases in malonyl-CoA occur in rat soleus muscles incubated with glucose or glucose plus insulin for 20 min [Saha, A. K., T. G. Kurowski, and N. B. Ruderman. Am. J. Physiol. 269 (Endocrinol. Metab. 32): E283-E289, 1995]. In addition, as reported here, acetoacetate in the presence of glucose increases malonyl-CoA levels in the incubated soleus. The increases in malonyl-CoA in all of these situations correlated closely with increases in the concentration of citrate (r2 = 0.64) and to an even greater extent the sum of citrate plus malate (r2 = 0.90), an antiporter for citrate efflux from the mitochondria. Where measured, no increase in the activity of acetyl-CoA carboxylase (ACC) was found. Inhibition of ATP citrate lyase with hydroxycitrate markedly diminished the increases in malonyl-CoA in these muscles, indicating that citrate was the major substrate for the malonyl-CoA precursor, cytosolic acetyl-CoA. Studies with enzyme purified by immunoprecipitation indicated that the observed increases in citrate could have also allosterically activated ACC. The results suggest that in the presence of glucose, insulin and acetoacetate acutely increase malonyl-CoA levels in the incubated soleus by increasing the cytosolic concentration of citrate. This novel mechanism could complement the glucose-fatty acid cycle in determining how muscle chooses its fuels. It could also provide a means by which glucose acutely modulates signal transduction in muscle and other cells (e.g., the pancreatic beta-cell) in which its metabolism is determined by substrate availability.

Oligomerization activates c-Raf-1 through a Ras-dependent mechanism.

The c-Raf-1 proto-oncoprotein is a Ras-GTP-regulated protein kinase that associates in situ with 14-3-3 proteins, which are naturally dimeric. In COS cells, recombinant Raf is found in oligomeric assemblies. To examine whether induced oligomerization can alter Raf kinase activity, sequences encoding the FK506-binding protein FKBP12 were fused to the amino terminus of c-Raf-1, introducing a binding site for FK506. Oligomerization of recombinant FKBP-Raf in situ, induced by the addition of the dimeric FK506 derivative FK1012A, activated Raf kinase activity at least half as well as epidermal growth factor (EGF). As with EGF, activation of FKBP-Raf by FK1012A is entirely Ras-GTP dependent. Thus oligomerization of Raf per se promotes Raf activation through a Ras-dependent mechanism.